Methods for generating engineered cells and compositions thereof

Abstract

To provide a method for genetically engineering T cells for use in a cell therapy without cell expansion, as well as compositions of the generated T cells and uses thereof for treating a subject.SOLUTION: Disclosed is a method for producing a composition of engineered T cells, the method comprising the steps of: (a) exposing a composition comprising primary T cells to a stimulator under conditions to stimulate T cells thereby generating a stimulated population, where the stimulator activates one or more intracellular signaling domains of one or more components of TCR complex, and activates one or more intracellular signaling domains of one or more co-stimulatory molecules; (b) introducing to the 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; and (d) following the incubation, harvesting T cells of the transformed cell population thereby producing a composition of the engineered cells.SELECTED DRAWING: Figure 13A

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 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., populations 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. [Background technology]

[0004] background Various 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 existing methods for producing genetically engineered cell compositions can be time-consuming or require different amounts of time to be successfully completed. In certain cases, some existing methods can result in compositions in which the cell population has low efficacy or persistence in vivo. Improved methods for producing and / or manipulating 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 for 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 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 harvest occurs (i) at 24 to 120 hours, or 1 day to 5 days, or about 1 day to about 5 days, inclusive, after exposure to the stimulatory reagent has begun; (ii) the integrated vector has evolved into a genomic vector. (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% of all T cells in the population, all CD3+ T cells in the population, all CD4+ T cells in the population, or 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 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 occurs between (i) 48 and 120 hours, or 2 to 5 days, or about 2 to 5 days after exposure to the stimulatory reagent began. (ii) at about 5 days inclusive; (ii) when integrated vector is detected in the genome but before achieving a stable integrated vector copy number per diploid genome (iVCN); (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% of 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 an integrated vector is detected in the genome, but before achieving a stable iVCN per diploid genome. In certain embodiments, stable iVCN per diploid genome is achieved when iVCN peaks and remains unchanged, or changes only within a margin of error, for a period of more than 24 hours or 1 day, or for a period of more than about 24 hours or 1 day. In certain embodiments, stable iVCN per diploid genome is achieved when the ratio of iVCN to 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 a margin of error. In some embodiments, harvesting is performed when the ratio of integrated vector copy number (iVCN) to total VCN in a population of transformed cells is, on average, less than 0.8.

[0009] In some embodiments, the T cells are harvested at a time when the percentage of naive-like T cells is greater than or greater than about 60% of 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 recombinant protein-expressing cells 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 to stimulate 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 a viral vector comprising a heterologous polynucleotide encoding a recombinant protein into 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 to 120 hours, or 2 to 5 days, or about 2 to about 5 days, inclusive, after initiation of exposure to the stimulatory reagent, and wherein at harvest, the population of transformed cells has an integrated vector copy number (iVCN) of between 0.4 and 2.0 copies per diploid genome, or between about 0.4 and 2.0 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, on average, less than or about less than 2.0 copies per diploid genome. In certain embodiments, harvesting is performed when the iVCN of the population of transformed cells is, on average, less than or about 1.5 copies per diploid genome. In certain embodiments, harvesting is performed when the iVCN of the population of transformed cells is, on average, less than or about 1.0 copies per diploid genome. In some embodiments, harvesting is performed when the iVCN of the population of transformed cells is, on average, less than or about 0.75 copies per diploid genome. In certain embodiments, harvesting is performed when the iVCN of the population of transformed cells is, on average, less than or about 0.5 copies per diploid genome.

[0012] Provided herein is a method for 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 producing 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. 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 initiation of exposure to the stimulatory reagent, and wherein at harvest, the percentage of naive-like cells is greater than or greater than about 60% of the total T cells in the population, total CD4+ T cells in the population, or total CD8+ T cells in the population, or cells expressing the recombinant protein thereof; thereby producing a composition of engineered cells.

[0013] In some embodiments, at the time of harvest in the provided methods, the percentage of central memory T 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 cells expressing a recombinant protein thereof. In certain embodiments, the central memory T cells are CCR7+CD45RA-.

[0014] Provided herein is a method for 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 producing 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 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 initiation of exposure to the stimulatory reagent, 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, total CD4+ T cells in the population, or total CD8+ T cells in the population, or 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 harvest, the percentage of naive-like T cells is greater than or greater than about 60% of all recombinant protein-expressing CD8+ T cells in the population, for example, greater than or greater than about 65%, 70%, 80%, 90%, or 95% of 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 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 CD27+CCR7+ 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 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 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 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. 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 stimuli has begun. In some embodiments, the harvesting is performed within 72 hours or 3 days after exposure to the stimuli has begun. In certain embodiments, the harvesting is performed within 48 hours or 2 days after exposure to the stimuli has begun.

[0020] In certain embodiments, one or both of the exposing and introducing is 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 for 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, 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; and (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 the initiation of exposure to the stimulatory reagent; 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 comprise 10-200 IU / mL of recombinant IL-2; 100 IU / mL to 1,000 IU / mL of recombinant IL-7; and / or 10-200 IU / mL of recombinant IL-15. In certain embodiments, the one or more recombinant cytokines are or comprise recombinant IL-2, recombinant IL-7, and recombinant IL-15. For example, in some cases, the one or more recombinant cytokines comprise 10-200 IU / mL of recombinant IL-2; 100 IU / mL to 1,000 IU / mL of recombinant IL-7; and 10-200 IU / mL of 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, where optionally, 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 and secondary agents 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: (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, wherein the bead:cell ratio 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 T cells of the stimulated population a viral vector comprising a heterologous polynucleotide encoding a recombinant protein, thereby producing a population of transformed cells; and (c) harvesting the T cells of the transformed population, wherein 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.

[0027] In some embodiments, the ratio of beads to 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 comprising a plurality of streptavidin or streptavidin mutein molecules.

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

[0030] In some embodiments, the exposure comprises 10 cells in the input composition. 6 The method 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 0.1 μg to 20 μg per particle 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; and, in the input composition, providing a mixture of cells 10 6 In some embodiments, the input composition comprises primary T cells, the method comprising exposing the T cells to a stimulatory reagent in an amount of at or about 0.1 μg to 20 μg per cell, inclusive, wherein the stimulatory reagent comprises an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules, and the exposing is 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; and, in the input composition, providing a mixture of cells 10 6 In some embodiments, the input composition comprises primary T cells, wherein the input composition comprises at least one of a plurality of streptavidin or streptavidin mutein molecules attached thereto, the 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, to CD3, and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule, the method comprising exposing the input composition to at least one of a plurality of streptavidin or streptavidin mutein molecules attached thereto, the 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, to CD3, the primary agent being a secondary agent that specifically binds to a T cell costimulatory molecule, the method comprising exposing the input composition to at least one of a plurality of streptavidin or streptavidin mutein molecules attached thereto, the plurality of streptavidin or streptavidin mutein molecules being attached to the plurality of streptavidin or streptavidin mutein molecules ...

[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 stimuli has begun. In certain embodiments, the harvesting is performed within 72 hours or 3 days after the exposure of the input composition to the stimuli has begun. In some embodiments, the harvesting is performed within 48 hours or 2 days after the exposure of the input composition to the stimuli has begun.

[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 carried out in the presence of one or more recombinant cytokines. In certain embodiments, the incubation is carried out in a basal medium lacking one or more recombinant cytokines.

[0037] Provided herein is a method for 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, wherein the bead:cell ratio 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 producing a stimulated population; and (b) transducing into the T cells of the stimulated population a viral vector comprising 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 to 120 hours or 2 to 5 days, inclusive, after the initiation of exposure to the stimulatory reagent; thereby producing a composition of engineered cells.

[0038] Provided herein is a method for 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, wherein the oligomeric particle reagent has 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, wherein the exposing is performed under conditions for stimulating the T cells, including the presence of one or more recombinant cytokines, thereby producing a stimulated population; (b) transducing 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 to 120 hours or 2 to 5 days, inclusive, after the initiation of exposure to the stimulatory reagent; 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 in the input composition. 6 In certain embodiments, the exposure is performed using an amount of stimulation reagent that is at or about 0.4 μg to 8 μg per cell in the input composition. 6 In certain embodiments, the exposure is performed using a stimulation reagent in an amount of 0.8 μg to 4 μg per cell, inclusive. 6The stimulation is carried out using an amount of stimulating 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 comprise 10-200 IU / mL of recombinant IL-2; 100 IU / mL to 1,000 IU / mL of recombinant IL-7; and / or 10-200 IU / mL of recombinant IL-15. In certain embodiments, the one or more recombinant cytokines are or comprise recombinant IL-2, recombinant IL-7, and recombinant IL-15. For example, in some cases, the one or more recombinant cytokines comprise 10-200 IU / mL of recombinant IL-2; 100 IU / mL to 1,000 IU / mL of recombinant IL-7; and 10-200 IU / mL of 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, e.g., anti-CD3 and anti-CD28 antibodies or antigen-binding fragments thereof, are present on or attached to the surface of beads. In certain embodiments, the ratio of beads to cells is 2:1 to 0.5:1 or about 2:1 to 0.5:1. In certain embodiments, the ratio of beads to 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 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 the 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 TIFF2025063150000002.tif37163.

[0045] In certain embodiments, one or both of the primary and secondary agents comprise an antibody or 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, hi some embodiments, the oligomeric particle reagent comprises a radius of 50 nm to 150 nm, 75 nm to 125 nm, 80 nm to 115 nm, or 90 nm to 110 nm (inclusive); or a radius of 90 nm ± 15 nm or 95 nm ± 20 to 25 nm.

[0047] In some embodiments, the oligomeric particle reagent is 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 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.

[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 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 signals 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 biologically active fragment. In certain embodiments, the substance is or comprises a streptavidin-binding peptide, and the streptavidin-binding peptide is TIFF2025063150000003.tif37162. In some embodiments, the agent is or comprises biotin or a biologically active fragment or a biotin analog or biologically active fragment.

[0050] In certain embodiments, the substance is added 42 to 120 hours, or about 42 to 120 hours, inclusive, after exposure to a stimulation reagent comprising oligomeric particles begins. In certain embodiments, the substance is added 48 to 120 hours, or about 48 to 120 hours, or 2 to 5 days, inclusive, after exposure to an oligomeric particle reagent begins. In certain embodiments, the substance is added 72 to 96 hours, or about 72 to 96 hours, or 3 to 4 days, inclusive, after exposure to an oligomeric particle reagent begins. In some embodiments, the substance is added 48 hours, or about 48 hours, or 2 days, inclusive, after exposure to an oligomeric particle reagent begins. In certain embodiments, the substance is added 72 hours, or about 72 hours, or 3 days, inclusive, after exposure to an oligomeric particle reagent begins. 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 carried out in the presence of a basal medium lacking recombinant cytokines.

[0052] Provided herein are methods for producing a composition of engineered T cells, comprising: (a) transferring an input composition comprising primary T cells to an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules, the oligomeric particle reagent comprising the cells or the oligomeric particle reagent comprising the ... 6 (b) 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, wherein the oligomeric particle reagent has attached thereto (i) a primary agent that specifically binds to CD3 and (ii) a secondary agent that specifically binds to CD28, and the exposing is performed in the presence of a serum-free medium containing recombinant IL-2, IL-7, and IL-15, thereby generating a stimulated population; (b) transducing the T cells of the stimulated population with a viral vector comprising a heterologous polynucleotide encoding a recombinant protein, thereby generating a population of transformed cells, wherein the transduction is performed in the presence of a serum-free medium containing 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, thereby producing a composition of engineered cells, wherein the harvesting is performed 72 to 96 hours or 3 to 4 days, inclusive, after exposure to the stimulatory reagent has begun; wherein the T cells of the transformed population are incubated for 24 to 96 hours or 1 to 4 days, inclusive, under conditions consistent with the stimulatory reagent.

[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 x 10 6 or approximately 450 x 10 6 500 x 10 viable primary T cells 6 pieces or approximately 500 x 10 6 viable primary T cells, 550 x 10 6 or approximately 550 x 10 6 viable primary T cells, 600 x 10 6 or approximately 600 x 10 6 700 x 10 viable primary T cells 6 or approximately 700 x 10 6 viable primary T cells, 800 x 10 6 or approximately 800 x 10 6 viable primary T cells, 900 x 10 6 or approximately 900 x 10 6 viable primary T cells or 1,000 x 10 6 or approximately 1,000 x 10 6 In certain embodiments, the input composition is 600 x 10 viable primary T cells, or any value between any of the foregoing. 6 pieces or approximately 600 x 10 6 In certain embodiments, the number of primary T cells in the input composition is up to 900 x 10 6 viable primary T cells or 900 x 10 6 or approximately 900 x 10 6 viable primary T cells.

[0054] In some embodiments, the primary T cells in the input composition comprise 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 the 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 the 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 the 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 the 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 are methods for producing a composition of engineered T cells, comprising: (a) transferring an input composition comprising primary T cells to an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules, the oligomeric particle reagent comprising the cells or the oligomeric particle reagent comprising the ... 6and exposing the oligomeric particle reagent to at or about 0.4 μg to 8 μg of stimulatory reagent per particle, wherein the oligomeric particle reagent has attached thereto (i) a primary agent that specifically binds to CD3 and (ii) a secondary agent that specifically binds to CD28, and the input composition comprises a ratio of CD4+ cells:CD8+ T cells of 2:1 to 1:2, and the input composition is at least 300×10 of CD4+ and CD8+ T cells. 6(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 introduction is carried out in the presence of a serum-free medium having recombinant IL-2, IL-7, and IL-15; and (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) adding, during at least a portion of the incubation, a substance comprising biotin or a biologically active fragment thereof, optionally D-biotin or a biotin analog or a biologically active fragment thereof, wherein the addition occurs 48 to 72 hours or 2 to 3 days, inclusive, after exposure to the stimulatory reagent is initiated; and (d) harvesting the T cells of the transformed population after the incubation, wherein the harvesting occurs 72 to 96 hours or 3 to 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 or about 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 or about 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 at least 300×10 6exposing an input composition comprising primary T cells to a stimulation reagent comprising paramagnetic beads at a beads:cell ratio of 2:1 to 0.5:1 or about 2:1 to 0.5:1, wherein the beads are inert paramagnetic beads comprising a polystyrene coating and a diameter of 4.5 μm or about 4.5 μm, and the beads have (i) an anti-CD3 antibody or antigen-binding fragment thereof and an anti-CD28 antibody or antigen-binding fragment thereof attached thereto, wherein the input composition comprises a ratio of CD4+ T cells:CD8+ T cells of 2:1 to 1:2, and wherein 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 to 72 hours or 2 to 3 days, inclusive, optionally wherein the incubation is performed at a temperature of about 37°C; and (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 to 96 hours or about 3 to 4 days, inclusive, after the initiation of exposure to the stimulatory reagent; 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 x 10 6 or approximately 450 x 10 6 500 x 10 viable primary T cells 6 pieces or approximately 500 x 10 6 viable primary T cells, 550 x 10 6 pieces or approximately 550 x 10 6viable primary T cells, 600 x 10 6 or approximately 600 x 10 6 700 x 10 viable primary T cells 6 or approximately 700 x 10 6 viable primary T cells, 800 x 10 6 or approximately 800 x 10 6 viable primary T cells, 900 x 10 6 or approximately 900 x 10 6 viable primary T cells or 1,000 x 10 6 or approximately 1,000 x 10 6 In certain embodiments, the input composition is 600 x 10 viable primary T cells, or any value between any of the foregoing. 6 pieces or approximately 600 x 10 6 In certain embodiments, the number of cells in the input composition is up to 900 x 10 6 viable primary T cells or 900 x 10 6 or approximately 900 x 10 6 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 containing 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, wherein the enrichment selects for cells that are CD3+ in a closed system, thereby generating a selected population. In some cases, the enrichment is a first selection, and the cells and the first selection are further selected for cells that are CD3+ to further enrich the selection, thereby generating a second selected population. In some embodiments, the input composition comprises enriched CD3+ cells. In some embodiments, the input composition comprises enriched CD3+ cells from the first selection. In some embodiments, the input composition comprises 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 the cells with an antibody immobilized on or attached to an affinity chromatography matrix, wherein the antibody can specifically bind 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 an unselected population; and (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 unselected population, thereby producing a second selected population, the enrichment producing separate enriched populations of CD4+ T cells and CD8+ T cells, 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, 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, enriching 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, enriching 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 to enrich for CD4+ or CD8+ cells.

[0064] In certain embodiments, the 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, where the antibody specifically binds 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 contacting cells in the sample with the affinity chromatography matrix in the first and / or second selection, followed by eluting the antibody-bound cells, thereby recovering the selected cells from the matrix. In certain embodiments, enriching CD4+ or CD8+ T cells from a biological sample comprises (a) enriching CD4+ cells by positive selection based on surface expression of CD4; and (b) enriching CD8+ cells by positive selection based on surface expression of CD8. In some embodiments, both (a) and (b) are performed.

[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 recovered 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 biotin, a biotin analog, and a peptide capable of binding to the binding reagent, and the binding reagent is selected from streptavidin, a streptavidin analog or mutein, avidin, and an avidin analog or mutein. In some embodiments, the binding partner comprises the amino acid sequence set forth in SEQ ID NO: 64; and / or the binding reagent is a streptavidin mutein comprising the amino acid sequence set forth in SEQ ID NO: 75, 80, 76, or 63. In some embodiments, after contacting the cells in the sample with an affinity chromatography matrix, the method for 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 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 antibodies in the selection have a -3 ~10 -7 in the range of or 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, where enrichment comprises contacting the cells of the sample with a first immunoaffinity reagent that specifically binds to CD4 in an incubation composition and a second immunoaffinity reagent that specifically binds to CD8 in an incubation composition under conditions in which the immunoaffinity reagents specifically bind to CD4 and CD8 molecules on the surface of 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, wherein the input composition comprises the cells of the enriched composition comprising CD4+ cells and CD8+ cells. In some embodiments, each of the immunoaffinity reagents comprises an antibody or 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 prior to enrichment.

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

[0069] In certain embodiments, harvesting is carried out when integrated vector is detected in genome, but before achieving stable iVCN per diploid genome.In some embodiments, harvesting is carried out 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 carried out 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, harvesting is performed when the iVCN of the population of transformed cells is, on average, less than 1.5 copies or less than about 1.5 copies per diploid genome. In certain embodiments, harvesting is performed when the iVCN of the population of transformed cells is, on average, less than 1.0 copies or less than about 1.0 copies per diploid genome. In certain embodiments, harvesting is performed when the iVCN of the population of transformed cells is, on average, less than 0.75 copies or less than about 0.75 copies per diploid genome. In some embodiments, harvesting is performed when the iVCN of the population of transformed cells is, on average, less than 0.5 copies or less than about 0.5 copies per diploid genome. In certain embodiments, harvesting 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 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, 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, or greater than or greater than about 60% of the total CD4+ T cells in the population, or greater than or greater than about 60% of 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% 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 harvest, the percentage of naive-like T cells is greater than or greater than about 60% of all recombinant protein-expressing CD8+ T cells in the population, for example, greater than or greater than about 65%, 70%, 80%, 90%, or 95% of 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 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, 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 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, 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 CD8+ T cells in the population, e.g., greater than or greater than about 65%, 70%, 80%, 90%, or 95% of 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 pharmaceutically 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 capable of binding 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 specific embodiments, the target antigen is a tumor antigen. In certain 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 T cells, 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 the intracellular signaling domain, or a signaling portion thereof, of a CD3 chain, optionally the CD3-zeta (CD3ζ) chain. In some embodiments, the costimulatory signaling region comprises the 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 contains 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 a heterologous polynucleotide encoding a recombinant protein into cells, such as T cells. Any of the provided embodiments of the methods described herein can be carried out using non-viral transduction methods to introduce a heterologous polynucleotide encoding a recombinant protein into primary T cells for the production of cell therapy. In certain aspects, the non-viral method is a method that facilitates the integration of a polynucleotide encoding a recombinant receptor, or a portion thereof, into the genome of a cell. A variety of non-viral transduction methods can be used, including any of the non-viral transduction 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 expression level is 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 T cells that are surface positive for markers 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%, or of the total number of T cells in the composition or the total number of T cells expressing the 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 T cells that are surface positive for a marker expressed on central memory T cells.In some embodiments, at least 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. %, 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, at least 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 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are 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 do not 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 markers 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 markers expressed on naive-like T cells, include 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 + / CD8 + At least 50%, 60%, 70%, 80%, 85%, or 90% of the cells are naive-like T cells or are surface positive for markers 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 markers 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 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 markers 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 compositions or the total number of T cells expressing a recombinant protein in the compositions 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 T cells that are surface positive for markers expressed on naive-like T cells. In some embodiments, among a plurality of T cell compositions produced by the methods disclosed herein, on average, at least 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 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, among a plurality of T cell compositions produced by the methods disclosed herein, on average, at least 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. 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, among a plurality of T cell compositions produced by the methods disclosed herein, on average, at least 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. %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or 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 foregoing embodiments, in a plurality of T cell compositions produced by the methods disclosed herein, on average, 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 foregoing embodiments, in a plurality of T cell compositions produced by the methods disclosed herein, on average, 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 + / CD8 + At least 50%, 60%, 70%, 80%, or 90% of the cells are naive-like T cells or are surface positive for markers 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 the 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% of the total number of T cells in the composition or the total number of T cells expressing the recombinant protein in the composition. In some embodiments, 0%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total receptors in the composition are naive-like T cells or T cells that are surface positive for a marker expressed on naive-like T cells. + / CD8 + At least 60% of the cells are naive-like T cells or are surface positive for markers expressed on naive-like T cells and express all receptors in the composition. + / CD4 + In some embodiments, 30% of the cells are naive-like T cells or are surface positive for a marker expressed on naive-like T cells. + / CD8 + At least 70% of the cells are naive-like T cells or are surface positive for markers expressed on naive-like T cells and express all receptors in the composition. + / CD4 + In some embodiments, 40% of the cells are naive-like T cells or are surface positive for a marker expressed on naive-like T cells. + / CD8 + At least 70% of the cells are naive-like T cells or are surface positive for markers expressed on naive-like T cells and express all receptors in the composition. + / CD4 +In some embodiments, 50% of the cells are naive-like T cells or are surface positive for a marker expressed on naive-like T cells. + / CD8 + At least 70% of the cells are naive-like T cells or are surface positive for markers expressed on naive-like T cells and express all receptors in the composition. + / CD4 + In some embodiments, 60% of the cells are naive-like T cells or are surface positive for a marker expressed on naive-like T cells. + / CD8 + At least 70% of the cells are naive-like T cells or are surface positive for markers 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 + / 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 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%, or of the total number of T cells in the composition or the total number of T cells expressing the 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% of the total receptor markers 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. + / CD8 + At least 25% of the cells are central memory T cells or are surface positive for markers 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. In some embodiments, the total receptor in the composition + / CD8 + At least 65% of the cells are central memory T cells or are surface positive for markers 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. In some embodiments, the total receptor in the composition + / CD8 + At least 70% of the cells are central memory T cells or are surface positive for markers 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. In some embodiments, the total receptor in the composition + / CD8 + At least 70% of the cells are central memory T cells or are surface positive for markers 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 + / 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 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. In some embodiments, the total receptor markers in the composition are 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. In some embodiments, the total receptor markers in the composition are 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 markers 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 markers expressed on the naive-like T cells are selected from the group consisting of CD45RA, CD27, CD28, and CCR7. In some embodiments, the receptor + / CD4 + T cells or naive-like T cells, or receptors that are surface positive for markers expressed on naive-like T cells + / CD4 + The T cells are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+. + / CD4 + T cells or naive-like T cells, or receptors that are surface positive for markers expressed on naive-like T cells + / CD4 + The T cells are CD27+CCR7+. + / CD8 +T cells or naive-like T cells, or receptors that are surface positive for markers expressed on naive-like T cells + / CD8 + The T cells are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+. + / CD8 + T cells or naive-like T cells, or receptors that are surface positive for markers 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 + / 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 + / 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 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 CD4+ T cells and CD8+ T cells. In some embodiments, at least about 90% of the cells in the composition are CD4+ T cells and CD8+ T cells, and at least about 90% of the cells in the composition are CD4+ T cells and CD8+ T cells, and at least about 90% 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 about 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 ... 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 about 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 ... 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 about 50%, at least about 55%, at least about 60%, or at least about 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 98% of the cells in the composition are CD4+ T cells and CD8+ T cells. + / CD8 + At least or at least about 75% of the cells are CD27+CCR7+ and all receptors in the composition + / CD4 + At least about 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 ... in the composition 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 about 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 ... 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 about 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 ... 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 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 at least about 70% of the cells in the composition are CD27+CCR7+.+ / 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 ...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 at least + / 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 ...50% of the cells in the composition are CD27+CCR7+. + / 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 ...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 at least + / 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 ...70% of the cells in the composition are CD27+CCR7+. + / 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%, 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 x 10 4 ~50×10 6 pieces or approximately 1 x 10 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 is capable of binding to a target protein associated with, specific for, 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 mutant (EGFR), or EGFR-specific cytotoxic T cell proliferation (TIC)-associated leukemia (LEV). 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 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-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 The antigen is selected from among: ROR1-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 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 antibody fragment thereof, which in some embodiments is a single-chain fragment. In some embodiments, the fragment comprises antibody variable regions 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 the 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 the intracellular signaling domain of a T cell costimulatory molecule, or a signaling portion thereof. In some embodiments, the costimulatory signaling domain comprises the 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 pharmaceutically acceptable excipient. In certain embodiments, the composition further comprises a pharmaceutically 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, 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, the method 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 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 x 10 4 ~50×10 6 pieces or approximately 1 x 10 4 ~50×10 6 In certain embodiments, the dose of T cells comprises 50 x 10 (inclusive) T cells. 6 Fewer than 10 x 10 T cells 6 Fewer than 5 x 10 T cells 6 T cells, less than 1 x 10 6 Fewer than 0.5 x 10 T cells 6 Fewer than 1 x 10 T cells or 1 x 10 5 In certain embodiments, the dose of T cells comprises less than 20 x 10 6 pieces or approximately 20 x 10 6 In some embodiments, the dose of T cells comprises 10 x 10 6 pieces or approximately 10 x 10 6 Contains T cells.

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

[0115] In some embodiments, the T cells in 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 a 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). [Brief explanation of the drawings]

[0116] [Figure 1] Figures 1A and 1B show the results of a WST metabolic assay of T cells from three different donors incubated with anti-CD3 / anti-CD28 multimerized onto different batches of oligomeric reagent. Figure 1A summarizes the WST metabolic activity for all test batches (pools) compared to reference batches containing anti-CD3 / anti-CD28 multimerized onto oligomeric scaffolds with average hydrodynamic radii 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. [Figure 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 expanded and non-expanded expansion processes involving bead-based stimulation reagents (Bead), incubation in basal media (Bead-Basal Media), or oligomer stimulation reagents (Oligomer) to generate T cell compositions containing CAR+ T cells. Results are shown from samples collected during the stimulation (Stim), transduction (Trans), incubation (Inc), collection (Collect), cell formulation (Form), and cryopreservation (Cryo) steps. [Figure 3] Figure 3 displays the cytolytic activity of exemplary anti-BCMA CAR T cells assessed when cell compositions generated from the expansion process (Expanded) and the non-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 4A]Figures 4A-4D depict results from CAR+ T cell compositions generated from the expansion process and the non-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). Figure 4A depicts the percentage of CD4+ and CD8+ T cells positive for both CCR7 and CD27. Figure 4B depicts the percentage of CCR7+CD27+ cells relative to CD4+ CAR+ T cells. Figure 4C depicts the percentage of CCR7+CD27+ cells relative to CD8+ CAR+ T cells. Figure 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 day 1 activation (d1 AMAT), day 2 transduction (d2 XMAT), and various times after initiation of culture (d4 INOC+2, d6 INOC+4, d7 INOC+5). [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 5-1] Figures 5A-5F depict tumor burden in in vivo mouse xenograft tumor models after treatment with engineered CAR-T cell compositions generated by the expansion expansion process and the non-expansion expansion process with (a) bead-based stimulation reagents (Beads), (c) oligomer stimulation reagents (Oligomers), or (d) bead-based stimulation reagents incubated in basal media (Bead-Basal Media), compared with tumor-only or mock-transduced controls, respectively, over a 50-day period. Figure 5A depicts the mean tumor burden per group. Figures 5B-5F display 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 5-2] See description of Figure 5-1. [Figure 6-1] Figures 6A-6E depict representative bioluminescence images of tumor burden in an in vivo mouse xenograft tumor model at select days after treatment with a mock-transduced control (Figure 6A) or an engineered CAR-T cell composition generated by the Expanded (Figure 6B) and non-Expansion expansion processes with oligomer (Figure 6C), bead (Figure 6D), or bead-based stimulation in basal media (Bead-Basal Media) (Figure 6E), each over a 50-day period. [Figure 6-2] See description of Figure 6-1. [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, as well as the percentage of CAR+ T cells within the total T cell population, 33 days after treatment with engineered CAR-T cell compositions generated by the Expanded and Non-Expanded Expansion processes with (a) oligomer stimulation reagent (Oligomer), (b) bead-based stimulation reagent (Bead), or (c) bead-based stimulation reagent incubated in basal media (Bead-Basal Media). Cells were stained with reagents specific for truncated receptor surrogate markers and analyzed by flow cytometry. [Figure 8A] Figures 8A and 8B depict the percentage of viable total cells and total cell numbers 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 stimulator. [Figure 8B] See legend to Figure 8A. [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-culture with 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 throughout long-term stimulation. ND - not detectable. Figure 11B depicts the percentage of positively staining cells from samples collected on days 11 and 19 from 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 12A]Figures 12A-12C show results from an in vivo tumor mouse model in which mice were injected on day 0 with 1 x 10, 0.5 x 10, or 0.25 x 10 anti-CD19 CAR-T cells generated using the expanded or non-expanded expansion process, or a PBS control. Figure 12A depicts quantification of tumor presence, measured as the mean radiance of tumor-associated bioluminescence signals, and the percentage of tumor cells in blood samples collected at different time points during the study. Figure 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. Figure 12C depicts the percent survival of mice up to 60 days post-injection. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 13A] Figures 13A-13B depict engineered T cell (EC) compositions generated by the expansion process involving spinoculation and bead-based stimulation reagents under static incubation for 18-30 hours (Arm 1), as well as non-expansion processes involving spinoculation and oligomeric stimulation reagents incubated in basal medium for 96 hours (Arm 4), spinoculation and bead-based stimulation reagents incubated in basal medium for 72 hours (Arm 2), oligomeric stimulation reagents incubated in basal medium for 72 hours (Arm 5), and bead-based stimulation reagents incubated in basal medium for 48 hours (Arm 3), as well as unstimulated cryopreserved T-cell compositions (CMAT). Figure 13A depicts the percentage of CCR7+CD27+ cells among CD4+ CAR+ T cells. Figure 13B depicts the percentage of CD4+ CAR+ T cells and CD8+ CAR+ T cells. [Figure 13B] See legend to Figure 13A. [Figure 14]Figure 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 process involving spinoculation and bead-based stimulation reagents under static incubation for 18 to 30 hours (Arm 1), as well as a process using a mock empty vector (Arm 6, Mock), and non-expansion processes involving spinoculation and bead-based stimulation reagents incubated in basal medium for 72 hours (Arm 2), spinoculation and bead-based stimulation reagents incubated in basal medium for 48 hours (Arm 3), spinoculation and oligomeric stimulation reagents incubated in basal medium for 96 hours (Arm 4), and spinoculation and oligomeric stimulation reagents and incubation in basal medium for 72 hours (Arm 5). Cytolytic activity was assessed by microscopy based on the loss of red fluorescent signal and normalized to cell counts at the beginning of each culture every 4 hours over the 48, 72, and 96 hour periods. The dotted line indicates the EC50 for killing. [Figure 15]Figures 15A-15C depict the activity 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), and spinoculation and oligomeric stimulation reagent and incubation in basal medium for 72 hours (5) in a long-term stimulation assay involving continuous incubation with paramagnetic beads conjugated to recombinant BCMA Fc fusion protein for 6 days. 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 incubation days 1 through 6. Figure 15C shows the percent viable cells (% viability) across T cells produced from different engineering processes observed at different time points during the course of long-term stimulation. [Figure 16A] Figures 16A-16C depict cytokine profiles after long-term stimulation of the T cell compositions depicted in Figures 15A-15C with continuous incubation for 6 days with paramagnetic beads conjugated to recombinant BCMA Fc fusion protein. Figure 16A shows the percentage of CD4 / CAR+ and CD8 / CAR+ T cells that stained positive for the cytokines TNFα, IFN-γ, and IL-2 on day 0 of long-term stimulation. Figure 16B shows the percentage of CD4 / CAR+ and CD8 / CAR+ T cells that stained positive for the cytokines TNFα, IFN-γ, and IL-2 on day 6 of long-term stimulation. Figure 16C shows the polyfunctional score and IL-2 global score from cumulative cytokine levels as determined in CD8+ cells, normalized by adjusting for donor. [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. [Figure 17-1] 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 17-2] Figure 17B shows the relationship between the percentage of central memory / naive-like CD4+ T cells in the therapeutic output T cell composition (e.g., drug) and the number of population doublings that achieve the harvest criterion (Spearman ρ: -0.54; p-value: <0.001). Similar results were observed for CD8+ T cells in the therapeutic output T cell composition (e.g., drug). [Figure 17-3] Figure 17C shows the number of population doublings to reach the harvest criteria for high (0.35x10^6 cells / mL) and low (0.05x10^6 cells / mL) seeding densities during the expansion step of the production process. 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 shows the effect of treatment duration on the amount of TCM cells in the output T cell composition. [Figure 18A] Figures 18A-18D show Kaplan-Meier survival curves for subjects administered CAR+ T cell compositions, separated into groups that received compositions containing percentages 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 18B] See legend to Figure 18A. [Figure 18C] See legend to Figure 18A. [Figure 18D] See legend to Figure 18A. [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 enriched CD4+ input compositions and the number of population doublings required to reach harvest criteria (Spearman ρ: 0.43; p-value: <0.001). Similar results were observed for enriched CD8+ input compositions. [Figure 19A] 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 compositions generated from the expansion process and the non-expansion process with bead-based stimulation reagents (Beads) or oligomer stimulation reagents (Oligomers) (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 a mock-transduced T cell composition (mock). [Figure 19B] See legend to Figure 19A. [Figure 20A] 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) after 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 containing tumor cells only (tumor without TX) and tumor cells with a mock-transduced T cell composition (Mock). [Figure 20B] See legend to Figure 20A. [Figure 20C] See legend to Figure 20A. [Figure 20D] See legend to Figure 20A. [Figure 21] Figure 21 shows copy numbers assessed by droplet digital PCR (ddPCR) before isolating high-molecular-weight DNA fractions (pregel; standard vector copy number (VCN) assay) or after isolating those above thresholds of 15 kb, 17.5 kb, or 20 kb by pulsed-field gel electrophoresis (PFGE) (integrated vector copy number (VCN) assay). Copy numbers were assessed using primers specifically amplifying a portion of the integrated transgene sequence; a packaging plasmid (a viral packaging plasmid encoding the vesicular stomatitis Indiana virus G protein (VSVg)); or a genomic control (a gene encoding the ribonuclease P protein subunit p30 (RRP30)). Assessment was performed on samples from transduced cells or on 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]Figures 22A-22B depict the copy number, 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 number (in high molecular weight DNA samples after PFGE) of the transgene sequence at various time points (before transduction ("Pre"), 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 (Figure 22A) or primary T cells isolated from human subjects (Figure 22B) transduced with a lentiviral preparation containing a transgene sequence encoding a CAR. Copy numbers for each gene were normalized to the number of diploid genomes (cp / diploid genome; using primers specific for the albumin gene as a reference). [Figure 23A] Figure 23A shows the integrated copy number assessed on days 3, 4, or 5 of an exemplary non-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 oligomer-streptavidin mutein reagent at a concentration of 4.0 μg per 10 cells, or (3) anti-CD3 / anti-CD28 Fab-conjugated oligomer-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] 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 24A]Figures 24A-24E depict the copy number per diploid genome (Figure 24A), the fraction of integrated transgene (Figure 4B), the fraction of non-integrated transgene (Figure 24C), the non-integrated transgene copy number per diploid genome (Figure 24D), and the 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. [Figure 24B] See legend to Figure 24A. [Figure 24C] See legend to Figure 24A. [Figure 24D] See legend to Figure 24A. [Figure 24E] See legend to Figure 24A. [Figure 25] Figure 25 depicts the copy number per diploid genome, unintegrated transgene copy number, unintegrated transgene fraction, and integrated transgene fraction 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 a chimeric antigen receptor (CAR). The time points assessed include days 0 through 8 of the expansion process, including at the time of thawing (TMAT; day 0), activation (AMAT; day 1), transduction (XMAT; day 2), or various times after the initiation of culture (inoc+1 through inoc+6; representing days 3 through 8 of the process). [Figure 26] Figure 26 shows the clonality of T cells (applied Shannon index) of the isolated CD4+ and CD8+ T cell composition (CMAT) before manipulation and the CD4+ and CD8+ therapeutic CAR+ T cell composition after manipulation. [Figure 27]Figure 27 shows the phenotypic differences of generated cell compositions in the presence of different concentrations of anti-CD3 / anti-CD28 Fab conjugate oligomeric streptavidin mutein reagent and / or 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 reagent and media composition. [Figure 28] Figure 28 shows the percentage of CD4+ CAR+ T cells and CD8+ CAR+ T cells positive for both CCR7 and CD27 staining in engineered compositions of primary T cells from three different donors using the non-expansion and expansion processes as controls. [Figure 29] Figure 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 non-expansion and expansion processes in contrast. [Figure 30] Figures 30A-30D show exemplary effects of incubating T cells with anti-CD3 / anti-CD28 oligomeric stimulatory reagents in the presence or absence of Compound 63 on mTor signaling and viability and growth kinetics. Figure 30A shows pS6 expression in live CD8+ T cells by the memory subset. 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 numbers, respectively, over time (indicated by days; e.g., d1) in culture after the start of stimulation ("input"). [Figure 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 stimulation reagents in the presence or absence of compound 63. Figure 31A shows intracellular expression of caspases upon thawing. Figures 31B-31D show the phenotypic profiles of CD8 and CD4 CAR-T cells, respectively, by subset expression of CD27 and / or CCR7. Figures 31C-31E show intracellular IL-2, IFNγ, or TNF (left panels) or a combination of IL-2 and / or IFNγ or TNF (right panels) in CD8 and CD4 CAR-T cells stimulated with antigen-bearing targets, respectively. Figure 31F shows expansion and survival over 12 days (left panel) and total expansion metrics calculated by area under the curve (right panel) for CAR-T cells stimulated with anti-CAR beads. [Figure 32-1] 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 processes using cryopreserved apheresis (CrAPH) or cryopreserved T cell selected material (CMAT) collected from three donors. D5_HRPO, D5_WPRO, and D5_FPRO refer to the day 5 harvested cell product, day 5 washed cell product, and day 5 formulated cell product (e.g., formulated drug product), respectively. [Figure 32-2]Figures 32D-32F show exemplary average percentages of cell phenotypes at the time of collection (day 4 or 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), total nucleated CAR+ cells in the collected product (HRPO) of 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). [Figure 32-3] See description of Figure 32-2. [Figure 32-4] See description of Figure 32-2. [Figure 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. [Figure 34A] 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 among live CD45+ cells (left panel), and the percentage of CD8+CAR+ and CD4+CAR+ cells among CD45+ cells (right panel). Figure 34B shows the ratios of CD4+CAR+ cells:CD8+CAR+ cells and CD4+ cells:CD8+ cells. [Figure 34B] See legend to Figure 34A. [Figure 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). [Figure 36A] Figures 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 36B] See legend to Figure 36A. [Figure 37A] 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 37B] See legend to Figure 37A. [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 or 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 an anti-ID antibody. Figure 39 shows the total viable cell count and AUC for cells engineered to express an anti-BCMA CAR. [Figure 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. [Figure 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 engineering 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 after treatment with higher dose (top panel) and lower dose (bottom panel) anti-CD19 CAR-T cell compositions generated from donor-matched non-expansion and expansion engineering 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 (top panel) and lower (bottom 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. [Figure 44A] Figures 44A-44D show total viable 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. [Figure 44B] See legend to Figure 44A. [Figure 44C] See legend to Figure 44A. [Figure 44D] See legend to Figure 44A. [Figure 45-1] 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 a CAR using an expansion process (○) or a non-expansion process (●). [Figure 45-2] Figures 45B-45C show the relationship between copy number per cell in the cell composition as assessed by standard VCN (Figure 45B) or iVCN (Figure 45C) and surface expression of the CAR as indicated by the percentage of CAR-expressing CD3+ cells among viable CD45+ cells (% CD3+CAR+) as assessed by flow cytometry. DETAILED DESCRIPTION OF THE INVENTION

[0117] Detailed Description Provided herein are methods for generating or producing a composition of engineered cells, e.g., engineered T cells, that express 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 period, such as within four days or within several days, after the start of stimulation of the cells in the input composition.

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

[0119] In certain embodiments, the provided methods are used in conjunction with processes for efficiently producing or generating engineered cells suitable for use in cell therapy. In some embodiments, the provided processes for generating engineered cells include 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, e.g., without an expansion unit operation and / or without including a step intended to cause cell expansion. 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 cell expansion.

[0120] In certain aspects, the duration of the provided processes can be measured from when the cells, e.g., T cells, of the input cell population or input composition are first contacted or exposed to stimulatory conditions (e.g., as described herein, such as in Section IB), which initial contact or exposure 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 period required to harvest or collect the output population containing engineered cells (also referred to herein as the output composition or 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 day. 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 in processes requiring longer times. 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 a composition of engineered T cells within a reduced time from collection of the biological sample 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 collection of the biological sample from a subject until the engineered T cells are collected, harvested, or formulated (e.g., for cryopreservation or administration), including any or all times the biological sample or enriched, isolated, or selected cells are cryopreserved and stored.

[0122] In certain embodiments, output compositions or populations of T cells containing engineered T cells, e.g., T cells expressing a recombinant receptor such as a chimeric antigen receptor, produced or generated by the provided processes are particularly effective or potent when utilized as cells for cell therapy. For example, in some aspects, output compositions containing engineered T cells, e.g., CAR+ T cells, produced from the provided processes have significantly greater potency and / or proliferative capacity than engineered T cells produced or generated by alternative, existing processes. In some aspects, 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, are not expected to achieve high levels of potency or efficacy in some aspects. Existing methods of manufacturing T cells for cell therapy include processes for expanding cells, or require expansion of cells, e.g., to a threshold density, e.g., processes that include an expansion unit operation and / or include steps intended to cause cell expansion. Certain embodiments contemplate that expansion is expected to be necessary to result in sufficient amounts of vector incorporation 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 integration 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 expansion unit operations and / or that include steps intended to cause expansion of the cells). Certain embodiments contemplate that vector integration can 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 an oligomeric stimulatory reagent. Existing reagents for use in stimulating T cells in vitro, such as in the absence or presence of small amounts of exogenous growth factors, are known (see, e.g., U.S. Patent No. 6,352,694 B1 and European Patent No. EP 0 700 430 B1). Generally, such reagents utilize beads with a diameter of more than 1 μm, e.g., magnetic beads, to which various binding agents (e.g., anti-CD3 antibodies 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, because it is necessary to ensure that these magnetic beads are completely removed before administering expanded T cells to a subject. In some aspects, such removal, such as by exposing the cells to a magnetic field, can reduce the yield of viable cells available for cell therapy. In certain cases, such reagents, for example, stimulating reagents containing magnetic beads, should 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 processes utilizing oligomeric stimulatory reagents overcome such potential limitations. For example, in some embodiments, the provided processes avoid or reduce the risk of residual stimulatory reagents, e.g., reagents 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 that require additional measures to ensure that the final engineered T cell population is bead-free. In some embodiments, this can be easily accomplished in this embodiment by adding a substance, e.g., a competing reagent, that dissociates the oligomeric stimulatory 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 stimulatory reagent from the cells, e.g., by adding a substance or competing reagent, results in little or no cell loss compared to removal or separation of bead-based stimulatory reagents. In some aspects, the timing of removal or separation of the oligomeric stimulatory reagent is not limited at all, or is less limited than removal or separation of the bead-based stimulatory 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 processes, cells are transduced or subjected to transduction in a process involving incubation in the presence of a basal medium, for example, incubation of T cells into which a viral vector encoding a heterologous or recombinant protein (e.g., a CAR) has been introduced. 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 a basal medium reduces differentiation that may occur during the manipulation process, compared to incubation in, for example, a supplemented medium, a complete medium, a medium containing serum, or a medium containing a serum substitute. 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, including a population of cells that contain CAR+ T cells.

[0127] Among other findings provided herein is the observation that weak or reduced stimulation, such as reduced amounts of stimulating reagents typically utilized to stimulate T cells, can produce engineered CAR+ T cells that are as potent, durable, or effective as CAR+ T cells produced by processes involving stronger stimulation conditions or greater amounts or concentrations of stimulating reagents. For example, in some embodiments, stimulating cells with a lower or relatively lower amount of oligomeric stimulating reagent can increase the potency, efficacy, or persistence of the resulting engineered cell population compared to processes using higher amounts of oligomeric stimulating reagents. Such embodiments contemplate that such effects can persist even at doses low enough to reduce expression of activation markers or the fraction 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 culturing, which can 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 cell expansion allows for tightly controlled overall process duration. In certain embodiments, variability in process duration is reduced or eliminated, which, in some aspects, may allow for 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 were individually incorporated by reference. To the extent that a definition set forth herein conflicts or is otherwise inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition 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 output populations of engineered cells, such as engineered CD4+ T and CD8+ T cells, that express a recombinant protein, e.g., a recombinant receptor such as a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In certain embodiments, the methods provided herein are used in connection with the manufacture, generation, or production of cell therapies and may be used in connection with additional processing steps, such as steps for isolating, separating, selecting, activating or stimulating, transducing, washing, suspending, diluting, concentrating, and / or formulating the cells. In some embodiments, methods of generating or producing engineered cells, e.g., engineered T cells, include 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, performed in order: first isolating (e.g., selecting or separating) input cells, e.g., primary CD3+ T cells, CD4+ T cells, and / or CD8+ T cells, from a biological sample; incubating under stimulatory conditions and genetically engineering the cells to introduce a recombinant polynucleotide encoding a recombinant receptor, e.g., by transduction or transfection; and then collecting, harvesting, or loading the output population into a container, e.g., a bag or vial. 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 provided methods are used in connection with generating 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 in CD3+ T cells from a starting sample, such as PBMCs or a 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 being 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 connection with one or more of the following steps: activating or stimulating cells, e.g., cells of an input population; genetically engineering the activated or stimulated cells, e.g., by transduction or transfection, to introduce a polynucleotide encoding a recombinant protein.In certain embodiments, the methods may also be used in connection 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 provided methods may be used in connection 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 process, stage, or condition that results in cell expansion. In certain embodiments, cells of an input population, e.g., an enriched starting population of 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 subjected to a provided method to generate a population of engineered cells but are not expanded. In certain embodiments, the cells of an output population, e.g., an output population of cells engineered by a provided process, are the same, approximately the same, reduced, or diminished compared to the amount of cells in the input population, e.g., at the start 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 culturing, 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 for tightly controllable duration of the entire process.

[0134] In some embodiments, the methods are used in conjunction with one or more steps for stimulating 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, e.g., a stimulatory reagent described herein, such as in Section IB-1. In certain embodiments, a set or fixed amount of cells is stimulated, e.g., at a set or fixed concentration. In certain embodiments, the stimulating step, e.g., culturing the cells under stimulatory conditions, is carried out for a set or fixed time, such as less than 2 days, or for a time between 18 and 30 hours. In some aspects, the stimulating step with the stimulatory reagent is carried out for 20 hours ± 4 hours or for 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 integration of a heterologous or recombinant polynucleotide encoding a recombinant protein or to allow expression of the recombinant protein. In certain embodiments, the cells are incubated for a set or fixed time, e.g., more than 18 hours or less than 4 days, e.g., 72 hours ± 6 hours. In any of the provided embodiments, the introducing can be performed on the cells after they have been stimulated with a stimulating reagent. In some embodiments, the manipulation step is initiated or initiated within a certain time from when the stimulating step is initiated 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 initiated between 18 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 performed 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 proteins. In certain embodiments, the serum-free medium may contain serum albumin, hydrolysates, growth factors, hormones, carrier proteins, and / or attachment factors.

[0137] In some embodiments, the provided methods are performed such that one, more, or all steps in preparing 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 more 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 collected, 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 10 days, or for a period of 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 including stimulating the cells and then introducing a polynucleotide encoding a recombinant receptor (e.g., a CAR) into the cells. In certain embodiments, stimulating is carried out for 18 to 30 hours, e.g., about 24 hours, and introduction of the polynucleotide is then carried out. In certain embodiments, the cells are harvested or collected within three days after introduction of the polynucleotide has begun, e.g., for formulation for cryopreservation or administration to a subject. In various embodiments, the cells are harvested or collected within four days after incubation under stimulating conditions has begun, e.g., for formulation for cryopreservation or administration 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 harvested from a population of T cells (such as an enriched population of T cells) while the population remains in a closed system, e.g., during isolation, incubation, manipulation, culture, and / or formulation. In certain embodiments, such cells may be analyzed for a marker, characteristic, or property, including but not limited to, viability, apoptosis, activation, stimulation, growth, and / or depletion. 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, analysis of sampled or collected cells is automated. In certain embodiments, 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 particular aspects but otherwise include similar or identical features, aspects, steps, stages, reagents, or conditions of the provided method or aspect being compared to the exemplary or alternative process. For example, an output population of engineered cells, e.g., non-expanded cells, produced by the provided methods may be compared to cells produced by a process involving 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 provided methods and alternative processes 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 with a particular disease or condition, in need of cell therapy, or to whom cell therapy will be administered. In some aspects, the subject is a human, e.g., a patient in need of a particular therapeutic intervention, such as adoptive cell therapy, for which cells are isolated, processed, and / or manipulated. Thus, the cells, in some embodiments, are 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, as well as processed samples derived therefrom.

[0144] In some aspects, the sample is a blood or blood-derived sample, or derived from an apheresis or leukapheresis product. 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, tonsil, or other organ, 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 wash solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, the wash step is accomplished by a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the wash step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are washed after washing, e.g., with a solution containing calcium. ++ / Mg ++ The cells are resuspended in a wide variety of biocompatible buffers, such as PBS, which does not contain 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, the 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 isolating, selecting, activating, stimulating, manipulating, transducing, transfecting, incubating, culturing, harvesting, formulating a population of cells, and / or administering 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 for ensuring adequate 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 to exploit 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 enable 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 or is about 2 L, 4 L, 6 L, 8 L, 10 L, 12 L, 14 L, 16 L, 18 L, or 20 L, or any value between any of the foregoing. In some embodiments, the volume of collected autologous plasma is or is about 10 mL, 50 mL, 100 mL, 150 mL, 200 mL, 250 mL, or 300 mL or more, or any value between any of the foregoing. In some embodiments, the leukapheresis product is subjected to procedures, e.g., 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, T cells selected from a thawed cryopreserved and / or cryoprotected apheresis product or leukapheresis product are not again cryopreserved and / or cryoprotected 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 frozen in a cryopreservation solution or buffer containing 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 (e.g., bags) that can each be used individually or in combination in processing the product. For example, when 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 15-30 x 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 between 15-30 x 10. 9 When the total number of cells is eight, the cryopreserved and / or cryoprotected apheresis product or leukapheresis product is divided into eight cryopreservation containers (e.g., bags).

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

[0154] In some embodiments, the sample (e.g., an apheresis or leukapheresis sample) is collected after the donor has been diagnosed with a disease or condition, either before cell selection or without prior cell selection (e.g., without prior T cell selection, such as chromatographic selection), and cryopreserved and / or cryoprotected. 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 therapy or any therapy designated for treatment of the disease or condition, or any therapy other than radiation and / or chemotherapy. In some embodiments, the sample is collected after the first recurrence of the disease after initial treatment for the disease and before the donor or subject receives 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 later require treatment.

[0155] In some embodiments, the sample (e.g., an apheresis or leukapheresis sample) is collected, cryopreserved, and / or cryoprotected, either before or without prior cell selection (e.g., without prior T cell selection, such as by chromatographic selection), at a time point after second-line treatment for the disease and after a second recurrence of the disease before the donor or subject receives subsequent treatment for the disease. In some embodiments, the patient is 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., an IPI (International Prognostic Index) > 2).

[0156] In some embodiments, a sample (e.g., an apheresis or leukapheresis sample) is collected and 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 never be considered at risk of developing or being diagnosed with any disease, but may choose to bank or preserve the cells in case cell therapy is needed at a later stage in life. 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 equal to 12, 24, 36, or 48 hours, or more than 0.5, 1, 1.5, or 2 days, or a period equal to 0.5, 1, 1.5, or 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 equal to 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 a period 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 collected from donors 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 by, for example, 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 before cryogenic storage of the sample. In some embodiments, processing is performed after thawing the sample following cryogenic storage.

[0159] By allowing a donor, and thus their cells, to store their cells at a stage when they have not received extensive treatment for a disease and / or before the onset or diagnosis of a disease or condition, such cells may have certain advantages for use in cell therapy compared to cells collected after one or more treatments. For example, cells collected before one or more treatments may be healthier, may exhibit higher levels of specific cellular activity, may proliferate more rapidly, and / or may be more amenable to genetic manipulation than cells that have undergone several treatments. Another example of an advantage of 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 if a recipient later needs them. This increases the capacity of apheresis laboratories and provides technicians with greater 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 sample before a patient requires cell therapy, then cryopreserving the apheresis sample for later use in a process for engineering cells (e.g., T cells) with a recombinant receptor (e.g., a 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 a formulated cell population to the subject. In such examples, the cryopreserved apheresis sample is thawed before 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 to manufacture 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 involves one or more preparative 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, or lyse or remove cells sensitive to a particular reagent. In some examples, cells are separated based on one or more characteristics, such as density, adhesion properties, size, sensitivity, and / or resistance to a particular component. In some embodiments, the method involves preparing white blood cells from peripheral blood by lysing red blood cells and a density-based cell separation method, such as centrifugation through a Percoll or Ficoll gradient. In certain embodiments, methods, techniques, and reagents for selection, isolation, and enrichment are described, for example, in WO2013124474 and WO2015164675, which are incorporated herein by reference in their entireties.

[0163] In some embodiments, a 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 optionally remove or reduce unwanted or undesirable components. In some cases, the dilution and / or washing removes or reduces the presence of cryoprotectants, such as DMSO, contained in the thawed sample, which can otherwise negatively affect cell viability, yield, and recovery rate upon prolonged exposure to room temperature. In some embodiments, the dilution and / or washing allows for medium exchange of the thawed cryopreserved product with serum-free medium, such as that described in Section II of this specification or that 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), e.g., the dipeptide in Glutamax™ (ThermoFisher). In some embodiments, the serum-free medium further comprises one or more recombinant cytokines, e.g., recombinant human IL-2, recombinant human IL-7, and / or recombinant human IL-15.

[0165] In some embodiments, at least a portion of the selection step involves incubation of cells with a selection reagent. The incubation with one or more selection reagents may be performed as part of a selection method using one or more selection reagents to select one or more different cell types based on the intracellular or cellular expression or presence of one or more specific molecules, 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 may be used. In some embodiments, the selection reagent(s) results in a separation based on affinity or immunoaffinity. For example, in some aspects, the selection involves 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, typically followed by a washing step and separating cells bound to the antibody or binding partner from cells not bound to the antibody or binding partner.

[0166] In some aspects of such processes, a volume of cells is mixed with a certain amount 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 on 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., bead) ratio to promote energetically favorable interactions. In other cases, the method involves cell selection, where all or part of the selection is performed within the internal cavity of a centrifuge chamber, e.g., under centrifugal rotation. In some embodiments, incubation of the cells with the selection reagent, e.g., an immunoaffinity-based selection reagent, is performed within the centrifuge chamber. In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus described in International Patent Application Publication No. WO2009 / 072003 or US20110003380A1, which are incorporated by reference in their entirety. In one example, the system is the system described in International Publication No. WO2016 / 073602, which is incorporated by reference in its entirety.

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

[0168] In some embodiments, at least a portion of the selection step, including incubation of cells with selection reagents, is performed in a centrifuge chamber. In some aspects of such processes, a volume of cells is mixed with a selection reagent based on the desired affinity in an amount that is significantly less than the amount typically 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 according to the manufacturer's instructions for the same number and / or volume of cells.

[0169] In some embodiments, for selection, e.g., based on the immunoaffinity of the cells, the 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 less 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 the amount of selection reagent that 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 selection reagent are premixed before addition to the cells. In some embodiments, the selection buffer and selection reagent are added to the cells separately. 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 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 80 g, at least 1000 rpm, or at least 1500 rpm or at least 1700 rpm), e.g., 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 a chamber or other container. In some embodiments, spinning is performed using repeated intermittents of spinning at such low speeds followed by rest periods, e.g., 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, or 10 seconds of spin and / or rest, e.g., about 1 second or 2 seconds of spin followed by about 5 seconds, about 6 seconds, about 7 seconds, or about 8 seconds of rest.

[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, push, and centrifuge cells, reagents, and other components into the chamber 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(s), the incubated cells are separated to select cells based on the presence or absence of a particular reagent(s). In some embodiments, the separation is performed in the same closed system in which the cells were incubated with the selection reagent. In some embodiments, after incubation with the selection reagent, the incubated cells, including the cells to which the selection reagent has bound, 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, in which cells that bind to a reagent, such as an antibody or binding partner, are retained for subsequent use, and / or negative selection, in which cells that do not bind to 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 (e.g., a marker) expressed in the positively or negatively selected cells, respectively. high This is achieved 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 nuclei of the cells.

[0176] Separation does not necessarily result in the enrichment or elimination 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, may indicate an increase in the number or proportion of such cells, but need not result in the complete absence of cells that do not express the marker. Similarly, negative selection, elimination, or depletion of a particular type of cell, such as cells expressing a marker, may indicate a decrease in the number or proportion of such cells, but need not result in the complete elimination 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 that simultaneously express multiple markers, 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 simultaneously positively selected 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 the 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 to 10, 1 to 5, or 3 to 5 times.

[0178] For example, in some aspects, specific subpopulations of T cells, e.g., cells that are positive for or have 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 for markers 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 in 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 in 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 PBMC samples by negative selection for markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, e.g., 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 on or relatively highly expressed in 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, combining TCM-enriched CD8+ T cells with CD4+ T cells further enhances efficacy.

[0182] In embodiments, memory T cells are present in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted for the CD62L-CD8+ and / or CD62L+CD8+ fractions, 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 that express or highly express 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, and 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 based on CD4 expression, which are subjected to negative selection based on CD14 and CD45RA expression and positive selection based on 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 a 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, methods for selecting cells may include those described in published U.S. Patent Application No. 20170037369, the entire contents of which are incorporated herein by reference. 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, e.g., a sample of PBMCs or other white blood cells, is subjected to selection of CD4+ T cells, which 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 selection of CD8+ T cells, which 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 PBMCs or a 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 PBMCs or a 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, which is used to generate a population enriched in CD8+ T cells. In some embodiments, cells from the enriched population in CD4+ T cells and cells from the enriched population in CD8+ T cells are mixed, combined, and / or pooled to generate an input population containing CD8+ T cells and CD4+ T cells. In certain embodiments, the enriched population in CD4+ T cells and the enriched population in CD8+ T cells 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, retaining both negative and positive fractions. 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 the positive and negative selections 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 bearing 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 for cell separation for positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) 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 containing a small magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, e.g., paramagnetic beads (e.g., Dynalbeads or MACS® beads). The magnetically responsive material, e.g., particles, are 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 is desired to be separated, e.g., negatively or positively selected.

[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 and European Patent Specification EP 452342 B, which are incorporated herein by reference. 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, 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, magnetic particles are attached to cells via coating with a primary antibody specific for one or more markers. In certain embodiments, cells, rather than 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, streptavidin-coated magnetic particles are used in conjunction with a biotinylated primary antibody or biotinylated secondary antibody.

[0195] In some aspects, separation is achieved by placing a sample in a magnetic field, and cells with attached magnetically responsive or magnetizable particles are attracted to a magnet to separate them 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 magnetically activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetically 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 in which non-target and target species are sequentially eluted after application of an external magnetic field. That is, cells attached to magnetized particles are held in place while unattached species are eluted. Then, after this initial elution step is complete, species trapped in the magnetic field and prevented from elution are somehow released so that they can be eluted and recovered. 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 the concentration used or required to achieve an optimal or maximal 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 (e.g., "yield" is the number of cells so recovered or selected compared to the total number of cells in the incubation that are targeted by, or for which the reagent is specific, or that bear a marker 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 total, e.g., 70% or less, of bound cells, e.g., CD4+ T cells and / or CD8+ T cells, upon recovery of the cells bound to the reagent. In some embodiments, a suboptimal concentration of 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 can 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 of two or more selection reagents with 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 reagents relative to cell types recognized by the other. For example, a reagent that specifically binds to a marker whose ratio it is desired to bias 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 relative to the other, depending on the degree to which it is desired to increase the ratio.

[0199] In some embodiments, when operating in a suboptimal range and / or with enough 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 resulting population containing enriched or selected CD4+ T cells and CD8+ T cells, can be determined as a matter of routine.

[0200] In some embodiments, the separation and / or isolation step is performed using magnetic beads to which the immunoaffinity reagent is reversibly bound, for example, via a peptide ligand interaction with a streptavidin mutein, as described in WO 2015 / 164675 (incorporated herein by reference in its entirety). An example of such a magnetic bead is Streptamers®. In some embodiments, the separation and / or isolation step is 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 antibodies are 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 selections are 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 enrich 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 starting 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 starting 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 a patient. In some embodiments, the magnetizable particles or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competing unlabeled antibodies, magnetizable particles, or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0203] In some embodiments, the isolation and / or selection results in one or more populations of enriched T cells, e.g., CD3+ T cells, CD4+ T cells, and / or CD8+ T cells. In some embodiments, two or more distinct populations of enriched T cells are isolated, selected, enriched, or obtained from a single biological sample. In some embodiments, the distinct populations are isolated, selected, enriched, and / or obtained from separate biological samples collected, harvested, and / or obtained from the same subject.

[0204] In certain embodiments, the isolation and / or selection results in one or more populations of enriched T cells that comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD3+ T cells. In certain embodiments, the enriched population of T cells consists essentially of CD3+ T cells.

[0205] In certain embodiments, the isolation and / or enrichment results in an enriched population of CD4+ T cells that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD4+ T cells. In certain embodiments, the input population of CD4+ T cells comprises less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and / or does not contain CD8+ T cells and / or is free or substantially free of CD8+ T cells. In some embodiments, the enriched population of T cells consists essentially of CD4+ T cells.

[0206] In certain embodiments, the isolation and / or enrichment results in an enriched population of CD8+ T cells that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD8+ T cells. In certain embodiments, the population of CD8+ T cells contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and / or no CD4+ T cells and / or is free or substantially free of CD4+ T cells. In some embodiments, the enriched population of T cells consists essentially of CD8+ T cells.

[0207] In some embodiments, one or more populations of enriched T cells are frozen, e.g., cryopreserved and / or cryoprotected, after isolation, selection, and / or enrichment. In certain embodiments, an enriched population of CD3+ T cells is frozen, e.g., cryopreserved and / or cryoprotected, after isolation, selection, and / or enrichment. In certain embodiments, an enriched population of CD4+ T cells is frozen, e.g., cryopreserved and / or cryoprotected, after isolation, selection, and / or enrichment. In certain embodiments, an enriched population of CD8+ T cells is frozen, e.g., cryopreserved and / or cryoprotected, after isolation, selection, and / or enrichment. In some embodiments, one or more populations of enriched T cells are frozen, e.g., cryopreserved and / or cryoprotected, before any step of incubating, activating, stimulating, manipulating, transducing, transfecting, culturing, expanding, harvesting, and / or formulating the population of cells. In certain embodiments, the enriched population of CD3+ T cells is frozen, e.g., cryopreserved and / or cryoprotected, prior to any step of incubation, activation, stimulation, manipulation, transduction, transfection, culture, expansion, harvesting, and / or formulation of the population of cells. In certain embodiments, the enriched population of CD4+ T cells is frozen, e.g., cryopreserved and / or cryoprotected, prior to any step of incubation, activation, stimulation, manipulation, transduction, transfection, culture, expansion, harvesting, and / or formulation of the population of cells. In some embodiments, the enriched population of CD8+ T cells is frozen, e.g., cryopreserved and / or cryoprotected, prior to any step of incubation, activation, stimulation, manipulation, transduction, transfection, culture, expansion, harvesting, and / or formulation of the population of cells. In some embodiments, one or more populations of enriched T cells, e.g., an enriched population of CD3+ T cells, are frozen, e.g., cryopreserved and / or cryoprotected, prior to any step of activating, stimulating, manipulating, transducing, transfecting, incubating, culturing, harvesting, and / or formulating the population of cells.In some embodiments, one or more populations of enriched T cells, e.g., a population of enriched CD4+ T cells and / or enriched CD8+ cells, are frozen, e.g., cryopreserved and / or cryoprotected, prior to any step of activating, stimulating, manipulating, transducing, transfecting, incubating, culturing, harvesting, and / or formulating the population of cells. In certain embodiments, one or more cryoprotected input populations are stored, e.g., at or about -80°C, for between 12 hours and 7 days, between 24 hours and 120 hours, or between 2 days and 5 days. In certain embodiments, one or more cryoprotected input populations are stored at or about -80°C for less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, or less than 5 days, less than 4 days, less than 3 days, less than 2 days, or less than 1 day. In some embodiments, one or more cryoprotected input populations are stored at or about -70°C or at or about -80°C for less than 3 days, e.g., about 2 days.

[0208] a. Selection agent or reagent In certain aspects, the methods provided herein utilize a selection agent. In some embodiments, the agent is a selection reagent. In some embodiments, the selection agent binds to a molecule on the surface of a cell, e.g., a cell surface molecule. In some cases, the cell surface molecule is a selection marker. In some embodiments, the selection agent is capable of specifically binding to a selection marker expressed by one or more of the cells in the sample.

[0209] In some embodiments, cells, e.g., target cells (e.g., T cells), have or express a molecule, e.g., a selection marker, on their cell surface, such that the cells to be selected are defined by the presence of at least one common specific molecule. In some embodiments, a sample containing target cells may also contain additional cells that lack the molecule (e.g., the selection marker). For example, in some embodiments, T cells may be selected from a sample containing multiple cell types, e.g., red blood cells or B cells.

[0210] In some aspects, the cell surface molecule, e.g., a selection marker, can be an antigen that defines a desired cell population or subpopulation, e.g., a blood cell, e.g., a lymphocyte (e.g., T cell, T helper cell, e.g., CD3+ T cell, CD8+ T cell, CD4+ T helper cell, B cell, or natural killer cell), monocyte, or stem cell, e.g., a population or subpopulation of CD34-positive peripheral stem cells or Nanog- or Oct-4-expressing stem cells. In some embodiments, the selection marker can be a marker expressed on the surface of T cells or a subset of T cells, e.g., CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA, and / or CD45RO. Examples of T cells include cells such as CMV-specific CD8+ T-lymphocytes, cytotoxic T cells, memory T cells, and regulatory T cells (Tregs). Examples of Tregs include CD4 CD25 CD45RA Treg cells, and examples of memory T cells include CD62L CD8+ specific central memory T cells.

[0211] In some embodiments, the selection marker can be CD4, and the selection agent specifically binds to CD4. In some aspects, the selection agent that specifically binds to CD4 can be selected from the group consisting of an anti-CD4 antibody, a bivalent antibody fragment of an anti-CD4 antibody, a monovalent antibody fragment of an anti-CD4 antibody, and a proteinaceous CD4-binding molecule with antibody-like binding properties. In some embodiments, the anti-CD4 antibody, e.g., a bivalent antibody fragment or a monovalent antibody fragment (e.g., a CD4 Fab fragment), can be derived from antibody 13B8.2 or a functionally active variant of 13B8.2 that retains specific binding to CD4. For example, exemplary variants of antibody 13B8.2 or m13B8.2 are described in U.S. Patent No. 7,482,000, U.S. Patent Application No. US2014 / 0295458, or International Patent Application No. WO2013 / 124474; and Bes, C, et al. J Biol Chem 278, 14265-14273 (2003). The mutant Fab fragment, designated "ml3B8.2," possesses the variable domain of the CD4-binding murine antibody 13B8.2 and a constant domain containing a gamma-type constant human CH1 domain for the heavy chain and a kappa-type constant human light chain domain, as described in U.S. Patent No. 7,482,000. In some embodiments, the anti-CD4 antibody, e.g., a variant of antibody 13B8.2, contains the amino acid substitution H91A in the variable light chain, the amino acid substitution Y92A in the variable light chain, the amino acid substitution H35A in the variable heavy chain, and / or the amino acid substitution R53A in the variable heavy chain (each according to Kabat numbering). In some aspects, compared to the variable domain of the 13B8.2 Fab fragment in ml3B8.2, the His residue at position 91 of the light chain (position 93 in SEQ ID NO:102) is mutated to Ala, and the Arg residue at position 53 of the heavy chain (position 55 in SEQ ID NO:101) is mutated to Ala. In some embodiments, the reagent that reversibly binds anti-CD4 or a fragment thereof is commercially available or derived from a commercially available reagent (e.g., catalog number 6-8000-206 or 6-8000-205 or 6-8002-100; IBA GmbH, Gottingen, Germany). In some embodiments, the selection agent comprises an anti-CD4 Fab fragment.In some embodiments, the anti-CD4 Fab fragment comprises a variable heavy chain having the sequence set forth in SEQ ID NO: 101 and a variable light chain having the sequence set forth in SEQ ID NO: 102. In some embodiments, the anti-CD4 Fab fragment comprises the CDRs of the variable heavy chain having the sequence set forth in SEQ ID NO: 101 and the CDRs of the variable light chain having the sequence set forth in SEQ ID NO: 102.

[0212] In some embodiments, the selection marker can be CD8, and the selection agent specifically binds to CD8. In some aspects, the selection agent that specifically binds to CD8 can be selected from the group consisting of an anti-CD8 antibody, a bivalent antibody fragment of an anti-CD8 antibody, a monovalent antibody fragment of an anti-CD8 antibody, and a proteinaceous CD8-binding molecule with antibody-like binding properties. In some embodiments, the anti-CD8 antibody, e.g., a bivalent or monovalent antibody fragment (e.g., a CD8 Fab fragment), can be derived from the antibody OKT8 (e.g., ATCC CRL-8014) or a functionally active variant thereof that retains specific binding to CD8. In some embodiments, the reagent that reversibly binds to anti-CD8 or a fragment thereof is commercially available or derived from a commercially available reagent (e.g., catalog no. 6-8003 or 6-8000-201; IBA GmbH, Gottingen, Germany). In some embodiments, the selection agent comprises an anti-CD8 Fab fragment. In some embodiments, the anti-CD8 Fab fragment comprises a variable heavy chain having the sequence set forth in SEQ ID NO: 104 and a variable light chain having the sequence set forth in SEQ ID NO: 105. In some embodiments, the anti-CD8 Fab fragment comprises the CDRs of the variable heavy chain having the sequence set forth in SEQ ID NO: 104 and the CDRs of the variable light chain having the sequence set forth in SEQ ID NO: 105.

[0213] In some embodiments, the selection marker can be CD3, and the selection agent specifically binds to CD3. In some aspects, the selection agent that specifically binds to CD3 can be selected from the group consisting of an anti-CD3 antibody, a bivalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, and a proteinaceous CD3-binding molecule with antibody-like binding properties. In some embodiments, the anti-CD3 antibody, e.g., a bivalent antibody fragment or a monovalent antibody fragment (e.g., a CD3 Fab fragment), can be derived from the antibody OKT3 (e.g., ATCC CRL-8001; see, e.g., Stemberger et al. PLoS One. 2012;7(4):e35798) or a functionally active variant thereof that retains specific binding to CD3. In some embodiments, the reagent that reversibly binds to anti-CD3 or a fragment thereof is commercially available or derived from a commercially available reagent (e.g., catalog number 6-8000-201, 6-8001-100; IBA GmbH, Gottingen, Germany). In some embodiments, the selection agent comprises an anti-CD3 Fab fragment. In some embodiments, the anti-CD3 Fab fragment comprises a variable heavy chain having the sequence set forth in SEQ ID NO:89 and a variable light chain having the sequence set forth in SEQ ID NO:90. In some embodiments, the anti-CD3 Fab fragment comprises the CDRs of the variable heavy chain having the sequence set forth in SEQ ID NO:89 and the CDRs of the variable light chain having the sequence set forth in SEQ ID NO:90.

[0214] In any of the above examples, the bivalent antibody fragment may be a (Fab)2'-fragment or a bivalent single-chain Fv fragment, while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv). In any of the above examples, the proteinaceous binding molecule with antibody-like binding properties may be an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, an ankyrin scaffold-based protein, a crystalline scaffold-based protein, an adnectin, and an avimer.

[0215] 2. T cell selection by chromatography In some embodiments, cells, e.g., T cells, are isolated, selected, or enriched by chromatographic isolation, such as by column chromatography, including affinity chromatography or gel permeation chromatography. In some embodiments, the method utilizes a receptor binding reagent that binds to a receptor molecule located on the surface of a target cell, e.g., the cell to be isolated, selected, or enriched. Such methods may be described as (traceless) cell affinity chromatography technology (CATCH) and may include any of the methods or techniques described in PCT Application Nos. WO2013124474 and WO2015164675 (incorporated herein by reference in their entireties).

[0216] In some embodiments, cells, e.g., target cells, have or express receptor molecules on their cell surfaces such that the cells to be isolated, selected, or enriched are defined by the presence of at least one common specific receptor molecule. In some embodiments, a sample containing target cells may also contain additional cells that lack receptor molecules. For example, in some embodiments, T cells are isolated, enriched, and / or selected from a sample containing multiple cell types, e.g., red blood cells or B cells.

[0217] In some embodiments, the selection agent is contained in a chromatography column, e.g., directly or indirectly bound to a chromatography matrix (e.g., stationary phase). In some embodiments, the selection agent is present on the chromatography matrix (e.g., stationary phase) when the sample is applied to the column. In some embodiments, the selection agent can be indirectly bound to the chromatography matrix (e.g., stationary phase) through a reagent, e.g., a selection reagent. In some embodiments, the selection reagent is covalently or non-covalently bound to the stationary phase of the column. In some embodiments, the selection reagent is reversibly immobilized on the chromatography matrix (e.g., stationary phase). In some cases, the selection reagent is immobilized on the chromatography matrix (e.g., stationary phase) via a covalent bond. In some aspects, the selection reagent is reversibly immobilized on the chromatography matrix (e.g., stationary phase) by a non-covalent bond.

[0218] In some aspects, a selection agent is added to the sample. In some aspects, a receptor-binding reagent is added to the sample. In certain embodiments, the receptor-binding reagent has a binding site B that specifically binds to a receptor molecule (e.g., CD3, CD4, and / or CD8) on the surface of a cell, e.g., a target cell. In some aspects, the receptor-binding reagent also includes a binding partner C that can specifically and reversibly bind to binding site Z of a selection agent, such as an affinity reagent.

[0219] In certain aspects, the affinity reagent may also contain two or more binding sites Z, to which the binding partner C can bind, resulting in multimerization of the receptor-binding reagent. Thus, the affinity reagent used herein may also contain a multimerization reagent. The affinity reagent may be, for example, streptavidin, streptavidin mutein, avidin, avidin mutein, or a mixture thereof. In some aspects, different chromatographic matrices may be coupled to different affinity reagents and stacked in a column to form a multicomponent system for separation.

[0220] In certain embodiments, a sample, e.g., a sample containing cells and a receptor-binding reagent, is loaded onto or contacted with a chromatography matrix containing an attached or immobilized affinity reagent. In certain aspects, the affinity reagent has multiple binding sites Z that specifically bind to the binding partner C of the receptor-binding reagent. In certain aspects, the receptor-binding reagent binds to the affinity reagent through an interaction between the binding partner C and the binding site Z. Thus, in some embodiments, cells, e.g., target cells, are immobilized on the chromatography matrix via a complex formed by one or more binding sites Z of the affinity reagent and the binding partner C of the receptor-binding reagent. In further aspects, cells, e.g., target cells, can be depleted from the sample, such as by rinsing, releasing, or washing the remaining sample from the chromatography matrix. In certain aspects, the receptor-binding reagent can be included in the cell-containing sample or applied or contacted to the chromatography matrix for binding to the attached affinity reagent or multimerization reagent, e.g., before the sample is added to the chromatography matrix.

[0221] In some aspects, a competing reagent is then loaded onto the chromatography column. In certain embodiments, the competing reagent has a binding site capable of binding to binding site Z of the affinity reagent. In certain embodiments, the competing reagent forms a complex with the affinity reagent, thereby immobilizing the competing reagent on the chromatography matrix. As a result of this competitive binding, binding partner C displaces binding between the receptor-binding reagent and the affinity reagent at binding site Z. In certain embodiments, the cells are eluted from the chromatography matrix by adding or loading a competing reagent onto the chromatography matrix to which the complex containing the affinity reagent, receptor-binding reagent, and cells (e.g., target cells) is attached. In some aspects, the receptor-binding reagent has low affinity for the cell's receptor molecule at binding site B, such that the receptor-binding reagent dissociates from the cell in the presence of the competing reagent. Thus, in some embodiments, the cells, e.g., target cells, are eluted from the chromatography matrix free or essentially free of bound receptor-binding molecules.

[0222] In some embodiments, the selection agent is present on the chromatography matrix (e.g., stationary phase) at the time the sample is added to the chromatography column (e.g., stationary phase), e.g., directly bound (e.g., covalently or non-covalently) or indirectly bound via a selection reagent. Thus, upon addition of the sample, the selection agent can bind to and immobilize target cells on the chromatography matrix (e.g., stationary phase) of the column. Alternatively, in some embodiments, the selection agent can be added to the sample. In this manner, the selection agent binds to target cells (e.g., T cells) in the sample, and the sample can then be added to a chromatography matrix (e.g., stationary phase) containing the selection reagent. In this case, the selection agent already bound to the target cells binds to the selection reagent, thereby immobilizing the target cells on the chromatography matrix (e.g., stationary phase). In some embodiments, the selection agent binds to a selection reagent, as described herein, via a binding partner C, as described herein, included in the selection agent.

[0223] In some embodiments, two or more selection agents associate, e.g., reversibly or irreversibly bind, with the selection reagent, e.g., via one or more binding sites Z present on the selection reagent. In some cases, this results in the selection agents being positioned closely together such that an avidity effect can occur when a target cell bearing a cell surface molecule (e.g., a selection marker) (at least two copies thereof) contacts a selection agent that is capable of binding to the particular molecule (e.g., selection marker).

[0224] In some embodiments, two or more different selection agents that are the same, i.e., have the same selection marker binding specificity, can reversibly bind to the selection reagent. In some embodiments, it is possible to use at least two different selection agents that bind to different selection markers, and in some cases, three or four different selection agents. In some aspects, each of the at least two selection agents can bind to a different molecule (e.g., selection marker), e.g., a first molecule, a second molecule, etc. In some cases, different molecules (e.g., selection markers), e.g., cell surface molecules, can be present on the same target cell. In other cases, different molecules (e.g., selection markers), e.g., cell surface molecules, can be present on different target cells present in the same cell population. In some cases, a third, fourth, etc. selection agent can associate with the same reagent, each further containing a different binding site.

[0225] In some embodiments, two or more different selection agents contain the same binding partner C. In some embodiments, two or more different selection agents contain different binding partners. In some aspects, a first selection agent can have a binding partner C1 that can specifically bind to a binding site Z1 present on the selection agent, and a second selection agent can have a binding partner C2 that can specifically bind to a binding site Z1 or a binding site Z2 present on the selection agent. Thus, in some cases, the multiple binding sites Z contained by the selection agent include binding sites Z1 and Z2 that can reversibly bind to binding partners C1 and C2 contained by the selection agent, respectively. In some embodiments, C1 and C2 are the same, and / or Z1 and Z2 are the same. In other aspects, one or more of the multiple binding sites Z can be different. In other cases, one or more of the multiple binding partners C can be different. It is within the skill of one of ordinary skill in the art to select any combination of different binding partners C that are compatible with a selection agent containing binding site Z, so long as each of the binding partners C is capable of interacting with, e.g., specifically binding to, one of the binding sites Z.

[0226] In some embodiments, the reversible bond formed between binding partner C and binding site Z can be disrupted by a competitor and / or free binder. In some embodiments, the competitor and / or free binder can be biotin, a biotin derivative or analog, or a streptavidin-binding peptide that can compete with one or more binding sites Z for binding to binding partner C. In some embodiments, binding partner C and the competitor and / or free binder are different, and the competitor and / or free binder exhibits a higher binding affinity for one or more binding sites Z compared to the affinity of the binding partner. In certain aspects of any of the methods provided herein, adding the competitor and / or free binder to the stationary phase of the chromatography column to disrupt binding of the selection agent to the selection reagent does not require displacing the target cells (e.g., T cells) from the chromatography matrix (e.g., stationary phase).

[0227] In some embodiments, cells of the sample, e.g., target cells, can be depleted from the sample, such as by rinsing, releasing, or washing the remaining sample from the chromatography matrix (e.g., stationary phase). In some embodiments, one or more (e.g., 2, 3, 4, 5, 6) washing steps are used to remove unbound cells and debris from the chromatography matrix (e.g., stationary phase). In some embodiments, the sample is allowed to permeate the matrix for at least or about 5, 10, 16, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, or 120 minutes before one or more washing steps are performed.

[0228] In some embodiments, an elution sample from the eluate of the first chromatography column is collected, containing cells, for example, target cells, a competing reagent, and a receptor-binding reagent. In certain embodiments, the elution sample is loaded onto a second chromatography column having a suitable stationary phase that can simultaneously act as an affinity chromatography matrix and a gel permeation matrix. In certain embodiments, the affinity chromatography matrix has an affinity reagent immobilized thereon. In some aspects, the receptor-binding reagent and the competing reagent bind to the binding site Z on the affinity reagent, thereby immobilizing it on the chromatography matrix. As a result, in certain aspects, the elution sample containing the isolated target cells is depleted of the receptor-binding reagent and the competing reagent. Thus, in some aspects, the target cells that do not contain any reactants are now ready for further use, for example, for processing by any of the methods described herein.

[0229] In some embodiments, multiple cell selection steps are performed, where a positively or negatively selected fraction from one step is subjected to another selection step, e.g., subsequent positive or negative selection. In certain embodiments, methods, techniques, and reagents for selection, isolation, and enrichment are described, for example, in PCT Application No. WO2015164675, which is incorporated herein by reference in its entirety.

[0230] In some embodiments, a single selection step can be used to isolate target cells (e.g., CD3+ T cells) from a sample. In some embodiments, the single selection step can be performed on a single chromatography column. In some examples, a single selection step can simultaneously deplete cells expressing multiple markers. Similarly, multiple cell types can be simultaneously positively selected. In certain embodiments, the selection step is repeated and / or performed more than once, where the positively or negatively selected fraction from one step is subjected to the same selection step, e.g., repeated positive or negative selection. In some examples, the single selection step is repeated and / or performed more than once, e.g., to increase the purity of the selected cells and / or to further remove and / or deplete negatively selected cells from the negatively selected fraction. In certain embodiments, one or more selection steps are performed two, three, four, five, six, seven, eight, nine, ten, or more than ten times. In certain embodiments, one or more selection steps are performed and / or repeated 1 to 10 times, 1 to 5 times, or 3 to 5 times.

[0231] Cell selection can be performed using one or more chromatography columns. In some embodiments, the one or more chromatography columns are included in a closed system. In some embodiments, the closed system is an automated closed system, e.g., requiring minimal or no user (e.g., human) input. In some embodiments, cell selection is performed continuously (e.g., sequential selection techniques). In some embodiments, one or more chromatography columns are arranged in series. For example, a first column can be oriented so that the column output (e.g., eluate) can be fed to a second chromatography column, e.g., via a connecting tube. In some embodiments, multiple chromatography columns can be arranged in series. In some embodiments, cell selection can be achieved by performing sequential positive and negative selection steps, where subsequent steps subject negative and / or positive fractions from the previous step to further selection. In this case, the entire process is performed within the same tube or set of tubes. In some embodiments, a sample containing target cells is subjected to sequential selection, where a first selection is accomplished to enrich for one of the CD4+ or CD8+ populations, and unselected cells from the first selection are used as the source of cells for a second selection to enrich for the other of the CD4+ or CD8+ populations. In some embodiments, one or more additional rounds of selection are performed to enrich for a subpopulation of one or both of the CD4+ or CD8+ populations, e.g., central memory T (T CM) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+). In some embodiments, a sample containing target cells is subjected to sequential selections, where a first selection is performed to enrich for a CD3+ population and the selected cells are used as a cell source for a second selection to enrich for a CD3+ population. In some embodiments, a sample containing target cells is subjected to sequential selections, where a first selection is performed on a first stationary phase (e.g., in a first chromatographic column) to enrich for a CD3+ population and the flow-through containing unbound cells is used as a cell source for a second selection to enrich for a CD3+ population on a second stationary phase (e.g., in a second chromatographic column), where the first and second stationary phases are arranged sequentially. In some embodiments, the selection is a positive selection for CD3+ T cells (e.g., by using an antibody or antigen-binding fragment thereof that specifically binds to cell surface CD3). In some embodiments, one or more additional rounds of selection are performed to select a subpopulation of the CD3+ population, e.g., central memory T (T CM ) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+). In some embodiments, a sample containing target cells is subjected to sequential selection, where a first selection is achieved to enrich for a CD3+ population, and the selected cells are used as a source of cells for a second selection to enrich for a CD4+ population. In some embodiments, one or more additional rounds of selection are performed to enrich for a subpopulation of the CD3+CD4+ population, e.g., central memory T (T CM) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+). In some embodiments, a sample containing target cells is subjected to sequential selection, where a first selection is achieved to enrich for a CD3+ population, and the selected cells are used as a source of cells for a second selection to enrich for a CD8+ population. In some embodiments, one or more additional rounds of selection are performed to enrich for a subpopulation of the CD3+CD8+ population, e.g., central memory T (T CM) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+). In some aspects, it is envisioned that specific subpopulations of T cells (e.g., CD3+ cells), for example, cells positive for or expressing high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells), are selected by positive or negative sequential selection techniques. In some embodiments, cell selection is performed in parallel (e.g., parallel selection techniques). In some embodiments, one or more chromatography columns are arranged in parallel. For example, two or more columns can be arranged so that a sample is loaded onto two or more columns simultaneously via a tube, allowing the sample to be applied to each column without the need for the sample to pass through the first column. For example, using parallel selection techniques, cell selection can be achieved by simultaneously performing positive and / or negative selection steps in a closed system, e.g., the entire process being carried out within the same tube or set of tubes. In some embodiments, a sample containing target cells is subjected to parallel selection, in which the sample is loaded onto two or more chromatography columns, each column achieving selection of a cell population. In some embodiments, two or more chromatography columns independently achieve selection of CD3+, CD4+, or CD8+ populations. In some embodiments, two or more chromatography columns, including affinity chromatography or gel permeation chromatography, independently achieve selection of the same cell population. For example, two or more chromatography columns may achieve selection of CD3+ cells. In some embodiments, two or more chromatography columns, including affinity chromatography or gel permeation chromatography, independently achieve selection of different cell populations. For example, two or more chromatography columns may independently achieve selection of CD3+ cells, CD4+ cells, and CD8+ cells.In some embodiments, one or more additional rounds of selection, e.g., using sequential selection techniques, can be accomplished to enrich for subpopulations of one or all of the cell populations selected via parallel selection. For example, selected cells can be enriched for central memory T (T). CM ) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+). In some embodiments, the sample containing the target cells is subjected to parallel selection performed on two or more columns to enrich for the CD3+ population. In some embodiments, one or more additional rounds of selection are performed to enrich for a subpopulation of the CD3+ population, such as central memory T (T CM ) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+). In some embodiments, a sample containing target cells is subjected to parallel selection, where selection is accomplished independently on two or more columns to enrich for CD3+ and CD4+ populations. In some embodiments, one or more additional rounds of selection are performed to enrich for subpopulations of the CD3+ and CD4+ populations, such as central memory T (T CM ) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+). In some embodiments, a sample containing target cells is subjected to parallel selections that are performed to enrich for CD3+ and CD8+ populations. In some embodiments, one or more additional rounds of selection are performed to enrich for subpopulations of the CD3+ and CD8+ populations, such as central memory T (T CM) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+). In some embodiments, a sample containing target cells is subjected to parallel selections that are performed to enrich for CD4+ and CD8+ populations. In some embodiments, one or more additional rounds of selection are performed to enrich for subpopulations of the CD4+ and CD8+ populations, such as central memory T (T CM ) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+). In some aspects, it is envisioned that specific subpopulations of T cells (e.g., CD3+, CD4+, CD8+ T cells), for example, cells positive for or expressing high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells), are selected by positive or negative parallel selection techniques. In some embodiments, a combination of serial and parallel selection techniques can be used.

[0232] Generally, the binding capacity of the stationary phase (e.g., selection resin) affects how much stationary phase is required to select a certain amount of target moiety, e.g., target cells such as T cells. Binding capacity, e.g., the number of target cells that can be immobilized per mL of stationary phase (e.g., selection resin), can be used to determine or control the number of target cells captured on one or more columns. One or more chromatography columns can be used for the on-column cell selection and stimulation disclosed herein. When multiple columns are used, they can be arranged in series, in parallel, or in any suitable combination thereof. Therefore, the binding capacity of the stationary phase (e.g., selection resin) can be used to standardize the amount of reagents in a single-column approach or the amount of reagents per column in a multi-column approach.

[0233] In some embodiments, the binding capacity of a stationary phase as used herein is the maximum number of target cells (e.g., CD3+ T cells, CD4+ T cells, or CD8+ T cells) that bind to the stationary phase under given solvent and cell concentration conditions when an excess of target cells is loaded onto the stationary phase. In one aspect, the binding capacity of a stationary phase as used herein for on-column cell selection and stimulation is the static binding capacity. In some embodiments, the static binding capacity of a stationary phase (e.g., a selection resin) disclosed herein ranges from about 50 million to about 100 million target cells per mL of stationary phase. In some embodiments, the static binding capacity of the stationary phase (e.g., a selection resin) is about 10 million to about 20 million cells per mL of stationary phase, about 20 million to about 30 million cells, about 30 million to about 40 million cells, about 40 million to about 50 million cells, about 50 million to about 60 million cells, about 60 million to about 70 million cells, about 70 million to about 80 million cells, about 80 million to about 90 million cells, or about 90 million to about 100 million cells. , about 110 million to about 120 million, about 120 million to about 130 million, about 130 million to about 140 million, about 140 million to about 150 million, about 150 million to about 160 million, about 160 million to about 170 million, about 170 million to about 180 million, about 180 million to about 190 million, or about 190 million to about 200 million.

[0234] In some embodiments, the binding capacity of a stationary phase, as used herein, is the number of target cells (e.g., CD3+ T cells, CD4+ T cells, or CD8+ T cells) that bind to the stationary phase under given flow conditions before significant breakthrough of unbound target cells occurs. In one aspect, the binding capacity of a stationary phase, as used herein for on-column cell selection and stimulation, is the dynamic binding capacity, i.e., the binding capacity under operating conditions in a packed chromatography column during sample application. In some embodiments, the dynamic binding capacity is determined by loading a sample containing a known concentration of target cells and monitoring the flow-through; target cells will bind to the stationary phase up to a certain breakthrough point before unbound target cells flow through the column. In some embodiments, the dynamic binding capacity of a stationary phase (e.g., a selection resin) disclosed herein ranges from about 50 million to about 100 million target cells per mL of stationary phase. In some embodiments, the dynamic binding capacity of the stationary phase (e.g., a selection resin) is about 10 million to about 20 million target cells per mL of stationary phase, about 20 million to about 30 million, about 30 million to about 40 million, about 40 million to about 50 million, about 50 million to about 60 million, about 60 million to about 70 million, about 70 million to about 80 million, about 80 million to about 90 million, or about 90 million to about 100 million target cells per mL of stationary phase. , about 110 million to about 120 million, about 120 million to about 130 million, about 130 million to about 140 million, about 140 million to about 150 million, about 150 million to about 160 million, about 160 million to about 170 million, about 170 million to about 180 million, about 180 million to about 190 million, or about 190 million to about 200 million.

[0235] Generally, the chromatography method is fluid chromatography, typically liquid chromatography. In some aspects, chromatography can be performed in a flow-through mode, where a fluid sample containing cells, e.g., target cells, is applied to one end of a column containing a chromatography matrix by gravity flow or by a pump, and the fluid sample exits the column at the other end. In addition, chromatography can be performed in an "up and down" mode, where a fluid sample containing cells to be isolated is applied to one end of a column containing a chromatography matrix packed in a pipette tip, for example, by a pipette, and the fluid sample enters the chromatography matrix / pipette tip and exits the other end of the column. Alternatively, chromatography can also be performed in a batch mode, where a chromatography material (stationary phase) is incubated with a sample containing cells, for example, under shaking, rotation, or repeated contact, and the fluid sample is removed, for example, using a pipette.

[0236] In some aspects, any material suitable for chromatographic isolation of cells may be utilized as a chromatography matrix in the context of the present invention. In certain aspects, suitable chromatography materials are at least non-toxic or essentially non-toxic, e.g., not detrimental to cell viability when used in a packed chromatography column under the desired conditions for cell isolation and / or cell separation. In some aspects, the chromatography matrix remains in a predetermined location, typically in a predetermined position, while the location of the sample to be separated and the components contained therein changes. Thus, in some aspects, the chromatography matrix is ​​a "stationary phase."

[0237] Typically, each chromatography matrix has a solid or semi-solid phase, while the sample containing the target cells to be isolated / separated is in a fluid phase. The mobile phase used to accomplish the chromatographic separation is also a fluid phase. The chromatography matrix can be a particulate material (of any suitable size and shape) or a monolithic chromatography material including a paper substrate or membrane (see the Examples section). Thus, chromatography can be both column chromatography and planar chromatography. In addition to standard chromatography columns, columns or pipette tips that allow bidirectional flow can be used for column-based / flow-through mode-based chromatographic separation of cells as described herein. In some aspects, particulate matrix materials are used, and the particulate matrix materials can have an average particle size of, for example, about 5 μm to about 200 μm, or about 5 μm to about 400 μm, or about 5 μm to about 600 μm. In some aspects, planar chromatography is used, and the matrix material can be any material suitable for planar chromatography, such as a conventional cellulosic or organic polymeric membrane (e.g., a paper membrane, a nitrocellulose membrane, or a polyvinylidene difluoride (PVDF) membrane) or a silica-coated glass plate.

[0238] In some aspects, the chromatography matrix / stationary phase is a non-magnetic or non-magnetizable material. Such materials can include derivatized silica or cross-linked gels. Cross-linked gels (typically manufactured in bead form) can be based on natural polymers such as cross-linked polysaccharides. Suitable examples include, but are not limited to, agarose gels or cross-linked dextran gels. Cross-linked gels can also be based on synthetic polymers, i.e., polymer classes that do not exist in nature. Typically, such synthetic polymers that form the basis of chromatographic stationary phases for cell separation are polymers that have polar monomer units and are therefore polar themselves.

[0239] Examples of suitable synthetic polymers are polyacrylamide, styrene-divinylbenzene gel, and copolymers of acrylates and diols or acrylamide and diols. An example is polymethacrylate gel commercially available as Fractogel®. A further example is copolymers of ethylene glycol and methacrylate commercially available as Toyopearl®. In some embodiments, the chromatographic stationary phase can also include natural and synthetic polymeric components, such as composite matrices or composites or copolymers of polysaccharides and agarose or polysaccharides and N,N'-methylenebisacrylamide, e.g., polyacrylamide / agarose composites. An example of a copolymer of dextran and N,N'-methylenebisacrylamide is the Sephacryl® series of materials mentioned above. Derivatized silica can include silica particles coupled to synthetic or natural polymers. Examples of such embodiments include, but are not limited to, polysaccharide-grafted silica, polyvinylpyrrolidone-grafted silica, polyethylene oxide-grafted silica, poly(2-hydroxyethylaspartamide) silica, and poly(N-isopropylacrylamide)-grafted silica.

[0240] In some embodiments, the chromatography matrix utilized in the present invention is a gel filtration (also known as size exclusion) matrix, for example, when used in a removal cartridge as described herein. Gel filtration can be characterized by its ability to be at least essentially free from interaction with the cells to be separated. Thus, gel filtration matrices enable separation of cells or other biological entities, as defined herein, primarily based on their size. Each chromatography matrix is ​​typically a particulate porous material, as described above. The chromatography matrix can have a specific exclusion limit, typically defined in terms of molecular weight, above which molecules are completely blocked from entering the pores. The molecular weight defining the size exclusion limit can be selected to be below the weight corresponding to the weight of the target cell (or biological entity) to be isolated. In such embodiments, the target cell is prevented from entering the pores of the size exclusion chromatography matrix. Similarly, the stationary phase, which is an affinity chromatography matrix, can have pores that are smaller than the size of the selected target cell. In exemplary embodiments, the affinity chromatography matrix and / or gel filtration matrix has an average pore size of 0 to about 500 nm.

[0241] Other components present in the sample, such as receptor-binding molecules or competing reagents, may have a size below the exclusion limit of the pores and can enter the pores of the size-exclusion chromatography matrix. Of those components that can partially or completely enter the pore volume, larger molecules that are inaccessible due to the pore volume typically elute first, and the smallest molecules typically elute last. In some embodiments, the exclusion limit of the size-exclusion chromatography matrix is ​​selected to be below the maximum width of the target cells. Thus, components that are accessible to the pore volume typically remain in / on the size-exclusion chromatography matrix longer than the target cells. Thus, the target cells can be recovered in the eluate of the chromatography column separately from other substances / components of the sample. Therefore, components such as the receptor-binding reagent, or, if applicable, the competing reagent, elute from the gel filtration matrix at a later time than the target cells. This separation effect can be further enhanced if the gel permeation matrix contains an affinity reagent (usually covalently bound thereto) containing a binding site, e.g., binding site Z, that can bind to a reagent, such as the receptor-binding reagent and / or competing reagent, present in the sample. The receptor-binding reagent and / or competing reagent binds to binding site Z of the affinity reagent, thereby immobilizing it on a gel permeation matrix. This method is typically performed in a removal cartridge as used in the present invention, and in some embodiments, the methods, combinations, and kits of the present invention include and / or utilize such a gel filtration matrix. In each method, cells are thus separated based on size.

[0242] The chromatography matrix utilized in the present invention may also include a magnetically attractable substance, such as one or more magnetically attractable particles or magnetic fluids. Each magnetically attractable particle may include a multimerization reagent or affinity reagent having a binding site capable of binding to target cells. The magnetically attractable particles may include diamagnetic, ferromagnetic, paramagnetic, or superparamagnetic materials. Superparamagnetic materials respond to magnetic fields by induced magnetic fields without producing permanent magnetization. Iron oxide-based magnetic particles are commercially available, for example, from Dynal Biotech as Dynabeads®, Miltenyi Biotec as magnetic MicroBeads, and CPG Inc. as magnetic porous glass beads, as well as from various other suppliers, such as Roche Applied Science, BIOCLON, BioSource International Inc., micromod, AMBION, Merck, Bangs Laboratories, Polysciences, or Novagen Inc., to name a few. Superparamagnetic Co and FeCo and ferromagnetic Co nanocrystalline magnetic nanoparticles have been described, for example, by Hutten, A. et al. (J. Biotech. (2004), 112, 47-63). However, in some embodiments, the chromatography matrix utilized in the present invention does not contain any magnetically attractable material.

[0243] In some embodiments, receptor molecules located on cell surfaces, e.g., target cell surfaces, can be any molecule that remains covalently or noncovalently bound to the cell surface during the chromatographic separation process in the methods of the present invention. The receptor molecule is a molecule that can be targeted by a receptor-binding reagent. In some embodiments, the receptor is a peptide or protein, such as a membrane receptor protein. In some embodiments, the receptor is a lipid, polysaccharide, or nucleic acid. Protein receptors can be peripheral or integral membrane proteins, which in some embodiments can have one or more membrane-spanning domains. In certain embodiments, the receptor molecule is an immune cell surface protein, e.g., CD3, CD4, or CD8. In some embodiments, the receptor molecule can be an antigen that defines a desired cell population or subpopulation, e.g., a blood cell population or subpopulation, e.g., lymphocytes (e.g., T cells, CD3+ T cells, CD4+ T cells, or CD8+ T cells).

[0244] In some embodiments, the receptor-binding reagent has or contains binding site B. In certain embodiments, binding site B is monovalent. In some aspects, the monovalent binding site B is or contains a monovalent antibody fragment or proteinaceous binding molecule with immunoglobulin-like function, an aptamer, or an MHC molecule. Examples of monovalent antibody fragments include, but are not limited to, Fab fragments, Fv fragments, and single-chain Fv fragments (scFv), including bivalent single-chain Fv fragments. Examples of (recombinant) antibody fragments are Fab fragments, Fv fragments, single-chain Fv fragments (scFv), bivalent antibody fragments such as (Fab)2'-fragments, diabodies, triabodies (Iliades, P., et al., FEBS Lett (1997) 409, 437-441), decabodies (Stone, E., et al., Journal of Immunological Methods (2007) 318, 88-94) and other domain antibodies (Holt, LJ, et al., Trends Biotechnol. (2003), 21, 11, 484-490). In some embodiments, one or more binding sites of the receptor molecule-binding reagent may be a bivalent proteinaceous artificial binding molecule, such as a dimeric lipocalin mutein, also known as "duocalin." In some embodiments, the receptor-binding reagent may have a single second binding site, i.e., it may be monovalent. Examples of monovalent receptor-binding reagents include, but are not limited to, monovalent antibody fragments, proteinaceous binding molecules with antibody-like binding properties, or MHC molecules.

[0245] Still further examples of suitable proteinaceous binding molecules include EGF-like domains, Kringle domains, fibronectin type I domains, fibronectin type II domains, fibronectin type III domains, PAN domains, G1a domains, SRCR domains, Kunitz / Bovine pancreatic trypsin inhibitor domains, tendamistat, Kazal-type serine protease inhibitor domains, Trefoil (P-type) domains, von Willebrand factor type C domains, anaphylatoxin-like domains, CUB domains, thyroglobulin type I repeats, LDL-receptor class A domains, Sushi domains, Link domains, thrombospondin type I domains, immunoglobulin domains or immunoglobulin-like domains (e.g., domain antibodies or camel heavy chain antibodies), C-type lectin domains, MAM domains, von Willebrand factor type A domains, somatomedin B domains, WAP-type 4 disulfide core domains, F5 / 8 C-type domains, hemopexin domains, SH2 domains, SH3 domains, laminin-type EGF-like domains, C2 domains, "kappabodies" (see Ill. et al., Protein Eng. (1997) 10, 949-57, so-called "minibodies" (Martin et al., EMBO J (1994) 13, 5303-5309), diabodies (see Holliger et al., PNAS USA (1993) 90, 6444-6448), so-called "Janusis" (Traunecker et al., EMBO J (1991) 10, 3655-3659, or Traunecker et al., Int J Cancer (1992) Suppl 7, 51-52), nanobodies, microbodies, affilins, affibodies, knottins, ubiquitin, zinc finger proteins, autofluorescent proteins, or leucine-rich repeat proteins. Examples of nucleic acid molecules with antibody-like functions are aptamers, which fold into defined three-dimensional motifs and exhibit high affinity for a given target structure.

[0246] In certain aspects, the receptor-binding protein contains a binding partner C. In some aspects, the binding partner C included in the receptor-binding reagent can be, for example, hydrocarbon-based (including polymeric) and can contain nitrogen, phosphorus, sulfur, carbon, halogen, or pseudohalogen groups. The binding partner can be an alcohol, organic acid, inorganic acid, amine, phosphine, thiol, disulfide, alkane, amino acid, peptide, oligopeptide, polypeptide, protein, nucleic acid, lipid, sugar, oligosaccharide, or polysaccharide. As a further example, the binding partner can be a cation, anion, polycation, polyanion, polycation, electrolyte, polyelectrolyte, carbon nanotube, or carbon nanofoam. Generally, such binding partners have a higher affinity for the binding site of the multimerization reagent than other substances. Examples of respective binding partners include, but are not limited to, crown ethers, immunoglobulins, fragments thereof, and proteinaceous binding molecules with antibody-like functions.

[0247] In some embodiments, the binding partner C included in the receptor binding reagent comprises biotin, and the affinity reagent comprises a streptavidin analog or an avidin analog that reversibly binds to biotin. In some embodiments, the binding partner C included in the receptor binding reagent comprises a biotin analog that reversibly binds to streptavidin or avidin, and the affinity reagent comprises streptavidin, avidin, a streptavidin analog, or an avidin analog that reversibly binds to the respective biotin analog. In some embodiments, the binding partner C included in the receptor binding reagent comprises a streptavidin or an avidin-binding peptide, and the affinity reagent comprises a streptavidin, avidin, a streptavidin analog, or an avidin analog that reversibly binds to the respective streptavidin or avidin-binding peptide.

[0248] In some embodiments, the binding partner included in the receptor binding reagent may comprise a streptavidin-binding peptide. In some embodiments, the peptide sequence contains a sequence having the general formula His-Pro-Xaa (wherein Xaa is glutamine, asparagine, or methionine), e.g., the sequence set forth in SEQ ID NO:78. In some embodiments, the peptide sequence contains a sequence set forth in SEQ ID NO:93. In some embodiments, the peptide sequence has the general formula set forth in SEQ ID NO:79, e.g., SEQ ID NO:69. As an example, the peptide sequence is Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (also called Strep-tag®, set forth in SEQ ID NO:70). In one example, the peptide sequence is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also called Strep-tag® II, shown in SEQ ID NO:64), which is described in U.S. Patent 6,103,493 and is commercially available, for example, under the trademark Strep-Tactin®. The streptavidin-binding peptide can be, for example, a single peptide, such as the "Strep-tag®" described in U.S. Patent 5,506,121, or a streptavidin-binding peptide having a sequential arrangement of two or more individual binding modules, such as those described in International Patent Publication WO 02 / 077018 or U.S. Patent 7,981,632.

[0249] In some embodiments, the binding partner C of the receptor-binding reagent comprises a moiety known to those skilled in the art as an affinity tag. In such embodiments, the affinity reagent comprises a corresponding binding partner, e.g., an antibody or antibody fragment, that is known to bind to the affinity tag. As some illustrative examples of known affinity tags, the binding partner included in the receptor binding reagent may be dinitrophenol or digoxigenin, oligohistidine, polyhistidine, an immunoglobulin domain, maltose binding protein, glutathione-S-transferase (GST), chitin-binding protein (CBP) or thioredoxin, calmodulin-binding peptide (CBP), FLAG'-peptide, HA-tag (e.g., SEQ ID NO:94), VSV-G-tag (e.g., SEQ ID NO:95), HSV-tag (e.g., SEQ ID NO:96), T7 epitope (e.g., SEQ ID NO:97), maltose binding protein (MBP), an HSV epitope in the sequence of herpes simplex virus glycoprotein D (e.g., SEQ ID NO:98), a "myc" epitope in the sequence of the transcription factor c-myc (e.g., SEQ ID NO:99), a V5-tag (e.g., SEQ ID NO:100), a nucleotide sequence similar to that of the nucleotide sequence ... The affinity reagent may comprise a nucleotide sequence encoding ...

[0250] Further examples of suitable binding partners C include, but are not limited to, lectins, protein A, protein G, metals, metal ions, nitrilotriacetic acid derivatives (NTA), RGD motifs, dextran, polyethyleneimine (PEI), redox polymers, glycoproteins, aptamers, dyes, amylose, maltose, cellulose, chitin, glutathione, calmodulin, gelatin, polymyxin, heparin, NAD, NADP, lysine, arginine, benzamidine, polyU, or oligo-dT. Lectins such as concavalin A are known to bind to polysaccharides and glycosylated proteins. An example of a dye is a triazine dye such as Cibacron blue F3G-A (CB) or Red HE-3B, which specifically binds to NADH-dependent enzymes. Typically, Green A binds to CoA proteins, human serum albumin, and dehydrogenases. In some cases, the dyes 7-aminoactinomycin D and 4',6-diamidino-2-phenylindole bind to DNA. Generally, cations of metals such as Ni, Cd, Zn, Co, or Cu are typically used to bind affinity tags, such as hexahistidine or oligohistidine-containing sequences, including His-Asn-His-Arg-His-Lys-His-Gly-Gly-Gly-Cys tag (MAT tag) (SEQ ID NO: 103), and N-methacryloyl-(L)-cysteine ​​methyl ester.

[0251] Consistent with International Patent Application PCT / EP2012 / 063969, published as WO 2013 / 011011 (the entire contents of which are incorporated herein by reference for all purposes), the strength of binding between a receptor-binding reagent and a receptor molecule on a target cell may not be essential for the reversibility of binding of the target cell to the affinity reagent via the receptor-binding reagent. Rather, it is preferred that the dissociation constant (Kd) for binding between the receptor-binding reagent and the receptor molecule via binding site B be low affinity, e.g., about 10 -3 ~about 10 -7 M or high affinity, e.g., about 10 -7 ~Approx. 1×10-10 Regardless of the strength of binding, meaning within the Kd of M, as long as the dissociation of the binding between the receptor-binding reagent and the receptor molecule via binding site B occurs sufficiently fast, the target cells can be reversibly stained. In this regard, the dissociation rate constant (k off ) is approximately 3 × 10 -5 sec -1 (This dissociation rate constant is a constant that characterizes the dissociation reaction of the complex formed between binding site B of the receptor-binding reagent and the receptor molecule on the surface of the target cell.) The association rate constant (k) for the association reaction between binding site B of the receptor-binding reagent and the receptor molecule on the surface of the target cell can have any value. To ensure sufficiently reversible binding between the receptor molecule and the receptor-binding reagent, it is recommended to use a value of about 3 × 10 -5 sec -1 That's about 5 x 10 -5 sec -1 or more, for example, 1×10 -4 sec -1 or more or about 1×10 -4 sec -1 That's it, 5 x 10 -4 sec -1 or more or about 5 x 10 -4 sec -1 That's it, 1×10 -3 sec -1 or more or about 1×10 -3 sec -1 That's it, 5 x 10 -3 sec -1 or more or about 5 x 10 -3 sec -1 That's it, 1×10 -2 sec -1 or more or about 1×10 -2 sec -1 That's it, 1×10 -1 sec -1 or more or about 1×10 -1 sec -1 or more, or 5 x 10 -1 sec -1 or more or about 5 x 10 -1 sec -1 k for binding equilibria with values ​​equal to or greater thanoff It is useful to select values ​​of kinetic and thermodynamic constants, where it is noted that the values ​​of the kinetic and thermodynamic constants as used herein refer to conditions of atmospheric pressure, i.e., 1.013 bar, and room temperature, i.e., 25°C.

[0252] In some embodiments, the receptor binding reagent has a single (monovalent) binding site B capable of specifically binding to a receptor molecule. In some embodiments, the receptor binding reagent has at least two (i.e., multiple binding sites B, including three, four, or five identical binding sites B) capable of binding to a receptor molecule. In any of these embodiments, the binding of receptor molecules via (each of) binding sites B is greater than or equal to about 3×10 -5 sec -1 More than k off Thus, a receptor-binding reagent can be a monovalent molecule (e.g., a monovalent antibody fragment or a monovalent artificial binding molecule (proteinaceous or otherwise), such as a mutein based on a polypeptide of the lipocalin family (also known as "Anticalin®"), or a bivalent molecule, such as an antibody or fragment in which both binding sites are retained, e.g., an F(ab')2 fragment. In some embodiments, k off Speed ​​is 3x10 -5 sec -1 If so, the receptor molecule may be a multivalent molecule, for example a pentameric IgE molecule.

[0253] In some embodiments of the present invention, the (traceless) isolation of biological materials via the reversible cell affinity chromatography techniques described herein is provided by, at the molecular level, the k of binding of a receptor binding reagent to a receptor molecule on a target cell via at least binding site B. off Speed ​​(3×10 -5 sec -1Rather, as described, for example, in U.S. Patent No. 7,776,562 or International Patent Application WO 02 / 054065, low affinity binding between the binding site B of a receptor-binding reagent bound to a receptor molecule, along with an avidity effect mediated by an immobilized affinity reagent, allows for reversible and traceless isolation of target cells. In these embodiments, complexes can be formed between two or more binding sites Z of the affinity reagent and at least two binding partners C of the receptor-binding reagent, thereby allowing for reversible immobilization and subsequent elution of target cells from the affinity chromatography matrix (via the addition of a competitor that disrupts the bond (complex) formed between binding partner C and binding site Z, dissociating the receptor-binding reagent from the target cell). As discussed above, such low binding affinity is less than about 1.0 × 10 for binding of the receptor-binding reagent to a receptor molecule on the surface of a target cell via binding site B. -3 M ~ approx. 1.0×10 -7 Dissociation constant (K D )

[0254] 3. Input Composition In certain embodiments, the provided methods are used in connection with producing or preparing an input population or composition of cells. In certain embodiments, the input cell composition comprises a population of cells for use in genetic engineering, e.g., cells to be genetically engineered or cells undergoing a process to produce genetically engineered cells. In certain embodiments, the cells are treated with, contacted with, or incubated with a nucleic acid encoding a recombinant receptor. In certain embodiments, the input composition contains T cells, viable T cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, and / or subpopulations thereof.

[0255] In some embodiments, cell viability is assessed with assays that may include, but are not limited to, dye uptake assays (e.g., calcein AM assays), XTT cell viability assays, and dye exclusion assays (e.g., trypan blue, eosin, or propidium dye exclusion assays). In certain embodiments, viable cells have negative expression of one or more apoptotic markers, such as annexin V or activated caspase 3. In some embodiments, viable cells are negative for expression of one or more apoptotic markers, which may include, but are not limited to, caspases or activated caspases, such as caspase 2, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, or caspase 10, Bcl-2 family members, such as Bax, Bad, and Bid, annexin V, or TUNEL staining. In certain embodiments, viable cells are negative for activated caspase 3. In certain embodiments, viable cells are negative for annexin V.

[0256] In some embodiments, the input composition comprises an enriched population of CD3+ T cells, e.g., viable CD3+ T cells. In some embodiments, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% of the cells in the input population are CD3+ T cells, e.g., viable CD3+ T cells. In some embodiments, the input population consists essentially of CD3+ T cells, e.g., viable CD3+ T cells.

[0257] In certain embodiments, the input population is a population of cells enriched for CD4+ T cells and CD8+ T cells, e.g., an enriched population of CD4+ T cells and CD8+ T cells. In certain embodiments, the input population comprises or contains at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% of cells that are CD4+ or CD8+ T cells. In some embodiments, the input population consists essentially of CD4+ and CD8+ T cells.

[0258] In certain embodiments, the input population is an enriched population of CD4+ T cells. In certain embodiments, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% of the cells in the input population are CD4+ T cells. In some embodiments, the input population consists essentially of CD4+ T cells.

[0259] In some embodiments, cells from an enriched population of CD4+ T cells and cells from an enriched population of CD8+ T cells are mixed, combined, and / or pooled to generate an input population containing CD4+ T cells and CD8+ T cells. In certain embodiments, the enriched populations of CD4+ T cells and CD8+ T cells 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 enriched populations of CD4+ and CD8+ T cells are pooled, mixed, and / or combined following isolation, enrichment, and / or selection of CD4+ and CD8+ T cells from a biological sample. In certain embodiments, the enriched populations of CD4+ and CD8+ T cells are pooled, mixed, and / or combined following freezing, e.g., cryopreservation, and thawing, of the enriched populations of CD4+ and CD8+ T cells.

[0260] In certain embodiments, the input population is produced, generated, or manufactured by mixing, pooling, and / or combining cells from an enriched population of CD4+ cells with cells from an enriched population of CD8+ cells. In certain embodiments, the enriched population of CD4+ T cells contains at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% CD4+ T cells. In certain embodiments, the enriched population of CD4+ T cells contains 100% or about 100% CD4+ T cells. In certain embodiments, the enriched population of T cells contains or contains less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and / or contains no CD8+ T cells and / or is free or substantially free of CD8+ T cells. In some embodiments, the population of cells consists essentially of CD4+ T cells. In certain embodiments, the enriched population of CD8+ T cells contains at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% CD8+ T cells, or 100% or about 100% CD8+ T cells. In certain embodiments, the enriched population of CD8+ T cells contains or contains less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and / or contains no CD4+ T cells and / or is free or substantially free of CD4+ T cells. In some embodiments, the population of cells consists essentially of CD8+ T cells.

[0261] In certain embodiments, CD4+ T cells and CD8+ T cells are pooled, mixed, and / or combined at 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 at about a 1:1 or 1:1 ratio of CD4+ T cells:CD8+ T cells. In certain embodiments, viable CD4+ T cells and viable CD8+ T cells are pooled, mixed and / or combined at 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 at about a 1:1 or 1:1 ratio of CD4+ T cells:CD8+ T cells.

[0262] In certain embodiments, the input composition comprises 50×10 6 , 100×10 6 , 150×10 6 , 200×10 6 , 250×10 6 , 300×10 6 , 350×10 6 , 400×10 6 , 450×10 6 , 500×10 6 , 550×10 6 , 600×10 6 , 700×10 6 , 800×10 6 , 900×10 6 , 1,000 x 10 6 , 1,100×10 6 Or 1,200 x 10 6 pieces, approximately 50×10 6 , 100×10 6 , 150×10 6 , 200×10 6 , 250×10 6 , 300×10 6 , 350×10 6 , 400×10 6 , 450×106 , 500×10 6 , 550×10 6 , 600×10 6 , 700×10 6 , 800×10 6 , 900×10 6 , 1,000 x 10 6 , 1,100×10 6 Or 1,200 x 10 6 pieces, or at least 50 x 10 6 , 100×10 6 , 150×10 6 , 200×10 6 , 250×10 6 , 300×10 6 , 350×10 6 , 400×10 6 , 450×10 6 , 500×10 6 , 550×10 6 , 600×10 6 , 700×10 6 , 800×10 6 , 900×10 6 , 1,000 x 10 6 , 1,100×10 6 Or 1,200 x 10 6 In certain embodiments, the input composition has a quantity of 50 x 10 T cells, e.g., viable T cells, viable CD3+ T cells, or viable mixed CD4+ and CD8+ T cells. 6 , 100×10 6 , 150×10 6 , 200×10 6 , 250×10 6 , 300×10 6 , 350×10 6 , 400×10 6 , 450×10 6 , 500×10 6 , 550×10 6 , 600×10 6 pieces, approximately 50×10 6 , 100×10 6 , 150×10 6 , 200×10 6, 250×10 6 , 300×10 6 , 350×10 6 , 400×10 6 , 450×10 6 , 500×10 6 , 550×10 6 , 600×10 6 pieces, or at least 50 x 10 6 , 100×10 6 , 150×10 6 , 200×10 6 , 250×10 6 , 300×10 6 , 350×10 6 , 400×10 6 , 450×10 6 , 500×10 6 , 550×10 6 , 600×10 6 In certain embodiments, the input composition has a quantity of 50 x 10 CD4+ T cells, e.g., viable CD4+ T cells. 6 , 100×10 6 , 150×10 6 , 200×10 6 , 250×10 6 , 300×10 6 , 350×10 6 , 400×10 6 , 450×10 6 , 500×10 6 , 550×10 6 , 600×10 6 pieces, approximately 50×10 6 , 100×10 6 , 150×10 6 , 200×10 6 , 250×10 6 , 300×10 6 , 350×10 6 , 400×10 6 , 450×10 6 , 500×10 6 , 550×10 6 , 600×10 6 pieces, or at least 50 x 10 6 , 100×10 6 , 150×106 , 200×10 6 , 250×10 6 , 300×10 6 , 350×10 6 , 400×10 6 , 450×10 6 , 500×10 6 , 550×10 6 , 600×10 6 The composition has a quantity of CD8+ T cells, e.g., viable CD8+ T cells. In some embodiments, the quantity of cells is the quantity of viable CD4+ and CD8+ T cells pooled, mixed, and / or combined together in the same composition. In such embodiments, the CD4+ and CD8+ T cells are present at a CD4+ T cell:CD8+ T cell ratio of 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 1:1.2, between 1.2:1 and 1:1.2, between 1.1:1 and 1:1.1, or about 1:1 or 1:1. In some embodiments, the quantity of cells is the quantity of viable CD4+ and CD8+ T cells pooled, mixed, and / or combined together in a CD4+ T cell:CD8+ T cell ratio of about 1:1 or 1:1.

[0263] In certain embodiments, the input composition comprises 300×10 6 ~600×10 6 or approximately 300 x 10 6 ~600×10 6 In some embodiments, the input population has a quantity of T cells, e.g., viable CD3+ cells, or a mixture of viable CD4+ and viable CD8+ cells (e.g., mixed at a 1:1 or about 1:1 ratio). In some embodiments, the input population has a quantity of 300 x 10 6 or approximately 300 x 10 6 In some embodiments, the input population has a concentration of, for example, 400 x 10 viable CD3+ cells, or a mixture of viable CD4+ and viable CD8+ cells (e.g., mixed at a 1:1 or about 1:1 ratio). ... 6 or approximately 400 x 10 6In some embodiments, the input population has a concentration of, for example, 500 x 10 viable CD3+ cells or a mixture of viable CD4+ and viable CD8+ cells (e.g., mixed at a 1:1 or about 1:1 ratio). ... 6 or approximately 500 x 10 6 In some embodiments, the input population has a concentration of, for example, 600 x 10 viable CD3+ cells or a mixture of viable CD4+ and viable CD8+ cells (e.g., mixed at a 1:1 or about 1:1 ratio). ... 6 or approximately 600 x 10 6 In some embodiments, the input population has a concentration of, for example, 700 x 10 viable CD3+ cells or a mixture of viable CD4+ and viable CD8+ cells (e.g., mixed at a 1:1 or about 1:1 ratio). ... 6 or approximately 700 x 10 6 In some embodiments, the input population has a concentration of, for example, 800 x 10 viable CD3+ cells or a mixture of viable CD4+ and viable CD8+ cells (e.g., mixed at a 1:1 or about 1:1 ratio). ... 6 or approximately 800 x 10 6 In some embodiments, the input population has a concentration of, for example, 900 x 10 viable CD3+ cells or a mixture of viable CD4+ and viable CD8+ cells (e.g., mixed at a 1:1 or about 1:1 ratio). ... 6 or approximately 900 x 10 6 In some embodiments, the input population has a concentration of, for example, 100 x 10 viable CD3+ cells or a mixture of viable CD4+ and viable CD8+ cells (e.g., mixed at a 1:1 or about 1:1 ratio). ... 7 or about 100 x 10 7 In some embodiments, the input population has a concentration of, for example, 110 x 10 viable CD3+ cells or a mixture of viable CD4+ and viable CD8+ cells (e.g., mixed at a 1:1 or about 1:1 ratio). ... 7 or approximately 110 x 10 7 In some embodiments, the input population has a concentration of, for example, 120 x 10 viable CD3+ cells or a mixture of viable CD4+ and viable CD8+ cells (e.g., mixed at a 1:1 or about 1:1 ratio). ...7 or approximately 120 x 10 7 amounts of, for example, viable CD3+ cells or a mixture of viable CD4+ and viable CD8+ cells (eg, mixed in a 1:1 or about 1:1 ratio).

[0264] In certain embodiments, CD4+ T cells and CD8+ T cells are pooled, mixed, and / or combined such that the input composition has at most a target number (2n) or at most about a target number (about 2n) of T cells, e.g., viable T cells, viable CD3+ T cells, or viable mixed CD4+ and CD8+ T cells. In certain embodiments where a composition comprising enriched CD4+ T cells contains at least n CD4+ T cells and a composition comprising enriched CD8+ T cells (e.g., from the same donor as the CD4+ T cell composition, e.g., from the same apheresis or leukapheresis sample from the donor) contains at least n CD8+ T cells, n CD4+ T cells from the CD4+ T cell composition and n CD8+ T cells from the CD8+ T cell composition are pooled, mixed, and / or combined (i.e., a CD4+:CD8+ ratio of 1:1) to generate an input composition containing the target number (2n) of T cells. In certain embodiments where the composition comprising enriched CD4+ T cells contains n or fewer (e.g., fewer than n) CD4+ T cells and the composition comprising enriched CD8+ T cells (e.g., from the same donor as the CD4+ T cell composition, e.g., from the same apheresis or leukapheresis sample from the donor) contains n or fewer (e.g., fewer than n) CD8+ T cells, all of the cells of the CD4+ T...

Claims

1. 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 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 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 after 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.

2. 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; (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 transduced population, the harvesting comprising: (i) at 24 to 120 hours (inclusive) after exposure to the stimulant agent was initiated; (ii) when an 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 in the population or cells expressing a recombinant protein thereof; A process to be carried out Including, Thereby producing a composition of engineered cells.

3. Input composition is at least 300×10 6 The method of claim 2, comprising viable primary T cells.

4. The method of claim 2 or 3, 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.

5. 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 transduced population, the harvesting comprising: (i) at 48 to 120 hours (inclusive) after exposure to the stimulant agent was initiated; (ii) when an 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.

6. The method of any one of claims 1 to 5, wherein harvesting is performed at the time integrated vector is detected in the genome but before achieving stable iVCN per diploid genome.

7. The method of claim 6, wherein a stable iVCN per diploid genome is achieved when the iVCN peaks and remains unchanged or changes only within an acceptable error range for a period of more than 24 hours.

8. The method of claim 6, 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, at or about 1.0, or within an acceptable error range thereof.

9. 9. The method of any one of claims 1 to 8, 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.

10. 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 48-120 hours, inclusive, after exposure to the stimulatory reagent was initiated, and at harvest, the population of transformed cells has, on average, an integrated vector copy number (iVCN) of at or about 0.4-2.0 copies per diploid genome, inclusive. Including, Thereby producing a composition of engineered cells.

11. The method of any one of claims 1 to 10, wherein harvesting is performed at a time when the population of transformed cells has an iVCN of less than or about 2.0 copies per diploid genome on average.

12. 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.

13. 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; The method of any one of claims 1 to 12.

14. 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 the total recombinant protein-expressing CD4+ 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 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; 14. The method of claim 12 or 13.

15. 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. The method of any one of claims 2 to 14.

16. 20. The method of claim 1 or 15, wherein after the introduction, incubation is carried out for up to 72 hours.

17. 20. The method of claim 1 or 15, wherein after the introduction, incubation is carried out for up to 48 hours.

18. 20. The method of claim 1 or 15, wherein after the introduction, incubation is carried out for up to 24 hours.

19. 19. The method of any one of claims 1 and 15 to 18, wherein the incubation is carried out for at least 18 hours.

20. 20. The method of any one of claims 1 to 19, wherein the harvesting is performed within 96 hours after exposure to the stimulant has begun.

21. 20. The method of any one of claims 1 to 19, wherein the harvesting is performed within 72 hours after exposure to the stimulant has begun.

22. 20. The method of any one of claims 1 to 19, wherein the harvesting is performed within 48 hours after exposure to the stimulant has begun.

23. The method of any one of claims 1 to 22, wherein one or both of the exposing in (a) and the introducing in (b) are performed in the presence of one or more recombinant cytokines.

24. The method of any one of claims 1 to 22, wherein the exposing in (a) and the introducing in (b) are performed in the presence of one or more recombinant cytokines.

25. The method of any one of claims 15 to 24, wherein the incubation is carried out in the presence of one or more recombinant cytokines.

26. The method of any one of claims 15 to 24, wherein the incubation is carried out in a basal medium lacking one or more recombinant cytokines.

27. The method of any one of claims 5-9 and 12-26, 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+.

28. The method of any one of claims 5 to 9 and 12 to 27, wherein the naive-like T cells comprise CD27+CCR7+ cells.

29. The method of any one of claims 5-9 and 12-28, wherein the naive-like T cells comprise CCR7+CD45RA+ T cells.

30. 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, comprising 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 after 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.

31. The method of any one of claims 1 and 23-30, wherein the one or more recombinant cytokines are human.

32. The method of any one of claims 1 and 23-31, wherein the one or more recombinant cytokines are selected from recombinant IL-2, recombinant IL-7, and recombinant IL-15, optionally from 10 to 200 IU / mL of recombinant IL-2, from 100 IU / mL to 1,000 IU / mL of recombinant IL-7, and / or from 10 to 200 IU / mL of recombinant IL-15.

33. The method of any one of claims 1 and 23-32, wherein the one or more recombinant cytokines are or comprise recombinant IL-2, recombinant IL-7, and recombinant IL-15, optionally 10-200 IU / mL of recombinant IL-2, 100 IU / mL to 1,000 IU / mL of recombinant IL-7, and 10-200 IU / mL of recombinant IL-15.

34. The method of any one of claims 1 to 33, wherein one or both of the exposing in (a) and the introducing in (b) are performed in serum-free medium.

35. The method of any one of claims 1 to 34, wherein the exposing in (a) and the introducing in (b) are performed in serum-free medium.

36. The stimulating agent is (i) a primary 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. The method according to any one of claims 1, 6 to 9, 11, 13, 14, 16 to 24 and 32 to 35.

37. The stimulating agent is (i) a primary 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. The method of any one of claims 2 to 35.

38. 38. The method of claim 36 or 37, wherein one or both of the primary agent and secondary agent comprises an antibody or an antigen-binding fragment thereof.

39. The method of any one of claims 36 to 38, wherein the primary and secondary agents comprise antibodies, and optionally, the stimulatory reagent comprises an anti-CD3 antibody and an anti-CD28 antibody or antigen-binding fragment thereof.

40. The method of any one of claims 37 to 39, wherein the primary agent and secondary agent are present on or attached to the surface of a solid support.

41. 41. The method of claim 40, wherein the solid support is or comprises a bead.

42. 42. The method of claim 41, wherein the ratio of beads to cells is less than 3:

1.

43. 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 transduced population, the harvesting being performed between 48 and 120 hours, inclusive, after exposure to the stimulatory agent has commenced. Including, Thereby producing a composition of engineered cells.

44. The method of claims 41-43, wherein the ratio of beads to cells is 1:1 or about 1:

1.

45. 45. The method of any one of claims 41 to 44, wherein the beads have anti-CD3 and anti-CD28 antibodies or antigen-binding fragments thereof attached to them.

46. 40. The method of claim 36, 38 or 39, 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.

47. 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.

48. (a) Exposure to cells 10 6 48. The method of claim 1, 46 or 47, 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.

49. 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:

50. 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, CD3, and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule, the exposing being carried out under conditions for stimulating the T cells, thereby generating a stimulated population. A method for stimulating T cells, comprising:

51. 51. The method of claim 49 or 50, wherein the input composition comprises primary T cells.

52. The amount of stimulation reagent in the input composition is 6 The method of claims 48, 49 and 51, wherein the amount is between or about 0.1 μg and 2 μg per individual, inclusive.

53. 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.

53. The method of any one of claims 43 to 48 and 52.

54. 54. The method of claim 53, wherein the introducing is followed by incubation for up to 72 hours.

55. 54. The method of claim 53, wherein the introducing is followed by incubation for up to 48 hours.

56. 54. The method of claim 53, wherein the introducing is followed by incubation for up to 24 hours.

57. The method of any one of claims 43-48 and 52-56, wherein the harvesting is performed within 96 hours after exposure of the input composition to the stimulant is initiated.

58. The method of any one of claims 43-48 and 52-57, wherein the harvesting is performed within 72 hours after exposure of the input composition to the stimulant is initiated.

59. The method of any one of claims 43-48 and 52-58, wherein the harvesting is performed within 48 hours after exposure of the input composition to the stimulant is initiated.

60. The method of any one of claims 43-48 and 52-59, wherein one or both of the exposing in (a) and the introducing in (b) are performed in the presence of one or more recombinant cytokines.

61. The method of any one of claims 43-48 and 52-59, wherein the exposing in (a) and the introducing in (b) are carried out in the presence of one or more recombinant cytokines.

62. The method of any one of claims 43-48 and 52-61, wherein the incubation is carried out in the presence of one or more recombinant cytokines.

63. The method of any one of claims 43-48 and 52-61, wherein the incubation is carried out in a basal medium lacking one or more recombinant cytokines.

64. 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.

65. 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 specifically binds 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.

66. The amount of stimulation reagent in the input composition is 6 The method of any one of claims 48-63 and 65, wherein the amount of the IgG antibody is between 0.4 μg and 8 μg per cell, or between about 0.4 μg and 8 μg per cell, inclusive.

67. The amount of stimulation reagent in the input composition is 6 The method of any one of claims 48-63, 65 and 66, wherein the amount of the IgG antibody is between 0.8 μg and 4 μg per cell (inclusive).

68. The amount of stimulation reagent in the input composition is 6 The method of any one of claims 48-63 and 65-67, wherein the amount is at or about 0.8 μg per individual.

69. The amount of stimulating agent is 10 6 The method of any one of claims 48-63 and 65-68, wherein the amount is at or about 1.2 μg per individual.

70. The amount of stimulating agent is 10 6 The method of any one of claims 48-63 and 65-68, wherein the amount is at or about 1.8 μg per individual.

71. The method of any one of claims 60-70, wherein the one or more recombinant cytokines are human.

72. The method of any one of claims 60-71, 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, from 100 IU / mL to 1,000 IU / mL of recombinant IL-7, and / or from 10 to 200 IU / mL of recombinant IL-15.

73. The method of any one of claims 60-72, wherein the one or more recombinant cytokines are or comprise recombinant IL-2, recombinant IL-7, and 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 between 10 and 200 IU / mL of recombinant IL-15.

74. The method of any one of claims 1-48 and 52-73, wherein one or both of the exposing in (a) and the introducing in (b) are performed in serum-free medium.

75. The method of any one of claims 1-48 and 52-74, wherein the exposing in (a) and the introducing in (b) are performed in serum-free medium.

76. The method of any one of claims 1, 15-48 and 52-75, wherein the incubation is performed in serum-free medium.

77. The method of any one of claims 41-45, 53-64 and 71-76, wherein the ratio of beads to cells is at or about 2:1 to 0.5:

1.

78. The method of any one of claims 41-45, 53-64 and 71-77, wherein the ratio of beads to cells is 1:1 or about 1:

1.

79. 8. The method of any one of claims 41-45, 53-64 and 71-78, 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.

80. The method of any one of claims 41-45, 53-64 and 71-79, wherein the beads comprise a diameter of 4.5 μm or about 4.5 μm.

81. The method of any one of claims 41-45, 53-64 and 71-80, wherein the beads are inert.

82. The method of any one of claims 41-45, 53-64 and 71-81, wherein the beads are or comprise a polystyrene surface.

83. The method of any one of claims 41-45, 53-64 and 71-82, wherein the beads are paramagnetic or superparamagnetic.

84. exposing the cells to a magnetic field either after or during the incubation, thereby removing the stimulatory reagent from the cells.

84. The method of claim 83, further comprising, prior to harvesting:

85. The method of any one of claims 1, 46-63 and 65-76, 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 biologically active fragments thereof.

86. Each of the plurality of 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 The method of any one of claims 1, 46-63, 65-76 and 85, comprising:

87. 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 which 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. The method of any one of claims 1, 46-63, 65-76, 85 and 86, comprising:

88. The method of any one of claims 1, 46-63, 65-76 and 85-87, wherein the plurality of streptavidin mutein molecules comprises a sequence of amino acids set forth in SEQ ID NO: 63 or 68.

89. The method of any one of claims 46-63, 65-76 and 85-88, wherein the primary agent and the secondary agent each comprise a streptavidin-binding peptide.

90. Streptavidin-binding peptides 90. The method of claim 89, selected from the group consisting of:

91. The method of any one of claims 46-63, 65-76 and 85-90, wherein one or both of the primary agent and secondary agent comprises an antibody or an antigen-binding fragment thereof.

92. 92. The method of claim 91, wherein said one or more agents is or comprises a monovalent antibody fragment.

93. 93. The method of claim 91 or 92, wherein one or both of the primary and secondary agents is or comprises a Fab.

94. The oligomeric particle reagent comprises: a radius 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 1, 46-63, 65-76 and 85-93, comprising:

95. 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 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 g / mol The method of any one of claims 1, 46-63, 65-76 and 85-94, comprising a molecular weight of

96. 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 1, 46-63, 65-76 and 85-88, comprising:

97. 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; The method of any one of claims 1, 46-63, 65-76 and 85-96.

98. 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; The method of any one of claims 46-63, 65-76 and 85-97.

99. 99. The method of claim 98, wherein the substance is a free binding partner and / or a competitive reagent.

100. The method of claim 98 or 99, wherein the presence of the substance terminates or reduces a signal induced or modulated by the primary and secondary agents in the T cell.

101. 101. The method of any one of claims 97 to 100, wherein the substance is or comprises a streptavidin binding peptide, biotin or a biologically active fragment, or a biotin analog or a biologically active fragment.

102. The substance is or comprises a streptavidin-binding peptide, the streptavidin-binding peptide comprising: The method of claim 101, wherein the compound is selected from the group consisting of:

103. 102. The method of claim 101, wherein the substance is or comprises biotin or a biologically active fragment or a biotin analog or a biologically active fragment.

104. The method of any one of claims 97-103, wherein the agent is added at or about 42 hours to 120 hours, inclusive, after exposure to the oligomeric particle stimulation reagent has begun.

105. 105. The method of any one of claims 97-104, wherein said agent is added at or about 72 hours to 96 hours after exposure to oligomeric particle reagent begins.

106. 106. The method of any one of claims 97-105, wherein said substance is added at or about 48 hours after exposure to oligomeric particle reagent begins.

107. 107. The method of any one of claims 97-106, wherein said substance is added at or about 72 hours after exposure to oligomeric particle reagent begins.

108. 108. The method of any one of claims 97-107, wherein said substance is added at or about 96 hours after exposure to oligomeric particle reagent begins.

109. The method of any one of claims 97 to 108, wherein the substance is added after incubation and before harvesting.

110. 110. The method of any one of claims 97-109, wherein the substance is added during at least a portion of the incubation, and the incubation continues after the substance is added.

111. 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 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 performed 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.

112. Input composition is at least 300×10 6 The method of any one of claims 1-111, comprising viable primary T cells.

113. Input composition is 600 x 10 6 600 x 10 pieces or approx. 6 The method of any one of claims 1-112, comprising viable primary T cells.

114. 114. The method of any one of claims 1-113, 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.

115. The method of any one of claims 1-114, 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.

116. 116. The method of any one of claims 1-115, wherein the input composition comprises a ratio of CD4+ cells to CD8+ cells of between 1.5:1 and 2.0:

1.

117. 117. The method of any one of claims 1-116, wherein the input composition comprises a ratio of CD4+ cells to CD8+ cells of between 1.2:1 and 0.8:1, optionally a ratio of CD4+ T cells to CD8+ T cells of 1:1 or about 1:

1.

118. 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 of an oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules. 6 exposing the oligomeric particles to at or about 0.4 μg to 8 μg 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 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 a stimulated population comprising CD4+ and CD8+ T cells, the exposing being 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 performed 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 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, during at least a portion of the incubation, the addition being performed between 48 and 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.

119. The method of any one of claims 111-118, wherein the substance is added 48 hours or about 48 hours after exposure to the stimulating agent is initiated.

120. The method of any one of claims 111-118, wherein the substance is added 72 hours or about 72 hours after exposure to the stimulating agent is initiated.

121. The method of any one of claims 111-118, wherein the substance is added 96 hours or about 96 hours after exposure to the stimulating agent has begun.

122. 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 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 to CD8+ T cells of 2:1 to 1:2, and a ratio of at least 300×10 6 a stimulated population comprising CD4+ and CD8+ T cells, the exposing being 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 performed 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.

123. The method of any one of claims 111-122, 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.

124. The method of any one of claims 111-123, wherein the incubation is performed in a basal medium lacking recombinant cytokines.

125. 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 one of claims 1-124, comprising: viable primary T cells, or any value in between.

126. Input composition is at least 600×10 6 or at least about 600 x 10 6 The method of any one of claims 1-125, comprising viable primary T cells.

127. Input composition is 900 x 10 6 pcs or max 900 x 10 6 The method of any one of claims 1-126, comprising viable primary T cells.

128. Input composition is 900 x 10 6 pcs or approx. 900 x 10 6 The method of any one of claims 1-125, comprising viable primary T cells.

129. The method of any one of claims 44-59, 61-69, 81-113, 115 and 116, wherein the incubation is carried out for 24 hours or about 24 hours after the introducing.

130. 130. The method of any one of claims 1 and 15-129, wherein the incubation is carried out for 48 hours or about 48 hours after the introducing.

131. The method of any one of claims 1 and 15 to 130, wherein the incubation is carried out for 72 hours or about 72 hours after the introducing.

132. 132. The method of any one of claims 1-131, comprising enriching CD3+ T cells or CD4+ T cells and / or CD8+ T cells from the biological sample to produce an input composition prior to exposure to the stimulatory reagent.

133. The method of any one of claims 1-132, comprising enriching CD3+ T cells from a biological sample to produce an input composition prior to exposure to a stimulatory reagent.

134. 134. The method of claim 132 or 133, wherein the enrichment is by immunoaffinity-based selection.

135. The method of claim 134, wherein the immunoaffinity-based selection is performed by contacting the 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 and performing positive selection of CD3+ cells.

136. The method of any one of claims 1-132, comprising enriching CD4+ or CD8+ T cells from a biological sample to produce an input composition prior to exposure to a stimulatory reagent.

137. 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 claim 136.

138. The method of claim 136 or 137, wherein separate enriched populations of CD4+ T cells and CD8+ T cells are mixed, thereby producing the input composition.

139. 139. The method of claim 138, wherein the mixing is performed in a closed system, and optionally, the closed system is automated.

140. 140. The method of any one of claims 136-139, wherein enriching cells in the first and / or second selection comprises performing positive or negative selection based on expression of cell surface markers.

141. The method of any one of claims 136-140, wherein enriching for cells in the first or second selection comprises performing multiple positive or negative selection steps based on expression of cell surface markers or markers for enriching for CD4+ or CD8+ cells.

142. 142. The method of any one of claims 136-141, wherein enrichment of cells in the first and / or second selection comprises immunoaffinity-based selection.

143. 143. The method of claim 142, wherein enrichment of cells in the first and second selections comprises immunoaffinity-based selection.

144. The method of claim 142 or 143, wherein the immunoaffinity-based selection is performed by contacting the 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 and performing positive or negative selection of CD4+ or CD8+ cells.

145. 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.

145. The method of claim 135 or 144.

146. 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 claim 145.

147. 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; 147. The method of claim 145 or 146.

148. The method of any one of claims 145 to 147, further comprising the step of contacting the cells in the sample with the affinity chromatography matrix, followed by adding a competing reagent to cleave the bond between the binding partner and the binding reagent, thereby recovering the selected cells from the matrix.

149. 149. The method of claim 148, wherein the competitive reagent is biotin or a biotin analogue.

150. 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 150. The method of any one of claims 135 and 144-149, comprising:

151. One or more antibodies in one or more selections are -3 ~10 -7 in the range of about 10 -7 ~about 10 -10 The dissociation constant (K d The method of any one of claims 135 and 144-150, wherein the antibody has an affinity for a cell surface marker,

152. The method of any one of claims 135 and 144-151, wherein the chromatography matrix is ​​packed in a separation vessel that is a column.

153. Enrichment of CD4+ or CD8+ T cells from a biological sample (a) enrichment of CD4+ cells comprises 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) The method of any one of claims 136 to 152, comprising:

154. 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; The method of any one of claims 1 to 153.

155. 155. The method of claim 154, wherein each of the immunoaffinity reagents comprises an antibody or an antigen-binding fragment thereof.

156. The method of claim 154 or 155, wherein the immunoaffinity reagent is immobilized on the outer surface of a bead.

157. The method of claim 156, wherein the beads are magnetic beads.

158. The method of claim 157, wherein recovering cells bound to the first or second immunoaffinity reagent comprises exposing the cells to a magnetic field.

159. The method of any one of claims 132-158, wherein the biological sample comprises primary T cells obtained from a subject, optionally a human subject.

160. The method of any one of claims 132-159, 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 leukapheresis product.

161. The method of any one of claims 132-160, wherein the biological sample is an apheresis product or a leukapheresis product that has been previously cryo-frozen prior to enrichment.

162. The method of any one of claims 1-48 and 52-161, wherein the harvesting is performed 96 hours or about 96 hours after exposure to the stimulating agent is initiated.

163. The method of any one of claims 1-48 and 52-161, 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.

164. The method of any one of claims 1-48 and 52-161, wherein the harvesting is performed 72 hours or about 72 hours after exposure to the stimulating agent is initiated.

165. The method of any one of claims 1-48 and 52-161, wherein the harvesting is performed 48 hours or about 48 hours after exposure to the stimulating agent is initiated.

166. The method of any one of claims 1 and 12-165, wherein harvesting is performed at the time integrated vector is detected in the genome but before achieving stable iVCN per diploid genome.

167. The method of any one of claims 1 and 12-166, wherein harvesting is performed at a time 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.

168. The method of any one of claims 1 and 12-167, wherein the harvesting is performed at a time when the population of transformed cells has an iVCN of less than or about 2.0 copies per diploid genome on average.

169. The method of any one of claims 1-168, wherein harvesting the cells comprises removing cellular debris by rinsing or washing the cells.

170. The method of any one of claims 1 and 30-169, wherein the cells are harvested at a time when the percentage of naive-like cells is greater than or greater than about 60% among all T cells in the population, among all CD4+ T cells in said population, or among all CD8+ T cells or cells expressing a recombinant protein thereof in said population.

171. The method of any one of claims 1 and 30-169, 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.

172. 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% 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; or the percentage of naive-like 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; 172. The method of claim 170 or 171.

173. 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 claim 171.

174. The method of any one of claims 170-173, wherein the naive-like T cells, or T cells that are surface positive for a marker expressed on naive-like T cells, are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+.

175. The method of any one of claims 170-174, wherein the naive-like T cells comprise CD27+CCR7+ T cells.

176. The method of any one of claims 170-175, wherein the naive-like T cells comprise CCR7+CD45RA+ T cells.

177. The method of any one of claims 1-176, further comprising formulating the cells of the engineered cell composition for cryopreservation and / or administration to a subject, optionally in the presence of a pharma- ceutically acceptable excipient.

178. The method of claim 177, wherein the cells of the engineered cell composition are formulated in the presence of a cryoprotectant.

179. 179. The method of claim 178, wherein the cryoprotectant comprises DMSO.

180. The method of any one of claims 177-179, wherein the cells of the engineered cell composition are formulated in a container, optionally a vial or bag.

181. The method of any one of claims 1 to 180, wherein a viral vector comprising a heterologous polynucleotide encoding a recombinant protein is introduced into the T cell.

182. The method of claim 181, wherein the viral vector is a retroviral vector.

183. The method of claim 180 or 181, wherein the viral vector is a lentiviral vector or a gamma retroviral vector.

184. The method of any one of claims 1-183, wherein said introducing is carried out in the presence of a transduction adjuvant.

185. 185. The method of claim 184, 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.

186. 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. The method of any one of claims 1 to 185,

187. The method of claim 186, wherein the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer.

188. The method of claim 187, wherein the target antigen is a tumor antigen.

189. 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), and 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 (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-related protein 1 (TRP1, also known as TYRP1 or gp75), tyrosinase-related tandem The method of any one of claims 186 to 188, wherein the antigen is selected from among 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.

190. The method of any one of claims 1-48 or 52-189, wherein the recombinant protein is or comprises a functional non-TCR antigen receptor or a TCR or an antigen-binding fragment thereof.

191. The method of any one of claims 1-48 or 52-190, wherein the recombinant protein is a chimeric antigen receptor (CAR).

192. 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 The method of any one of claims 34-42 or 74-191, wherein the method is serum-free.

193. The method of any one of claims 1 and 26-192, 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.

194. The method of any one of claims 1 and 26-191 and 193, wherein the basal medium lacking one or more recombinant cytokines is serum-free.

195. The method of any one of claims 1, 26-191 and 193-194, 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.

196. 203. A therapeutic T cell composition produced by the method of any one of claims 1-195.

197. On average, for a plurality of compositions produced by the method, 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.

197. The therapeutic T cell composition of claim 196, wherein at 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.

198. The therapeutic T cell composition of claim 196, wherein on average in a plurality of compositions produced by said 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 the 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.

199. The therapeutic T cell composition of claim 196, 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.

200. The therapeutic T cell composition of claim 197 or 199, wherein the marker expressed on naive-like T cells is selected from the group consisting of CD45RA, CD27, CD28, and CCR7.

201. The therapeutic T cell composition of any one of claims 197, 199 and 200, 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+.

202. 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 one of claims 197 and 199-201, wherein at least 70%, 80%, 85%, or 90% of the cells are CD27+CCR7+.

203. 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 one of claims 197 and 199-202, wherein at least 50%, 60%, 70%, 80%, 85%, or 90% of the cells are CD27+CCR7+.

204. 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 one of claims 197 and 199-203, wherein at least 50%, 60%, 70%, 80%, 85%, or 90% of the cells are CD27+CCR7+.

205. 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.

206. The therapeutic T cell composition of any one of claims 196-205, 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 said composition are CD3+ T cells.

207. The therapeutic T cell composition of any one of claims 196-206, 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 said composition are CD4+ T cells and CD8+ T cells.

208. 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 + The therapeutic T cell composition of any one of claims 205-207, wherein at least, or at least about 60% of the cells are CD27+CCR7+.

209. at least 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, The therapeutic T cell composition of any one of claims 205-208, wherein, or about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are CD27+CCR7+.

210. The therapeutic T cell composition of any one of claims 197-209, wherein the ratio of receptor+ / CD4+ T cells to receptor+ / CD8+ T cells in said composition is from about 1:3 to about 3:

1.

211. The therapeutic T cell composition of any one of claims 196-210, wherein the ratio of receptor+ / CD4+ T cells to receptor+ / CD8+ T cells in said composition is from about 1:2 to about 2:

1.

212. The therapeutic T cell composition of any one of claims 196-211, wherein the ratio of receptor+ / CD4+ T cells to receptor+ / CD8+ T cells in said composition is 1:1 or about 1:

1.

213. 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.

213. The therapeutic composition of any one of claims 196-212, which is or comprises:

214. The therapeutic composition of any one of claims 196-213, wherein the recombinant protein is a chimeric antigen receptor (CAR).

215. 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 10 6 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 claims 196-214, wherein each of the two end values ​​is inclusive.

216. The therapeutic composition of any one of claims 196-215, wherein the amount of said composition is between 1.0 mL and 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.

217. An article of manufacture comprising the composition of any one of claims 196-216 and instructions for administering said composition to a subject.

218. The article of manufacture of claim 217, 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.

219. A method of treating a subject having or suspected of having a disease or condition comprising administering to said subject a dose of T cells from the composition of any one of claims 196-216.

220. The dose of T cells is 1×10 4 pieces ~ 50×10 6 pcs or approx. 1×10 4 pieces ~ 50×10 6 220. The method of claim 219, comprising (inclusive) T cells.

221. The method of claim 219 or 220, wherein said 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.

222. The method of any one of claims 219-221, wherein the disease or condition is cancer.

223. The composition of any one of claims 196-216 for use in a method of treating a subject having a disease or condition, optionally wherein the disease or condition is cancer.

224. 220. Use of the composition of any one of claims 196-216 for the manufacture of a medicament for treating a subject having a disease or condition, optionally wherein the disease or condition is cancer.

225. 225. The composition for use of claim 223 or the use of claim 224, wherein the cells of the composition express a recombinant receptor that specifically recognizes or 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.

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