Methods for non-viral production of engineered immune cells

Abstract

Provided herein is a method for producing genetically engineered immune cells, such as T cells. In some aspects, immune cells are genetically engineered by targeted integration of transgenes into the target site of genes in immune cells. In some embodiments, the genetically engineered immune cells are produced from whole blood samples. In some aspects, immune cells are genetically engineered after on-column stimulation of immune cells. In some aspects, immune cells are genetically engineered by non-viral gene delivery methods. Also provided herein are related cells, compositions, and uses. TIFF2026504491000039.tif70170

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 443,358, entitled "METHODS FOR NON-VIRAL MANUFACTURING OF ENGINEERED IMMUNE CELLS," filed February 3, 2023; U.S. Provisional Patent Application No. 63 / 443,702, entitled "METHODS FOR NON-VIRAL MANUFACTURING OF ENGINEERED IMMUNE CELLS," filed February 6, 2023; and U.S. Provisional Patent Application No. 63 / 465,200, entitled "METHODS FOR NON-VIRAL MANUFACTURING OF ENGINEERED IMMUNE CELLS," filed May 9, 2023, which are incorporated by reference in their entireties for all purposes.

[0002] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING This application is submitted with an electronic Sequence Listing, which is provided as a file named 735042027240SeqList.xml, created on February 1, 2024, and having a size of 347,616 bytes. The information in this electronic Sequence Listing is incorporated herein by reference in its entirety.

[0003] Field The present disclosure relates to a method for producing genetically engineered immune cells, such as T cells. In some aspects, immune cells are genetically engineered by targeted integration of a transgene into a gene target site in immune cells. In some embodiments, genetically engineered immune cells are produced from a whole blood sample. In some aspects, immune cells are genetically engineered following on-column stimulation of immune cells. In some aspects, immune cells are genetically engineered by non-viral gene delivery methods. Also provided herein are related cells, compositions, and uses. [Background technology]

[0004] background A variety of cell therapies are available for treating diseases and pathological conditions.Cell therapy includes methods that utilize immune cells, such as T cells (for example, CD4+ and CD8+ T cells), and these immune cells can be genetically engineered with recombinant receptors, such as chimeric antigen receptors (CARs).For example, there is a need for improved methods for producing engineered cells suitable for use in cell therapy.Methods, cells, compositions, and uses that meet this need are provided. Summary of the Invention

[0005] overview In some aspects, provided herein are methods for producing genetically engineered T cells, the methods comprising: (a) applying a whole blood sample comprising a plurality of T cells to a stationary phase within an interior cavity of a chromatography column, wherein the stationary phase comprises a selection agent that specifically binds to a selection marker expressed on the surface of the plurality of T cells, wherein the selection agent specifically binds to the selection marker, thereby immobilizing the plurality of T cells on the stationary phase; (b) adding a T cell stimulatory reagent to the plurality of T cells immobilized on the stationary phase, wherein the T cell stimulatory reagent comprises: (i) a primary agent that specifically binds to a member of the TCR / CD3 complex, and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule. (c) incubating the plurality of T cells immobilized on the stationary phase in the presence of a T cell stimulatory reagent under conditions for stimulating the T cells, the plurality of T cells; (d) collecting the T cells, the plurality of T cells that are no longer immobilized after the incubating step, from the chromatography column; and (e) introducing, by non-viral gene delivery, a nucleic acid molecule comprising a transgene, under conditions for targeted integration of the transgene encoding the recombinant protein into a genetic target site in one or more of the collected T cells, whereby genetically engineered T cells that express the recombinant protein are produced.

[0006] In some of either embodiment, the method includes further incubating the collected T cells prior to introduction of the nucleic acid molecule.

[0007] In some of either embodiment, the nucleic acid molecule is a DNA molecule. In some embodiments, the nucleic acid molecule is a modified DNA molecule. In some embodiments, the nucleic acid molecule is modified to enhance its stability.

[0008] In some of either embodiment, the nucleic acid molecule is a single-stranded DNA molecule or a double-stranded DNA molecule. In some of either embodiment, the nucleic acid molecule is a single-stranded DNA molecule. In some of either embodiment, the nucleic acid molecule is a double-stranded DNA molecule.

[0009] In some of any of the embodiments, the targeted integration is by Programmable Addition via Site-specific Targeting Element (PASTE). In some of any of the embodiments, PASTE comprises introducing one or more gene editing agents to edit a gene in one or more of the collected T cells.

[0010] In some of any of the embodiments, the targeted integration is by homology-directed repair (HDR). In some of any of the embodiments, HDR comprises introducing one or more gene editing agents to induce gene disruption of a gene in one or more of the collected T cells.

[0011] In some of any of the embodiments, introducing the nucleic acid molecule and / or one or more gene editing agents is by electroporation. In some of any of the embodiments, introducing the nucleic acid molecule is by electroporation. In some of any of the embodiments, introducing the one or more gene editing agents is by electroporation. In some of any of the embodiments, introducing the nucleic acid molecule and one or more gene editing agents is by electroporation.

[0012] In some of either embodiment, the conditions for targeted integration include culturing the collected T cells under conditions for integration of the transgene into the target site.

[0013] Also provided in some embodiments are methods for producing genetically engineered T cells, the methods comprising: (a) applying a whole blood sample comprising a plurality of T cells to a stationary phase within an interior cavity of a chromatography column, the stationary phase comprising a selection agent that specifically binds to a selection marker expressed on the surface of the plurality of T cells, wherein the selection agent specifically binds to the selection marker, thereby immobilizing the plurality of T cells on the stationary phase; (b) adding a T cell stimulatory reagent to the plurality of T cells immobilized on the stationary phase, the T cell stimulatory reagent comprising (i) a primary agent that specifically binds to a member of the TCR / CD3 complex, and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule; (c) incubating the plurality of T cells immobilized on the stationary phase in the presence of the T cell stimulatory reagent under conditions to stimulate the T cells, the plurality of T cells; and (d) removing the plurality of T cells that are no longer immobilized after the incubating step. (e) further incubating the collected T cells; (f) after the further incubation, introducing into the T cells, the collected T cells, (i) a nucleic acid molecule comprising a transgene encoding a recombinant protein, wherein the nucleic acid molecule is a DNA molecule, and introduction of the nucleic acid molecule is by non-viral gene delivery, and (ii) one or more gene editing agents to induce gene disruption of a gene in the T cells, the collected T cells, wherein introduction of the nucleic acid molecule and / or the one or more gene editing agents is by electroporation; and (g) culturing the collected T cells under conditions to integrate the transgene into a target site of the gene in one or more of the collected T cells by homology directed repair (HDR), whereby the method produces genetically engineered T cells that express the recombinant protein.

[0014] In some of any of the embodiments, introducing the nucleic acid molecule is by electroporation. In some of any of the embodiments, introducing the one or more gene editing agents is by electroporation. In some of any of the embodiments, introducing the nucleic acid molecule and the one or more gene editing agents is by electroporation.

[0015] Also provided in some embodiments are methods for producing genetically engineered T cells, the methods comprising: (a) applying a whole blood sample comprising a plurality of T cells to a stationary phase within an interior cavity of a chromatography column, the stationary phase comprising a selection agent that specifically binds to a selection marker expressed on the surface of the plurality of T cells, wherein the selection agent specifically binds to the selection marker, thereby immobilizing the plurality of T cells on the stationary phase; (b) adding a T cell stimulatory reagent to the plurality of T cells immobilized on the stationary phase, the T cell stimulatory reagent comprising (i) a primary agent that specifically binds to a member of the TCR / CD3 complex, and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule; (c) incubating the plurality of T cells immobilized on the stationary phase in the presence of the T cell stimulatory reagent under conditions to stimulate the T cells, the plurality of T cells; and (d) removing the plurality of T cells that are no longer immobilized after the incubating step. (e) further incubating the collected T cells; (f) after the further incubation, introducing into the T cells, the collected T cells, (i) a nucleic acid molecule comprising a transgene encoding a recombinant protein, wherein the nucleic acid molecule is a DNA molecule, and introduction of the nucleic acid molecule is by non-viral gene delivery, and (ii) one or more gene editing agents for inducing gene disruption of a gene in the T cells, the collected T cells, wherein introduction of the nucleic acid molecule and the one or more gene editing agents is by electroporation; and (g) culturing the collected T cells under conditions for integration of the transgene into a target site of the gene in one or more of the collected T cells by homology directed repair (HDR), whereby the method produces genetically engineered T cells that express the recombinant protein.

[0016] In some embodiments, the nucleic acid molecule is a modified DNA molecule. In some embodiments, the nucleic acid molecule is modified to enhance its stability.

[0017] In some of either embodiment, the nucleic acid molecule is a single-stranded DNA molecule or a double-stranded DNA molecule. In some of either embodiment, the nucleic acid molecule is a single-stranded DNA molecule. In some of either embodiment, the nucleic acid molecule is a double-stranded DNA molecule.

[0018] In some of any of the embodiments, the nucleic acid molecule is a double-stranded DNA molecule, a naked DNA molecule, and / or a closed DNA molecule. In some of any of the embodiments, the nucleic acid molecule is a double-stranded DNA molecule. In some of any of the embodiments, the nucleic acid molecule is a naked DNA molecule. In some of any of the embodiments, the nucleic acid molecule is a closed DNA molecule.

[0019] In some of any of the embodiments, the nucleic acid molecule is a naked DNA molecule and / or a closed DNA molecule. In some of any of the embodiments, the nucleic acid molecule is a naked DNA molecule. In some of any of the embodiments, the nucleic acid molecule is a closed DNA molecule. In some of any of the embodiments, the nucleic acid molecule is a naked closed DNA molecule.

[0020] In some of any of the embodiments, the nucleic acid molecule is a naked closed double-stranded DNA molecule. In some of any of the embodiments, the nucleic acid molecule is a closed linear duplex (CELiD) DNA molecule, a minicircle DNA molecule, a minimalistic immunologically-defined gene expression (MIDGE) DNA molecule, a ministring DNA molecule, a dumbbell-shaped linear double-stranded closed DNA molecule, or a doggybone™ DNA molecule. In some of any of the embodiments, the nucleic acid molecule is a closed linear double-stranded (CELiD) DNA molecule. In some of any of the embodiments, the nucleic acid molecule is a minicircle DNA molecule. In some of any of the embodiments, the nucleic acid molecule is a minimalistic immunologically-defined gene expression (MIDGE) DNA molecule. In some of any of the embodiments, the nucleic acid molecule is a ministring DNA molecule. In some of any of the embodiments, the nucleic acid molecule is a dumbbell-shaped linear double-stranded closed DNA molecule. In some of any of the embodiments, the nucleic acid molecule is a doggybone™ DNA molecule.

[0021] In some embodiments, the T cell stimulating reagent is added to the cell culture medium. In some embodiments, the cell culture medium is a basal medium. In some embodiments, the cell culture medium is a serum-free medium. In some embodiments, the cell culture medium is cytokine-free or contains recombinant IL-2, IL-7, and IL-15. In some embodiments, the cell culture medium is cytokine-free. In some embodiments, the cell culture medium contains recombinant IL-2, IL-7, and IL-15.

[0022] In some of the embodiments, the T cell stimulating reagent comprises an oligomer of streptavidin or streptavidin mutein molecules; a primary agent; and a secondary agent. In some of the embodiments, the primary agent binds to the oligomeric streptavidin or streptavidin mutein molecules. In some of the embodiments, the primary agent comprises a first streptavidin binding partner that binds to the oligomeric streptavidin or streptavidin mutein molecules. In some of the embodiments, the secondary agent binds to the oligomeric streptavidin or streptavidin mutein molecules. In some of the embodiments, the secondary agent comprises a second streptavidin binding partner that binds to the oligomeric streptavidin or streptavidin mutein molecules.

[0023] In some of either embodiment, the T cell stimulating reagent comprises an oligomer of streptavidin or streptavidin mutein molecule; the primary agent comprises a first streptavidin binding partner that binds to the oligomeric streptavidin or streptavidin mutein molecule; and the secondary agent comprises a second streptavidin binding partner that binds to the oligomeric streptavidin or streptavidin mutein molecule.

[0024] In some of either embodiment, the primary agent is an anti-CD3 agent. In some embodiments, the secondary agent is an anti-CD28 agent.

[0025] In some of either embodiment, the T cell stimulating reagent is in a soluble form.

[0026] Such T cell stimulating reagents are further described below and throughout the description provided.

[0027] In some of the embodiments, the T cells 10 are a plurality of T cells, each including the respective endpoints, immobilized on the stationary phase or expected to be immobilized on the stationary phase.6 The T cell stimulatory reagent is added in an amount of 0.1 μg to 20 μg, 0.4 μg to 8 μg, 0.8 μg to 4 μg, or 1 μg to 2 μg per cell. In some of either embodiment, the T cell stimulatory reagent is added to a plurality of T cells, 10 of which are immobilized on a stationary phase or are expected to be immobilized on a stationary phase. 6 In some of the embodiments, the T cell stimulatory reagent is added in an amount of 0.1 μg to 20 μg, or about 0.1 μg to 20 μg, inclusive, per T cell. ... inclusive, per T cell. In some of the embodiments, the T cell stimulatory reagent is added in an amount of 0.1 μg to 20 μg, inclusive, per T cell. 6 In some of the embodiments, the T cell stimulatory reagent is added in an amount of 0.4 μg to 8 μg, or about 0.4 μg to 8 μg, inclusive, per T cell. In some of the embodiments, the T cell stimulatory reagent is added in an amount of ... 6 In some of the embodiments, the T cell stimulatory reagent is added in an amount of 0.8 μg to 4 μg, or about 0.8 μg to 4 μg, inclusive, per T cell. In some of the embodiments, the T cell stimulatory reagent is added in an amount of ... 6 The amount added is 1 μg to 2 μg, or about 1 μg to 2 μg (inclusive) per piece.

[0028] In some of either embodiment, the T cell stimulatory reagent comprises a plurality of T cells, 10 T cells, which are expected to be immobilized on a stationary phase. 6 In some of the embodiments, the T cell stimulatory reagent is added in an amount of 0.1 μg to 20 μg, or about 0.1 μg to 20 μg, inclusive, per T cell. In some of the embodiments, the T cell stimulatory reagent is added in an amount of 0.1 μg to 20 μg, inclusive, per T cell, which is the plurality of T cells expected to be immobilized on the stationary phase. 6 In some of the embodiments, the T cell stimulatory reagent is added in an amount of 0.4 μg to 8 μg, or about 0.4 μg to 8 μg, inclusive, per T cell. In some of the embodiments, the T cell stimulatory reagent is added in an amount of ... 6In some of the embodiments, the T cell stimulatory reagent is added in an amount of 0.8 μg to 4 μg, or about 0.8 μg to 4 μg, inclusive, per T cell. In some of the embodiments, the T cell stimulatory reagent is added in an amount of ... 6 The amount added is 1 μg to 2 μg, or about 1 μg to 2 μg (inclusive) per piece.

[0029] In some of any of the embodiments, the binding capacity of the stationary phase is 500 to 5 billion or about 500 to 5 billion T cells expressing the selection marker, 500 to 3 billion or about 500 to 3 billion T cells expressing the selection marker, or 1 to 2 billion or about 1 to 2 billion T cells expressing the selection marker, inclusive. In some of any of the embodiments, the binding capacity of the stationary phase is 500 to 5 billion or about 500 to 5 billion T cells expressing the selection marker, inclusive. In some of any of the embodiments, the binding capacity of the stationary phase is 500 to 3 billion or about 500 to 3 billion T cells expressing the selection marker, inclusive. In some of any of the embodiments, the binding capacity of the stationary phase is 1 to 2 billion or about 1 to 2 billion T cells expressing the selection marker, inclusive.

[0030] In some of the embodiments, the T cell stimulating reagent is added in an amount of 0.1 mg to 20 mg, 0.4 mg to 8 mg, 0.8 mg to 4 mg, or 1 mg to 3 mg, or about 0.1 mg to 20 mg, 0.4 mg to 8 mg, 0.8 mg to 4 mg, or 1 mg to 3 mg, inclusive. In some of the embodiments, the T cell stimulating reagent is added in an amount of 0.1 mg to 20 mg, or about 0.1 mg to 20 mg, inclusive. In some of the embodiments, the T cell stimulating reagent is added in an amount of 0.4 mg to 8 mg, or about 0.4 mg to 8 mg, inclusive. In some of the embodiments, the T cell stimulating reagent is added in an amount of 0.8 mg to 4 mg, or about 0.8 mg to 4 mg, inclusive. In some of the embodiments, the T cell stimulating reagent is added in an amount of 1 mg to 3 mg, or about 1 mg to 3 mg, inclusive. In some of either embodiment, the T cell stimulating reagent is added in an amount of 1 mg to 2 mg, or about 1 mg to 2 mg, inclusive.

[0031] In some of any of the embodiments, the step of adding the T cell stimulating reagent occurs within 60 minutes, 30 minutes, or 15 minutes, or within about 60 minutes, 30 minutes, or 15 minutes, after addition of the sample. In some of any of the embodiments, the step of adding the T cell stimulating reagent occurs within 60 minutes or within about 60 minutes, after addition of the whole blood sample. In some of any of the embodiments, the step of adding the T cell stimulating reagent occurs within 30 minutes or within about 30 minutes, after addition of the whole blood sample. In some of any of the embodiments, the step of adding the T cell stimulating reagent occurs within 15 minutes or within about 15 minutes, after addition of the whole blood sample.

[0032] In some embodiments, the incubating step is carried out in cell culture medium. In some embodiments, the cell culture medium is a basal medium. In some embodiments, the cell culture medium is a serum-free medium. In some embodiments, the cell culture medium is cytokine-free or comprises recombinant IL-2, IL-7, and IL-15. In some embodiments, the cell culture medium is cytokine-free. In some embodiments, the cell culture medium comprises recombinant IL-2, IL-7, and IL-15.

[0033] In some of either embodiment, the incubating step is carried out at a temperature of 35° C. to 39° C., or about 35° C. to about 39° C. In some of either embodiment, the incubating step is carried out at a temperature of 37° C. or about 37° C.

[0034] In some embodiments, the incubating step is carried out for 0.5 to 8 hours, 2 to 6 hours, or 3 to 5 hours, or for about 0.5 to 8 hours, 2 to 6 hours, or 3 to 5 hours, inclusive. In some embodiments, the incubating step is carried out for 0.5 to 8 hours, or for about 0.5 to 8 hours, inclusive. In some embodiments, the incubating step is carried out for 2 to 6 hours, or for about 2 to 6 hours, inclusive. In some embodiments, the incubating step is carried out for 3 to 5 hours, or for about 3 to 5 hours, inclusive. In some embodiments, the incubating step is carried out for 4 hours or about 4 hours.

[0035] In some of either embodiment, the collecting step includes adding a wash buffer to the stationary phase to collect the T cells, the plurality of T cells.

[0036] In some embodiments, the wash buffer is cell culture medium. In some embodiments, the cell culture medium is basal medium. In some embodiments, the cell culture medium is serum-free medium. In some embodiments, the cell culture medium is cytokine-free or contains recombinant IL-2, IL-7, and IL-15. In some embodiments, the cell culture medium is cytokine-free. In some embodiments, the cell culture medium contains recombinant IL-2, IL-7, and IL-15.

[0037] In some of either embodiment, the wash buffer does not contain a competitor. In some of either embodiment, the competitor is biotin.

[0038] In some of the embodiments, the harvesting step occurs 0.5 to 8 hours, 2 to 6 hours, or 3 to 5 hours, or about 0.5 to 8 hours, 2 to 6 hours, or 3 to 5 hours, inclusive, after addition of the T cell stimulating reagent. In some of the embodiments, the harvesting step occurs 0.5 to 8 hours, or about 0.5 to 8 hours, inclusive, after addition of the T cell stimulating reagent. In some of the embodiments, the harvesting step occurs 2 to 6 hours, or about 2 to 6 hours, inclusive, after addition of the T cell stimulating reagent. In some of the embodiments, the harvesting step occurs 3 to 5 hours, or about 3 to 5 hours, inclusive, after addition of the T cell stimulating reagent. In some of the embodiments, the harvesting step occurs 4 hours or about 4 hours after addition of the T cell stimulating reagent.

[0039] In some of either embodiment, the further incubation step is carried out in the presence of a T cell stimulating reagent.

[0040] In some embodiments, the further incubation step is performed in cell culture medium. In some embodiments, the cell culture medium is a basal medium. In some embodiments, the cell culture medium is a serum-free medium. In some embodiments, the cell culture medium is cytokine-free or contains recombinant IL-2, IL-7, and IL-15. In some embodiments, the cell culture medium is cytokine-free. In some embodiments, the cell culture medium contains recombinant IL-2, IL-7, and IL-15.

[0041] In some of either embodiment, the further incubation step is carried out at a temperature of 35° C. to 39° C., or about 35° C. to about 39° C. In some of either embodiment, the further incubation step is carried out at a temperature of 37° C. or about 37° C.

[0042] In some embodiments, the further incubation step is carried out for 10 to 30 hours, 16 to 24 hours, or 18 to 22 hours, or for about 10 to 30 hours, 16 to 24 hours, or 18 to 22 hours, inclusive. In some embodiments, the further incubation step is carried out for 10 to 30 hours, or for about 10 to 30 hours, inclusive. In some embodiments, the further incubation step is carried out for 16 to 24 hours, or for about 16 to 24 hours, inclusive. In some embodiments, the further incubation step is carried out for 18 to 22 hours, or for about 18 to 22 hours, inclusive. In some embodiments, the further incubation step is carried out for 20 hours or about 20 hours.

[0043] In some of any of the embodiments, the method includes removing the T cell stimulating reagent from the collected T cells before introducing the one or more gene editing agents. In some of any of the embodiments, the method includes removing the T cell stimulating reagent from the collected T cells before introducing the nucleic acid molecule. In some of any of the embodiments, the removing step is performed after a further incubation step. In some of any of the embodiments, the removing step includes washing the collected T cells.

[0044] In some of any of the embodiments, the T cell stimulating reagent comprises an oligomer of streptavidin or streptavidin mutein molecule; the primary agent comprises a first streptavidin binding partner that binds to the oligomeric streptavidin or streptavidin mutein molecule; and the secondary agent comprises a second streptavidin binding partner that binds to the oligomeric streptavidin or streptavidin mutein molecule; and the method comprises disrupting binding between the first and second streptavidin binding partners and the streptavidin or streptavidin mutein molecule prior to introduction of the one or more gene editing agents. In some of either embodiment, the T cell stimulating reagent comprises an oligomer of streptavidin or streptavidin mutein molecule; the primary agent comprises a first streptavidin binding partner that binds to the oligomeric streptavidin or streptavidin mutein molecule; the secondary agent comprises a second streptavidin binding partner that binds to the oligomeric streptavidin or streptavidin mutein molecule; and the method includes disrupting binding between the first and second streptavidin binding partners and the streptavidin or streptavidin mutein molecule prior to introducing the nucleic acid molecule. In some of either embodiment, the disrupting step is performed after a further incubation step. In some of either embodiment, the disrupting step is by adding to the collected T cells a competitor that competes for binding between the first and second streptavidin binding partners and the streptavidin or streptavidin mutein molecule. In some of either embodiment, the competitor is biotin.

[0045] In some of any of the embodiments, the step of introducing one or more gene editing agents occurs before or simultaneously with the introduction of the nucleic acid molecule. In some of any of the embodiments, the step of introducing one or more gene editing agents occurs before the introduction of the nucleic acid molecule. In some of any of the embodiments, the step of introducing one or more gene editing agents occurs simultaneously with the introduction of the nucleic acid molecule.

[0046] In some of any of the embodiments, the step of introducing the one or more gene editing agents occurs 12 to 36 hours, 18 to 30 hours, or 22 to 26 hours, or about 12 to 36 hours, 18 to 30 hours, or 22 to 26 hours, inclusive, after addition of the T cell stimulating reagent. In some of any of the embodiments, the step of introducing the one or more gene editing agents occurs 12 to 36 hours, or about 12 to 36 hours, inclusive, after addition of the T cell stimulating reagent. In some of any of the embodiments, the step of introducing the one or more gene editing agents occurs 18 to 30 hours, or about 18 to 30 hours, inclusive, after addition of the T cell stimulating reagent. In some of any of the embodiments, the step of introducing the one or more gene editing agents occurs 22 to 26 hours, or about 22 to 26 hours, inclusive, after addition of the T cell stimulating reagent. In some of either embodiment, the step of introducing the one or more gene editing agents occurs 24 hours or about 24 hours after addition of the T cell stimulating reagent.

[0047] In some embodiments, the nucleic acid molecule is introduced into a cell culture medium containing the nucleic acid molecule. In some embodiments, the cell culture medium is a basal medium. In some embodiments, the cell culture medium is a serum-free medium. In some embodiments, the cell culture medium is cytokine-free or contains recombinant IL-2, IL-7, and IL-15. In some embodiments, the cell culture medium is cytokine-free. In some embodiments, the cell culture medium contains recombinant IL-2, IL-7, and IL-15.

[0048] In some of the embodiments, the step of introducing the nucleic acid molecule is performed 12 to 36 hours, 18 to 30 hours, or 22 to 26 hours, or about 12 to 36 hours, 18 to 30 hours, or 22 to 26 hours, inclusive, after addition of the T cell stimulating reagent. In some of the embodiments, the step of introducing the nucleic acid molecule is performed 12 to 36 hours, or about 12 to 36 hours, inclusive, after addition of the T cell stimulating reagent. In some of the embodiments, the step of introducing the nucleic acid molecule is performed 18 to 30 hours, or about 18 to 30 hours, inclusive, after addition of the T cell stimulating reagent. In some of the embodiments, the step of introducing the nucleic acid molecule is performed 22 to 26 hours, or about 22 to 26 hours, inclusive, after addition of the T cell stimulating reagent. In some of either embodiment, the step of introducing the nucleic acid molecule occurs 24 hours or about 24 hours after addition of the T cell stimulatory reagent.

[0049] In some of either embodiment, the culturing is performed in the presence of a nucleic acid molecule.

[0050] In some embodiments, the culturing is performed in a cell culture medium. In some embodiments, the cell culture medium is a basal medium. In some embodiments, the cell culture medium is a serum-free medium. In some embodiments, the cell culture medium is cytokine-free or contains recombinant IL-2, IL-7, and IL-15. In some embodiments, the cell culture medium is cytokine-free. In some embodiments, the cell culture medium contains recombinant IL-2, IL-7, and IL-15.

[0051] In some of either embodiment, the culture is carried out at a temperature of 35° C. to 39° C., or about 35° C. to about 39° C. In some of either embodiment, the culture is carried out at a temperature of 37° C. or about 37° C.

[0052] In some embodiments, culturing is carried out for 12 to 36 hours, 18 to 30 hours, or 22 to 26 hours, or for about 12 to 36 hours, 18 to 30 hours, or 22 to 26 hours (both inclusive). In some embodiments, culturing is carried out for 12 to 36 hours or about 12 to 36 hours (both inclusive). In some embodiments, culturing is carried out for 18 to 30 hours or about 18 to 30 hours (both inclusive). In some embodiments, culturing is carried out for 22 to 26 hours or about 22 to 26 hours (both inclusive). In some embodiments, culturing is carried out for 24 hours or about 24 hours.

[0053] In some of either embodiment, the method includes harvesting the engineered T cells that express the recombinant protein.

[0054] In some embodiments, the collecting step occurs 36 to 60 hours, 42 to 54 hours, or 46 to 50 hours, or about 36 to 60 hours, 42 to 54 hours, or 46 to 50 hours, inclusive, after addition of the whole blood sample. In some embodiments, the collecting step occurs 36 to 60 hours, or about 36 to 60 hours, inclusive, after addition of the whole blood sample. In some embodiments, the collecting step occurs 42 to 54 hours, or about 42 to 54 hours, inclusive, after addition of the whole blood sample. In some embodiments, the collecting step occurs 46 to 50 hours, or about 46 to 50 hours, inclusive, after addition of the whole blood sample. In some embodiments, the collecting step occurs 48 hours or about 48 hours after addition of the whole blood sample.

[0055] In some of the embodiments, the harvesting step occurs 36 to 60 hours, 42 to 54 hours, or 46 to 50 hours, or about 36 to 60 hours, 42 to 54 hours, or 46 to 50 hours, inclusive, after addition of the T cell stimulating reagent. In some of the embodiments, the harvesting step occurs 36 to 60 hours, or about 36 to 60 hours, inclusive, after addition of the T cell stimulating reagent. In some of the embodiments, the harvesting step occurs 42 to 54 hours, or about 42 to 54 hours, inclusive, after addition of the T cell stimulating reagent. In some of the embodiments, the harvesting step occurs 46 to 50 hours, or about 46 to 50 hours, inclusive, after addition of the T cell stimulating reagent. In some of the embodiments, the harvesting step occurs 48 hours or about 48 hours after addition of the T cell stimulating reagent.

[0056] In some of any of the embodiments, the harvesting step occurs 12 to 36 hours, 18 to 30 hours, or 22 to 26 hours, or about 12 to 36 hours, 18 to 30 hours, or 22 to 26 hours, inclusive, after the introduction of the one or more gene editing agents. In some of any of the embodiments, the harvesting step occurs 12 to 36 hours, or about 12 to 36 hours, inclusive, after the introduction of the one or more gene editing agents. In some of any of the embodiments, the harvesting step occurs 18 to 30 hours, or about 18 to 30 hours, inclusive, after the introduction of the one or more gene editing agents. In some of any of the embodiments, the harvesting step occurs 22 to 26 hours, or about 22 to 26 hours, inclusive, after the introduction of the one or more gene editing agents. In some of any of the embodiments, the harvesting step occurs 24 hours or about 24 hours after the introduction of the one or more gene editing agents.

[0057] In some of the embodiments, the harvesting step is performed 12 to 36 hours, 18 to 30 hours, or 22 to 26 hours, or about 12 to 36 hours, 18 to 30 hours, or 22 to 26 hours, inclusive, after introduction of the nucleic acid molecule. In some of the embodiments, the harvesting step is performed 12 to 36 hours, or about 12 to 36 hours, inclusive, after introduction of the nucleic acid molecule. In some of the embodiments, the harvesting step is performed 18 to 30 hours, or about 18 to 30 hours, inclusive, after introduction of the nucleic acid molecule. In some of the embodiments, the harvesting step is performed 22 to 26 hours, or about 22 to 26 hours, inclusive, after introduction of the nucleic acid molecule. In some of the embodiments, the harvesting step is performed 24 hours or about 24 hours after introduction of the nucleic acid molecule.

[0058] In some of any of the embodiments, the methods include formulating the harvested, genetically engineered T cells for cryopreservation or for administration to a subject. In some of any of the embodiments, the methods include formulating the harvested, genetically engineered T cells for cryopreservation. In some of any of the embodiments, the methods include formulating the harvested, genetically engineered T cells for administration to a subject.

[0059] In some of any of the embodiments, the harvested, genetically engineered T cells are formulated in the presence of a cryoprotectant or a pharmaceutically acceptable excipient. In some of any of the embodiments, the harvested, genetically engineered T cells are formulated in the presence of a cryoprotectant. In some of any of the embodiments, the harvested, genetically engineered T cells are formulated in the presence of a pharmaceutically acceptable excipient.

[0060] In some of any of the embodiments, the plurality of T cells are primary T cells from a human subject.

[0061] In some of any of the embodiments, the selection marker is selected from the group consisting of CD3, CD4, CD8, CD45RA, CD27, CD28, and CCR7. In some of any of the embodiments, the selection marker is CD3, CD4, or CD8. In some of any of the embodiments, the selection marker is CD3. In some of any of the embodiments, the selection marker is CD4. In some of any of the embodiments, the selection marker is CD8.

[0062] In some of either embodiments, the selection agent comprises an antibody or antibody fragment that specifically binds to the selection marker. In some of either embodiments, the antibody or antibody fragment that is the selection agent is a monovalent antibody fragment. In some of either embodiments, the antibody or antibody fragment that is the selection agent is a Fab fragment.

[0063] In some of either embodiment, the T cell stimulating reagent comprises an oligomer of streptavidin or streptavidin mutein molecule; the primary agent comprises a first streptavidin binding partner that binds to the oligomeric streptavidin or streptavidin mutein molecule; and the secondary agent comprises a second streptavidin binding partner that binds to the oligomeric streptavidin or streptavidin mutein molecule.

[0064] In some of either embodiment, the T cell stimulatory reagent consists of or consists essentially of an oligomer, a primary agent, and a secondary agent.

[0065] In some embodiments, the oligomer comprises 500-5000 or about 500-5000 tetramers, 1000-4000 or about 1000-4000 tetramers, or 2000-3000 or about 2000-3000 tetramers (inclusive) of streptavidin or streptavidin mutein molecules. In some embodiments, the oligomer comprises 500-5000 or about 500-5000 tetramers (inclusive) of streptavidin or streptavidin mutein molecules. In some embodiments, the oligomer comprises 1000-4000 or about 1000-4000 tetramers (inclusive) of streptavidin or streptavidin mutein molecules. In some of either embodiment, the oligomer comprises at or about 2000-3000 tetramers of streptavidin or streptavidin mutein molecules, inclusive, hi some of either embodiment, the oligomer comprises at or about 2400 tetramers of streptavidin or streptavidin mutein molecules.

[0066] In some of either embodiment, the oligomer is an oligomer of a streptavidin mutein molecule.

[0067] In some of either embodiment, the streptavidin mutein molecule comprises the amino acid sequence IGAR (SEQ ID NO:133) or VTAR (SEQ ID NO:134) at a sequence position corresponding to positions 44-47 of the amino acid sequence set forth in SEQ ID NO:1. In some of either embodiment, the streptavidin mutein molecule comprises the amino acid sequence IGAR (SEQ ID NO:133) at a sequence position corresponding to positions 44-47 of the amino acid sequence set forth in SEQ ID NO:1. In some of either embodiment, the streptavidin mutein molecule comprises the amino acid sequence VTAR (SEQ ID NO:134) at a sequence position corresponding to positions 44-47 of the amino acid sequence set forth in SEQ ID NO:1.

[0068] In some of either embodiment, the streptavidin mutein molecule begins at the N-terminus in the region of amino acid positions 10-16 of SEQ ID NO:1 and ends at the C-terminus in the region of amino acid positions 133-142 of SEQ ID NO:1.

[0069] In some of any of the embodiments, the streptavidin mutein molecule comprises the amino acid sequence set forth in any one of SEQ ID NOs:3-6, 27, 28, 104, 105, and 136. In some of any of the embodiments, the streptavidin mutein molecule comprises the amino acid sequence set forth in SEQ ID NO:6.

[0070] In some of any of the embodiments, the first streptavidin binding partner is present at the C-terminus of the primary agent; and / or the second streptavidin binding partner is present at the C-terminus of the secondary agent. In some of any of the embodiments, the first streptavidin binding partner is present at the C-terminus of the primary agent. In some of any of the embodiments, the second streptavidin binding partner is present at the C-terminus of the secondary agent. In some of any of the embodiments, the first streptavidin binding partner is present at the C-terminus of the primary agent; and the second streptavidin binding partner is present at the C-terminus of the secondary agent.

[0071] In some of any of the embodiments, the first and / or second streptavidin-binding partner is a streptavidin-binding peptide. In some of any of the embodiments, the first streptavidin-binding partner is a streptavidin-binding peptide. In some of any of the embodiments, the second streptavidin-binding partner is a streptavidin-binding peptide. In some of any of the embodiments, the first and second streptavidin-binding partners are streptavidin-binding peptides.

[0072] In some of any of the embodiments, the streptavidin-binding peptide that is the first and / or second streptavidin-binding partner comprises the amino acid sequence set forth in any one of SEQ ID NOs:7, 8, and 15-19. In some of any of the embodiments, the streptavidin-binding peptide that is the first streptavidin-binding partner comprises the amino acid sequence set forth in any one of SEQ ID NOs:7, 8, and 15-19. In some of any of the embodiments, the streptavidin-binding peptide that is the second streptavidin-binding partner comprises the amino acid sequence set forth in any one of SEQ ID NOs:7, 8, and 15-19. In some of any of the embodiments, the streptavidin-binding peptide that is the first and second streptavidin-binding partner comprises the amino acid sequence set forth in any one of SEQ ID NOs:7, 8, and 15-19.

[0073] In some of any of the embodiments, the streptavidin-binding peptide that is the first and / or second streptavidin-binding partner comprises the amino acid sequence set forth in SEQ ID NO: 16. In some of any of the embodiments, the streptavidin-binding peptide that is the first streptavidin-binding partner comprises the amino acid sequence set forth in SEQ ID NO: 16. In some of any of the embodiments, the streptavidin-binding peptide that is the second streptavidin-binding partner comprises the amino acid sequence set forth in SEQ ID NO: 16. In some of any of the embodiments, the streptavidin-binding peptides that are the first and second streptavidin-binding partners comprise the amino acid sequence set forth in SEQ ID NO: 16.

[0074] In some of either embodiment, the member of the TCR / CD3 complex is CD3.

[0075] In some of either embodiment, the T cell costimulatory molecule is CD28, CD90 (Thy-1), CD95 (Apo- / Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM. In some of either embodiment, the T cell costimulatory molecule is CD28.

[0076] In some of any of the embodiments, the primary agent comprises an antibody or antibody fragment that specifically binds to a member of the TCR / CD3 complex; and / or the secondary agent comprises an antibody or antibody fragment that specifically binds to a T cell costimulator. In some of any of the embodiments, the primary agent comprises an antibody or antibody fragment that specifically binds to a member of the TCR / CD3 complex. In some of any of the embodiments, the secondary agent comprises an antibody or antibody fragment that specifically binds to a T cell costimulator. In some of any of the embodiments, the primary agent comprises an antibody or antibody fragment that specifically binds to a member of the TCR / CD3 complex; and the secondary agent comprises an antibody or antibody fragment that specifically binds to a T cell costimulator.

[0077] In some of either embodiment, the antibody or antibody fragment that is the primary agent comprises a heavy chain and a first streptavidin-binding partner is fused to the C-terminus of the heavy chain of the primary agent; and / or the antibody or antibody fragment that is the secondary agent comprises a heavy chain and a second streptavidin-binding partner is fused to the C-terminus of the heavy chain of the secondary agent. In some of either embodiment, the antibody or antibody fragment that is the primary agent comprises a heavy chain and a first streptavidin-binding partner is fused to the C-terminus of the heavy chain of the primary agent. In some of either embodiment, the antibody or antibody fragment that is the secondary agent comprises a heavy chain and a second streptavidin-binding partner is fused to the C-terminus of the heavy chain of the secondary agent. In some of either embodiment, the antibody or antibody fragment that is the primary agent comprises a heavy chain and a first streptavidin binding partner is fused to the C-terminus of the heavy chain of the primary agent; the antibody or antibody fragment that is the secondary agent comprises a heavy chain and a second streptavidin binding partner is fused to the C-terminus of the heavy chain of the secondary agent.

[0078] In some of any embodiments, the antibody or antibody fragment that is the primary and / or secondary agent is a monovalent antibody fragment. In some of any embodiments, the antibody or antibody fragment that is the primary agent is a monovalent antibody fragment. In some of any embodiments, the antibody or antibody fragment that is the secondary agent is a monovalent antibody fragment. In some of any embodiments, the antibody or antibody fragment that is the primary and secondary agent is a monovalent antibody fragment.

[0079] In some of any of the embodiments, the antibody or antibody fragment that is the primary and / or secondary agent is a Fab fragment. In some of any of the embodiments, the antibody or antibody fragment that is the primary agent is a Fab fragment. In some of any of the embodiments, the antibody or antibody fragment that is the secondary agent is a Fab fragment. In some of any of the embodiments, the antibody or antibody fragment that is the primary and secondary agent is a Fab fragment.

[0080] In some of either embodiment, the primary agent comprises an anti-CD3 antibody or antibody fragment and the secondary agent comprises an anti-CD28 antibody or antibody fragment. In some of either embodiment, the primary agent comprises an anti-CD3 Fab fragment and the secondary agent comprises an anti-CD28 Fab fragment.

[0081] In some of any of the embodiments, the gene is the T cell receptor alpha constant (TRAC) gene. In some of any of the embodiments, the target site is within the sequence shown in SEQ ID NO:250.

[0082] In some of either embodiment, the nucleic acid molecule comprises a 5' homology arm and a 3' homology arm that comprise sequences homologous to nucleic acid sequences surrounding the target site, and the nucleic acid molecule comprises the structure [5' homology arm]-[transgene]-[3' homology arm].

[0083] In some of either embodiment, the 5' homology arm and the 3' homology arm comprise sequences homologous to sequences of the TRAC gene surrounding the target site.

[0084] In some of either embodiment, the 5' homology arm comprises a sequence comprising at least or at least about 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in SEQ ID NO:248. In some of either embodiments, the 5' homology arm comprises at least or at least about 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of the sequence set forth in SEQ ID NO: 248. In some of either embodiments, the 5' homology arm comprises the sequence set forth in SEQ ID NO: 248.

[0085] In some of either embodiment, the 3' homology arm comprises a sequence comprising at least or at least about 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in SEQ ID NO:249. In some of either embodiments, the 3' homology arm comprises at least or at least about 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of the sequence set forth in SEQ ID NO: 249. In some of either embodiments, the 3' homology arm comprises the sequence set forth in SEQ ID NO: 249.

[0086] In some of either embodiment, the 5' homology arm comprises the sequence shown in SEQ ID NO:248 and the 3' homology arm comprises the sequence shown in SEQ ID NO:249.

[0087] In some of any of the embodiments, transcription of the integrated transgene is under the control of a promoter contained in the nucleic acid molecule. In some of any of the embodiments, the promoter is the human elongation factor 1 alpha (EF1 alpha) promoter. In some of any of the embodiments, the promoter comprises the sequence set forth in SEQ ID NO:247.

[0088] In some of any of the embodiments, the recombinant protein is a recombinant receptor. In some of any of the embodiments, the recombinant receptor is a T cell receptor or a chimeric antigen receptor. In some of any of the embodiments, the recombinant receptor is a T cell receptor. In some of any of the embodiments, the recombinant receptor is a chimeric antigen receptor.

[0089] In some of any of the embodiments, the one or more gene editing agents comprise (i) a gene editing nuclease or nuclease combination, or (ii) a nucleic acid molecule comprising one or more sequences encoding a gene editing nuclease or nuclease combination. In some of any of the embodiments, the one or more gene editing agents comprise a gene editing nuclease or nuclease combination.

[0090] In some of either embodiment, the gene-editing nuclease or nuclease combination specifically recognizes a nucleic acid sequence near or including the target site. In some of either embodiment, the gene-editing nuclease or nuclease combination specifically recognizes a nucleic acid sequence including the target site.

[0091] In some of either embodiment, the nucleic acid sequence comprising the target site comprises the sequence shown in SEQ ID NO:250.

[0092] In some of either embodiment, the gene-editing nuclease or nuclease combination is a zinc finger nuclease, a transcription activator-like effector nuclease, or a CRISPR-Cas combination. In some of either embodiment, the gene-editing nuclease or nuclease combination is a CRISPR-Cas combination.

[0093] In some of any of the embodiments, the CRISPR-Cas combination comprises a CRISPR-Cas nickase, a reverse transcriptase, and a serine integrase.

[0094] In some of the embodiments, the CRISPR-Cas combination comprises a guide RNA comprising a targeting sequence complementary to a nucleic acid sequence comprising the target site. In some of the embodiments, the CRISPR-Cas combination is a ribonucleoprotein complex comprising the guide RNA and a Cas protein. In some of the embodiments, the Cas protein is a S. pyogenes Cas protein.

[0095] In some of any of the embodiments, the CRISPR-Cas combination is a CRISPR-Cas9 combination or a CRISPR-Cas12 combination. In some of any of the embodiments, the CRISPR-Cas combination is a CRISPR-Cas9 combination. In some of any of the embodiments, the CRISPR-Cas combination is a CRISPR-Cas12 combination.

[0096] In some of any of the embodiments, the targeting sequence comprises the sequence set forth in any one of SEQ ID NOs: 144 to 175. In some of any of the embodiments, the targeting sequence comprises the sequence set forth in SEQ ID NO: 148.

[0097] In some of either embodiment, the method is performed ex vivo.

[0098] Also provided herein, in some embodiments, are genetically engineered T cells produced by any of the provided methods and expressing a recombinant protein.

[0099] In some of any of the embodiments, the transgene is integrated into a target site of a gene in the genetically engineered T cell. In some of any of the embodiments, the gene is the T cell receptor alpha constant (TRAC) gene. In some of any of the embodiments, the target site is within the sequence set forth in SEQ ID NO:250.

[0100] In some of any of the embodiments, the recombinant protein is a recombinant receptor. In some of any of the embodiments, the recombinant receptor is a T cell receptor or a chimeric antigen receptor. In some of any of the embodiments, the recombinant receptor is a T cell receptor. In some of any of the embodiments, the recombinant receptor is a chimeric antigen receptor.

[0101] Also provided herein, in some embodiments, is a population of T cells comprising a plurality of any of the provided engineered T cells.

[0102] In some of any of the embodiments, the plurality of engineered T cells is at least 10%, 15%, or 20% of the population of T cells. In some of any of the embodiments, the plurality of engineered T cells is at least 10% of the population of T cells. In some of any of the embodiments, the plurality of engineered T cells is at least 15% of the population of T cells. In some of any of the embodiments, the plurality of engineered T cells is at least 20% of the population of T cells.

[0103] In some of any of the embodiments, the gene is disrupted in at least 85%, 90%, or 95% of T cells in the population of T cells. In some of any of the embodiments, the gene is disrupted in at least 85% of T cells in the population of T cells. In some of any of the embodiments, the gene is disrupted in at least 90% of T cells in the population of T cells. In some of any of the embodiments, the gene is disrupted in at least 95% of T cells in the population of T cells.

[0104] In some of either embodiment, the gene is the T cell receptor alpha constant (TRAC) gene.

[0105] Also provided herein, in some embodiments, are pharmaceutical compositions comprising any of the populations of provided T cells and a pharmaceutically acceptable excipient.

[0106] Also provided herein, in some aspects, are methods of treatment comprising administering any of the provided pharmaceutical compositions to a subject having a disease or condition.

[0107] In some of either embodiment, the recombinant protein is a recombinant receptor that targets an antigen expressed on a target cell associated with a disease or pathological condition.

[0108] Also provided herein, in some aspects, are methods for cytolytic killing of target cells, comprising contacting the target cells with any of the provided populations.

[0109] Also provided herein, in some aspects, are methods for cytolytic killing of target cells comprising contacting the target cells with any of the provided pharmaceutical compositions.

[0110] In some of either embodiment, the contacting step is performed ex vivo.

[0111] In some of any of the embodiments, the contacting step is performed in vivo. In some of any of the embodiments, the contacting step is by administering the pharmaceutical composition to a subject having a disease or condition. In some of any of the embodiments, the target cell is associated with a disease or condition and the recombinant protein is a recombinant receptor that targets an antigen expressed on the target cell.

[0112] In some of any of the embodiments, the recombinant receptor is a T cell receptor or a chimeric antigen receptor. In some of any of the embodiments, the recombinant receptor is a T cell receptor. In some of any of the embodiments, the recombinant receptor is a chimeric antigen receptor.

[0113] In some of either embodiment, the pharmaceutical composition is for use in treating a disease or disorder in a subject.

[0114] In some of either embodiment, the recombinant protein is a recombinant receptor that targets an antigen expressed on cells associated with a disease or pathological condition.

[0115] In some of any of the embodiments, the recombinant receptor is a T cell receptor or a chimeric antigen receptor. In some of any of the embodiments, the recombinant receptor is a T cell receptor. In some of any of the embodiments, the recombinant receptor is a chimeric antigen receptor.

[0116] Also provided herein, in some aspects, is the use of any of the provided pharmaceutical compositions for treating a disease or disorder in a subject.

[0117] Also provided herein, in some aspects, is the use of any of the provided pharmaceutical compositions for the manufacture of a medicament for treating a disease or disorder in a subject.

[0118] In some of either embodiment, the recombinant protein is a recombinant receptor that targets an antigen expressed on cells associated with a disease or pathological condition.

[0119] In some of any of the embodiments, the recombinant receptor is a T cell receptor or a chimeric antigen receptor. In some of any of the embodiments, the recombinant receptor is a T cell receptor. In some of any of the embodiments, the recombinant receptor is a chimeric antigen receptor. [Brief explanation of the drawings]

[0120] [Figure 1A] Depletion, total nucleated cell count (TNC) and purity of the positive fraction collected from multiple whole blood sample loads onto an anti-CD3 affinity chromatography column are shown. [Figure 1B] Shown is CD3 and CAR expression on cells after on-column stimulation, elution, and non-viral manipulation of T cells selected from whole blood samples loaded onto an anti-CD3 affinity chromatography column. [Figure 1C] The graph shows the survival rate, knockout (KO) efficiency, and knock-in (KI) efficiency over time in engineered cells. [Figure 1D] Cell growth of total cells, KO cells, and KI cells over time after manipulation is shown. DETAILED DESCRIPTION OF THE INVENTION

[0121] Detailed Description In some embodiments, provided herein are methods for producing genetically engineered immune cells, e.g., T cells. In some embodiments, the provided methods are any described herein, e.g., in Section I. In some embodiments, the provided methods are performed ex vivo.

[0122] In some embodiments, the provided methods include stimulating and manipulating immune cells, e.g., T cells. In some embodiments, the provided methods include selecting, stimulating, and manipulating immune cells, e.g., T cells. In some embodiments, the stimulating step is by on-column stimulation of immune cells, e.g., T cells, wherein the immune cells, e.g., T cells, are immobilized on a stationary phase within the interior cavity of a chromatography column during at least a portion of the incubation period in the presence of a stimulating reagent, e.g., a T cell stimulating reagent, added to the stationary phase. In some embodiments, the immune cells, e.g., T cells, are immobilized by a selection agent contained in the stationary phase that specifically binds to a selection marker expressed on the surface of the immune cells, e.g., T cells. In some embodiments, the provided methods include selecting immune cells by adding a sample containing immune cells, e.g., T cells, to the stationary phase before stimulation, thereby allowing the immune cells, e.g., T cells, to be immobilized on the stationary phase via the selection agent for on-column stimulation. In some embodiments, the sample is a whole blood sample.

[0123] In some embodiments, immune cells, e.g., T cells, are collected from the chromatography column after on-column stimulation. In some embodiments, the collected immune cells, e.g., T cells, are no longer immobilized after on-column stimulation. In some aspects, on-column stimulation promotes detachment of immobilized immune cells, e.g., T cells, from the stationary phase.

[0124] In some embodiments, the collected immune cells, e.g., T cells, are engineered outside of a chromatography column. In some embodiments, the collected immune cells, e.g., T cells, are engineered by targeted integration of a transgene encoding a recombinant protein. In some embodiments, the targeted integration comprises inducing gene disruption in the immune cells, e.g., T cells. In some embodiments, the targeted integration is by homology-directed repair (HDR). In some embodiments, the transgene is introduced by non-viral gene delivery. In some embodiments, the nucleic acid molecule is a DNA molecule. In some embodiments, the nucleic acid molecule is a naked DNA molecule.

[0125] In some embodiments, the nucleic acid molecule is a double-stranded DNA molecule. In some embodiments, the nucleic acid molecule is a single-stranded DNA molecule.

[0126] In some embodiments, the nucleic acid molecule is a modified DNA molecule. In some embodiments, the nucleic acid molecule is modified to enhance its stability.

[0127] In some embodiments, the nucleic acid molecule is a closed DNA molecule. In some embodiments, the nucleic acid molecule is a naked closed DNA molecule.

[0128] Some available methods for producing engineered cells for use in cell therapy, such as recombinant receptor-expressing cells, can require a significant amount of time to accomplish. In some aspects, the time required to produce the engineered cells can affect the in vivo activity of the engineered cells after administration. Longer production times can result in reduced in vivo potency, persistence, or proliferation capacity of the engineered cells.

[0129] In some aspects, some available methods may result in inefficient cell engineering, such that, for example, only a small fraction of the cells subjected to the engineering procedure ultimately express the recombinant protein, e.g., the recombinant receptor.

[0130] Certain available methods for manipulating cells can also affect the in vivo activity of manipulated cells.For example, the method that causes random or semi-random integration of transgene into the genome of manipulated cells, such as lentiviral transduction, can also affect the in vivo activity of manipulated cells.For example, random or semi-random integration events can cause transcription activation or inactivation effects or the introduction of new splice variants.There is a need for an improved method for producing manipulated cells.

[0131] The provided embodiments offer various advantages. In some aspects, the provided methods reduce the time required to produce engineered cells, e.g., to within 48 hours from the start of immune cell stimulation prior to their manipulation. In some aspects, the provided methods allow for more rapid production of engineered cells, e.g., improving the production turnaround time of engineered cells and ultimately reducing production costs. In some aspects, the provided methods allow sufficient time for transgene integration, but limit the time for stimulating or ex vivo expanding engineered cells.

[0132] In some aspects, provided methods improve the efficiency of manipulating cells. In some aspects, provided methods include manipulating cells from a whole blood sample rather than an apheresis or leukapheresis sample. In some aspects, provided methods including manipulating cells from a whole blood sample result in higher recombinant protein expression than when manipulating cells from an apheresis or leukapheresis sample. In some embodiments, recombinant protein expression is at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold higher in cells produced from a whole blood sample than in cells produced from an apheresis or leukapheresis sample. In some aspects, the use of a whole blood sample can reduce the handling or processing of the cells to be manipulated. Reduced handling or processing may improve cell health so that transgene delivery, integration, and expression can be more easily achieved during manipulations, including, for example, manipulations involving electroporation of cells.

[0133] In some aspects, provided methods employ methods for targeted integration of a transgene into the genome of an engineered cell, hi some aspects, provided methods avoid effects that may be associated with random or semi-random integration events.

[0134] In some aspects, engineered cells produced from whole blood samples by the provided methods have improved long-term effects on cytotoxic activity and increased in vivo proliferation capacity after administration compared to engineered cells produced from apheresis or leukapheresis samples. In some aspects, engineered cells produced by the provided methods, as well as provided genetically engineered cells, have improved in vivo potency, persistence, and / or proliferation capacity.

[0135] In some embodiments, provided methods include manipulating one or more immune cells, e.g., T cells. In some embodiments, the manipulating is by any of the methods described herein, e.g., in Section IC. In some embodiments, provided methods include targeted integration of a transgene into one or more immune cells, e.g., T cells, at a target site of the gene. In some embodiments, provided methods include introducing a nucleic acid molecule containing the transgene into one or more immune cells, e.g., T cells. In some embodiments, introducing the nucleic acid molecule is under conditions for targeted integration of the transgene into the target site. In some embodiments, introducing the nucleic acid molecule is by non-viral gene delivery. In some embodiments, the nucleic acid molecule is a DNA molecule. In some embodiments, the nucleic acid molecule is a naked DNA molecule.

[0136] In some embodiments, the nucleic acid molecule is a double-stranded DNA molecule. In some embodiments, the nucleic acid molecule is a single-stranded DNA molecule.

[0137] In some embodiments, the nucleic acid molecule is a modified DNA molecule. In some embodiments, the nucleic acid molecule is modified to enhance its stability.

[0138] In some embodiments, the nucleic acid molecule is a closed DNA molecule. In some embodiments, the nucleic acid molecule is a naked closed DNA molecule.

[0139] In some embodiments, introducing the one or more gene editing agents is by electroporation.

[0140] In some embodiments, the provided methods include introducing one or more gene editing agents to edit genes in one or more immune cells, such as T cells. In some embodiments, the introducing one or more gene editing agents is by electroporation. In some embodiments, the introducing one or more gene editing agents is performed before the introduction of the nucleic acid molecule. In some embodiments, the introducing one or more gene editing agents is performed simultaneously with the introduction of the nucleic acid molecule.

[0141] In some embodiments, the targeted integration is by homology-directed repair (HDR). In some embodiments, HDR involves introducing one or more gene editing agents to induce gene disruption of a gene in one or more immune cells, such as T cells.

[0142] In some embodiments, the transgene encodes a recombinant protein. In some embodiments, the provided methods produce genetically engineered immune cells, such as T cells, that express the recombinant protein. In some embodiments, the recombinant protein is a recombinant receptor. In some embodiments, the recombinant receptor is a T cell receptor (TCR). In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR).

[0143] In some embodiments, the provided methods include stimulating a plurality of immune cells (e.g., T cells) containing one or more immune cells (e.g., T cells). In some embodiments, the stimulating is by any of the methods described herein, e.g., in Section IB. In some embodiments, the stimulating is by on-column stimulation of the plurality of immune cells, e.g., T cells. In some embodiments, the provided methods include incubating the plurality of immune cells (e.g., T cells) under conditions for stimulating the immune cells (e.g., T cells), wherein the immune cells (e.g., T cells) are the plurality of immune cells (e.g., T cells). In some embodiments, the incubating is by any of the methods described herein, e.g., in Section IB-2.

[0144] In some embodiments, the incubating step is performed in the presence of a stimulatory reagent. In some embodiments, the stimulatory reagent is any described herein, e.g., in Section IB-1. In some embodiments, the provided methods include adding the stimulatory reagent to the plurality of immune cells, e.g., T cells.

[0145] In some embodiments, the stimulatory reagent contains a primary agent that specifically binds to a molecule to provide a primary activation signal to an immune cell, e.g., a T cell. In some embodiments, the stimulatory reagent contains a secondary agent that specifically binds to a costimulatory molecule to provide a costimulatory signal to an immune cell, e.g., a T cell. In some embodiments, the stimulatory reagent contains a primary agent and a secondary agent.

[0146] In some embodiments, the stimulatory reagent is a T cell stimulatory reagent. In some embodiments, the T cell stimulatory reagent contains a primary agent that specifically binds to a member of the TCR / CD3 complex. In some embodiments, the T cell stimulatory reagent contains a secondary agent that specifically binds to a T cell costimulatory molecule. In some embodiments, the T cell stimulatory reagent contains a primary agent and a secondary agent.

[0147] In some embodiments, the incubating step occurs in the internal cavity of a chromatography column. In some embodiments, the stimulating step is by on-column stimulation of a plurality of immune cells, e.g., T cells. In some embodiments, the plurality of immune cells, e.g., T cells, are immobilized on a stationary phase within the internal cavity of the chromatography column. In some embodiments, the stationary phase is any of those described herein, e.g., in Section IA-1.

[0148] In some embodiments, the immobilized plurality of immune cells, e.g., T cells, is incubated in the presence of a stimulating reagent, e.g., a T cell stimulating reagent. In some embodiments, the stimulating reagent, e.g., a T cell stimulating reagent, is added to the plurality of immune cells, e.g., T cells, immobilized on a stationary phase.

[0149] In some embodiments, the stationary phase contains a selection agent that specifically binds to a selection marker expressed on the surface of a plurality of immune cells, e.g., T cells, hi some embodiments, the selection agent specifically binds to the selection marker, thereby immobilizing the plurality of immune cells, e.g., T cells, on the stationary phase.

[0150] In some embodiments, provided methods include selecting a plurality of immune cells, e.g., T cells. In some embodiments, the selecting step is by any of the methods described herein, e.g., in Section IA. In some embodiments, provided methods include adding a sample containing a plurality of immune cells, e.g., T cells, to an interior cavity of a chromatography column. In some embodiments, the sample is a whole blood sample. In some embodiments, the plurality of immune cells, e.g., T cells, are immobilized on a stationary phase. In some embodiments, the plurality of immune cells, e.g., T cells, are immobilized on the stationary phase by a selection agent specifically binding to a selection marker.

[0151] In some embodiments, the plurality of immune cells are a plurality of lymphocytes. In some embodiments, the plurality of immune cells are a plurality of T cells, B cells, or NK cells. In some embodiments, the plurality of immune cells are a plurality of T cells. In some embodiments, the plurality of T cells are CD4+ T cells. In some embodiments, the plurality of T cells are CD8+ T cells. In some embodiments, the plurality of T cells comprises CD4+ T cells and CD8+ T cells.

[0152] In some embodiments, the plurality of immune cells, e.g., T cells, are primary cells from the subject, e.g., primary T cells. In some embodiments, the subject is a human subject.

[0153] In some embodiments, provided methods include collecting immune cells, e.g., T cells, that are the plurality of immune cells. In some embodiments, the collecting step is by any of the methods described herein, e.g., in Section IB-3. In some embodiments, the collected immune cells, e.g., T cells, are collected from a chromatography column. In some embodiments, the collected immune cells, e.g., T cells, are immune cells, e.g., T cells, that are no longer immobilized on a stationary phase. In some embodiments, the collected immune cells, e.g., T cells, are immune cells, e.g., T cells, that are no longer immobilized on a stationary phase after the incubating step. In some embodiments, as a result of the incubation, the immune cells (e.g., T cells) that are the plurality of immune cells (e.g., T cells) are no longer immobilized on the stationary phase.

[0154] In some embodiments, the collected immune cells, e.g., T cells, contain one or more immune cells, e.g., T cells, that have been engineered. In some embodiments, the engineering is of one or more of the collected immune cells, e.g., T cells.

[0155] In some embodiments, the nucleic acid molecule is introduced into the collected immune cells (e.g., T cells), which are immune cells (e.g., T cells). In some embodiments, the nucleic acid molecule is introduced into one or more of the collected immune cells, e.g., T cells.

[0156] In some embodiments, the one or more gene editing agents are introduced into the collected immune cells (e.g., T cells), which are immune cells (e.g., T cells). In some embodiments, the one or more gene editing agents are introduced into one or more of the collected immune cells, e.g., T cells.

[0157] In some embodiments, the provided methods include further incubating the collected immune cells, e.g., T cells. In some embodiments, the further incubation is by any of the methods described herein, e.g., in Section IB-4. In some embodiments, the further incubation is performed prior to manipulation. In some embodiments, the further incubation is performed prior to introduction of a nucleic acid molecule. In some embodiments, the further incubation is performed prior to introduction of one or more gene editing agents.

[0158] In some embodiments, the further incubating step is performed in the presence of a stimulating reagent. In some embodiments, the further incubating step is not performed within the internal cavity of the chromatography column. In some embodiments, the further incubating step is performed external to the chromatography column.

[0159] In some embodiments, the conditions for targeted integration include culturing one or more immune cells, e.g., T cells, under conditions for integrating the transgene into the target site. In some embodiments, the culturing is by any of the methods described herein, e.g., in Section IC-5. In some embodiments, the culturing is under conditions for integrating the transgene by HDR. In some embodiments, the culturing is of collected immune cells, e.g., T cells. In some embodiments, the culturing is in the presence of a nucleic acid molecule.

[0160] In some embodiments, the provided methods include harvesting engineered immune cells, e.g., T cells, that express a recombinant protein, e.g., a recombinant receptor, e.g., a TCR or a CAR. In some embodiments, the harvesting is by any of the methods described herein, e.g., in Section I.D.

[0161] In some embodiments, the methods provided include formulating the harvested, genetically engineered immune cells, e.g., T cells, in some embodiments, the formulation is by any of the methods described herein, e.g., in Section IE.

[0162] Also provided herein, in some embodiments, are genetically engineered immune cells produced by any of the provided methods. In some embodiments, the genetically engineered immune cells are genetically engineered lymphocytes. In some embodiments, the genetically engineered immune cells are genetically engineered T cells.

[0163] Also provided herein in some embodiments are pharmaceutical compositions containing any of the provided engineered immune cells, e.g., T cells. In some embodiments, the provided pharmaceutical compositions are any of those described herein, e.g., in Section II. In some embodiments, the pharmaceutical composition contains a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is for use in treating a disease or condition in a subject.

[0164] Also provided herein, in some embodiments, are methods of treatment comprising administering any of the provided pharmaceutical compositions to a subject having a disease or condition. In some embodiments, the provided methods are any described herein, e.g., in Section II. Also provided herein, in some embodiments, are uses of the provided pharmaceutical compositions for treating a disease or condition in a subject. Also provided herein, in some embodiments, are uses of the provided pharmaceutical compositions for the manufacture of a medicament for treating a disease or condition in a subject. In some embodiments, the provided uses are any described herein, e.g., in Section II.

[0165] 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 the definitions set forth herein conflict or are otherwise inconsistent with the definitions set forth in the patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over the definitions incorporated herein by reference.

[0166] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0167] I. Methods for Producing Engineered Immune Cells Sections IA-IE describe exemplary steps of the provided methods. In some embodiments, the provided methods include one or more of selecting, stimulating, and manipulating immune cells, e.g., T cells. In some embodiments, the provided methods include stimulating and manipulating immune cells, e.g., T cells. In some embodiments, the provided methods include selecting, stimulating, and manipulating immune cells, e.g., T cells.

[0168] In some embodiments, immune cells, e.g., T cells, are stimulated on-column after selecting the immune cells, e.g., T cells, based on surface expression of a selection marker via column chromatography. In some embodiments, the immune cells, e.g., T cells, are collected from the chromatography column after stimulation, and the collected immune cells, e.g., T cells, are then engineered by targeted integration of a transgene encoding a recombinant protein.

[0169] In some embodiments, the provided methods also include one or more of the steps of harvesting and formulating the immune cells, e.g., T cells.

[0170] In some embodiments, any of the steps of the provided methods are performed in a closed system. In some embodiments, any of the steps of the provided methods are automated.

[0171] A. Selection In some embodiments, the provided methods include selecting immune cells, e.g., T cells. In some embodiments, the selecting is based on expression of a selectable marker on the surface of the immune cells, e.g., T cells.

[0172] In some embodiments, the selecting step is by column chromatography. In some embodiments, the selecting step achieves immobilization of the immune cells, e.g., T cells, onto a stationary phase within the interior cavity of a chromatography column. In some embodiments, the selection agent specifically binds to a selection marker, thereby immobilizing the immune cells, e.g., T cells, onto the stationary phase. In some embodiments, the stationary phase is any of those described herein, e.g., in Section IA-1.

[0173] In some embodiments, the selecting step is performed before stimulating the immune cells, e.g., T cells. In some embodiments, the immune cells, e.g., T cells, are immobilized on a stationary phase during stimulation. In some embodiments, the selecting step is performed subsequent to stimulating the immune cells, e.g., T cells.

[0174] In some embodiments, the selecting step occurs prior to the step of engineering the immune cells, e.g., T cells. In some embodiments, the selecting step occurs subsequent to the step of engineering the immune cells, e.g., T cells.

[0175] In some embodiments, the selecting step is carried out using any of the methods described in WO2013 / 124474, WO2015 / 164675, WO2017 / 068425, WO2020 / 089343, WO2021 / 084050, US2015 / 0024411, US2017 / 0037369, US2019 / 0112576, and US2022 / 0002669.

[0176] In some embodiments, the selecting step is performed at a temperature above room temperature. In some embodiments, the selecting step is performed at physiological temperature. In some embodiments, the selecting step is performed at a temperature of 30°C to 39°C, or about 30°C to 39°C. In some embodiments, the selecting step is performed at a temperature of 35°C to 39°C, or about 35°C to 39°C. In some embodiments, the selecting step is performed at 37°C or about 37°C.

[0177] In some embodiments, the temperature is regulated by one or more heating elements configured to supply heat to the stationary phase. In some embodiments, the temperature is regulated using any of the methods or devices described in WO2020 / 089343, WO2021 / 084050, and US2022 / 0002669.

[0178] In some embodiments, immune cells, such as T cells, are contained in a sample. In some embodiments, the provided method includes adding the sample to a stationary phase. In some embodiments, the sample contains cell types other than immune cells, such as T cells. In some embodiments, the sample contains additional cells, such as non-T cells, that do not express the selection marker.

[0179] In some embodiments, the sample is a biological sample. In some embodiments, the immune cells are primary cells from a subject, such as T cells. In some embodiments, the subject is a human subject.

[0180] Exemplary samples include body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat; tissue; and organ samples. Further exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsils, and other organs. In some embodiments, the sample is obtained directly from a subject. In some embodiments, the sample is a processed sample. In some embodiments, the sample is derived from any of the aforementioned samples.

[0181] The sample can contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells; red blood cells; and / or platelets. In some embodiments, the sample contains T cells.

[0182] In some embodiments, the sample is 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 sample is a whole blood sample. In some embodiments, the sample is an apheresis product. In some embodiments, the sample is a leukapheresis product. In some embodiments, the whole blood sample from a subject is not processed before addition to the stationary phase.

[0183] In some aspects, the sample is blood or a blood-derived sample. In particular embodiments, the sample is a whole blood sample. In aspects of the provided methods, producing cells directly from a whole blood source sample can result in improved overall transduction efficiency compared to methods in which cells are first processed from a subject-derived blood sample, for example, by apheresis or leukapheresis.

[0184] In some embodiments, the immune cells, e.g., T cells, are obtained from the subject's circulating blood, e.g., by apheresis or leukapheresis. In some embodiments, the sample is or is derived from an apheresis or leukapheresis product.

[0185] In some embodiments, immune cells, e.g., T cells, obtained from the circulating blood of a subject are washed, e.g., to remove the plasma fraction and to place the immune cells, e.g., T cells, in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the immune cells, e.g., T cells, are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution lacks calcium, magnesium, and / or many or all divalent cations. In some aspects, the washing step is accomplished using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 Cell Processor, Baxter) according to the manufacturer's instructions. In some aspects, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the immune cells, e.g., T cells, after washing, are washed in various biocompatible buffers, e.g., Ca, Mg, Mn ... 2+ / Mg 2+ In some embodiments, the blood sample is washed to remove one or more anticoagulants, such as heparin, added during apheresis or leukapheresis.

[0186] 1. Stationary phase In some embodiments, one or more steps of the provided methods involve the use of a stationary phase. In some embodiments, the stationary phase contains a selection agent. In some embodiments, the selection agent is any described herein, e.g., in Section IA-1-a.

[0187] In some embodiments, the stationary phase comprises a chromatography matrix. In some embodiments, the chromatography matrix is ​​suitable for cell separation using column chromatography. In some embodiments, the chromatography matrix is ​​any described herein, e.g., in Section IA-1-b.

[0188] In some embodiments, the selection agent comprises a binding partner. In some embodiments, the binding partner is for immobilizing the selection agent to a chromatography matrix.

[0189] In some embodiments, the stationary phase contains a selection reagent. In some embodiments, the selection reagent is any described herein, e.g., in Section IA-1-c. In some embodiments, the selection reagent contains one or more molecules of streptavidin, avidin, a streptavidin analog or mutein, or an avidin analog or mutein.

[0190] In some embodiments, the selection agent is immobilized on a chromatography matrix. In some embodiments, the selection agent is immobilized directly on the chromatography matrix. In some embodiments, the binding partner of the selection agent is immobilized directly on the chromatography matrix.

[0191] In some embodiments, the selection reagent is immobilized on a chromatography matrix.

[0192] Methods for immobilizing a selection substance or selection reagent on a chromatography matrix can be identified and selected by those skilled in the art. In some cases, the material of the chromatography matrix, e.g., a resin, can be activated to form covalent bonds with ligands containing amine, thiol, or hydroxyl groups. Such activated materials include commercially available epoxy-activated materials, e.g., epoxy-activated agarose.

[0193] In some embodiments, the selection agent is indirectly immobilized on the chromatography matrix. In some embodiments, the selection agent is immobilized on the chromatography matrix via binding of the selection agent to a selection reagent immobilized on the chromatography matrix. In some embodiments, a binding partner of the selection agent binds to the selection reagent. In some embodiments, the binding partner binds to a molecule of the selection reagent that is streptavidin, avidin, a streptavidin analog or mutein, or an avidin analog or mutein.

[0194] In some aspects, the binding capacity of the stationary phase affects how much of the stationary phase is required to select a certain number of immune cells, e.g., T cells, that express a selectable marker. Binding capacity can be used to determine or control the number of immune cells, e.g., T cells, that are immobilized. In some aspects, binding capacity of the stationary phase can be used to standardize the amount of reagent, e.g., the amount of stimulating reagent, used in a single column.

[0195] In some embodiments, 1 mL of stationary phase can accommodate up to 100 million ± 25 million immune cells, e.g., T cells, that express a selection marker. In some embodiments, the stationary phase is 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, or 40 mL, or is about 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, or 40 mL. In some embodiments, the stationary phase is 10 mL or about 10 mL and can accommodate up to 1 billion ± 250 million immune cells, e.g., T cells, that express a selection marker. In some embodiments, the stationary phase is 20 mL or about 20 mL and can accommodate up to 2 billion ± 500 million immune cells, e.g., T cells, that express a selection marker. In some embodiments, the stationary phase is 40 mL or about 40 mL and can accommodate about 3 billion to about 5 billion immune cells, e.g., T cells, that express a selection marker.

[0196] In some embodiments, the stationary phase has the capacity to bind immune cells expressing a selection marker, e.g., 500 to 5 billion or about 500 to 5 billion T cells. In some embodiments, the stationary phase has the capacity to bind immune cells expressing a selection marker, e.g., 500 to 4 billion or about 500 to 4 billion T cells. In some embodiments, the stationary phase has the capacity to bind immune cells expressing a selection marker, e.g., 500 to 3 billion or about 500 to 3 billion T cells. In some embodiments, the stationary phase has the capacity to bind immune cells expressing a selection marker, e.g., 500 to 2 billion or about 500 to 2 billion T cells. In some embodiments, the stationary phase has the capacity to bind immune cells expressing a selection marker, e.g., 1 to 5 billion or about 1 to 5 billion T cells. In some embodiments, the stationary phase has the capacity to bind immune cells expressing a selection marker, e.g., 1 to 4 billion or about 1 to 4 billion T cells. In some embodiments, the stationary phase has a binding capacity of 1 to 3 billion or about 1 to 3 billion immune cells, e.g., T cells, that express the selection marker. In some embodiments, the stationary phase has a binding capacity of 1 to 2 billion or about 1 to 2 billion (inclusive) immune cells, e.g., T cells, that express the selection marker. In some embodiments, the stationary phase is 20 mL. In some embodiments, the stationary phase has a binding capacity of 2 billion ± 500 million immune cells, e.g., T cells, that express the selection marker.

[0197] In some embodiments, the binding capacity of the stationary phase is the maximum number of immune cells, e.g., T cells, expressing the selection marker that bind to the stationary phase under given solvent and cell concentration conditions when an excess of immune cells, e.g., T cells, expressing the selection marker are loaded onto the stationary phase. In some embodiments, the binding capacity is or is about 100 million ± 25 million immune cells, e.g., T cells, expressing the selection marker per mL of stationary phase.

[0198] In some embodiments, the static binding capacity is the maximum number of immune cells, e.g., T cells, expressing a selectable marker that can be immobilized on a stationary phase under, for example, certain solvent and cell concentration conditions. In some embodiments, the static binding capacity of the stationary phase is in the range of about 75 million to about 125 million immune cells, e.g., T cells, expressing a selectable marker per mL of stationary phase. In some embodiments, the static binding capacity of the stationary phase is in the range of about 50 million to about 100 million immune cells, e.g., T cells, expressing a selectable marker per mL of stationary phase. In some embodiments, the static binding capacity is 100 million ± 25 million immune cells, e.g., T cells, expressing a selectable marker, or about 100 million ± 25 million immune cells, e.g., T cells, expressing a selectable marker per mL of stationary phase. In some embodiments, the static binding capacity of the stationary phases disclosed herein is in the range of about 75 million to about 125 million immune cells, e.g., T cells, expressing a selectable marker per mL of stationary phase. In some embodiments, the static binding capacity of the stationary phase is about 10 million to about 20 million, 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, about 90 million, or about 100 million immune cells expressing a selection marker, e.g., T cells, per mL of stationary phase. 10,000 to about 100 million, 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.

[0199] In some embodiments, the binding capacity of a stationary phase is the number of immune cells, e.g., T cells, expressing a selection marker that bind to the stationary phase under given flow conditions before significant breakthrough of unbound immune cells, e.g., T cells, expressing the selection marker occurs. In one aspect, the binding capacity of a stationary phase is the dynamic binding capacity, e.g., 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 immune cells, e.g., T cells, expressing a selection marker and monitoring the flow-through, where the immune cells, e.g., T cells, expressing the selection marker bind to the stationary phase to a certain breakthrough point, after which unbound immune cells, e.g., T cells, expressing the selection marker pass through the column. In some embodiments, the dynamic binding capacity is at or about 100 million ± 25 million immune cells, e.g., T cells, expressing the selection marker per mL of stationary phase. In some embodiments, the dynamic binding capacity of the stationary phase is between 75 million and 125 million immune cells, e.g., T cells, that express the selection marker per mL of stationary phase, or between about 75 million and about 125 million immune cells, e.g., T cells, that express the selection marker per mL of stationary phase. In some embodiments, the dynamic binding capacity of the stationary phase is in the range of between about 50 million and about 100 million immune cells, e.g., T cells, that express the selection marker per mL of stationary phase. In some embodiments, the dynamic binding capacity of the stationary phase is about 10 million to about 20 million, 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, about 90 million, or about 100 million immune cells, e.g., T cells, expressing a selection marker per mL of stationary phase. 10,000 to about 100 million, 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.

[0200] a. Selected substances In some embodiments, the selectable marker is a lipid, polysaccharide, or nucleic acid. In some embodiments, the selectable marker is a peptide or protein, such as a receptor, e.g., a membrane receptor protein. In some embodiments, the selectable marker is a peripheral membrane protein or an integral membrane protein. In some embodiments, the selectable marker can have one or more membrane-spanning domains. As some illustrative examples, membrane proteins having a transmembrane domain include G protein-coupled receptors, e.g., olfactory receptors, rhodopsin receptors, rhodopsin pheromone receptors, peptide hormone receptors, taste receptors, GABA receptors, opioid receptors, serotonin receptors, Ca2+ receptors, melanopsin, neurotransmitter receptors, e.g., ligand-gated, voltage-gated or mechano-gated receptors including acetylcholine, nicotinic, adrenergic, norepinephrine, catecholamine, L-DOPA-, dopamine and serotonin (biogenic amines, endorphins / enkephalins) neuropeptide receptors, receptor kinases, e.g., serine / threonine kinases, tyrosine kinases, porins / channels, e.g., chloride channels, potassium channels, sodium channels, OMP proteins, ABC transporters (ATP-binding cassette-transporters), e.g., amino acid transporters, Na-glucose transporters, Na / iodine transporters, ion transporters, e.g., light-harvesting complexes, cytochrome c oxidases, ATPases. It may be Na / K, H / K, Ca, a cell adhesion receptor, such as a metalloprotease, an integrin, or a catherin.

[0201] In some embodiments, the selection marker is a molecule expressed by or defining a cell population, e.g., a population or subpopulation of blood cells, e.g., lymphocytes (e.g., T cells, B cells, or NK cells), monocytes, or stem cells (e.g., CD34-positive peripheral blood stem cells or Nanog- or Oct-4-expressing stem cells). In some embodiments, the selection marker is expressed on the surface of a target cell, e.g., a cell targeted for genetic manipulation. In some embodiments, the selection marker is a molecule expressed on the surface of an immune cell. In some embodiments, the selection marker is a molecule expressed on the surface of a lymphocyte. In some embodiments, the selection marker is a molecule expressed on the surface of a T cell, a B cell, or a NK cell. In some embodiments, the selection marker is a molecule expressed on the surface of a T cell. Examples of T cells include cells such as CMV-specific CD8+ T cells, 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. In some embodiments, the selection marker is CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD57, CD45RA, or CD45RO. In some embodiments, the selection marker is CD3. In some embodiments, the selection marker is CD28. In some embodiments, the selection marker is CD4. In some embodiments, the selection marker is CD8.

[0202] In some embodiments, the selection agent comprises an antibody, an antibody fragment, a proteinaceous molecule with antibody-like binding properties, an Ig domain-containing molecule, a cytokine, a chemokine, an MHC molecule, an MHC-peptide complex, a receptor ligand, or a binding fragment of any of the foregoing that specifically binds to the selection marker. In some embodiments, the selection agent comprises an antibody. In some embodiments, the selection agent comprises an antibody fragment. In some embodiments, the antibody fragment is selected from a Fab fragment, an Fv fragment, a single-chain Fv fragment (scFv), a bivalent antibody fragment such as an F(ab')2-fragment, a diabody, a triabody (Iliades, P., et al., FEBS Lett (1997) 409, 437-441), a decabody (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).

[0203] In some embodiments, the selection agent binds to the selection marker in a monovalent manner. In some embodiments, the selection agent comprises a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties, an aptamer, or an MHC molecule. In some embodiments, the selection agent comprises a monovalent antibody fragment. In some embodiments, the monovalent antibody fragment is a Fab fragment, an Fv fragment, or a single-chain Fv fragment (scFv). In some embodiments, the monovalent antibody fragment is a Fab fragment.

[0204] In some embodiments, the selection agent comprises an antibody fragment that is a bivalent antibody fragment. In some embodiments, the bivalent antibody fragment is a F(ab')2-fragment or a bivalent single-chain Fv fragment.

[0205] In some embodiments, the selection agent comprises a proteinaceous molecule with antibody-like binding properties, hi some embodiments, the proteinaceous molecule with antibody-like binding properties is an aptamer, a lipocalin-based polypeptide-based mutein, a glubody, an ankyrin scaffold-based protein, a crystalline scaffold-based protein, an adnectin, or an avimer.Other exemplary proteinaceous 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 four disulfide core domains, F5 / 8 C-type domain, hemopexin domain, SH2 domain, SH3 domain, laminin-type EGF-like domain, C2 domain, "kappabody" (see below, Ill. et al., Protein Eng (1997) 10, 949-57, also known as "minibody" (Martin et al., EMBO J (1994) 13, 5303-5309), diabody (see below, Holliger et al., PNAS USA (1993) 90, 6444-6448), so-called "Janusis" (see below, 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, ubiquitins, zinc finger proteins, autofluorescent proteins, and leucine-rich repeat proteins. In some embodiments, the selection agent is a bivalent artificial proteinaceous binding molecule, such as a dimeric lipocalin mutein, also known as a "duocalin."

[0206] In some embodiments, the dissociation constant (K) of binding between the selection agent and the selection marker is D ) is about 10 -2 M ~ about 10 -13 M, about 10 -3 M ~ about 10 -12 M, about 10 -4 M ~ about 10 -11 M, or about 10 -5 M ~ about 10 -10 In some embodiments, the dissociation constant (K D ) is about 10 -3 ~about 10 -7 M, e.g., low affinity K D In some embodiments, the dissociation constant (K D ) is about 10 -7 ~Approx. 1×10 -10 M, e.g., high affinity K D is.

[0207] In some embodiments, the binding between the selection agent and the selection marker is k off When expressed in units of rate (also called dissociation rate constant), k off The speed is about 0.5 x 10 -4 sec -1 or more, about 1 x 10 -4 sec -1 or more, about 2 x 10 -4 sec -1 or more, about 3 x 10 -4 sec -1 or more, about 4 x 10 -4 sec -1 or more, about 5 x 10 -4 sec -1 or more, about 1 x 10 -3 sec -1 or more, about 1.5 x 10 -3 sec -1 or more, about 2 x 10 -3 sec -1 or more, about 3 x 10 -3 sec -1or more, about 4 x 10 -3 sec -1 , about 5×10 -3 sec -1 or more, about 1 x 10 -2 sec or more, or approximately 5 x 10 -1 sec -1 or higher. The k suitable for the interaction of a particular selection agent and selection marker off It is within the skill of one in the art to empirically determine the rate range (see, e.g., US 9,023,604). The K D , k off and k on The rate can be determined by any suitable means, for example, by fluorescence titration, equilibrium dialysis, or surface plasmon resonance.

[0208] In some embodiments, the selection marker is a co-receptor. In some embodiments, the selection marker is a T cell co-receptor. In some embodiments, the selection marker is CD4. In some embodiments, the selection agent comprises an anti-CD4 antibody, a bivalent antibody fragment of an anti-CD4 antibody, a monovalent antibody fragment of an anti-CD4 antibody, or a proteinaceous CD4-binding molecule with antibody-like binding properties. In some embodiments, the anti-CD4 antibody, a bivalent antibody fragment of an anti-CD4 antibody, or a monovalent antibody fragment of an anti-CD4 antibody (e.g., an anti-CD4 Fab fragment) is derived from antibody 13B8.2 or a functionally active variant of 13B8.2 that retains specific binding to CD4. 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, International Patent Application WO2013 / 124474, and Bes, C, et al. J Biol Chem 278, 14265-14273 (2003). The variant Fab fragment designated "ml3B8.2" possesses the variable domains of the CD4-binding murine antibody 13B8.2 and constant domains containing a gamma-type constant human CH1 domain of 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 variant of an anti-CD4 antibody, e.g., antibody 13B8.2, contains the amino acid substitution H91A in the variable light chain, Y92A in the variable light chain, H35A in the variable heavy chain, and / or R53A in the variable heavy chain, each according to Kabat numbering. In some embodiments, 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:30) is mutated to Ala, and the Arg residue at position 53 of the heavy chain (position 55 in SEQ ID NO:29) is mutated to Ala. In some embodiments, the selection agent contains an anti-CD4 Fab. In some embodiments, the anti-CD4 Fab contains a variable heavy chain having the sequence set forth in SEQ ID NO:29 and a variable light chain having the sequence set forth in SEQ ID NO:30.In some embodiments, the anti-CD4 Fab contains the CDRs of a variable heavy chain having the sequence shown in SEQ ID NO:29 and the CDRs of a variable light chain having the sequence shown in SEQ ID NO:30.

[0209] In some embodiments, the selection marker is CD8. In some embodiments, the selection agent contains an anti-CD8 antibody, a bivalent antibody fragment of an anti-CD8 antibody, a monovalent antibody fragment of an anti-CD8 antibody, or a proteinaceous CD8-binding molecule with antibody-like binding properties. In some embodiments, the anti-CD8 antibody, a bivalent antibody fragment of an anti-CD8 antibody, or a monovalent antibody fragment of an anti-CD8 antibody (e.g., an anti-CD8 Fab fragment) is 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 selection agent contains an anti-CD8 Fab. In some embodiments, the anti-CD8 Fab contains a variable heavy chain having the sequence set forth in SEQ ID NO:36 and a variable light chain having the sequence set forth in SEQ ID NO:37. In some embodiments, the anti-CD8 Fab contains the CDRs of a variable heavy chain having the sequence set forth in SEQ ID NO:36 and the CDRs of a variable light chain having the sequence set forth in SEQ ID NO:37.

[0210] In some embodiments, the selection marker is a molecule containing an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the selection marker is a member of the T cell antigen receptor complex. In some embodiments, the selection marker is a member of the TCR / CD3 complex. In some embodiments, the selection marker is CD3. In some embodiments, the selection marker is a CD3 chain. In some embodiments, the selection marker is a CD3 zeta chain.

[0211] In some embodiments, the selection marker is CD3. In some embodiments, the selection agent contains an anti-CD3 antibody, a bivalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, or a proteinaceous CD3-binding molecule with antibody-like binding properties. In some embodiments, the anti-CD3 antibody, a bivalent antibody fragment of an anti-CD3 antibody, or a monovalent antibody fragment of an anti-CD3 antibody (e.g., an anti-CD3 Fab fragment) is 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 selection agent contains an anti-CD3 Fab. In some embodiments, the anti-CD3 Fab contains a variable heavy chain having the sequence set forth in SEQ ID NO:31 and a variable light chain having the sequence set forth in SEQ ID NO:32. In some embodiments, the anti-CD3 Fab contains the CDRs of a variable heavy chain having the sequence shown in SEQ ID NO:31 and the CDRs of a variable light chain having the sequence shown in SEQ ID NO:32.

[0212] In some embodiments, the selection marker is CD25. In some embodiments, the selection agent comprises an anti-CD25 antibody, a bivalent antibody fragment of an anti-CD25 antibody, a monovalent antibody fragment of an anti-CD25 antibody, or a proteinaceous CD25-binding molecule with antibody-like binding properties. In some embodiments, the selection agent comprises an anti-CD25 Fab. In some embodiments, the anti-CD25 antibody, a bivalent antibody fragment of an anti-CD25 antibody, or a monovalent antibody fragment of an anti-CD25 antibody (e.g., an anti-CD25 Fab) is derived from the antibody FRT5 (see, e.g., Stemberger et al. 2012. pLoS One. 2012; 7(4): e35798) or a functionally active variant thereof that retains specific binding to CD25.

[0213] In some embodiments, the selection marker is CD62L. In some embodiments, the selection agent comprises an anti-CD62L antibody, a bivalent antibody fragment of an anti-CD62L antibody, a monovalent antibody fragment of an anti-CD62L antibody, or a proteinaceous CD62L-binding molecule with antibody-like binding properties. In some embodiments, the selection agent comprises an anti-CD62L Fab. In some embodiments, the anti-CD62L antibody, a bivalent antibody fragment of an anti-CD62L antibody, or a monovalent antibody fragment of an anti-CD62L antibody (e.g., an anti-CD62L Fab) is derived from the antibody DREG56 (e.g., ATCC HB300; see, e.g., Stemberger et al. 2012, pLoS One. 2012; 7(4): e35798) or a functionally active variant thereof that retains specific binding to CD62L.

[0214] In some embodiments, the selection marker is CD45RA. In some embodiments, the selection agent comprises an anti-CD45RA antibody, a bivalent antibody fragment of an anti-CD45RA antibody, a monovalent antibody fragment of an anti-CD45RA antibody, or a proteinaceous CD45RA binding molecule with antibody-like binding properties. In some embodiments, the selection agent comprises an anti-CD45RA Fab. In some embodiments, the anti-CD45RA antibody, a bivalent antibody fragment of an anti-CD45RA antibody, or a monovalent antibody fragment of an anti-CD45RA antibody (e.g., an anti-CD45RA Fab fragment) is derived from the antibody MEM56 (e.g., Millipore 05-1413; see, e.g., Stemberger et al. 2012, pLoS One. 2012; 7(4): e35798) or a functionally active variant thereof that retains specific binding to CD45RA.

[0215] In some embodiments, the selectable marker is a costimulatory molecule, an accessory molecule, a cytokine receptor, a chemokine receptor, an immune checkpoint molecule, or a member of the TNF family or TNF receptor family. In some embodiments, the selectable marker is a costimulatory molecule. In some embodiments, the costimulatory molecule is CD28, CD90 (Thy-1), CD95 (Apo- / Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM.

[0216] In some embodiments, the selection marker is CD28. In some embodiments, the selection agent contains an anti-CD28 antibody, a bivalent antibody fragment of an anti-CD28 antibody, a monovalent antibody fragment of an anti-CD28 antibody, or a proteinaceous CD28-binding molecule with antibody-like binding properties. In some embodiments, the anti-CD28 antibody, bivalent antibody fragment of an anti-CD28 antibody, or monovalent antibody fragment of an anti-CD28 antibody (e.g., an anti-CD28 Fab fragment) is derived from the antibody CD28.3 (deposited as a synthetic single-chain Fv construct under GenBank accession number AF451974.1; see also Vanhove et al., BLOOD, 15 July 2003, Vol. 102, No. 2, pages 564-570), the variable heavy and light chains of which contain the amino acid sequences set forth in SEQ ID NOs: 33 and 34, respectively. In some embodiments, the selection agent contains an anti-CD28 Fab. In some embodiments, the anti-CD28 Fab contains a variable heavy chain having the sequence shown in SEQ ID NO: 33 and a variable light chain having the sequence shown in SEQ ID NO: 34. In some embodiments, the anti-CD28 Fab contains the CDRs of the variable heavy chain having the sequence shown in SEQ ID NO: 33 and the CDRs of the variable light chain having the sequence shown in SEQ ID NO: 34.

[0217] In some embodiments, the selection marker is CD90. In some embodiments, the selection agent comprises an anti-CD90 antibody, a bivalent antibody fragment of an anti-CD90 antibody, a monovalent antibody fragment of an anti-CD90 antibody, or a proteinaceous CD90 binding molecule with antibody-like binding properties. In some embodiments, the selection agent comprises an anti-CD90 Fab. In some embodiments, the anti-CD90 antibody, a bivalent antibody fragment of an anti-CD90 antibody, or a monovalent antibody fragment of an anti-CD90 antibody (e.g., an anti-CD90 Fab fragment) is derived from the anti-CD90 antibody G7 (Biolegend, catalog number 105201).

[0218] In some embodiments, the selection marker is CD95. In some embodiments, the selection agent contains an anti-CD95 antibody, a bivalent antibody fragment of an anti-CD95 antibody, a monovalent antibody fragment of an anti-CD95 antibody, or a proteinaceous CD95 binding molecule with antibody-like binding properties. In some embodiments, the selection agent contains an anti-CD95 Fab. In some embodiments, the anti-CD95 antibody, a bivalent antibody fragment of an anti-CD95 antibody, or a monovalent antibody fragment of an anti-CD95 antibody (e.g., an anti-CD95 Fab fragment) is derived from monoclonal mouse anti-human CD95 CH11 (Upstate Biotechnology, Lake Placid, NY), anti-CD95 mAb 7C11, or anti-APO-1, e.g., as described in Paulsen et al. Cell Death & Differentiation 18.4 (2011): 619-631.

[0219] In some embodiments, the selection marker is CD137. In some embodiments, the selection agent comprises an anti-CD137 antibody, a bivalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment of an anti-CD137 antibody, or a proteinaceous CD137-binding molecule with antibody-like binding properties. In some embodiments, the selection agent comprises an anti-CD137 Fab. In some embodiments, the anti-CD137 antibody, a bivalent antibody fragment of an anti-CD137 antibody, or a monovalent antibody fragment of an anti-CD137 antibody (e.g., an anti-CD137 Fab fragment) is derived from LOB12, IgG2a, or LOB12.3, IgG1, as described in Taraban et al. Eur J Immunol. 2002 Dec; 32(12): 3617-27. See also, e.g., US6569997, US6303121, and Mittler et al. Immunol Res. 2004; 29(1-3): 197-208.

[0220] In some embodiments, the selection marker is CD40. In some embodiments, the selection agent comprises an anti-CD40 antibody, a bivalent antibody fragment of an anti-CD40 antibody, a monovalent antibody fragment of an anti-CD40 antibody, or a proteinaceous CD40 binding molecule with antibody-like binding properties. In some embodiments, the selection agent comprises an anti-CD40 Fab.

[0221] In some embodiments, the selection marker is CD40L. In some embodiments, the selection agent contains an anti-CD40L antibody, a bivalent antibody fragment of an anti-CD40L antibody, a monovalent antibody fragment of an anti-CD40L antibody, or a proteinaceous CD40L-binding molecule with antibody-like binding properties. In some embodiments, the selection agent contains an anti-CD40L Fab. In some embodiments, the anti-CD40L antibody, a bivalent antibody fragment of an anti-CD40L antibody, or a monovalent antibody fragment of an anti-CD40L antibody (e.g., an anti-CD40L Fab fragment) is derived from Hu5C8, as described in Blair et al. JEM vol. 191 no. 4 651-660. See also, e.g., WO1999061065, US20010026932, US7547438, and WO2001056603.

[0222] In some embodiments, the selection marker is ICOS. In some embodiments, the selection agent contains an anti-ICOS antibody, a bivalent antibody fragment of an anti-ICOS antibody, a monovalent antibody fragment of an anti-ICOS antibody, or a proteinaceous ICOS-binding molecule with antibody-like binding properties. In some embodiments, the selection agent contains an anti-ICOS Fab. In some embodiments, the anti-ICOS antibody, a bivalent antibody fragment of an anti-ICOS antibody, or a monovalent antibody fragment of an anti-ICOS antibody (e.g., an anti-ICOS Fab fragment) is derived from any of the antibodies described in US20080279851 and Deng et al. Hybrid Hybridomics. 2004 Jun;23(3):176-82.

[0223] In some embodiments, the selection marker is linker for T cell activation (LAT). In some embodiments, the selection agent comprises an anti-LAT antibody, a bivalent antibody fragment of an anti-LAT antibody, a monovalent antibody fragment of an anti-LAT antibody, or a proteinaceous LAT-binding molecule with antibody-like binding properties. In some embodiments, the selection agent comprises an anti-LAT Fab.

[0224] In some embodiments, the selection marker is CD27. In some embodiments, the selection agent comprises an anti-CD27 antibody, a bivalent antibody fragment of an anti-CD27 antibody, a monovalent antibody fragment of an anti-CD27 antibody, or a proteinaceous CD27-binding molecule with antibody-like binding properties. In some embodiments, the selection agent comprises an anti-CD27 Fab. In some embodiments, the anti-CD27 antibody, a bivalent antibody fragment of an anti-CD27 antibody, or a monovalent antibody fragment of an anti-CD27 antibody (e.g., an anti-CD27 Fab fragment) is derived from any of the antibodies described in WO2008051424.

[0225] In some embodiments, the selection marker is OX40. In some embodiments, the selection agent contains an anti-OX40 antibody, a bivalent antibody fragment of an anti-OX40 antibody, a monovalent antibody fragment of an anti-OX40 antibody, or a proteinaceous OX40 binding molecule with antibody-like binding properties. In some embodiments, the selection agent contains an anti-OX40 Fab. In some embodiments, the anti-OX40 antibody, a bivalent antibody fragment of an anti-OX40 antibody, or a monovalent antibody fragment of an anti-OX40 antibody (e.g., an anti-OX40 Fab fragment) is derived from any of the antibodies described in WO2013038191 and Melero et al. Clin Cancer Res. 2013 Mar 1; 19(5): 1044-53.

[0226] In some embodiments, the selection marker is HVEM. In some embodiments, the selection agent contains an anti-HVEM antibody, a bivalent antibody fragment of an anti-HVEM antibody, a monovalent antibody fragment of an anti-HVEM antibody, or a proteinaceous HVEM-binding molecule with antibody-like binding properties. In some embodiments, the selection agent contains an anti-HVEM Fab. In some embodiments, the anti-HVEM antibody, a bivalent antibody fragment of an anti-HVEM antibody, or a monovalent antibody fragment of an anti-HVEM antibody (e.g., an anti-HVEM Fab fragment) is derived from any of the antibodies described in WO2006054961, WO2007001459, and Park et al. Cancer Immunol Immunother. 2012 Feb;61(2):203-14.

[0227] In some embodiments, the selection agent further comprises a binding partner. In some embodiments, the selection agent comprises 1 to 5, 1 to 4, 1 to 3, or 1 to 2 (inclusive) binding partners. In some embodiments, the selection agent comprises exactly one binding partner. In some embodiments, the selection agent comprises exactly two binding partners. In some embodiments, the selection agent comprises exactly three binding partners. In some embodiments, the selection agent comprises exactly four binding partners. In some embodiments, the selection agent comprises exactly five binding partners.

[0228] In some embodiments, each binding partner of a selection agent containing multiple binding partners is individually selected from among the binding partners described herein, e.g., any of those described in this section. In some embodiments, each binding partner of a selection agent containing multiple binding partners is the same and is any one of the binding partners described herein, e.g., any of those described in this section.

[0229] In some embodiments, the binding partner is hydrocarbon-based (including polymeric) and contains nitrogen, phosphorus, sulfur, carbon, halogen, or pseudohalogen groups. In some embodiments, the binding partner is an alcohol, organic acid, inorganic acid, amine, phosphine, thiol, disulfide, alkane, amino acid, peptide, oligopeptide, polypeptide, protein, nucleic acid, lipid, monosaccharide, oligosaccharide, or polysaccharide. By way of further example, in some embodiments, the binding partner is a cation, anion, polycation, polyanion, polycation, electrolyte, polyelectrolyte, carbon nanotube, or carbon nanofoam. By way of still further example, in some embodiments, the binding partner is a crown ether, an immunoglobulin or fragment thereof, or a proteinaceous binding molecule with antibody-like function.

[0230] In some embodiments, the binding partner comprises a moiety known to those skilled in the art as an affinity tag, hi some embodiments, the selection reagent comprises a corresponding binding partner, e.g., an antibody or antibody fragment known to bind to the affinity tag. As some illustrative examples of known affinity tags, in some embodiments, the affinity tag comprises dinitrophenol or digoxigenin, oligohistidine, polyhistidine, an immunoglobulin domain, glutathione-S-transferase (GST), chitin-binding protein (CBP) or thioredoxin, calmodulin-binding peptide (CBP), FLAG'-peptide, an HA tag (SEQ ID NO:20), a VSV-G tag (SEQ ID NO:21), an HSV tag (SEQ ID NO:22), a T7 epitope (SEQ ID NO:23), maltose-binding protein (MBP), an HSV epitope of herpes simplex virus glycoprotein D (SEQ ID NO:24), a "myc" epitope of the transcription factor c-myc (SEQ ID NO:25), or a V5 tag (SEQ ID NO:26). In some embodiments, a complex formed between the binding site of a selection reagent and an affinity tag, e.g., between the selection reagent's corresponding binding partner, e.g., an antibody or antibody fragment, and an affinity tag, can be competitively disrupted by contacting the complex with the free binding partner, e.g., an unbound affinity tag.

[0231] In some embodiments, the affinity tag comprises an oligonucleotide tag that hybridizes to an oligonucleotide linked to or contained in a selection reagent having a complementary sequence.

[0232] In some embodiments, the binding partner is a lectin, protein A, protein G, metal, metal ion, nitrilotriacetic acid derivative (NTA), RGD-motif, dextran, polyethyleneimine (PEI), redox polymer, glycoprotein, aptamer, dye, amylose, maltose, cellulose, chitin, glutathione, calmodulin, gelatin, polymyxin, heparin, NAD, NADP, lysine, arginine, benzamidine, polyU, or oligodT. Lectins such as concanavalin A are known to bind to polysaccharides and glycosylated proteins. Examples of dyes are triazine dyes, such as cibacron blue F3G-A (CB) or Red HE-3B, which specifically bind to NADH-dependent enzymes. Green A is known to bind to CoA protein, human serum albumin, and dehydrogenase. The dyes 7-aminoactinomycin D and 4',6-diamidino-2-phenylindole are known to bind to DNA. Cations of metals such as Ni, Cd, Zn, Co, or Cu can also be used to bind affinity tags, such as hexahistidine or oligohistidine-containing sequences including the MAT tag (SEQ ID NO:35) and N-methacryloyl-(L)-cysteine ​​methyl ester.

[0233] In some embodiments, binding between the binding partner and the binding site of the selection reagent occurs in the presence of divalent, trivalent, or tetravalent cations. In some embodiments, the selection reagent comprises a divalent, trivalent, or tetravalent cation, e.g., held, e.g., complexed, by a suitable chelator. In some embodiments, the binding partner comprises a moiety that complexes with a divalent, trivalent, or tetravalent cation. Metal chelators include ethylenediamine, ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), N,N-bis(carboxymethyl)glycine (also known as nitrilotriacetic acid, NTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 2,3-dimercapto-1-propanol (dimercaprol), porphyrins, and heme. As an example, EDTA chelates most monovalent, divalent, trivalent, and tetravalent metal ions, e.g., silver (Ag + ), calcium (Ca 2+ ), manganese (Mn 2+ ), copper (Cu 2+ ), iron (Fe 2+ ), cobalt (Co + ), and zirconium (Zr 4+ ), but BAPTA can form complexes with Ca 2+ As an example, one skilled in the art can use the chelating agent nitrilotriacetic acid (NTA) to chelate oligohistidine tags and copper (Cu) 2+ ), Nickel (Ni 2+ ), cobalt (Co 2+ ) or zinc (Zn 2+ ) ions.

[0234] In some embodiments, the binding partner comprises a calmodulin-binding peptide and the selection reagent comprises multimeric calmodulin, e.g., as described in U.S. Patent No. 5,985,658. In some embodiments, the binding partner comprises a FLAG peptide and the selection reagent comprises an antibody that binds to the FLAG peptide. For example, in some embodiments, the selection reagent comprises monoclonal antibody 4E11 that binds to the FLAG peptide, e.g., as described in U.S. Patent No. 4,851,341. In some embodiments, the binding partner comprises an oligohistidine tag and the selection reagent comprises an antibody or a transition metal ion that binds to the oligohistidine tag. In some embodiments, calmodulin, antibodies such as 4E11, chelated metal ions, and free chelators can be multimerized by methods including, for example, biotinylation and complexation with streptavidin, avidin, or oligomers thereof, or by, in a first step, introducing carboxyl residues into a polysaccharide, such as dextran, as described, for example, in Noguchi et al. (1992), Bioconjugate Chemistry 3: 132-137, and, in a second step, using carbodiimide chemistry to link calmodulin, antibodies, chelated metal ions, or free chelators to the carboxyl groups in the polysaccharide, such as dextran, via primary amino groups. In some embodiments, binding between the binding partner and the binding site of the selection reagent can be disrupted by metal ion chelation. Metal chelation can be achieved, for example, by the addition of EGTA or EDTA.

[0235] In some embodiments, the binding partner binds to a biotin-binding molecule, hi some embodiments, the binding partner binds to the biotin-binding site of the molecule.

[0236] In some embodiments, the binding partner is streptavidin or an avidin binding partner. In some embodiments, the binding partner is a streptavidin binding partner. In some embodiments, the streptavidin binding partner is also an avidin binding partner.

[0237] In some embodiments, the binding partner binds to a molecule that is streptavidin, avidin, a streptavidin analog or mutein, or an avidin analog or mutein. In some embodiments, the molecule is any of the streptavidin, avidin, streptavidin analog or mutein and avidin analog or mutein molecules described herein, e.g., in Section IA-1-c. In some embodiments, the selection reagent contains the molecule. In some embodiments, the binding partner binds to the biotin-binding site of the molecule. In some embodiments, the binding partner binds to the molecule's natural biotin binding site (see, e.g., Qureshi et al. (2001), Journal of Biological Chemistry 276(49): 46422-46428; and Livnah et al. (1993), Proc Natl Acad Sci 90: 5076-5080, which describe the interaction of biotin with streptavidin and avidin, respectively). In some embodiments, the binding partner allows for functionalization of streptavidin, avidin, streptavidin analogs or muteins, or reagents containing avidin analogs or muteins.

[0238] Binding partners that bind to streptavidin, avidin, streptavidin analogs or muteins, or avidin analogs or muteins can be identified and selected by one of skill in the art, including those that bind to the biotin binding site of these molecules. In some embodiments, the binding partner binds to a molecule that is streptavidin.

[0239] In some embodiments, the binding partner contains biotin. In some embodiments, the binding partner is biotin. In some embodiments, the biotin is D-biotin. In some embodiments, the binding partner contains a biotin analog or derivative. In some embodiments, the binding partner is a biotin analog or derivative. In some embodiments, the biotin analog or derivative is a structural analog of biotin. In some embodiments, the biotin analog or derivative binds to the biotin binding site of streptavidin, avidin, a streptavidin analog or mutein, or an avidin analog or mutein. In some embodiments, the biotin analog or derivative binds to the biotin binding site of streptavidin. In some embodiments, the biotin analog or derivative is desthiobiotin, iminobiotin, guanidinobiotin, diaminobiotin, lipoic acid, HABA (hydroxyazobenzene-benzoic acid), dimethyl-HABA, biotin sulfone, caproylamidobiotin, or biocytin (or any of the biotin analogs and derivatives described, for example, in International Published PCT Application WO2008140573).

[0240] In some embodiments, the binding partner comprises a streptavidin-binding peptide. In some embodiments, the binding partner is a streptavidin-binding peptide. In some embodiments, the streptavidin-binding peptide binds to streptavidin, avidin, a streptavidin analog or mutein, or the biotin-binding site of an avidin analog or mutein. In some embodiments, the streptavidin-binding peptide binds to the biotin-binding site of streptavidin. In some embodiments, the streptavidin-binding peptide comprises an amino acid sequence having the formula set forth in SEQ ID NO:9, e.g., the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the streptavidin-binding peptide comprises an amino acid sequence set forth in SEQ ID NO:11, e.g., the formula set forth in SEQ ID NO:12. In some embodiments, the streptavidin-binding peptide comprises the amino acid sequence set forth in SEQ ID NO:7, also referred to as Strep-tag®. In some embodiments, the sequence of the streptavidin-binding peptide is set forth in SEQ ID NO: 7. In some embodiments, the streptavidin-binding peptide contains the amino acid sequence set forth in SEQ ID NO: 8, also referred to as Strep-tag® II. In some embodiments, the sequence of the streptavidin-binding peptide is set forth in SEQ ID NO: 8.

[0241] In some embodiments, the streptavidin-binding peptide may be further modified. In some embodiments, the streptavidin-binding peptide contains the amino acid sequence set forth in SEQ ID NO:8 conjugated to nickel-charged trisNTA, also referred to as His-STREPPER or His / Strep-tag® II Adapter.

[0242] In some embodiments, the streptavidin-binding peptide contains a concatenated sequence of two streptavidin-binding modules. In some embodiments, the streptavidin-binding peptide contains exactly two concatenated sequences of streptavidin-binding modules. In some embodiments, the streptavidin-binding modules are separated from each other by 50 or fewer amino acids, e.g., 45, 40, 35, 30, 25, 20, 15, 10, or 5 or fewer amino acids. In some embodiments, the streptavidin-binding modules are directly connected to each other. In some embodiments, one streptavidin-binding module has 3 to 8 amino acids and contains at least the sequence His-Pro-Xaa (SEQ ID NO:9), where Xaa is glutamine, asparagine, or methionine. In some embodiments, the other streptavidin binding module has the same or a different sequence as the first streptavidin binding module, e.g., as set forth in SEQ ID NO:11 (see, e.g., International Published PCT Application WO 02 / 077018; and U.S. Patent No. 7,981,632). In some embodiments, one of the streptavidin binding modules contains the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, each of the streptavidin binding modules contains the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, one of the streptavidin binding modules contains the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, each of the streptavidin binding modules contains the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the streptavidin binding peptide contains an amino acid sequence having a formula set forth in SEQ ID NO:13 or SEQ ID NO:14. In some embodiments, the streptavidin-binding peptide comprises an amino acid sequence set forth in any of SEQ ID NOs: 15-19. In some embodiments, the sequence of the streptavidin-binding peptide is set forth in any of SEQ ID NOs: 15-19.In some embodiments, the streptavidin-binding peptide contains the amino acid sequence set forth in SEQ ID NO: 16, also referred to as Twin-Strep-tag®. In some embodiments, the sequence of the streptavidin-binding peptide is set forth in SEQ ID NO: 16.

[0243] b. Chromatography matrix In some embodiments, the chromatography matrix is ​​essentially non-toxic, eg, does not harm the health or viability of cells that are applied to the chromatography matrix.

[0244] In some embodiments, the chromatography matrix comprises a non-magnetic or non-magnetizable material. In some embodiments, the chromatography matrix does not comprise any magnetically attractive substances.

[0245] In some embodiments, the chromatography matrix comprises a monolithic matrix. In some embodiments, the chromatography matrix comprises a membrane matrix. In some embodiments, the chromatography matrix comprises a granular matrix. In some embodiments, the chromatography matrix comprises a bead matrix.

[0246] In some embodiments, the chromatography matrix comprises derivatized silica or a cross-linked gel. In some embodiments, the cross-linked gel is based on a natural polymer, e.g., a polysaccharide. In some embodiments, the polysaccharide is cross-linked. Examples of polysaccharide matrices include agarose gels (e.g., Superflow™ agarose or Sepharose® materials, e.g., Superflow™ Sepharose®, which are commercially available in a variety of bead sizes and pore sizes) or gels of cross-linked dextran. Further examples include particulate cross-linked agarose matrices to which dextran is covalently attached, such as those commercially available as Sephadex® or Superdex® (various bead sizes, with various pore sizes), both available from GE Healthcare. Further examples include Sephacryl®, also available from GE Healthcare in a variety of bead sizes and pore sizes.

[0247] In some embodiments, the cross-linked gel is based on a synthetic polymer. In some embodiments, the synthetic polymer is a polymer that has polar monomer units and is therefore itself polar. In some embodiments, the synthetic polymer is hydrophilic. Examples of synthetic polymers include polyacrylamide, styrene-divinylbenzene gels, and copolymers of acrylates and diols or acrylamides 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 chromatography matrix comprises natural and synthetic polymeric components, such as composite matrices or copolymers of polysaccharides and agarose, e.g., polyacrylamide / agarose composites, or composite matrices or copolymers of polysaccharides and N,N'-methylenebisacrylamide. An example of copolymers of dextran and N,N'-methylenebisacrylamide is the Sephacryl® series of materials. Derivatized silica can include silica particles coupled to synthetic or natural polymers, examples of which include polysaccharide-modified silica, polyvinylpyrrolidone-modified silica, polyethylene oxide-modified silica, poly(2-hydroxyethylaspartamide)-modified silica, and poly(N-isopropylacrylamide)-modified silica.

[0248] In some embodiments, the chromatography matrix comprises a particulate matrix. In some embodiments, the chromatography matrix comprises a polymeric resin, a metal oxide, a semi-metal oxide, or a mixed oxide. In some embodiments, the particles of the granular matrix have a diameter of 5 μm to 600 μm, 5 μm to 400 μm, 5 μm to 200 μm, 5 μm to 150 μm, 5 μm to 125 μm, 5 μm to 100 μm, 5 μm to 75 μm, 5 μm to 50 μm, 5 μm to 25 μm, 25 μm to 600 μm, 25 μm to 400 μm, 25 μm to 200 μm, 25 μm to 150 μm, 25 μm to 125 μm, 25 μm to 100 μm, 25 μm to 75 μm, 25 μm to 50 μm, 50 μm to 600 μm, 50 μm to 400 μm, 50 μm to 20 0μm, 50μm~150μm, 50μm~125μm, 50μm~100μm, 50μm~75μm, 75μm~600μm, 75μm~400μm, 75μm~200μm, 75μm~150μm, 75μm~125μm, 75μm~100μm , 100μm~600μm, 100μm~400μm, 100μm~200μm, 100μm~150μm, 100μm~125μm, 125μm~600μm, 125μm~400μm, 125μm~200μm, 125μm~150μm, 150μm m~600μm, 150μm~400μm, 150μm~200μm, 200μm~600μm, 200μm~400μm, or 400μm~600μm, or about 5μm~600μm, 5μm~400μm, 5μm~200μm, 5μm~15 0μm, 5μm~125μm, 5μm~100μm, 5μm~75μm, 5μm~50μm, 5μm~25μm, 25μm~600μm, 25μm~400μm, 25μm~200μm, 25μm~150μm, 25μm~125μm, 25μm~10 0μm, 25μm~75μm, 25μm~50μm, 50μm~600μm, 50μm~400μm, 50μm~200μm, 50μm~150μm, 50μm~125μm, 50μm~100μm, 50μm~75μm, 75μm~600μm, 75 μm~400μm, 75μm~200μm, 75μm~150μm, 75μm~125μm, 75μm~100μm, 100μm~600μm, 100μm~400μm, 100μm~200μm, 100μm~150μm, 100μm~125μm,The particles have an average particle size of 125 μm to 600 μm, 125 μm to 400 μm, 125 μm to 200 μm, 125 μm to 150 μm, 150 μm to 600 μm, 150 μm to 400 μm, 150 μm to 200 μm, 200 μm to 600 μm, 200 μm to 400 μm, or 400 μm to 600 μm (inclusive). In some embodiments, the particles of the granular matrix are 50 μm to 150 μm or about 50 μm to 150 μm in diameter (inclusive). In some embodiments, the particles of the granular matrix are 75 μm to 125 μm or about 75 μm to 125 μm in diameter (inclusive). In some embodiments, the particles of the granular matrix are between 90 μm and 110 μm in diameter, or between about 90 μm and 110 μm inclusive.

[0249] In some embodiments, the chromatography matrix comprises a chromatography resin. In some embodiments, the chromatography matrix comprises chromatography resin beads, such as those commercially available as CytoSorb® (CytoSorbents™). In some embodiments, the resin comprises a polystyrene resin. In some embodiments, the chromatography resin beads have a diameter of 5 μm to 600 μm, 5 μm to 400 μm, 5 μm to 200 μm, 5 μm to 150 μm, 5 μm to 125 μm, 5 μm to 100 μm, 5 μm to 75 μm, 5 μm to 50 μm, 5 μm to 25 μm, 25 μm to 600 μm, 25 μm to 400 μm, 25 μm to 200 μm, 25 μm to 150 μm, 25 μm to 125 μm, 25 μm to 100 μm, 25 μm to 75 μm, 25 μm to 50 μm, 50 μm to 60 0μm, 50μm~400μm, 50μm~200μm, 50μm~150μm, 50μm~125μm, 50μm~100μm, 50μm~75μm, 75μm~600μm, 75μm~400μm, 75μm~200μm, 75 μm~150μm, 75μm~125μm, 75μm~100μm, 100μm~600μm, 100μm~400μm, 100μm~200μm, 100μm~150μm, 100μm~125μm, 125μm~600μm, 12 5μm to 400μm, 125μm to 200μm, 125μm to 150μm, 150μm to 600μm, 150μm to 400μm, 150μm to 200μm, 200μm to 600μm, 200μm to 400μm, or 400μm to 600μm, or about 5μm to 600μm, 5μm to 400μm, 5μm to 200μm, 5μm to 150μm, 5μm to 125μm, 5μm to 100μm, 5μm to 75μm, 5μm to 50μm, 5μm to 25μm, 25μm to 600 μm, 25μm~400μm, 25μm~200μm, 25μm~150μm, 25μm~125μm, 25μm~100μm, 25μm~75μm, 25μm~50μm, 50μm~600μm, 50μm~400μm, 50μm ~200μm, 50μm~150μm, 50μm~125μm, 50μm~100μm, 50μm~75μm, 75μm~600μm, 75μm~400μm, 75μm~200μm, 75μm~150μm, 75μm~125μm,In some embodiments, the chromatography resin beads are 75 μm to 100 μm, 100 μm to 600 μm, 100 μm to 400 μm, 100 μm to 200 μm, 100 μm to 150 μm, 100 μm to 125 μm, 125 μm to 600 μm, 125 μm to 400 μm, 125 μm to 200 μm, 125 μm to 150 μm, 150 μm to 600 μm, 150 μm to 400 μm, 150 μm to 200 μm, 200 μm to 600 μm, 200 μm to 400 μm, or 400 μm to 600 μm (inclusive). In some embodiments, the chromatography resin beads are 50 μm to 150 μm or about 50 μm to 150 μm in diameter (inclusive). In some embodiments, the chromatography resin beads have a diameter of 75 μm to 125 μm, or about 75 μm to 125 μm, inclusive. In some embodiments, the chromatography resin beads have a diameter of 90 μm to 110 μm, or about 90 μm to 110 μm, inclusive.

[0250] In some embodiments, the chromatography matrix contains a magnetically attractive substance, such as one or more magnetically attractive particles or magnetic fluids. The magnetically attractive particles can be diamagnetic, ferromagnetic, paramagnetic, or superparamagnetic materials. Superparamagnetic materials generate an induced magnetic field in response to a magnetic field, but do not result in permanent magnetization. Iron oxide-based magnetic particles are commercially available, for example, from Dynal Biotech as Dynabeads®, Miltenyi Biotec as magnetic MicroBeads, CPG Inc. as magnetic porous glass beads, and from various other suppliers, such as Roche Applied Science, BIOCLON, BioSource International Inc., micromod, AMBION, Merck, Bangs Laboratories, Polysciences, or Novagen Inc. Superparamagnetic Co and FeCo based magnetic nanoparticles, as well as ferromagnetic Co nanocrystals, are described, for example, in Hutten, A. et al. (J. Biotech. (2004), 112, 47-63).

[0251] c. Selection reagent In some embodiments, the selection reagent contains a molecule to which a binding partner of the selection agent can bind.

[0252] In some cases, the selection reagent contains at least two chelating groups, K, that may be capable of binding to a transition metal ion. In some embodiments, the selection reagent may be capable of binding to an oligohistidine affinity tag, glutathione-S-transferase, calmodulin or an analog thereof, calmodulin-binding peptide (CBP), FLAG peptide, HA tag, maltose-binding protein (MBP), an HSV epitope, a myc epitope, or a biotinylated carrier protein.

[0253] In some embodiments, the molecule is avidin, e.g., wild-type avidin. In some embodiments, the molecule is an avidin analog. In some embodiments, an avidin analog is a variant of wild-type avidin that has one or more modified functional groups but contains a biotin-binding site. In some embodiments, the molecule is an avidin mutein. In some embodiments, an avidin mutein is a polypeptide that is distinguished from the sequence of wild-type avidin by one or more amino acid substitutions, deletions, or additions, but contains a biotin-binding site. In some embodiments, the avidin analog is neutravidin, i.e., a deglycosylated avidin with a modified arginine, which can exhibit a more neutral pi and can be used in place of wild-type avidin. In some embodiments, the avidin analog is any of those commercially available as ExtrAvidin® available through Sigma Aldrich, NeutrAvidin available from Thermo Scientific or Invitrogen, and CaptAvidin™ available from Molecular Probes. In some embodiments, the avidin analog or mutein is any of those described in International Published PCT Application WO2008 / 140573.

[0254] In some embodiments, the molecule is streptavidin, e.g., wild-type streptavidin. In some embodiments, the streptavidin has the amino acid sequence set forth in SEQ ID NO:1, as disclosed in Argarana et al., Nucleic Acids Res. 14 (1986) 1871-1882, or has an amino acid sequence that is a sequence present in its homologs from other Streptomyces species. In some embodiments, the streptavidin has the amino acid sequence set forth in SEQ ID NO:1.

[0255] In some embodiments, the molecule is a streptavidin analog. In some embodiments, a streptavidin analog is a variant of wild-type streptavidin that has one or more modified functional groups but contains a biotin-binding site. In some embodiments, the molecule is a streptavidin mutein. In some embodiments, a streptavidin mutein is a polypeptide that differs from the sequence of wild-type streptavidin by one or more amino acid substitutions, deletions, or additions, but contains a biotin-binding site.

[0256] In some embodiments, the streptavidin mutein binds to a streptavidin-binding peptide, such as any of those described herein. In some embodiments, the streptavidin mutein binds to any of the streptavidin-binding peptides set forth in SEQ ID NOs: 7, 8, and 15-19. In some embodiments, the binding affinity of the streptavidin-binding peptide to the streptavidin mutein is greater than or equal to 1×10 -13 M, 1 x 10 -12 M, or 1 x 10 -11 Over M and 1 x 10 -4 M, 5 x 10 -4 M, 1 x 10 -5 M, 5 x 10 -5 M, 1 x 10 -6 M, 5 x 10 -6 M, or 1 x 10 -7In some embodiments, the streptavidin mutein binds to biotin, e.g., D-biotin. In some embodiments, the streptavidin mutein binds to a biotin analog or derivative, e.g., any of those described herein. In some embodiments, the streptavidin mutein binds to biotin or a biotin analog or derivative with greater affinity than the streptavidin-binding peptide. In some embodiments, binding of a streptavidin-binding peptide to a streptavidin mutein, e.g., the biotin-binding site of a streptavidin mutein, can be disrupted by the presence of biotin or a biotin analog or derivative. In some embodiments, binding of a streptavidin mutein to the streptavidin-binding peptide of any of SEQ ID NOs:7, 8, and 15-19 is disrupted by the presence of biotin, e.g., D-biotin.

[0257] In some embodiments, the streptavidin mutein contains only a portion of wild-type streptavidin. In some embodiments, the streptavidin mutein is a minimal streptavidin (sometimes referred to as recombinant core streptavidin) in which wild-type streptavidin has been truncated at the N-terminus and / or C-terminus. In some embodiments, the streptavidin mutein is any of the recombinant core streptavidins described in Sano et al. (1995), Journal of Biological Chemistry 270(47): 28204-28209. In some embodiments, the streptavidin mutein begins at the N-terminus in the region of amino acid positions 10-16 of SEQ ID NO:1 and ends at the C-terminus in the region of amino acid positions 133-142 of SEQ ID NO:1. References to residue positions in streptavidin or streptavidin muteins are with reference to the numbering of residues in SEQ ID NO:1. In some embodiments, the sequence of the streptavidin mutein is set forth in any of SEQ ID NOs:2, 103, and 135. In some embodiments, the streptavidin mutein is the amino acid sequence from Ala 13 to Ser 139 of SEQ ID NO:1. In some embodiments, the sequence of the streptavidin mutein is set forth in SEQ ID NO:135. In some embodiments, the streptavidin mutein contains an N-terminal methionine and the amino acid sequence from Glu 14 to Ser 139 of SEQ ID NO:1. In some embodiments, the sequence of the streptavidin mutein is set forth in SEQ ID NO:2.

[0258] In some embodiments, the streptavidin mutein contains one or more amino acid substitutions compared to wild-type streptavidin, e.g., compared to the wild-type streptavidin sequence set forth in SEQ ID NO:1. In some embodiments, the streptavidin mutein contains one or more amino acid substitutions compared to a streptavidin mutein that is a minimal streptavidin. In some embodiments, the streptavidin contains one or more amino acid substitutions compared to a streptavidin mutein that begins at the N-terminus in the region of amino acid positions 10-16 of SEQ ID NO:1 and ends at the C-terminus in the region of amino acid positions 133-142 of SEQ ID NO:1, e.g., a minimal streptavidin. In some embodiments, the streptavidin contains one or more amino acid substitutions compared to a streptavidin mutein set forth in any of SEQ ID NOs:2, 103, and 135.

[0259] In some embodiments, the streptavidin mutein binds biotin and contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid differences compared to the sequence of amino acids set forth in SEQ ID NO: 1, 2, 103, or 135. In some embodiments, the streptavidin mutein binds biotin and contains an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of amino acids set forth in SEQ ID NO: 1, 2, 103, or 135. In some embodiments, the amino acid substitutions are conservative or non-conservative mutations. In some embodiments, the streptavidin mutein is any of those described in U.S. Patent Nos. 5,168,049; 5,506,121; 6,022,951; 6,156,493; 6,165,750; 6,103,493; 6,368,813; and International Published PCT Applications WO2014 / 076277, WO2008 / 140573, WO 86 / 02077, WO 98 / 40396, and WO 96 / 24606. In some embodiments, the streptavidin mutein is any of those described in DE 19641876 A1; Howarth et al. (2006) Nat. Methods, 3:267-73; Zhang et al. (2015) Biochem. Biophys. Res. Commun., 463:1059-63; Fairhead et al. (2013) J. Mol. Biol., 426: 199-214; Wu et al. (2005) J. Biol. Chem., 280: 23225-31; Lim et al. (2010) Biochemistry, 50: 8682-91); and Qureshi et al. (2001), Journal of Biological Chemistry 276(49): 46422-46428.

[0260] In some embodiments, the streptavidin mutein is any as described in U.S. Patent No. 6,103,493. In some embodiments, the streptavidin mutein contains at least one mutation within a region corresponding to amino acid positions 44-53 of wild-type streptavidin, e.g., as shown in SEQ ID NO:1. In some embodiments, "corresponding to" refers to an amino acid position with reference to the amino acid sequence of wild-type streptavidin, e.g., as shown in SEQ ID NO:1. One of skill in the art will be able to identify these residues using methods including, for example, sequence alignment. In some embodiments, the streptavidin mutein contains a mutation at one or more of residues 44, 45, 46, and 47 of wild-type streptavidin. In some embodiments, the streptavidin mutein contains a substitution of Glu at position 44 with a hydrophobic aliphatic amino acid, e.g., Val, Ala, Ile, or Leu. In some embodiments, the streptavidin mutein contains any amino acid at position 45. In some embodiments, the streptavidin mutein contains an aliphatic amino acid, e.g., a hydrophobic aliphatic amino acid, at position 46. In some embodiments, the streptavidin mutein contains a substitution of Val with a basic amino acid, e.g., Arg or Lys, e.g., Arg, at position 47. In some embodiments, Ala is present at position 46, Arg is present at position 47, and Val or Ile is present at position 44. In some embodiments, the streptavidin mutein contains a substitution of residue Val, as shown in exemplary streptavidin muteins containing the sequence of amino acids set forth in SEQ ID NO:3, 4, or 104, at sequence positions corresponding to positions 44-47 of the sequence of amino acids set forth in SEQ ID NO:1. 44 -Thr 45 -Ala 46 -Arg 47In some embodiments, the streptavidin mutein contains residues Ile, as shown in exemplary streptavidin muteins containing the sequence of amino acids set forth in SEQ ID NO:5, 6, or 104 at sequence positions corresponding to positions 44-47 of the sequence of amino acids set forth in SEQ ID NO:1. 44 -Gly 45 -Ala 46 -Arg 47 (SEQ ID NO:133). In some embodiments, the streptavidin mutein contains the amino acid sequence set forth in any of SEQ ID NOs:3-6, 104, and 105. In some embodiments, the streptavidin mutein is commercially available under the trademark Strep-Tactin® m1. In some embodiments, the streptavidin mutein is commercially available under the trademark Strep-Tactin® m2. In some embodiments, the streptavidin mutein contains the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the streptavidin mutein contains the amino acid sequence set forth in SEQ ID NO:6.

[0261] In certain embodiments, the streptavidin mutein is any as described in International Published PCT Application WO 2014 / 076277. In some embodiments, the streptavidin mutein contains at least two cysteine ​​residues in a region corresponding to amino acid positions 44-53 of the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, cysteine ​​residues are present at positions 45 and 52, creating a disulfide bridge connecting these amino acids. In some embodiments, amino acid 44 is glycine or alanine; amino acid 46 is alanine or glycine; and amino acid 47 is arginine. In some embodiments, the streptavidin mutein contains at least one mutation in a region corresponding to amino acid residues 115-121 of the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the streptavidin mutein contains at least one mutation at amino acid position 117, 120, or 121 and / or a deletion of amino acids 118 and 119 and a substitution at least at amino acid position 121.

[0262] In some embodiments, the streptavidin mutein contains a mutation at a position corresponding to position 117, which can be to a large hydrophobic residue such as Trp, Tyr, or Phe; to a charged residue such as Glu, Asp, or Arg; to a hydrophilic residue such as Asn or Gln; to the hydrophobic residues Leu, Met, or Ala; or to the polar residues Thr, Ser, or His. In some embodiments, the mutation at position 117 is combined with a mutation at a position corresponding to position 120 (which can be to a small residue such as Ser, Ala, or Gly) and a mutation at a position corresponding to position 121 (which can be to a hydrophobic residue, e.g., a bulky hydrophobic residue such as Trp, Tyr, or Phe). In some embodiments, the mutation at position 117 is combined with a mutation at a position corresponding to position 120 of wild-type streptavidin shown in SEQ ID NO:1 (which mutation can be to a hydrophobic residue such as Leu, Ile, Met, or Val; or Tyr or Phe), and a mutation at a position corresponding to position 121 of SEQ ID NO:1 (which mutation can be to a small residue such as Gly, Ala, or Ser, or with Gln, or with a hydrophobic residue such as Leu, Val, Ile, Trp, Tyr, Phe, or Met). In some embodiments, the streptavidin mutein contains residues Glu117, Gly120, and Tyr121, with reference to the positions in the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the streptavidin mutein also contains residues Val at sequence positions corresponding to positions 44-47 of the amino acid sequence shown in SEQ ID NO:1. 44 -Thr 45 -Ala 46 -Arg 47 or residue Ile 44 -Gly 45 -Ala 46 -Arg 47In some embodiments, the streptavidin mutein contains residues Val44, Thr45, Ala46, Arg47, Glu117, Gly120, and Tyr121. In some embodiments, the mutein streptavidin contains a sequence of amino acids set forth in any of SEQ ID NOs:27, 28, and 136, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of amino acids set forth in any of SEQ ID NOs:27, 28, and 136, contains residues Val44, Thr45, Ala46, Arg47, Glu117, Gly120, and Tyr121, and binds biotin. In some embodiments, the streptavidin mutein contains the sequence of amino acids set forth in SEQ ID NO: 27. In some embodiments, the streptavidin mutein contains the sequence of amino acids set forth in SEQ ID NO: 28. In some embodiments, the streptavidin mutein contains the sequence of amino acids set forth in SEQ ID NO: 136.

[0263] In some embodiments, the streptavidin mutein contains a sequence of amino acids set forth in any of SEQ ID NOs: 3-6, 27, 28, 104, 105, and 136, and the binding partner contains a streptavidin-binding peptide, wherein the sequence of the streptavidin-binding peptide is set forth in any of SEQ ID NOs: 7, 8, and 15-19. In some embodiments, the streptavidin mutein contains a sequence of amino acids set forth in SEQ ID NO: 6, and the binding partner contains a streptavidin-binding peptide, wherein the sequence of the streptavidin-binding peptide is set forth in any of SEQ ID NOs: 7, 8, and 15-19. In some embodiments, the streptavidin mutein contains a sequence of amino acids set forth in any of SEQ ID NOs:3-6, 27, 28, 104, 105, and 136, and the binding partner contains a streptavidin-binding peptide, wherein the sequence of the streptavidin-binding peptide is set forth in SEQ ID NO: 16. In some embodiments, the streptavidin mutein contains a sequence of amino acids set forth in SEQ ID NO: 6, and the binding partner contains a streptavidin-binding peptide, wherein the sequence of the streptavidin-binding peptide is set forth in SEQ ID NO: 16.

[0264] B. stimulation In some embodiments, the provided methods include stimulating immune cells, e.g., T cells. In some embodiments, the provided methods include incubating immune cells, e.g., T cells, under conditions for stimulating immune cells, e.g., T cells. In some embodiments, the incubating step is performed in the presence of a stimulatory reagent. In some embodiments, the provided methods include adding a stimulatory reagent to the immune cells, e.g., T cells.

[0265] In some embodiments, the stimulating step occurs prior to the step of manipulating the immune cells, e.g., T cells. In some embodiments, the stimulating step occurs subsequent to the step of manipulating the immune cells, e.g., T cells.

[0266] 1. Stimulating Reagents In some embodiments, the stimulating reagent is any of those described in US 11,274,278 and US 2021 / 0032297, e.g., any of the soluble multimerization reagents or oligomeric particle reagents described therein suitable for stimulating immune cells, e.g., T cells, including any that can be added to a stationary phase within the internal cavity of a chromatography column for stimulation of immune cells, e.g., T cells, immobilized on the stationary phase. In some embodiments, the T cell stimulating reagent is an oligomeric particle reagent composed of oligomers of streptavidin or streptavidin mutein molecules to which one or more binding agents for stimulating T cells are attached. In some embodiments, a primary agent (e.g., an anti-CD3 agent) and a secondary agent (e.g., an anti-CD28 agent) for stimulating T cells are attached to the oligomers of streptavidin or streptavidin mutein molecules. In some embodiments, the T cell stimulating reagent is in a soluble form.

[0267] In some embodiments, the stimulating reagent contains one or more binding agents. In some embodiments, the one or more binding agents are any described herein, e.g., in Section IB-1-b. In some embodiments, the one or more binding agents are selected from any of the selection agents described herein, e.g., in Section IA-1-a. In some embodiments, each of the one or more binding agents specifically binds to a molecule expressed on the surface of an immune cell, e.g., a T cell. In some embodiments, the stimulating reagent contains multiple binding agents that specifically bind to different molecules expressed on the surface of an immune cell, e.g., a T cell. In some embodiments, the one or more binding agents include a primary agent and a secondary agent.

[0268] In some embodiments, the stimulatory reagent contains a primary agent that specifically binds to a molecule to provide a primary activation signal to an immune cell, e.g., a T cell. In some embodiments, the stimulatory reagent contains a secondary agent that specifically binds to a costimulatory molecule to provide a costimulatory signal to an immune cell, e.g., a T cell. In some embodiments, the stimulatory reagent contains a primary agent and a secondary agent.

[0269] In some embodiments, the stimulatory reagent is a T cell stimulatory reagent. In some embodiments, the T cell stimulatory reagent contains a primary agent that specifically binds to a member of the TCR / CD3 complex. In some embodiments, the T cell stimulatory reagent contains a secondary agent that specifically binds to a T cell costimulatory molecule. In some embodiments, the T cell stimulatory reagent contains a primary agent and a secondary agent.

[0270] In some embodiments, one or more binding substances, e.g., the primary and secondary agents, each contain a binding partner. The binding partners can be the same or different between one or more binding substances, e.g., the primary and secondary agents. In some embodiments, the stimulating reagent contains one or more binding substances, e.g., a protein reagent having binding sites for the binding partners of each of the primary and secondary agents. In some embodiments, the protein reagent is any described herein, e.g., in Section IB-1-a. In some embodiments, one or more binding substances, e.g., the binding partners of each of the primary and secondary agents, bind to the protein reagent.

[0271] In some embodiments, the protein reagent contains multiple binding sites for one or more binding agents, e.g., binding partners of the first and second agents, respectively. Thus, in some embodiments, the protein reagent allows one or more binding agents, e.g., the first and second agents, to multimerize thereon, and in some aspects, causes an avidity effect for one or more binding agents, e.g., the first and second agents, to bind to target molecules. The multiple binding sites can be the same or different between the protein reagents.

[0272] In some embodiments, the stimulating reagent is a protein reagent to one or more binding agents, e.g., each of the primary and secondary agents, in a weight ratio of about 10:1 to 2:1, 9:1 to 2:1, 8:1 to 2:1, 7:1 to 2:1, 6:1 to 2:1, 5:1 to 2:1, 4:1 to 2:1, 3:1 to 2:1, 10:1 to 3:1, 9:1 to 3:1, 8:1 to 3:1, 7:1 to 3:1, 6:1 to 3:1, 5:1 to 3:1, 4:1 to 3:1, 10:1 to 4:1, 5:1 to 4:1, 6:1 to 4:1, 7:1 to 4:1, 8:1 to 4:1, 9:1 to 4:1, 10 ... In some embodiments, the stimulating reagent comprises a weight ratio of protein reagent to one or more binding agents, e.g., each of a primary and secondary agent, of about 10:1 to 2:1, inclusive. In some embodiments, the stimulation reagent contains a weight ratio of the protein reagent to one or more binding substances, e.g., each of the primary and secondary agents, of about 8:1 to 2:1, inclusive. In some embodiments, the stimulation reagent contains a weight ratio of the protein reagent to one or more binding substances, e.g., each of the primary and secondary agents, of about 8:1 to 4:1, inclusive. In some embodiments, the weight ratio to the protein reagent is different between the one or more binding substances, e.g., the primary and secondary agents. In some embodiments, the weight ratio to the protein reagent is the same between the one or more binding substances, e.g., the primary and secondary agents. In some embodiments, the stimulation reagent contains a weight ratio of the protein reagent to one or more binding substances, e.g., each of the primary and secondary agents, of about 6:1.

[0273] In some embodiments, the stimulating reagent comprises a ratio of about 10:1 to 2:1, 9:1 to 2:1, 8:1 to 2:1, 7:1 to 2:1, 6:1 to 2:1, 5:1 to 2:1, 4:1 to 2:1, 3:1 to 2:1, 10:1 to 3:1, 9:1 to 3:1, 8:1 to 3:1, 7:1 to 3:1, 6:1 to 3:1, 5:1 to 3:1, 4:1 to 3:1, 10:1 to 4:1, 9:1 to 4:1 , 8:1 to 4:1, 7:1 to 4:1, 6:1 to 4:1, 5:1 to 4:1, 10:1 to 5:1, 9:1 to 5:1, 8:1 to 5:1, 7:1 to 5:1, 6:1 to 5:1, 10:1 to 6:1, 9:1 to 6:1, 8:1 to 6:1, 7:1 to 6:1, 10:1 to 7:1, 9:1 to 7:1, 8:1 to 7:1, 10:1 to 8:1, 9:1 to 8:1, or 10:1 to 9:1 (inclusive). In some embodiments, the stimulating reagent is prepared by mixing the protein reagent and one or more binding agents, e.g., each of the primary and secondary agents, in a relative weight ratio of about 10:1 to 2:1 (inclusive). In some embodiments, the stimulation reagent is prepared by mixing the protein reagent with one or more binding substances, e.g., each of the primary and secondary agents, in a relative weight ratio of about 8:1 to 2:1, inclusive. In some embodiments, the stimulation reagent is prepared by mixing the protein reagent with one or more binding substances, e.g., each of the primary and secondary agents, in a relative weight ratio of about 8:1 to 4:1, inclusive. In some embodiments, the weight ratio to the protein reagent is different between the one or more binding substances, e.g., the primary and secondary agents. In some embodiments, the weight ratio to the protein reagent is the same between the one or more binding substances, e.g., the primary and secondary agents. In some embodiments, the stimulation reagent is prepared by mixing the protein reagent with one or more binding substances, e.g., each of the primary and secondary agents, in a relative weight ratio of about 6:1. In some embodiments, the mixing is performed at room temperature.

[0274] In some embodiments, the stimulating reagent contains one or more binding substances, e.g., primary and secondary agents, in a relative weight ratio of 4:1 to 1:1. In some embodiments, the stimulating reagent contains one or more binding substances, e.g., primary and secondary agents, in a relative weight ratio of 3:1 to 1:1. In some embodiments, the stimulating reagent contains one or more binding substances, e.g., primary and secondary agents, in a relative weight ratio of 2:1 to 1:1. In some embodiments, the stimulating reagent contains one or more binding substances, e.g., primary and secondary agents, in a relative weight ratio of about 1:1, e.g., equal parts by weight of one or more binding substances, e.g., primary and secondary agents.

[0275] In some embodiments, the stimulating reagent is prepared by mixing one or more binding substances, e.g., primary and secondary agents, in a relative weight ratio of 4:1 to 1:1. In some embodiments, the stimulating reagent is prepared by mixing one or more binding substances, e.g., primary and secondary agents, in a relative weight ratio of 3:1 to 1:1. In some embodiments, the stimulating reagent is prepared by mixing one or more binding substances, e.g., primary and secondary agents, in a relative weight ratio of 2:1 to 1:1. In some embodiments, the stimulating reagent is prepared by mixing one or more binding substances, e.g., primary and secondary agents, in a relative weight ratio of about 1:1, e.g., by mixing one or more binding substances, e.g., primary and secondary agents, in equal parts by weight.

[0276] In some embodiments, the binding partner binds to a biotin-binding site of one or more molecules of the protein reagent to which it binds. In some embodiments, the biotin-binding site is the native biotin-binding site of one or more of said molecules (see, e.g., Qureshi et al. (2001), Journal of Biological Chemistry 276(49): 46422-46428; and Livnah et al. (1993), Proc Natl Acad Sci 90: 5076-5080, which describe the interaction of biotin with streptavidin and avidin, respectively).

[0277] In some embodiments, the complex formed between one or more binding substances, such as the binding partners of the primary and secondary agents, and the protein reagent can be of any desired strength and affinity. In some embodiments, the complex is reversible. In some embodiments, the binding partner reversibly binds to one or more molecules of the protein reagent to which it binds. Exemplary binding partners and molecules for reversible binding are described herein and in, for example, U.S. Patent Nos. 5,168,049; 5,506,121; 6,103,493; 7,776,562; 7,981,632; 8,298,782; 8,735,540; and 9,023,604; and International Published PCT Applications WO2013 / 124474 and WO2014 / 076277.

[0278] In some embodiments, the binding affinity of the binding partner for one or more molecules of the protein reagent to which it binds is reduced relative to the binding affinity of biotin for streptavidin, and is about 10 -14 The dissociation constant (K d ) Binding affinity can be determined by any suitable method. In some embodiments, the binding affinity of a binding partner for one or more molecules of a protein reagent to which it binds is greater than or equal to 1×10 -13 M, 1 x 10 -12 M, or 1 x 10 -11 Over M and 1 x 10 -4 M, 5 x 10 -4 M, 1 x 10 -5 M, 5 x 10 -5 M, 1 x 10 -6 M, 5 x 10 -6 M, or 1 x 10 -7 It is less than M.

[0279] In some embodiments, the binding partner contains biotin, e.g., D-biotin, and one or more molecules of the protein reagent to which it binds are analogs or muteins of streptavidin or avidin that have reduced affinity for biotin compared to streptavidin or avidin.

[0280] In some embodiments, the binding partner contains a biotin analog or derivative, e.g., any as described herein, that has a reduced affinity for streptavidin or avidin compared to biotin, and one or more molecules of the protein reagent to which it binds are streptavidin or avidin. In some embodiments, the binding partner contains a biotin analog or derivative, e.g., any as described herein, that has a reduced affinity for streptavidin or avidin compared to biotin, and one or more molecules of the protein reagent to which it binds are streptavidin or avidin analogs or muteins that have a reduced affinity for the biotin analog or derivative compared to biotin.

[0281] In some embodiments, the binding partner contains a streptavidin-binding peptide, e.g., any as described herein, that has a reduced affinity for streptavidin or avidin compared to biotin, and one or more molecules of the protein reagent to which it binds are streptavidin or avidin. In some embodiments, the binding partner contains a streptavidin-binding peptide, e.g., any as described herein, and one or more molecules of the protein reagent to which it binds are analogs or muteins of streptavidin or avidin that have a reduced affinity for the streptavidin-binding peptide compared to biotin. In some embodiments, the binding partner contains a streptavidin-binding peptide, e.g., any as described herein, and one or more molecules of the protein reagent to which it binds are muteins of streptavidin that have a reduced affinity for the streptavidin-binding peptide compared to biotin.

[0282] In some embodiments, binding of a binding partner to one or more molecules of a protein reagent is disrupted by the presence of biotin, e.g., D-biotin. In some embodiments, binding of a binding partner to one or more molecules of a protein reagent is disrupted by the presence of a biotin analog or derivative, e.g., any of those described herein. For example, binding of the streptavidin-binding peptides known as Strep-tag®, Strep-tag® II, and Twin-Strep-tag® to the streptavidin mutein known as StrepTactin® m1 or m2 or StrepTactin XT® is disrupted by the presence of biotin, e.g., D-biotin, iminobiotin, lipoic acid, desthiobiotin, diaminobiotin, HABA, and dimethyl-HABA (see, e.g., U.S. Pat. Nos. 5,506,121 and 6,103,493, and International Published PCT Application WO2014 / 076277). Other combinations of molecules and binding partners whose binding can be disrupted by the presence of biotin or a biotin analog or derivative can be identified and selected by one of skill in the art.

[0283] In some embodiments, the stimulating reagent is not immobilized on a solid support. In some embodiments, the stimulating reagent is in a soluble form. In some embodiments, the stimulating reagent is soluble in cell culture medium, such as any of those described herein.

[0284] In some embodiments, the stimulation reagent contains a weight ratio of protein reagent:primary agent (e.g., anti-CD3 binding agent):secondary agent (e.g., anti-CD28 binding agent) of about 10:1:1 to 2:1:1, inclusive. In some embodiments, the stimulation reagent contains a weight ratio of protein reagent:primary agent:secondary agent of about 8:1:1 to 2:1:1, inclusive. In some embodiments, the stimulation reagent contains a weight ratio of protein reagent:primary agent:secondary agent of about 8:1:1 to 4:1:1, inclusive. In some embodiments, the stimulation reagent contains a weight ratio of protein reagent:primary agent:secondary agent of about 6:1:1. In some embodiments, 4 μg of stimulation reagent contains about 3 μg of protein reagent, 0.5 μg of anti-CD3 binding agent, and 0.5 μg of anti-CD28 binding agent.

[0285] a. Protein reagents In some embodiments, the protein reagent contains a molecule to which one or more binding agents, e.g., binding partners of the primary and secondary agents, can bind. In some embodiments, the protein reagent contains a plurality of molecules to which a binding partner can bind. In some embodiments, the binding partner binds to one of the plurality of molecules. In some embodiments, the binding partner binds to two of the plurality of molecules.

[0286] In some embodiments, the molecule is any of those described herein that can bind to one or more binding partners of the binding substance. In some embodiments, the molecule is any of the streptavidin, avidin, streptavidin analogues or muteins and avidin analogues or muteins molecules described herein. In some embodiments, the molecule is streptavidin. In some embodiments, the molecule is any of the streptavidin mutein molecules described herein.

[0287] In some embodiments, each molecule of the protein reagent is individually selected from among any of the streptavidin, avidin, streptavidin analog or mutein and avidin analog or mutein molecules described herein, hi some embodiments, the protein reagent contains a mixture of any of the streptavidin, avidin, streptavidin analog or mutein and avidin analog or mutein molecules described herein.

[0288] In some embodiments, each molecule of the protein reagent is individually selected from among any of the streptavidin and streptavidin analog or mutein molecules described herein, hi some embodiments, the protein reagent contains a mixture of any of the streptavidin and streptavidin analog or mutein molecules described herein.

[0289] In some embodiments, each molecule of the protein reagent is the same and is any one of the streptavidin, avidin, streptavidin analog or mutein, and avidin analog or mutein molecules described herein. In some embodiments, each molecule of the protein reagent is the same and is streptavidin. In some embodiments, each molecule of the protein reagent is the same and is any one of the streptavidin mutein molecules described herein.

[0290] In some cases, the protein reagent contains at least two chelating groups, K, that may be capable of binding to a transition metal ion. In some embodiments, the protein reagent may be capable of binding to an oligohistidine affinity tag, glutathione-S-transferase, calmodulin or an analog thereof, calmodulin-binding peptide (CBP), FLAG peptide, HA tag, maltose-binding protein (MBP), an HSV epitope, a myc epitope, or a biotinylated carrier protein.

[0291] In some embodiments, the molecule is avidin, e.g., wild-type avidin. In some embodiments, the molecule is an avidin analog. In some embodiments, an avidin analog is a variant of wild-type avidin that has one or more modified functional groups but contains a biotin-binding site. In some embodiments, the molecule is an avidin mutein. In some embodiments, an avidin mutein is a polypeptide that is distinguished from the sequence of wild-type avidin by one or more amino acid substitutions, deletions, or additions, but contains a biotin-binding site. In some embodiments, the avidin analog is neutravidin, i.e., a deglycosylated avidin with a modified arginine, which can exhibit a more neutral pi and can be used in place of wild-type avidin. In some embodiments, the avidin analog is any of those commercially available as ExtrAvidin® available through Sigma Aldrich, NeutrAvidin available from Thermo Scientific or Invitrogen, and CaptAvidin™ available from Molecular Probes. In some embodiments, the avidin analog or mutein is any of those described in International Published PCT Application WO2008 / 140573.

[0292] In some embodiments, the molecule is streptavidin, e.g., wild-type streptavidin. In some embodiments, the streptavidin has the amino acid sequence set forth in SEQ ID NO:1, as disclosed in Argarana et al., Nucleic Acids Res. 14 (1986) 1871-1882, or has an amino acid sequence that is a sequence present in its homologs from other Streptomyces species. In some embodiments, the streptavidin has the amino acid sequence set forth in SEQ ID NO:1.

[0293] In some embodiments, the molecule is a streptavidin analog. In some embodiments, a streptavidin analog is a variant of wild-type streptavidin that has one or more modified functional groups but contains a biotin-binding site. In some embodiments, the molecule is a streptavidin mutein. In some embodiments, a streptavidin mutein is a polypeptide that differs from the sequence of wild-type streptavidin by one or more amino acid substitutions, deletions, or additions, but contains a biotin-binding site.

[0294] In some embodiments, the streptavidin mutein binds to a streptavidin-binding peptide, such as any of those described herein. In some embodiments, the streptavidin mutein binds to any of the streptavidin-binding peptides set forth in SEQ ID NOs: 7, 8, and 15-19. In some embodiments, the binding affinity of the streptavidin-binding peptide to the streptavidin mutein is greater than or equal to 1×10 -13 M, 1 x 10 -12 M, or 1 x 10 -11 Over M and 1 x 10 -4 M, 5 x 10 -4 M, 1 x 10 -5 M, 5 x 10 -5 M, 1 x 10 -6 M, 5 x 10 -6 M, or 1 x 10 -7In some embodiments, the streptavidin mutein binds to biotin, e.g., D-biotin. In some embodiments, the streptavidin mutein binds to a biotin analog or derivative, e.g., any of those described herein. In some embodiments, the streptavidin mutein binds to biotin or a biotin analog or derivative with greater affinity than the streptavidin-binding peptide. In some embodiments, binding of a streptavidin-binding peptide to a streptavidin mutein, e.g., the biotin-binding site of a streptavidin mutein, can be disrupted by the presence of biotin or a biotin analog or derivative. In some embodiments, binding of a streptavidin mutein to the streptavidin-binding peptide of any of SEQ ID NOs:7, 8, and 15-19 is disrupted by the presence of biotin, e.g., D-biotin.

[0295] In some embodiments, the streptavidin mutein contains only a portion of wild-type streptavidin. In some embodiments, the streptavidin mutein is a minimal streptavidin (sometimes referred to as recombinant core streptavidin) in which wild-type streptavidin has been truncated at the N-terminus and / or C-terminus. In some embodiments, the streptavidin mutein is any of the recombinant core streptavidins described in Sano et al. (1995), Journal of Biological Chemistry 270(47): 28204-28209. In some embodiments, the streptavidin mutein begins at the N-terminus in the region of amino acid positions 10-16 of SEQ ID NO:1 and ends at the C-terminus in the region of amino acid positions 133-142 of SEQ ID NO:1. References to residue positions in streptavidin or streptavidin muteins are with reference to the numbering of residues in SEQ ID NO:1. In some embodiments, the sequence of the streptavidin mutein is set forth in any of SEQ ID NOs:2, 103, and 135. In some embodiments, the streptavidin mutein is the amino acid sequence from Ala 13 to Ser 139 of SEQ ID NO:1. In some embodiments, the sequence of the streptavidin mutein is set forth in SEQ ID NO:135. In some embodiments, the streptavidin mutein contains an N-terminal methionine and the amino acid sequence from Glu 14 to Ser 139 of SEQ ID NO:1. In some embodiments, the sequence of the streptavidin mutein is set forth in SEQ ID NO:2.

[0296] In some embodiments, the streptavidin mutein contains one or more amino acid substitutions compared to wild-type streptavidin, e.g., compared to the wild-type streptavidin sequence set forth in SEQ ID NO:1. In some embodiments, the streptavidin mutein contains one or more amino acid substitutions compared to a streptavidin mutein that is a minimal streptavidin. In some embodiments, the streptavidin contains one or more amino acid substitutions compared to a streptavidin mutein that begins at the N-terminus in the region of amino acid positions 10-16 of SEQ ID NO:1 and ends at the C-terminus in the region of amino acid positions 133-142 of SEQ ID NO:1, e.g., a minimal streptavidin. In some embodiments, the streptavidin contains one or more amino acid substitutions compared to a streptavidin mutein set forth in any of SEQ ID NOs:2, 103, and 135.

[0297] In some embodiments, the streptavidin mutein binds biotin and contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid differences compared to the sequence of amino acids set forth in SEQ ID NO: 1, 2, 103, or 135. In some embodiments, the streptavidin mutein binds biotin and contains an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of amino acids set forth in SEQ ID NO: 1, 2, 103, or 135. In some embodiments, the amino acid substitutions are conservative or non-conservative mutations. In some embodiments, the streptavidin mutein is any of those described in U.S. Patent Nos. 5,168,049; 5,506,121; 6,022,951; 6,156,493; 6,165,750; 6,103,493; 6,368,813; and International Published PCT Applications WO2014 / 076277, WO2008 / 140573, WO 86 / 02077, WO 98 / 40396, and WO 96 / 24606. In some embodiments, the streptavidin mutein is any of those described in DE 19641876 A1; Howarth et al. (2006) Nat. Methods, 3:267-73; Zhang et al. (2015) Biochem. Biophys. Res. Commun., 463: 1059-63; Fairhead et al. (2013) J. Mol. Biol., 426: 199-214; Wu et al. (2005) J. Biol. Chem., 280: 23225-31; Lim et al. (2010) Biochemistry, 50: 8682-91; and Qureshi et al. (2001), Journal of Biological Chemistry 276(49): 46422-46428.

[0298] In some embodiments, the streptavidin mutein is any as described in U.S. Patent No. 6,103,493. In some embodiments, the streptavidin mutein contains at least one mutation within a region corresponding to amino acid positions 44-53 of wild-type streptavidin, e.g., as shown in SEQ ID NO:1. In some embodiments, "corresponding to" refers to an amino acid position with reference to the amino acid sequence of wild-type streptavidin, e.g., as shown in SEQ ID NO:1. One of skill in the art will be able to identify these residues using methods including, for example, sequence alignment. In some embodiments, the streptavidin mutein contains a mutation at one or more of residues 44, 45, 46, and 47 of wild-type streptavidin. In some embodiments, the streptavidin mutein contains a substitution of Glu at position 44 with a hydrophobic aliphatic amino acid, e.g., Val, Ala, Ile, or Leu. In some embodiments, the streptavidin mutein contains any amino acid at position 45. In some embodiments, the streptavidin mutein contains an aliphatic amino acid, e.g., a hydrophobic aliphatic amino acid, at position 46. In some embodiments, the streptavidin mutein contains a substitution of Val with a basic amino acid, e.g., Arg or Lys, e.g., Arg, at position 47. In some embodiments, Ala is present at position 46, Arg is present at position 47, and Val or Ile is present at position 44. In some embodiments, the streptavidin mutein contains a substitution of residue Val, as shown in exemplary streptavidin muteins containing the sequence of amino acids set forth in SEQ ID NO:3, 4, or 104, at sequence positions corresponding to positions 44-47 of the sequence of amino acids set forth in SEQ ID NO:1. 44 -Thr 45 -Ala 46 -Arg 47In some embodiments, the streptavidin mutein contains residues Ile, as shown in exemplary streptavidin muteins containing the sequence of amino acids set forth in SEQ ID NO:5, 6, or 104 at sequence positions corresponding to positions 44-47 of the sequence of amino acids set forth in SEQ ID NO:1. 44 -Gly 45 -Ala 46 -Arg 47 (SEQ ID NO:133). In some embodiments, the streptavidin mutein contains the amino acid sequence set forth in any of SEQ ID NOs:3-6, 104, and 105. In some embodiments, the streptavidin mutein is commercially available under the trademark Strep-Tactin® m1. In some embodiments, the streptavidin mutein is commercially available under the trademark Strep-Tactin® m2. In some embodiments, the streptavidin mutein contains the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the streptavidin mutein contains the amino acid sequence set forth in SEQ ID NO:6.

[0299] In certain embodiments, the streptavidin mutein is any as described in International Published PCT Application WO 2014 / 076277. In some embodiments, the streptavidin mutein contains at least two cysteine ​​residues in a region corresponding to amino acid positions 44-53 of the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, cysteine ​​residues are present at positions 45 and 52, creating a disulfide bridge connecting these amino acids. In some embodiments, amino acid 44 is glycine or alanine; amino acid 46 is alanine or glycine; and amino acid 47 is arginine. In some embodiments, the streptavidin mutein contains at least one mutation in a region corresponding to amino acid residues 115-121 of the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the streptavidin mutein contains at least one mutation at amino acid position 117, 120, or 121 and / or a deletion of amino acids 118 and 119 and a substitution at least at amino acid position 121.

[0300] In some embodiments, the streptavidin mutein contains a mutation at a position corresponding to position 117, which can be to a large hydrophobic residue such as Trp, Tyr, or Phe; to a charged residue such as Glu, Asp, or Arg; to a hydrophilic residue such as Asn or Gln; to the hydrophobic residues Leu, Met, or Ala; or to the polar residues Thr, Ser, or His. In some embodiments, the mutation at position 117 is combined with a mutation at a position corresponding to position 120 (which can be to a small residue such as Ser, Ala, or Gly) and a mutation at a position corresponding to position 121 (which can be to a hydrophobic residue, e.g., a bulky hydrophobic residue such as Trp, Tyr, or Phe). In some embodiments, the mutation at position 117 is combined with a mutation at a position corresponding to position 120 of wild-type streptavidin shown in SEQ ID NO:1 (which mutation can be to a hydrophobic residue such as Leu, Ile, Met, or Val; or Tyr or Phe), and a mutation at a position corresponding to position 121 of SEQ ID NO:1 (which mutation can be to a small residue such as Gly, Ala, or Ser, or with Gln, or with a hydrophobic residue such as Leu, Val, Ile, Trp, Tyr, Phe, or Met). In some embodiments, the streptavidin mutein contains residues Glu117, Gly120, and Tyr121, with reference to the positions in the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the streptavidin mutein also contains residues Val at sequence positions corresponding to positions 44-47 of the amino acid sequence shown in SEQ ID NO:1. 44 -Thr 45 -Ala 46 -Arg 47 or residue Ile 44 -Gly 45 -Ala 46 -Arg 47In some embodiments, the streptavidin mutein contains residues Val44, Thr45, Ala46, Arg47, Glu117, Gly120, and Tyr121. In some embodiments, the mutein streptavidin contains a sequence of amino acids set forth in any of SEQ ID NOs:27, 28, and 136, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of amino acids set forth in any of SEQ ID NOs:27, 28, and 136, contains residues Val44, Thr45, Ala46, Arg47, Glu117, Gly120, and Tyr121, and binds biotin. In some embodiments, the streptavidin mutein contains the sequence of amino acids set forth in SEQ ID NO: 27. In some embodiments, the streptavidin mutein contains the sequence of amino acids set forth in SEQ ID NO: 28. In some embodiments, the streptavidin mutein contains the sequence of amino acids set forth in SEQ ID NO: 136.

[0301] In some embodiments, the streptavidin mutein contains a sequence of amino acids set forth in any of SEQ ID NOs: 3-6, 27, 28, 104, 105, and 136, and the binding partner contains a streptavidin-binding peptide, wherein the sequence of the streptavidin-binding peptide is set forth in any of SEQ ID NOs: 7, 8, and 15-19. In some embodiments, the streptavidin mutein contains a sequence of amino acids set forth in SEQ ID NO: 6, and the binding partner contains a streptavidin-binding peptide, wherein the sequence of the streptavidin-binding peptide is set forth in any of SEQ ID NOs: 7, 8, and 15-19. In some embodiments, the streptavidin mutein contains a sequence of amino acids set forth in any of SEQ ID NOs:3-6, 27, 28, 104, 105, and 136, and the binding partner contains a streptavidin-binding peptide, wherein the sequence of the streptavidin-binding peptide is set forth in SEQ ID NO: 16. In some embodiments, the streptavidin mutein contains a sequence of amino acids set forth in SEQ ID NO: 6, and the binding partner contains a streptavidin-binding peptide, wherein the sequence of the streptavidin-binding peptide is set forth in SEQ ID NO: 16.

[0302] In some embodiments, the protein reagent contains a plurality of molecules, e.g., a plurality of any of the described streptavidin, avidin, streptavidin analog or mutein and avidin analog or mutein molecules. In some embodiments, the plurality of molecules is a mixture of molecules each independently selected from any of the streptavidin, avidin, streptavidin analog or mutein and avidin analog or mutein molecules described herein. In some embodiments, the plurality of molecules is a mixture of any of the streptavidin and streptavidin mutein molecules described herein.

[0303] In some embodiments, each of the plurality of molecules is the same and is any one of the streptavidin, avidin, streptavidin analog or mutein, and avidin analog or mutein molecules described herein. In some embodiments, each of the plurality of molecules is the same and is streptavidin. In some embodiments, each of the plurality of molecules is the same and is any one of the streptavidin mutein molecules described herein.

[0304] In some embodiments, the plurality of molecules contains 100 to 50,000, 500 to 10,000, 1,000 to 20,000, 500 to 5,000, 300 to 7,500, 1,500 to 7,500, 500 to 3,500, 1,000 to 5,000, 1,500 to 2,500, 1,500 to 2,500, 2,000 to 3,000, 2,500 to 3,500, 2,000 to 4,000, or 2,000 to 5,000 tetramers, inclusive, of the molecule or mixture of molecules. In some embodiments, the plurality of molecules contains 500 to 7,500, or about 500 to 7,500 tetramers, inclusive, of the molecule or mixture of molecules. In some embodiments, the plurality of molecules contains 500-5000 or about 500-5000 tetramers, 1000-4000 or about 1000-4000 tetramers, or 2000-3000 or about 2000-3000 tetramers (inclusive) of the molecule or mixture of molecules. In some embodiments, the plurality of molecules contains 500-5000 or about 500-5000 tetramers (inclusive) of the molecule or mixture of molecules. In some embodiments, the plurality of molecules contains 1000-4000 or about 1000-4000 tetramers (inclusive) of the molecule or mixture of molecules. In some embodiments, the plurality of molecules contains 2000-3000 or about 2000-3000 tetramers (inclusive) of the molecule or mixture of molecules. In some embodiments, the plurality of molecules contains about 2500 tetramers of the molecule or mixture of molecules. In any of the foregoing embodiments, the number of tetramers is the number of tetramers of the molecule. In any of the foregoing embodiments, the number of tetramers is the number of tetramers of the mixture of molecules.

[0305] In some embodiments, the protein reagent has a radius of 5 nm to 150 nm, 25 nm to 150 nm, 50 nm to 150 nm, 75 nm to 125 nm, 80 nm to 140 nm, 85 nm to 135 nm, 80 nm to 120 nm, 80 nm to 115 nm, or 90 nm to 110 nm (inclusive). In some embodiments, the protein reagent has a radius of 50 nm to 150 nm (inclusive). In some embodiments, the protein reagent has a radius of 75 nm to 125 nm (inclusive). In some embodiments, the protein reagent has a radius of 80 nm to 120 nm (inclusive). In some embodiments, the protein reagent has a radius of 90 nm to 110 nm (inclusive).

[0306] In some embodiments, the radius is a hydrodynamic radius, a radius of gyration, a Stokes radius, a Stokes-Einstein radius, or an effective hydration radius in solution. In some embodiments, the radius is a hydrodynamic radius. In some embodiments, the radius is a Stokes radius.

[0307] In some embodiments, the protein reagent is an oligomer of multiple molecules. In some embodiments, the oligomer is made by linking individual molecules of the protein reagent. In some embodiments, the oligomer is made by linking monomers, dimers, trimers, or tetramers of the molecule. In some embodiments, the molecules are directly linked to one another. In some embodiments, the molecules are indirectly linked to one another. The oligomer can be made using any suitable method, for example, any of those described in U.S. Pat. No. 7,776,562 and published U.S. patent application Ser. No. 2021 / 0032297. In some embodiments, the molecules of the multiple molecules are crosslinked by a polysaccharide or bifunctional linker.

[0308] In some cases, the molecules of the plurality of molecules are cross-linked by a polysaccharide. In some embodiments, the oligomer is prepared in a first step by introducing carboxyl residues into a polysaccharide, such as dextran, as described, for example, in Noguchi et al., Bioconjugate Chemistry (1992) 3, 132-137. In some embodiments, then, in a second step, a protein reagent molecule, such as streptavidin, avidin, streptavidin analog or mutein, or avidin analog or mutein molecule, can be linked to the carboxyl groups in the dextran backbone via internal lysine residues and / or free N-terminal primary amino groups using carbodiimide chemistry.

[0309] In some embodiments, the molecules of the plurality of molecules are crosslinked by a bifunctional linker. Suitable bifunctional linkers can be identified and selected by those skilled in the art. In some embodiments, the linker is a heterobifunctional linker. In some embodiments, the molecules of the plurality of molecules, for example, streptavidin, avidin, streptavidin analog or mutein, or avidin analog or mutein molecules, for example, streptavidin mutein molecules, are crosslinked by an amine-thiol crosslinker. Exemplary crosslinking reagents include sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) or succinimidyl-6-[(β-maleimidopropionamido)hexanoate (SMPH), the use of which in the preparation of oligomers is described, for example, in US 2021 / 0032297.

[0310] b. Binding substance In some embodiments, the one or more binding substances are suitable for stimulating immune cells, such as T cells. In some embodiments, the one or more binding substances are immobilized on a protein reagent of the stimulating reagent. In some embodiments, the one or more binding substances are individually selected from any of the binding substances described herein. In some embodiments, the one or more binding substances include 2, 3, 4, 5, 6, 7, 8, 9, or 10 different binding substances that can target the same or different molecules. For example, in some embodiments, the one or more binding substances include a primary agent and a secondary agent that target different molecules from each other.

[0311] In some embodiments, one of the one or more binding agents is a primary agent that binds to a molecule expressed on the surface of an immune cell, e.g., a T cell, thereby providing a primary activation signal to the immune cell, e.g., a T cell. In some embodiments, the molecule is a member of the TCR / CD3 complex. In some embodiments, the molecule is CD3.

[0312] In some embodiments, one of the one or more binding agents is a secondary agent that binds to a second molecule expressed on the surface of an immune cell, e.g., a T cell. In some embodiments, the second molecule is a costimulatory molecule. In some embodiments, the secondary agent binds to an immune cell, e.g., a T cell, thereby providing a costimulatory signal. In some embodiments, the costimulatory molecule is CD28, CD90 (Thy-1), CD95 (Apo- / Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM. In some embodiments, the costimulatory molecule is CD28.

[0313] In some embodiments, the binding agent binds to a molecule expressed on the surface of an immune cell, e.g., a T cell. For example, a wide variety of antibodies or antibody fragments that target cell surface molecules are available and suitable for use as part of the binding agent herein, and can be appropriately identified and selected for use by one of skill in the art.

[0314] In some embodiments, the binding agent is monovalent. In some embodiments, the binding agent contains two or more binding sites for binding to molecules expressed on the surface of immune cells (also referred to herein as cell surface molecules). In some embodiments, the binding agent is bivalent.

[0315] In some embodiments, the dissociation constant (K D ) is about 10 -2 M ~ about 10 -13 M, about 10 -3 M ~ about 10 -12 M, about 10 -4 M ~ about 10 -11 M, or about 10 -5 M ~ about 10 -10 In some embodiments, the dissociation constant (K D ) is about 10 -3 ~about 10 -7 M, e.g., low affinity K D In some embodiments, the dissociation constant (K D ) is about 10 -7 ~Approx. 1×10 -10 M, e.g., high affinity K D is.

[0316] In some embodiments, dissociation of the binding between the binding agent and the cell surface molecule occurs sufficiently rapidly to allow immune cells, e.g., T cells, to associate with the binding agent only transiently, e.g., after disruption of the reversible binding between the protein reagent and the binding agent. off When expressed in units of rate (also called dissociation rate constant), k off The speed is about 0.5 x 10 -4 sec -1 or more, about 1 x 10 -4 sec -1 or more, about 2 x 10 -4 sec -1 or more, about 3 x 10 -4 sec-1 or more, about 4 x 10 -4 sec -1 or more, about 5 x 10 -4 sec -1 or more, about 1 x 10 -3 sec -1 or more, about 1.5 x 10 -3 sec -1 or more, about 2 x 10 -3 sec -1 or more, about 3 x 10 -3 sec -1 or more, about 4 x 10 -3 sec -1 , about 5×10 -3 sec -1 or more, about 1 x 10 -2 sec or more, or approximately 5 x 10 -1 sec -1 or higher. off It is within the skill of one in the art to empirically determine the rate range (see, e.g., U.S. Pat. No. 9,023,604). For example, a rate of, e.g., 4.0×10 to allow removal or dissociation of the majority of bound material from immune cells, e.g., T cells, within 1 hour after disruption of binding to the protein reagent may be used. -4 sec -1 Ultra-higher k off In other cases, a binding agent with a fast binding rate, e.g., 1.0×10, may be used such that the majority of the binding agent is removed or dissociated from immune cells, e.g., T cells, within about three and a half hours after blocking binding to the protein reagent. -4 sec -1 Lower k off Kinetic binding agents may also be used.

[0317] The K of the bond formed between the binding substance and the cell surface molecule D , k off and k on The rate can be determined by any suitable means, for example, by fluorescence titration, equilibrium dialysis, or surface plasmon resonance.

[0318] In some embodiments, the receptor is a lipid, polysaccharide, or nucleic acid. In some embodiments, the cell surface molecule is a peptide or protein, e.g., a receptor, e.g., a membrane receptor protein. In some embodiments, the cell surface molecule is a peripheral membrane protein or an integral membrane protein. In some embodiments, the cell surface molecule can have one or more membrane-spanning domains. As some illustrative examples, membrane proteins having a transmembrane domain include G protein-coupled receptors, e.g., olfactory receptors, rhodopsin receptors, rhodopsin pheromone receptors, peptide hormone receptors, taste receptors, GABA receptors, opioid receptors, serotonin receptors, Ca2+ receptors, melanopsin, neurotransmitter receptors, e.g., ligand-gated, voltage-gated or mechano-gated receptors including acetylcholine, nicotinic, adrenergic, norepinephrine, catecholamine, L-DOPA-, dopamine and serotonin (biogenic amines, endorphins / enkephalins) neuropeptide receptors, receptor kinases, e.g., serine / threonine kinases, tyrosine kinases, porins / channels, e.g., chloride channels, potassium channels, sodium channels, OMP proteins, ABC transporters (ATP-binding cassette-transporters), e.g., amino acid transporters, Na-glucose transporters, Na / iodine transporters, ion transporters, e.g., light-harvesting complexes, cytochrome c oxidases, ATPases. It can be Na / K, H / K, Ca, a cell adhesion receptor, such as a metalloprotease, an integrin, or a cathelin.

[0319] In some embodiments, the cell surface molecule is a molecule expressed by or defining a cell population, e.g., a population or subpopulation of blood cells, e.g., lymphocytes (e.g., T cells, B cells, or NK cells), monocytes, or stem cells (e.g., CD34-positive peripheral blood stem cells or Nanog- or Oct-4-expressing stem cells). In some embodiments, the cell surface molecule is expressed on the surface of a target cell, e.g., a cell targeted for genetic manipulation. In some embodiments, the cell surface molecule is a molecule expressed on the surface of an immune cell. In some embodiments, the cell surface molecule is a molecule expressed on the surface of a lymphocyte. In some embodiments, the cell surface molecule is a molecule expressed on the surface of a T cell, a B cell, or an NK cell. In some embodiments, the cell surface molecule is a molecule expressed on the surface of a T cell. Examples of T cells include cells such as CMV-specific CD8+ T cells, 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.

[0320] In some embodiments, the binding agent comprises an antibody, an antibody fragment, a proteinaceous molecule with antibody-like binding properties, an Ig domain-containing molecule, a cytokine, a chemokine, an MHC molecule, an MHC-peptide complex, a receptor ligand, or a binding fragment of any of the foregoing that specifically binds to a cell surface molecule. In some embodiments, the binding agent comprises an antibody. In some embodiments, the binding agent comprises an antibody fragment. In some embodiments, the antibody fragment is selected from a Fab fragment, an Fv fragment, a single-chain Fv fragment (scFv), a bivalent antibody fragment such as an F(ab')2-fragment, a diabody, a triabody (Iliades, P., et al., FEBS Lett (1997) 409, 437-441), a decabody (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).

[0321] In some embodiments, the binding agent binds to the cell surface molecule in a monovalent manner. In some embodiments, the binding agent comprises a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties, an aptamer, or an MHC molecule. In some embodiments, the binding agent comprises a monovalent antibody fragment. In some embodiments, the monovalent antibody fragment is a Fab fragment, an Fv fragment, or a single-chain Fv fragment (scFv). In some embodiments, the monovalent antibody fragment is a Fab fragment.

[0322] In some embodiments, the binding agent comprises an antibody fragment that is a bivalent antibody fragment. In some embodiments, the bivalent antibody fragment is a F(ab')2-fragment or a bivalent single-chain Fv fragment.

[0323] In some embodiments, the binding agent comprises a proteinaceous molecule with antibody-like binding properties, hi some embodiments, the proteinaceous molecule with antibody-like binding properties is an aptamer, a lipocalin-based polypeptide-based mutein, a glubody, an ankyrin scaffold-based protein, a crystalline scaffold-based protein, an adnectin, or an avimer.Other exemplary proteinaceous 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 four disulfide core domains, F5 / 8 C-type domains, hemopexin domains, SH2 domains, SH3 domains, laminin-type EGF-like domains, C2 domains, "kappa bodies" (see below, Ill. et al., Protein Eng (1997) 10, 949-57, also known as "minibodies" (Martin et al., EMBO J (1994) 13, 5303-5309), diabodies (see below, Holliger et al., PNAS USA (1993) 90, 6444-6448), so-called "Janusis" (see below, 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, ubiquitins, zinc finger proteins, autofluorescent proteins, and leucine-rich repeat proteins. In some embodiments, the binding agent is a bivalent artificial proteinaceous binding molecule, such as a dimeric lipocalin mutein, also known as a "duocalin."

[0324] In some embodiments, the cell surface molecule is an immunoreceptor tyrosine-based activation motif (ITAM)-containing molecule. In some embodiments, the cell surface molecule is a member of the T cell antigen receptor complex. In some embodiments, the cell surface molecule is a member of the TCR / CD3 complex. In some embodiments, the cell surface molecule is CD3. In some embodiments, the cell surface molecule is a CD3 chain. In some embodiments, the cell surface molecule is a CD3 zeta chain.

[0325] In some embodiments, the cell surface molecule is CD3. In some embodiments, the binding agent, e.g., the primary agent, contains an anti-CD3 antibody, a bivalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, or a proteinaceous CD3-binding molecule with antibody-like binding properties. In some embodiments, the anti-CD3 antibody, a bivalent antibody fragment of an anti-CD3 antibody, or a monovalent antibody fragment of an anti-CD3 antibody (e.g., an anti-CD3 Fab fragment) is 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 binding agent, e.g., the primary agent, contains an anti-CD3 Fab. In some embodiments, the anti-CD3 Fab contains a variable heavy chain having the sequence set forth in SEQ ID NO:31 and a variable light chain having the sequence set forth in SEQ ID NO:32. In some embodiments, the anti-CD3 Fab contains the CDRs of a variable heavy chain having the sequence shown in SEQ ID NO:31 and the CDRs of a variable light chain having the sequence shown in SEQ ID NO:32.

[0326] In some embodiments, the cell surface molecule is a costimulatory molecule, an accessory molecule, a cytokine receptor, a chemokine receptor, an immune checkpoint molecule, or a member of the TNF family or TNF receptor family. In some embodiments, the cell surface molecule is a costimulatory molecule. In some embodiments, the costimulatory molecule is CD28, CD90 (Thy-1), CD95 (Apo- / Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM.

[0327] In some embodiments, the cell surface molecule is CD28. In some embodiments, the binding agent, e.g., secondary agent, contains an anti-CD28 antibody, a bivalent antibody fragment of an anti-CD28 antibody, a monovalent antibody fragment of an anti-CD28 antibody, or a proteinaceous CD28-binding molecule with antibody-like binding properties. In some embodiments, the anti-CD28 antibody, bivalent antibody fragment of an anti-CD28 antibody, or monovalent antibody fragment of an anti-CD28 antibody (e.g., an anti-CD28 Fab fragment) is derived from the antibody CD28.3 (deposited as a synthetic single-chain Fv construct under GenBank accession number AF451974.1; see also Vanhove et al., BLOOD, 15 July 2003, Vol. 102, No. 2, pages 564-570), the variable heavy and light chains of which contain the amino acid sequences set forth in SEQ ID NOs:33 and 34, respectively. In some embodiments, the binding agent, e.g., secondary agent, contains an anti-CD28 Fab. In some embodiments, the anti-CD28 Fab contains a variable heavy chain having the sequence shown in SEQ ID NO: 33 and a variable light chain having the sequence shown in SEQ ID NO: 34. In some embodiments, the anti-CD28 Fab contains the CDRs of the variable heavy chain having the sequence shown in SEQ ID NO: 33 and the CDRs of the variable light chain having the sequence shown in SEQ ID NO: 34.

[0328] In some embodiments, the cell surface molecule is CD90. In some embodiments, the binding agent, e.g., secondary agent, comprises an anti-CD90 antibody, a bivalent antibody fragment of an anti-CD90 antibody, a monovalent antibody fragment of an anti-CD90 antibody, or a proteinaceous CD90 binding molecule with antibody-like binding properties. In some embodiments, the binding agent, e.g., secondary agent, comprises an anti-CD90 Fab. In some embodiments, the anti-CD90 antibody, a bivalent antibody fragment of an anti-CD90 antibody, or a monovalent antibody fragment of an anti-CD90 antibody (e.g., an anti-CD90 Fab fragment) is derived from the anti-CD90 antibody G7 (Biolegend, catalog number 105201).

[0329] In some embodiments, the cell surface molecule is CD95. In some embodiments, the binding agent, e.g., secondary agent, contains an anti-CD95 antibody, a bivalent antibody fragment of an anti-CD95 antibody, a monovalent antibody fragment of an anti-CD95 antibody, or a proteinaceous CD95 binding molecule with antibody-like binding properties. In some embodiments, the binding agent, e.g., secondary agent, contains an anti-CD95 Fab. In some embodiments, the anti-CD95 antibody, a bivalent antibody fragment of an anti-CD95 antibody, or a monovalent antibody fragment of an anti-CD95 antibody (e.g., an anti-CD95 Fab fragment) is derived from monoclonal mouse anti-human CD95 CH11 (Upstate Biotechnology, Lake Placid, NY), anti-CD95 mAb 7C11, or anti-APO-1, e.g., as described in Paulsen et al. Cell Death & Differentiation 18.4 (2011): 619-631.

[0330] In some embodiments, the cell surface molecule is CD137. In some embodiments, the binding agent, e.g., secondary agent, comprises an anti-CD137 antibody, a bivalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment of an anti-CD137 antibody, or a proteinaceous CD137-binding molecule with antibody-like binding properties. In some embodiments, the binding agent, e.g., secondary agent, comprises an anti-CD137 Fab. In some embodiments, the anti-CD137 antibody, a bivalent antibody fragment of an anti-CD137 antibody, or a monovalent antibody fragment of an anti-CD137 antibody (e.g., an anti-CD137 Fab fragment) is derived from LOB12, IgG2a, or LOB12.3, IgG1, as described in Taraban et al. Eur J Immunol. 2002 Dec; 32(12): 3617-27. See also, e.g., US6569997, US6303121, and Mittler et al. Immunol Res. 2004; 29(1-3): 197-208.

[0331] In some embodiments, the cell surface molecule is CD40. In some embodiments, the binding agent, e.g., secondary agent, comprises an anti-CD40 antibody, a bivalent antibody fragment of an anti-CD40 antibody, a monovalent antibody fragment of an anti-CD40 antibody, or a proteinaceous CD40 binding molecule with antibody-like binding properties. In some embodiments, the binding agent, e.g., secondary agent, comprises an anti-CD40 Fab.

[0332] In some embodiments, the cell surface molecule is CD40L. In some embodiments, the binding agent, e.g., secondary agent, contains an anti-CD40L antibody, a bivalent antibody fragment of an anti-CD40L antibody, a monovalent antibody fragment of an anti-CD40L antibody, or a proteinaceous CD40L-binding molecule with antibody-like binding properties. In some embodiments, the binding agent, e.g., secondary agent, contains an anti-CD40L Fab. In some embodiments, the anti-CD40L antibody, a bivalent antibody fragment of an anti-CD40L antibody, or a monovalent antibody fragment of an anti-CD40L antibody (e.g., an anti-CD40L Fab fragment) is derived from Hu5C8, as described in Blair et al. JEM vol. 191 no. 4 651-660. See also, e.g., WO1999061065, US20010026932, US7547438, and WO2001056603.

[0333] In some embodiments, the cell surface molecule is ICOS. In some embodiments, the binding agent, e.g., secondary agent, contains an anti-ICOS antibody, a bivalent antibody fragment of an anti-ICOS antibody, a monovalent antibody fragment of an anti-ICOS antibody, or a proteinaceous ICOS-binding molecule with antibody-like binding properties. In some embodiments, the binding agent, e.g., secondary agent, contains an anti-ICOS Fab. In some embodiments, the anti-ICOS antibody, a bivalent antibody fragment of an anti-ICOS antibody, or a monovalent antibody fragment of an anti-ICOS antibody (e.g., an anti-ICOS Fab fragment) is derived from any of the antibodies described in US20080279851 and Deng et al. Hybrid Hybridomics. 2004 Jun;23(3):176-82.

[0334] In some embodiments, the cell surface molecule is linker for activation of T cells (LAT). In some embodiments, the binding agent, e.g., secondary agent, contains an anti-LAT antibody, a bivalent antibody fragment of an anti-LAT antibody, a monovalent antibody fragment of an anti-LAT antibody, or a proteinaceous LAT-binding molecule with antibody-like binding properties. In some embodiments, the binding agent, e.g., secondary agent, contains an anti-LAT Fab.

[0335] In some embodiments, the cell surface molecule is CD27. In some embodiments, the binding agent, e.g., secondary agent, comprises an anti-CD27 antibody, a bivalent antibody fragment of an anti-CD27 antibody, a monovalent antibody fragment of an anti-CD27 antibody, or a proteinaceous CD27-binding molecule with antibody-like binding properties. In some embodiments, the binding agent, e.g., secondary agent, comprises an anti-CD27 Fab. In some embodiments, the anti-CD27 antibody, a bivalent antibody fragment of an anti-CD27 antibody, or a monovalent antibody fragment of an anti-CD27 antibody (e.g., an anti-CD27 Fab fragment) is derived from any of the antibodies described in WO2008051424.

[0336] In some embodiments, the cell surface molecule is OX40. In some embodiments, the binding agent, e.g., secondary agent, contains an anti-OX40 antibody, a bivalent antibody fragment of an anti-OX40 antibody, a monovalent antibody fragment of an anti-OX40 antibody, or a proteinaceous OX40 binding molecule with antibody-like binding properties. In some embodiments, the binding agent, e.g., secondary agent, contains an anti-OX40 Fab. In some embodiments, the anti-OX40 antibody, a bivalent antibody fragment of an anti-OX40 antibody, or a monovalent antibody fragment of an anti-OX40 antibody (e.g., an anti-OX40 Fab fragment) is derived from any of the antibodies described in WO2013038191 and Melero et al. Clin Cancer Res. 2013 Mar 1; 19(5): 1044-53.

[0337] In some embodiments, the cell surface molecule is HVEM. In some embodiments, the binding agent, e.g., secondary agent, contains an anti-HVEM antibody, a bivalent antibody fragment of an anti-HVEM antibody, a monovalent antibody fragment of an anti-HVEM antibody, or a proteinaceous HVEM-binding molecule with antibody-like binding properties. In some embodiments, the binding agent, e.g., secondary agent, contains an anti-HVEM Fab. In some embodiments, the anti-HVEM antibody, a bivalent antibody fragment of an anti-HVEM antibody, or a monovalent antibody fragment of an anti-HVEM antibody (e.g., an anti-HVEM Fab fragment) is derived from any of the antibodies described in WO2006054961, WO2007001459, and Park et al. Cancer Immunol Immunother. 2012 Feb;61(2):203-14.

[0338] In some embodiments, the binding agent further comprises a binding partner. In some embodiments, the binding agent comprises 1 to 5, 1 to 4, 1 to 3, or 1 to 2 (inclusive) binding partners. In some embodiments, the binding agent comprises exactly one binding partner. In some embodiments, the binding agent comprises exactly two binding partners. In some embodiments, the binding agent comprises exactly three binding partners. In some embodiments, the binding agent comprises exactly four binding partners. In some embodiments, the binding agent comprises exactly five binding partners.

[0339] Exemplary binding partners are described in this section. In some embodiments, each binding partner of a binding substance that contains multiple binding partners is individually selected from among the described binding partners. In some embodiments, each binding partner of a binding substance that contains multiple binding partners is the same and is any one of the binding partners described herein.

[0340] In some embodiments, the binding partner is hydrocarbon-based (including polymeric) and contains nitrogen, phosphorus, sulfur, carbon, halogen, or pseudohalogen groups. In some embodiments, the binding partner is an alcohol, organic acid, inorganic acid, amine, phosphine, thiol, disulfide, alkane, amino acid, peptide, oligopeptide, polypeptide, protein, nucleic acid, lipid, monosaccharide, oligosaccharide, or polysaccharide. By way of further example, in some embodiments, the binding partner is a cation, anion, polycation, polyanion, polycation, electrolyte, polyelectrolyte, carbon nanotube, or carbon nanofoam. By way of still further example, in some embodiments, the binding partner is a crown ether, an immunoglobulin or fragment thereof, or a proteinaceous binding molecule with antibody-like function.

[0341] In some embodiments, the binding partner comprises a moiety known to those skilled in the art as an affinity tag, hi some embodiments, the protein reagent comprises a corresponding binding partner, e.g., an antibody or antibody fragment, known to bind to the affinity tag. As some illustrative examples of known affinity tags, in some embodiments, the affinity tag comprises dinitrophenol or digoxigenin, oligohistidine, polyhistidine, an immunoglobulin domain, glutathione-S-transferase (GST), chitin-binding protein (CBP) or thioredoxin, calmodulin-binding peptide (CBP), FLAG'-peptide, an HA tag (SEQ ID NO:20), a VSV-G tag (SEQ ID NO:21), an HSV tag (SEQ ID NO:22), a T7 epitope (SEQ ID NO:23), maltose-binding protein (MBP), an HSV epitope of herpes simplex virus glycoprotein D (SEQ ID NO:24), a "myc" epitope of the transcription factor c-myc (SEQ ID NO:25), or a V5 tag (SEQ ID NO:26). In some embodiments, a complex formed between a binding site of a protein reagent and an affinity tag, e.g., between a corresponding binding partner of a selection reagent, e.g., an antibody or antibody fragment, and an affinity tag, can be competitively disrupted by contacting the complex with a free binding partner, e.g., an unbound affinity tag.

[0342] In some embodiments, the affinity tag comprises an oligonucleotide tag that hybridizes to an oligonucleotide linked to or contained within a protein reagent having a complementary sequence.

[0343] In some embodiments, the binding partner is a lectin, protein A, protein G, metal, metal ion, nitrilotriacetic acid derivative (NTA), RGD-motif, dextran, polyethyleneimine (PEI), redox polymer, glycoprotein, aptamer, dye, amylose, maltose, cellulose, chitin, glutathione, calmodulin, gelatin, polymyxin, heparin, NAD, NADP, lysine, arginine, benzamidine, polyU, or oligodT. Lectins such as concanavalin A are known to bind to polysaccharides and glycosylated proteins. Examples of dyes are triazine dyes, such as cibacron blue F3G-A (CB) or Red HE-3B, which specifically bind to NADH-dependent enzymes. Green A is known to bind to CoA protein, human serum albumin, and dehydrogenase. The dyes 7-aminoactinomycin D and 4',6-diamidino-2-phenylindole are known to bind to DNA. Cations of metals such as Ni, Cd, Zn, Co, or Cu can also be used to bind affinity tags, such as hexahistidine or oligohistidine-containing sequences including the MAT tag (SEQ ID NO:35) and N-methacryloyl-(L)-cysteine ​​methyl ester.

[0344] In some embodiments, binding between the binding partner and the binding site of the protein reagent occurs in the presence of divalent, trivalent, or tetravalent cations. In some embodiments, the protein reagent comprises a divalent, trivalent, or tetravalent cation, e.g., held, e.g., complexed, by a suitable chelating agent. In some embodiments, the binding partner comprises a moiety that complexes with a divalent, trivalent, or tetravalent cation. Metal chelators include ethylenediamine, ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), N,N-bis(carboxymethyl)glycine (also known as nitrilotriacetic acid, NTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 2,3-dimercapto-1-propanol (dimercaprol), porphyrins, and heme. As an example, EDTA chelates most monovalent, divalent, trivalent, and tetravalent metal ions, e.g., silver (Ag + ), calcium (Ca 2+ ), manganese (Mn 2+ ), copper (Cu 2+ ), iron (Fe 2+ ), cobalt (Co + ), and zirconium (Zr 4+ ), but BAPTA can form complexes with Ca 2+ As an example, one skilled in the art can use the chelating agent nitrilotriacetic acid (NTA) to chelate oligohistidine tags and copper (Cu) 2+ ), Nickel (Ni 2+ ), cobalt (Co 2+ ) or zinc (Zn 2+ ) ions.

[0345] In some embodiments, the binding partner comprises a calmodulin-binding peptide and the protein reagent comprises multimeric calmodulin, e.g., as described in U.S. Patent No. 5,985,658. In some embodiments, the binding partner comprises a FLAG peptide and the protein reagent comprises an antibody that binds to the FLAG peptide. For example, in some embodiments, the protein reagent comprises monoclonal antibody 4E11 that binds to the FLAG peptide, e.g., as described in U.S. Patent No. 4,851,341. In some embodiments, the binding partner comprises an oligohistidine tag and the protein reagent comprises an antibody or a transition metal ion that binds to the oligohistidine tag. In some embodiments, calmodulin, antibodies such as 4E11, chelated metal ions, and free chelators can be multimerized by methods including, for example, biotinylation and complexation with streptavidin, avidin, or oligomers thereof, or by, in a first step, introducing carboxyl residues into a polysaccharide, such as dextran, as described, for example, in Noguchi et al. (1992), Bioconjugate Chemistry 3: 132-137, and, in a second step, using carbodiimide chemistry to link calmodulin, antibodies, chelated metal ions, or free chelators to the carboxyl groups in the polysaccharide, such as dextran, via primary amino groups. In some embodiments, the bond between the binding partner and the binding site of the protein reagent can be disrupted by metal ion chelation. Metal chelation can be achieved, for example, by the addition of EGTA or EDTA.

[0346] In some embodiments, the binding partner binds to a biotin-binding molecule, hi some embodiments, the binding partner binds to the biotin-binding site of the molecule.

[0347] In some embodiments, the binding partner is streptavidin or an avidin binding partner. In some embodiments, the binding partner is a streptavidin binding partner. In some embodiments, the streptavidin binding partner is also an avidin binding partner.

[0348] In some embodiments, the binding partner binds to a molecule that is streptavidin, avidin, a streptavidin analog or mutein, or an avidin analog or mutein. In some embodiments, the molecule is any of the streptavidin, avidin, streptavidin analog or mutein and avidin analog or mutein molecules described in Section IB-1-a. In some embodiments, the protein reagent contains the molecule. In some embodiments, the binding partner binds to the biotin binding site of the molecule. In some embodiments, the binding partner binds to the molecule's natural biotin binding site (see, e.g., Qureshi et al. (2001), Journal of Biological Chemistry 276(49): 46422-46428; and Livnah et al. (1993), Proc Natl Acad Sci 90: 5076-5080, which describe the interaction of biotin with streptavidin and avidin, respectively). In some embodiments, the binding partner allows for functionalization of streptavidin, avidin, streptavidin analogs or muteins, or reagents containing avidin analogs or muteins.

[0349] Binding partners that bind to streptavidin, avidin, streptavidin analogs or muteins, or avidin analogs or muteins can be identified and selected by one of skill in the art, including those that bind to the biotin binding site of these molecules. In some embodiments, the binding partner binds to a molecule that is streptavidin.

[0350] In some embodiments, the binding partner contains biotin. In some embodiments, the binding partner is biotin. In some embodiments, the biotin is D-biotin. In some embodiments, the binding partner contains a biotin analog or derivative. In some embodiments, the binding partner is a biotin analog or derivative. In some embodiments, the biotin analog or derivative is a structural analog of biotin. In some embodiments, the biotin analog or derivative binds to the biotin binding site of streptavidin, avidin, a streptavidin analog or mutein, or an avidin analog or mutein. In some embodiments, the biotin analog or derivative binds to the biotin binding site of streptavidin. In some embodiments, the biotin analog or derivative is desthiobiotin, iminobiotin, guanidinobiotin, diaminobiotin, lipoic acid, HABA (hydroxyazobenzene-benzoic acid), dimethyl-HABA, biotin sulfone, caproylamidobiotin, or biocytin (or any of the biotin analogs and derivatives described, for example, in International Published PCT Application WO2008140573).

[0351] In some embodiments, the binding partner comprises a streptavidin-binding peptide. In some embodiments, the binding partner is a streptavidin-binding peptide. In some embodiments, the streptavidin-binding peptide binds to streptavidin, avidin, a streptavidin analog or mutein, or the biotin-binding site of an avidin analog or mutein. In some embodiments, the streptavidin-binding peptide binds to the biotin-binding site of streptavidin. In some embodiments, the streptavidin-binding peptide comprises an amino acid sequence having the formula set forth in SEQ ID NO:9, e.g., the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the streptavidin-binding peptide comprises an amino acid sequence set forth in SEQ ID NO:11, e.g., the formula set forth in SEQ ID NO:12. In some embodiments, the streptavidin-binding peptide comprises the amino acid sequence set forth in SEQ ID NO:7, also referred to as Strep-tag®. In some embodiments, the sequence of the streptavidin-binding peptide is set forth in SEQ ID NO: 7. In some embodiments, the streptavidin-binding peptide contains the amino acid sequence set forth in SEQ ID NO: 8, also referred to as Strep-tag® II. In some embodiments, the sequence of the streptavidin-binding peptide is set forth in SEQ ID NO: 8.

[0352] In some embodiments, the streptavidin-binding peptide may be further modified. In some embodiments, the streptavidin-binding peptide contains the amino acid sequence set forth in SEQ ID NO:8 conjugated to nickel-charged trisNTA, also referred to as His-STREPPER or His / Strep-tag® II Adapter.

[0353] In some embodiments, the streptavidin-binding peptide contains a concatenated sequence of two streptavidin-binding modules. In some embodiments, the streptavidin-binding peptide contains exactly two concatenated sequences of streptavidin-binding modules. In some embodiments, the streptavidin-binding modules are separated from each other by 50 or fewer amino acids, e.g., 45, 40, 35, 30, 25, 20, 15, 10, or 5 or fewer amino acids. In some embodiments, the streptavidin-binding modules are directly connected to each other. In some embodiments, one streptavidin-binding module has 3 to 8 amino acids and contains at least the sequence His-Pro-Xaa (SEQ ID NO:9), where Xaa is glutamine, asparagine, or methionine. In some embodiments, the other streptavidin binding module has the same or a different sequence as the first streptavidin binding module, e.g., as set forth in SEQ ID NO:11 (see, e.g., International Published PCT Application WO 02 / 077018; and U.S. Patent No. 7,981,632). In some embodiments, one of the streptavidin binding modules contains the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, each of the streptavidin binding modules contains the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, one of the streptavidin binding modules contains the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, each of the streptavidin binding modules contains the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the streptavidin binding peptide contains an amino acid sequence having a formula set forth in SEQ ID NO:13 or SEQ ID NO:14. In some embodiments, the streptavidin-binding peptide comprises an amino acid sequence set forth in any of SEQ ID NOs: 15-19. In some embodiments, the sequence of the streptavidin-binding peptide is set forth in any of SEQ ID NOs: 15-19.In some embodiments, the streptavidin-binding peptide contains the amino acid sequence set forth in SEQ ID NO: 16, also referred to as Twin-Strep-tag®. In some embodiments, the sequence of the streptavidin-binding peptide is set forth in SEQ ID NO: 16.

[0354] 2. Incubation In some aspects, the incubation step is carried out according to techniques such as those described in US 6,040,177; Klebanoff et al. (2012) J Immunother. 35(9): 651-660; Terakura et al. (2012) Blood 1: 72-82; and Wang et al. (2012) J Immunother. 35(9): 689-701. In some embodiments, the incubation step is carried out using any of the methods described in WO2021 / 084050, US 11,274,278, US2019 / 0112576, US2021 / 0032297, and US2022 / 0002669.

[0355] In some embodiments, the provided methods involve on-column stimulation of immune cells, e.g., T cells. In some embodiments, "on-column" refers to one or more immune cells, e.g., T cells, being immobilized on a stationary phase contained in the interior cavity of a chromatography column during at least a portion of the incubation period. For example, in some embodiments, the stationary phase contains a selection agent that specifically binds to a selection marker expressed on the surface of the immune cells, e.g., T cells. In some embodiments, the selection agent specifically binds to the selection marker, thereby immobilizing the immune cells, e.g., T cells, on the stationary phase.

[0356] In some embodiments, the immune cells, e.g., T cells, are not or no longer immobilized on the stationary phase during a portion of the incubation. In some embodiments, a portion of the incubation is performed while the immune cells, e.g., T cells, are present in the internal cavity, but not necessarily immobilized on the stationary phase. In some embodiments, stimulation can be continued after elution of the immune cells, e.g., T cells, from the chromatography column, e.g., by further incubating the immune cells, e.g., T cells, after elution, e.g., in the presence of a stimulating reagent.

[0357] In some embodiments, the incubating step is initiated within or about 120 minutes after adding the sample to the internal cavity. In some embodiments, the incubating step is initiated within or about 90 minutes after adding the sample to the internal cavity. In some embodiments, the incubating step is initiated within or about 60 minutes after adding the sample to the internal cavity. In some embodiments, the incubating step is initiated within or about 45 minutes after adding the sample to the internal cavity. In some embodiments, the incubating step is initiated within or about 30 minutes after adding the sample to the internal cavity. In some embodiments, the incubating step is initiated within or about 20 minutes after adding the sample to the internal cavity. In some embodiments, the incubating step is initiated within or about 15 minutes after adding the sample to the internal cavity. In some embodiments, the incubating step begins within 10 minutes or within about 10 minutes after adding the sample to the internal cavity.

[0358] In some embodiments, the immune cells, e.g., T cells, of the sample are allowed sufficient time to penetrate the stationary phase before the start of incubation, e.g., before adding a stimulating reagent to the stationary phase. In some embodiments, the immune cells, e.g., T cells, of the sample are allowed sufficient time to become immobilized on the stationary phase, e.g., via binding to a selection agent on the stationary phase, before the start of incubation. In some embodiments, the incubation step begins at least 5, 10, or 15 minutes after the addition of the sample. In some embodiments, the stationary phase is washed at least once after the addition of the sample and before the start of incubation.

[0359] In some embodiments, the incubating step is initiated by the addition of a stimulatory reagent to the immune cells, eg, T cells.

[0360] In some embodiments, the incubating step comprises incubating 10 cells of immune cells, immune cells immobilized on a stationary phase, or cells of any of the aforementioned estimated cell counts. 6 In some embodiments, the incubation step is performed in the presence of 0.1 μg to 20 μg of stimulating reagent per 10 cells of immune cells, immune cells immobilized on a stationary phase, or cells of any of the aforementioned estimated cell counts. 6 In some embodiments, the incubation step is performed in the presence of 0.1 μg to 16 μg of stimulating reagent per 10 cells of immune cells, immune cells immobilized on a stationary phase, or cells of any of the aforementioned estimated cell counts. 6 In some embodiments, the incubation step is performed in the presence of 0.1 μg to 12 μg of stimulating reagent per 10 cells of immune cells, immune cells immobilized on a stationary phase, or cells of any of the aforementioned estimated cell counts. 6 In some embodiments, the incubation step is performed in the presence of 0.1 μg to 8 μg of stimulating reagent per 10 cells of immune cells, immune cells immobilized on a stationary phase, or cells of any of the aforementioned estimated cell counts. 6In some embodiments, the incubation step is performed in the presence of 0.1 μg to 6 μg of stimulating reagent per 10 cells of immune cells, immune cells immobilized on a stationary phase, or cells of any of the aforementioned estimated cell counts. 6 In some embodiments, the incubation step is performed in the presence of 0.5 μg to 20 μg of stimulating reagent per 10 cells of immune cells, immune cells immobilized on a stationary phase, or any of the aforementioned estimated cell counts. 6 In some embodiments, the incubation step is performed in the presence of 0.5 μg to 16 μg of stimulating reagent per 10 cells of immune cells, immune cells immobilized on a stationary phase, or cells of any of the aforementioned estimated cell counts. 6 In some embodiments, the incubation step is performed in the presence of 0.5 μg to 12 μg of stimulating reagent per 10 cells of immune cells, immune cells immobilized on a stationary phase, or cells of any of the aforementioned estimated cell counts. 6 In some embodiments, the incubation step is performed in the presence of 0.5 μg to 8 μg of stimulating reagent per 10 cells of immune cells, immune cells immobilized on a stationary phase, or cells of any of the aforementioned estimated cell counts. 6 In some embodiments, the incubation step is performed in the presence of 0.5 μg to 6 μg of stimulating reagent per 10 cells of immune cells, immune cells immobilized on a stationary phase, or cells of any of the aforementioned estimated cell counts. 6 In some embodiments, the incubation step is performed in the presence of 1 μg to 20 μg of stimulating reagent per 10 cells, or about 1 μg to 20 μg of stimulating reagent per 10 cells. In some embodiments, the incubation step is performed in the presence of 10 cells, 10 cells immobilized on a stationary phase, or 10 cells of any of the aforementioned estimated cell counts. 6 In some embodiments, the incubation step is performed in the presence of 1 μg to 16 μg of stimulating reagent per 10 cells, or about 1 μg to 16 μg of stimulating reagent per 10 cells. In some embodiments, the incubation step is performed in the presence of 10 cells, 10 cells immobilized on a stationary phase, or 10 cells of any of the aforementioned estimated cell counts. 6In some embodiments, the incubation step is performed in the presence of 1 μg to 12 μg of stimulating reagent per 10 cells, or about 1 μg to 12 μg of stimulating reagent per 10 cells. In some embodiments, the incubation step is performed in the presence of 10 cells, 10 cells immobilized on a stationary phase, or 10 cells of any of the aforementioned estimated cell counts. 6 In some embodiments, the incubation step is performed in the presence of 1 μg to 8 μg of stimulating reagent per 10 cells, or about 1 μg to 8 μg of stimulating reagent per 10 cells. In some embodiments, the incubation step is performed in the presence of 1 μg to 8 μg of stimulating reagent per 10 cells, or about ... 6 In some embodiments, the incubation step is performed in the presence of 1 μg to 6 μg of stimulating reagent per 10 cells, or about 1 μg to 6 μg of stimulating reagent per 10 cells. In some embodiments, the incubation step is performed in the presence of 10 cells, 10 cells immobilized on a stationary phase, or 10 cells of any of the aforementioned estimated cell counts. 6 In some embodiments, the incubation step is performed in the presence of 2 μg to 20 μg of stimulating reagent per 10 cells of immune cells, immune cells immobilized on a stationary phase, or cells of any of the aforementioned estimated cell counts. 6 In some embodiments, the incubation step is performed in the presence of 2 μg to 16 μg of stimulating reagent per 10 cells, or about 2 μg to 16 μg of stimulating reagent per 10 cells. In some embodiments, the incubation step is performed in the presence of 10 cells of immune cells, immune cells immobilized on a stationary phase, or any of the aforementioned estimated cell counts. 6 In some embodiments, the incubation step is performed in the presence of 2 μg to 12 μg of stimulating reagent per 10 cells of immune cells, immune cells immobilized on a stationary phase, or cells of any of the aforementioned estimated cell counts. 6 In some embodiments, the incubation step is performed in the presence of 2 μg to 8 μg of stimulating reagent per 10 cells, or about 2 μg to 8 μg of stimulating reagent per 10 cells. In some embodiments, the incubation step is performed in the presence of 10 cells of immune cells, immune cells immobilized on a stationary phase, or any of the aforementioned estimated cell counts. 6 In some embodiments, the incubation step is performed in the presence of 2 μg to 6 μg of stimulating reagent per 10 cells, or about 2 μg to 6 μg of stimulating reagent per 10 cells. In some embodiments, the incubation step is performed in the presence of 10 cells of immune cells, immune cells immobilized on a stationary phase, or any of the aforementioned estimated cell counts. 6In some embodiments, the incubation step is performed in the presence of 3 μg to 5 μg of stimulating reagent per 10 cells of immune cells, immune cells immobilized on a stationary phase, or any of the aforementioned estimated cell counts. 6 In some embodiments, the incubation step is performed in the presence of 3.5 μg to 4.5 μg of stimulating reagent per 10 cells of immune cells, immune cells immobilized on a stationary phase, or cells of any of the aforementioned estimated cell counts. 6 In any of the foregoing embodiments, the amount of stimulatory reagent is about 100 μg per 100 cells of immune cells. 6 In any of the foregoing embodiments, the amount of stimulatory reagent is per 10 cells of the estimated number of immune cells immobilized on the stationary phase. 6 In any of the foregoing embodiments, the amount of stimulating reagent is per 10 cells of stationary phase binding capacity. 6 In some embodiments, the amount of stimulatory reagent present in a described composition containing a stimulatory reagent, e.g., either contacted with an immune cell or added to an internal cavity, is any of those described in the previous embodiments.

[0361] In some embodiments, the stimulatory reagent is an immune cell (e.g., T cell) that is immobilized on a stationary phase or that is expected to be immobilized on a stationary phase. 6 In some embodiments, the stimulatory reagent is added in an amount of 0.1 μg to 20 μg, or about 0.1 μg to 20 μg, inclusive, per 10 immune cells (e.g., T cells) that are immobilized on a stationary phase or that are expected to be immobilized on a stationary phase. 6 In some embodiments, the stimulatory reagent is added in an amount of 0.4 μg to 8 μg, or about 0.4 μg to 8 μg, inclusive, per 10 immune cells (e.g., T cells) that are immobilized on a stationary phase or that are expected to be immobilized on a stationary phase. 6In some embodiments, the stimulatory reagent is added in an amount of 0.8 μg to 4 μg, or about 0.8 μg to 4 μg, inclusive, per 10 immune cells (e.g., T cells) that are immobilized on a stationary phase or that are expected to be immobilized on a stationary phase. 6 The amount added is 1 μg to 2 μg, or about 1 μg to 2 μg (inclusive) per piece.

[0362] In some embodiments, the stimulating reagent is 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 9 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 6 mg, 0.1 mg to 5 mg, 0.1 mg to 4 mg, 0.1 mg to 3 mg, 0.1 mg to 2 mg, 0.1 mg to 1 mg, 0.4 mg to 20 mg, 0.4 mg to 15 mg, 0.4 mg to 10 mg, 0.4 mg to 9 mg, 0.4 mg to 8 mg, 0.4 mg to 7 mg, 0.4 mg to 6 mg, 0.4 mg to 5 mg, 0.4 mg to 4 mg, 0.4 mg to 3 mg, 0.4 mg to 2 mg mg, 0.4mg~1mg, 0.8mg~20mg, 0.8mg~15mg, 0.8mg~10mg, 0.8mg~9mg, 0.8mg~8mg, 0.8mg~7mg, 0.8mg~6mg, 0.8mg~5mg, 0.8mg~4mg, 0.8mg~3mg, 0.8mg~2mg , 0.8mg~1mg, 1mg~20mg, 1mg~15mg, 1mg~10mg, 1mg~9mg, 1mg~8mg, 1mg~7mg, 1mg~6mg, 1mg~5mg, 1mg~4mg, 1mg~3mg, 1mg~2mg, 2mg~20mg, 2mg~15mg, 2mg~1 0mg, 2mg~9mg, 2mg~8mg, 2mg~7mg, 2mg~6mg, 2mg~5mg, 2mg~4mg, 2mg~3mg, 3mg~20mg, 3mg~15mg, 3mg~10mg, 3mg~9mg, 3mg~8mg, 3mg~7mg, 3mg~6mg, 3mg~5m g, 3mg~4mg, 4mg~20mg, 4mg~15mg, 4mg~10mg, 4mg~9mg, 4mg~8mg, 4mg~7mg, 4mg~6mg, 4mg~5mg, 5mg~20mg, 5mg~15mg, 5mg~10mg, 5mg~9mg, 5mg~8mg, 5mg~7 mg, 5mg to 6mg, 6mg to 20mg, 6mg to 15mg, 6mg to 10mg, 6mg to 9mg, 6mg to 8mg, 6mg to 7mg, 7mg to 20mg, 7mg to 15mg, 7mg to 10mg, 7mg to 9mg, 7mg to 8mg, 8mg to 20mg, 8mg to 15mg, 8mg to 10mg, 8mg to 9mg, 9mg to 20mg, 9mg to 15mg, 9mg to 10mg, 10mg to 20mg, 10mg to 15mg, or 15mg to 20mg, or about 0.1mg to 20mg, 0.1mg to 15mg, 0.1mg to 10mg, 0.1mg to 9mg, 0.1mg~8mg, 0.1mg~7mg, 0.1mg~6mg, 0.1mg~5mg, 0.1mg~4mg, 0.1mg~3mg, 0.1mg~2mg, 0.1mg~1mg, 0.4mg~20mg, 0.4mg~15mg, 0.4mg~10mg, 0. 4mg~9mg, 0.4mg~8mg, 0.4mg~7mg, 0.4mg~6mg, 0.4mg~5mg, 0.4mg~4mg, 0.4mg~3mg, 0.4mg~2mg, 0.4mg~1mg, 0.8mg~20mg, 0.8mg~15mg, 0.8m g~10mg, 0.8mg~9mg, 0.8mg~8mg, 0.8mg~7mg, 0.8mg~6mg, 0.8mg~5mg, 0.8mg~4mg, 0.8mg~3mg, 0.8mg~2mg, 0.8mg~1mg, 1mg~20mg, 1mg~15mg , 1mg~10mg, 1mg~9mg, 1mg~8mg, 1mg~7mg, 1mg~6mg, 1mg~5mg, 1mg~4mg, 1mg~3mg, 1mg~2mg, 2mg~20mg, 2mg~15mg, 2mg~10mg, 2mg~9mg, 2mg~8 mg, 2mg~7mg, 2mg~6mg, 2mg~5mg, 2mg~4mg, 2mg~3mg, 3mg~20mg, 3mg~15mg, 3mg~10mg, 3mg~9mg, 3mg~8mg, 3mg~7mg, 3mg~6mg, 3mg~5mg, 3mg ~4mg, 4mg~20mg, 4mg~15mg, 4mg~10mg, 4mg~9mg, 4mg~8mg, 4mg~7mg, 4mg~6mg, 4mg~5mg, 5mg~20mg, 5mg~15mg, 5mg~10mg, 5mg~9mg, 5mg~8mg , 5mg-7mg, 5mg-6mg, 6mg-20mg, 6mg-15mg, 6mg-10mg, 6mg-9mg, 6mg-8mg, 6mg-7mg, 7mg-20mg, 7mg-15mg, 7mg-10mg, 7mg-9mg, 7mg-8mg, 8mg-20mg, 8mg-15mg, 8mg-10mg, 8mg-9mg, 9mg-20mg, 9mg-15mg, 9mg-10mg, 10mg-20mg, 10mg-15mg, or 15mg-20mg (each inclusive). In some embodiments, the stimulating reagent is added in an amount of 0.1mg-20mg, or about 0.1mg-20mg (inclusive). In some embodiments, the stimulating reagent is added in an amount of 0.4mg-8mg, or about 0.In some embodiments, the stimulating reagent is added in an amount of 0.8 mg to 4 mg, or about 0.8 mg to 4 mg, inclusive. In some embodiments, the stimulating reagent is added in an amount of 1 mg to 3 mg, or about 1 mg to 3 mg, inclusive.

[0363] In some embodiments, the incubating step can include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to stimulate immune cells, e.g., T cells.

[0364] In some embodiments, the incubating step is performed in cell culture medium. In some embodiments, the stimulating reagent is added to the stationary phase in the cell culture medium.

[0365] In some embodiments, the cell culture medium is a serum-free medium. In some embodiments, the serum-free medium is a defined or fully defined cell culture medium. In certain embodiments, the serum-free medium is a conditioned culture medium that has been processed, e.g., filtered, to remove inhibitors and / or growth factors. In some embodiments, the serum-free medium contains protein. In some embodiments, the serum-free medium contains serum albumin, hydrolysates, growth factors, hormones, carrier proteins, and / or attachment factors. In some embodiments, the serum-free medium contains glutamine.

[0366] In some embodiments, the cell culture medium is a basal medium. In some embodiments, the basal medium does not contain any recombinant cytokines. In some embodiments, the basal medium is serum-free. In some embodiments, the basal medium does not contain human-derived serum. In some embodiments, the basal medium contains a mixture of inorganic salts, sugars, amino acids, and optionally vitamins, organic acids, and / or buffers or other well-known cell culture nutrients. In addition to nutrients, the basal medium can also help maintain pH and osmolality. A wide variety of commercially available basal media are well known to those skilled in the art, including Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), Iscove's Modified Dulbecco's Medium, and Ham's Medium. In some embodiments, the basal medium is Iscove's Modified Dulbecco's Medium, RPMI-1640, or α-MEM.

[0367] In some embodiments, the basal medium is a balanced salt solution (e.g., PBS, DPBS, HBSS, EBSS). In some embodiments, the basal medium is selected from Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), F-10, F-12, RPMI 1640, Glasgow Minimum Essential Medium (GMEM), alpha Minimum Essential Medium (alphaMEM), Iscove's Modified Dulbecco's Medium, and M199. In some embodiments, the basal medium is a complex medium (e.g., RPMI-1640, IMDM). In some embodiments, the basal medium is OpTmizer™ CTS™ T-Cell Expansion Basal Medium (ThermoFisher).

[0368] In certain embodiments, the basal medium is supplemented with additional additives. In some embodiments, the basal medium is not supplemented with any additional additives. Additives to cell culture media include nutrients, sugars such as glucose, amino acids, vitamins, or additives such as ATP and NADH.

[0369] In some embodiments, the cell culture medium contains one or more cytokines. In certain embodiments, the one or more cytokines are recombinant cytokines. In certain embodiments, the one or more cytokines are human recombinant cytokines. In certain embodiments, the one or more cytokines bind to a receptor expressed by T cells. In certain embodiments, the one or more cytokines comprise a member of the four-α-helical bundle family of cytokines. In some embodiments, the members of the four-α-helical bundle family of cytokines comprise interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-12 (IL-12), interleukin-15 (IL-15), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the one or more cytokines comprise IL-15. In certain embodiments, the one or more cytokines comprise IL-7. In certain embodiments, the one or more cytokines include IL-2. In certain embodiments, the one or more cytokines are selected from IL-2, IL-15, and IL-7. In certain embodiments, the cell culture medium contains recombinant IL-2, IL-15, and IL-7.

[0370] In certain embodiments, the amount or concentration of one or more cytokines is measured and / or quantified using international units (IU). International units can be used to quantify vitamins, hormones, cytokines, vaccines, blood products, and similar biologically active substances. In some embodiments, an IU is or includes a unit of measure of the potency of a biological preparation compared to an international reference standard of a specific weight and strength (e.g., WHO 1st International Standard for Human IL-2, 86 / 504). International units are the only recognized standardized method for reporting biological activity units that are published and obtained from international collaborative research efforts. In certain embodiments, IUs for a population, sample, or source of cytokines can be obtained by product comparison testing using a similar WHO standard. For example, in some embodiments, the IU / mg of a population, sample, or source of human recombinant IL-2, IL-7, or IL-15 is compared to a WHO standard IL-2 product (NIBSC code: 86 / 500), a WHO standard IL-17 product (NIBSC code: 90 / 530), and a WHO standard IL-15 product (NIBSC code: 95 / 554), respectively.

[0371] In certain embodiments, the ED50 of recombinant human IL-2 or IL-15 is equivalent to the concentration required for half-maximal stimulation of cell proliferation (XTT cleavage) using CTLL-2 cells. In certain embodiments, the ED50 of recombinant human IL-7 is equivalent to the concentration required for half-maximal stimulation of proliferation of PHA-activated human peripheral blood lymphocytes. Details of assays and IU calculations for IL-2 are discussed in Wadhwa et al., Journal of Immunological Methods (2013), 379 (1-2): 1-7; and Gearing and Thorpe, Journal of Immunological Methods (1988), 114 (1-2): 3-9; details of assays and IU calculations for IL-15 are discussed in Soman et al., Journal of Immunological Methods (2009) 348 (1-2): 83-94.

[0372] In some embodiments, the cell culture medium contains IL-2, e.g., human recombinant IL-2, at a concentration of 1 IU / mL to 500 IU / mL, 10 IU / mL to 250 IU / mL, 50 IU / mL to 200 IU / mL, 50 IU / mL to 150 IU / mL, 75 IU / mL to 125 IU / mL, 100 IU / mL to 200 IU / mL, or 10 IU / mL to 100 IU / mL. In certain embodiments, the cell culture medium contains recombinant IL-2 at 50 IU / mL, 60 IU / mL, 70 IU / mL, 80 IU / mL, 90 IU / mL, 100 IU / mL, 110 IU / mL, 120 IU / mL, 130 IU / mL, 140 IU / mL, 150 IU / mL, 160 IU / mL, 170 IU / mL, 180 IU / mL, 190 IU / mL or 100 IU / mL. In some embodiments, the cell culture medium contains about 100 IU / mL of recombinant IL-2, e.g., human recombinant IL-2.

[0373] In some embodiments, the cell culture medium contains recombinant IL-7, e.g., human recombinant IL-7, at a concentration of 100 IU / mL to 2,000 IU / mL, 500 IU / mL to 1,000 IU / mL, 100 IU / mL to 500 IU / mL, 500 IU / mL to 750 IU / mL, 750 IU / mL to 1,000 IU / mL, or 550 IU / mL to 650 IU / mL. In certain embodiments, the cell culture medium contains IL-7 at 50 IU / mL, 100 IU / mL, 150 IU / mL, 200 IU / mL, 250 IU / mL, 300 IU / mL, 350 IU / mL, 400 IU / mL, 450 IU / mL, 500 IU / mL, 550 IU / mL, 600 IU / mL, 650 IU / mL, 700 IU / mL, 750 IU / mL, 800 IU / mL, 750 IU / mL, 750 IU / mL, or 1,000 IU. In certain embodiments, the cell culture medium contains about 600 IU / mL of IL-7, e.g., human recombinant IL-7.

[0374] In some embodiments, the cell culture medium contains recombinant IL-15, e.g., human recombinant IL-15, at a concentration of 1 IU / mL to 500 IU / mL, 10 IU / mL to 250 IU / mL, 50 IU / mL to 200 IU / mL, 50 IU / mL to 150 IU / mL, 75 IU / mL to 125 IU / mL, 100 IU / mL to 200 IU / mL, or 10 IU / mL to 100 IU / mL. In certain embodiments, the cell culture medium contains recombinant IL-15 at 50 IU / mL, 60 IU / mL, 70 IU / mL, 80 IU / mL, 90 IU / mL, 100 IU / mL, 110 IU / mL, 120 IU / mL, 130 IU / mL, 140 IU / mL, 150 IU / mL, 160 IU / mL, 170 IU / mL, 180 IU / mL, 190 IU / mL or 200 IU / mL. In some embodiments, the cell culture medium contains about 100 IU / mL of recombinant IL-15, e.g., human recombinant IL-15.

[0375] In some embodiments, the cell culture medium does not contain cytokines.

[0376] In some embodiments, after the start of incubation, the immune cells, e.g., T cells, are incubated within the internal cavity of the chromatography. In some embodiments, the incubation step is carried out for 1 day, about 1 day, or less than 1 day. In some embodiments, the incubating step is carried out for about 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours, or for less than 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours. In some embodiments, the incubating step is performed for 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 7 to 24, 8 to 24, 9 to 24, 10 to 24, 11 to 24, 12 to 24, 13 to 24, 14 to 24, 15 to 24, 16 to 24, 17 to 24, 18 to 24, 19 to 24, 20 to 24, 21 to 24, 22 to 24, 23 to 24, 2 to 23, 2 to 22, 2 to 21, 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or The test is typically performed for 2 to 3 hours, or for approximately 2 to 24 hours, 3 to 24 hours, 4 to 24 hours, 5 to 24 hours, 6 to 24 hours, 7 to 24 hours, 8 to 24 hours, 9 to 24 hours, 10 to 24 hours, 11 to 24 hours, 12 to 24 hours, 13 to 24 hours, 14 to 24 hours, 15 to 24 hours, 16 to 24 hours, 17 to 24 hours, 18 to 24 hours, 19 to 24 hours, 20 to 24 hours, 21 to 24 hours, 22 to 24 hours, 23 to 24 hours, 2 to 23 hours, 2 to 22 hours, 2 to 21 hours, 2 to 20 hours, 2 to 19 hours, 2 to 18 hours, 2 to 17 hours, 2 to 16 hours, 2 to 15 hours, 2 to 14 hours, 2 to 13 hours, 2 to 12 hours, 2 to 11 hours, 2 to 10 hours, 2 to 9 hours, 2 to 8 hours, 2 to 7 hours, 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, or 2 to 3 hours. In some embodiments, the incubating step is carried out for 24 hours, about 24 hours, or less than 24 hours. In some embodiments, the incubating step is carried out for 12 hours, about 12 hours, or less than 12 hours. In some embodiments, the incubating step is carried out for 5 hours, about 5 hours, or less than 5 hours.In some embodiments, the incubating step is carried out for 4 hours, about 4 hours, or less than 4 hours. In some embodiments, the incubating step is carried out for 2 hours, about 2 hours, or less than 2 hours.

[0377] In some embodiments, the incubating step is carried out for 1 hour to 8 hours, or for about 1 hour to 8 hours, inclusive. In some embodiments, the incubating step is carried out for 2 hours to 6 hours, or for about 2 hours to 6 hours, inclusive. In some embodiments, the incubating step is carried out for 3 hours to 5 hours, or for about 3 hours to 5 hours, inclusive. In some embodiments, the incubating step is carried out for 4 hours or for about 4 hours. In some embodiments, the incubating step is carried out for 4.5 hours or for about 4.5 hours.

[0378] In some embodiments, the incubating step is performed at a temperature above room temperature. In some embodiments, the incubating step is performed at physiological temperature. In some embodiments, the incubating step is performed at a temperature of 35°C to 39°C, or about 35°C to 39°C. In some embodiments, the incubating step is performed at 37°C or about 37°C.

[0379] In some embodiments, the temperature is regulated by one or more heating elements configured to supply heat to the stationary phase. In some embodiments, the oxygen and carbon dioxide content of the stationary phase is controlled using gas exchange. In some embodiments, the temperature or gas exchange is regulated using any of the methods or devices described in WO2020 / 089343, WO2021 / 084050, and US2022 / 0002669.

[0380] In some embodiments, the incubation step promotes downregulation of a selection marker used in immune cell selection, e.g., T cell selection, resulting in the spontaneous release or detachment of immune cells, e.g., T cells, from the stationary phase in some cases. The release or detachment of immune cells, e.g., T cells, can be performed without any additional steps or reagents. In some aspects, immune cells, e.g., T cells, can be collected, for example, using a wash buffer that does not contain a competitor to promote the release of immune cells, e.g., T cells, from the stationary phase.

[0381] 3. Collection In some embodiments, the provided methods include collecting immune cells, e.g., T cells. In some embodiments, the immune cells, e.g., T cells, are collected from a chromatography column. In some embodiments, the collecting step includes eluting the immune cells, e.g., T cells, from the chromatography column.

[0382] In some embodiments, the harvesting step occurs 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after, about, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after addition of the stimulation reagent. In some embodiments, the harvesting step occurs within 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours of adding the stimulating reagent, or within about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the harvesting step occurs about 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 7 to 24, 8 to 24, 9 to 24, 10 to 24, 11 to 24, 12 to 24, 13 to 24, 14 to 24, 15 to 24, 16 to 24, 17 to 24, 18 to 24, 19 to 24, 20 to 24, 21 to 24, 22 to 24, 23 to 24, 2 to 23, 2 to 22, 2 to 21, 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 hours after addition of the stimulation reagent. In some embodiments, the collecting step occurs about 2 to 24 hours after the addition of the stimulating reagent. In some embodiments, the collecting step occurs about 2 to 12 hours after the addition of the stimulating reagent. In some embodiments, the collecting step occurs about 1 to 8 hours after the addition of the stimulating reagent. In some embodiments, the collecting step occurs about 2 to 6 hours after the addition of the stimulating reagent. In some embodiments, the collecting step occurs about 3 to 5 hours after the addition of the stimulating reagent. In some embodiments, the collecting step occurs about 4.5 hours after the addition of the stimulating reagent.

[0383] In some embodiments, the harvesting step occurs 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after the start of incubation. In some embodiments, the harvesting step occurs within 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours of the start of incubation. In some embodiments, the harvesting step occurs about 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 7 to 24, 8 to 24, 9 to 24, 10 to 24, 11 to 24, 12 to 24, 13 to 24, 14 to 24, 15 to 24, 16 to 24, 17 to 24, 18 to 24, 19 to 24, 20 to 24, 21 to 24, 22 to 24, 23 to 24, 2 to 23, 2 to 22, 2 to 21, 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 hours after the start of the incubation. In some embodiments, the collecting step occurs about 2-24 hours after the start of incubation. In some embodiments, the collecting step occurs about 2-12 hours after the start of incubation. In some embodiments, the collecting step occurs about 1-8 hours after the start of incubation. In some embodiments, the collecting step occurs about 2-6 hours after the start of incubation. In some embodiments, the collecting step occurs about 3-5 hours after the start of incubation. In some embodiments, the collecting step occurs about 4.5 hours after the start of incubation.

[0384] In some embodiments, the harvesting step comprises adding a wash buffer to the stationary phase to harvest the immune cells, e.g., T cells. In some embodiments, the wash buffer is a cell culture medium. In some embodiments, the cell culture medium is any of those described in Section IB-2.

[0385] In some embodiments, the collecting step can be performed without adding a competitor to elute immune cells, e.g., T cells, from the stationary phase. In some embodiments, the wash buffer does not contain a competitor to elute immune cells, e.g., T cells, from the stationary phase. In some embodiments, the wash buffer contains a competitor to elute immune cells, e.g., T cells, from the stationary phase.

[0386] In some embodiments, the competitor promotes the release of immune cells, e.g., T cells, from the stationary phase. In some embodiments, the competitor interferes with the immobilization of immune cells, e.g., T cells, on the stationary phase. In some embodiments, the competitor interferes with the immobilization of the selection agent on the stationary phase chromatography matrix. For example, in some embodiments, the stationary phase contains a molecule that is streptavidin, avidin, a streptavidin analog or mutein, or any of the avidin analog or mutein molecules described herein, and the binding partner of the selection agent is a binding partner that reversibly binds to the molecule, e.g., with a lower binding affinity compared to the binding affinity of streptavidin to biotin, e.g., any of the streptavidin or avidin binding partners described herein, e.g., streptavidin-binding peptides, or a binding partner whose binding is disrupted in the presence of biotin. In some embodiments, the competitor has a higher binding affinity for the molecule than the binding partner of the selection agent. In some embodiments, the competitor disrupts the binding of the binding partner of the selection agent to the molecule. In some embodiments, the competitor is biotin, e.g., D-biotin. In some embodiments, the competitor is a biotin analog or derivative, e.g., any described herein.

[0387] In some embodiments, the chromatography column and collection vessel are connected in a closed system. In some embodiments, the closed system is sterile. In some embodiments, the selection, stimulation, and / or elution steps are performed by an automated system with minimal or no manual operation or intervention, e.g., by a human.

[0388] 4. Further incubation In some embodiments, the provided methods include further incubating the immune cells, e.g., T cells. In some embodiments, the further incubating is performed in the presence of a stimulating reagent. In some embodiments, the further incubating is performed after the immune cells, e.g., T cells, are collected from the chromatography column. Thus, in some aspects, the stimulation of the immune cells, e.g., T cells, continues after the immune cells, e.g., T cells, are collected from the chromatography column. In some embodiments, the further incubating is performed before manipulating the immune cells, e.g., T cells.

[0389] In some embodiments, the further incubation step is performed in the presence of a stimulating reagent. In some embodiments, the stimulating reagent is present at any concentration or amount described in Section IB-2. In some embodiments, the stimulating reagent added for the incubating step is not removed before the further incubation. In some embodiments, the further incubation step is performed in the same medium that was present during the incubation. In some embodiments, the further incubation step is performed in the cell culture medium present in the chromatography column eluted by collecting, including any stimulating reagent present therein.

[0390] In some embodiments, the incubating step can further include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to stimulate immune cells.

[0391] In some embodiments, the further incubation step is performed in a cell culture medium, hi some embodiments, the cell culture medium is any of those described in Section IB-2.

[0392] In some embodiments, the further incubating step is performed at a temperature above room temperature. In some embodiments, the further incubating step is performed at physiological temperature. In some embodiments, the further incubating step is performed at a temperature of 35°C to 39°C, or about 35°C to 39°C. In some embodiments, the further incubating step is performed at 37°C or about 37°C.

[0393] In some embodiments, the further incubation step is 2 hours to 30 hours, 2 hours to 26 hours, 2 hours to 22 hours, 2 hours to 18 hours, 2 hours to 14 hours, 2 hours to 10 hours, 2 hours to 6 hours, 2 hours to 4 hours, 4 hours to 30 hours, 4 hours to 26 hours, 4 hours to 22 hours, 4 hours to 18 hours, 4 hours to 14 hours, 4 hours to 10 hours, 4 hours to 6 hours, 6 hours to 30 hours, 6 hours to 26 hours, 6 hours to 22 hours, 6-18 hours, 6-14 hours, 6-10 hours, 10-30 hours, 10-26 hours, 10-22 hours, 10-18 hours, 10-14 hours, 14-30 hours, 14-26 hours, 14-22 hours, 14-18 hours, 18-30 hours, 18-26 hours, 18-22 hours, 22-30 hours, 22-26 hours, or 26 hours or more 30 hours, or approximately 2 hours to 30 hours, 2 hours to 26 hours, 2 hours to 22 hours, 2 hours to 18 hours, 2 hours to 14 hours, 2 hours to 10 hours, 2 hours to 6 hours, 2 hours to 4 hours, 4 hours to 30 hours, 4 hours to 26 hours, 4 hours to 22 hours, 4 hours to 18 hours, 4 hours to 14 hours, 4 hours to 10 hours, 4 hours to 6 hours, 6 hours to 30 hours, 6 hours to 26 hours, 6 hours to 22 hours, 6 hours to 18 hours, 6 hours or more The incubation is continued for 14 hours, 6 to 10 hours, 10 to 30 hours, 10 to 26 hours, 10 to 22 hours, 10 to 18 hours, 10 to 14 hours, 14 to 30 hours, 14 to 26 hours, 14 to 22 hours, 14 to 18 hours, 18 to 30 hours, 18 to 26 hours, 18 to 22 hours, 22 to 30 hours, 22 to 26 hours, or 26 to 30 hours (inclusive). In some embodiments, the further incubation is continued for 10 to 30 hours or about 10 to 30 hours (inclusive). In some embodiments, the further incubation is continued for 16 to 24 hours or about 16 to 24 hours (inclusive). In some embodiments, the further incubation is continued for 18 to 22 hours or about 18 to 22 hours (inclusive). In some embodiments, the further incubating step is carried out for 20 hours or about 20 hours.

[0394] In some embodiments, the further incubating step is performed in an incubator. In some embodiments, the immune cells, e.g., T cells, are transferred into a container for further incubation. In some embodiments, the container is a vial. In certain embodiments, the container is a bag. In some embodiments, the immune cells, e.g., T cells, are transferred into the container under sealed or sterile conditions. In some embodiments, the container, e.g., a vial or bag, is then placed in an incubator for all or a portion of the further incubating step. In certain embodiments, the incubator is set to 16°C, 24°C, or 35°C, about 16°C, 24°C, or 35°C, or at least 16°C, 24°C, or 35°C. In some embodiments, the incubator is set to 37°C, about 37°C, or 37°C ± 2°C, ± 1°C, ± 0.5°C, or ± 0.1°C.

[0395] In certain embodiments, the further incubating step is performed under static conditions, e.g., without centrifugation, shaking, rotation, rocking, or perfusion of the medium. In some embodiments, the further incubating step is performed under gentle mixing conditions, e.g., with rocking.

[0396] In some embodiments, the provided methods include removing the stimulatory reagent from the immune cells, e.g., T cells. In some embodiments, the removing step is followed by a further incubation step. In some embodiments, the removing step occurs before manipulating. In some embodiments, the removing step occurs before introducing a nucleic acid molecule. In some embodiments, the removing step occurs before introducing one or more gene editing agents. In some embodiments, the removing step terminates stimulation of the immune cells, e.g., T cells. In some embodiments, the removing step includes washing the immune cells, e.g., T cells, using any of the cell media described herein, e.g., in the presence or absence of a competitor.

[0397] In some embodiments, the provided methods include disrupting binding of one or more binding substances to a protein reagent of the stimulation reagent. In some embodiments, the disrupting step is further followed by an incubating step. In some embodiments, the disrupting step is performed before manipulating. In some embodiments, the disrupting step is performed before introducing a nucleic acid molecule. In some embodiments, the disrupting step is performed before introducing one or more gene editing agents. In some embodiments, the disrupting step terminates stimulation of immune cells, e.g., T cells. In some embodiments, the disrupting step is performed by adding a competitor to compete for binding of one or more binding substances to the protein reagent of the stimulation reagent. In some embodiments, the competitor is biotin or a biotin analog, such as any described herein, and the one or more binding substances and the protein reagent are any described herein that are capable of disrupting binding with biotin or a biotin analog.

[0398] C. Operation In some embodiments, the provided methods include manipulating immune cells, such as T cells. In some embodiments, the provided methods include targeted integration of a transgene into a target site of the gene in immune cells, such as T cells. In some embodiments, the provided methods include introducing a nucleic acid molecule containing the transgene into immune cells, such as T cells. In some embodiments, the step of introducing the nucleic acid molecule is under conditions for targeted integration of the transgene into the target site. In some embodiments, the step of introducing the nucleic acid molecule is by non-viral gene delivery.

[0399] In some embodiments, provided methods do not include the step of inducing gene disruption.In some embodiments, targeted integration is by a method that does not induce gene disruption.In some embodiments, targeted integration is by programmable addition of site-specific targeting elements (PASTE), such as those described in WO2022 / 159892 and US20220154224.In some embodiments, PASTE involves introducing one or more gene editing agents to edit genes in immune cells.

[0400] In some embodiments, the methods provided include inducing a targeted gene disruption. In some embodiments, the methods provided include homology-dependent repair (HDR) using a nucleic acid molecule containing a transgene, thereby targeting integration of the transgene at a target site.

[0401] In some cases, the provided method includes introducing one or more targeted gene disruptions, such as DNA breaks, at target sites by gene editing techniques combined with targeted integration of transgenes by HDR. In some aspects, the one or more targeted gene disruptions are carried out by introducing one or more gene editing agents capable of introducing gene disruptions. In some embodiments, the HDR step requires DNA disruption or cleavage, such as double-strand breaks, at the target sites. In some embodiments, the DNA cleavage is induced by gene editing methods, such as targeted nucleases. In some embodiments, the method generates engineered immune cells, such as T cells, that are knocked out for the expression of genes containing the target sites. In some aspects, after carrying out the method, the engineered immune cells, such as T cells, contain transgenes that are operably linked to the endogenous transcriptional regulatory elements of the genes.

[0402] In some aspects, provided methods include introducing one or more gene editing agents and introducing a nucleic acid molecule containing a transgene and one or more homology arms into immune cells, such as T cells. In some aspects, the transgene contains a sequence of nucleotides encoding a recombinant protein. In some embodiments, the nucleic acid sequence is targeted for integration into the target site via homology-directed repair (HDR).

[0403] In some aspects, provided methods include introducing a nucleic acid molecule comprising a transgene into an immune cell, e.g., a T cell, having a gene disruption in a gene having a target site, wherein the gene disruption has been induced by one or more gene editing agents capable of inducing a gene disruption in the gene, and wherein the nucleic acid sequence is targeted for integration into the gene via HDR.

[0404] In some aspects, the provided methods include using gene editing methods and / or targeted nucleases to generate targeted DNA breaks, followed by HDR based on one or more nucleic acid molecules containing nucleic acid sequences encoding homologous sequences, and optionally other molecules, that are homologous to sequences within or near the gene linked to the transgene, to specifically target and integrate the transgene at or near the DNA break. Thus, in some aspects, the provided methods include inducing targeted gene disruption and introducing (e.g., by HDR) the nucleic acid molecule containing the transgene into immune cells, such as T cells.

[0405] In some embodiments, the targeted integration of transgene by HDR occurs at one or more target sites within gene.In some aspects, targeted integration occurs within the open reading frame sequence of gene.In some aspects, the targeted integration of transgene results in gene knockout, for example, thereby eliminating the expression of gene.

[0406] In some aspects, the transgene is integrated into the gene, for example, by homology-directed repair (HDR), within an exon of the gene's open reading frame or a subsequence thereof, such that the transgene is in-frame with the sequence of the exon. In some aspects, all or a portion of the gene at the modified locus, for example, the upstream portion of the integrated transgene and the recombinant protein, are expressed, optionally separated by a polycistronic element.

[0407] In some aspects, the provided methods allow the recombinant protein to be expressed under the control of the gene's endogenous transcriptional regulatory element, for example, the gene's endogenous promoter. In some aspects, the provided methods allow the transgene to be operably linked to the endogenous regulatory or control element, for example, a cis-regulatory element, for example, a promoter, or the 5' and / or 3' untranslated region (UTR) of the gene. Thus, in some aspects, the provided methods allow the recombinant protein, for example, a CAR, to be expressed, and / or expression is conditionally, temporally, and / or quantitatively regulated in the same way as the gene.

[0408] In some embodiments, the nucleic acid molecule is introduced into an immune cell, e.g., a T cell, prior to, concurrently with, or subsequent to the introduction of one or more gene editing agents. In the presence of one or more targeted gene disruptions, e.g., DNA breaks, the nucleic acid molecule can be used as a DNA repair template to effectively integrate a transgene at or near the site of the targeted gene disruption by HDR based on homology between the endogenous gene sequence surrounding the gene disruption and one or more homology arms, such as 5' and / or 3' homology arms, contained in the nucleic acid molecule.

[0409] In some aspects, the nucleic acid molecule and one or more gene editing agents are introduced simultaneously. In some embodiments, the introduction of one or more gene editing agents is carried out simultaneously with the introduction of the nucleic acid molecule. In some aspects, the two introduction steps can be carried out sequentially. The introduction of one or more gene editing agents is carried out before the introduction of the nucleic acid molecule.

[0410] In some embodiments, gene editing step and HDR step are carried out simultaneously and / or in one experimental reaction.In some embodiments, gene editing step and HDR step are carried out consecutively or sequentially in one or consecutive experimental reaction.In some embodiments, gene editing step and HDR step are carried out simultaneously or at different times in separate experimental reaction.

[0411] Any method for introducing one or more gene editing agents can be used as described, depending on the specific agent used. In some aspects, the agent is a gene-specific RNA-guided nuclease, such as a clustered regularly interspaced short batch sequence nucleic acid (CRISPR)-Cas system, such as a CRISPR-Cas9 or CRISPR-Cas12 system. In some embodiments, an agent containing a Cas, such as Cas9 or Cas12, and a guide RNA (gRNA) containing a targeting sequence that targets a region of a gene are introduced into immune cells. In some embodiments, the agent is or includes a ribonucleoprotein (RNP) complex (Cas / gRNA RNP) of a Cas, such as Cas9 or Cas12, and a gRNA containing a targeting sequence that targets a gene. In some embodiments, the introduction includes contacting the agent with immune cells in vitro. In some embodiments, the introduction can further include causing delivery of the agent and / or a nucleic acid molecule, such as a template for HDR, into the immune cells. In various embodiments, the provided methods utilize direct delivery of a ribonucleoprotein (RNP) complex of a Cas, e.g., Cas9 or Cas12, and a gRNA into immune cells, e.g., by electroporation. In some cases, electroporation of the immune cells to be modified includes subjecting the cells to a cold shock, e.g., at 32°C, after electroporation of the immune cells and prior to plating.

[0412] In some embodiments, the steps of introducing the nucleic acid molecule and introducing the one or more gene editing agents (e.g., Cas / gRNA RNPs) can be performed simultaneously or sequentially in any order. In some of the optional embodiments, the nucleic acid molecule is introduced into the immune cell, e.g., T cell, after introducing the one or more gene editing agents (e.g., Cas / gRNA RNPs).

[0413] Any method for introducing a nucleic acid molecule can be used as described, depending on the particular method used for delivery of the nucleic acid molecule to immune cells. In some of the optional embodiments, non-viral gene delivery methods are used.

[0414] In some embodiments, the nucleic acid molecule is introduced into the collected one or more T cells by non-viral gene delivery. In some embodiments, the nucleic acid molecule comprises a transgene encoding a recombinant protein. In some embodiments, the introducing is under conditions for targeted integration of the transgene into a gene target site in the collected one or more T cells.

[0415] In some embodiments, the methods provided comprise introducing, by non-viral gene delivery, a nucleic acid molecule comprising a transgene under conditions for targeted integration of the transgene encoding a recombinant protein into a genetic target site in one or more collected T cells.

[0416] In some embodiments, the nucleic acid molecule is a DNA molecule. In some embodiments, the nucleic acid molecule is a naked DNA molecule. In some embodiments, the nucleic acid molecule, e.g., the naked DNA molecule, is a non-viral, capsid-free DNA molecule.

[0417] In some embodiments, the nucleic acid molecule is a double-stranded DNA molecule. In some embodiments, the nucleic acid molecule is a single-stranded DNA molecule.

[0418] In some embodiments, the nucleic acid molecule, e.g., the naked DNA molecule, is a modified DNA molecule. In some embodiments, the nucleic acid molecule, e.g., the naked DNA molecule, is modified to increase its stability.

[0419] In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, is a non-viral capsid-free DNA molecule with covalently closed ends (also referred to herein as a "closed-end DNA" or "ceDNA" molecule). In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, is a ceDNA molecule. In some embodiments, the nucleic acid molecule is a naked ceDNA molecule.

[0420] In some embodiments, the nucleic acid molecule, eg, the naked DNA molecule, is a capsid-free, linear, double-stranded DNA molecule.

[0421] In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, comprises at least one sequence (also referred to herein as a "homology arm") that is homologous to a nucleic acid sequence surrounding the target site. In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, comprises a sequence that includes a 5' homology arm and a 3' homology arm that comprise sequences that are homologous to a nucleic acid sequence surrounding the target site. In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, comprises the structure [5' homology arm]-[transgene]-[3' homology arm].

[0422] In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, has a nick or gap. In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, comprises at least one inverted tandem repeat (also referred to herein as "ITR"). In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, comprises at least one homology arm. In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, comprises at least one ITR, at least one homology arm, and a transgene. In some embodiments, the structure of the nucleic acid molecule, e.g., naked DNA molecule, is [5'ITR]-[homology arm (5')]-[transgene]-[homology arm (3')]-[ITR3'].

[0423] In some embodiments, the ITR is an ITR derived from an AAV serotype, an ITR derived from a goose virus ITR, an ITR derived from a B19 virus ITR, or a wild-type ITR derived from a parvovirus. In some embodiments, the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In some embodiments, the ITR is a mutant ITR.

[0424] In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, comprises an additional ITR that is different from the first ITR. In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, comprises two mutant ITRs at both the 5' and 3' ends of the transgene. In some embodiments, the two mutant ITRs are symmetric mutants.

[0425] In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, is selected from the group consisting of a closed-end linear double-stranded (CELiD) DNA molecule, a minicircle DNA molecule, a minimalistic immunologically-defined gene expression (MIDGE) DNA molecule, a ministring DNA molecule, a dumbbell-shaped linear double-stranded closed-end DNA molecule, or a doggybone™ DNA molecule. In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, is a CELiD DNA molecule. In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, is a MIDGE DNA molecule. In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, is a ministring DNA. In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, is a dumbbell-shaped linear double-stranded closed-end DNA molecule. In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, is a doggybone™ DNA molecule.

[0426] In some embodiments, the nucleic acid molecule, e.g., the naked DNA molecule, is a doggybone™ DNA molecule. In some embodiments, the doggybone™ DNA (dbDNA™) molecule is a unique synthetic closed, linear, double-stranded DNA molecule. In some embodiments, the closed linear DNA molecule is double-stranded DNA covalently closed at each end. The double-stranded sections of the closed linear DNA molecule can be complementary. When denatured, the closed linear DNA may form a single-stranded circle. The DNA may be closed at each end by any suitable structure, including a cruciform, hairpin, or hairpin loop, as desired. The ends of the closed linear DNA may consist of non-complementary sequences. In some embodiments, the non-complementary sequences force the DNA into a single-stranded configuration at the cruciform, hairpin, or hairpin loop.

[0427] In some embodiments, the nucleic acid molecule, e.g., a naked DNA molecule, is a closed-end linear double-stranded (CELiD) DNA molecule. In some embodiments, the CELiD DNA molecule is a linear double-stranded molecule. In some embodiments, the CELiD DNA molecule is double-stranded DNA covalently closed at each end. In some embodiments, the CELiD DNA molecule can contain heterologous DNA flanked by ITRs. In some embodiments, the heterologous DNA can encode a protein. In some embodiments, the CELiD DNA molecule is a linear double-stranded DNA molecule containing heterologous DNA flanked by inverted terminal repeats (ITRs), at least one of which contains an AAV Rep protein binding site and an AAV trs site, wherein the linear double-stranded DNA molecule has covalently closed ends. In some embodiments, because the ends are covalently closed, the CELiD DNA molecule is exonuclease resistant. In some embodiments, each single linear strand of heterologous DNA in the double-stranded molecule is located between two full-length AAV ITRs. In some embodiments, each linear strand of a CELiD DNA molecule pairs with a complementary strand, such that the final double-stranded CELiD molecule contains a total of four ITRs.

[0428] In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, is a minicircle DNA molecule. In some embodiments, the minicircle DNA molecule is a circular DNA molecule. In some embodiments, the circular DNA molecule contains at least one gene encoding a recombinant protein. In some embodiments, the minicircle DNA molecule is a circular DNA having an attR site and a genetically engineered gene expression cassette, wherein the gene expression cassette comprises a promoter, a nucleotide sequence encoding an immunoglobulin κ chain signal peptide, a nucleotide sequence encoding a Flag tag, a gene encoding a recombinant protein, a nucleotide sequence encoding a His6 tag, a stop codon, and a polyA tail signal, which are sequentially linked.

[0429] In some embodiments, the nucleic acid molecule, e.g., the naked DNA molecule, is a minimalist immunologically defined gene expression (MIDGE) DNA molecule. In some embodiments, the MIDGE DNA molecule is a circular DNA molecule. In some embodiments, the MIDGE DNA molecule is a double-stranded circular DNA molecule. In some embodiments, the MIDGE DNA molecule comprises an expression cassette containing a promoter, a gene of interest, and an RNA stabilizing sequence, e.g., a polyA sequence. The complementary sense and antisense strands encoding the transgene can be connected at both the 5' and 3' ends of the double-stranded MIDGE DNA molecule by single-stranded hairpin DNA with a non-complementary sequence loop structure, so that the MIDGE DNA molecule has a "dumbbell" shape. The MIDGE DNA molecule can be resistant to enzymatic digestion and relatively stable in cells and serum.

[0430] In some embodiments, the nucleic acid molecule, e.g., the naked DNA molecule, is a ministring DNA molecule. In some embodiments, the ministring DNA molecule is a reinforced linear covalently closed (LCC) minivector. In some embodiments, the ministring DNA molecule is double-stranded. In some embodiments, the ministring DNA molecule comprises an LCC end, a minimal transgene expression cassette element, and a DNA targeting sequence (DTS) at both ends.

[0431] In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, is a dumbbell-shaped linear double-stranded closed-end DNA molecule. In some embodiments, the dumbbell-shaped linear double-stranded closed-end DNA molecule is a linear double-stranded molecule. In some embodiments, the dumbbell-shaped linear double-stranded closed-end DNA molecule is covalently closed at each end. In some embodiments, the dumbbell-shaped linear double-stranded closed-end DNA molecule comprises two hairpin structures of ITRs at the 5' and 3' ends of the expression cassette.

[0432] In some embodiments, provided methods include introducing a nucleic acid molecule under conditions for targeted integration of the transgene into a target site of the gene in one or more collected T cells. Such methods can be referred to as a "DNA knock-in system." In some embodiments, provided methods allow for insertion of a transgene into a defined target site. In some embodiments, provided methods use large transgenes (<5 kb) to allow gene editing techniques to insert into a defined target site in the genome of a host cell. In some embodiments, the homology arms disclosed herein can be, for example, 50 base pairs to 2000 base pairs. In some embodiments, targeted insertion of the transgene into the target site has excellent efficiency (higher on-target) and excellent specificity (lower off-target).

[0433] In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, has covalently closed ends. In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, is resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III), e.g., for more than 1 hour at 37°C.

[0434] In some embodiments, following electroporation and entry into the T cell, the nucleic acid molecule, e.g., naked DNA molecule, translocates to the nucleus where expression of the transgene can occur. In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, translocates to the nucleus where expression of the transgene located between two ITRs can occur. In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, is present in an amount sufficient to transfect a T cell or multiple T cells and to provide sufficient levels of gene transduction and expression without undue adverse effects.

[0435] In some embodiments, nucleic acid molecule, for example, naked DNA molecule, is introduced by electroporation.In some embodiments, electroporation, also called electric gene transfer, gene electroinjection, gene electrotransfer or electrically mediated gene therapy, causes temporary destabilization of cell membrane.In some embodiments, the DNA molecule in the medium surrounding the destabilized membrane can penetrate into the cytoplasm and nucleoplasm of T cell.

[0436] In some embodiments, the nucleic acid molecule comprises about 500-1000 base pairs of homology on either side of the transgene and / or target site. In some embodiments, the nucleic acid molecule comprises about 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology 5' of the target site or transgene, 3' of the target site or transgene, or both 5' and 3' of the target site or transgene. In some embodiments, the nucleic acid molecule comprises at least 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology 5' of the target site or transgene, 3' of the target site or transgene, or both 5' and 3' of the target site or transgene. In some embodiments, the nucleic acid molecule contains 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology at the 5' end of the target site or transgene, at the 3' end of the target site or transgene, or at both the 5' and 3' end of the target site or transgene. In some embodiments, the nucleic acid molecule contains one or more mutations, e.g., silent mutations, that prevent Cas, e.g., Cas9 or Cas12, from recognizing and cleaving the nucleic acid molecule. The nucleic acid molecule can contain, for example, at least 1, 2, 3, 4, 5, 10, 20, or 30 silent mutations compared to the corresponding sequence in the genome of the cell to be modified. In some embodiments, the nucleic acid molecule contains up to 2, 3, 4, 5, 10, 20, 30, or 50 silent mutations compared to the corresponding sequence in the genome of the cell to be modified. In some embodiments, the cDNA contains one or more mutations, e.g., silent mutations, that prevent a Cas, e.g., Cas9 or Cas12, from recognizing and cleaving the nucleic acid molecule. The nucleic acid molecule can contain, for example, at least 1, 2, 3, 4, 5, 10, 20, or 30 silent mutations compared to the corresponding sequence in the genome of the cell to be modified. In some embodiments, the nucleic acid molecule contains up to 2, 3, 4, 5, 10, 20, 30, or 50 silent mutations compared to the corresponding sequence in the genome of the cell to be modified.

[0437] In some embodiments, the nucleic acid molecule comprises about 150-1000 nucleotides of homology on either side of the transgene and / or target site. In some embodiments, the nucleic acid molecule comprises about 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5' of the target site or transgene, 3' of the target site or transgene, or both 5' and 3' of the target site or transgene. In some embodiments, the nucleic acid molecule comprises at least 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5' of the target site or transgene, 3' of the target site or transgene, or both 5' and 3' of the target site or transgene. In some embodiments, the nucleic acid molecule comprises up to 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5' of the target site or transgene, 3' of the target site or transgene, or both 5' and 3' of the target site or transgene.

[0438] In some embodiments, the nucleic acid molecule is introduced into the immune cell after introduction of one or more gene editing agents, e.g., Cas / gRNA RNPs, introduced, for example, via electroporation. In some embodiments, the nucleic acid molecule is introduced immediately after introduction of one or more gene editing agents capable of inducing gene disruption. In some embodiments, the nucleic acid molecule is introduced into the immune cell within at or about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 3 hours, 4 hours, 4 hours, 5 hours, 6 hours, 8 hours, 9 minutes, 9 minutes, 10 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 30 minutes, 40 minutes, 50 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 3 hours, 4 hours, 4 hours, 5 hours, 5 hours, 6 hours, 6 hours, 6 hours, 6 hours, 7 hours, 7 hours, 8 hours, 8 hours, 9 minutes, 9 minutes, 10 minutes, 10 minutes, 15 minutes, 15 minutes, 20 minutes, 20 minutes, 30 minutes, 30 minutes, 40 minutes, 40 minutes, 50 minutes, 50 minutes, 60 minutes, 60 minutes, 90 minutes, 90 minutes, 2 hours, 2 hours, 3 hours, 3 hours, 3 hours, 4 hours, 4 hours, 5 hours, 7 hours, 8 hours, 9 hours, 10 minutes, 10 minutes, 10 minutes, 15 minutes, 15 minutes, 20 minutes, 20 minutes, 30 minutes, 30 minutes, 30 minutes, 40 minutes, 40 minutes, 50 minutes, 50 minutes, 60 minutes, 60 minutes, 90 minutes, 90 minutes, 2 hours, 2 hours, 3 hours, 3 hours, 3 hours, 3 hours, 4 hours, 4 hours, 5 hours In some embodiments, the nucleic acid molecule is transfected at or about 15 minutes to 4 hours or about 4 hours after introduction of the one or more gene editing agents, e.g., from 15 minutes or about 15 minutes to 3 hours or about 3 hours, from 15 minutes or about 15 minutes to 2 hours or about 2 hours, from 15 minutes or about 15 minutes to 1 hour or about 1 hour, from 15 minutes or about 15 minutes to 30 minutes or about 30 minutes, from 30 minutes or about 30 minutes to 4 hours or about 4 hours, from 30 minutes or about 30 minutes to 3 hours or about 3 hours, The antibody is introduced into immune cells after 30 minutes or about 30 minutes, 2 hours or about 2 hours, 30 minutes or about 30 minutes, 1 hour or about 1 hour, 1 hour or about 1 hour, 4 hours or about 4 hours, 1 hour or about 1 hour, 3 hours or about 3 hours, 1 hour or about 1 hour, 2 hours or about 2 hours, 2 hours or about 2 hours, 4 hours or about 4 hours, 2 hours or about 2 hours, 3 hours or about 3 hours, or 3 hours or about 3 hours, 4 hours or about 4 hours.In some embodiments, the nucleic acid molecule is introduced into the immune cell 2 hours or about 2 hours after introduction of one or more gene editing agents, e.g., Cas / gRNA RNPs, introduced, e.g., via electroporation.

[0439] In some embodiments, the introduction of one or more gene editing substances is carried out simultaneously with the introduction of nucleic acid molecules.In some embodiments, the one or more gene editing substances and nucleic acid molecules are introduced simultaneously by electroporation.In some embodiments, the one or more gene editing substances and nucleic acid molecules are introduced into a cell culture medium that contains the one or more gene editing substances and nucleic acid molecules together.In some embodiments, the cell culture medium is present during electroporation.

[0440] In some embodiments, introducing the one or more gene editing agents occurs after addition of the stimulating reagent. In some embodiments, introducing the one or more gene editing agents occurs 6 hours to 36 hours, 6 hours to 30 hours, 6 hours to 24 hours, 6 hours to 18 hours, 6 hours to 12 hours, 12 hours to 36 hours, 12 hours to 30 hours, 12 hours to 24 hours, 12 hours to 18 hours, 18 hours to 36 hours, 18 hours to 30 hours, 18 hours to 24 hours, 24 hours to 36 hours, 24 hours to 30 hours, or if In some embodiments, introducing the one or more gene editing agents occurs 12 hours to 36 hours, or about 12 hours to 36 hours, or about 6 hours to 36 hours, or abou...

Claims

1. 1. A method for producing genetically engineered T cells, comprising: (a) applying a whole blood sample containing a plurality of T cells to a stationary phase within an interior cavity of a chromatography column, wherein the stationary phase comprises a selection substance that specifically binds to a selection marker expressed on the surface of the plurality of T cells, and wherein the selection substance specifically binds to the selection marker, thereby immobilizing the plurality of T cells on the stationary phase; (b) adding a T cell stimulatory reagent to the plurality of T cells immobilized on the stationary phase, the T cell stimulatory reagent comprising (i) a primary agent that specifically binds to a member of the TCR / CD3 complex, and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule; (c) incubating the plurality of T cells immobilized on the stationary phase in the presence of the T cell stimulatory reagent under conditions to stimulate T cells, the plurality of T cells; (d) collecting the plurality of T cells from the chromatography column that are no longer immobilized after the incubating step; and (e) introducing a nucleic acid molecule comprising a transgene encoding a recombinant protein by non-viral gene delivery under conditions for targeted integration of the transgene into a genetic target site in one or more of the collected T cells. Including, The method produces genetically engineered T cells that express the recombinant protein. The method.

2. 10. The method of claim 1, further comprising incubating the collected T cells prior to the introduction of the nucleic acid molecule.

3. 3. The method of claim 1 or claim 2, wherein the nucleic acid molecule is a DNA molecule, optionally a single-stranded or double-stranded DNA molecule.

4. 4. The method of any one of claims 1 to 3, wherein the targeted integration is by Programmable Addition via Site-specific Targeting Element (PASTE).

5. 5. The method of claim 4, wherein said PASTE comprises introducing one or more gene editing agents to edit said gene in one or more of said collected T cells.

6. The method of any one of claims 1 to 3, wherein the targeted integration is by homology-directed repair (HDR).

7. 7. The method of claim 6, wherein the HDR comprises introducing one or more gene editing agents to induce gene disruption of said gene in one or more of the collected T cells.

8. The method of any one of claims 1 to 7, wherein the introduction of the nucleic acid molecule and / or the one or more gene editing substances is by electroporation.

9. 9. The method of any one of claims 1 to 8, wherein said conditions for targeted integration comprise culturing the collected T cells under conditions for integration of the transgene into the target site.

10. 1. A method for producing genetically engineered T cells, comprising: (a) applying a whole blood sample containing a plurality of T cells to a stationary phase within an interior cavity of a chromatography column, wherein the stationary phase comprises a selection substance that specifically binds to a selection marker expressed on the surface of the plurality of T cells, and wherein the selection substance specifically binds to the selection marker, thereby immobilizing the plurality of T cells on the stationary phase; (b) adding a T cell stimulatory reagent to the plurality of T cells immobilized on the stationary phase, the T cell stimulatory reagent comprising (i) a primary agent that specifically binds to a member of the TCR / CD3 complex, and (ii) a secondary agent that specifically binds to a T cell costimulatory molecule; (c) incubating the plurality of T cells immobilized on the stationary phase in the presence of the T cell stimulatory reagent under conditions to stimulate T cells, the plurality of T cells; (d) collecting from the chromatography column the T cells, the plurality of T cells, that are no longer immobilized after the incubating step; (e) further incubating the collected T cells; (f) after said further incubation step, administering to said collected T cells: (i) a nucleic acid molecule comprising a transgene encoding a recombinant protein, wherein the nucleic acid molecule is a DNA molecule, optionally a single-stranded DNA molecule or a double-stranded DNA molecule, and wherein introduction of the nucleic acid molecule is by non-viral gene delivery; and (ii) one or more gene editing agents to induce gene disruption of a gene in a T cell, the T cell being the collected T cell. wherein the introduction of the nucleic acid molecule and / or the one or more gene editing agents is by electroporation; and (g) culturing the collected T cells under conditions for integrating the transgene into the target site of the gene in one or more of the collected T cells by homologous recombination repair (HDR). Including, The method produces genetically engineered T cells that express the recombinant protein. The method.

11. The method of any one of claims 1 to 10, wherein the nucleic acid molecule is a double-stranded DNA molecule, a naked DNA molecule, and / or a closed DNA molecule.

12. 12. The method of any one of claims 1 to 11, wherein the nucleic acid molecule is a closed linear double-stranded (CELiD) DNA molecule, a minicircle DNA molecule, a minimalistic immunological-defined gene expression (MIDGE) DNA molecule, a ministring DNA molecule, a dumbbell-shaped closed linear double-stranded DNA molecule, or a doggybone™ DNA molecule.

13. The method of any one of claims 1 to 12, wherein the T cell stimulating reagent is added to the cell culture medium.

14. T cells 10, which are the plurality of T cells that are immobilized on the stationary phase or are expected to be immobilized on the stationary phase, respectively, including the values ​​at both ends. 6 14. The method of any one of claims 1 to 13, wherein the T cell stimulatory reagent is added in an amount of at or about 0.1 μg to 20 μg, 0.4 μg to 8 μg, 0.8 μg to 4 μg, or 1 μg to 2 μg per cell.

15. 15. The method of any one of claims 1 to 14, wherein the binding capacity of the stationary phase is at or about 500 to 5 billion T cells expressing the selection marker, at or about 500 to 5 billion T cells expressing the selection marker, at or about 500 to 3 billion T cells expressing the selection marker, or at or about 1 to 2 billion T cells expressing the selection marker, inclusive.

16. 16. The method of any one of claims 1-15, wherein the T cell stimulating reagent is added in an amount of at or about 0.1 mg to 20 mg, 0.4 mg to 8 mg, 0.8 mg to 4 mg, or 1 mg to 3 mg, inclusive.

17. 17. The method of any one of claims 1 to 16, wherein the step of adding the T cell stimulating reagent occurs within or about 60 minutes, 30 minutes, or 15 minutes after adding the whole blood sample.

18. 18. The method of any one of claims 1 to 17, wherein the incubating step is carried out in cell culture medium.

19. 19. The method of any one of claims 1 to 18, wherein the incubating step is carried out at a temperature of 35°C to 39°C, or about 35°C to about 39°C.

20. 20. The method of any one of claims 1-19, wherein said incubating step is carried out for at or about 0.5 hours to 8 hours, 2 hours to 6 hours, or 3 hours to 5 hours, inclusive.

21. 21. The method of any one of claims 1 to 20, wherein said collecting step comprises adding a wash buffer to said stationary phase to collect T cells, said plurality of T cells.

22. 22. The method of claim 21, wherein the wash buffer is cell culture medium.

23. 23. The method of claim 21 or claim 22, wherein the wash buffer does not contain a competitor.

24. 24. The method of any one of claims 1-23, wherein the harvesting step is performed at or about 0.5 hours to 8 hours, 2 hours to 6 hours, or 3 hours to 5 hours, inclusive, after addition of the T cell stimulatory reagent.

25. The method of any one of claims 2 to 24, wherein the further incubation step is carried out in the presence of the T cell stimulating reagent.

26. 26. The method of any one of claims 2 to 25, wherein said further incubating step is carried out in cell culture medium.

27. 27. The method of any one of claims 2 to 26, wherein the further incubating step is carried out at a temperature of 35°C to 39°C, or about 35°C to about 39°C.

28. 28. The method of any one of claims 2 to 27, wherein the further incubation step is carried out for at or about 10 to 30 hours, 16 to 24 hours, or 18 to 22 hours, inclusive.

29. 29. The method of any one of claims 5 and 7-28, comprising removing the T cell stimulating reagent from the collected T cells prior to introduction of the one or more gene editing agents.

30. 30. The method of any one of claims 1 to 29, comprising removing the T cell stimulating reagent from the collected T cells prior to introducing the nucleic acid molecule.

31. 31. The method of claim 29 or claim 30, wherein the removing step occurs after the further incubating step.

32. 32. The method of any one of claims 29 to 31, wherein the removing step comprises washing the collected T cells.

33. the T cell stimulating reagent comprises an oligomer of streptavidin or a streptavidin mutein molecule; the primary agent comprises a first streptavidin binding partner that binds to an oligomeric streptavidin or streptavidin mutein molecule; the secondary agent comprises a second streptavidin binding partner that binds to the oligomeric streptavidin or streptavidin mutein molecule; the method comprising, prior to introduction of the one or more gene editing agents, disrupting binding between the first streptavidin binding partner and the second streptavidin binding partner and the streptavidin or streptavidin mutein molecule.

33. The method of any one of claims 5 and 7 to 32.

34. the T cell stimulating reagent comprises an oligomer of streptavidin or a streptavidin mutein molecule; the primary agent comprises a first streptavidin binding partner that binds to an oligomeric streptavidin or streptavidin mutein molecule; the secondary agent comprises a second streptavidin binding partner that binds to the oligomeric streptavidin or streptavidin mutein molecule; the method comprising, prior to introduction of the nucleic acid molecule, disrupting binding between the first streptavidin binding partner and the second streptavidin binding partner and the streptavidin or streptavidin mutein molecule; 34. The method of any one of claims 1 to 33.

35. 35. The method of claim 33 or claim 34, wherein said disrupting step occurs after said further incubating step.

36. 36. The method of any one of claims 33 to 35, wherein said interfering step is by adding to said collected T cells a competitor that competes for binding between said first streptavidin binding partner and said second streptavidin binding partner and said streptavidin or streptavidin mutein molecule.

37. The method of any one of claims 23 to 36, wherein the competitor is biotin.

38. 38. The method of any one of claims 5 and 7-37, wherein the introduction of the one or more gene editing agents is performed before or simultaneously with the introduction of the nucleic acid molecule.

39. 39. The method of any one of claims 5 and 7-38, wherein the introduction of the one or more gene editing agents occurs at or about 12 to 36 hours, 18 to 30 hours, or 22 to 26 hours, inclusive, after addition of the T cell stimulating reagent.

40. 40. The method of any one of claims 1 to 39, wherein the nucleic acid molecule is introduced in a cell culture medium containing the nucleic acid molecule.

41. 41. The method of any one of claims 1 to 40, wherein the step of introducing the nucleic acid molecule occurs at or about 12 to 36 hours, 18 to 30 hours, or 22 to 26 hours, inclusive, after addition of the T cell stimulatory reagent.

42. The method of any one of claims 9 to 41, wherein the culturing is carried out in the presence of the nucleic acid molecule.

43. The method of any one of claims 9 to 42, wherein the culturing is carried out in a cell culture medium.

44. 44. The method of any one of claims 9 to 43, wherein the culturing is carried out at a temperature of 35°C to 39°C, or about 35°C to about 39°C.

45. 45. The method of any one of claims 9 to 44, wherein the culturing is for or about 12 to 36 hours, 18 to 30 hours, or 22 to 26 hours, inclusive.

46. 46. ​​The method of any one of claims 13 to 45, wherein the cell culture medium is a basal medium.

47. 47. The method of any one of claims 13 to 46, wherein the cell culture medium is a serum-free medium.

48. 48. The method of any one of claims 13 to 47, wherein the cell culture medium is cytokine-free or contains recombinant IL-2, IL-7, and IL-15.

49. 49. The method of any one of claims 1 to 48, comprising harvesting said engineered T cells that express said recombinant protein.

50. 50. The method of claim 49, wherein said collecting step occurs at or about 36 to 60 hours, 42 to 54 hours, or 46 to 50 hours, inclusive, after addition of said whole blood sample.

51. 51. The method of claim 49 or claim 50, wherein the harvesting step occurs at or about 36 to 60 hours, 42 to 54 hours, or 46 to 50 hours, inclusive, after addition of the T cell stimulatory reagent.

52. 52. The method of any one of claims 49-51, wherein said harvesting occurs at or about 12 to 36 hours, 18 to 30 hours, or 22 to 26 hours, inclusive, after introduction of the one or more gene editing agents.

53. 53. The method of any one of claims 49-52, wherein said harvesting step is performed at or about 12 to 36 hours, 18 to 30 hours, or 22 to 26 hours, inclusive, after introduction of said nucleic acid molecule.

54. 54. The method of any one of claims 49-53, comprising formulating the harvested engineered T cells for cryopreservation or for administration to a subject.

55. 55. The method of claim 54, wherein the harvested genetically engineered T cells are formulated in the presence of a cryoprotectant or a pharmaceutically acceptable excipient.

56. 56. The method of any one of claims 1 to 55, wherein said plurality of T cells are primary T cells from a human subject.

57. 57. The method of any one of claims 1 to 56, wherein the selection marker is selected from the group consisting of CD3, CD4, CD8, CD45RA, CD27, CD28, and CCR7.

58. 58. The method of any one of claims 1 to 57, wherein the selection marker is CD3, CD4, or CD8.

59. The method of any one of claims 1 to 58, wherein the selection marker is CD3.

60. 60. The method of any one of claims 1 to 59, wherein said selection agent comprises an antibody or antibody fragment that specifically binds to said selection marker.

61. 61. The method of claim 60, wherein the antibody or antibody fragment that is the selection agent is a monovalent antibody fragment.

62. 62. The method of claim 60 or claim 61, wherein the antibody or antibody fragment that is the selection agent is a Fab fragment.

63. the T cell stimulating reagent comprises an oligomer of streptavidin or a streptavidin mutein molecule; the primary agent comprises a first streptavidin binding partner that binds to an oligomeric streptavidin or streptavidin mutein molecule; the secondary agent comprises a second streptavidin binding partner that binds to an oligomeric streptavidin or streptavidin mutein molecule; 63. The method of any one of claims 1 to 32 and 37 to 62.

64. 64. The method of any one of claims 33-63, wherein said T cell stimulating reagent consists of or consists essentially of said oligomer, a primary agent, and a secondary agent.

65. 65. The method of any one of claims 33-64, wherein said oligomer comprises at or about 500-5000 tetramers, at or about 1000-4000 tetramers, or at or about 2000-3000 tetramers, inclusive, of said streptavidin or streptavidin mutein molecules.

66. The method of any one of claims 33 to 65, wherein said oligomer is an oligomer of said streptavidin mutein molecule.

67. 67. The method of any one of claims 33 to 66, wherein the streptavidin mutein molecule comprises the amino acid sequence IGAR (SEQ ID NO: 133) or VTAR (SEQ ID NO: 134) at sequence positions corresponding to positions 44 to 47 of the sequence of amino acids shown in SEQ ID NO:

1.

68. The method of any one of claims 33 to 67, wherein the streptavidin mutein molecule starts at the N-terminus in the region of amino acid positions 10 to 16 of SEQ ID NO: 1 and ends at the C-terminus in the region of amino acid positions 133 to 142 of SEQ ID NO:

1.

69. 69. The method of any one of claims 33 to 68, wherein said streptavidin mutein molecule comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 27, 28, 104, 105, and 136.

70. 70. The method of any one of claims 33 to 69, wherein said streptavidin mutein molecule comprises the amino acid sequence shown in SEQ ID NO:

6.

71. the first streptavidin binding partner is at the C-terminus of the primary agent; and / or the second streptavidin binding partner is at the C-terminus of the secondary agent; 71. The method of any one of claims 33 to 70.

72. 72. The method of any one of claims 33 to 71, wherein the first streptavidin-binding partner and / or the second streptavidin-binding partner is a streptavidin-binding peptide.

73. 73. The method of claim 72, wherein the streptavidin-binding peptide that is the first streptavidin-binding partner and / or the second streptavidin-binding partner comprises an amino acid sequence set forth in any one of SEQ ID NOs: 7, 8, and 15-19.

74. 74. The method of claim 72 or claim 73, wherein the streptavidin-binding peptide that is the first streptavidin-binding partner and / or the second streptavidin-binding partner comprises the amino acid sequence shown in SEQ ID NO:

16.

75. The method of any one of claims 1 to 74, wherein the member of the TCR / CD3 complex is CD3.

76. 76. The method of any one of claims 1 to 75, wherein the T cell costimulatory molecule is CD28, CD90 (Thy-1), CD95 (Apo- / Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM.

77. The method of any one of claims 1 to 76, wherein the T cell costimulatory molecule is CD28.

78. the primary agent comprises an antibody or antibody fragment that specifically binds to a member of the TCR / CD3 complex; and / or the secondary agent comprises an antibody or antibody fragment that specifically binds to the T cell costimulator.

78. The method of any one of claims 1 to 77.

79. the antibody or antibody fragment that is the primary agent comprises a heavy chain and the first streptavidin binding partner is fused to the C-terminus of the heavy chain of the primary agent; and / or the antibody or antibody fragment that is the secondary agent comprises a heavy chain, and the second streptavidin binding partner is fused to the C-terminus of the heavy chain of the secondary agent; 79. The method of claim 78.

80. 80. The method of claim 78 or claim 79, wherein the antibody or antibody fragment that is the primary agent and / or the secondary agent is a monovalent antibody fragment.

81. 81. The method of any one of claims 78-80, wherein the antibody or antibody fragment that is the primary and / or secondary agent is a Fab fragment.

82. 82. The method of any one of claims 1-81, wherein the primary agent comprises an anti-CD3 antibody or antibody fragment and the secondary agent comprises an anti-CD28 antibody or antibody fragment.

83. 83. The method of any one of claims 1-82, wherein the primary agent comprises an anti-CD3 Fab fragment and the secondary agent comprises an anti-CD28 Fab fragment.

84. 84. The method of any one of claims 1 to 83, wherein the gene is the T-cell receptor alpha constant (TRAC) gene.

85. 85. The method of any one of claims 1 to 84, wherein the target site is present within the sequence set forth in SEQ ID NO:

250.

86. 86. The method of any one of claims 1 to 3 and 6 to 85, wherein the nucleic acid molecule comprises a 5' homology arm and a 3' homology arm comprising sequences homologous to nucleic acid sequences surrounding the target site, and the nucleic acid molecule comprises the structure [5' homology arm]-[transgene]-[3' homology arm].

87. 87. The method of claim 86, wherein the 5' homology arm and the 3' homology arm comprise sequences homologous to sequences of the TRAC gene surrounding the target site.

88. 88. The method of claim 86 or claim 87, wherein the 5' homology arm comprises a sequence comprising at least or at least about 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in SEQ ID NO:

248.

89. 89. The method of any one of claims 86-88, wherein the 5' homology arm comprises at least or at least about 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of the sequence set forth in SEQ ID NO:

248.

90. 90. The method of any one of claims 86 to 89, wherein the 5' homology arm comprises the sequence shown in SEQ ID NO:

248.

91. 91. The method of any one of claims 86-90, wherein the 3' homology arm comprises a sequence comprising at least or at least about 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in SEQ ID NO:

249.

92. 92. The method of any one of claims 86-91, wherein the 3' homology arm comprises at least or at least about 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of the sequence set forth in SEQ ID NO:

249.

93. 93. The method of any one of claims 86 to 92, wherein the 3' homology arm comprises the sequence shown in SEQ ID NO:

249.

94. 94. The method of any one of claims 1 to 93, wherein transcription of the integrated transgene is under the control of a promoter comprised in said nucleic acid molecule.

95. 95. The method of claim 94, wherein the promoter is a human elongation factor 1 alpha (EF1α) promoter.

96. 96. The method of claim 94 or claim 95, wherein the promoter comprises the sequence shown in SEQ ID NO:

247.

97. 97. The method of any one of claims 1 to 96, wherein the recombinant protein is a recombinant receptor.

98. 98. The method of claim 97, wherein the recombinant receptor is a T cell receptor or a chimeric antigen receptor.

99. 99. The method of any one of claims 5 and 7-98, wherein the one or more gene-editing substances comprise (i) a gene-editing nuclease or nuclease combination, or (ii) a nucleic acid molecule comprising one or more sequences encoding the gene-editing nuclease or nuclease combination.

100. 100. The method of any one of claims 5 and 7-99, wherein the one or more gene editing agents comprise a gene editing nuclease or nuclease combination.

101. 101. The method of Claim 99 or Claim 100, wherein the gene-editing nuclease or nuclease combination specifically recognizes a nucleic acid sequence near or including the target site.

102. The method of any one of claims 99 to 101, wherein the gene-editing nuclease or nuclease combination specifically recognizes a nucleic acid sequence comprising the target site.

103. 103. The method of claim 101 or claim 102, wherein the nucleic acid sequence comprising the target site comprises the sequence set forth in SEQ ID NO:

250.

104. The method of any one of claims 99-103, wherein the gene-editing nuclease or nuclease combination is a zinc finger nuclease, a transcription activator-like effector nuclease, or a CRISPR-Cas combination.

105. The method of any one of claims 99 to 104, wherein the gene-editing nuclease or nuclease combination is a CRISPR-Cas combination.

106. 106. The method of claim 104 or claim 105, wherein the CRISPR-Cas combination comprises a CRISPR-Cas nickase, a reverse transcriptase, and a serine integrase.

107. 107. The method of claim 105 or claim 106, wherein the CRISPR-Cas combination comprises a guide RNA comprising a targeting sequence complementary to a nucleic acid sequence comprising the target site.

108. 108. The method of claim 105 or claim 107, wherein the CRISPR-Cas combination is a ribonucleoprotein complex comprising the guide RNA and a Cas protein.

109. 109. The method of claim 108, wherein the Cas protein is a S. pyogenes Cas protein.

110. The method of any one of claims 104 to 109, wherein the CRISPR-Cas combination is a CRISPR-Cas9 combination or a CRISPR-Cas12 combination.

111. The method of any one of claims 107 to 110, wherein the targeting sequence comprises a sequence set forth in any one of SEQ ID NOs: 144 to 175.

112. The method of any one of claims 107 to 111, wherein the targeting sequence comprises the sequence set forth in SEQ ID NO:

148.

113. 113. A genetically engineered T cell produced by the method of any one of claims 1 to 112, wherein the T cell expresses said recombinant protein.

114. 114. The genetically engineered T cell of claim 113, wherein said transgene is integrated into said target site of said gene in said genetically engineered T cell.

115. 115. The genetically engineered T cell of claim 114, wherein the gene is the T cell receptor alpha constant (TRAC) gene.

116. 116. The genetically engineered T cell of claim 114 or claim 115, wherein the target site is present within the sequence set forth in SEQ ID NO:

250.

117. 117. The genetically engineered T cell of any one of claims 113-116, wherein the recombinant protein is a recombinant receptor.

118. 118. The genetically engineered T cell of claim 117, wherein the recombinant receptor is a T cell receptor or a chimeric antigen receptor.

119. 119. A population of T cells comprising a plurality of the genetically engineered T cells of any one of claims 113-118.

120. 120. The population of claim 119, wherein said plurality of genetically engineered T cells is at least 10%, 15%, or 20% of said population of T cells.

121. 121. The population of claim 119 or claim 120, wherein the gene is disrupted in at least 85%, 90%, or 95% of the T cells in said population of T cells.

122. 122. The population of claim 121, wherein the gene is the T cell receptor alpha constant (TRAC) gene.

123. 123. A pharmaceutical composition comprising the population of T cells of any one of claims 119-122 and a pharmaceutically acceptable excipient.

124. 124. A method of treatment comprising administering to a subject having a disease or condition the pharmaceutical composition of claim 123.

125. 125. The method of claim 124, wherein said recombinant protein is a recombinant receptor that targets an antigen expressed on a target cell associated with said disease or condition.

126. 123. A method of cytolytic killing of target cells comprising contacting the target cells with the population of any one of claims 119-122.

127. 124. A method of cytolytic killing of a target cell comprising contacting the target cell with the pharmaceutical composition of claim 123.

128. 128. The method of claim 126 or claim 127, wherein said contacting step is performed ex vivo.

129. 128. The method of claim 126 or claim 127, wherein said contacting step is performed in vivo.

130. 130. The method of claim 129, wherein said contacting is by administering said pharmaceutical composition to a subject having a disease or condition.

131. 131. The method of claim 130, wherein said target cell is associated with said disease or condition and said recombinant protein is a recombinant receptor that targets an antigen expressed on said target cell.

132. 132. The method of claim 125 or claim 131, wherein the recombinant receptor is a T cell receptor or a chimeric antigen receptor.

133. 124. The pharmaceutical composition of claim 123 for use in treating a disease or disorder in a subject.

134. 134. The pharmaceutical composition of claim 133, wherein said recombinant protein is a recombinant receptor that targets an antigen expressed on a cell associated with said disease or condition.

135. 135. The pharmaceutical composition of claim 134, wherein the recombinant receptor is a T cell receptor or a chimeric antigen receptor.

136. 124. Use of the pharmaceutical composition of claim 123 for treating a disease or disorder in a subject.

137. 124. Use of the pharmaceutical composition of claim 123 for the manufacture of a medicament for treating a disease or disorder in a subject.

138. 138. The use of claim 136 or claim 137, wherein the recombinant protein is a recombinant receptor that targets an antigen expressed on a cell associated with the disease or condition.

139. 139. The use of claim 138, wherein the recombinant receptor is a T cell receptor or a chimeric antigen receptor.

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