Methods for Producing Engineered Cells and Compositions Thereof

Abstract

The present disclosure provides methods for genetically engineering T cells, e.g., primary CD4+ T cells and / or CD8+ T cells, for use in cell therapy without cell expansion. In certain aspects, the provided methods successfully produce compositions of engineered T cells, e.g., compositions comprising a population of engineered T cells, that express a chimeric antigen receptor (CAR) within a reduced timeframe compared to alternative engineering methods, e.g., methods involving cell expansion. In certain aspects, the provided methods successfully produce compositions of engineered T cells suitable for use in cell therapy within four days from the time the cell stimulation or activation process is initiated. In some aspects, the resulting engineered cell composition is comprised of a cell population that is less differentiated, less exhausted, and more potent than engineered T cell compositions produced by other means, e.g., methods involving cell expansion. Also provided are compositions of T cells produced by the provided methods and their use for treating subjects.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Application No. 62 / 716,981, entitled "Processes for Generating Engineered Cells and Compositions Thereof," filed August 9, 2018; U.S. Provisional Application No. 62 / 742,249, entitled "Processes for Generating Engineered Cells and Compositions Thereof," filed October 5, 2018; U.S. Provisional Application No. 62 / 798,433, entitled "Processes for Generating Engineered Cells and Compositions Thereof," filed January 29, 2019; U.S. Provisional Application No. 62 / 842,402, entitled "Processes for Generating Engineered Cells and Compositions Thereof," filed May 2, 2019; and U.S. Provisional Application No. 62 / 842,402, entitled "Processes for Generating Engineered Cells and Compositions Thereof," filed June 13, 2019. This application claims priority from U.S. Provisional Application No. 62 / 861,308, entitled "Thereof," the contents of which are incorporated herein by reference in their entireties.

[0002] Incorporation by reference of sequence listing This application is filed with an electronic sequence listing. The sequence listing is provided as a file entitled 735042017440SeqList.txt, created on August 5, 2019, and is 93,105 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

[0003] Field The present disclosure provides a method for genetically engineering T cells, e.g., primary CD4+ T cells and / or CD8+ T cells, for use in cell therapy without cell expansion. In certain aspects, the provided methods successfully generate compositions of engineered T cells, e.g., populations of engineered T cells, that express a chimeric antigen receptor (CAR) in a reduced time compared to alternative engineering methods, e.g., methods involving cell expansion. In certain aspects, the provided methods successfully generate compositions of engineered T cells suitable for use in cell therapy within 4 days from when the process of stimulating or activating the cells is initiated. In some aspects, the resulting engineered cell composition is composed of a cell population that is less differentiated, less exhausted, and more potent than engineered T cell compositions generated by other means, e.g., methods involving cell expansion. Also provided are compositions of T cells generated by the provided methods and their use for treating subjects. [Background technology]

[0004] background A variety of cell therapies are available for the treatment of diseases and illnesses. Cell therapies include methods involving immune cells, such as T cells, genetically engineered with recombinant receptors, such as chimeric antigen receptors. However, in some cases, some of the existing methods for generating genetically engineered cell compositions can be time consuming or require different amounts of time to be successfully completed. In certain cases, some of the existing methods can also result in compositions in which the cell population has low efficacy or persistence in vivo. Improved methods for manufacturing and / or engineering such cell therapies are needed, including providing more efficient methods and / or improved cell composition products. Summary of the Invention

[0005] overview Provided herein is a method of producing a composition of engineered T cells, comprising the steps of: (a) exposing an input composition comprising primary T cells to a stimulatory reagent under conditions to stimulate the T cells, thereby generating a stimulated population; (b) introducing into the T cells of the stimulated population a viral vector comprising a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and (c) harvesting the T cells of the transformed population, the harvesting being (i) at a time point between 24 and 120 hours, or between 1 and 5 days, or between about 1 and about 5 days, inclusive, after exposure to the stimulatory reagent has begun; (ii) detecting whether the integrated vector has been integrated into the genome; (iii) at a time before the total number of viable cells at harvest is 3x, 2x or about 3x, about 2x or the same or about the same as the number of total viable cells in the stimulated population; and / or (iv) at a time when the percentage of CD27+CCR7+ is greater than or greater than about 60% among all T cells in the population, among all CD3+ T cells in the population, among all CD4+ T cells in the population, or among all CD8+ T cells in the population or recombinant protein-expressing cells thereof; thereby producing a composition of engineered cells.

[0006] In some embodiments, the input composition is at least 300×10 6 In certain embodiments, the stimulatory reagent is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules.

[0007] Provided herein is a method for producing a composition of engineered T cells, comprising: (a) administering to the patient at least 300×10 6(b) exposing an input composition comprising viable primary T cells to a stimulatory reagent under conditions for stimulating the T cells, thereby generating a stimulated population, wherein the stimulatory reagent is capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules; (b) introducing into the T cells of the stimulated population a viral vector comprising a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and (c) harvesting the T cells of the transformed population, wherein the harvesting is performed within the following steps: (i) 48 to 120 hours or 2 to 5 days or about 2 to 5 days after exposure to the stimulatory reagent has begun. (ii) at about 5 days inclusive; (ii) when integrated vector is detected in the genome but before a stable integrated vector copy number per diploid genome (iVCN) is achieved; (iii) before the total number of viable cells at harvest is 3x, 2x or about 3x, about 2x or the same or about the same as the number of total viable cells in the stimulated population; and / or (iv) when the percentage of naive-like T cells is greater than or greater than about 60% among the total T cells in the population, the total CD3+ cells in the population, the total CD4+ T cells in the population, or the total CD8+ T cells in the population or recombinant protein-expressing cells thereof; thereby producing a composition of engineered cells.

[0008] In some embodiments, harvesting is performed when integrated vector is detected in genome, but before achieving stable iVCN per diploid genome. In certain embodiments, stable iVCN per diploid genome is achieved when iVCN reaches a peak and remains unchanged or changes only within a margin of error for a period of more than 24 hours or 1 day or more than about 24 hours or 1 day. In certain embodiments, stable iVCN per diploid genome is achieved when the fraction of iVCN to total vector copy number (VCN) in diploid genome of a population of transformed cells is 1.0 or about 1.0 on average, or within a margin of error. In some embodiments, harvesting is performed when the fraction of integrated vector copy number (iVCN) to total VCN in a population of transformed cells is less than 0.8 on average.

[0009] In some embodiments, the T cells are harvested at a time point where the percentage of naive-like T cells is greater than or greater than about 60% among the total T cells in the population, the total CD3+ cells in the population, the total CD4+ T cells in the population, or the total CD8+ T cells in the population or cells expressing a recombinant protein thereof. In certain embodiments, the naive-like T cells comprise CD27+CCR7+ T cells.

[0010] Provided herein is a method of producing a composition of engineered T cells, comprising the steps of: (a) exposing an input composition comprising primary T cells to a stimulatory reagent under conditions for stimulating the T cells, thereby generating a stimulated population, wherein the stimulatory reagent is capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules; (b) introducing a viral vector comprising a heterologous polynucleotide encoding a recombinant protein into the T cells of the stimulated population, thereby generating a population of transformed cells; and (c) harvesting the T cells of the transformed population, wherein the harvesting is performed 48-120 hours, or 2-5 days, or about 2-5 days, inclusive, after exposure to the stimulatory reagent has begun, and wherein at harvest, the population of transformed cells has an integrated vector copy number (iVCN) of between 0.4 copies per diploid genome or between about 0.4 copies per diploid genome; thereby producing a composition of engineered cells.

[0011] In some embodiments, harvesting is performed when the iVCN of the population of transformed cells is less than or about 2.0 copies per diploid genome on average. In certain embodiments, harvesting is performed when the iVCN of the population of transformed cells is less than or about 1.5 copies per diploid genome on average. In certain embodiments, harvesting is performed when the iVCN of the population of transformed cells is less than or about 1.0 copies per diploid genome on average. In some embodiments, harvesting is performed when the iVCN of the population of transformed cells is less than or about 0.75 copies per diploid genome on average. In certain embodiments, harvesting is performed when the iVCN of the population of transformed cells is less than or about 0.5 copies per diploid genome on average.

[0012] Provided herein is a method of producing a composition of engineered T cells, comprising the steps of: (a) exposing an input composition comprising primary T cells to a stimulatory reagent under conditions for stimulating the T cells, thereby generating a stimulated population, where the stimulatory reagent is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules; (b) introducing into the T cells of the stimulated population a viral vector comprising a heterologous polynucleotide encoding a recombinant protein. and (c) harvesting the T cells of the transformed population, wherein the harvesting is performed at 48 to 120 hours, inclusive, or 2 to 5 days, or about 2 to about 5 days, inclusive, after exposure to the stimulatory reagent is initiated, and wherein at the time of harvest, the percentage of naive-like cells is greater than or greater than about 60% of the total T cells in the population, of the total CD4+ T cells in the population, or of the total CD8+ T cells in the population, or of cells expressing a recombinant protein thereof; thereby producing a composition of engineered cells.

[0013] In some embodiments, at the time of harvest in the methods provided, the percentage of central memory T cells is greater than or greater than about 60% among the total T cells in the population, the total CD4+ T cells in the population, or the total CD8+ T cells in the population or recombinant protein-expressing cells thereof. In certain embodiments, the central memory T cells are CCR7+CD45RA-.

[0014] Provided herein is a method of producing a composition of engineered T cells, comprising the steps of: (a) exposing an input composition comprising primary T cells to a stimulatory reagent under conditions for stimulating the T cells, thereby generating a stimulated population, wherein the stimulatory reagent is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules; (b) introducing into the T cells of the stimulated population a viral vector comprising a heterologous polynucleotide encoding a recombinant protein, thereby generating a stimulated population of T cells. and (c) harvesting T cells of the transformed population, wherein the harvesting is performed 48 to 120 hours, inclusive, or 2 to 5 days, or about 2 to about 5 days, inclusive, after exposure to the stimulatory reagent is initiated, and wherein at the time of harvest, the percentage of naive-like cells and / or central memory cells is greater than or greater than about 60% of the total T cells in the population, of the total CD4+ T cells in the population, or of the total CD8+ T cells in the population, or of cells expressing a recombinant protein thereof; thereby producing a composition of engineered cells.

[0015] In some embodiments, at the time of harvest, the percentage of naive-like T cells is greater than or greater than about 60% of all recombinant protein-expressing cells in the population, e.g., greater than or greater than about 65%, 70%, 80%, 90% or 95% of all recombinant protein-expressing T cells in the population. In some embodiments, at the time of harvest, the percentage of naive-like T cells is greater than or greater than about 60% of all recombinant protein-expressing CD4+ T cells in the population, e.g., greater than or greater than about 65%, 70%, 80%, 90% or 95% of all recombinant protein-expressing CD4+ cells in the population. In some embodiments, at the time of harvesting, the percentage of naive-like T cells is greater than or about 60% among all recombinant protein-expressing CD8+ T cells in the population, for example, greater than or about 65%, 70%, 80%, 90% or 95% among all recombinant protein-expressing CD8+ cells in the population. In some embodiments, naive-like T cells, or T cells that are surface positive for markers expressed on naive-like T cells, are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+. In some embodiments, naive-like T cells comprise CCR7+CD45RA+. In certain embodiments, naive-like T cells comprise CD27+CCR7+ cells.

[0016] In some embodiments, at the time of harvest, the percentage of CD27+CCR7+ is greater than or about 60% of all recombinant protein-expressing cells in the population, e.g., greater than or about 65%, 70%, 80%, 90% or 95% of all recombinant protein-expressing T cells in the population. In some embodiments, at the time of harvest, the percentage of CD27+CCR7+ is greater than or about 40% of all recombinant protein-expressing CD4+ T cells in the population, e.g., greater than or about 50%, 60%, 70%, 80%, 90% or 95% of all recombinant protein-expressing CD4+ cells in the population. In some embodiments, at the time of harvest, the percentage of CD27+CCR7+ is greater than or greater than about 40% of all recombinant protein-expressing CD8+ T cells in the population, e.g., greater than or greater than about 50%, 60%, 70%, 80%, 90% or 95% of all recombinant protein-expressing CD8+ cells in the population.

[0017] In some embodiments, at the time of harvest, the percentage of naive-like T cells and / or central memory T cells is greater than or greater than about 60% of all recombinant protein-expressing cells in the population, e.g., greater than or greater than about 65%, 70%, 80%, 90% or 95% of all recombinant protein-expressing T cells in the population. In some embodiments, at the time of harvest, the percentage of naive-like T cells and / or central memory T cells is greater than or greater than about 40% of all recombinant protein-expressing CD4+ T cells in the population, e.g., greater than or greater than about 50%, 60%, 70%, 80%, 90% or 95% of all recombinant protein-expressing CD4+ cells in the population. In some embodiments, at the time of harvesting, the percentage of naive-like T cells and / or central memory T cells is greater than or about 40% among all recombinant protein-expressing CD8+ T cells in the population, e.g., greater than or about 50%, 60%, 70%, 80%, 90% or 95% among all recombinant protein-expressing CD8+ cells in the population. In some embodiments, naive-like T cells, or T cells that are surface positive for markers expressed on naive-like T cells, are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+. In some embodiments, naive-like T cells comprise CCR7+CD45RA+. In certain embodiments, naive-like T cells comprise CD27+CCR7+ cells. In certain embodiments, central memory T cells are CCR7+CD45RA-.

[0018] In some embodiments, after the introduction and before harvesting, the method further comprises incubating the population of transformed cells for up to 96 hours or up to 4 days. In certain embodiments, the incubation is carried out at a temperature of about 37° C. In certain embodiments, the incubation is carried out for up to 72 hours or up to 3 days after the introduction. In certain embodiments, the incubation is carried out for up to 48 hours or up to 2 days after the introduction. In some embodiments, the incubation is carried out for up to 24 hours or up to 1 day after the introduction. In certain embodiments, the incubation is carried out for at least 18 hours. In certain embodiments, the incubation is carried out under static conditions. In certain embodiments, static conditions are conditions that do not include centrifugation, shaking, rotation, rocking or perfusion, such as continuous or semi-continuous perfusion of medium.

[0019] In certain embodiments, the harvesting is performed within 96 hours or 4 days after exposure to the stimulant is initiated. In some embodiments, the harvesting is performed within 72 hours or 3 days after exposure to the stimulant is initiated. In certain embodiments, the harvesting is performed within 48 hours or 2 days after exposure to the stimulant is initiated.

[0020] In certain embodiments, one or both of the exposing and introducing are performed in the presence of one or more recombinant cytokines. In some embodiments, the incubation is performed in the presence of one or more recombinant cytokines. In certain embodiments, the incubation is performed in a basal medium lacking one or more recombinant cytokines.

[0021] Provided herein is a method of producing a composition of engineered T cells, comprising the steps of: (a) exposing an input composition comprising primary T cells to a stimulatory reagent under conditions for stimulating the T cells, including the presence of one or more cytokines, thereby generating a stimulated population, where the stimulatory reagent is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules; (b) introducing into the T cells of the stimulated population a viral vector comprising a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells. wherein the introducing is performed in the presence of one or more recombinant cytokines; (c) incubating the population of transformed cells for up to 96 hours or up to 4 days, wherein the incubation is performed at a temperature of about 37° C. and wherein the incubation is performed in a basal medium lacking the one or more recombinant cytokines; and (c) harvesting T cells of the transformed population after incubation, thereby producing a composition of engineered cells, wherein the harvesting is performed 48 to 120 hours or 2 to 5 days, inclusive, after exposure to the stimulatory reagent is initiated, thereby producing a composition of engineered cells.

[0022] In some embodiments, the one or more recombinant cytokines are human. In certain embodiments, the one or more recombinant cytokines are selected from recombinant IL-2, recombinant IL-7, and recombinant IL-15. In certain embodiments, the one or more recombinant cytokines include 10-200 IU / mL recombinant IL-2; 100 IU / mL-1,000 IU / mL recombinant IL-7; and / or 10-200 IU / mL recombinant IL-15. In certain embodiments, the one or more recombinant cytokines are or include recombinant IL-2, recombinant IL-7, and recombinant IL-15. For example, in some cases, the one or more recombinant cytokines include 10-200 IU / mL recombinant IL-2; 100 IU / mL-1,000 IU / mL recombinant IL-7; and 10-200 IU / mL recombinant IL-15.

[0023] In some embodiments, one or both of the exposing and introducing is performed in serum-free medium.

[0024] In certain embodiments, the stimulatory reagent comprises (i) a primary agent that specifically binds to a member of the TCR, and optionally specifically binds to CD3, and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule, optionally where the costimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS. In certain embodiments, one or both of the primary and secondary agents comprise an antibody or antigen-binding fragment thereof. In some embodiments, the primary and secondary agents comprise an antibody or antigen-binding fragment thereof. In certain embodiments, the stimulatory reagent comprises an anti-CD3 antibody and an anti-CD28 antibody.

[0025] In some embodiments, the primary agent and secondary agent are present on or attached to the surface of a solid support. In certain embodiments, the solid support is or comprises beads. In certain embodiments, the ratio of beads:cells is less than 3:1.

[0026] Provided herein is a method of producing a composition of engineered T cells, comprising the steps of: (a) exposing an input composition comprising primary T cells to a stimulatory reagent comprising beads having attached thereto (i) a primary agent that specifically binds a member of the TCR complex, and optionally, CD3, and (ii) a secondary agent that specifically binds a T cell costimulatory molecule, where the ratio of beads:cells is less than 3:1, and the exposure is performed under conditions for stimulating the T cells, thereby producing a stimulated population; (b) introducing into the T cells of the stimulated population a viral vector that comprises a heterologous polynucleotide encoding a recombinant protein, thereby producing a population of transformed cells; and (c) harvesting the T cells of the transformed population, where the harvesting is performed 48-120 hours or 2-5 days, inclusive, after exposure to the stimulatory reagent has begun; thereby producing a composition of engineered cells.

[0027] In some embodiments, the ratio of beads:cells is 1: 1 or about 1: 1. In certain embodiments, the beads have anti-CD3 and anti-CD28 antibodies or antigen-binding fragments thereof attached to them.

[0028] In certain embodiments, the primary and secondary agents are reversibly bound to the surface of an oligomeric particle reagent that comprises a plurality of streptavidin or streptavidin mutein molecules.

[0029] Provided herein is a method of producing a composition of engineered T cells, comprising the steps of: (a) exposing an input composition comprising primary T cells to a stimulatory reagent comprising an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules, the oligomeric particle reagent having (i) a primary agent that specifically binds a member of the TCR complex, and optionally a primary agent that specifically binds CD3, and (ii) a secondary agent that specifically binds a T cell costimulatory molecule, in reversible association, where the exposure is performed under conditions for stimulating the T cells, thereby producing a stimulated population; (b) introducing into the T cells of the stimulated population a viral vector that comprises a heterologous polynucleotide encoding a recombinant protein, thereby producing a population of transformed cells; and (c) harvesting the T cells of the transformed population, where the harvest is performed 48 to 120 hours or 2 to 5 days, inclusive, after exposure to the stimulatory reagent has begun; thereby producing a composition of engineered cells.

[0030] In some embodiments, the exposure comprises 10 cells in the input composition. 6 This is carried out using a stimulation reagent, such as an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules, in an amount of at or about 0.1 μg to 20 μg per particle, inclusive.

[0031] Provided herein is a method for stimulating T cells, comprising: providing an input composition comprising T cells, the input composition comprising at least 10 cells. 6 In some embodiments, the input composition comprises a step of exposing the T cells to a stimulatory reagent in an amount of at or about 0.1 μg to 20 μg per cell, inclusive, the stimulatory reagent comprising an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules, the exposing being performed under conditions for stimulating the T cells, thereby generating a stimulated population.

[0032] Provided herein is a method for stimulating T cells, comprising: providing an input composition comprising T cells, the input composition comprising at least 10 cells. 6 A method comprising exposing the T cells to a stimulatory reagent in an amount of 0.1 μg to 20 μg per particle, inclusive, the stimulatory reagent comprising an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules having attached thereto (i) a primary agent that specifically binds to a member of the TCR complex, and optionally, CD3, and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule, the exposing being performed under conditions for stimulating the T cells, thereby generating a stimulated population. In some embodiments, the input composition comprises primary T cells.

[0033] In some embodiments, after the introduction and before harvesting, the method further comprises incubating the population of transformed cells for up to 96 hours or up to 4 days. In some embodiments, the incubation is carried out at a temperature of about 37° C. In certain embodiments, the incubation is carried out for up to 72 hours or up to 3 days after the introduction. In certain embodiments, the incubation is carried out for up to 48 hours or up to 2 days after the introduction. In some embodiments, the incubation is carried out for up to 24 hours or up to 1 day after the introduction. In certain embodiments, the incubation is carried out under static conditions. In certain embodiments, static conditions are conditions that do not include centrifugation, shaking, rotation, rocking or perfusion, such as continuous or semi-continuous perfusion of medium.

[0034] In certain embodiments, the harvesting is performed within 96 hours or 4 days after the exposure of the input composition to the stimulant is initiated. In certain embodiments, the harvesting is performed within 72 hours or 3 days after the exposure of the input composition to the stimulant is initiated. In some embodiments, the harvesting is performed within 48 hours or 2 days after the exposure of the input composition to the stimulant is initiated.

[0035] In certain embodiments, one or both of the exposing and introducing is performed in the presence of one or more recombinant cytokines.

[0036] In some embodiments, the incubation is performed in the presence of one or more recombinant cytokines. In certain embodiments, the incubation is performed in a basal medium lacking one or more recombinant cytokines.

[0037] Provided herein is a method of producing a composition of engineered T cells, comprising the steps of: (a) exposing an input composition comprising primary T cells to a stimulatory reagent comprising beads having attached thereto (i) a primary agent that specifically binds to a member of the TCR complex, and optionally, CD3, and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule, where the ratio of beads:cells is less than 3:1, and the exposure is performed under conditions for stimulating the T cells, including the presence of one or more recombinant cytokines, thereby producing a stimulated population; (b) introducing into the T cells of the stimulated population a viral vector that comprises a heterologous polynucleotide encoding a recombinant protein, thereby producing a population of transformed cells. (c) incubating the population of transformed cells for up to 96 hours or up to 4 days, optionally wherein the incubation is performed at a temperature of about 37° C. and wherein the incubation is performed in a basal medium lacking one or more recombinant cytokines; and (d) harvesting T cells of the transformed population after incubation, thereby producing a composition of engineered cells, wherein the harvesting is performed 48-120 hours or 2-5 days, inclusive, after exposure to the stimulatory reagent has begun; thereby producing a composition of engineered cells.

[0038] Provided herein is a method of producing a composition of engineered T cells, comprising the steps of: (a) exposing an input composition comprising primary T cells to a stimulation reagent comprising an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules, the oligomeric particle reagent having attached thereto (i) a primary agent that specifically binds to a member of the TCR complex, and optionally, CD3, and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule, the exposure being performed under conditions for stimulating the T cells, including the presence of one or more recombinant cytokines, thereby producing a stimulated population; (b) introducing into the T cells of the stimulated population a viral vector comprising a heterologous polynucleotide encoding a recombinant protein; (c) incubating the population of transformed cells for up to 96 hours or up to 4 days, optionally wherein the incubation is performed at a temperature of about 37° C. and wherein the incubation is performed in a basal medium lacking the one or more recombinant cytokines; and (d) harvesting T cells of the transformed population after incubation, thereby producing a composition of engineered cells, wherein the harvesting is performed 48-120 hours or 2-5 days, inclusive, after exposure to the stimulatory reagent is initiated, thereby producing a composition of engineered cells.

[0039] In some embodiments, exposure to an oligomeric particle stimulation reagent comprising a plurality of streptavidin or streptavidin mutein molecules stimulates cells 10 6 In certain embodiments, the exposure is performed with an amount of stimulation reagent that is between 0.4 μg and 8 μg per cell, inclusive. In certain embodiments, the exposure is performed with an amount of stimulation reagent that is between 0.4 μg and 8 μg per cell, inclusive. 6 In certain embodiments, the exposure is performed with an amount of stimulation reagent that is between 0.8 μg and 4 μg per cell, inclusive. In certain embodiments, the exposure is performed with an amount of stimulation reagent that is between 0.8 μg and 4 μg per cell, inclusive. 6The stimulation is carried out using an amount of stimulation reagent that is at or about 0.8 μg per cell.

[0040] In some embodiments, the one or more recombinant cytokines are human. In some embodiments, the one or more recombinant cytokines are selected from recombinant IL-2, recombinant IL-7, and / or recombinant IL-15. For example, in some cases, the one or more recombinant cytokines include 10-200 IU / mL recombinant IL-2; 100 IU / mL-1,000 IU / mL recombinant IL-7; and / or 10-200 IU / mL recombinant IL-15. In certain embodiments, the one or more recombinant cytokines are or include recombinant IL-2, recombinant IL-7, and recombinant IL-15. For example, in some cases, the one or more recombinant cytokines include 10-200 IU / mL recombinant IL-2; 100 IU / mL-1,000 IU / mL recombinant IL-7; and 10-200 IU / mL recombinant IL-15. In certain embodiments, one or both of the exposing and introducing are performed in serum-free medium. In some embodiments, the incubation is performed in serum-free medium.

[0041] In certain embodiments, the stimulating reagent is a stimulating reagent in which the primary and secondary agents, such as anti-CD3 and anti-CD28 antibodies or antigen-binding fragments thereof, are present on or attached to the surface of the beads. In certain embodiments, the ratio of beads:cells is 2:1 to 0.5:1 or about 2:1 to 0.5:1. In certain embodiments, the ratio of beads:cells is 1:1 or about 1:1. In some embodiments, the beads comprise a diameter greater than or greater than about 3.5 μm and less than or equal to about 9 μm or less than or equal to about 8 μm or less than or equal to about 7 μm or less than or equal to about 6 μm or less than or equal to about 5 μm. In certain embodiments, the beads comprise a diameter of 4.5 μm or about 4.5 μm. In some embodiments, the beads are inert. In certain embodiments, the beads are or comprise a polystyrene surface. In some embodiments, the beads are paramagnetic or superparamagnetic. For example, in some cases, the beads are beads that are attracted to a magnetic field. In some embodiments, prior to harvesting, the method further comprises exposing the cells to a magnetic field after or during incubation, thereby removing the stimulatory reagent from the cells.

[0042] In some embodiments, the stimulating reagent is a stimulating reagent in which a primary agent and a secondary agent, e.g., anti-CD3 and anti-CD28 antibodies or antigen-binding fragments thereof, are reversibly bound to an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules. In certain embodiments, the streptavidin or streptavidin mutein molecules bind or are capable of binding to biotin, avidin, biotin analogs or muteins, avidin analogs or muteins, and / or biologically active fragments thereof. In certain embodiments, each of the plurality of streptavidin mutein molecules comprises the amino acid sequence Va1 at sequence positions corresponding to positions 44-47 relative to the positions in streptavidin in the sequence of amino acids set forth in SEQ ID NO:61. 44 -Thr 45 -Ala 46 -Arg 47 or Ile 44 -Gly 45 -Ala 46-Arg 47 Includes.

[0043] In some embodiments, each of the plurality of streptavidin mutein molecules exhibits: a) a sequence of amino acids set forth in any of SEQ ID NOs: 62, 63, 68, 75-77 or 80-83; b) at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to any of SEQ ID NOs: 62, 63, 68, 75-77 or 80-83; 44 -Thr 45 -Ala 46 -Arg 47 or Ile 44 -Gly 45 -Ala 46 -Arg 47 and / or a sequence of amino acids that reversibly binds to biotin, a biotin analog, or a streptavidin binding peptide; or c) a functional fragment of a) or b) that reversibly binds to biotin, a biotin analog, or a streptavidin binding peptide. In certain embodiments, the plurality of streptavidin mutein molecules comprises a sequence of amino acids set forth in SEQ ID NO: 63 or 68.

[0044] In certain embodiments, the primary and secondary agents reversibly bound to the surface of the oligomeric particle reagent each comprise a streptavidin-binding peptide. In some embodiments, the streptavidin-binding peptide is TIFF0007620542000001.tif37163.

[0045] In certain embodiments, one or both of the primary and secondary agents comprise an antibody or an antigen-binding fragment thereof. In certain embodiments, one or more of the primary / secondary agents is or comprises a monovalent antibody fragment. In some embodiments, one or both of the primary and secondary agents is or comprises a Fab.

[0046] In some embodiments, the oligomeric particle reagent comprises a radius greater than 60 nm, greater than 70 nm, greater than 80 nm, or greater than 90 nm. In some embodiments, the oligomeric particle reagent comprises a radius between 50 nm and 150 nm, between 75 nm and 125 nm, between 80 nm and 115 nm, or between 90 nm and 110 nm, inclusive; or a radius of 90 nm ± 15 nm or 95 nm ± 20-25 nm.

[0047] In some embodiments, the oligomeric particle reagent comprises at least 5×10 7 g / mol or at least 1×10 8 g / mol; and / or 5 × 10 7 g / mol~5×10 8 g / mol, 1×10 8 g / mol~5×10 8 g / mol or 1×10 8 g / mol~2×10 8 Includes molecular weight in g / mol.

[0048] In certain embodiments, the oligomeric particle reagent comprises at least 500 streptavidin or streptavidin mutein tetramers, at least 1,000 streptavidin or streptavidin mutein tetramers, at least 1,500 streptavidin or streptavidin mutein tetramers or at least 2,000 streptavidin or streptavidin mutein tetramers; and / or between 1,000 and 20,000 streptavidin or streptavidin mutein tetramers, between 1,000 and 10,000 streptavidin or streptavidin mutein tetramers or between 2,000 and 5,000 streptavidin or streptavidin mutein tetramers.

[0049] In some embodiments, the method further comprises adding a substance to the cells after or during at least a portion of the incubation, the substance being capable of disrupting the binding between the primary and secondary agents, e.g., anti-CD3 and anti-CD28 antibodies or antigen-binding fragments thereof, and the oligomeric particle reagent. In certain embodiments, the substance is a free binding partner and / or a competitor. In certain embodiments, the presence of the substance terminates or reduces the signal induced or modulated by the primary and secondary agents in the T cells. In some embodiments, the substance is or comprises a streptavidin-binding peptide, biotin or a biologically active fragment, or a biotin analog or a biologically active fragment. In certain embodiments, the substance is or comprises a streptavidin-binding peptide, the streptavidin-binding peptide being TIFF0007620542000002.tif37162. In some embodiments, the agent is or comprises biotin or a biologically active fragment or a biotin analog or a biologically active fragment.

[0050] In certain embodiments, the substance is added 42 hours to 120 hours or about 42 hours to 120 hours, inclusive, after exposure to the stimulating reagent comprising oligomeric particles is initiated. In certain embodiments, the substance is added 48 hours to 120 hours or about 48 hours to 120 hours, or 2 days to 5 days, inclusive, after exposure to the oligomeric particle reagent is initiated. In certain embodiments, the substance is added 72 hours to 96 hours or about 72 hours to 96 hours, or 3 days to 4 days, inclusive, after exposure to the oligomeric particle reagent is initiated. In some embodiments, the substance is added 48 hours or about 48 hours, or 2 days to 2 days, inclusive, after exposure to the oligomeric particle reagent is initiated. In certain embodiments, the substance is added 72 hours or about 72 hours, or 3 days to 3 days, inclusive, after exposure to the oligomeric particle reagent is initiated. In certain embodiments, the agent is added at or about 96 hours or 4 days or about 4 days after exposure to the oligomeric particle reagent has begun.

[0051] In some embodiments, the substance is added after incubation and before harvesting. In certain embodiments, the substance is added during at least a portion of the incubation, and the incubation continues after the substance is added. In certain embodiments, after the substance is added, the incubation is performed in the presence of basal medium lacking recombinant cytokines.

[0052] Provided herein is a method for producing a composition of engineered T cells, comprising: (a) mixing an input composition comprising primary T cells with an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules, the oligomeric particle reagent comprising: 6 exposing the T cells of the stimulated population to at or about 0.02 μg to 8 μg per T cell of a stimulatory reagent, the oligomeric particle reagent having attached thereto (i) a primary agent that specifically binds to CD3 and (ii) a secondary agent that specifically binds to CD28, the exposure being carried out in the presence of serum-free medium having recombinant IL-2, IL-7 and IL-15, thereby generating a stimulated population; (b) introducing into the T cells of the stimulated population a viral vector comprising a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells, the introduction being carried out in the presence of serum-free medium having recombinant IL-2, IL-7 and IL-15; (c) subjecting the population of transformed cells to stasis. and (d) harvesting the T cells of the transformed population after incubation, wherein the harvesting is performed 72 to 96 hours or 3 to 4 days, inclusive, after exposure to the stimulatory agent was initiated, thereby producing a composition of engineered cells.

[0053] In some embodiments, the input composition is at least 300×10 6 In some embodiments, the number of primary T cells in the input composition is 450×10 6 pcs or approx. 450 x 10 6 viable primary T cells, 500 x 10 6 pcs or approx. 500 x 10 6 viable primary T cells, 550 x 10 6 pcs or approx. 550 x 10 6 viable primary T cells, 600 x 10 6 pcs or approx. 600 x 10 6 viable primary T cells, 700 x 10 6 pcs or approx. 700 x 10 6 viable primary T cells, 800 x 10 6 800 x 10 6 viable primary T cells, 900 x 10 6 pcs or approx. 900 x 10 6 1,000 x 10 viable primary T cells or 1,000 x 10 6 Pieces or about 1,000 x 10 6 In certain embodiments, the input composition is 600×10 6 pcs or approx. 600 x 10 6 In certain embodiments, the number of primary T cells in the input composition is up to 900 x 10 6 10 viable primary T cells or 900 x 10 6 pcs or approx. 900 x 10 6 These are viable primary T cells.

[0054] In some embodiments, the primary T cells in the input composition include primary CD3+ T cells, or primary CD4+ T cells and / or primary CD8+ T cells. In some embodiments, the cells in the input composition are enriched, such as by selection or isolation, from a biological sample from a subject. In some embodiments, the primary T cells are enriched in primary CD3+ T cells isolated or selected, such as by immunoaffinity-based selection for CD3+ T cells from a biological sample. In some embodiments, the primary T cells are enriched in primary CD4+ T cells isolated or selected, such as by immunoaffinity-based selection for primary CD4+ T cells from a biological sample. In some embodiments, the primary T cells are enriched in primary CD8+ T cells isolated or selected, such as by immunoaffinity-based selection for CD8+ T cells from a biological sample. In some embodiments, the primary T cells are enriched primary CD4+ T cells and enriched primary CD8+ T cells isolated or selected, such as by immunoaffinity-based selection for CD4+ T cells and CD8+ T cells from a biological sample.

[0055] In certain embodiments, the input composition comprises a ratio of CD4+ cells:CD8+ cells of 1.5:1 to 2.0:1. In some embodiments, the input composition comprises a ratio of CD4+ cells:CD8+ cells of 1.2:1 to 0.8:1. In some embodiments, the ratio is at or about 1:1 CD4+ T cells:CD8+ T cells.

[0056] Provided herein is a method for producing a composition of engineered T cells, comprising: (a) mixing an input composition comprising primary T cells with an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules, the oligomeric particle reagent comprising: 6and exposing the oligomeric particle reagent to at or about 0.4 μg to 8 μg of stimulatory reagent, the oligomeric particle reagent having attached thereto (i) a primary agent that specifically binds to CD3 and (ii) a secondary agent that specifically binds to CD28, the input composition comprising a ratio of CD4+ cells:CD8+ T cells of 2:1 to 1:2, and at least 300×10 6(b) introducing into the stimulated population of T cells a viral vector comprising a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells, wherein the introduction is carried out in the presence of a serum-free medium having recombinant IL-2, IL-7 and IL-15; (c) incubating the population of transformed cells under static conditions for at or about 24 hours to 72 hours, 1 day to 3 days, or about 1 day to 3 days, wherein optionally the incubation is carried out at a temperature of about 37° C. and (d) during at least a portion of the incubation, adding a substance comprising biotin or a biologically active fragment thereof, optionally D-biotin or a biotin analog or a biologically active fragment thereof, the addition occurring 48-72 hours or 2-3 days, inclusive, after exposure to the stimulatory reagent is initiated; and (d) harvesting the T cells of the transformed population after the incubation, the harvesting occurring 72-96 hours or 3-4 days, inclusive, after exposure to the stimulatory reagent is initiated, thereby producing a composition of engineered cells. In some embodiments, the primary T cells comprise a ratio of CD4+ T cells:CD8+ T cells of 1:1 or about 1:1. In some embodiments, the substance is added 48 hours or about 48 hours or 2 days or about 2 days after exposure to the oligomeric particle reagent is initiated. In certain embodiments, the substance is added at or about 72 hours or 3 days after exposure to the oligomeric particle reagent has begun. In certain embodiments, the substance is added at or about 96 hours or 4 days after exposure to the oligomeric particle reagent has begun.

[0057] Provided herein is a method for producing a composition of engineered T cells, comprising: (a) administering to the patient at least 300×10 6exposing an input composition comprising primary T cells to a stimulation reagent comprising paramagnetic beads at a ratio of beads:cells of 2:1 to 0.5:1 or about 2:1 to 0.5:1, the beads being inert paramagnetic beads comprising a polystyrene coating and a diameter of 4.5 μm or about 4.5 μm, the beads having (i) an anti-CD3 antibody or antigen-binding fragment thereof and an anti-CD28 antibody or antigen-binding fragment thereof attached thereto, the input composition comprising a ratio of CD4+ T cells:CD8+ T cells of 2:1 to 1:2, and at least 300×10 6 (b) introducing a viral vector comprising a heterologous polynucleotide encoding a recombinant protein into the T cells of the stimulated population, thereby generating a population of transformed cells, wherein the introduction is performed in the presence of serum-free medium with recombinant IL-2, IL-7 and IL-15; (c) incubating the population of transformed cells under static conditions for 48-72 hours or 2-3 days, inclusive, optionally wherein the incubation is performed at a temperature of about 37° C.; (d) after incubation, exposing the cells to a magnetic field to remove the stimulatory reagent from the cells and then harvesting the T cells, thereby producing a composition of engineered cells, wherein the harvesting is performed 72-96 hours or about 3-4 days, inclusive, after exposure to the stimulatory reagent has begun; thereby producing a composition of engineered cells. In some embodiments, the primary T cells comprise a 1:1 or about 1:1 ratio of CD4+ T cells:CD8+ T cells.

[0058] In some embodiments, the number of primary T cells in the input composition is 450×10 6 pcs or approx. 450 x 10 6 viable primary T cells, 500 x 10 6 pcs or approx. 500 x 10 6 viable primary T cells, 550 x 10 6 pcs or approx. 550 x 10 6viable primary T cells, 600 x 10 6 pcs or approx. 600 x 10 6 viable primary T cells, 700 x 10 6 pcs or approx. 700 x 10 6 viable primary T cells, 800 x 10 6 800 x 10 6 viable primary T cells, 900 x 10 6 pcs or approx. 900 x 10 6 1,000 x 10 viable primary T cells or 1,000 x 10 6 Pieces or about 1,000 x 10 6 In certain embodiments, the input composition is 600×10 6 pcs or approx. 600 x 10 6 In certain embodiments, the number of cells in the input composition is up to 900 x 10 6 10 viable primary T cells or 900 x 10 6 pcs or approx. 900 x 10 6 These are viable primary T cells.

[0059] In some embodiments, at least a portion of the incubation is carried out in the presence of serum-free medium comprising recombinant IL-2, IL-7 and IL-15. In certain embodiments, the incubation is carried out in a basal medium lacking recombinant cytokines. In certain embodiments, the incubation is carried out 24 hours or about 24 hours or 1 day or about 1 day after the introduction. In some embodiments, the incubation is carried out 48 hours or about 48 hours or 2 days or about 2 days after the introduction. In certain embodiments, the incubation is carried out 72 hours or about 72 hours or 3 days or about 3 days after the introduction.

[0060] In some embodiments, prior to exposure to the stimulatory reagent, the method includes enriching CD3+ T cells from the biological sample, where enrichment is selecting for cells that are CD3+ in a closed system, thereby generating a selected population. In some cases, enrichment is a first selection, and the cells and the first selection are further selected for cells that are CD3+ to further enrich for the selection, thereby generating a second selected population. In some embodiments, the input composition includes enriched CD3+ cells. In some embodiments, the input composition includes enriched CD3+ cells from the first selection. In some embodiments, the input composition includes enriched CD3+ cells from the second selection.

[0061] In certain embodiments, the enrichment of cells comprises immunoaffinity-based selection. In some embodiments, the immunoaffinity-based selection is carried out by contacting cells with an antibody immobilized on or attached to an affinity chromatography matrix, the antibody being capable of specifically binding to a cell surface marker on the cells. In some embodiments, the immunoaffinity-based selection is a positive selection of CD3+ T cells. In certain embodiments, the method further comprises contacting the cells in the sample with the affinity chromatography matrix, followed by eluting the cells bound to the antibody, thereby recovering the selected cells from the matrix.

[0062] In some embodiments, prior to exposure to the stimulatory reagent, the method comprises enriching CD4+ or CD8+ T cells from the biological sample, the enrichment comprising: (a) performing a first selection in a closed system, the first selection comprising enrichment of one of (i) CD4+ cells, and (ii) CD8+ cells, from the sample comprising primary human T cells, thereby producing a first selected population and a non-selected population; (b) performing a second selection in the closed system, the second selection comprising enrichment of the other of (i) CD4+ cells, and (ii) CD8+ cells, from the non-selected population, thereby producing a second selected population, the enrichment producing separate enriched populations of CD4+ T cells and CD8+ T cells, the separate enriched populations each comprising cells from one of the first selected population or the second selected population, and the input composition comprising cells from one or more of the enriched population of CD4+ T cells and the enriched population of CD8+ T cells.

[0063] In some embodiments, the separate enriched populations of CD4+ T cells and CD8+ T cells are mixed, thereby producing an input composition. In certain embodiments, the mixing is performed in a closed system, and optionally, the closed system is automated. In certain embodiments, the enrichment of cells in the first and / or second selection comprises performing positive or negative selection based on the expression of cell surface markers. In some embodiments, the enrichment of cells in the first or second selection comprises performing multiple positive or negative selection steps based on the expression of cell surface markers or markers for enriching CD4+ or CD8+ cells.

[0064] In certain embodiments, enrichment of cells in the first and / or second selection comprises immunoaffinity-based selection. In some embodiments, immunoaffinity-based selection is performed by contacting cells with an antibody immobilized on or attached to an affinity chromatography matrix, which can specifically bind to a cell surface marker on the cells to perform positive or negative selection of CD4+ or CD8+ cells. In certain embodiments, the method further comprises a step of eluting the antibody-bound cells after contacting cells in the sample with the affinity chromatography matrix in the first and / or second selection, thereby recovering the selected cells from the matrix. In certain embodiments, enrichment of CD4+ or CD8+ T cells from the biological sample comprises (a) enriching CD4+ cells by positive selection based on surface expression of CD4; (b) enriching CD8+ cells by positive selection based on surface expression of CD8. In some embodiments, both (a) and (b).

[0065] In some embodiments, immunoaffinity-based selection on an affinity chromatography matrix is ​​performed using an antibody that further comprises one or more binding partners capable of forming a reversible bond with a binding reagent immobilized on the matrix, and the antibody reversibly binds to the chromatography matrix during the contact. In such embodiments, cells expressing a cell surface marker on the matrix to which the antibody specifically binds can be retrieved from the matrix by cleavage of the reversible bond between the binding reagent and the binding partner. In some embodiments, the binding partner is selected from among biotin, biotin analogs and peptides capable of binding to the binding reagent, and the binding reagent is selected from among streptavidin, streptavidin analogs or muteins, avidin and avidin analogs or muteins. In some embodiments, the binding partner comprises the sequence of amino acids set forth in SEQ ID NO: 64; and / or the binding reagent is a streptavidin mutein comprising the sequence of amino acids set forth in SEQ ID NO: 75, 80, 76 or 63. In some embodiments, after contacting the cells in the sample with the affinity chromatography matrix, the method of enriching the cells includes adding a competing reagent to cleave the bond between the binding partner and the binding reagent, thereby recovering the selected cells from the matrix. In some embodiments, the competing reagent is biotin or a biotin analog. In some embodiments, the antibody in the selection is at least 3×10 -5 sec -1 or about 3 x 10 -5 sec -1 The binding and dissociation rate constants (k off In some embodiments, the antibody in the selection has a -3 ~10 -7 in the range of about 10 -7 ~about 10 -10 Dissociation constants (K d In some embodiments, the chromatography matrix for selection is packed into a separation vessel that is a column.

[0066] In some embodiments, prior to exposure to the stimulatory reagent, the method includes enriching CD4+ or CD8+ T cells from the biological sample, the enrichment comprising contacting the cells of the sample with a first immunoaffinity reagent that specifically binds CD4 in an incubation composition and a second immunoaffinity reagent that specifically binds CD8 in an incubation composition under conditions in which the immunoaffinity reagents specifically bind to CD4 and CD8 molecules on the surface of the cells in the sample, respectively; and recovering the cells bound to the first and / or second immunoaffinity reagent, thereby producing an enriched composition comprising CD4+ cells and CD8+ cells, the input composition comprising the cells of the enriched composition comprising CD4+ cells and CD8+ cells. In some embodiments, each of the immunoaffinity reagents comprises an antibody or an antigen-binding fragment thereof. In certain embodiments, the immunoaffinity reagents are immobilized on the outer surface of beads. In certain embodiments, the beads are magnetic beads. In some embodiments, recovering the cells bound to the first or second immunoaffinity reagent comprises exposing the cells to a magnetic field.

[0067] In certain embodiments, the biological sample comprises primary T cells obtained from a subject, optionally a human subject. In certain embodiments, the biological sample is or comprises a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukapheresis product. In some embodiments, the biological sample is an apheresis product or a leukapheresis product that has been previously cryo-frozen before enrichment.

[0068] In certain embodiments, the harvest is performed 48 hours or about 48 hours or 2 days or about 2 days after exposure to the stimulating reagent is initiated. In some embodiments, the harvest is performed 72 hours or about 72 hours or 3 days or about 3 days after exposure to the stimulating reagent is initiated. In certain embodiments, the harvest is performed 48 hours or about 48 hours or 2 days or about 2 days after exposure to the stimulating reagent is initiated.

[0069] In certain embodiments, harvesting is performed when integrated vector is detected in genome, but before achieving stable iVCN per diploid genome.In some embodiments, harvesting is performed when the ratio of integrated vector copy number (iVCN) to total VCN in the population of transformed cells is less than 0.8 on average.In certain embodiments, harvesting is performed when the iVCN of the population of transformed cells is less than 2.0 copies or less than about 2.0 copies per diploid genome on average.

[0070] In some embodiments, the harvesting is performed when the iVCN of the population of transformed cells is less than or about 1.5 copies per diploid genome on average. In certain embodiments, the harvesting is performed when the iVCN of the population of transformed cells is less than or about 1.0 copies per diploid genome on average. In certain embodiments, the harvesting is performed when the iVCN of the population of transformed cells is less than or about 0.75 copies per diploid genome on average. In some embodiments, the harvesting is performed when the iVCN of the population of transformed cells is less than or about 0.5 copies per diploid genome on average. In certain embodiments, the harvesting of the cells includes rinsing or washing the cells to remove cell debris. In certain embodiments, the cells are harvested when the percentage of naive-like cells is greater than or about 60% of the total T cells in the population, the total CD4+ T cells in the population, or the total CD8+ T cells in the population or recombinant protein-expressing cells thereof. In some embodiments, the cells are harvested at a time when the percentage of naive-like T cells and / or central memory T cells is greater than or greater than about 60% of the total T cells in the population, the total CD4+ T cells in the population, or the total CD8+ T cells in the population or recombinant protein-expressing cells thereof. In some embodiments, at the time of harvest, the percentage of naive-like T cells is greater than or greater than about 60% of the total recombinant protein-expressing cells in the population, e.g., greater than or greater than about 65%, 70%, 80%, 90% or 95% of the total recombinant protein-expressing T cells in the population. In some embodiments, at the time of harvest, the percentage of naive-like T cells is greater than or greater than about 60% among all recombinant protein-expressing CD4+ T cells in the population, e.g., greater than or greater than about 65%, 70%, 80%, 90% or 95% among all recombinant protein-expressing CD4+ cells in the population.In some embodiments, at the time of harvesting, the percentage of naive-like T cells is greater than or about 60% among all recombinant protein-expressing CD8+ T cells in the population, for example, greater than or about 65%, 70%, 80%, 90% or 95% among all recombinant protein-expressing CD8+ cells in the population. In some embodiments, naive-like T cells, or T cells that are surface positive for markers expressed on naive-like T cells, are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+. In some embodiments, naive-like T cells comprise CCR7+CD45RA+. In certain embodiments, naive-like T cells comprise CD27+CCR7+ cells.

[0071] In some embodiments, at the time of harvest, the percentage of naive-like T cells and / or central memory T cells is greater than or greater than about 60% of the total recombinant protein-expressing cells in the population, e.g., greater than or greater than about 65%, 70%, 80%, 90% or 95% of the total recombinant protein-expressing T cells in the population. In some embodiments, the percentage of naive-like T cells and / or central memory T cells is greater than or greater than about 60% of the total recombinant protein-expressing CD4+ T cells in the population, e.g., greater than or greater than about 65%, 70%, 80%, 90% or 95% of the total recombinant protein-expressing CD4+ cells in the population. In some embodiments, the percentage of naive-like T cells and / or central memory T cells is greater than or about 60% among all recombinant protein-expressing CD8+ T cells in the population, e.g., greater than or about 65%, 70%, 80%, 90% or 95% among all recombinant protein-expressing CD8+ cells in the population. In some embodiments, naive-like T cells, or T cells that are surface positive for markers expressed on naive-like T cells, are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+. In some embodiments, naive-like T cells comprise CCR7+CD45RA+. In certain embodiments, naive-like T cells comprise CD27+CCR7+ cells. In certain embodiments, central memory T cells are CCR7+CD45RA-.

[0072] In certain embodiments, the method further comprises the step of formulating the cells of the output composition for cryopreservation and / or administration to a subject.In some embodiments, the cells are formulated in the presence of a pharma- ceutically acceptable excipient.In certain embodiments, the cells of the output composition are formulated in the presence of a cryoprotectant.In some embodiments, the cryoprotectant comprises DMSO.In some cases, DMSo can prevent intracellular crystals from forming during the freezing process.

[0073] In some embodiments, the cells of the output composition are formulated in a container, optionally a vial or bag. In certain embodiments, the recombinant protein is a recombinant receptor that can bind to a target antigen associated with, specific for, and / or expressed on a disease, disorder, or diseased cell or tissue. In certain embodiments, the disease, disorder, or disease is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer.

[0074] In some particular embodiments, the target antigen is a tumor antigen. In particular embodiments, the target antigen is αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), cancer testis antigen, cancer testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutant (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp10O), glypican-3 (GPC3), G protein-coupled receptor class C group 5 member D (GPRC5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimer, human high molecular weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, leucine-rich repeat-containing 8 family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16,Natural killer group 2 member D (NKG2D) ligand, Melan-A (MART-1), neural cell adhesion molecule (NCAM), carcinoembryonic antigen, preferentially expressed antigen in melanoma (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-related protein 1 (TRP 1, also known as TYRP1 or gp75), tyrosinase-related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms' tumor 1 (WT-1), pathogen-specific or pathogen-expressed antigens or antigens associated with universal tags and / or biotinylated molecules and / or molecules expressed by HIV, HCV, HBV or other pathogens,

[0075] In certain embodiments, the recombinant protein is or comprises a functional non-TCR antigen receptor or a TCR or an antigen-binding fragment thereof. In certain embodiments, the recombinant protein is a chimeric antigen receptor (CAR). In some embodiments, the recombinant receptor is an anti-BCMA CAR. In certain embodiments, the recombinant protein is an anti-CD19 CAR.

[0076] In certain embodiments, the chimeric antigen receptor comprises an extracellular domain comprising an antigen binding domain, a spacer and / or a hinge region, a transmembrane domain, and an intracellular signaling domain comprising a costimulatory signaling region. In some embodiments, the extracellular domain comprising the antigen binding domain comprises an scFv. In certain embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM). In certain embodiments, the intracellular signaling domain is or comprises an intracellular signaling domain or a signaling portion thereof of a CD3 chain, optionally a CD3-zeta (CD3ζ) chain. In some embodiments, the costimulatory signaling region comprises an intracellular signaling domain or a signaling portion thereof of CD28, 4-1BB, or ICOS.

[0077] In certain embodiments, the serum-free medium comprises 0.5 mM to 5 mM of a dipeptide form of L-glutamine in a basal medium; 0.5 mM to 5 mM L-glutamine; and at least one protein, and is serum-free.

[0078] In some embodiments, the basal medium lacking one or more recombinant cytokines comprises L-glutamine. In some embodiments, L-glutamine is present at a concentration of about 0.5 mM to about 5 mM. In some embodiments, L-glutamine is present at a concentration of about 2 mM.

[0079] In some embodiments, the basal medium lacking one or more recombinant cytokines is serum-free.

[0080] In some embodiments, the basal medium further comprises at least one protein selected from one or more of albumin, insulin or transferrin, optionally one or more of human or recombinant albumin, insulin or transferrin.

[0081] In some embodiments, any of the methods described herein can be carried out using non-viral genetic engineering methods, for example, to introduce heterologous polynucleotides encoding recombinant proteins into cells, for example, T cells. Any of the provided embodiments of the methods described herein can be carried out using non-viral introduction methods to introduce heterologous polynucleotides encoding recombinant proteins into primary T cells for the manufacture of cell therapy. In certain aspects, the non-viral method is a method that facilitates the integration of the polynucleotides encoding recombinant receptors or portions thereof into the genome in cells. A variety of non-viral introduction methods can be used, including any of the non-viral introduction methods described herein.

[0082] In one aspect, provided herein is a composition comprising engineered cells produced by any of the methods provided herein.In some embodiments, the composition is a therapeutic composition.Provided herein is a therapeutic T cell composition produced by any of the methods provided herein.

[0083] In some embodiments, the recombinant protein is expressed by at least or at least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total number of T cells in the composition or the total number of T cells expressing the recombinant protein in the composition. 00%, or 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, are naive-like T cells or are T cells that are surface positive for a marker expressed on naive-like cells. In some embodiments, at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, of the total number of T cells in the composition or the total number of T cells expressing a recombinant protein in the composition. or about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are central memory T cells or are T cells that are surface positive for a marker expressed on central memory T cells.In some embodiments, the total number of T cells in the composition or the total number of T cells expressing a recombinant protein in the composition is at least or about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total number of T cells in the composition or the total number of T cells expressing a recombinant protein in the composition. %, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, are naive-like T cells or central memory T cells, or are T cells that are surface positive for a marker expressed on naive-like T cells or central memory T cells. In some embodiments, the recombinant protein is expressed by at least or at least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total number of T cells in the composition or the total number of T cells expressing the recombinant protein in the composition. 97%, 98%, 99% or 100% or about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% are CD27+CCR7+ T cells.

[0084] In some embodiments, the naive-like T cells are surface positive for T cell activation markers selected from the group consisting of CD45RA, CD27, CD28 and CCR7; and / or are surface negative for markers selected from the group consisting of CD25, CD45RO, CD56, CD62L, KLRG1; and / or have low CD95 expression; and / or have low CD95 expression; and / or are negative for intracellular expression of cytokines selected from the group consisting of IL-2, IFN-γ, IL-4, IL-10.

[0085] In some embodiments, the markers expressed on naive-like T cells are selected from the group consisting of CD45RA, CD27, CD28 and CCR7. In some embodiments, the naive-like T cells, or T cells that are surface positive for markers expressed on naive-like T cells, are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+. In some embodiments, the naive-like T cells, or T cells that are surface positive for markers expressed on naive-like T cells, comprise CD27+CCR7+ T cells and express all receptors in the composition. + / CD8 + At least 70%, 80%, 85% or 90% of the cells are CD27+CCR7+. In some embodiments, naive-like T cells, or T cells that are surface positive for a marker expressed on naive-like T cells, comprise CD27+CCR7+ T cells and do not express all receptors in the composition. + / CD4 + At least 50%, 60%, 70%, 80%, 85% or 90% of the cells are CD27+CCR7+.

[0086] In some embodiments, naive-like T cells, or T cells that are surface positive for a marker expressed on naive-like T cells, comprise CD27+CCR7+ T cells and do not express all receptors in the composition. + / CD8 + At least 50%, 60%, 70%, 80%, 85% or 90% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4+ At least 50%, 60%, 70%, 80%, 85% or 90% of the cells are CD27+CCR7+. In some embodiments, all receptors in the composition are + / CD8 + At least 50%, 60%, 70%, 80%, 85% or 90% of the cells are naive-like T cells or are surface positive for a marker expressed on naive-like T cells and express all receptors in the composition. + / CD4 + At least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the cells are naive-like T cells or are surface positive for a marker expressed on naive-like T cells.

[0087] In some embodiments, the total receptor in the composition + / CD8 + At least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the cells are central memory T cells or are surface positive for a marker expressed on central memory T cells and express all receptors in the composition. + / CD4 + At least 50%, 60%, 70%, 80% or 90% of the cells are central memory T cells or are surface positive for a marker expressed on central memory T cells. + / CD8 + At least 40%, 50%, 60%, 70%, 80% or 90% of the cells are naive-like T cells or central memory T cells, or are surface positive for a marker expressed on naive-like T cells or central memory T cells, and express all receptors in the composition. + / CD4 + At least 50%, 60%, 70%, 80% or 90% of the cells are naive-like T cells or central memory T cells, or are surface positive for a marker expressed on naive-like T cells or central memory T cells.

[0088] In some embodiments, on average, among a plurality of T cell compositions produced by the methods disclosed herein, at least about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total number of T cells in the composition or the total number of T cells expressing a recombinant protein in the composition are expressed. 97%, 98%, 99%, or 100%, or 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, are naive-like T cells or are T cells that are surface positive for a marker expressed on naive-like T cells. In some embodiments, on average, among a plurality of T cell compositions produced by the methods disclosed herein, at least about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total number of T cells in the composition or the total number of T cells expressing a recombinant protein in the composition are expressed. 98%, 99%, or 100%, or 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, are central memory T cells or are T cells that are surface positive for a marker expressed on central memory T cells.In some embodiments, on average, among a plurality of T cell compositions produced by the methods disclosed herein, at least about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total number of T cells in the composition or the total number of T cells expressing a recombinant protein in the composition. 100%, or 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% or about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are naive-like T cells or central memory T cells, or are T cells that are surface positive for a marker expressed on naive-like T cells or central memory T cells. In some embodiments, on average, among a plurality of T cell compositions produced by the methods disclosed herein, at least about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total number of T cells in the composition or the total number of T cells expressing a recombinant protein in the composition are expressed. %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are CD27+CCR7+ T cells.In any of the above embodiments, on average, in a plurality of T cell compositions produced by the methods disclosed herein, at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, at least or at least about 99%, about 100%, or 100% of the cells in the compositions can be CD4+ T cells and CD8+ T cells. In any of the above embodiments, on average, in a plurality of T cell compositions produced by the methods disclosed herein, at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, at least or at least about 99%, about 100%, or 100% of the cells in the compositions can be CD3+ T cells.

[0089] In one aspect, provided herein is a therapeutic T cell composition comprising CD4+ T cells expressing a recombinant receptor and CD8+ T cells expressing a recombinant receptor, wherein the total receptor in the composition is 0.01 to 0.1%. + / CD8 + At least 50%, 60%, 70%, 80% or 90% of the cells are naive-like T cells or are surface positive for a marker expressed on naive-like T cells and express all receptors in the composition. + / CD4 + A therapeutic T cell composition, wherein at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the cells are naive-like T cells or are surface positive for a marker expressed on naive-like T cells.

[0090] In some embodiments, the total number of T cells in the composition or the total number of T cells expressing a recombinant protein in the composition is at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. At or about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, are naive-like T cells or are T cells that are surface positive for a marker expressed on naive-like T cells. In some embodiments, the total receptor in the composition + / CD8 + At least 60% of the cells are naive-like T cells or are surface positive for a marker expressed on naive-like T cells and express all receptors in the composition. + / CD4 + 30% of the cells are naive-like T cells or are surface positive for a marker expressed on naive-like T cells. In some embodiments, the total receptor in the composition + / CD8 + At least 70% of the cells are naive-like T cells or are surface positive for a marker expressed on naive-like T cells and express all receptors in the composition. + / CD4 + 40% of the cells are naive-like T cells or are surface positive for a marker expressed on naive-like T cells. In some embodiments, the total receptor in the composition + / CD8 + At least 70% of the cells are naive-like T cells or are surface positive for a marker expressed on naive-like T cells and express all receptors in the composition. + / CD4 +50% of the cells are naive-like T cells or are surface positive for a marker expressed on naive-like T cells. In some embodiments, the total receptor in the composition is 50%. + / CD8 + At least 70% of the cells are naive-like T cells or are surface positive for a marker expressed on naive-like T cells and express all receptors in the composition. + / CD4 + 60% of the cells are naive-like T cells or are surface positive for a marker expressed on naive-like T cells. In some embodiments, the total receptor in the composition + / CD8 + At least 70% of the cells are naive-like T cells or are surface positive for a marker expressed on naive-like T cells and express all receptors in the composition. + / CD4 + 70% of the cells are naive-like T cells or are surface positive for markers expressed on naive-like T cells.

[0091] In one aspect, provided herein is a therapeutic T cell composition comprising CD4+ T cells expressing a recombinant receptor and CD8+ T cells expressing a recombinant receptor, wherein the total receptor in the composition is 0.01 to 0.1%. + / CD8 + At least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the cells are central memory T cells or are surface positive for a marker expressed on central memory T cells and express all receptors in the composition. + / CD4 + A therapeutic T cell composition, wherein at least 50%, 60%, 70%, 80% or 90% of the cells are central memory T cells or are surface positive for a marker expressed on central memory T cells.

[0092] In some embodiments, at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, of the total number of T cells in the composition or the total number of T cells expressing a recombinant protein in the composition. or about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are central memory T cells or are T cells that are surface positive for a marker expressed on central memory T cells. + / CD8 + At least 25% of the cells are central memory T cells or are surface positive for a marker expressed on central memory T cells and express all receptors in the composition. + / CD4 + At least 60% of the cells are central memory T cells or are surface positive for a marker expressed on central memory T cells. + / CD8 + At least 65% of the cells are central memory T cells or are surface positive for a marker expressed on central memory T cells and express all receptors in the composition. + / CD4 + At least 60% of the cells are central memory T cells or are surface positive for a marker expressed on central memory T cells. + / CD8 + At least 70% of the cells are central memory T cells or are surface positive for a marker expressed on central memory T cells and express all receptors in the composition.+ / CD4 + 65% of the cells are central memory T cells or are surface positive for a marker expressed on central memory T cells. + / CD8 + At least 70% of the cells are central memory T cells or are surface positive for a marker expressed on central memory T cells and express all receptors in the composition. + / CD4 + 70% of the cells are central memory T cells or are surface positive for markers expressed on central memory T cells.

[0093] In one aspect, provided herein is a therapeutic T cell composition comprising CD4+ T cells expressing a recombinant receptor and CD8+ T cells expressing a recombinant receptor, wherein the total receptor in the composition is 0.01 to 0.1%. + / CD8 + At least 40%, 50%, 60%, 70%, 80% or 90% of the cells are naive-like T cells or central memory T cells, or are surface positive for a marker expressed on naive-like T cells or central memory T cells, and express all receptors in the composition. + / CD4 + A therapeutic T cell composition, wherein at least 50%, 60%, 70%, 80% or 90% of the cells are naive-like T cells or central memory T cells, or are surface positive for a marker expressed on naive-like T cells or central memory T cells.

[0094] In some embodiments, at least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total number of T cells in the composition or the total number of T cells expressing the recombinant receptor in the composition. , 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, are naive-like T cells or central memory T cells, or are T cells that are surface positive for a marker expressed on naive-like T cells or central memory T cells. + / CD8 + At least 50% of the cells are naive-like T cells or central memory T cells, or are surface positive for a marker expressed on naive-like T cells or central memory T cells, and express all receptors in the composition. + / CD4 + At least 65% of the cells are naive-like T cells or central memory T cells, or are surface positive for a marker expressed on naive-like T cells or central memory T cells. + / CD8 + At least 70% of the cells are naive-like T cells or central memory T cells, or are surface positive for a marker expressed on naive-like T cells or central memory T cells, and express all receptors in the composition. + / CD4 +At least 70% of the cells are naive-like T cells or central memory T cells, or are surface positive for a marker expressed on naive-like T cells or central memory T cells. + / CD8 + At least 75% of the cells are naive-like T cells or central memory T cells, or are surface positive for a marker expressed on naive-like T cells or central memory T cells, and express all receptors in the composition. + / CD4 + At least 75% of the cells are naive-like T cells or central memory T cells, or are surface positive for a marker expressed on naive-like T cells or central memory T cells.

[0095] In some embodiments, the naive-like T cells are surface positive for a T cell activation marker selected from the group consisting of CD45RA, CD27, CD28 and CCR7; and / or are surface negative for a marker selected from the group consisting of CD25, CD45RO, CD56, CD62L, KLRG1; and / or have low CD95 expression. In some embodiments, the marker expressed on the naive-like T cells is selected from the group consisting of CD45RA, CD27, CD28 and CCR7. In some embodiments, the receptor + / CD4 + are T cells or naive-like T cells, or are surface positive for a marker expressed on naive-like T cells + / CD4 + The T cells are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+. + / CD4 + are T cells or naive-like T cells, or are surface positive for a marker expressed on naive-like T cells + / CD4 + The T cells are CD27+CCR7+. In some embodiments, the receptor + / CD8 +are T cells or naive-like T cells, or are surface positive for a marker expressed on naive-like T cells + / CD8 + The T cells are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+. + / CD8 + are T cells or naive-like T cells, or are surface positive for a marker expressed on naive-like T cells + / CD8 + T cells are CD27+CCR7+.

[0096] In one aspect, provided herein is a therapeutic T cell composition comprising CD4+ T cells expressing a recombinant receptor and CD8+ T cells expressing a recombinant receptor, wherein the total receptor in the composition is 0.01 to 0.1%. + / CD8 + At least 50%, 60%, 70%, 80% or 90% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4 + A therapeutic T cell composition, wherein at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the cells are CD27+CCR7+.

[0097] In one aspect, provided herein is a therapeutic T cell composition comprising CD4+ T cells expressing a recombinant receptor and CD8+ T cells expressing a recombinant receptor, wherein the total receptor in the composition is 0.01 to 0.1%. + / CD8 + At least 50%, 60%, 70%, 80% or 90% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4 + A therapeutic T cell composition, wherein at least 50%, 60%, 70%, 80% or 90% of the cells are CD27+CCR7+.

[0098] In some embodiments, at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, at least or at least about 99%, about 100%, or 100% of the cells in the composition are CD4+ T cells and CD8+ T cells. In some embodiments, at least or at least about 90% of the cells in the composition are CD4+ T cells and CD8+ T cells, and at least about 100% of the cells in the composition are CD4+ T cells and CD8+ T cells. + / CD8 + At least about 60% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4 + At least or at least about 40% of the cells are CD27+CCR7+. In some embodiments, at least or at least about 95% of the cells in the composition are CD4+ T cells and CD8+ T cells, and ...40% of the cells are CD27+CCR7+. In some embodiments, at least about 95% of the cells in the composition are CD4+ T cells and CD8+ T cells, and at least about 40% of the cells in the composition are CD27+CCR7+. + / CD8 + At least about 65% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4 + At least or at least about 45% of the cells are CD27+CCR7+. In some embodiments, at least or at least about 98% of the cells in the composition are CD4+ T cells and CD8+ T cells, and ...45% of the cells are CD27+CCR7+. In some embodiments, at least about 98% of the cells in the composition are CD4+ T cells and CD8+ T cells, and at least about 45% of the cells in the composition are CD27+CCR7+. + / CD8 + At least or at least about 70%, at least or at least about 75%, at least or at least about 80%, or at least or at least about 85% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4 +At least or at least about 50%, at least or at least about 55%, at least or at least about 60%, or at least or at least about 65% of the cells are CD27+CCR7+. In some embodiments, at least or at least about 98% of the cells in the composition are CD4+ T cells and CD8+ T cells, and the total receptor activator in the composition is CD4+ T cells and CD8+ T cells. + / CD8 + At least about 75% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4 + At least or at least about 55% of the cells are CD27+CCR7+. In some embodiments, at least or at least about 98% of the cells in the composition are CD4+ T cells and CD8+ T cells, and ...55% of the cells are CD27+CCR7+. In some embodiments, at least about 98% of the cells in the composition are CD4+ T cells and CD8+ T cells, and at least about 55% of the cells are CD27+CCR7+. + / CD8 + At least or at least about 80% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4 + At least or at least about 60% of the cells are CD27+CCR7+. In some embodiments, at least or at least about 98% of the cells in the composition are CD4+ T cells and CD8+ T cells, and ...60% of the cells are CD27+CCR7+. In some embodiments, at least about 98% of the cells in the composition are CD4+ T cells and CD8+ T cells, and at least about 60% of the cells in the composition are CD27+CCR7+. + / CD8 + At least or at least about 85% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4 + At least or at least about 65% of the cells are CD27+CCR7+. In some embodiments, at least or at least about 98% of the cells in the composition are CD4+ T cells and CD8+ T cells, and ...65% of the cells are CD27+CCR7+. In some embodiments, at least about 98% of the cells in the composition are CD4+ T cells and CD8+ T cells, and at least about 65% of the cells in the composition are CD27+CCR7+. + / CD8 + At least or at least about 90% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4 + At least or at least about 70% of the cells are CD27+CCR7+. In some embodiments, at least 90% of the cells in the composition are CD4+ T cells and CD8+ T cells, and the total receptors in the composition are+ / CD8 + At least 60% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4 + At least 40% of the cells are CD27+CCR7+. In some embodiments, at least 90% of the cells in the composition are CD4+ T cells and CD8+ T cells, and ... CCR7+ T cells. + / CD8 + At least 70% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4 + At least 50% of the cells are CD27+CCR7+. In some embodiments, at least 90% of the cells in the composition are CD4+ T cells and CD8+ T cells, and ... CCR7+ T cells. + / CD8 + At least 70% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4 + At least 60% of the cells are CD27+CCR7+. In some embodiments, at least 95% of the cells in the composition are CD4+ T cells and CD8+ T cells, and ... CCR7+ T cells. + / CD8 + At least 70% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4 + At least 70% of the cells are CD27+CCR7+. In some embodiments, at least 95% of the cells in the composition are CD4+ T cells and CD8+ T cells, and ... CCR7+ T cells. + / CD8 + At least 80% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4 + At least 80% of the cells are CD27+CCR7+.

[0099] In some embodiments, at least or at least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total number of T cells in the composition or the total number of T cells expressing the recombinant receptor in the composition. 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% or about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are CD27+CCR7+.

[0100] In some embodiments, the ratio of receptor+ / CD4+ T cells:receptor+ / CD8+ T cells in the composition is about 1:3 to about 3:1. In some embodiments, the ratio of receptor+ / CD4+ T cells:receptor+ / CD8+ T cells in the composition is about 1:2 to about 2:1. In some embodiments, the ratio of receptor+ / CD4+ T cells:receptor+ / CD8+ T cells in the composition is 1:1 or about 1:1.

[0101] In some embodiments, the composition comprises one or more unit doses of cells. In some embodiments, the unit dose is 1×10 4 ~50×10 6 1 x 10 pieces or approx. 4 ~50×10 6 Contains T cells.

[0102] In some embodiments, the recombinant protein is or comprises a chimeric receptor and / or a recombinant antigen receptor. In some embodiments, the recombinant receptor can bind to a target protein associated with, specific to, and / or expressed on a disease, disorder, or diseased cell or tissue. In some embodiments, the disease, disorder, or disease is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer. In some embodiments, the target antigen is a tumor antigen. In some embodiments, the target antigen is αvβ6 integrin (avb6 integrin), B-cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), cancer testis antigen, cancer testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD138, CD171, epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutated (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like 5 (FCRL5;Also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp10O), G protein-coupled receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimer, human high molecular weight black Chromoma-associated antigen (HMW-MAA), Hepatitis B surface antigen, Human leukocyte antigen A1 (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Rα), IL-13 receptor alpha 2 (IL-13Ra2), Kinase insert domain receptor (kdr), Kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, Leucine-rich repeat-containing 8 family member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAA) GE)-A1, MAGE-A3, MAGE-A6, mesothelin, c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand, melan A (MART-1), neural cell adhesion molecule (NCAM), carcinoembryonic antigen, preferentially expressed antigen in melanoma (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein (TPBG, also known as 5T4), tumor associated glycoprotein 72 (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms' tumor 1 (WT-1), pathogen specific antigens or antigens associated with universal tags and / or biotinylated molecules and / or molecules expressed by HIV, HCV, HBV or other pathogens;

[0103] In some embodiments, the recombinant receptor is or comprises a functional non-TCR antigen receptor or a TCR or an antigen-binding fragment thereof.

[0104] In some embodiments, the recombinant protein comprises an extracellular domain comprising an antigen-binding domain. In some embodiments, the antigen-binding domain is or comprises an antibody or an antibody fragment thereof, which in some embodiments is a single chain fragment. In some embodiments, the fragment comprises an antibody variable region linked by a flexible linker. In some embodiments, the fragment comprises an scFv.

[0105] In some embodiments, the recombinant protein comprises an intracellular signaling region. In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain is or comprises an intracellular signaling domain of a CD3 chain. In some embodiments, the CD3 chain is a CD3-zeta (CD3ζ) chain or a signaling portion thereof.

[0106] In some embodiments, the recombinant protein further comprises a transmembrane domain located between the extracellular domain and the intracellular signaling region. In some embodiments, the intracellular signaling region further comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain comprises an intracellular signaling domain of a T cell costimulatory molecule, or a signaling portion thereof. In some embodiments, the costimulatory signaling domain comprises an intracellular signaling domain of CD28, 4-1BB, or ICOS, or a signaling portion thereof. In some embodiments, the costimulatory signaling domain is between the transmembrane domain and the intracellular signaling domain.

[0107] In some embodiments, the T cells are primary T cells obtained from the subject. In some embodiments, the T cells are autologous cells of the subject. In some embodiments, the T cells are allogeneic cells of the subject.

[0108] In some embodiments, the composition comprises a pharma- ceutically acceptable excipient. In certain embodiments, the composition further comprises a pharma- ceutically acceptable carrier. In certain embodiments, the composition further comprises a cryoprotectant. In some embodiments, the cryoprotectant is DMSO.

[0109] In certain embodiments, at least 60% of the total T cells, CD4+ T cells, or CD8+ T cells or recombinant protein-expressing cells thereof of the composition are CD27+CCR7+.At least 65%, 70%, 80%, 90% or 95% of the total recombinant protein-expressing cells of the composition are CD27+CCR7+, at least 65%, 70%, 80%, 90% or 95% of the recombinant protein-expressing CD4+ T cells of the composition are CD27+CCR7+, or at least 65%, 70%, 80%, 90% or 95% of the recombinant protein-expressing CD8+ T cells of the composition are CD27+CCR7+.

[0110] In one aspect, provided herein is an article of manufacture comprising a composition of a method or composition provided herein and instructions for administering the output composition to a subject.

[0111] In certain embodiments, the subject has a disease or condition. In some embodiments, the recombinant receptor specifically recognizes or specifically binds to an antigen associated with, or expressed or present on, the disease or condition cells.

[0112] In one aspect, provided herein is a method of treating a subject having or suspected of having a disease or condition, comprising administering to the subject a dose of T cells from any of the compositions provided herein.

[0113] In certain embodiments, the dose of T cells is administered to the subject as a single dose, or is administered only once within a period of 2 weeks, 1 month, 3 months, 6 months, 1 year, or longer. In some embodiments, the dose of T cells is 1×10 4 ~50×10 6 pcs or approx. 1×10 4 ~50×10 6 In certain embodiments, the dose of T cells comprises 50×10 6 T cells, less than 10 x 10 6 < 5 x 10 T cells 6 < 1 x 10 T cells 6 < 0.5 x 10 T cells 6 < 1 x 10 T cells or 1 x 10 5 In certain embodiments, the dose of T cells is less than 20×10 6 Pieces or about 20 x 10 6 In some embodiments, the dose of T cells comprises 10×10 6 Pieces or about 10 x 10 6 Contains T cells.

[0114] In certain embodiments, the dose of T cells is 2×10 6 pcs or approx. 2 x 10 6 In certain embodiments, the dose of T cells comprises 1×10 6 pcs or approx. 1×10 6 Contains T cells.

[0115] In some embodiments, the T cells of the dose of T cells are total T cells, total viable T cells, total viable recombinant receptor-expressing T cells, total viable recombinant receptor-expressing CD4+ T cells, or total viable recombinant receptor-expressing CD8+ T cells. In certain embodiments, the disease or condition is cancer. In certain embodiments, the disease or condition is myeloma, leukemia, or lymphoma. In some embodiments, the disease or condition is B-cell malignancy and / or is acute lymphocytic leukemia (ALL), adult ALL, chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and diffuse large B-cell lymphoma (DLBCL). [The present invention 1001] 1. A method of producing a composition of engineered T cells, comprising: (a) exposing an input composition comprising primary T cells to a stimulatory reagent comprising an oligomeric particulate reagent comprising a plurality of streptavidin or streptavidin mutein molecules under conditions for stimulating the T cells, thereby generating a stimulated population, wherein the stimulatory reagent is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules; (b) introducing into T cells of the stimulated population a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; (c) incubating the population of transformed cells for up to 96 hours, optionally wherein the incubation is performed at a temperature of about 37° C. and wherein the incubation is performed in a basal medium lacking one or more recombinant cytokines; and (c) harvesting the T cells of the transformed population following said incubation, thereby producing a composition of engineered cells, said harvesting being performed between 48 and 120 hours, inclusive, after exposure to said stimulatory reagent was initiated. Including, Thereby producing a composition of engineered cells. [The present invention 1002] 1. A method of producing a composition of engineered T cells, comprising: (a) exposing an input composition comprising primary T cells to a stimulatory reagent under conditions to stimulate the T cells, thereby generating a stimulated population; (b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and (c) harvesting the T cells of the transformed population, the harvesting comprising: (i) at 24 to 120 hours (inclusive) after exposure to the stimulant agent was initiated; (ii) when integrated vector is detected in the genome, but before achieving a stable integrated vector copy number per diploid genome (iVCN); (iii) at a time before the total number of viable cells at the time of harvest is 3-fold, 2-fold, or about 3-fold, about 2-fold, or the same or about the same as the number of total viable cells of the stimulated population; and / or (iv) the percentage of CD27+CCR7+ is greater than or greater than about 60% among all T cells in the population, among all CD3+ T cells in the population, among all CD4+ T cells in the population, or among all CD8+ T cells or cells expressing a recombinant protein thereof in the population; A process to be carried out Including, Thereby producing a composition of engineered cells. [The present invention 1003] Input composition is at least 300×10 6 The method of claim 1002, comprising administering to said patient a sample of said subject the subject's live primary T cells. [The present invention 1004] The method of claim 1002 or 1003, wherein the stimulatory reagent is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules. [The present invention 1005] 1. A method of producing a composition of engineered T cells, comprising: (a) At least 300 × 10 6 exposing an input composition comprising viable primary T cells to a stimulatory reagent under conditions for stimulating the T cells, thereby generating a stimulated population, wherein the stimulatory reagent is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules; (b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and (c) harvesting the T cells of the transformed population, the harvesting comprising: (i) at 48 to 120 hours (inclusive) after exposure to the stimulant agent was initiated; (ii) when integrated vector is detected in the genome of the transformed population, but before a stable integrated vector copy number per diploid genome (iVCN) is achieved; (iii) at a time before the total number of viable cells at the time of harvest is 3-fold, 2-fold, or about 3-fold, about 2-fold, or the same or about the same as the number of total viable cells of the stimulated population; and / or (iv) the percentage of naive-like T cells is greater than or greater than about 60% among the total T cells in the population, the total CD3+ cells in the population, the total CD4+ T cells in the population, or the total CD8+ T cells or cells expressing a recombinant protein thereof in the population; A process to be carried out Including, Thereby producing a composition of engineered cells. [The present invention 1006] Any of the methods of 1001 to 1005, wherein harvesting is performed at the time when an integrated vector is detected in the genome but before achieving stable iVCN per diploid genome. [The present invention 1007] The method of the present invention 1006, wherein a stable iVCN per diploid genome is achieved when the iVCN peaks and remains unchanged for a period of more than 24 hours or changes only within an acceptable margin of error. [The present invention 1008] The method of the present invention 1006, wherein a stable iVCN per diploid genome is achieved when the fraction of iVCN relative to the total vector copy number (VCN) in the diploid genome of a population of transformed cells is on average 1.0 or about 1.0, or within an acceptable error range thereof. [The present invention 1009] Any of the methods of claims 1001 to 1008, wherein harvesting is performed at a time when the fraction of integrated vector copy number (iVCN) to total VCN of the population of transformed cells is on average less than 0.8. [The present invention 1010] 1. A method of producing a composition of engineered T cells, comprising: (a) exposing an input composition comprising primary T cells to a stimulatory reagent under conditions for stimulating the T cells, thereby generating a stimulated population, wherein the stimulatory reagent is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules; (b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and (c) harvesting the T cells of the transformed population, the harvesting being performed 48 to 120 hours, inclusive, after exposure to the stimulatory reagent is initiated, and at harvest, the population of transformed cells has, on average, an integrated vector copy number (iVCN) of between or about 0.4 and 2.0 copies per diploid genome, inclusive. Including, Thereby producing a composition of engineered cells. [The present invention 1011] Any of the methods of 1001 to 1010, wherein the harvesting is performed at a time when the iVCN of the population of transformed cells is, on average, less than or about 2.0 copies per diploid genome. [The present invention 1012] 1. A method of producing a composition of engineered T cells, comprising: (a) exposing an input composition comprising primary T cells to a stimulatory reagent under conditions for stimulating the T cells, thereby generating a stimulated population, wherein the stimulatory reagent is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules; (b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and (c) harvesting the T cells of the transformed population, the harvesting being performed between 48 and 120 hours, inclusive, after exposure to the stimulatory reagent is initiated, and at the time of harvesting, the percentage of naive-like cells and / or central memory T cells is greater than or greater than about 60% of the total T cells in the population, the total CD4+ T cells in the population, or the total CD8+ T cells or cells expressing the recombinant protein thereof in the population. Including, Thereby producing a composition of engineered cells. [The present invention 1013] At the time of collection, the percentage of naive-like T cells is greater than or greater than about 60% among all recombinant protein-expressing cells in the population, optionally greater than or greater than about 65%, 70%, 80%, 90%, or 95% among all recombinant protein-expressing T cells in the population; the percentage of naive-like T cells is greater than or greater than about 40% among all recombinant protein-expressing CD4+ T cells in the population, optionally greater than or greater than about 50%, 60%, 70%, 80%, 90%, or 95% among all recombinant protein-expressing CD4+ cells in the population; or the percentage of naive-like T cells is greater than or greater than about 40% of the total recombinant protein-expressing CD8+ T cells in the population, optionally greater than or greater than about 50%, 60%, 70%, 80%, 90%, or 95% of the total recombinant protein-expressing CD8+ cells in the population; Any of the methods according to 1001 to 1012 of the present invention. [The present invention 1014] At the time of collection, the percentage of naive-like T cells and / or central memory T cells is greater than or greater than about 60% among all recombinant protein-expressing cells in the population, optionally greater than or greater than about 65%, 70%, 80%, 90%, or 95% among all recombinant protein-expressing T cells in the population; the percentage of naive-like T cells and / or central memory T cells is greater than or greater than about 40% of all recombinant protein-expressing CD4+ T cells in the population, optionally greater than or greater than about 50%, 60%, 70%, 80%, 90%, or 95% of all recombinant protein-expressing CD4+ cells in the population; or the percentage of naive-like T cells and / or central memory T cells is greater than or greater than about 40% of the total recombinant protein-expressing CD8+ T cells in the population, optionally greater than or greater than about 50%, 60%, 70%, 80%, 90%, or 95% of the total recombinant protein-expressing CD8+ cells in the population; The method of the present invention 1012 or 1013. [The present invention 1015] further comprising incubating the population of transformed cells for up to 96 hours after the introduction and prior to harvesting; Optionally, the incubation is carried out at a temperature of about 37° C. Any of the methods 1002 to 1014 of the present invention. [The present invention 1016] The method of any one of claims 1001 to 1015, wherein after the introduction, incubation is carried out for up to 72 hours. [The present invention 1017] The method of any one of claims 1001 to 1015, wherein after the introduction, incubation is carried out for up to 48 hours. [The present invention 1018] The method of any one of claims 1001 to 1015, wherein after the introduction, incubation is carried out for up to 24 hours. [The present invention 1019] The method of any of claims 1001 and 1015 to 1018, wherein the incubation is carried out for at least 18 hours. [The present invention 1020] The method of any of claims 1001 to 1019, wherein the harvesting is performed within 96 hours after exposure to the irritant has begun. [The present invention 1021] Any of the methods of claims 1001 to 1019, wherein the harvesting is performed within 72 hours after exposure to the irritant has begun. [The present invention 1022] The method of any of claims 1001 to 1019, wherein the collection is performed within 48 hours after exposure to the irritant has begun. [The present invention 1023] The method of any of claims 1001 to 1022, wherein one or both of the exposing in (a) and the introducing in (b) are carried out in the presence of one or more recombinant cytokines. [The present invention 1024] The method of any of claims 1001 to 1022, wherein the exposing in (a) and the introducing in (b) are carried out in the presence of one or more recombinant cytokines. [The present invention 1025] The method of any of claims 1015 to 1024, wherein the incubation is carried out in the presence of one or more recombinant cytokines. [The present invention 1026] The method of any of claims 1015 to 1024, wherein the incubation is carried out in a basal medium lacking one or more recombinant cytokines. [The present invention 1027] The method of any of claims 1005 to 1009 and 1012 to 1026, wherein the naive-like T cells, or T cells that are surface positive for a marker expressed on the naive-like T cells, are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+. [The present invention 1028] Any of the methods of claims 1005 to 1009 and 1012 to 1027, wherein the naive-like T cells comprise CD27+CCR7+ cells. [The present invention 1029] Any of the methods of claims 1005 to 1009 and 1012 to 1028, wherein the naive-like T cells comprise CCR7+CD45RA+T cells. [The present invention 1030] 1. A method of producing a composition of engineered T cells, comprising: (a) exposing an input composition comprising primary T cells to a stimulatory reagent under conditions for stimulating the T cells, including the presence of one or more recombinant cytokines, thereby generating a stimulated population, wherein the stimulatory reagent is capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules; (b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells, wherein the introduction is performed in the presence of one or more recombinant cytokines; (c) incubating the population of transformed cells for up to 96 hours, optionally wherein the incubation is performed at a temperature of about 37° C. and wherein the incubation is performed in a basal medium lacking one or more recombinant cytokines; and (c) harvesting the T cells of the transformed population following said incubation, thereby producing a composition of engineered cells, said harvesting being performed between 48 and 120 hours, inclusive, after exposure to said stimulatory reagent was initiated. Including, Thereby producing a composition of engineered cells. [The present invention 1031] The method of any of claims 1001 and 1023-1030, wherein the one or more recombinant cytokines are human. [The present invention 1032] The method of any of claims 1001 and 1023 to 1031, wherein the one or more recombinant cytokines are selected from recombinant IL-2, recombinant IL-7, and recombinant IL-15, optionally 10 to 200 IU / mL of recombinant IL-2, 100 IU / mL to 1,000 IU / mL of recombinant IL-7, and / or 10 to 200 IU / mL of recombinant IL-15. [The present invention 1033] The method of any of claims 1001 and 1023 to 1032, wherein the one or more recombinant cytokines are or comprise recombinant IL-2, recombinant IL-7, and recombinant IL-15, optionally 10 to 200 IU / mL of recombinant IL-2, 100 IU / mL to 1,000 IU / mL of recombinant IL-7, and 10 to 200 IU / mL of recombinant IL-15. [The present invention 1034] The method of any of claims 1001 to 1033, wherein one or both of the exposing in (a) and the introducing in (b) are carried out in a serum-free medium. [The present invention 1035] The method according to any one of claims 1001 to 1034, wherein the exposing in (a) and the introducing in (b) are carried out in a serum-free medium. [The present invention 1036] The stimulating agent is (i) a principal agent that specifically binds to a member of the TCR complex, and optionally specifically binds to CD3; and (ii) an adjunct that specifically binds to a T cell costimulatory molecule; Including, Optionally, the costimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS. Any of the methods of 1001, 1006 to 1009, 1011, 1013, 1014, 1016 to 1024, and 1032 to 1035 of the present invention. [The present invention 1037] The stimulating agent is (i) a principal agent that specifically binds to a member of the TCR complex, and optionally specifically binds to CD3; and (ii) an adjunct that specifically binds to a T cell costimulatory molecule; Including, Optionally, the costimulatory molecule is selected from CD28, CD137 (4-1-BB), OX40, or ICOS. Any of the methods of 1002 to 1035 of the present invention. [The present invention 1038] The method of claim 1036 or 1037, wherein one or both of the main agent and the auxiliary agent comprises an antibody or an antigen-binding fragment thereof. [The present invention 1039] The method of any one of claims 1036 to 1038, wherein the main agent and the accessory agent comprise antibodies, and optionally, the stimulating reagent comprises an anti-CD3 antibody and an anti-CD28 antibody or antigen-binding fragments thereof. [The present invention 1040] The method of any of claims 1037 to 1039, wherein the main agent and the auxiliary agent are present on the surface of or attached to a solid support. [The present invention 1041] The method of claim 1040, wherein the solid support is or comprises a bead. [The present invention 1042] The method of claim 1041, wherein the ratio of beads to cells is less than 3:1. [The present invention 1043] 1. A method of producing a composition of engineered T cells, comprising: (a) exposing an input composition comprising primary T cells to a stimulatory reagent comprising beads having attached thereto (i) a primary agent that specifically binds to a member of the TCR complex, and optionally, CD3, and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule, wherein the ratio of beads to cells is less than 3:1, and the exposing is carried out under conditions for stimulating the T cells, thereby generating a stimulated population; (b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and (c) harvesting the T cells of the transformed population, the harvesting being performed between 48 and 120 hours, inclusive, after exposure to the stimulatory reagent is initiated. Including, Thereby producing a composition of engineered cells. [The present invention 1044] The method of any one of claims 1041 to 1043, wherein the ratio of beads to cells is 1:1 or approximately 1:1. [The present invention 1045] The method according to any one of claims 1041 to 1044, wherein an anti-CD3 antibody and an anti-CD28 antibody or an antigen-binding fragment thereof are attached to the beads. [The present invention 1046] The method of any one of claims 1036, 1038 or 1039, wherein the primary and secondary agents are reversibly bound to the surface of an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules. [The present invention 1047] 1. A method of producing a composition of engineered T cells, comprising: (a) exposing an input composition comprising primary T cells to a stimulatory reagent comprising an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules reversibly associated with (i) a primary agent that specifically binds to a member of the TCR complex, and optionally, to CD3, and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule, wherein the exposure is performed under conditions for stimulating the T cells, thereby generating a stimulated population; (b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; and (c) harvesting the T cells of the transformed population, the harvesting being performed between 48 and 120 hours, inclusive, after exposure to the stimulatory agent is initiated. Including, Thereby producing a composition of engineered cells. [The present invention 1048] (a) Exposure to cells 10 6 8. The method of claim 1001, 1046 or 1047, carried out using an amount of stimulating reagent that is between or about 0.1 μg and 20 μg per cell (inclusive). [The present invention 1049] An input composition containing T cells is prepared by subjecting the input composition to at least 10 cells. 6 exposing the T cells to a stimulating reagent in an amount of 0.1 μg to 20 μg per cell, inclusive, the stimulating reagent comprising an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules, the exposing being carried out under conditions for stimulating the T cells, thereby generating a stimulated population. A method for stimulating T cells, comprising: [The present invention 1050] An input composition containing T cells is prepared by subjecting the input composition to at least 10 cells. 6 exposing the T cells to a stimulatory reagent in an amount of 0.1 μg to 2 μg per particle, inclusive, the stimulatory reagent comprising an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules having attached thereto (i) a primary agent that specifically binds to a member of the TCR complex and optionally specifically binds to CD3, and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule, the exposing being performed under conditions for stimulating the T cells, thereby generating a stimulated population. A method for stimulating T cells, comprising: [The present invention 1051] The method of any one of claims 1049 to 1050, wherein the input composition comprises primary T cells. [The present invention 1052] The amount of stimulation reagent in the input composition is 6 The method of any one of claims 1048, 1049 and 1051, wherein the amount of the antibody is 0.1 µg to 2 µg or about 0.1 µg to 2 µg (inclusive) per cell. [The present invention 1053] further comprising incubating the population of transformed cells for up to 96 hours after the introduction and prior to harvesting; Optionally, the incubation is carried out at a temperature of about 37° C. Any of the methods of 1043 to 1048 and 1052 of the present invention. [The present invention 1054] The method of claim 1053, wherein after the introduction, incubation is carried out for up to 72 hours. [The present invention 1055] The method of claim 1053, wherein after the introduction, incubation is carried out for up to 48 hours. [The present invention 1056] The method of claim 1053, wherein the introduction is followed by incubation for up to 24 hours. [The present invention 1057] Any of the methods of claims 1043 to 1048 and 1052 to 1056, wherein the harvesting is performed within 96 hours after exposure of the input composition to the stimulant is initiated. [The present invention 1058] Any of the methods of claims 1043 to 1048 and 1052 to 1057, wherein the harvesting is performed within 72 hours after exposure of the input composition to the stimulant is initiated. [The present invention 1059] Any of the methods of claims 1043 to 1048 and 1052 to 1058, wherein the harvesting is performed within 48 hours after exposure of the input composition to the stimulant is initiated. [The present invention 1060] The method of any of claims 1043 to 1048 and 1052 to 1059, wherein one or both of the exposing in (a) and the introducing in (b) are carried out in the presence of one or more recombinant cytokines. [The present invention 1061] The method of any of claims 1043 to 1048 and 1052 to 1059, wherein the exposing in (a) and the introducing in (b) are carried out in the presence of one or more recombinant cytokines. [The present invention 1062] The method of any of claims 1043 to 1048 and 1052 to 1061, wherein the incubation is carried out in the presence of one or more recombinant cytokines. [The present invention 1063] The method of any of claims 1043 to 1048 and 1052 to 1061, wherein the incubation is carried out in a basal medium lacking one or more recombinant cytokines. [The present invention 1064] 1. A method of producing a composition of engineered T cells, comprising: (a) exposing an input composition comprising primary T cells to a stimulatory reagent comprising beads having attached thereto (i) a primary agent that specifically binds to a member of the TCR complex, and optionally, CD3, and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule, wherein the ratio of beads to cells is less than 3:1, and the exposure is carried out under conditions for stimulating the T cells, including the presence of one or more recombinant cytokines, thereby generating a stimulated population; (b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells, wherein the introduction is performed in the presence of one or more recombinant cytokines; (c) incubating the population of transformed cells for up to 96 hours, optionally wherein the incubation is performed at a temperature of about 37° C. and wherein the incubation is performed in a basal medium lacking one or more recombinant cytokines; and (d) harvesting the T cells of the transformed population following said incubation, thereby producing a composition of engineered cells, said harvesting being performed between 48 and 120 hours, inclusive, after exposure to said stimulatory reagent was initiated. Including, Thereby producing a composition of engineered cells. [The present invention 1065] 1. A method of producing a composition of engineered T cells, comprising: (a) exposing an input composition comprising primary T cells to a stimulatory reagent comprising an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules to which (i) a primary agent that specifically binds to a member of the TCR complex and optionally binds specifically to CD3, and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule, the exposure being carried out under conditions for stimulating the T cells, including the presence of one or more recombinant cytokines, thereby generating a stimulated population; (b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells, wherein the introduction is performed in the presence of one or more recombinant cytokines; (c) incubating the population of transformed cells for up to 96 hours, optionally wherein the incubation is performed at a temperature of about 37° C. and wherein the incubation is performed in a basal medium lacking one or more recombinant cytokines; and (d) harvesting the T cells of the transformed population following said incubation, thereby producing a composition of engineered cells, said harvesting being performed between 48 and 120 hours, inclusive, after exposure to said stimulatory reagent was initiated. Including, Thereby producing a composition of engineered cells. [The present invention 1066] The amount of stimulation reagent in the input composition is 6 The method of any one of claims 1048 to 1063 and 1065, wherein the amount is 0.4 μg to 8 μg or about 0.4 μg to 8 μg (inclusive) per cell. [The present invention 1067] The amount of stimulation reagent in the input composition is 6 Any of the methods of claims 1048 to 1063, 1065 and 1066, wherein the amount is 0.8 μg to 4 μg or about 0.8 μg to 4 μg (inclusive) per cell. [The present invention 1068] The amount of stimulation reagent in the input composition is 6 Any of the methods of claims 1048 to 1063 and 1065 to 1067, wherein the amount is 0.8 μg or about 0.8 μg per cell. [The present invention 1069] The amount of stimulating agent is 10 6 Any of the methods of claims 1048 to 1063 and 1065 to 1068, wherein the amount is 1.2 μg or about 1.2 μg per cell. [The present invention 1070] The amount of stimulating agent is 10 6 Any of the methods of claims 1048 to 1063 and 1065 to 1068, wherein the amount is 1.8 μg or about 1.8 μg per cell. [The present invention 1071] The method of any of claims 1060 to 1070, wherein the one or more recombinant cytokines are human. [The present invention 1072] The method of any of claims 1060 to 1071, wherein the one or more recombinant cytokines are selected from recombinant IL-2, recombinant IL-7, and / or recombinant IL-15, optionally from 10 to 200 IU / mL of recombinant IL-2, 100 IU / mL to 1,000 IU / mL of recombinant IL-7, and / or 10 to 200 IU / mL of recombinant IL-15. [The present invention 1073] Any of the methods of inventions 1060 to 1072, wherein the one or more recombinant cytokines are or comprise recombinant IL-2, recombinant IL-7, and recombinant IL-15, optionally 10 to 200 IU / mL of recombinant IL-2, 100 IU / mL to 1,000 IU / mL of recombinant IL-7, and 10 to 200 IU / mL of recombinant IL-15. [The present invention 1074] The method of any one of claims 1001 to 1048 and 1052 to 1073, wherein one or both of the exposure in (a) and the introduction in (b) are carried out in a serum-free medium. [The present invention 1075] The method according to any one of claims 1001 to 1048 and 1052 to 1074, wherein the exposure in (a) and the introduction in (b) are carried out in a serum-free medium. [The present invention 1076] The method according to any one of claims 1001, 1015 to 1048 and 1052 to 1075, wherein the incubation is carried out in a serum-free medium. [The present invention 1077] The method of any one of claims 1041 to 1045, 1053 to 1064 and 1071 to 1076, wherein the ratio of beads to cells is 2:1 to 0.5:1 or about 2:1 to 0.5:1. [The present invention 1078] Any of the methods of claims 1041 to 1045, 1053 to 1064 and 1071 to 1077, wherein the ratio of beads to cells is 1:1 or about 1:1. [The present invention 1079] The method of any of claims 1041-1045, 1053-1064 and 1071-1078, wherein the beads comprise a diameter greater than or greater than about 3.5 μm, but not greater than about 9 μm, or not greater than about 8 μm, or not greater than about 7 μm, or not greater than about 6 μm, or not greater than about 5 μm. [The present invention 1080] The method of any of claims 1041-1045, 1053-1064 and 1071-1079, wherein the beads comprise a diameter of 4.5 μm or about 4.5 μm. [The present invention 1081] The method of any one of claims 1041 to 1045, 1053 to 1064 and 1071 to 1080, wherein the beads are inert. [The present invention 1082] The method of any one of claims 1041 to 1045, 1053 to 1064 and 1071 to 1081, wherein the beads are or include a polystyrene surface. [The present invention 1083] The method of any one of claims 1041 to 1045, 1053 to 1064 and 1071 to 1082, wherein the beads are paramagnetic or superparamagnetic. [The present invention 1084] exposing the cells to a magnetic field either after or during the incubation, thereby removing the stimulatory reagent from the cells. The method of claim 1083, further comprising, prior to harvesting, [The present invention 1085] The method of any of claims 1001, 1046-1063 and 1065-1076, wherein the streptavidin or streptavidin mutein molecule binds or is capable of binding to biotin, avidin, a biotin analogue or mutein, an avidin analogue or mutein, and / or a biologically active fragment thereof. [The present invention 1086] Each of the streptavidin mutein molecules has the amino acid sequence Va1 at a sequence position corresponding to positions 44-47 in streptavidin in the sequence of amino acids set forth in SEQ ID NO:61. 44 -Thr 45 -Ala 46 -Arg 47 or Ile 44 -Gly 45 -Ala 46 -Arg 47 Any of the methods of the present invention 1001, 1046 to 1063, 1065 to 1076 and 1085, comprising the steps of: [The present invention 1087] Each of the plurality of streptavidin mutein molecules (a) a sequence of amino acids set forth in any of SEQ ID NOs: 62, 63, 68, 75-77, or 80-83; (b) a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to any of SEQ ID NOs: 62, 63, 68, 75-77, or 80-83, and 44 -Thr 45 -Ala 46 -Arg 47 Or Ile 44 -Gly 45 -Ala 46 -Arg 47 and / or a sequence of amino acids which reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide; or (c) a functional fragment of (a) or (b) that reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide. Any of the methods of the present invention 1001, 1046 to 1063, 1065 to 1076, 1085 and 1086, comprising the steps of: [The present invention 1088] The method of any one of claims 1001, 1046-1063, 1065-1076 and 1085-1087, wherein the plurality of streptavidin mutein molecules comprises a sequence of amino acids set forth in SEQ ID NO: 63 or 68. [The present invention 1089] The method according to any one of claims 1046 to 1063, 1065 to 1076 and 1085 to 1088, wherein the main agent and the auxiliary agent each contain a streptavidin-binding peptide. [The present invention 1090] Streptavidin-binding peptides TIFF0007620542000003.tif37164 The method of claim 1089, wherein the compound is selected from the group consisting of: [The present invention 1091] The method according to any one of claims 1046 to 1063, 1065 to 1076 and 1085 to 1090, wherein one or both of the main agent and the auxiliary agent comprise an antibody or an antigen-binding fragment thereof. [The present invention 1092] The method of claim 1091, wherein said one or more agents is or comprises a monovalent antibody fragment. [The present invention 1093] The method of claim 1091 or 1092, wherein one or both of the main agent and the accessory agent is or comprises a Fab. [The present invention 1094] The oligomeric particle reagent comprises: a radius of greater than 60 nm, greater than 70 nm, greater than 80 nm, or greater than 90 nm; A radius between 50 nm and 150 nm, between 75 nm and 125 nm, between 80 nm and 115 nm, or between 90 nm and 110 nm (inclusive); or Radius of 90nm±15nm or 95nm±20~25nm The method of any one of claims 1001, 1046 to 1063, 1065 to 1076 and 1085 to 1093. [The present invention 1095] The oligomeric particle reagent comprises: At least 5×10 7 g / mol or at least 1×10 8 g / mol; and / or 5×10 7 g / mol~5×10 8 g / mol, 1×10 8 g / mol~5×10 8 g / mol, or 1×10 8 g / mol~2×10 8 g / mol The method of the present invention, wherein the molecular weight of the polymer is 1001, 1046 to 1063, 1065 to 1076, or 1085 to 1094. [The present invention 1096] The oligomeric particle reagent comprises: at least 500 streptavidin or streptavidin mutein tetramers, at least 1,000 streptavidin or streptavidin mutein tetramers, at least 1,500 streptavidin or streptavidin mutein tetramers, or at least 2,000 streptavidin or streptavidin mutein tetramers; and / or 1,000 to 20,000 streptavidin or streptavidin mutein tetramers, 1,000 to 10,000 streptavidin or streptavidin mutein tetramers, or 2,000 to 5,000 streptavidin or streptavidin mutein tetramers The method of any one of claims 1001, 1046 to 1063, 1065 to 1076 and 1085 to 1088. [The present invention 1097] adding a substance to the cells either after or during at least a portion of the incubation; the agent terminates or reduces stimulation of T cells by a stimulatory agent; Any of the methods of 1001, 1046 to 1063, 1065 to 1076 and 1085 to 1096 of the present invention. [The present invention 1098] adding a substance to the cells either after or during at least a portion of the incubation; The substance can break the bond between the main agent and the auxiliary agent and the oligomeric particle reagent; Any of the methods of 1046 to 1063, 1065 to 1076 and 1085 to 1097 of the present invention. [This invention 1099] The method of any one of claims 1098 to 1109, wherein said agent is a free binding partner and / or a competitive reagent. [The present invention 1100] The method of any one of claims 1098 to 1099, wherein the presence of said substance terminates or reduces the signal induced or modulated by the primary and secondary agents in the T cell. [The present invention 1101] The method of any of claims 1097 to 1100, wherein said substance is or comprises a streptavidin-binding peptide, biotin or a biologically active fragment, or a biotin analog or a biologically active fragment. [The present invention 1102] The substance is or comprises a streptavidin-binding peptide, the streptavidin-binding peptide comprising: TIFF0007620542000004.tif37163 The method of the present invention 1101, wherein the compound is selected from the group consisting of: [The present invention 1103] 1101. The method of claim 1101, wherein said substance is or comprises biotin or a biologically active fragment or a biotin analogue or a biologically active fragment. [The present invention 1104] The method of any of claims 1097 to 1103, wherein the agent is added at or about 42 hours to 120 hours, inclusive, after exposure to the oligomeric particle stimulant reagent is initiated. [The present invention 1105] The method of any of claims 1097 to 1104, wherein the agent is added at or about 72 to 96 hours after exposure to the oligomeric particle reagent is initiated. [The present invention 1106] The method of any of claims 1097 to 1105, wherein said agent is added 48 hours or about 48 hours after exposure to the oligomeric particle reagent is initiated. [The present invention 1107] The method of any of claims 1097 to 1106, wherein said agent is added 72 hours or about 72 hours after exposure to the oligomeric particle reagent is initiated. [The present invention 1108] The method of any of claims 1097 to 1107, wherein the agent is added 96 hours or about 96 hours after exposure to the oligomeric particle reagent has begun. [The present invention 1109] The method of any of claims 1097 to 1108, wherein said substance is added after incubation and before harvesting. [The present invention 1110] The method of any of claims 1097 to 1109, wherein the substance is added during at least a portion of the incubation, and the incubation continues after the substance has been added. [The present invention 1111] 1. A method of producing a composition of engineered T cells, comprising: (a) An input composition comprising primary T cells is mixed with 10 cells / mL of an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules. 6 exposing the cells to at or about 0.2 μg to 8 μg per cell of a stimulatory reagent, the oligomeric particle reagent having attached thereto (i) a primary agent that specifically binds to CD3 and (ii) a secondary agent that specifically binds to CD28, the exposure being carried out in the presence of serum-free medium having recombinant IL-2, IL-7, and IL-15, thereby generating a stimulated population; (b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells, wherein the introduction is carried out in the presence of serum-free medium having recombinant IL-2, IL-7, and IL-15; (c) incubating the population of transformed cells under static conditions for 24 hours to 96 hours, optionally with the incubation at a temperature of about 37° C. and during at least a portion of the incubation with the addition of a substance comprising biotin or a biologically active fragment thereof, optionally D-biotin or a biotin analog or a biologically active fragment thereof; and (d) harvesting the T cells of the transformed population following said incubation, thereby producing a composition of engineered cells, said harvesting being performed between 72 and 96 hours, inclusive, after exposure to said stimulatory reagent was initiated. Including, Thereby producing a composition of engineered cells. [The present invention 1112] Input composition is at least 300×10 6 The method of any one of claims 1001 to 1111, comprising the viable primary T cells. [The present invention 1113] Input composition is 600 x 10 6 600 x 10 pieces or approx. 6 The method of any of claims 1001 to 1112, comprising the viable primary T cells. [The present invention 1114] Any of the methods of inventions 1001 to 1113, wherein at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, at least or at least about 99%, about 100% or 100% of the cells in the input composition are CD3+ T cells. [The present invention 1115] Any of the methods of inventions 1001 to 1114, wherein at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, at least or at least about 99%, about 100% or 100% of the cells in the composition are CD4+ T cells and CD8+ T cells. [The present invention 1116] The method of any of claims 1001 to 1115, wherein the input composition comprises a ratio of CD4+ cells to CD8+ cells of between 1.5:1 and 2.0:1. [The present invention 1117] The method of any of claims 1001 to 1116, wherein the input composition comprises a ratio of CD4+ cells to CD8+ cells of between 1.2:1 and 0.8:1, and optionally a ratio of CD4+ T cells to CD8+ T cells of 1:1 or about 1:1. [The present invention 1118] 1. A method of producing a composition of engineered T cells, comprising: (a) An input composition comprising primary T cells is mixed with 10 cells / mL of an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules. 6 a step of exposing the oligomer particles to 0.4 μg to 8 μg or about 0.4 μg to 8 μg of a stimulating reagent, the oligomer particles having attached thereto (i) a main agent that specifically binds to CD3 and (ii) a secondary agent that specifically binds to CD28; The input composition comprises a ratio of CD4+ T cells to CD8+ T cells of 2:1 to 1:2 and is at least 300×10 6 comprising CD4+ and CD8+ T cells, wherein the exposure is carried out in the presence of serum-free medium containing recombinant IL-2, IL-7, and IL-15, thereby generating a stimulated population; (b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells, wherein the introduction is carried out in the presence of serum-free medium having recombinant IL-2, IL-7, and IL-15; (c) incubating the population of transformed cells under static conditions for 24 hours to 72 hours or about 24 hours to 72 hours, optionally with the incubation at a temperature of about 37° C. and during at least a portion of the incubation with the addition of a substance comprising biotin or a biologically active fragment thereof, optionally D-biotin or a biotin analog or a biologically active fragment thereof, the addition being performed 48 to 72 hours (inclusive) after exposure to the stimulating agent has begun; and (d) harvesting the T cells of the transformed population following said incubation, thereby producing a composition of engineered cells, said harvesting being performed between 72 and 96 hours, inclusive, after exposure to said stimulatory reagent was initiated. Including, Thereby producing a composition of engineered cells. [The present invention 1119] The method of any of claims 1111 to 1118, wherein said agent is added 48 hours or about 48 hours after exposure to the stimulating agent has begun. [The present invention 1120] The method of any of claims 1111 to 1118, wherein the substance is added 72 hours or about 72 hours after exposure to the stimulating agent has begun. [The present invention 1121] The method of any of claims 1111 to 1118, wherein the substance is added 96 hours or about 96 hours after exposure to the stimulating agent has begun. [The present invention 1122] 1. A method of producing a composition of engineered T cells, comprising: (a) exposing an input composition comprising primary T cells to a stimulation reagent comprising paramagnetic beads at a ratio of beads to cells of 2:1 to 0.5:1 or about 2:1 to 0.5:1, the beads being inert paramagnetic beads comprising a polystyrene coating and a diameter of 4.5 μm or about 4.5 μm, the beads having (i) an anti-CD3 antibody, or an antigen-binding fragment thereof, and an anti-CD28 antibody, or an antigen-binding fragment thereof, attached thereto, the input composition comprising a ratio of CD4+ T cells to CD8+ T cells of 2:1 to 1:2, and a total of at least 300×10 6 comprising CD4+ and CD8+ T cells, wherein the exposure is carried out in the presence of serum-free medium containing recombinant IL-2, IL-7, and IL-15, thereby generating a stimulated population; (b) introducing into the stimulated population of T cells a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells, wherein the introduction is carried out in the presence of serum-free medium having recombinant IL-2, IL-7, and IL-15; (c) incubating the population of transformed cells under static conditions for 48 to 72 hours, inclusive, optionally wherein the incubation is carried out at a temperature of about 37°C; (d) after said incubation, exposing the cells to a magnetic field to remove the stimulatory reagent from the cells and then harvesting the T cells, thereby producing a composition of engineered cells, wherein harvesting is performed between 72 and 96 hours, inclusive, after exposure to the stimulatory reagent began. Including, Thereby producing a composition of engineered cells. [The present invention 1123] The method of any of claims 1111 to 1122, wherein at least a portion of the incubation is carried out in the presence of serum-free medium comprising recombinant IL-2, IL-7, and IL-15. [The present invention 1124] The method of any of claims 1111 to 1123, wherein the incubation is carried out in a basal medium lacking recombinant cytokines. [The present invention 1125] The input composition is 450 x 10 6 Pieces or approx. 450 x 10 6 viable primary T cells, 500 x 10 6 pcs or approx. 500 x 10 6 viable primary T cells, 550 x 10 6 pcs or approx. 550 x 10 6 viable primary T cells, 600 x 10 6 pcs or approx. 600 x 10 6 viable primary T cells, 700 x 10 6 pcs or approx. 700 x 10 6 viable primary T cells, 800 x 10 6 800 x 10 6 viable primary T cells, 900 x 10 6 pcs or approx. 900 x 10 6 viable primary T cells, or 1,000 x 10 6 Pieces or about 1,000 x 10 6 The method of any of claims 1001 to 1124, comprising: [The present invention 1126] Input composition is at least 600×10 6 or at least about 600 x 10 6 The method of any one of claims 1001 to 1125, comprising the viable primary T cells. [The present invention 1127] Input composition is 900 x 10 6 pcs or max 900 x 10 6 The method of any of claims 1001 to 1126, comprising the viable primary T cells. [The present invention 1128] Input composition is 900 x 10 6 pcs or approx. 900 x 10 6 The method of any one of claims 1001 to 1125, comprising the viable primary T cells. [The present invention 1129] The method of any one of claims 1044 to 1059, 1061 to 1069, 1081 to 1113, 1115 and 1116, wherein the incubation is carried out for 24 hours or about 24 hours after the introduction. [The present invention 1130] The method of any of claims 1001 and 1015 to 1129, wherein the incubation is carried out for 48 hours or about 48 hours after the introduction. [The present invention 1131] The method of any of claims 1001 and 1015 to 1130, wherein the incubation is carried out for 72 hours or about 72 hours after the introducing. [The present invention 1132] The method of any one of claims 1001 to 1131, comprising the step of enriching CD3+ T cells or CD4+ T cells and / or CD8+ T cells from the biological sample prior to exposure to the stimulatory reagent to produce an input composition. [The present invention 1133] The method of any one of claims 1001 to 1132, comprising the step of enriching CD3+ T cells from the biological sample to produce the input composition prior to exposure to the stimulatory reagent. [The present invention 1134] The method of any one of claims 1132 to 1133, wherein the enrichment is by immunoaffinity-based selection. [This invention 1135] The method of the present invention 1134, wherein the immunoaffinity-based selection is performed by contacting the cells with an antibody immobilized on or attached to an affinity chromatography matrix, which antibody is capable of specifically binding to a cell surface marker and performing positive selection of CD3+ cells. [The present invention 1136] The method of any of claims 1001 to 1132, comprising the step of enriching CD4+ or CD8+ T cells from the biological sample to produce the input composition prior to exposure to the stimulatory reagent. [This invention 1137] Concentration, (a) performing a first selection in a closed system, the first selection comprising enrichment of one of (i) CD4+ cells and (ii) CD8+ cells from a sample comprising primary human T cells, whereby the enrichment produces a first selected population and a non-selected population; and (b) performing a second selection in a closed system, the second selection comprising enrichment of the other of (i) CD4+ cells and (ii) CD8+ cells from the unselected population, whereby the enrichment produces a second selected population, the enrichment producing separate enriched populations of CD4+ T cells and CD8+ T cells. Including, each of the separate enriched populations comprises cells from either the first selected population or the second selected population; the input composition comprises cells from one or more of an enriched population of CD4+ T cells and an enriched population of CD8+ T cells; The method of the present invention 1136. [The present invention 1138] The method of any one of claims 1136 to 1137, wherein separate enriched populations of CD4+ T cells and CD8+ T cells are mixed, thereby producing the input composition. [The present invention 1139] The method of claim 1138, wherein the mixing is carried out in a closed system, and optionally, the closed system is automated. [The present invention 1140] The method of any of claims 1136 to 1139, wherein enriching the cells in the first and / or second selection comprises performing a positive or negative selection based on the expression of a cell surface marker. [This invention 1141] Any of the methods of claims 1136 to 1140, wherein the enrichment of cells in the first or second selection comprises performing multiple positive or negative selection steps based on the expression of a cell surface marker or a marker for enriching CD4+ or CD8+ cells. [This invention 1142] 1136. The method of any of claims 1136 to 1141, wherein enriching for cells in the first and / or second selection comprises selection based on immune affinity. [This invention 1143] The method of claim 1142, wherein enriching for cells in the first and second selections comprises immunoaffinity-based selection. [This invention 1144] The method of claim 1142 or 1143, wherein the immunoaffinity-based selection is performed by contacting the cells with an antibody immobilized on or attached to an affinity chromatography matrix, which antibody specifically binds to a cell surface marker and is capable of performing positive or negative selection of CD4+ or CD8+ cells. [This invention 1145] the antibody further comprises one or more binding partners capable of forming a reversible bond with a binding reagent immobilized on the matrix, the antibody reversibly binding to the chromatographic matrix during contact; Cells expressing a cell surface marker on the matrix to which the antibody specifically binds can be recovered from the matrix by cleavage of the reversible bond between the binding reagent and the binding partner. The method of the present invention 1135 or 1144. [The present invention 1146] the binding partner is selected from biotin, a biotin analog, and a peptide capable of binding to the binding agent; and the binding reagent is selected from streptavidin, streptavidin analogues or muteins, avidin, and avidin analogues or muteins; The method of the present invention 1145. [This invention 1147] the binding partner comprises the sequence of amino acids set forth in SEQ ID NO:64; and / or The binding agent is a streptavidin mutein comprising a sequence of amino acids set forth in SEQ ID NO: 75, 80, 76 or 63; The method of the present invention 1145 or 1146. [This invention 1148] Any of the methods of claims 1145 to 1147, further comprising the step of contacting the cells in the sample with the affinity chromatography matrix, and then adding a competing reagent to cleave the bond between the binding partner and the binding reagent, thereby recovering the selected cells from the matrix. [This invention 1149] 1148. The method of claim 1148, wherein the competing reagent is biotin or a biotin analogue. [The present invention 1150] One or more antibodies in one or more selections are more than 3×10 -5 sec -1 Greater than or about 3 x 10 -5 sec -1 The binding and dissociation rate constants (k off Any of the methods of the present invention 1135 and 1144 to 1149, comprising the steps of: [This invention 1151] One or more antibodies in one or more selections are -3 ~10 -7 in the range of about 10 -7 ~about 10 -10 Dissociation constants (K d ) and having affinity for a cell surface marker. [This invention 1152] The method of any one of claims 1135 and 1144 to 1151, wherein the chromatography matrix is ​​packed in a separation vessel which is a column. [This invention 1153] Enrichment of CD4+ or CD8+ T cells from a biological sample (a) Enrichment of CD4+ cells involves positive selection based on surface expression of CD4; (b) the enrichment of CD8+ cells comprises positive selection based on surface expression of CD8; or Both (a) and (b) Any of the methods of the present invention 1136 to 1152, comprising: [This invention 1154] enriching for CD4+ or CD8+ T cells from the biological sample prior to exposure to the stimulatory reagent, the enrichment comprising contacting cells of the sample with a first immunoaffinity reagent that specifically binds CD4 in an incubation composition and a second immunoaffinity reagent that specifically binds CD8 in an incubation composition under conditions in which the immunoaffinity reagents specifically bind to CD4 and CD8 molecules, respectively, on the surface of cells in the sample; and recovering the cells that bind to the first and / or second immunoaffinity reagents, thereby producing an enriched composition comprising CD4+ cells and CD8+ cells. Including, The input composition comprises cells of an enriched composition comprising CD4+ cells and CD8+ cells; Any of the methods of 1001 to 1153 of the present invention. [This invention 1155] The method of any one of claims 1154 to 1156, wherein each of the immunoaffinity reagents comprises an antibody or an antigen-binding fragment thereof. [This invention 1156] The method of any one of claims 1154 to 1155, wherein the immunoaffinity reagent is immobilized on the outer surface of a bead. [This invention 1157] The method of claim 1156, wherein the beads are magnetic beads. [This invention 1158] The method of claim 1157, wherein recovering the cells bound to the first or second immunoaffinity reagent comprises exposing the cells to a magnetic field. [This invention 1159] The method of any of claims 1132 to 1158, wherein the biological sample comprises primary T cells obtained from a subject, optionally a human subject. [The present invention 1160] Any of the methods of claims 1132 to 1159, wherein the biological sample is or comprises a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukocyte apheresis product. [The present invention 1161] The method of any of claims 1132 to 1160, wherein the biological sample is an apheresis product or a leukapheresis product that has been pre-cryo-frozen prior to enrichment. [The present invention 1162] The method of any of claims 1001 to 1048 and 1052 to 1161, wherein the harvesting is performed 96 hours or about 96 hours after exposure to the stimulating agent is initiated. [The present invention 1163] The method of any of claims 1001 to 1048 and 1052 to 1161, wherein the harvesting is performed 2 days or about 2 days or 3 days or about 3 days after exposure to the stimulating agent is initiated. [The present invention 1164] The method of any of claims 1001 to 1048 and 1052 to 1161, wherein the harvesting is performed 72 hours or about 72 hours after exposure to the stimulating agent is initiated. [The present invention 1165] The method of any of claims 1001 to 1048 and 1052 to 1161, wherein the harvesting is performed 48 hours or about 48 hours after exposure to the stimulating agent is initiated. [The present invention 1166] Any of the methods of claims 1001 and 1012 to 1165, wherein harvesting is performed at the time when an integrated vector is detected in the genome but before achieving stable iVCN per diploid genome. [The present invention 1167] Any of the methods of claims 1001 and 1012 to 1166, wherein harvesting is performed at a time point when the fraction of integrated vector copy number (iVCN) to total VCN in the population of transformed cells is on average less than 0.8. [The present invention 1168] Any of the methods of claims 1001 and 1012 to 1167, wherein the harvesting is performed at a time when the iVCN of the population of transformed cells is, on average, less than or about 2.0 copies per diploid genome. [The present invention 1169] The method of any one of claims 1001 to 1168, wherein harvesting the cells comprises removing cell debris by rinsing or washing the cells. [The present invention 1170] Any of the methods of claims 1001 and 1030 to 1169, wherein the cells are harvested at a time point when the percentage of naive-like cells is greater than or greater than about 60% among the total T cells in the population, among the total CD4+ T cells in the population, or among the total CD8+ T cells or recombinant protein-expressing cells thereof in the population. [This invention 1171] Any of the methods of claims 1001 and 1030 to 1169, wherein the cells are harvested at a time when the percentage of naive-like cells and / or central memory T cells is greater than or greater than about 60% among the total T cells in the population, among the total CD4+ T cells in the population, or among the total CD8+ T cells or cells expressing a recombinant protein thereof in the population. [This invention 1172] At the time of collection, the percentage of naive-like T cells is greater than or greater than about 60% among all recombinant protein-expressing cells in the population, optionally greater than or greater than about 65%, 70%, 80%, 90%, or 95% among all recombinant protein-expressing T cells in the population; the percentage of naive-like T cells is greater than or greater than about 60% of all recombinant protein-expressing CD4+ T cells in the population, optionally greater than or greater than about 65%, 70%, 80%, 90%, or 95% of all recombinant protein-expressing CD4+ cells in the population; or the percentage of naive-like T cells is greater than or greater than about 60% of the total recombinant protein-expressing CD8+ T cells in the population, optionally greater than or greater than about 65%, 70%, 80%, 90%, or 95% of the total recombinant protein-expressing CD8+ cells in the population; The method of the present invention 1170 or 1171. [This invention 1173] At the time of collection, the percentage of naive-like T cells and / or central memory T cells is greater than or greater than about 60% among all recombinant protein-expressing cells in the population, optionally greater than or greater than about 65%, 70%, 80%, 90%, or 95% among all recombinant protein-expressing T cells in the population; the percentage of naive-like T cells and / or central memory T cells is greater than or greater than about 60% of the total recombinant protein-expressing CD4+ T cells in the population, optionally greater than or greater than about 65%, 70%, 80%, 90%, or 95% of the total recombinant protein-expressing CD4+ cells in the population; or the percentage of naive-like T cells and / or central memory T cells is greater than or greater than about 60% of the total recombinant protein-expressing CD8+ T cells in the population, optionally greater than or greater than about 65%, 70%, 80%, 90%, or 95% of the total recombinant protein-expressing CD8+ cells in the population; The method of the present invention 1171. [This invention 1174] The method of any of claims 1170 to 1173, wherein the naive-like T cells, or T cells that are surface positive for a marker expressed on the naive-like T cells, are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+. [This invention 1175] The method of any one of claims 1170 to 1174, wherein the naive-like T cells comprise CD27+CCR7+T cells. [The present invention 1176] The method of any one of claims 1170 to 1175, wherein the naive-like T cells comprise CCR7+CD45RA+T cells. [This invention 1177] Any of the methods of claims 1001 to 1176, further comprising the step of formulating the cells of the engineered cell composition, optionally in the presence of a pharma- ceutically acceptable excipient, for cryopreservation and / or administration to a subject. [This invention 1178] The method of any one of claims 1177 to 1179, wherein the cells of the engineered cell composition are formulated in the presence of a cryoprotectant. [This invention 1179] The method of any one of claims 1178 to 1179, wherein the cryoprotectant comprises DMSO. [The present invention 1180] The method of any of claims 1177 to 1179, wherein the cells of the engineered cell composition are formulated in a container, optionally a vial or bag. [This invention 1181] The method according to any of claims 1001 to 1180, wherein a viral vector comprising a heterologous polynucleotide encoding a recombinant protein is introduced into a T cell. [The present invention 1182] 1181. The method of claim 1181, wherein the viral vector is a retroviral vector. [The present invention 1183] 1182. The method of claim 1180 or 1181, wherein the viral vector is a lentiviral vector or a gamma retroviral vector. [This invention 1184] The method of any of claims 1001 to 1183, wherein the transduction is carried out in the presence of a transduction adjuvant. [This invention 1185] The method of the invention 1184, wherein the transduction adjuvant is protamine sulfate, optionally at or about 1 μg / ml to 50 μg / ml of protamine sulfate, a fibronectin derived transduction adjuvant or RetroNectin. [The present invention 1186] The recombinant protein is A recombinant receptor capable of binding to a target antigen associated with, specific for, and / or expressed on a disease, disorder, or diseased cell or tissue. Any of the methods of the present invention 1001 to 1185. [This invention 1187] The method of claim 1186, wherein the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer. [The present invention 1188] The method of claim 1187, wherein the target antigen is a tumor antigen. [The present invention 1189] Target antigens include αvβ6 integrin (avb6 integrin), B-cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), cancer testis antigen, cancer testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutant (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp10O), glypican-3 (GPC3), G protein-coupled receptor class C group 5 member D (GPRC5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimer, human high molecular weight black Chromoma-associated antigen (HMW-MAA), Hepatitis B surface antigen, Human leukocyte antigen A1 (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), Kinase insert domain receptor (kdr), Kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, Leucine-rich repeat-containing 8 family member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, Mesothelin (MSLN), c-Met, Murine cytomegalovirus (CMV), Mucin 1 (MUC1), MUC16, Natural killer group 2 member D (NKG2D) ligand,Melan-A (MART-1), neural cell adhesion molecule (NCAM), carcinoembryonic antigen, preferentially expressed antigen in melanoma (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein G (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-related protein 1 (TRP1, also known as TYRP1 or gp75), tyrosinase-associated ta The method according to any one of claims 1186 to 1188, wherein the antigen is selected from among: transmembrane protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms' tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen, or an antigen associated with a universal tag, and / or a biotinylated molecule, and / or a molecule expressed by HIV, HCV, HBV, or other pathogens. [The present invention 1190] The method of any of claims 1001 to 1048 or 1052 to 1189, wherein the recombinant protein is or comprises a functional non-TCR antigen receptor or a TCR or an antigen-binding fragment thereof. [The present invention 1191] The method of any one of claims 1001 to 1048 or 1052 to 1190, wherein the recombinant protein is a chimeric antigen receptor (CAR). [This invention 1192] Serum-free medium, Dipeptide form of L-glutamine at 0.5 mM to 5 mM in basal medium; 0.5 mM to 5 mM L-glutamine; and At least one protein The method of any one of claims 1034 to 1042 or 1074 to 1191, comprising the steps of: [The present invention 1193] The method of any of claims 1001 and 1026 to 1192, wherein the basal medium lacking one or more recombinant cytokines comprises L-glutamine, optionally at a concentration of about 0.5 mM to about 5 mM, optionally at a concentration of about 2 mM. [This invention 1194] The method of any of claims 1001 and 1026 to 1191 and 1193, wherein the basal medium lacking one or more recombinant cytokines is serum-free. [The present invention 1195] The method of any of claims 1001, 1026 to 1191 and 1193 to 1194, wherein the basal medium comprises at least one protein selected from one or more of albumin, insulin, or transferrin, optionally one or more of human or recombinant albumin, insulin, or transferrin. [The present invention 1196] A therapeutic T cell composition produced by any one of the methods of the present inventions nos. 1001 to 1195. [The present invention 1197] or at least about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total number of T cells in the compositions or the total number of T cells expressing a recombinant protein in the compositions, on average across a plurality of compositions produced by the method. %, or about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are naive-like T cells or T cells that are surface positive for a marker expressed on naive-like cells. [The present invention 1198] The therapeutic T cell composition of the present invention 1196, wherein on average in a plurality of compositions produced by the method, at least 50% or at least about 50% or 50% or about 50% of the total number of T cells in the composition or the total number of T cells expressing a recombinant protein in the composition are central memory T cells or are T cells that are surface positive for a marker expressed on central memory T cells. [The present invention 1199] A therapeutic T cell composition of the present invention 1196, wherein on average, at least 50% or at least about 50% or 50% or about 50% of the total number of T cells in the composition or the total number of T cells expressing a recombinant protein in the composition are naive-like T cells or central memory T cells, or are T cells that are surface positive for a marker expressed on naive-like T cells or central memory T cells. [The present invention 1200] The therapeutic T cell composition of the present invention 1197 or 1199, wherein the marker expressed on naive-like T cells is selected from the group consisting of CD45RA, CD27, CD28, and CCR7. [The present invention 1201] The therapeutic T cell composition of any of claims 1197, 1199 and 1200, wherein the naive-like T cells, or T cells that are surface positive for markers expressed on naive-like T cells, are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+. [The present invention 1202] Naive-like T cells, or T cells that are surface positive for a marker expressed on naive-like T cells, include CD27+CCR7+ T cells and are expressed by all receptors in the composition. + / CD8 + The therapeutic T cell composition of any of claims 1197 and 1199 to 1201, wherein at least 70%, 80%, 85%, or 90% of the cells are CD27+CCR7+. [The present invention 1203] Naive-like T cells, or T cells that are surface positive for a marker expressed on naive-like T cells, include CD27+CCR7+ T cells and are expressed by all receptors in the composition. + / CD4 + The therapeutic T cell composition of any of claims 1197 and 1199 to 1202, wherein at least 50%, 60%, 70%, 80%, 85%, or 90% of the cells are CD27+CCR7+. [The present invention 1204] The naive-like T cells, or T cells that are surface positive for a marker expressed on the naive-like T cells, include CD27+CCR7+ T cells, and the total receptor markers in the composition are + / CD8 + At least 50%, 60%, 70%, 80%, 85%, or 90% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4 + The therapeutic T cell composition of any of claims 1197 and 1199 to 1203, wherein at least 50%, 60%, 70%, 80%, 85%, or 90% of the cells are CD27+CCR7+. [The present invention 1205] 1. A therapeutic T cell composition comprising CD4+ T cells expressing a recombinant receptor and CD8+ T cells expressing a recombinant receptor, All receptors in the composition + / CD8 + At least 50%, 60%, 70%, 80%, or 90% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4 + At least 50%, 60%, 70%, 80%, or 90% of the cells are CD27+CCR7+, Therapeutic T cell composition. [The present invention 1206] Therapeutic T cell compositions according to any of claims 1196 to 1205, wherein at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100% or 100% of the cells in the composition are CD3+ T cells. [The present invention 1207] Therapeutic T cell compositions according to any of claims 1196 to 1206, wherein at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, at least or at least about 99%, about 100% or 100% of the cells in the composition are CD4+ T cells and CD8+ T cells. [The present invention 1208] At least or at least about 90% of the cells in the composition are CD4+ T cells and CD8+ T cells, and all receptors in the composition + / CD8 + At least or at least about 60% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4 + Therapeutic T cell composition according to any of claims 1205 to 1207, wherein at least or at least about 60% of the cells are CD27+CCR7+. [The present invention 1209] at least about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total number of T cells in the composition or the total number of T cells expressing a recombinant receptor in the composition %, or about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are CD27+CCR7+. [The present invention 1210] The therapeutic T cell composition of any of claims 1197 to 1209, wherein the ratio of receptor+ / CD4+ T cells to receptor+ / CD8+ T cells in the composition is about 1:3 to about 3:1. [The present invention 1211] The therapeutic T cell composition of any of claims 1196 to 1210, wherein the ratio of receptor+ / CD4+ T cells to receptor+ / CD8+ T cells in the composition is about 1:2 to about 2:1. [The present invention 1212] The therapeutic T cell composition of any of claims 1196 to 1211, wherein the ratio of receptor+ / CD4+ T cells to receptor+ / CD8+ T cells in said composition is 1:1 or about 1:1. [The present invention 1213] The recombinant protein is A recombinant receptor that is associated with, specific for, and / or expressed on a disease, disorder, or diseased cell or tissue and is capable of binding to a target protein. A therapeutic composition according to any one of claims 1196 to 1212, which is or contains the same. [The present invention 1214] The therapeutic composition of any of claims 1196 to 1213, wherein the recombinant protein is a chimeric antigen receptor (CAR). [The present invention 1215] The number of viable T cells in the composition is 25×10 6 Pieces or about 25 x 10 6 From 200 x 10 6 Pieces or about 200 x 10 6 and optionally the number of viable T cells in the composition is 25×10 6 Pieces or about 25 x 106 From 100 x 10 6 Pieces or about 100 x 10 6 pieces, 25×10 6 Pieces or about 25 x 10 6 From 70 x 10 6 pcs or approx. 70 x 10 6 pieces, 25×10 6 Pieces or about 25 x 10 6 From 50 x 10 6 Pieces or about 50 x 10 6 pieces, 50×10 6 Pieces or about 50 x 10 6 From 200 x 10 6 Pieces or about 200 x 10 6 pieces, 50×10 6 Pieces or about 50 x 10 6 From 100 x 10 6 Pieces or about 100 x 10 6 pieces, 50×10 6 Pieces or about 50 x 10 6 From 70 x 10 6 pcs or approx. 70 x 10 6 pieces, 70×10 6 pcs or approx. 70 x 10 6 From 200 x 10 6 Pieces or about 200 x 10 6 pieces, 70×10 6 pcs or approx. 70 x 10 6 From 100 x 10 6 Pieces or about 100 x 10 6 pcs or 100 x 10 6 Pieces or about 100 x 10 6 From 200 x 10 6 Pieces or about 200 x 10 6 The therapeutic composition of any one of 1196 to 1214, wherein the number of the compounds is 1 or more (each including both end values). [The present invention 1216] The therapeutic composition of any of claims 1196 to 1215, wherein the amount of the composition is 1.0 mL to 10 mL (inclusive), optionally 2 mL or about 2 mL, 3 mL or about 3 mL, 4 mL or about 4 mL, 5 mL or about 5 mL, 6 mL or about 6 mL, 7 mL or about 7 mL, 8 mL or about 8 mL, 9 mL or about 9 mL, or 10 mL or about 10 mL, or any value therebetween. [The present invention 1217] An article of manufacture comprising any one of the compositions of inventions 1196 to 1216 and instructions for administering the composition to a subject. [The present invention 1218] The article of manufacture of the invention 1217, wherein the subject has a disease or condition, and optionally the recombinant receptor specifically recognizes or specifically binds to an antigen associated with or expressed or present on cells of the disease or condition. [The present invention 1219] A method of treating a subject having or suspected of having a disease or condition, comprising administering to the subject a dose of T cells from any of the compositions of inventions 1196-1216. [The present invention 1220] The dose of T cells is 1×10 4 pieces~50×10 6 1 x 10 pieces or approx. 4 pieces~50×10 6 1219. The method of claim 1219, comprising administering to said patient a total of 1219 T cells (inclusive). [The present invention 1221] The method of claim 1219 or 1220, wherein the T cells of said dose of T cells are total T cells, total viable T cells, total viable recombinant receptor-expressing T cells, total viable recombinant receptor-expressing CD4+ T cells, or total viable recombinant receptor-expressing CD8+ T cells. [The present invention 1222] The method of any one of claims 1219 to 1221, wherein the disease or condition is cancer. [The present invention 1223] 13. A composition according to any of claims 1196 to 1216 for use in a method of treating a subject having a disease or condition, optionally wherein the disease or condition is cancer. [The present invention 1224] 23. The use of any of the compositions of claims 1196 to 1216 for the manufacture of a medicament for treating a subject having a disease or condition, optionally wherein the disease or condition is cancer. [The present invention 1225] A composition for use of the invention 1223 or use 1224, wherein the cells of said composition express a recombinant receptor that specifically recognizes or specifically binds to an antigen associated with or expressed or present on diseased or diseased cells, and optionally the recombinant receptor is a chimeric antigen receptor. [Brief description of the drawings]

[0116] [Figure 1] Figures 1A and 1B show the results of WST metabolic assay of T cells from three different donors incubated with anti-CD3 / anti-CD28 multimerized on different batches of oligomer reagent. Figure 1A summarizes the WST metabolic activity for all test batches (pools) compared to a reference batch containing anti-CD3 / anti-CD28 multimerized on an oligomer scaffold with an average hydrodynamic radius of 36 nm or 101 nm. The average WST metabolic activity among T cells from different donors for each test batch and the reference reagent is shown in Figure 1B. [Diagram 2]Figures 2A and 2B depict the total cell count (TNC) (Figure 2A) and percentage of viable cells (Viability) (Figure 2B) assessed from cell compositions generated from the expansion expansion process (Expanded) and non-expansion expansion process with bead-based stimulation reagent (Bead), bead-based stimulation reagent and incubation in basal media (Bead-Basal Media) or oligomer stimulation reagent (Oligomer) to generate T cell compositions containing CAR+ T cells. Results are shown from samples collected during the Stim, Transduction, Incubation, Collection, Cell Formulation, and Cryopreservation steps. [Diagram 3] FIG. 3 displays the cytolytic activity of exemplary anti-BCMA CAR T cells assessed when cell compositions generated from the expansion expansion process (Expanded) and the non-expansion expansion process with (a) bead-based stimulation reagent (Bead), (b) bead-based stimulation reagent and incubation in basal media (Bead-Basal Media) or (c) oligomer stimulation reagent (Oligomer) were incubated with a BCMA-expressing target cell line (RPMI-8226) at effector:target ratios of 27:1, 9:1, 3:1 or 1:1, respectively. [Figure 4]4A-4D depict results from CAR+ T cell compositions generated from the expansion expansion process and the non-expansion expansion process with (a) bead-based stimulation reagent (Bead), (b) bead-based stimulation reagent and incubation in basal media (Bead-Basal Media) or (c) oligomer stimulation reagent (Oligomer). FIG. 4A depicts the percentage of CD4+ and CD8+ T cells positive for both CCR7 and CD27. FIG. 4B depicts the percentage of CCR7+CD27+ cells relative to CD4+CAR+T cells. FIG. 4C depicts the percentage of CCR7+CD27+ cells relative to CD8+CAR+T cells. FIG. 4D displays the percentage of CCR7+CD27+ cells generated from a representative donor from the expansion process at various days during the manufacturing process, including activation on day 1 (d1 AMAT), transduction on day 2 (d2 XMAT) and various times after initiation of culture (d4 INOC+2, d6 INOC+4, d7 INOC+5). [Diagram 5] Figures 5A-5F depict tumor burden in an in vivo mouse xenograft tumor model following treatment with engineered CAR-T cell compositions generated by the expansion expansion process and the non-expansion expansion process with (a) bead-based stimulation reagent (Bead), (c) oligomer stimulation reagent (Oligomer) or (d) bead-based stimulation reagent incubated in basal media (Bead-Basal Media), respectively, over a 50-day period, compared to tumor only or mock-transduced controls. Figure 5A depicts the mean tumor burden per group. Figures 5B-5F display the results for individual mice in each evaluated treatment group: Figure 5B (Control), Figure 5C (Expansion Expansion Process), Figure 5D (Non-Expansion Expansion Oligomer Process), Figure 5E (Non-Expansion Expansion Bead Process) and Figure 5F (Non-Expansion Expansion Bead Process, Basal Media). [Figure 6]Figures 6A-6E depict representative bioluminescence images of tumor burden in an in vivo mouse xenograft tumor model at select days after treatment with mock-transduced controls (Figure 6A) or engineered CAR-T cell compositions generated by the Expanded (Figure 6B) and non-Expanded expansion processes with Oligomer (Figure 6C), Bead (Figure 6D) or Bead-Basal Media (Figure 6E), each over a 50-day period. [Figure 7] Figure 7 depicts the total number of T cells and CAR+ T cells per μL of blood collected from mice receiving xenograft tumors at 33 days after treatment with engineered CAR-T cell compositions generated by the Expanded and Non-Expansion Expansion processes with (a) Oligomer stimulation reagent (Oligomer), (b) Bead-based stimulation reagent (Bead) or (c) Bead-Basal Media-incubated bead-based stimulation reagent (Bead-Basal Media). Cells were stained with a reagent specific for a truncated receptor surrogate marker and analyzed by flow cytometry. [Figure 8] Figures 8A and 8B depict the percentage of total viable cells and the total number of cells quantified from anti-CD19 CAR-T cell compositions transduced with a lentivirus encoding an anti-CD19 CAR and generated using either an Expanded or Non-Expanded expansion process using an anti-CD3 / anti-CD28 Fab-conjugated streptavidin mutein oligomer as a stimulatory agent. [Figure 9]Figures 9A and 9B depict the viability or total cell number of anti-CD19 CAR-T cells during co-culture with CD19-expressing cells. Figure 9A shows the percentage of viable cells and the total number of CAR+ T cells from anti-CD19 CAR-T cell compositions generated using an expansion or non-expansion process. Figure 9B shows the percentage of viable cells and the total number of CAR+ T cells from anti-CD19 CAR-T cell compositions generated from T cells stimulated directly after selection (non-frozen) or cryopreserved prior to stimulation (frozen). [Figure 10] Figure 10 shows the cytolytic activity of anti-CD19 CAR+ T cell compositions containing cells generated using an expansion or non-expansion process and cryopreserved prior to stimulation. Cells were co-cultured with CD19-expressing cells at an effector:target cell ratio of 10:1 on days 0, 1, 3, or 6 after thawing. [Figure 11] Figures 11A and 11B depict anti-CD19 CAR-T cells generated using the Expanded or Non-expanded expansion process and cultured under long-term stimulation involving co-culturing HEK cells engineered to express CD19 at an effector:target cell ratio of 5:1 in medium lacking additional recombinant cytokines. Figure 11A depicts the number of viable anti-CD19 CAR-T cells assessed at various time points over the long-term stimulation. ND-not detectable. Figure 11B depicts the percentage of positively stained cells from samples collected on days 11 and 19 from the long-term stimulation cultures and stained with antibodies against surface proteins including CD25, CD69, PD-1, LAG-3, TIM-3, CD45RA and TIGIT as well as for surrogate markers of CAR expression. [Figure 12]12A-12C show results from an in vivo tumor mouse model in which mice were injected on day 0 with 1×10 6 , 0.5×10 6 or 0.25×10 6 anti-CD19 CAR-T cells or PBS control generated using the Expanded or Non-Expanded expansion process. FIG. 12A depicts quantification of tumor presence measured as the mean radiance of tumor-associated bioluminescence signal and the percentage of tumor cells in blood samples collected at different time points during the study. FIG. 12B depicts the percentage of both total T cells and total CAR-T cells in blood samples collected at different time points during the study. FIG. 12C depicts the percent survival of mice up to 60 days post-injection. [Figure 13] 13A-13B depict the engineered T-cell (EC) composition generated by the expansion growth process with spinoculated and bead-based stimulation reagent under static incubation for 18-30 hours (arm 1) and non-expansion growth process with spinoculated and oligomeric stimulation reagent incubated in basal medium for 96 hours (arm 4), spinoculated and bead-based stimulation reagent incubated in basal medium for 72 hours (arm 2), oligomeric stimulation reagent incubated in basal medium for 72 hours (arm 5), bead-based stimulation reagent incubated in basal medium for 48 hours (arm 3), and unstimulated cryopreserved T-cell composition (CMAT). FIG. 13A depicts the percentage of CCR7+CD27+ cells of CD4+CAR+T cells. FIG. 13B depicts the percentage of CD4+CAR+T cells and CD8+CAR+T cells. [Figure 14]FIG. 14 shows the cytolytic activity (% specific lysis) against K562-BCMA target cells at various effector:target ratios (E:T) of engineered T cell compositions generated by an expansion growth process with bead-based stimulation reagents spunoculated and under static incubation for 18-30 hours (arm 1) as well as a process using mock empty vector (arm 6, mock) and a non-expansion growth process with bead-based stimulation reagents spunoculated and incubated in basal medium for 72 hours (arm 2), bead-based stimulation reagents spunoculated and incubated in basal medium for 48 hours (arm 3), oligomeric stimulation reagents spunoculated and incubated in basal medium for 96 hours (arm 4), spinoculated and oligomeric stimulation reagents and incubation in basal medium for 72 hours (arm 5). Cytolytic activity was assessed by microscopy by loss of red fluorescent signal and normalized to cell counts at the beginning of each culture every 4 hours over a period of 48 hours, 72 hours and 96 hours. The dotted line indicates the EC50 for killing. [Figure 15]Figures 15A-15C depict the activity in a long-term stimulation assay involving continuous incubation with recombinant BCMA Fc fusion protein and paramagnetic beads for 6 days of T cell compositions generated by an expansion process involving spinoculation and bead-based stimulation reagent under static incubation for 18-30 hours (1) as well as a process using a mock empty vector (arm 6, mock) and a non-expansion expansion process involving spinoculation and bead-based stimulation reagent incubated in basal medium for 72 hours (2), spinoculation and bead-based stimulation reagent incubated in basal medium for 48 hours (3), spinoculation and oligomeric stimulation reagent incubated in basal medium for 96 hours (4), spinoculation and oligomeric stimulation reagent and incubation in basal medium for 72 hours (5). Figure 15A shows the total number of viable cells observed during the course of long-term stimulation. Figure 15B shows the expansion of different T cell compositions as quantified as the area under the curve for total viable cells measured from days 1 through 6 of incubation. Figure 15C shows the percent of viable cells (% viability) across T cells produced from different engineering processes observed at different time points during the course of long-term stimulation. [Figure 16]

[0023] Figures 16A-16C depict cytokine profiles following chronic stimulation of the T cell compositions depicted in Figures 15A-15C with continuous incubation for 6 days with paramagnetic beads conjugated with recombinant BCMA Fc fusion protein. Figure 16A shows the percentage of CD4 / CAR+ and CD8 / CAR+ T cells staining positive for the cytokines TNFa, IFN-g, and IL-2 on day 0 of chronic stimulation. Figure 16B shows the percentage of CD4 / CAR+ and CD8 / CAR+ T cells staining positive for the cytokines TNFa, IFN-g, and IL-2 on day 6 of chronic stimulation. Figure 16C shows the polyfunctional score and IL-2 global score from cumulative levels of cytokines as determined on CD8+ cells, normalized by adjusting for donor. [Figure 17A]Figure 17A shows the correlation between the number of doublings in the process for producing a therapeutic composition and the percentage of CD4+CAR+ cells that are positive for CD27+. [Figure 17B] Figure 17B shows the relationship between the percentage of central memory / naive-like CD4+ T cells in a therapeutic output T cell composition (e.g., drug) and the number of population doublings that achieve harvest criteria (Spearman ρ: -0.54; p-value: <0.001). Similar results were observed for CD8+ T cells in a therapeutic output T cell composition (e.g., drug). [Figure 17C] FIG. 17C shows the number of population doublings reaching harvest criteria for high (0.35×10^6 cells / mL) and low (0.05×10^6 cells / mL) seeding densities during the expansion step of the production process. [Figure 17D] Figure 17D shows the percentage of CD27+CAR+CD8+ T cells in the output therapeutic composition as a function of seeding density during the expansion step of the production process. [Figure 17E] FIG. 17E shows the effect of treatment duration on the amount of TCM cells in the output T cell composition. [Figure 18] Figures 18A-18D show Kaplan-Meier survival curves for subjects administered CAR+ T cell compositions separated into groups administered compositions containing a percentage of CCR7+CD27+ CAR+ T cells above or below certain threshold levels among CD4+ CAR+ T cells (Figure 18A for progression-free survival, Figure 18C for duration of response) and CD8+ CAR+ T cells (Figure 18B for progression-free survival, Figure 18D for duration of response). [Figure 18E] Figure 18E shows PFS curves based on the optimal split log-rank test for patients with "high" or "low" population doubling numbers (PDL) in CD8+ / CAR+ T cells. Low PDL refers to <6 PDL and >6 PDL refers to high PDL. [Figure 18F]FIG. 18F shows the percentage of CD27+CD28+ T cells in enriched CD4+ (left panel) and CD8+ (right panel) input compositions derived from non-Hodgkin's lymphoma patients. [Figure 18G] Figure 18G shows the relationship between the percentage of effector memory T cells in the enriched CD4+ input composition and the number of population doublings required to reach harvest criteria (Spearman ρ: 0.43; p-value: <0.001). Similar results were observed for the enriched CD8+ input composition. [Figure 19] Figures 19A and 19B display the tumor burden in mice treated with three different treatment doses: high (2x10), medium (4x10) and low (8x10) of anti-BCMA CAR+ T cell composition generated from the expansion process and from the non-expansion process with bead-based stimulation reagents (Bead) or oligomer stimulation reagents (Oligomer) (cells were harvested on day 5 (D5; Figure 19A) or day 4 (D4; Figure 19B) of the process). Controls: mice injected with tumor cells only (tumor without TX) and mice injected with tumor cells and mock-transduced T cell composition (mock). [Figure 20] Figures 20A-20D show the total number of T cells and CAR+ T cells per μL of blood collected from mice 5 days (Figure 20A), 13 days (Figure 20B), 19 days (Figure 20C), or 26 days (Figure 20D) following administration of anti-BCMA CAR+ T cell compositions generated from the Expanded and Non-Expanded Expansion process with bead-based stimulation reagents (Bead) or oligomer stimulation reagents (Oligomer), compared to controls including tumor cells only (tumor without TX) and tumor cells with mock-transduced T cell composition (Mock). [Figure 21]FIG. 21 shows the copy numbers before isolating the high molecular weight DNA fraction (pregel; standard vector copy number (VCN) assay) or after isolating those above the 15 kb, 17.5 kb or 20 kb threshold by pulsed-field gel electrophoresis (PFGE) (integrated vector copy number (VCN) assay), as assessed by droplet digital PCR (ddPCR). Copy numbers were assessed using primers that specifically amplify a portion of the integrated transgene sequence; a packaging plasmid (viral packaging plasmid encoding the Vesicular Stomatitis Indiana Virus G protein (VSVg)), or a genomic control (gene encoding the ribonuclease P protein subunit p30 (RRP30)). Assessment was performed in samples from transduced cells or in non-transduced controls, i.e. spiked CAR and VSVg plasmids. Copy numbers of each gene were normalized to the number of diploid genomes (cp / diploid genome; using primers specific for the albumin gene as a reference) or per 50 ng of genomic DNA. [Figure 22] 22A-22B depict copy numbers, as assessed by a standard vector copy number (VCN) assay (genomic DNA samples not subjected to PFGE, both high and low molecular weight DNA) and integrated copy numbers (in high molecular weight DNA samples after PFGE) of the transgene sequence at various time points (before transduction ("Pre"), at 5 minutes, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, and 96 hours after transduction, or at completion of the engineering process "Complete") for Jurkat T cells (FIG. 22A) or primary T cells isolated from human subjects (FIG. 22B) transduced with a lentiviral preparation containing a transgene sequence encoding a CAR. Copy numbers of each gene were normalized to the number of diploid genomes (cp / diploid genome; using primers specific for the albumin gene as a reference). [Figure 23]FIG. 23A shows the integrated copy numbers assessed on days 3, 4, or 5 of an exemplary non-expanded expanded T cell composition manufacturing process using primary T cells from two different human subjects (donor A and donor B) stimulated by incubation with (1) anti-CD3 / anti-CD28 antibody conjugated paramagnetic beads ("beads"), (2) anti-CD3 / anti-CD28 Fab conjugated oligomeric streptavidin mutein reagent at a concentration of 4.0 μg per 10 cells, or (3) anti-CD3 / anti-CD28 Fab conjugated oligomeric streptavidin mutein reagent at a concentration of 0.8 μg per 10 cells, incubated in basal medium without serum or growth factors ("Basal") or serum-free medium containing IL-2, IL-5, and IL-15 ("Complete") after transduction. Figure 23B depicts the correlation between copy number, as determined by iVCN, and the percentage of CAR-expressing cells (as determined by the percentage of CD3+ / activated Cas3- / CAR+ cells among CD3+ cells by flow cytometry). [Figure 24] Figures 24A-24E depict the copy number per diploid genome (Figure 24A), fraction of integrated transgene (Figure 4B), fraction of non-integrated transgene (Figure 24C), non-integrated transgene copy number per diploid genome (Figure 24D), and integrated copy number per CAR+ cell (Figure 24E) as assessed by standard VCN (without PFGE) and iVCN (with PFGE) during various exemplary expanded or non-expanded T cell composition manufacturing processes utilizing different stimulation reagents and harvest times as shown in Table E9. [Diagram 25]Figure 25 depicts copy number per diploid genome, unintegrated transgene copy number, fraction of unintegrated transgene, and fraction of integrated transgene as assessed by standard VCN (without PFGE) and iVCN (with PFGE) during various time points during an exemplary engineering process for engineering primary T cells from various donors to express chimeric antigen receptors (CARs). The time points assessed include days 0 to 8 of the expansion process, including at the time of thawed feedstock (TMAT; day 0), activation (AMAT; day 1), transduction (XMAT; day 2) or various times after the initiation of culture (inoc+1 to inoc+6; representing days 3 to 8 of the process). [Figure 26] FIG. 26 shows the clonality of T cells of the isolated CD4+ and CD8+ T cell composition before manipulation (CMAT) and the CD4+ and CD8+ therapeutic CAR+ T cell composition after manipulation (applied Shannon index). [Figure 27] Figure 27 shows the phenotypic differences of the generated cell compositions in the presence of different concentrations of anti-CD3 / anti-CD28 Fab conjugate oligomeric streptavidin mutein reagents and / or in the presence of different media compositions. The percentage of CD8+ CAR+ T cells that represented effector memory CD45RA+ cells (CCR7-CD45RA+; EMRA), naive-like T cells (CCR7+CD45RA+; NAIVE), effector memory (CCR7-CD45RA-; EM) and central memory (CCR7+CD45RA-; CM) is shown for various combinations of mutein reagents and media compositions. [Figure 28] FIG. 28 shows the percentage of CD4+ CAR+ and CD8+ CAR+ T cells positive for both CCR7 and CD27 staining in engineered compositions of primary T cells from three different donors using control non-expansion and expansion processes. [Figure 29]FIG. 29 shows the percentage of CD4+CAR+ T cells and CD8+CAR+ T cells that represented effector memory CD45RA+ cells (CCR7-CD45RA+; EMRA), naive-like T cells (CCR7+CD45RA+; NAIVE), effector memory (CCR7-CD45RA-; EM), and central memory (CCR7+CD45RA-; CM) in engineered compositions of primary T cells from three different donors using contrasting non-expansion and expansion processes. [Diagram 30] Figures 30A-30D show exemplary effects on mTor signaling and viability and growth kinetics of incubating T cells with anti-CD3 / anti-CD28 oligomeric stimulatory reagents in the presence or absence of compound 63. Figure 30A shows pS6 expression in live CD8+ T cells by memory subsets. Figure 30B shows mean fluorescence intensity (mfi) of total CD8 T cells with treatment as indicated. Figures 30C-30D show viability and total T cell counts, respectively, over time (indicated by days; e.g., d1) in culture following the initiation of stimulation ("input"). [Diagram 31] Figures 31A-31F show exemplary functional and phenotypic characteristics of cryopreserved CAR-T cells generated using a method utilizing incubation with anti-CD3 / anti-CD28 oligomer stimulating reagents in the presence or absence of compound 63. Figure 31A shows intracellular expression of caspases upon thawing. Figures 31B-31D show phenotypic profiles of CD8 and CD4 CAR-T cells by subset expression of CD27 and / or CCR7, respectively. Figures 31C-31E show intracellular IL-2, IFNγ or TNF (left panel) or a combination of IL-2 and / or IFNγ or TNF (right panel) in CD8 and CD4 CAR-T cells stimulated with antigen-bearing targets, respectively. Figure 31F shows expansion growth and survival over 12 days (left panel) and total expansion growth metrics calculated by area under the curve (right panel) for CAR-T cells stimulated with anti-CAR beads. [Diagram 32]Figures 32A-32C show exemplary viability (Figure 32A), total viable nucleated cells (Figure 32B) and cumulative downstream yield (Figure 32C) at each processing step for a process using cryopreserved apheresis (CrAPH) or cryopreserved T cell selected material (CMAT) collected from three donors. D5_HRPO, D5_WPRO, D5_FPRO refer to the cell product collected on day 5, the cell product washed on day 5, and the cell product formulated on day 5 (e.g., formulated drug product), respectively. Figures 32D-32F show exemplary average percentages of cell phenotypes at time of collection (day 4 or day 5) from processed cryopreserved (CrAPH) or fresh apheresis (CMAT) samples from three donors. Figures 32D-32F show exemplary average percentages of CAR+ cells in CD3+aCas3- cells (Figure 32D, top panel), CAR+ cells in aCas3-CD4+ cells (Figure 32D, bottom left panel), CAR+ cells in aCas3-CD8+ cells (Figure 32D, bottom right panel), harvest product (HRPO) total nucleated CAR+ cells in CD3+aCas3- cells (Figure 32E), CAR+CD25+ cells in aCas3-CD4+ cells (Figure 32F, left panel), and CAR+CD25+ cells in aCas3-CD8+ cells (Figure 32F, right panel). [Diagram 33] Figure 33 shows exemplary memory phenotype profiles of aCas3-CD4+CAR+ cells (top panel) and aCas3-CD8+CAR+ cells (bottom panel) generated at stimulation incubation times of 0, 16, 24 or 48 hours from the start of stimulation. [Diagram 34]Figures 34A-34B show exemplary quantification of cell phenotypes determined by flow cytometry for the expansion and non-expansion engineered processes using different donor types (reference, patient). Cells were engineered to express an exemplary anti-CD19 CAR (CD19), an exemplary anti-BCMA CAR (BCMA), or mock transduced (mock). Figure 34A shows the percentage of CD3+CD8+ and CD3+CD4+ cells of live CD45+ cells (left panel), and the percentage of CD8+CAR+ and CD4+CAR+ cells of CD45+ cells (right panel). Figure 34B shows the ratios of CD4+CAR+ cells:CD+CAR+ cells and CD4+ cells:CD8+ cells. [Diagram 35] Figure 35 shows the fold expansion of cell compositions from different donor types (reference, patient) generated by expansion or non-expansion engineered processes. Cells were engineered to express an exemplary anti-CD19 CAR (CD19) or an exemplary anti-BCMA CAR (BCMA). [Diagram 36] 36A-36B show exemplary proportions of cell phenotypes resulting from the expansion and non-expansion engineered processes using different donor types (reference, patient). Cells were engineered to express an exemplary anti-CD19 CAR (CD19), an exemplary anti-BCMA CAR (BCMA), or mock transduced (mock). Figures 36A-36B show exemplary percentages of CD45RA+CCR7+ cells in aCAS-CD8+CAR+ and aCAS-CD4+CAR+ cells (Figure 36A, upper left panel), CD45RA-CCR7+ cells in aCAS-CD8+CAR+ and aCas-CD4+CAR+ cells (Figure 36A, upper right panel), CD45RA-CCR7- cells in aCas-CD8+CAR+ and aCas-CD4+CAR+ cells (Figure 36A, lower left panel), CD45RA+CCR7- cells in aCAS-CD8+CAR+ and aCas-CD4+CAR+ cells (Figure 36A, lower right panel), and CD27+CCR7+ cells in aCAS-CD8+CAR+ and aCas-CD4+CAR+ cells (Figure 36B). [Figure 37]Figures 37A-37B show exemplary quantification of cell phenotype as determined by flow cytometry for donor-matched expansion and non-expansion engineered processes in which cells were engineered to express an exemplary anti-CD19 CAR (CD19 CAR T) or an exemplary anti-BCMA CAR (BCMA CAR T). [Figure 38] Figure 38 shows the total viable cell count and area under the curve (AUC) over time for cells generated from either a non-expansion expansion process or an expansion expansion process after long-term CAR-dependent stimulation with an anti-ID antibody. Figure 38 shows the total viable cell count and AUC for cells engineered to express an anti-CD19 CAR. [Figure 39] Figure 39 shows the total viable cell count and area under the curve (AUC) over time for cells generated from either a non-expansion or expansion process after long-term CAR-dependent stimulation with anti-ID antibody. Figure 39 shows the total viable cell count and AUC for cells engineered to express anti-BCMA CAR. [Diagram 40] Figures 40A-40B show the cytotoxic potential of anti-CD19 CAR T cells engineered by non-expansion or expansion expansion processes before (Figure 40A) and after (Figure 40B) chronic stimulation at different effector:target ratios. [Diagram 41] Figure 41 shows exemplary percentages of CAR+CD4+ and CAR+CD8+ cells engineered by the non-expansion expansion or expansion expansion process that express IL-2, IFNγ, TNF, and IL-2 / IFNγ / TNF as measured by flow cytometry before and after chronic stimulation. [Figure 42A] Figure 42A (left panel) shows tumor burden over time following treatment with donor-matched anti-BCMA CAR-T cell compositions generated from non-expansion and expansion engineered processes. Figure 42A (right panel) shows tumor growth from day -1 (pre-treatment) to day 50 post-treatment, calculated from the area under the curve (AUC) of BLI for each group. [Figure 42B]Figure 42B shows the absolute number of anti-BCMA CAR-T cells per μL of blood over several days for cells generated from donor-matched non-expansion and expansion manipulation processes. [Figure 43A] Figure 43A shows tumor burden over several days following treatment with higher (upper panel) and lower dose (lower panel) anti-CD19 CAR-T cell compositions generated from donor-matched non-expansion and expansion engineered processes. [Figure 43B] Figure 43B shows tumor growth from day -1 (pre-treatment) to day 50 after treatment, calculated from the area under the curve (AUC) of BLI for each group treated with higher (upper panel) and lower (lower panel) doses of anti-CD19 CAR-T cells. [Figure 43C] Figure 43C shows the absolute number of anti-CD19 CAR-T cells per μL of blood over several days for cells generated from donor-matched non-expansion and expansion manipulation processes. [Diagram 44] Figures 44A-44D show total live cells (Figure 44A), viability (Figure 44B) and phenotype (Figures 44C and 44D) during and after a production run involving a variety of non-expanded and expanded growth manipulation processes. [Diagram 45] Figure 45A shows the relationship between copy number per cell among total cells as assessed by standard VCN (without PFGE) and iVCN (with PFGE) in cell compositions produced from primary T cells from different human donors engineered to express CAR using an expansion expansion process (○) or a non-expansion expansion process (●). Figures 45B-45C show the relationship between copy number per cell in cell compositions as assessed by standard VCN (Figure 45B) or iVCN (Figure 45C) and surface expression of CAR as indicated by the percentage of CAR-expressing CD3+ cells among viable CD45+ cells (% of CD3+CAR+) assessed by flow cytometry. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0117] Detailed Description Provided herein is a method for generating or producing a composition of engineered cells, e.g., engineered T cells, expressing a recombinant protein, e.g., a recombinant receptor, such as a chimeric antigen receptor (CAR) or a recombinant T cell receptor (TCR). In some embodiments, the method comprises or includes one or more steps of incubating an input population of cells (also referred to herein as an input composition), e.g., a population of primary T cells, under stimulatory conditions, and then introducing a polynucleotide encoding the recombinant protein into the cells. In certain embodiments, the method is used in connection with a process that is completed within a certain time, such as within 4 days or within a few days, after the start of stimulation of cells in the input composition.

[0118] A variety of processes are available for generating compositions containing genetically engineered T cell populations, including processes for generating engineered T cells that express CAR. However, in certain aspects, some of these processes may require long or relatively long periods to generate engineered cells. In addition, in various aspects, some existing processes may require varying amounts of time to successfully produce engineered T cells suitable for cell therapy, making it difficult to adjust the administration of cell therapy. In certain aspects, some of these processes may produce populations of cells that contain a relatively high percentage or amount of exhausted cells, differentiated cells, or cells with low potency. Additional methods are needed to generate engineered T cells.

[0119] In certain embodiments, the provided methods are used in conjunction with a process for efficiently producing or generating engineered cells suitable for use in cell therapy. In some embodiments, the provided processes for generating engineered cells contain one or more steps for stimulating and genetically engineering (e.g., transforming, transducing or transfecting) T cells to produce a population of engineered T cells that can be collected or formulated for use as a composition for cell therapy. In some aspects, the processes provided herein successfully generate engineered cells without the need for any additional steps for expanding cells, for example, without an expansion unit operation and / or without including a step intended to cause the expansion of cells. In some aspects, the provided methods generate engineered T cells with enhanced potency compared to engineered T cell compositions produced from alternative processes, such as processes involving expanding cells.

[0120] In certain aspects, the duration of the provided processes can be measured from when the cells of the input cell population or input composition, e.g., T cells, are first contacted or exposed to stimulatory conditions (e.g., as described herein, such as in Section IB), which is referred to herein as stimulation or initiation of stimulating, and also referred to herein as exposure to a stimulatory reagent (e.g., when exposure to a stimulatory reagent begins). In some embodiments, the time period required to harvest or collect an output population containing engineered cells (also referred to herein as an output composition or a composition of engineered cells, e.g., engineered T cells) is measured from the initiation of stimulation. In certain embodiments, the duration of the process is, is about, or is less than 120, 108, 96, 84, 72, 60, 48, 36, or 30 hours. In certain embodiments, the duration of the process is, is about, or is less than 5, 4, 3, 2, or 1 days. In certain embodiments, the engineered cells, e.g., cells of an output composition or population, are more potent, persistent, or naive-like than cells engineered with a process requiring a longer time. In some aspects, the duration of the provided processes, e.g., the time required to generate or produce an engineered population of T cells, is 2, 3, 4, 5, 6, 7 or more than 7 days, about 2, 3, 4, 5, 6, 7 or more than 7 days, or at least 2, 3, 4, 5, 6, 7 or more than 7 days shorter than the time of some existing processes.In some embodiments, the duration of the provided process is, is about, or is less than 75%, 60%, 50%, 40%, 30%, 25%, 15% or 10% of an alternative or existing process.

[0121] In certain embodiments, the provided processes are performed on a population of cells isolated, enriched, or selected from a biological sample, e.g., CD3+, CD4+, and / or CD8+ T cells. In some aspects, the provided methods can produce or generate compositions of engineered T cells within a reduced time from when the biological sample is collected from a subject compared to other methods or processes. In some embodiments, the provided methods can produce or generate engineered T cells within or about 10, 9, 8, 7, 6, 5, 4, 3, or 2 days from when the biological sample is collected from a subject until the engineered T cells are collected, harvested, or formulated (e.g., for cryopreservation or administration), including any or all times that the biological sample or enriched, isolated, or selected cells are cryopreserved and stored.

[0122] In certain embodiments, the output compositions or populations of engineered T cells, e.g., T cells expressing recombinant receptors such as chimeric antigen receptors, produced or generated by the provided processes are particularly effective or potent when utilized as cells for cell therapy. For example, in some aspects, the output compositions containing engineered T cells, e.g., CAR+ T cells, produced from the provided processes have a much higher potency and / or proliferation capacity than engineered T cells produced or generated by alternative existing processes. In some aspects, the output compositions containing engineered T cells, e.g., CAR+ T cells, produced by the provided processes have enhanced anti-tumor or anti-cancer cell activity than engineered T cells, e.g., CAR+ T cells, produced by alternative or existing methods.

[0123] Certain embodiments contemplate that a population of engineered T cells, e.g., CAR+ T cells, produced by the provided processes, e.g., processes that do not include or require T cell expansion during the process, is not expected to achieve a high degree of potency or efficacy in some aspects. Existing methods of manufacturing T cells for cell therapy include processes for expanding cells, e.g., processes that include an expansion unit operation and / or include steps intended to cause cell expansion, or require cell expansion, e.g., to a threshold density. Certain embodiments contemplate that expansion is expected to be necessary to provide sufficient amounts of vector integration into the engineered T cells, which is expected to be necessary to generate a potent or effective population of CAR+ T cells. What is demonstrated herein is that potent or effective CAR+ T cell compositions can be produced by processes that lack steps or conditions for expansion, e.g., processes that lack an expansion unit operation and / or do not include steps intended to cause cell expansion, such as the provided processes. In some aspects, engineered cells that are harvested, collected, or formulated prior to stable incorporation of a vector, e.g., a viral vector, still result in compositions of potent or effective CAR+ T cells. In certain aspects, the CAR+ T cells produced by the provided methods are more potent, persistent, or effective than CAR+ T cells produced or generated from alternative processes that include expansion (e.g., processes that include an expansion unit operation and / or that include steps intended to cause expansion of cells). Certain embodiments contemplate that vector incorporation may continue to occur while or even after the cells are harvested, collected, or formulated.

[0124] Also provided herein is a process for producing or manipulating a T cell population, comprising stimulating cells with oligomeric stimulating reagents. Existing reagents are known for use in stimulating T cells in vitro, such as in the absence of exogenous growth factors or in the presence of small amounts of exogenous growth factors (see, for example, U.S. Patent 6,352,694 B1 and European Patent EP 0 700 430 B1). In general, such reagents may utilize beads, such as magnetic beads, with a diameter of more than 1 μm, to which various binding agents (e.g., anti-CD3 and / or anti-CD28 antibodies) are immobilized. However, in some cases, such magnetic beads are difficult to incorporate into methods for stimulating cells under conditions required, for example, for clinical trials or therapeutic purposes. This is because it must be ensured that these magnetic beads are completely removed before administering the expanded T cells to a subject. In some aspects, such removal, such as by exposing the cells to a magnetic field, may reduce the yield of viable cells available for cell therapy. In certain cases, such reagents, e.g., stimulating reagents containing magnetic beads, must be allowed to incubate with the cells for a minimum amount of time to allow separation of a sufficient amount of T cells from the stimulating reagent.

[0125] The provided process utilizing oligomeric stimulating reagent overcomes such potential limitations. For example, in some embodiments, the provided process avoids or reduces the risk of residual stimulating reagent, e.g., reagent containing magnetic beads, in the output cells generated or produced by the process. In some embodiments, this also means that it is easier to establish a process that complies with GMP standards, compared to other methods where additional measures must be taken to ensure that the final engineered T cell population is bead-free. In some embodiments, this can be easily achieved in this embodiment by adding a substance, e.g., a competing reagent, that dissociates the oligomeric stimulating reagent from the cells, e.g., by simply rinsing or washing the cells, e.g., by centrifugation. Thus, in some aspects, removal or separation of the oligomeric stimulating reagent from the cells, e.g., by adding a substance or a competing reagent, results in little or no cell loss compared to removal or separation of bead-based stimulating reagent. In some aspects, the timing of removal or separation of the oligomeric stimulating reagent is not limited at all, or is less limited than removal or separation of the bead-based stimulating reagent. Thus, in some aspects, the oligomeric stimulatory reagent may be removed or separated from the cells at any time or stage during the process provided.

[0126] In some aspects of the provided process, cells are transduced or subjected to transduction in a process involving incubation in the presence of basal medium, for example, incubation of T cells with a viral vector encoding a heterologous or recombinant protein (e.g., CAR). In certain aspects, the basal medium does not contain additional additives (e.g., nutrients, vitamins, or amino acids) or any additional recombinant cytokines or growth factors. In certain aspects, the basal medium does not contain recombinant cytokines or growth factors. In certain aspects, the basal medium may contain certain essential amino acids such as L-glutamine, but does not contain other further additional additives or recombinant cytokines. In some aspects, incubation in the presence of basal medium reduces differentiation that may occur during the manipulation process, for example, compared to incubation in the presence of supplemented medium, complete medium, medium containing serum, or medium containing serum replacement. Thus, in some aspects, incubation in the presence of basal medium, e.g., after transduction or spinoculation, increases the proportion of less differentiated naive-like cells in the output composition, e.g., the composition of produced cells that contains a population of cells that contain CAR+ T cells.

[0127] Among the findings provided herein is the observation that weak or reduced stimulation, such as a reduced amount of stimulating reagent typically utilized to stimulate T cells, can generate engineered CAR+ T cells that are as potent, persistent, or effective as CAR+ T cells generated by stronger stimulation conditions or processes involving greater amounts or concentrations of stimulating reagent. For example, in some embodiments, stimulating cells with a lower or relatively low amount of oligomeric stimulating reagent can increase the potency, efficacy, or persistence of the resulting engineered cell population compared to a process using a higher amount of oligomeric stimulating reagent. Such embodiments contemplate that such effects may persist even at doses low enough to reduce expression of activation markers or the portion of cells positive for activation markers during and after the process.

[0128] In certain embodiments, the provided methods do not include a step of expanding cells or expanding cells to a threshold amount or concentration. In some aspects, protocols that do not rely on expanding cells to increase the number or concentration of cells from a starting cell population, e.g., an input population, do not require incubation or culture, which may vary between cell populations. For example, some embodiments contemplate that cell populations obtained from different subjects, such as subjects with different diseases or disease subtypes, may divide or expand at different rates. In certain aspects, eliminating the potentially variable step of requiring cell expansion allows the duration of the entire process to be tightly controlled. In certain embodiments, the variability of the process duration is reduced or eliminated, which may allow, in some aspects, improved coordination of appointments and procedures between physicians, patients and technicians to facilitate autologous cell therapy.

[0129] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. To the extent that the definitions set forth herein conflict or are otherwise inconsistent with the definitions set forth in the patents, applications, published applications and other publications incorporated herein by reference, the definitions set forth herein shall take precedence over the definitions incorporated herein by reference.

[0130] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0131] I. Process for generating engineered cells Provided herein are methods for generating an output population of engineered cells, such as engineered CD4+ T and CD8+ T cells, expressing recombinant proteins, e.g., recombinant receptors, such as T cell receptors (TCRs) or chimeric antigen receptors (CARs). In certain embodiments, the methods provided herein are used in conjunction with the manufacture, production or production of cell therapies, and may be used in conjunction with additional processing steps, such as steps for isolating, separating, selecting, activating or stimulating, transducing, washing, suspending, diluting, concentrating and / or formulating cells. In some embodiments, the method of generating or producing engineered cells, e.g., engineered T cells, includes one or more of the steps of isolating cells from a subject, incubating the cells under stimulatory conditions, and genetically engineering the cells. In some embodiments, the method includes the following processing steps: first isolate, e.g., select or separate, input cells, e.g., primary CD3+ T cells, CD4+ T cells and / or CD8+ T cells, from a biological sample; incubate under stimulatory conditions, genetically engineer, e.g., introduce a recombinant polynucleotide encoding a recombinant receptor into the cells by transduction or transfection; and then collect, harvest or fill a container, e.g., a bag or vial, as an output population. In some aspects of the method, the biological sample can be a cryopreserved biological sample, e.g., a cryopreserved apheresis product. In some embodiments, the cells of the output population are reintroduced into the same subject, optionally after cryopreserving and storing the cells. In some embodiments, the output population of engineered cells is suitable for use in therapy, e.g., autologous cell therapy.

[0132] In certain embodiments, the methods provided are used in connection with the generation of an output population of cells expressing a recombinant receptor from an initial or input population of cells. In certain embodiments, the input population is produced, generated and / or manufactured by combining, mixing and / or pooling cells, including those derived from a population of cells containing enriched T cells, enriched CD3+ T cells, enriched CD4+ T cells and / or enriched CD8+ T cells (hereinafter also referred to as an enriched population of T cells, an enriched population of CD3+ T cells, an enriched population of CD4+ T cells and an enriched population of CD8+ T cells, respectively). In some embodiments, the input population of cells is a population of CD3+ T cells, e.g., without CD4+ and / or CD8+ selection. In some embodiments, the input population of cells is a population enriched for CD3+ T cells from a starting sample, such as a PBMC or leukapheresis sample, where the starting sample has not been subjected to CD4+ and / or CD8+ selection. In some embodiments, the starting sample is selected for CD3 without any prior, simultaneous or subsequent selection for another marker (e.g., CD4 and / or CD8) to generate an input population enriched for CD3+ T cells. In some embodiments, the input population of cells is enriched for CD3+ T cells and the input population is not subjected to further selection, e.g., CD4+ and / or CD8+ selection, before the input population is subjected to a manufacturing process step, such as activating or stimulating or manipulating the input population. In some embodiments, the input population of cells is a combined, mixed and / or pooled population of CD4+ and CD8+ T cells. In certain embodiments, the provided methods are used in conjunction with one or more of the steps of activating or stimulating cells, e.g., cells of an input population; genetically manipulating the activated or stimulated cells to introduce, e.g., a polynucleotide encoding a recombinant protein, by transduction or transfection.In certain embodiments, the methods may also be used in conjunction with isolating or selecting cells from a biological sample, e.g., from a biological sample taken, collected, and / or obtained from a subject, to generate an input population of enriched T cells. In certain embodiments, the methods provided may be used in conjunction with harvesting, collecting, and / or formulating an enriched population of T cells after the cells have been incubated, activated, stimulated, manipulated, transduced, and / or cultured.

[0133] In certain embodiments, the provided methods do not contain any steps, stages or conditions that result in the expansion of cells. In certain embodiments, the cells of an input population, e.g., a starting population of enriched CD3+ T cells (e.g., without prior or co-selection for CD4 and / or CD8 markers), a starting sample of CD4+ T cells (e.g., substantially no CD8+ T cells), a starting sample of CD8+ T cells (e.g., substantially no CD4+ T cells), or a starting population of CD4+ T cells and CD8+ T cells, are not expanded while undergoing the provided methods to generate a population of engineered cells. In certain embodiments, the cells of an output population, e.g., an output population of cells engineered by the provided processes, are the same, about the same, reduced or decreased compared to the amount of cells in the input population, such as at the beginning of culture under stimulatory conditions. In some aspects, protocols that do not rely on expanding cells to increase the number or concentration of cells from a starting cell population, e.g., an input population, do not require incubation or culture, which may vary between cell populations. For example, some embodiments contemplate that cell populations obtained from different subjects, such as subjects with different diseases or disease subtypes, may divide or expand at different rates. In certain aspects, eliminating the potentially variable step of requiring cell expansion allows the duration of the entire process to be tightly controlled.

[0134] In some embodiments, the method is used in conjunction with one or more steps to stimulate the cells, such as by incubating the cells under stimulatory conditions. In some aspects, such stimulatory conditions are or include culturing the cells with a stimulatory reagent, such as a stimulatory reagent described herein, such as in Section IB-1. In certain embodiments, a set or fixed amount of cells is stimulated, for example, at a set or fixed concentration. In certain embodiments, the stimulating step, e.g., culturing the cells under stimulatory conditions, is performed for a set or fixed time, such as less than 2 days, or for a time between 18 hours and 30 hours. In some aspects, the stimulating step with the stimulatory reagent is performed for 20 hours ± 4 hours or about 20 hours ± 4 hours.

[0135] In certain embodiments, the methods provided herein are carried out in connection with introducing a heterologous or recombinant polynucleotide into a cell, e.g., transducing or transfecting a cell, e.g., by a method described herein (e.g., in Section IC). In certain embodiments, the cells are incubated, either during or after genetically engineering the cells, for a time sufficient to allow for the incorporation of a heterologous or recombinant polynucleotide encoding a recombinant protein or to allow for the expression of a recombinant protein. In certain embodiments, the cells are incubated for a set or fixed time, e.g., for a time greater than 18 hours or less than 4 days, e.g., 72 hours ± 6 hours. In any of the embodiments provided, the introducing can be performed on the cells after the cells have been stimulated with a stimulating reagent. In some embodiments, the manipulation step is initiated or started within a certain time from when the stimulating is started or initiated, e.g., within 30 hours from when the stimulating reagent is added to, cultured or contacted with the cells. In certain embodiments, the manipulation step is initiated or started between 18 hours and 30 hours, e.g., 20 hours ± 4 hours, after the stimulating reagent is added to, cultured or contacted with the cells.

[0136] In some embodiments, one or more process steps are carried out at least partially in serum-free medium. In some embodiments, the serum-free medium is a defined or precisely defined cell culture medium. In certain embodiments, the serum-free medium is a controlled culture medium that has been processed, for example, filtered to remove inhibitors and / or growth factors. In some embodiments, the serum-free medium contains protein. In certain embodiments, the serum-free medium can contain serum albumin, hydrolysates, growth factors, hormones, carrier proteins and / or attachment factors.

[0137] In some embodiments, the methods provided are performed such that one, several or all steps in the preparation of cells for clinical use (e.g., in adoptive cell therapy) are performed without exposing the cells to non-sterile conditions. In some embodiments, the cells are isolated, separated or selected, transduced, washed, optionally activated or stimulated and formulated, all within a closed, sterile system or device. In some embodiments, one or several of the steps are performed separately from the closed system or device. In some such embodiments, the cells are transferred away from the closed system or device under sterile conditions, such as by sterile transfer to a separate closed system.

[0138] In certain embodiments, the enriched population of T cells may be harvested, formulated for cryoprotection, frozen (e.g., cryoprotected), and / or stored at below 0° C., below −20° C., or below −70° C. or −80° C. or below −70° C. or −80° C. before, during, or after any stage or step of the process to generate an output population of enriched T cells expressing the recombinant receptor. In some embodiments, the cells may be stored for a period of less than 1 day, less than 2 days, less than 3 days, less than 4 days, less than 5 days, less than 6 days, less than 7 days, less than 8 days, less than 9 days, or less than 1 week, less than 2 weeks, less than 3 weeks, less than 4 weeks, less than 5 weeks, less than 6 weeks, less than 7 weeks, less than 8 weeks, or for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks, or for more than 8 weeks. After storage, the enriched population of T cells may be thawed and processing may resume from the same point in the process. In some embodiments, the enriched input population of T cells is cryoprotected and stored prior to further processing, e.g., incubation under stimulatory conditions. In certain embodiments, the cultured and / or formulated population of enriched T cells is cryoprotected and stored prior to administration to a subject, e.g., as an autologous cell therapy.

[0139] In certain embodiments, the methods provided herein are used in connection with a process in which engineered cells are generated by a process that includes a step for stimulating the cells, followed by a step for introducing a polynucleotide encoding a recombinant receptor (e.g., a CAR) into the cells. In certain embodiments, the stimulating is performed for 18-30 hours, e.g., about 24 hours, and the introduction of the polynucleotide is performed thereafter. In certain embodiments, the cells are harvested or collected within 3 days after the introduction of the polynucleotide has begun, e.g., to be formulated for cryopreservation or administered to a subject. In various embodiments, the cells are harvested or collected within 4 days after the incubation under stimulation conditions has begun, e.g., to be formulated for cryopreservation or administered to a subject.

[0140] In certain embodiments, a portion of cells may be sampled or collected at any stage or step of the process, e.g., cells may be taken from a population of T cells (such as an enriched population of T cells) during isolation, incubation, manipulation, culture, and / or formulation, while the population remains in a closed system. In certain embodiments, such cells may be analyzed for makers, characteristics or properties, including but not limited to viability, apoptosis, activation, stimulation, growth and / or exhaustion. In some embodiments, cells are sampled or collected by an automated process while the enriched population of T cells remains in a closed system. In some embodiments, the analysis of the sampled or collected cells is automated. In certain embodiments, the analysis is performed in a closed system under sterile conditions.

[0141] In some embodiments, cells or populations of cells produced and / or processed by the provided methods may be compared to cells or populations of cells processed or produced by the exemplary and / or alternative processes. In certain embodiments, the alternative and / or exemplary processes may differ in one or more specific aspects, but otherwise include similar or the same features, aspects, steps, stages, reagents, or conditions of the aspects or aspects of the provided methods that are compared to the exemplary or alternative processes. For example, an output population of engineered cells, e.g., non-expanded cells, produced by the provided methods may be compared to cells produced in a process that involves one or more steps of expanding the cells. In some embodiments, unless otherwise specified, the provided methods and the exemplary or alternative processes will be similar and / or identical in other respects, such as using similar or identical steps for isolation, selection, enrichment, activation, stimulation, manipulation, transfection, transduction, culture, and / or formulation. In some embodiments, unless otherwise specified, the methods and alternative processes provided isolate, select and / or enrich cells from the same or similar type of biological sample and / or process cells and / or input cells of the same cell type.

[0142] Also provided are cells and populations prepared by the methods, including pharmaceutical populations and formulations, as well as kits, systems and devices for carrying out the methods. Additionally provided are methods of using the cells and populations prepared by the methods, including therapeutic methods, such as methods for adoptive cell therapy, and pharmaceutical populations for administration to a subject.

[0143] A. Sample and cell preparation In certain embodiments, the provided methods are used in connection with isolating, selecting or enriching cells from a biological sample to generate one or more input populations of enriched cells, e.g., T cells. In some embodiments, the provided methods include isolating cells or populations thereof from a biological sample, e.g., a biological sample obtained or derived from a subject, such as a subject having a particular disease or condition, in need of cell therapy or to whom cell therapy is administered. In some aspects, the subject is a human subject, e.g., a patient in need of a particular therapeutic intervention, such as adoptive cell therapy, for which the cells are isolated, processed and / or manipulated. Thus, the cells are, in some embodiments, primary cells, e.g., primary human cells. Samples include tissues, body fluids, and other samples taken directly from a subject. Biological samples can be samples obtained directly from a biological source or samples that are processed. Biological samples include, but are not limited to, body fluids, e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0144] In some aspects, the sample is a blood or blood-derived sample, or is derived from apheresis or leukapheresis products. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsils, or other organs, and / or cells derived therefrom. Samples include samples from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0145] In some examples, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. The sample, in some aspects, contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects, contains cells other than red blood cells and platelets.

[0146] In some embodiments, blood cells collected from a subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, the washing step is accomplished by a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) following the manufacturer's instructions. In some aspects, the washing step is accomplished by tangential flow filtration (TFF) following the manufacturer's instructions. In some embodiments, the cells are washed after washing, e.g., with Ca ++ / Mg ++ The cells are resuspended in a wide variety of biocompatible buffers, such as PBS free of erythrocytes. In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

[0147] In some embodiments, a cell-containing sample (e.g., an apheresis product or leukapheresis product) is washed to remove one or more anticoagulants, such as heparin, that were added during apheresis or leukapheresis.

[0148] In some embodiments, a cell-containing sample (e.g., a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product) is cryopreserved and / or cryoprotected (e.g., frozen) and then thawed and optionally washed prior to any step for isolation, selection, activation, stimulation, manipulation, transduction, transfection, incubation, culture, harvesting, formulation of a population of cells, and / or administration of the formulated cell population to a subject.

[0149] In some embodiments, a sample containing autologous peripheral blood mononuclear cells (PBMCs) from a subject is collected in a manner suitable to ensure proper manufacturing quality. In one aspect, the sample containing PBMCs is derived from fractionated whole blood. In some embodiments, whole blood from a subject is fractionated by leukapheresis using centrifugal force and utilizing density differences between cell phenotypes, whereby autologous mononuclear cells (MNCs) are preferentially enriched while other cell phenotypes, such as red blood cells, are reduced in the collected cell composition. In some embodiments, autologous plasma is collected simultaneously during MNC collection, which in some aspects can allow for extended stability of the leukapheresis product. In one aspect, autologous plasma is added to the leukapheresis product to improve the buffering capacity of the leukapheresis product matrix. In some aspects, the total volume of whole blood processed to generate the leukapheresis product is at or about 2L, 4L, 6L, 8L, 10L, 12L, 14L, 16L, 18L, or 20L, or any value between any of the foregoing. In some embodiments, the volume of autologous plasma collected is at or about 10mL, 50mL, 100mL, 150mL, 200mL, 250mL, or 300mL or more, or any volume between any of the foregoing. In some embodiments, the leukapheresis product is subjected to procedures, such as washing and formulation for in-process cryopreservation, within about 48 hours of completing the leukapheresis collection. In some embodiments, the leukapheresis product is subjected to one or more washing steps, e.g., within about 2, 6, 12, 18, 24, 36, or 48 hours of completing the leukapheresis collection. In some aspects, the one or more washing steps remove anticoagulant during the leukapheresis collection, cellular waste products that may accumulate in the leukapheresis product, residual platelets, and / or cellular debris. In some embodiments, one or more buffer exchanges are performed between the one or more washing steps.

[0150] In certain embodiments, the apheresis product or leukapheresis product is cryopreserved and / or cryoprotected (e.g., frozen) as described below, and then thawed before being subjected to a cell selection or isolation step (e.g., a T cell selection or isolation step). In some embodiments, after the cryopreserved and / or cryoprotected apheresis product or leukapheresis product is subjected to a T cell selection or isolation step, no additional cryopreservation and / or cryoprotection step is performed during or between any subsequent steps, such as activating, stimulating, manipulating, transducing, transfecting, incubating, culturing, harvesting, formulating, and / or administering the formulated cell population to a subject. For example, the T cells selected from the thawed cryopreserved and / or cryoprotected apheresis product or leukapheresis product are not cryopreserved and / or cryoprotected again before being thawed and optionally washed for downstream processes, such as T cell activation / stimulation or transduction.

[0151] In certain embodiments, the apheresis product or leukapheresis product is cryopreserved in a cryopreservation solution or buffer at a concentration of 5×10 6 Cells / mL, 10 x 10 6 cells / mL, 20 x 10 6 cells / mL, 30 x 10 6 cells / mL, 40 x 10 6 Cells / mL, 50 x 10 6 cells / mL, 60 x 10 6 Cells / mL, 70 x 10 6 Cells / mL, 80 x 10 6 Cells / mL, 90 x 10 6 Cells / mL, 100 x 10 6 cells / mL, 110 x 10 6 cells / mL, 120 x 10 6 cells / mL, 130 x 10 6 cells / mL, 140 x 10 6 cells / mL or 150 x 10 6 cells / mL, approximately 5 x 106 Cells / mL, 10 x 10 6 cells / mL, 20 x 10 6 cells / mL, 30 x 10 6 cells / mL, 40 x 10 6 Cells / mL, 50 x 10 6 cells / mL, 60 x 10 6 Cells / mL, 70 x 10 6 Cells / mL, 80 x 10 6 Cells / mL, 90 x 10 6 Cells / mL, 100 x 10 6 cells / mL, 110 x 10 6 cells / mL, 120 x 10 6 cells / mL, 130 x 10 6 cells / mL, 140 x 10 6 cells / mL or 150 x 10 6 cells / mL, or at least 5 x 10 6 Cells / mL, 10 x 10 6 cells / mL, 20 x 10 6 cells / mL, 30 x 10 6 cells / mL, 40 x 10 6 Cells / mL, 50 x 10 6 cells / mL, 60 x 10 6 Cells / mL, 70 x 10 6 Cells / mL, 80 x 10 6 Cells / mL, 90 x 10 6 Cells / mL, 100 x 10 6 cells / mL, 110 x 10 6 cells / mL, 120 x 10 6 cells / mL, 130 x 10 6 cells / mL, 140 x 10 6 cells / mL or 150 x 10 6 The cells are cryopreserved and / or cryoprotected (e.g., frozen) at a density of 10 ...

[0152] In certain embodiments, the cryopreserved and / or cryoprotected apheresis or leukapheresis product is banked (e.g., without T cell selection prior to freezing the sample), which in some aspects can provide more flexibility in subsequent manufacturing steps. In some aspects, the cryopreserved and / or cryoprotected apheresis or leukapheresis product is divided into multiple cryopreservation containers (such as bags) that can each be used individually or in combination in the processing of the product. For example, the total number of viable cells in the apheresis or leukapheresis product is 15×10 9 When the total number of viable cells in the apheresis product or leukapheresis product is less than 10×10, the cryopreserved and / or cryoprotected apheresis product or leukapheresis product is divided into four cryopreservation containers (e.g., bags). In some embodiments, the total number of viable cells in the apheresis product or leukapheresis product is less than 15-30×10 9 When the total number of cells is 8, the cryopreserved and / or cryoprotected apheresis product or leukapheresis product is divided into 8 cryopreservation containers (e.g., bags).

[0153] In one aspect, banking the cells prior to selection may increase the cell yield of downstream processes, and banking the cells earlier may mean that they are healthier and easier to meet manufacturing success criteria. In another aspect, once thawed, the cryopreserved and / or cryoprotected apheresis product or leukapheresis product can be subjected to one or more different selection methods. The advantage of this approach is, among other things, to enhance the availability, efficacy and / or other aspects of the cells of the cell therapy for the treatment of a disease or condition of interest, such as in the donor of the sample and / or another recipient.

[0154] In some embodiments, the sample (e.g., apheresis or leukapheresis sample) is collected and cryopreserved and / or cryoprotected at a time after the donor is diagnosed with any disease or condition, before cell selection or without prior cell selection (e.g., without prior T cell selection, such as chromatographic selection). In some aspects, the time of cryopreservation is also before the donor receives one or more of any initial treatment for the disease or condition, any targeted treatment or any treatment labeled for treatment for the disease or condition, or any treatment other than radiation and / or chemotherapy. In some embodiments, the sample is collected after the first recurrence of the disease after the initial treatment of the disease and before the donor or subject receives a subsequent treatment for the disease. The initial treatment and / or subsequent treatment can be a therapy other than cell therapy. In some embodiments, the collected cells may be used for cell therapy after the initial treatment and / or subsequent treatment. In one aspect, cryopreserved and / or cryoprotected samples without prior cell selection can help reduce initial costs, such as those associated with untreated patients in randomized clinical trials who may cross over and subsequently require treatment.

[0155] In some embodiments, samples (e.g., apheresis or leukapheresis samples) are collected, cryopreserved and / or cryoprotected, either prior to cell selection or without prior cell selection (e.g., without prior T cell selection, such as chromatographic selection), at a time point after second line treatment of the disease and after a second recurrence of the disease before the donor or subject undergoes a subsequent treatment for the disease. In some embodiments, patients are identified as likely to relapse after second line treatment, for example, by assessing certain risk factors. In some embodiments, the risk factors are based on disease type and / or genetic traits, such as double hit lymphoma, primary refractory cancer, or activated B cell lymphoma. In some embodiments, the risk factors are based on clinical features, such as early recurrence after first line treatment, or other poor prognostic indicators after treatment (e.g., IPI (International Prognostic Index)>2).

[0156] In some embodiments, the sample (e.g., apheresis or leukapheresis sample) is collected, cryopreserved and / or cryoprotected, before cell selection or without prior cell selection (e.g., without prior T cell selection, such as chromatographic selection) at a time before the donor or subject is diagnosed with any disease. In some aspects, the donor or subject may be determined to be at risk of developing any disease. In some aspects, the donor or subject may be a healthy subject. In certain cases, the donor or subject may choose to bank or preserve the cells in case cell therapy is needed at a later stage in life, even if they are never considered to be at risk of developing any disease or being diagnosed with any disease. In some embodiments, the donor or subject may be considered at risk of developing any disease based on factors such as gene mutations, gene abnormalities, gene disruptions, family history, protein abnormalities (such as defects in protein production and / or processing), and lifestyle choices that may increase the risk of developing any disease. In some embodiments, the cells are collected as a prophylactic.

[0157] In some embodiments, cryopreserved and / or cryoprotected samples of cells (e.g., apheresis or leukapheresis samples), e.g., samples of cells that have not been subjected to prior cell selection (e.g., without prior T cell selection, such as chromatographic selection), are stored or banked for a period of more than 12 hours, more than 24 hours, more than 36 hours, or more than 48 hours, or a period of more than 0.5 days, more than 1 day, more than 1.5 days, or more than 2 days, or a period of more than 0.5 days, more than 1 day, more than 1.5 days, or more than 2 days. In some embodiments, samples are stored or banked for a period of more than 1 week, more than 2 weeks, more than 3 weeks, or more than 4 weeks, or a period of more than 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, samples are stored or banked long term. In some aspects, the sample is greater than 1 month, greater than 2 months, greater than 3 months, greater than 4 months, greater than 5 months, greater than 6 months, greater than 7 months, greater than 8 months, greater than 9 months, greater than 10 months, greater than 11 months, greater than 1 year, greater than 2 years, greater than 3 years, greater than 4 years, greater than 5 years, greater than 6 years, greater than 7 years, greater than 8 years, greater than 9 years, greater than 10 years, greater than 11 years, greater than 12 years, greater than 13 years, greater than 14 years, greater than 15 years, greater than 16 years, greater than 17 years, greater than 18 years, greater than 19 years, greater than 20 years, greater than 25 years, greater than 30 years, greater than 35 years, greater than 36 years, greater than 37 years, greater than 38 years, greater than 39 years, greater than 40 years, greater than 41 years, greater than 42 years, greater than 43 years, greater than 44 years, greater than 45 years, greater than 46 years, greater than 47 years, greater than 48 years, greater than 49 years, greater than 50 years, greater than 51 years, greater than 52 years, greater than 53 years, greater than 54 years, greater than 55 years, greater than 56 years, greater than 57 years, greater than 58 years, greater than 59 years, greater than 60 years, greater than 61 years, greater than 62 years, greater than 63 years, greater than 64 years, greater than 65 years, greater than 66 years, greater than 67 years, greater than 68 years, greater than 69 years, greater than 70 years, greater than 71 years, greater than 72 years, greater than 73 years, greater than 74 years, greater than 75 years, greater than 7 years, greater than 40 years or more, or equal to 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years or more.

[0158] In some embodiments, apheresis or leukapheresis samples taken from a donor are transported to a storage or processing facility in a refrigerated environment and / or cryogenically stored at a storage facility or processed at a processing facility. In some embodiments, the sample is processed prior to transport, e.g., by selecting T cells, such as CD3+ T cells, CD4+ T cells and / or CD8+ T cells. In some embodiments, such processing is performed after transport and prior to cryogenic storage of the sample. In some embodiments, processing is performed after thawing the sample following cryogenic storage.

[0159] By allowing a donor to store their cells at a stage when the donor, and thus the cells, have not undergone extensive treatment for a disease and / or prior to the onset or diagnosis of a disease or condition, such cells may have certain advantages for use in cell therapy compared to cells harvested after one or more treatments. For example, cells harvested before one or more treatments may be healthier, may exhibit higher levels of specific cellular activity, may grow faster, and / or may be more receptive to genetic manipulation than cells that have undergone several treatments. Another example of an advantage according to the embodiments described herein may include convenience. For example, by collecting, optionally processing, and storing a donor's cells before they are needed for cell therapy, the cells are readily available when the recipient needs them later. This increases the capacity of apheresis laboratories and allows technicians more flexibility in scheduling the apheresis collection process.

[0160] Exemplary methods and systems for cryogenically storing and processing cells from a sample, such as an apheresis sample, may include those described in WO2018170188. In some embodiments, the methods and systems involve collecting an apheresis before a patient requires cell therapy, then cryopreserving the apheresis sample for later use in a process of engineering cells (e.g., T cells) with a recombinant receptor (e.g., CAR). In some cases, such a process may include a process described herein. In some embodiments, an apheresis sample is collected from a subject and cryopreserved prior to subsequent T cell selection, activation, stimulation, manipulation, transduction, transfection, incubation, culture, harvesting, formulation, and / or administration of the formulated cell population to the subject. In such examples, the cryopreserved apheresis sample is thawed prior to subjecting the sample to one or more selection steps, such as any described herein.

[0161] In some embodiments, a cryopreserved and / or cryoprotected sample of cells (e.g., an apheresis or leukapheresis sample), e.g., a sample of cells that has not been subjected to prior cell selection (e.g., without prior T cell selection, such as chromatographic selection), is thawed prior to use in downstream processes for manufacturing a cell population for cell therapy, e.g., a T cell population containing CAR+ T cells. In some embodiments, such a cryopreserved and / or cryoprotected sample of cells (e.g., an apheresis or leukapheresis sample) is used in connection with the processes provided herein for engineered T cell therapy, such as CAR+ T cell therapy. In certain examples, no further step of cryopreservation is performed prior to or during the harvesting / formulation step.

[0162] 1. T Cell Selection In some embodiments, the selection, isolation or enrichment of cells or populations includes one or more preparation and / or non-affinity-based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, and lyse or remove cells that are sensitive to a particular reagent. In some examples, cells are separated based on one or more properties, such as density, adhesion properties, size, sensitivity and / or resistance to a particular component. In some embodiments, the method includes preparation of white blood cells from peripheral blood by lysing red blood cells, and density-based cell separation methods, such as centrifugation through Percoll or Ficoll gradients. In certain embodiments, methods, techniques and reagents for selection, isolation and enrichment are described, for example, in WO2013124474 and WO2015164675, which patents are incorporated herein by reference in their entirety.

[0163] In some embodiments, the cryopreserved and / or cryoprotected apheresis product or leukapheresis product is thawed. In some embodiments, the thawed cell composition is subjected to dilution (e.g., with serum-free medium) and / or washing (e.g., with serum-free medium), which can remove or reduce unwanted or undesirable components in some cases. In some cases, the dilution and / or washing removes or reduces the presence of cryoprotectants, e.g., DMSO, contained in the thawed sample, which can otherwise negatively affect cell viability, yield, and recovery rate upon prolonged room temperature exposure. In some embodiments, the dilution and / or washing allows medium exchange of the thawed cryopreserved product to serum-free medium, e.g., as described in Section II herein or as described in PCT / US2018 / 064627, which is incorporated herein by reference.

[0164] In some embodiments, the serum-free medium comprises a basal medium supplemented with one or more supplements (e.g., OpTmizer™ T-Cell Expansion Basal Medium (ThermoFisher)). In some embodiments, the one or more supplements are serum-free. In some embodiments, the serum-free medium comprises a basal medium supplemented with one or more additional components for the maintenance, expansion and / or activation of cells (e.g., T cells), e.g., provided by additional supplements (e.g., OpTmizer™ T-Cell Expansion Supplement (ThermoFisher)). In some embodiments, the serum-free medium further comprises a serum replacement supplement, e.g., an immune cell serum replacement, e.g., CTS™ Immune Cell Serum Replacement (ThermoFisher, #A2596101), or an immune cell serum replacement as described in Smith et al. Clin Transl Immunology. 2015 Jan; 4(1): e31. In some embodiments, the serum-free medium further comprises a free form of an amino acid, such as L-glutamine. In some embodiments, the serum-free medium further comprises a dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine), such as the dipeptide in Glutamax™ (ThermoFisher). In some embodiments, the serum-free medium further comprises one or more recombinant cytokines, such as recombinant human IL-2, recombinant human IL-7, and / or recombinant human IL-15.

[0165] In some embodiments, at least a part of the selection step includes incubation of the cells with a selection reagent. The incubation with one or more selection reagents as part of the selection method can be performed using one or more selection reagents to select one or more different cell types based on the expression or presence of one or more specific molecules in or on the cells, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method using one or more selection reagents for separation based on such markers can be used. In some embodiments, the selection reagent or reagents result in a separation that is an affinity or immunoaffinity based separation. For example, in some aspects, the selection includes incubation with one or more reagents to separate cells and cell populations based on the cellular expression or expression level of one or more markers, typically cell surface markers, for example by incubation with an antibody or a binding partner that specifically binds to such marker, generally followed by a washing step, and separating the cells that are bound to the antibody or binding partner from the cells that are not bound to the antibody or binding partner.

[0166] In some aspects of such processes, a volume of cells is mixed with a quantity of a desired affinity-based selection reagent. Immunoaffinity-based selection can be performed using any system or method that results in favorable energetic interactions between the cells to be separated and a marker on the cells, e.g., an antibody or other binding partner on a solid surface, e.g., a molecule that specifically binds to a particle. In some embodiments, the method is performed using particles such as beads, e.g., magnetic beads, coated with a selection agent (e.g., an antibody) specific to a marker of the cells. The particles (e.g., beads) can be incubated or mixed with the cells in a container such as a tube or bag, with shaking or mixing, at a certain cell density to particle (e.g., beads) ratio to promote energetically favorable interactions. In other cases, the method includes selection of cells, where all or part of the selection is performed in the internal cavity of a centrifuge chamber, e.g., under centrifugal rotation. In some embodiments, incubation of the cells with a selection reagent, e.g., an immunoaffinity-based selection reagent, is performed in the centrifuge chamber. In certain embodiments, the isolation or separation is performed using a system, device, or apparatus described in International Patent Application Publication No. WO2009 / 072003 or US20110003380A1, the entireties of which are incorporated herein by reference. In one example, the system is a system described in International Publication No. WO2016 / 073602, the entireties of which are incorporated herein by reference.

[0167] In some embodiments, by performing such a selection step or a part thereof (e.g., incubation with antibody-coated particles, e.g., magnetic beads) in the cavity of the centrifuge chamber, the user can control certain parameters such as the volume of various solutions, the addition of solutions during processing, and the timing thereof, which can provide advantages compared to other available methods. For example, the ability to reduce the volume of liquid in the cavity during incubation can increase the concentration of the particles (e.g., bead reagents) used for selection, and thus the chemical potential of the solution, without affecting the total number of cells in the cavity. This can enhance pairwise interactions between the cells being processed and the particles used for selection. In some embodiments, for example, performing the incubation step in the chamber when associated with the systems, circuits, and controls described herein can also allow the user to agitate the solution at desired times during incubation, which can improve interactions.

[0168] In some embodiments, at least a portion of the selection step, including incubation of cells with the selection reagent, is performed in a centrifuge chamber. In some aspects of such a process, a volume of cells is mixed with a selection reagent based on the desired affinity in an amount that is much less than the amount that is normally used when performing a similar selection in a tube or container to select the same number and / or volume of cells according to the manufacturer's instructions. In some embodiments, an amount of one or more selection reagents is used that is 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, 50% or less, 60% or less, 70% or less, or 80% or less of the amount of the same selection reagent used to select cells in a tube or container-based incubation for the same number and / or volume of cells according to the manufacturer's instructions.

[0169] In some embodiments, for selection, e.g., based on the immunoaffinity of cells, cells are incubated in the cavity of the chamber in a population that also contains a selection buffer containing a selection reagent, such as a molecule, e.g., an antibody optionally bound to a scaffold, such as a polymer or surface, e.g., beads, e.g., magnetic beads, e.g., magnetic beads bound to monoclonal antibodies specific for CD3, CD4, and CD8, that specifically binds to a surface marker on the cells desired to be enriched and / or depleted, but not to surface markers on other cells in the population. In some embodiments, the selection reagent is added to the cells in the cavity of the chamber in a substantially lesser amount (e.g., 5% or less, 10% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% or less, or 80% or less of that amount) than would normally be used or required to achieve about the same or similar efficiency of selecting the same number of cells or the same volume of cells when selection is performed in a tube with shaking or rotation, as described. In some embodiments, incubation is performed by adding a selection buffer to the cells and selection reagent, e.g., 10 mL to 200 mL, e.g., at least 10 mL, at least 20 mL, at least 30 mL, at least 40 mL, at least 50 mL, at least 60 mL, at least 70 mL, at least 80 mL, at least 90 mL, at least 100 mL, at least 150 mL, or at least 200 mL, or at least about 10 mL, at least about 20 mL, at least about 30 mL, at least about 40 mL, at least about 50 mL, or less. The target volume is achieved by incubation of at least about 60 mL, at least about 70 mL, at least about 80 mL, at least about 90 mL, at least about 100 mL, at least about 150 mL or at least about 200 mL, or about 10 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 60 mL, about 70 mL, about 80 mL, about 90 mL, about 100 mL, about 150 mL or about 200 mL, or 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL or 200 mL of reagent.In some embodiments, the selection buffer and the selection reagent are premixed prior to addition to the cells. In some embodiments, the selection buffer and the selection reagent are added separately to the cells. In some embodiments, the selection incubation is performed with periodic gentle mixing conditions that help promote energetically favorable interactions, which may allow for the use of less overall selection reagent while achieving high selection efficiency.

[0170] In some embodiments, the total duration of incubation with the selection reagent is between 5 minutes and 6 hours, or between about 5 minutes and 6 hours, e.g., between 30 minutes and 3 hours, e.g., at least 30 minutes, at least 60 minutes, at least 120 minutes or at least 180 minutes, or at least about 30 minutes, at least about 60 minutes, at least about 120 minutes or at least about 180 minutes.

[0171] In some embodiments, incubation is generally under mixing conditions, such as in the presence of a spin, generally at a relatively low force or speed, such as a speed slower than that used to pellet the cells, e.g., at or about 600 rpm to 1700 rpm (e.g., 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm, or about 600 rpm, about 1000 rpm, or about 1500 rpm or about 1700 rpm, or at least 60 0 rpm, at least 1000 rpm, or at least 1500 rpm or at least 1700 rpm), for example, 80 g to 100 g or about 80 g to about 100 g (e.g., 80 g, 85 g, 90 g, 95 g or 100 g, or about 80 g, about 85 g, about 90 g, about 95 g or about 100 g, or at least 80 g, at least 85 g, at least 90 g, at least 95 g or at least 100 g) of sample or RCF at the wall of the chamber or other container. In some embodiments, the spinning is performed using repeated intermittences of spinning at such low speeds followed by rest periods, for example, 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, or 10 seconds of spinning and / or resting, for example, about 1 second or 2 seconds of spinning followed by about 5 seconds, about 6 seconds, about 7 seconds, or about 8 seconds of resting.

[0172] In some embodiments, such a process is performed within a completely closed system in which the chamber is integrated. In some embodiments, this process (and in some aspects also one or more additional steps, such as a preceding washing step to wash a cell-containing sample, such as an apheresis sample) is performed automatically using an automated program to draw cells, reagents and other components into the chamber, push them out, and centrifuge them at the appropriate times to complete the washing and binding steps in a single closed system.

[0173] In some embodiments, after incubation and / or mixing of the cells and the selection reagent and / or reagents, the incubated cells are separated to select cells based on the presence or absence of a particular reagent or reagents. In some embodiments, the separation is performed in the same closed system in which the incubation of the cells with the selection reagent was performed. In some embodiments, after incubation with the selection reagent, the incubated cells, including the cells bound by the selection reagent, are transferred to a system for immunoaffinity-based separation of the cells. In some embodiments, the system for immunoaffinity-based separation is or includes a magnetic separation column.

[0174] Such separation steps can be based on positive selection, where cells that bind a reagent, such as an antibody or binding partner, are retained for subsequent use, and / or negative selection, where cells that do not bind a reagent, such as an antibody or binding partner, are retained. In some instances, both fractions are retained for subsequent use. In some aspects, negative selection, which specifically identifies cell types within a heterogeneous population, can be particularly useful when antibodies are not available, such that separation is best performed based on markers expressed by cells other than the desired population.

[0175] In some embodiments, the process steps further comprise negative and / or positive selection of the incubated cells, e.g., using a system or device capable of affinity-based selection. In some embodiments, isolation is achieved by enrichment of a particular cell population by positive selection, or depletion of a particular cell population by negative selection. In some embodiments, positive or negative selection is achieved by detecting a specific or relatively high level of a marker expressed in the positively or negatively selected cells, respectively. high This is accomplished by incubating the cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers expressed (marker+) on the cells.

[0176] Separation does not necessarily result in the enrichment or removal of a particular cell population, or 100% of cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, such as cells expressing a marker, indicates an increase in the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, such as cells expressing a marker, indicates a decrease in the number or percentage of such cells, but does not necessarily result in the complete removal of any such cells.

[0177] In some examples, multiple separation steps are performed, in which a positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with multiple antibodies or binding partners, each specific for a marker targeted by negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating cells with multiple antibodies or binding partners expressed on different cell types. In certain embodiments, the separation step is repeated and / or performed more than once, in which a positively or negatively selected fraction from one step is subjected to the same separation step, such as repeated positive or negative selection. In some examples, a single separation step is repeated and / or performed more than once, for example, to increase the purity of selected cells and / or to further remove and / or deplete negatively selected cells from the negatively selected fraction. In certain embodiments, one or more separation steps are performed 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times. In certain embodiments, one or more selection steps are performed and / or repeated 1-10, 1-5, or 3-5 times.

[0178] For example, in some aspects, specific subpopulations of T cells, e.g., cells with positive or high expression levels of one or more surface markers, e.g., CD28+ T cells, CD62L+ T cells, CCR7+ T cells, CD27+ T cells, CD127+ T cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, CD45RA+ T cells and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some embodiments, such cells are selected by incubation with one or more antibodies or binding partners that specifically bind to such markers. In some embodiments, the antibodies or binding partners can be conjugated, e.g., directly or indirectly, to a solid support or matrix to effect selection, such as magnetic or paramagnetic beads. For example, CD3+ T cells, CD28+ T cells can be positively selected using CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander, and / or Exp ACT® beads).

[0179] In some embodiments, T cells are separated from a PBMC sample by negative selection of a marker expressed on B cells, monocytes or other leukocyte cells, e.g., non-T cells, such as CD14. In some aspects, a CD3+ selection step is used to generate a population enriched for CD3+ T cells from a starting sample, such as a PBMC or leukapheresis sample, where the starting sample has not been subjected to positive or negative selection based on another marker, such as CD4 and / or CD8. In some embodiments, the starting sample is selected for CD3 without any prior, simultaneous or subsequent selection for another marker (e.g., CD4 and / or CD8) to generate an input population enriched for CD3+ T cells. In some embodiments, an input population of cells is enriched for CD3+ T cells, and the input population is not subjected to further selection, e.g., CD4+ and / or CD8+ selection, before being subjected to a manufacturing process step, such as activating or stimulating or manipulating the input population. In certain embodiments, the input population enriched for CD3+ T cells has a CD4+ T cell:CD8+ T cell ratio of between 1:10 and 10:1, between 1:5 and 5:1, between 4:1 and 1:4, between 1:3 and 3:1, between 2:1 and 1:2, between 1.5:1 and 1:1.5, between 1.25:1 and 1:1.25, between 1.2:1 and 1:1.2, between 1.1:1 and 1:1.1, or about 1:1 or 1:1.

[0180] In some embodiments, T cells are separated from the PBMC sample by negative selection of markers expressed on B cells, monocytes or other white blood cells, e.g., non-T cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper T cells and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further separated into subpopulations by positive or negative selection for markers expressed or relatively highly expressed on one or more naive-like, memory and / or effector T cell subpopulations.

[0181] In some embodiments, CD8+ cells are further enriched or depleted for naive stem cells, central memory stem cells, effector memory stem cells and / or central memory stem cells, for example, by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment of central memory T (TCM) cells is performed to enhance efficacy, such as improving long-term survival, expansion and / or engraftment after administration, which in some aspects is particularly robust in such subpopulations. See Terakura et al., (2012) Blood. 1: 72-82; Wang et al. (2012) J Immunother. 35(9): 689-701. In some embodiments, TCM-enriched CD8+ T cells are combined with CD4+ T cells to further enhance efficacy.

[0182] In embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. In PBMCs, the CD62L-CD8+ and / or CD62L+CD8+ fractions can be enriched or depleted, such as by using anti-CD8 and anti-CD62L antibodies.

[0183] In some embodiments, the central memory T(T CMEnrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3 and / or CD127, and in some aspects on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is performed by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment of cells expressing CD62L. In one aspect, enrichment of central memory T (TCM) cells is performed starting from the negative fraction of cells selected on the basis of CD4 expression, which is subjected to negative selection on the basis of CD14 and CD45RA expression, and positive selection on the basis of CD62L. Such selections are performed simultaneously in some aspects, and sequentially in either order in other aspects. In some aspects, the same CD4 expression-based selection step used to prepare the CD8+ cell population or subpopulation is also used to generate the CD4+ cell population or subpopulation, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the method, optionally followed by one or more additional positive or negative selection steps. In some embodiments, the selection of the CD4+ cell population and the selection of the CD8+ cell population are performed simultaneously. In some embodiments, the selection of the CD4+ cell population and the CD8+ cell population are performed sequentially in either order. In some embodiments, the method for selecting cells may include those described in published US Patent Application No. 20170037369, which is incorporated herein by reference in its entirety. In some embodiments, the selected CD4+ cell population and the selected CD8+ cell population may be combined after selection. In some aspects, the selected CD4+ cell population and the selected CD8+ cell population may be combined in a bioreactor bag as described herein.

[0184] In certain embodiments, a biological sample, such as a sample of PBMCs or other white blood cells, is subjected to a selection of CD4+ T cells that retain both negative and positive fractions. In certain embodiments, CD8+ T cells are selected from the negative fraction. In some embodiments, a biological sample is subjected to a selection of CD8+ T cells that retain both negative and positive fractions. In certain embodiments, CD4+ T cells are selected from the negative fraction.

[0185] In some aspects, a CD8-based positive selection step is used to generate a population enriched in CD8+ T cells from a starting sample, such as a PBMC or leukapheresis sample, where the starting sample has not been subjected to selection based on another marker, such as CD3+ and / or CD4+. In some embodiments, both the negative and positive fractions from the CD8 positive selection step are retained, and the CD8 negative fraction is further subjected to a CD4-based positive selection step to generate a population enriched in CD4+ T cells. In some embodiments, cells from the CD8+ T cell enriched population and cells from the CD4+ T cell enriched population are mixed, combined and / or pooled to generate an input population containing CD4+ T cells and CD8+ T cells. In certain embodiments, the CD8+ T cell enriched population and the CD4+ T cell enriched population are pooled, mixed and / or combined before stimulating the cells, e.g., culturing the cells under stimulatory conditions as described in section IB. In certain embodiments, the pooled, mixed and / or combined cells or populations have a CD4+ T cell:CD8+ T cell ratio of between 1:10 and 10:1, between 1:5 and 5:1, between 4:1 and 1:4, between 1:3 and 3:1, between 2:1 and 1:2, between 1.5:1 and 1:1.5, between 1.25:1 and 1:1.25, between 1.2:1 and 1:1.2, between 1.1:1 and 1:1.1, or about or at 1:1. In certain embodiments, the cells or populations are pooled, mixed and / or combined to have a CD4+ T cell:CD8+ T cell ratio of 1:1 or about 1:1 in the pooled, mixed and / or combined cell composition.

[0186] In some aspects, a CD4-based positive selection step is used to generate a population enriched in CD4+ T cells from a starting sample, such as a PBMC or leukapheresis sample, where the starting sample has not been subjected to selection based on another marker, such as CD3+ and / or CD8+. In some embodiments, both the negative and positive fractions from the CD4-positive selection step are retained, and the CD4-negative fraction is further subjected to a CD8-based positive selection step that is used to generate a population enriched in CD8+ T cells. In some embodiments, cells from the CD4+ T cell enriched population and cells from the CD8+ T cell enriched population are mixed, combined and / or pooled to generate an input population containing CD8+ T cells and CD4+ T cells. In certain embodiments, the CD4+ T cell enriched population and the CD8+ T cell enriched population are pooled, mixed and / or combined before stimulating the cells, e.g., culturing the cells under stimulatory conditions as described in section IB. In certain embodiments, the pooled, mixed and / or combined cells or populations have a CD4+ T cell:CD8+ T cell ratio of between 1:10 and 10:1, between 1:5 and 5:1, between 4:1 and 1:4, between 1:3 and 3:1, between 2:1 and 1:2, between 1.5:1 and 1:1.5, between 1.25:1 and 1:1.25, between 1.2:1 and 1:1.2, between 1.1:1 and 1:1.1, or about or at 1:1. In certain embodiments, the cells or populations are pooled, mixed and / or combined to have a CD4+ T cell:CD8+ T cell ratio of 1:1 or about 1:1 in the pooled, mixed and / or combined cell composition.

[0187] In some embodiments, a selection agent that specifically binds CD4 and a selection agent that specifically binds CD8 are used to generate a population enriched in CD4+ T cells and a population enriched in CD8+ T cells, respectively. In some embodiments, the volumes of the CD4-specific selection agent and the CD8-specific selection agent are the same or substantially the same, e.g., a unit volume or unit weight of selection agent (e.g., ClinicMACS CD4 selection reagent and CD8 selection reagent) can be used to select CD4 or CD8 cells from the same number of total cells. In some embodiments, a greater amount of CD4-specific selection agent can be used than a CD8-specific selection agent having the same or substantially the same volume. For example, the volumes or weights of the CD4-specific selection agent and the CD8-specific selection agent can be in a ratio of about 5:1, 4:1, 3:1, 2:1, or 1:5:1.

[0188] In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both negative and positive fractions are retained. The negative fraction is then subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on markers characteristic of central memory T cells, such as CD62L or CCR7, where positive and negative selection are performed in either order.

[0189] CD4+ T helper cells may be classified into naive cells, central memory cells, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO- T cells, CD45RA+ T cells, CD62L+ T cells, or CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO-.

[0190] In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR and CD8. In some embodiments, the antibodies or binding partners are bound to a solid support or matrix, such as magnetic or paramagnetic beads, to allow cell separation for positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol.2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: SA Brooks and U. Schumacher (Copyright) Humana Press Inc., Totowa, NJ).

[0191] In some aspects, the sample to be incubated, or the cell population to be separated, is incubated with a selection reagent that contains a small magnetizable or magnetically responsive material, such as a magnetically responsive particle or microparticle, e.g., paramagnetic beads (e.g., Dynalbeads or MACS® beads). The magnetically responsive material, e.g., particle, is generally attached, directly or indirectly, to a binding partner, such as an antibody, that specifically binds to a molecule, such as a surface marker, present on the cell, cells, or cell population that it is desired to separate, e.g., negatively or positively select.

[0192] In some embodiments, the magnetic particles or beads comprise a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner.Many well-known magnetically responsive materials are known for use in magnetic separation methods, such as the magnetic particles described in U.S. Patent No. 4,452,773 to Molday, which is incorporated herein by reference, and European Patent Specification EP 452342 B.Colloidal-sized particles such as those described in U.S. Patent No. 4,795,698 to Owen and U.S. Patent No. 5,200,084 to Liberti et al., which are incorporated herein by reference, may also be used.

[0193] Incubation is generally carried out under conditions in which the antibody or binding partner, or a molecule, e.g., a secondary antibody or other reagent that specifically binds to such an antibody or binding partner attached to a magnetic particle or bead, specifically binds to the cell surface molecule if present on cells in the sample.

[0194] In certain embodiments, the magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, the magnetic particles are attached to the cells via coating with a primary antibody specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then magnetic particles coated with a cell type-specific secondary antibody or other binding partner (e.g., streptavidin) are added. In certain embodiments, magnetic particles coated with streptavidin are used in conjunction with a biotinylated primary antibody or a biotinylated secondary antibody.

[0195] In some aspects, separation is accomplished by placing the sample in a magnetic field and allowing cells with attached magnetically responsive or magnetizable particles to be attracted to the magnet and separated from unlabeled cells. In the case of positive selection, cells that are attracted to the magnet are retained. In the case of negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subjected to additional separation steps.

[0196] In some embodiments, affinity-based selection is by magnetic activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetic activated cell sorting (MACS), for example the CliniMACS system, allows for high purity selection of cells with magnetized particles attached to them. In certain embodiments, MACS operates in a mode where non-target and target species are eluted sequentially after application of an external magnetic field. That is, cells attached to magnetized particles are held in place while non-attached species are eluted. Then, after this first elution step is completed, species trapped in the magnetic field and prevented from eluting are released in some way so that they can be eluted and collected. In certain embodiments, non-target cells are labeled and depleted from the heterogeneous cell population.

[0197] In some embodiments, a suboptimal yield concentration of an affinity reagent is a concentration less than that used or required to achieve an optimal or maximum yield of bound cells in a given selection or enrichment, which involves incubating cells with the reagent and recovering or separating the cells bound to the reagent (wherein "yield" is, for example, the number of cells so recovered or selected, compared to the total number of cells in the incubation, which bear a marker targeted by or to which the reagent is specific or to which the reagent is specific and can bind). A suboptimal yield concentration is generally a concentration or amount of reagent in such a process or step that achieves a yield of less than the total of bound cells, e.g., CD4+ T cells and / or CD8+ T cells, e.g., 70% or less, upon recovery of the cells bound to the reagent. In some embodiments, a suboptimal concentration of an affinity reagent achieves a yield of 50% or less, 45% or less, 40% or less, 30% or less, or 25% or less, or about 50%, about 45%, about 40%, about 30%, or about 25%. Concentration may be expressed in terms of number or mass of particles or surfaces per cell, and / or mass or number of molecules of agent (e.g., antibody, such as an antibody fragment) per cell. In certain embodiments, the suboptimal yield concentration is sufficient to derive or achieve a fixed, controlled and / or defined ratio of naive-like CD4+ T cells to naive-like CD8+ T cells.

[0198] In some embodiments, for example, when operating at suboptimal yield concentrations for each or one or more of two or more selection reagents having affinity for CD4+ T cells and / or CD8+ T cells, one or more of such reagents are used at a higher concentration than one or more of the other such reagents to bias the ratio of cell types recognized by such reagent compared to cell types recognized by the other. For example, a reagent that specifically binds to a marker for which it is desired to bias the ratio may be included at a concentration (e.g., agent, or mass per cell) that is increased by half, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to the other, depending on the degree to which it is desired to increase the ratio.

[0199] In some embodiments, when operating in the suboptimal range and / or with sufficient cells to achieve saturation of the reagent, the amount of immunoaffinity reagent is proportional to the approximate yield of enriched cells. In certain embodiments, the appropriate amount or concentration of immunoaffinity reagent, depending on the desired ratio of the generated population containing enriched or selected CD4+ and CD8+ T cells, can be determined as a matter of routine.

[0200] In some embodiments, the separation and / or isolation steps are performed using magnetic beads to which the immunoaffinity reagents are reversibly bound, for example, via peptide ligand interactions with streptavidin muteins as described in WO 2015 / 164675, which is incorporated herein by reference in its entirety. An example of such magnetic beads is Streptamers®. In some embodiments, the separation and / or isolation steps are performed using magnetic beads, such as those commercially available from Miltenyi Biotec.

[0201] In some embodiments, the first selection or enrichment of CD4+ and CD8+ cells from the sample is performed using an immunoaffinity-based reagent comprising at least a first and a second affinity chromatography matrix to which an antibody is immobilized, respectively. In some embodiments, one or both of the first and / or second selections can utilize multiple affinity chromatography matrices and / or antibodies, whereby the multiple matrices and / or antibodies utilized in the same selection, i.e., the first selection or the second selection, are connected in series. In some embodiments, the affinity chromatography matrix or matrices utilized in the first and / or second selection have a density of at least about 50×10 6 Cells / mL, 100 x 10 6 Cells / mL, 200 x 10 6 cells / mL or 400 x 10 6It is possible to adsorb, select or concentrate cells / mL. In some embodiments, the adsorption capacity can be adjusted based on the diameter and / or length of the column. In some embodiments, the culture start ratio of the selected or enriched population is achieved by selecting a sufficient amount of matrix and / or a sufficient relative amount to achieve the culture start ratio, for example, based on the adsorption capacity of one or more columns for selecting cells.

[0202] In some embodiments, the magnetically responsive particles remain attached to the cells to be subsequently incubated, cultured and / or manipulated; in some aspects, the particles remain attached to the cells for administration to...

Claims

1. 1. A method for producing a composition of engineered T cells, the method comprising: (a) exposing an input composition comprising primary T cells to a stimulatory reagent to stimulate the T cells, thereby generating a stimulated population, wherein the stimulatory reagent comprises an anti-CD3 antibody and an anti-CD28 antibody or antigen-binding fragment thereof; (b) introducing into the stimulated population of T cells a viral vector comprising a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells; (c) incubating the population of transformed cells for up to 96 hours after said introduction and prior to harvesting; (d) harvesting T cells of the population of transformed cells, thereby producing a composition of engineered cells, said harvesting being performed between 48 and 120 hours, inclusive, after exposure to the stimulatory agent has commenced; and (e) formulating the cells of the engineered cell composition for cryopreservation and / or administration to a subject. A method comprising:

2. The method of claim 1, wherein the stimulatory reagent comprises an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules and capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and one or more intracellular signaling domains of one or more costimulatory molecules.

3. The method of claim 1 or 2, wherein said exposing and introducing is performed in the presence of one or more recombinant cytokines.

4. The method of any one of claims 1 to 3, wherein the incubation is carried out in the presence of one or more recombinant cytokines.

5. The method of any one of claims 1 to 3, wherein the incubation is carried out in a basal medium lacking one or more recombinant cytokines.

6. The collection, (a) performed when an integrated vector is detected in the genome but before achieving a stable integrated vector copy number (iVCN) per diploid genome, optionally comprising: (i) is achieved when the iVCN reaches a peak and remains unchanged or changes only within an acceptable margin of error for a period of more than 24 hours; or (ii) is achieved when the fraction of iVCN to total vector copy number (VCN) in the diploid genome of a population of transformed cells is, on average, at or about 1.0, or within an acceptable error thereof; (b) performed at a time when the fraction of iVCNs to total VCNs in the population of transformed cells is, on average, less than 0.8; and / or (c) performed at a time point when the population of transformed cells has an average iVCN of less than or about less than 2.0 copies per diploid genome; The method according to any one of claims 1 to 5.

7. The method of any one of claims 1 to 6, wherein the anti-CD3 and anti-CD28 antibodies or antigen-binding fragments thereof are present on or attached to the surface of a solid support.

8. 8. The method of claim 7, wherein the solid support is or comprises a bead.

9. 7. The method of any one of claims 1 to 6, wherein the stimulating reagent is an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules, and the anti-CD3 and anti-CD28 antibodies or antigen-binding fragments thereof are reversibly bound to the surface of the oligomeric particle reagent.

10. The exposure comprises 10 cells in the input composition. 6 10. The method of claim 9, wherein the method is carried out using an amount of stimulating reagent that is at or about 0.1 μg to 20 μg per cell, inclusive.

11. The method of any one of claims 1 to 10, wherein the incubation is carried out at a temperature of about 37°C.

12. A method described in any one of claims 1 to 11, wherein the cells are incubated for about 1 day, 2 days, or 3 days, or for at least 1 day, 2 days, or 3 days after introduction of the heterologous polynucleotide encoding the recombinant protein.

13. The method of any one of claims 1 to 12, wherein one or both of said exposing and said introducing are performed in the presence of one or more recombinant cytokines.

14. a basal medium lacking any recombinant cytokines, comprising L-glutamine at a concentration of about 0.5 mM to about 5 mM; the basal medium lacking any recombinant cytokines is serum-free, and / or The basal medium lacking one or more recombinant cytokines comprises at least one protein selected from albumin, insulin, and transferrin; 6. The method of claim 5.

15. 14. The method of claim 3 or 13, wherein the one or more recombinant cytokines are selected from recombinant IL-2, recombinant IL-7, and / or recombinant IL-15, optionally between 10 and 200 IU / mL of recombinant IL-2, between 100 IU / mL and 1,000 IU / mL of recombinant IL-7, and / or between 10 and 200 IU / mL of recombinant IL-15, and optionally wherein the one or more recombinant cytokines are human.

16. The method of any one of claims 1 to 15, wherein one or more of the exposing, introducing, and incubation are performed in serum-free medium.

17. The method of claim 1, wherein the stimulation reagent comprises an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules; and (a) the streptavidin or streptavidin mutein molecule binds or is capable of binding to biotin, avidin, a biotin analog or mutein, an avidin analog or mutein, and / or biologically active fragments thereof; (b) a plurality of streptavidin mutein molecules, each of which is (i) the amino acid sequence Va1 at sequence positions corresponding to positions 44 to 47 in streptavidin in the sequence of amino acids set forth in SEQ ID NO: 61; 44 -Thr 45 -Ala 46 -Arg 47 or Ile 44 -Gly 45 -Ala 46 -Arg 47 ; (ii) a sequence of amino acids set forth in any of SEQ ID NOs: 62, 63, 68, 75-77, and 80-83; (iii) a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher to any of SEQ ID NOs: 62, 63, 68, 75-77, and 80-83, and wherein the sequence identity is at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, based on the position in streptavidin of the amino acid sequence set forth in SEQ ID NO: 61, 44 -Thr 45 -Ala 46 -Arg 47 Or Ile 44 -Gly 45 -Ala 46 -Arg 47 and / or a sequence of amino acids which reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide; and / or (iv) a functional fragment of (ii) or (iii) that reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide. Including, The method of any one of claims 2 to 6 and 9 to 16.

18. The method of any one of claims 1 to 17, wherein the anti-CD3 antibody and the anti-CD28 antibody or antigen-binding fragment thereof each comprise a streptavidin-binding peptide.

19. The method of claim 18, wherein the streptavidin-binding peptide is an amino acid sequence set forth in any one of SEQ ID NOs: 64-67, 70, 73, and 74.

20. The method of any one of claims 1 to 19, wherein one or both of the anti-CD3 and anti-CD28 antibodies or antigen-binding fragments thereof are or comprise monovalent antibody fragments, optionally Fab.

21. The method of claim 20, wherein the stimulation reagent comprises an oligomeric particle reagent, and The oligomeric particle reagent comprises: A radius between 50 nm and 150 nm, between 75 nm and 125 nm, between 80 nm and 115 nm, or between 90 nm and 110 nm (inclusive); 5×10 7 g / mol to 5×10 8 g / mol, 1×10 8 g / mol to 5×10 8 g / mol, or 1×10 8 g / mol to 2×10 8 Molecular weight in g / mol; and / or 1,000 to 20,000 streptavidin or streptavidin mutein tetramers, 1,000 to 10,000 streptavidin or streptavidin mutein tetramers, or 2,000 to 5,000 oligomers of streptavidin or streptavidin mutein tetramers. The method of any one of claims 2 to 6 and 9 to 20, comprising:

22. adding a substance to the cells either after or during at least a portion of the incubation; the substance is capable of terminating or reducing stimulation of T cells by a stimulatory reagent and / or abolishing the binding between anti-CD3 and anti-CD28 antibodies or antigen-binding fragments thereof and the oligomeric particle reagent; 22. The method of any one of claims 1 to 21.

23. the substance is or comprises a streptavidin-binding peptide, biotin or a biologically active fragment, or a biotin analog or a biologically active fragment, Optionally, the substance is or comprises a streptavidin-binding peptide, the streptavidin-binding peptide comprising: Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 64), Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 3 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:73), Trp-Ser-His-Pro-GlnPhe-Glu-Lys-(GlyGlyGlySer) 3 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:65), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 2 -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:66), and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) 2 Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:67) Selected from the group consisting of:

23. The method of claim 22.

24. 23. The method of claim 22, wherein the substance is added 42 to 120 hours or about 42 to 120 hours, inclusive, after exposure to the oligomeric particle stimulation reagent is initiated, and optionally, the substance is added 48 hours or about 48 hours after exposure to the oligomeric particle reagent is initiated.

25. The method according to any one of claims 1 to 24, wherein said incubation is carried out under static conditions, optionally in an incubator.

26. 26. The method of claim 25, wherein said static conditions are conditions that do not include centrifugation, shaking, rotation, rocking, or perfusion.

27. The exposure 6 0.2μg-8μg per cell or about 0.2μg-8μg per cell, 10 6 0.4μg to 8μg per cell or about 0.4μg to 8μg per cell, or about 10 cells 6 The method of any one of claims 1 to 7 and 10 to 26, wherein the method is performed at or about 0.8 μg to 4 μg of stimulating reagent per cell.

28. 28. The method of any one of claims 1-27, wherein at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, at least or at least about 99%, about 100%, or 100% of the cells in the input composition are CD3+ T cells, or are CD4+ T cells and CD8+ T cells.

29. 29. The method of any one of claims 1-28, wherein said input composition comprises a ratio of CD4+ cells to CD8+ cells of between 1.5:1 and 2.0:1, or between 1.2:1 and 0.8:

1.

30. The exposure 6 the stimulation reagent is at or about 0.4 μg to 8 μg per cell, the stimulation reagent comprising an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules, to which (i) an anti-CD3 antibody, or antigen-binding fragment thereof, and (ii) an anti-CD28 antibody, or antigen-binding fragment thereof, are attached, the exposing being in the presence of serum-free medium comprising recombinant IL-2, IL-7, and IL-15; The input composition comprises a ratio of CD4+ T cells to CD8+ T cells of 2:1 to 1:2 and is at least 300×10 6 containing CD4+ and CD8+ T cells, said introducing being carried out in the presence of serum-free medium having recombinant IL-2, IL-7, and IL-15; The incubation is carried out under static conditions for 24 hours to 72 hours or about 24 hours to 72 hours, The collection is performed between 72 and 96 hours (inclusive) after exposure to the stimulating agent has begun; the method further comprises the step of adding a substance comprising biotin or a biologically active fragment thereof, optionally D-biotin or a biotin analog or a biologically active fragment thereof, during at least a portion of the incubation, said addition occurring between 48 and 72 hours (inclusive) after exposure to the stimulating agent has commenced; 30. The method of any one of claims 1 to 7 and 10 to 29.

31. the exposing is with a stimulating reagent comprising paramagnetic beads at a bead to cell ratio of 2:1 to 0.5:1 or about 2:1 to 0.5:1, the beads being inert paramagnetic beads comprising a polystyrene coating and a diameter of 4.5 μm or about 4.5 μm, to which (i) an anti-CD3 antibody or antigen-binding fragment thereof and an anti-CD28 antibody or antigen-binding fragment thereof are attached, the exposing is in the presence of serum-free medium comprising recombinant IL-2, IL-7, and IL-15; The input composition comprises a ratio of CD4+ T cells to CD8+ T cells of 2:1 to 1:2 and is at least 300×10 6 containing CD4+ and CD8+ T cells, said introducing being carried out in the presence of serum-free medium having recombinant IL-2, IL-7, and IL-15; The incubation is carried out under static conditions for 48 to 72 hours, inclusive; The collection is performed between 72 and 96 hours (inclusive) after exposure to the stimulating agent has begun; The method further comprises, after incubation and prior to harvesting, exposing the cells to a magnetic field to remove the stimulatory reagent from the cells.

30. The method of any one of claims 1, 2, 4-17, 21, 26, 28, and 29.

32. The input composition is 450 x 10 6 pcs or approx. 450 x 10 6 Viable primary T cells, 500 x 10 6 pcs or approx. 500 x 10 6 viable primary T cells, 550 x 10 6 pcs or approx. 550 x 10 6 viable primary T cells, 600 x 10 6 pcs or approx. 600 x 10 6 viable primary T cells, 700 x 10 6 pcs or approx. 700 x 10 6 viable primary T cells, 800 x 10 6 800 x 10 6 viable primary T cells, 900 x 10 6 pcs or approx. 900 x 10 6 Viable primary T cells, 1,000 x 10 6 Pieces or about 1,000 x 10 6 viable primary T cells, 1,100 x 10 6 Pieces or about 1,100 x 10 6 viable primary T cells, or 1,200 x 10 6 Pieces or approx. 1,200 x 10 6 viable primary T cells, or At least 450 x 10 6 or at least about 450×10 6 10 viable primary T cells, at least 500 x 10 6 or at least about 500×10 6 Viable primary T cells, at least 550 x 10 6 or at least about 550×10 6 10 viable primary T cells, at least 600 x 10 6 or at least about 600×10 6 10 viable primary T cells, at least 700 x 10 6 or at least about 700×10 6 10 viable primary T cells, at least 800 x 10 6 or at least about 800×10 6 Viable primary T cells, at least 900 x 10 6 or at least about 900×10 6 1,000 x 10 viable primary T cells 6 or at least about 1,000×10 6 Viable primary T cells, at least 1,100 x 10 6 or at least about 1,100×10 6 viable primary T cells, or at least 1,200 x 10 6 or at least about 1,200×10 6 Viable primary T cells The method of any one of claims 1 to 31, comprising:

33. Prior to exposure to the stimulant agent, enriching CD3+ T cells or CD4+ T cells and / or CD8+ T cells from the biological sample to produce an input composition; and / or enriching CD4+ or CD8+ T cells from the biological sample to produce an input composition, optionally wherein said enrichment produces separate enriched populations of CD4+ T cells and CD8+ T cells, and said separate enriched populations are mixed to produce the input composition. The method of any one of claims 1 to 32, comprising:

34. Concentration, (a) performing a first selection in a closed system, the first selection comprising enrichment of one of (i) CD4+ cells and (ii) CD8+ cells from a sample comprising primary human T cells, whereby the enrichment produces a first selected population and a non-selected population; and (b) performing a second selection in a closed system, the second selection comprising enrichment of the other of (i) CD4+ cells and (ii) CD8+ cells from the unselected population, whereby the enrichment produces a second selected population, the enrichment producing separate enriched populations of CD4+ T cells and CD8+ T cells. Including, each of the separate enriched populations comprises cells from either the first selected population or the second selected population; the input composition comprises cells from one or more of an enriched population of CD4+ T cells and an enriched population of CD8+ T cells; 34. The method of claim 33.

35. The enrichment of cells in the first and / or second selection comprises immunoaffinity-based selection.

35. The method of claim 34.

36. The immunoaffinity-based selection is performed by contacting the cells with an antibody immobilized on or attached to an affinity chromatography matrix, which antibody is capable of specifically binding to a cell surface marker and performing positive or negative selection of CD4+ or CD8+ cells.

36. The method of claim 35.

37. enriching for CD4+ or CD8+ T cells from the biological sample prior to exposure to the stimulatory reagent, the enrichment comprising contacting cells of the sample with a first immunoaffinity reagent that specifically binds CD4 in an incubation composition and a second immunoaffinity reagent that specifically binds CD8 in an incubation composition under conditions in which the first and second immunoaffinity reagents specifically bind to CD4 and CD8 molecules, respectively, on the surface of cells in the sample; and recovering the cells that bind to the first and / or second immunoaffinity reagents, thereby producing an enriched composition comprising CD4+ cells and CD8+ cells. Including, the input composition comprises cells of an enriched composition comprising CD4+ cells and CD8+ cells; Optionally, each of the immunoaffinity reagents comprises an antibody or an antigen-binding fragment thereof and / or the immunoaffinity reagents are immobilized on the outer surface of a bead.

37. The method of any one of claims 1 to 36.

38. each of said immunoaffinity reagents comprises an antibody or an antigen-binding fragment thereof and / or said immunoaffinity reagents are immobilized on the outer surface of a bead; 38. The method of claim 37.

39. the biological sample comprises primary T cells obtained from a subject, optionally a human subject; the biological sample is or comprises a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukapheresis product; and / or The biological sample is an apheresis product or a leukapheresis product that has been previously cryo-frozen prior to enrichment; The method of any one of claims 33 to 38.

40. when the percentage of naive-like T cells and / or central memory T cells is greater than or greater than about 60% among the total T cells in the population, among the total CD4+ T cells in the population, among the total CD8+ T cells in the population, or among the cells expressing the recombinant protein in the population, at a time point where the percentage of naive-like T cells and / or central memory T cells is greater than or greater than about 60% of all recombinant protein-expressing cells in the population, optionally greater than or greater than about 65%, 70%, 80%, 90%, or 95% of all recombinant protein-expressing T cells in the population; at a time point where the percentage of naive-like T cells and / or central memory T cells is greater than or greater than about 60% among all recombinant protein-expressing CD4+ T cells in the population, optionally greater than or greater than about 65%, 70%, 80%, 90%, or 95% among all recombinant protein-expressing CD4+ cells in the population; at a time point when the percentage of naive-like T cells and / or central memory T cells is greater than or greater than about 60% among all recombinant protein-expressing CD8+ T cells in the population, optionally greater than or greater than about 65%, 70%, 80%, 90%, or 95% among all recombinant protein-expressing CD8+ cells in the population; and / or When naive-like T cells, or T cells that are surface positive for markers expressed on naive-like T cells, are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+, The method of any one of claims 1 to 39, wherein the cells are harvested.

41. 41. The method of any one of claims 1 to 40, wherein the harvesting is performed at a time point when the percentage of CD27+CCR7+ is greater than or greater than about 60% among the total T cells in the population, the total CD3+ T cells in the population, the total CD4+ T cells in the population, the total CD8+ T cells in the population, or the cells expressing the recombinant protein thereof, optionally at a time point when the percentage of CD27+CCR7+ is greater than or greater than about 60% among the total T cells in the population, the total CD3+ T cells in the population, the total CD4+ T cells in the population, the total CD8+ T cells in the population, or the cells expressing the recombinant protein thereof,

42. The composition of engineered cells is collected as an output composition in a container, and the output composition is collected in one or more containers at about or at least about 10×10 6 , about or at least about 20×10 6 , about or at least about 25×10 6 , about or at least about 50×10 6 , about or at least about 100×10 6 , about or at least about 200×10 6 , about or at least about 400×10 6 , about or at least about 600×10 6 , about or at least about 800×10 6 , about or at least about 1000×10 6 , about or at least about 1200×10 6 , about or at least about 1400×10 6 , about or at least about 1600×10 6 , about or at least about 1800×10 6 , about or at least about 2000×10 6 , about or at least about 2500×10 6 , about or at least about 3000×10 6 or about or at least about 4000×10 6 42. The method of any one of claims 1 to 41, comprising total cells, optionally total viable cells.

43. 43. The method of any one of claims 1 to 42, further comprising formulating the cells of the engineered cell composition in the presence of a pharma- ceutically acceptable excipient or cryoprotectant for cryopreservation and / or administration to a subject.

44. 44. The method of any one of claims 1 to 43, further comprising formulating the cells of the engineered cell composition in the presence of DMSO for cryopreservation and / or administration to a subject.

45. The method of any one of claims 1 to 44, wherein the viral vector is a retroviral vector, a lentiviral vector, or a gamma retroviral vector.

46. The method of any one of claims 1 to 45, wherein the transduction is carried out in the presence of a transduction adjuvant.

47. the transduction adjuvant is protamine sulfate, a fibronectin-derived transduction adjuvant, or RetroNectin; and / or The introduction is carried out in the presence of 1 μg / ml to 50 μg / ml or about 1 μg / ml to 50 μg / ml of protamine sulfate; 47. The method of claim 46.

48. The method of any one of claims 1 to 47, wherein the recombinant protein is a recombinant receptor capable of binding to a target antigen associated with, specific for, and / or expressed on a disease, disorder, or diseased cell or tissue.

49. and / or the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer; The target antigen is a tumor antigen; 49. The method of claim 48.

50. the recombinant protein is or comprises a functional non-TCR antigen receptor or a TCR or an antigen-binding fragment thereof; and / or The recombinant protein is a chimeric antigen receptor (CAR); 50. The method of any one of claims 1 to 49.

51. Serum-free medium, 0.5 mM to 5 mM of the dipeptide form of L-glutamine in the basal medium; 0.5 mM to 5 mM L-glutamine; and At least one protein Contains no serum, 32. The method of any one of claims 16, 30, and 31.

52. The basal medium comprises at least one protein selected from one or more of albumin, insulin, or transferrin, optionally one or more of human or recombinant albumin, insulin, or transferrin; 52. The method of claim 51.

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