Methods for stimulating and transducing cells

Abstract

Provided herein is a method for selecting, stimulating and transducing cells in a sample using column chromatography, and collecting and / or eluting cells from the column without using additional steps or reagents to facilitate cell detachment from the column.In some embodiments, the method provided herein ultimately reduces the time required to generate a population of selected, stimulated and transduced cells useful for cell therapy compared to existing methods.Also provided are articles of manufacture and devices thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 185,240, filed May 6, 2021, which is incorporated by reference in its entirety for all purposes.

[0002] Incorporation by reference to sequence listing This application is submitted with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042025540SEQLIST.TXT, created on May 3, 2022, and is 92.5 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] The present disclosure provides a method for selecting, stimulating, and manipulating cells in a sample using column chromatography, and collecting and / or eluting the cells from the column without using additional steps or reagents to facilitate cell detachment from the column. In some embodiments, the methods provided herein ultimately reduce the time required to generate a population of selected, stimulated, and manipulated cells useful for cell therapy compared to existing methods. Also provided are articles of manufacture and devices thereof. [Background technology]

[0004] A variety of cell therapy methods are available for treating diseases and conditions. Among cell therapy methods, there are methods involving immune cells, such as T cells (e.g., CD4+ and CD8+ T cells), which can be genetically engineered with recombinant receptors, such as chimeric antigen receptors. For example, there is a need for improved methods for generating cell populations suitable for use in cell therapy. Methods, articles of manufacture, and devices that meet this need are provided. Summary of the Invention [Means for solving the problem]

[0005] In some embodiments, provided herein is a method for on-column transduction of T cells, the method comprising: (a) simultaneously contacting a plurality of T cells with a T cell stimulating reagent and a viral vector comprising a nucleic acid sequence encoding a recombinant protein, wherein the plurality of T cells is immobilized on a stationary phase contained within the interior cavity of a chromatography column; (b) incubating the plurality of T cells in the presence of the T cell stimulating reagent and the viral vector; and (c) collecting the plurality of T cells from the chromatography column within 24 hours of contacting, thereby producing a composition comprising T cells transduced with the recombinant protein.

[0006] In some of any of the embodiments, the stationary phase comprises a selection agent that specifically binds to a selection marker expressed on the surface of the plurality of T cells, and the specific binding of the selection agent to the selection marker achieves immobilization of the plurality of T cells on the stationary phase.

[0007] Also provided herein in some embodiments is a method for on-column transduction of T cells, the method comprising: (a) adding a sample comprising a plurality of T cells to an internal cavity of a chromatography column, the internal cavity comprising a stationary phase comprising a selection agent that specifically binds to a selection marker expressed on the surface of the plurality of T cells, thereby immobilizing the plurality of T cells on the stationary phase; (b) simultaneously contacting the plurality of T cells immobilized on the chromatography column with a T cell stimulating reagent and a viral vector comprising a nucleic acid sequence encoding the recombinant protein; (c) incubating the plurality of T cells in the presence of the T cell stimulating reagent and the viral vector; and (d) collecting the plurality of T cells from the chromatography column within 24 hours of contacting, thereby producing a composition comprising T cells transduced with the recombinant protein.

[0008] In some of any of the embodiments, the stimulatory reagent and the viral vector are contacted with the plurality of T cells as separate compositions. In some of any of the embodiments, the stimulatory reagent and the viral vector are contacted with the plurality of T cells as a mixture in the same composition.

[0009] In some embodiments, provided herein are methods for on-column transduction of T cells, the methods including: (a) preparing a mixture comprising a T cell stimulation reagent and a viral vector preparation; (b) contacting a plurality of T cells with the mixture in a chromatography column, wherein the plurality of T cells is immobilized on a stationary phase contained within the interior cavity of the chromatography column; (c) incubating the plurality of T cells in the presence of the T cell stimulation reagent and the viral vector; and (d) collecting the plurality of T cells from the chromatography column within 24 hours of contacting, thereby producing a composition comprising T cells transduced with the recombinant protein.

[0010] In some of any of the embodiments, the stationary phase comprises a selection agent that specifically binds to a selection marker expressed on the surface of the plurality of T cells, and the specific binding of the selection agent to the selection marker achieves immobilization of the plurality of T cells on the stationary phase.

[0011] Also provided herein in some embodiments is a method for on-column transduction of T cells, the method comprising: (a) adding a sample comprising a plurality of T cells to an internal cavity of a chromatography column, the internal cavity comprising a stationary phase comprising a selection agent that specifically binds to a selection marker expressed on the surface of the plurality of T cells, thereby immobilizing the plurality of T cells on the stationary phase; (b) contacting the plurality of T cells with a T cell stimulatory agent, e.g., a viral vector comprising a stimulatory reagent and a nucleic acid sequence encoding a recombinant protein, by adding a mixture comprising a T cell stimulatory reagent and a viral vector to the internal cavity of the chromatography column; (c) incubating the plurality of T cells in the presence of the T cell stimulatory reagent and the viral vector; and (d) collecting the plurality of T cells from the chromatography column within 24 hours of adding the mixture, thereby producing a composition comprising T cells transduced with the recombinant protein.

[0012] In some of any of the embodiments, the method includes mixing the stimulating reagent and the viral vector to form a mixture comprising the stimulating reagent and the recombinant nucleic acid molecule, eg, the viral vector.

[0013] In some of any of the embodiments, the contacting is initiated within at or about 10 minutes, within at or about 20 minutes, within at or about 30 minutes, within at or about 45 minutes, within at or about 60 minutes, within at or about 90 minutes, or within at or about 120 minutes after adding the sample to the internal cavity. In some of any of the embodiments, the contacting is initiated within at or about 60 minutes after adding the sample to the internal cavity.

[0014] In some of any of the embodiments, at least a portion of the incubating is carried out at a temperature between about 35° C. and about 39° C. In some of any of the embodiments, at least a portion of the incubating is carried out at a temperature of 37° C. or about 37° C.

[0015] In some optional embodiments, the temperature of the stationary phase is regulated by one or more heating elements configured to provide heat to the stationary phase.

[0016] In some embodiments, the T cell stimulatory agent, e.g., the stimulatory reagent and the viral vector, are contacted with the plurality of T cells in serum-free medium, and the incubation, e.g., incubating, is carried out in serum-free medium. In some embodiments, the serum-free medium comprises one or more recombinant T cell stimulatory cytokines.

[0017] In some of any of the embodiments, the T cell stimulatory agents, eg, stimulatory reagents and viral vectors, are contacted with a plurality of T cells in a medium that includes one or more recombinant T cell stimulatory cytokines.

[0018] In some of any of the embodiments, the mixture is a medium that includes one or more recombinant T cell stimulatory cytokines.

[0019] In some of any of the embodiments, the medium is a serum-free medium.

[0020] In some of any of the embodiments, the one or more recombinant cytokines are selected from IL-2, IL-15, and IL-7. In some of any of the embodiments, the one or more recombinant cytokines are IL-2, IL-15, and IL-7.

[0021] In some of the embodiments, the T cell stimulatory agents, e.g., stimulatory reagents, are each 10 6 In some embodiments, the T cell stimulator, e.g., stimulatory reagent, is contacted with the plurality of T cells in an amount of between or about 0.1 μg and 20 μg, inclusive, between or about 0.1 μg and 20 μg, between or about 0.4 μg and 8 μg, inclusive, or between or about 0.4 μg and 8 μg, inclusive, per plurality of T cells immobilized on a stationary phase of 10 cells or per estimated plurality of T cells immobilized on a stationary phase. 6 The T cells are contacted in an amount of between or about 1 μg and 2 μg, inclusive, per T cell immobilized on a stationary phase or per estimated T cell immobilized on a stationary phase.

[0022] In some optional embodiments, the mixtures each contain 10 6 The mixture comprises an amount of T cell stimulatory agent, e.g., stimulatory reagent, of between or about 0.1 μg and 20 μg, inclusive, between or about 0.1 μg and 20 μg, between or about 0.4 μg and 8 μg, inclusive, or between or about 0.4 μg and 8 μg, inclusive, or between or about 0.8 μg and 4 μg, inclusive, per plurality of T cells immobilized on a stationary phase of 10 cells or per estimated plurality of T cells immobilized on a stationary phase. 6The amount of T cell stimulatory agent, e.g., stimulatory reagent, may be between or about 1 μg and 2 μg, inclusive, per plurality of T cells immobilized on a stationary phase of cells or per estimated plurality of T cells immobilized on a stationary phase.

[0023] In some of the optional embodiments, the viral vector is administered to a plurality of T cells and 10 6 For a plurality of T cells immobilized on a stationary phase of 10 cells or an estimated plurality of T cells immobilized on a stationary phase, a volume of each viral vector preparation is between or about 0.1 μL and 100 μL, inclusive, between or about 0.1 μL and 100 μL, between or about 0.5 μL and 50 μL, inclusive, or between or about 1 μL and 25 μL, inclusive. In some of any of the embodiments, the viral vector is contacted with a plurality of T cells, inclusive, for a volume of 10 6 For a plurality of T cells immobilized on a stationary phase of 10 cells or a predicted plurality of T cells immobilized on a stationary phase, a volume of each viral vector preparation between or about 2 μL and 10 μL, inclusive, is used, as appropriate. 6 In some embodiments, the viral vector is contacted with the plurality of T cells at or about a volume of 6 μL per 10 T cells immobilized on a stationary phase or an estimated plurality of T cells immobilized on a stationary phase. 6 A volume of 6 μL or about 6 μL is contacted per T cell immobilized on the stationary phase or per estimated T cell immobilized on the stationary phase.

[0024] In some of the optional embodiments, the mixture comprises 10 6For each T cell population immobilized on a stationary phase of cells or a predicted number of T cells immobilized on a stationary phase, a volume of each viral vector preparation is comprised between or about 0.1 μL and 100 μL, inclusive, between or about 0.1 μL and 100 μL, between or about 0.5 μL and 50 μL, inclusive, or between or about 1 μL and 25 μL, inclusive. In some embodiments, the mixture comprises 10 6 A volume of viral vector preparation between or about 2 μL and 10 μL, inclusive, per T cell population immobilized on a stationary phase of 10 cells or per estimated T cell population immobilized on a stationary phase, optionally 10 6 In some embodiments, the mixture comprises a volume of 6 μL or about 6 μL of viral vector preparation per 10 T cells immobilized on a stationary phase or per estimated number of T cells immobilized on a stationary phase. 6 The viral vector preparation comprises a volume of 6 μL or about 6 μL per plurality of T cells immobilized on a stationary phase of cells or per estimated plurality of T cells immobilized on a stationary phase.

[0025] In some of the optional embodiments, the viral vector preparation comprises 1 x 10 6 TU / mL to 1 x 10 9 Between TU / mL or approximately 1 x 10 6 TU / mL to approximately 1 x 10 9 Between TU / mL and 1 x 10 6 TU / mL to 1 x 10 8 Between TU / mL or approximately 1 x 10 6 TU / mL to approximately 1 x 10 8 Between TU / mL and 1 x 10 6 TU / mL to 1 x 10 7 Between TU / mL or approximately 1 x 10 6 TU / mL to approximately 1 x 10 7 Between TU / mL and 1 x 10 7 TU / mL to 1 x 10 9 Between TU / mL or approximately 1 x 10 7TU / mL to approximately 1 x 10 9 Between TU / mL and 1 x 10 7 TU / mL to 1 x 10 8 Between TU / mL or approximately 1 x 10 7 TU / mL to approximately 1 x 10 8 Between TU / mL or 1 x 10 8 TU / mL to 1 x 10 9 Between TU / mL or approximately 1 x 10 8 TU / mL to approximately 1 x 10 9 It has a titer between 100 and 1000 TU / mL.

[0026] In some embodiments, the collecting is performed within 22, 20, 18, 16, 16, 14, 12, 10, 9, 8, 7, 6, or 5 hours after the contacting. In some embodiments, the collecting is performed within 2 to 24 hours, 2 to 22 hours, 2 to 20 hours, 2 to 18 hours, 2 to 16 hours, 2 to 14 hours, 2 to 12 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, 3 to 6 hours, 3 to 5 hours, 4 to 6 hours, or 4 to 5 hours, inclusive, of the contacting. The collecting is performed after between 2 hours and about 24 hours, about 2 hours and about 22 hours, about 2 hours and about 20 hours, about 2 hours and about 18 hours, about 2 hours and about 16 hours, about 2 hours and about 14 hours, about 2 hours and about 12 hours, about 2 hours and about 10 hours, about 2 hours and about 9 hours, about 2 hours and about 8 hours, about 2 hours and about 7 hours, about 2 hours and about 6 hours, about 2 hours and about 5 hours, about 3 hours and about 6 hours, about 3 hours and about 5 hours, about 4 hours and about 6 hours, or about 4 hours and about 5 hours. In some of any of the embodiments, the collecting is performed 4.5 hours or about 4.5 hours after the contacting.

[0027] In some of any of the embodiments, incubating in the presence of a T cell stimulatory reagent releases one or more of the plurality of immobilized T cells from the stationary phase.

[0028] In some embodiments, the collecting step includes adding a wash buffer to the column to collect one or more cells released from immobilization on the stationary phase during incubation. In some embodiments, the wash buffer is a cell culture medium. In some embodiments, the cell culture medium includes one or more recombinant T cell stimulatory cytokines, which may be selected from IL-2, IL-15, and IL-7. In some embodiments, the cell culture medium is a serum-free medium. In some embodiments, the cell culture medium does not include a competitor or free binder for eluting the T cells from the stationary phase.

[0029] In some of any of the embodiments, the cell culture medium comprises one or more recombinant T cell stimulatory cytokines selected from IL-2, IL-15, and IL-7. In some of any of the embodiments, the cell culture medium comprises one or more recombinant T cell stimulatory cytokines that are IL-2, IL-15, and IL-7.

[0030] In some optional embodiments, the collecting does not include adding a medium to the stationary phase that includes a competitor or free binder to elute the plurality of T cells from the stationary phase.

[0031] In some of any of the embodiments, the compositions comprising T cells transduced with a recombinant protein do not include a competitor or a free binder.

[0032] In some embodiments, the competitor or free binder is or comprises biotin or a biotin analog. In some embodiments, the competitor or free binder is or comprises D-biotin. In some embodiments, the biotin analog is desthiobiotin.

[0033] In some of any of the embodiments, the method further comprises incubating the composition comprising the transduced T cells in a solution. In some of any of the embodiments, the further incubating is carried out at a temperature of at or about 37° C.±2° C. In some of any of the embodiments, the further incubating is carried out for 14 days or less, 12 days or less, 10 days or less, 8 days or less, 6 days or less, or 5 days or less.

[0034] In some embodiments, the further incubation is carried out under conditions to induce proliferation or expansion of the transduced T cells, and the incubation, e.g., the further incubation, may be carried out in a cell culture medium comprising one or more recombinant T cell stimulating cytokines, which may be selected from IL-2, IL-15, and IL-7. In some embodiments, the further incubation is carried out in a cell culture medium comprising one or more recombinant T cell stimulating cytokines. In some embodiments, the recombinant T cell stimulating cytokines are selected from IL-2, IL-15, and IL-7. In some embodiments, the recombinant T cell stimulating cytokines are IL-2, IL-15, and IL-7.

[0035] In some embodiments, the further incubation is carried out under conditions where there is minimal or no further expansion or proliferation of the T cells, e.g., transduced T cells. In some embodiments, the further incubation is carried out in basal medium that does not contain any recombinant T cell stimulatory cytokines.

[0036] In some embodiments, the T cell stimulatory reagent comprises one or more stimulatory agents capable of delivering a stimulatory signal to the T cell. In some embodiments, at least one of the one or more stimulatory agents can deliver the stimulatory signal via the TCR / CD3 complex of the T cell, a CD3-containing complex of the T cell, and / or an ITAM-containing molecule of the T cell. In some embodiments, at least one of the one or more stimulatory agents can deliver a primary activation signal to the T cell.

[0037] In some embodiments, the at least one stimulating agent is a first stimulating agent, and the stimulating reagent further includes a second stimulating agent capable of enhancing the stimulatory signal delivered by the first stimulating agent. In some embodiments, the second stimulating agent binds to a costimulatory molecule of T cells. In some embodiments, the costimulatory molecule is selected from among CD28, CD90 (Thy-1), CD95 (Apo- / Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, and HVEM. In some embodiments, the second stimulating agent binds to CD28.

[0038] In some of any of the embodiments, the first stimulating agent specifically binds CD3 and the second stimulating agent specifically binds CD28.

[0039] In some of any of the embodiments, one or more stimulating agents independently comprise a monovalent antibody fragment.

[0040] In some of any of the embodiments, the first stimulating agent comprises a monovalent antibody fragment that binds to CD3 and the second stimulating agent comprises a monovalent antibody fragment that binds to CD28.

[0041] In some of any of the embodiments, the monovalent antibody fragment is selected from the group consisting of a Fab fragment, an Fv fragment, and a single chain Fv fragment (scFv).

[0042] In some of any of the embodiments, the first stimulating agent is an anti-CD3 Fab and the second stimulating agent is an anti-CD28 Fab.

[0043] In some of any of the embodiments, the T cell stimulating reagent comprises a first stimulating agent that is an anti-CD3 Fab and a second stimulating agent that is an anti-CD28 Fab.

[0044] In some of any of the embodiments, one or more stimulating agents, optionally the first stimulating agent and the second stimulating agent, are immobilized on a solid surface, optionally a bead. In some of any of the embodiments, the solid surface is a bead.

[0045] In some embodiments, one or more stimulating agents, optionally a first stimulating agent and a second stimulating agent, are reversibly bound to, e.g., bound to, a soluble oligomeric reagent. In some embodiments, the soluble oligomeric reagent comprises a plurality of streptavidin or streptavidin mutein tetramers. In some embodiments, the soluble oligomeric reagent is an oligomer comprising a plurality of streptavidin or streptavidin mutein tetramers.

[0046] In some of any of the embodiments, the soluble oligomeric reagent comprises a plurality of streptavidin mutein tetramers.In some of any of the embodiments, the soluble oligomeric reagent comprises an oligomer comprising a plurality of streptavidin mutein tetramers.

[0047] In some optional embodiments, the size of the oligomeric particle reagent, e.g., oligomeric reagent, is: i) greater than 50 nm in radius; ii) at least 5×10 6g / mol and / or (iii) at least 100 streptavidin or streptavidin mutein tetramers. In some of any of the embodiments, the soluble oligomeric particle reagent, e.g., oligomeric reagent, comprises, on average, between or about 1000 and 3000 streptavidin or streptavidin mutein tetramers, inclusive, optionally between or about 2000 and 3000 streptavidin or streptavidin mutein tetramers, inclusive, optionally 2500 or about 2500 streptavidin mutein tetramers. In some optional embodiments, the soluble oligomeric particle reagent, eg, the oligomeric reagent, comprises between or about 2000 and 3000 streptavidin or streptavidin mutein tetramers, inclusive.

[0048] In some of the embodiments, the soluble oligomeric particle reagent, e.g., the oligomeric reagent, comprises between 1000 and 3000 or between about 1000 and about 3000 streptavidin mutein tetramers, inclusive. In some of the embodiments, the soluble oligomeric particle reagent, e.g., the oligomeric reagent, comprises between 2000 and 3000 or between about 2000 and about 3000 streptavidin mutein tetramers, inclusive. In some of the embodiments, the soluble oligomeric particle reagent, e.g., the oligomeric reagent, comprises at or about 2500 streptavidin mutein tetramers.

[0049] In some of the embodiments, the molecules of the soluble oligomeric particle reagent are cross-linked to one another. In some of the embodiments, the molecules of the soluble oligomeric particle reagent are cross-linked to one another by polysaccharides. In some of the embodiments, the molecules of the soluble oligomeric particle reagent are cross-linked to one another by bifunctional linkers. In some of the embodiments, the molecules of the soluble oligomeric particle reagent are cross-linked to one another by heterobifunctional linkers. In some of the embodiments, the molecules of the soluble oligomeric particle reagent are cross-linked to one another by amine-to-thiol cross-links.

[0050] In some embodiments, each of the one or more stimulating agents, optionally both the first stimulating agent and the second stimulating agent, comprises a binding partner that reversibly binds to the soluble oligomeric reagent. In some embodiments, the binding partner is a streptavidin-binding peptide. In some of any of the embodiments, the streptavidin-binding peptide is selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 15), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), S

[0023] In some embodiments, the streptavidin-binding peptide has the sequence SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16). In some embodiments, the sequence of the streptavidin-binding peptide is set forth in any of SEQ ID NOs: 7, 8, and 15-19. In some of any of the embodiments, the streptavidin binding peptide is SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16).

[0051] In some embodiments, the binding partner reversibly binds to the biotin-binding site of streptavidin or a streptavidin mutein tetramer. In some embodiments, the binding partner reversibly binds to the biotin-binding site of a streptavidin mutein tetramer. In some embodiments, the binding partner is biotin, a biotin analog, or a streptavidin-binding peptide. In some embodiments, the binding partner is a streptavidin-binding peptide. In some embodiments, 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-binding peptide is SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16).

[0052] In some embodiments, the streptavidin or streptavidin mutein tetramer reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide. In some embodiments, the streptavidin-binding peptide is selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 15), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18), and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19). In some of any of the embodiments, the sequence of the streptavidin-binding peptide is set forth in any of SEQ ID NOs: 7, 8, and 15-19. In some of any of the embodiments, the streptavidin binding peptide is SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16).

[0053] In some embodiments, the streptavidin tetramer reversibly binds to a biotin analog or a streptavidin-binding peptide. In some embodiments, the streptavidin tetramer reversibly binds to a streptavidin-binding peptide. In some embodiments, the streptavidin-binding peptide reversibly binds to the biotin-binding site of the streptavidin tetramer. In some embodiments, 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-binding peptide is SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16).

[0054] In some embodiments, the streptavidin mutein tetramer reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide. In some embodiments, the streptavidin mutein tetramer reversibly binds to a streptavidin-binding peptide. In some embodiments, the streptavidin-binding peptide reversibly binds to the biotin-binding site of the streptavidin mutein tetramer. In some embodiments, 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-binding peptide is SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16).

[0055] In some optional 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.

[0056] In some optional embodiments, the streptavidin mutein comprises the amino acid sequence Ile in streptavidin at a sequence position corresponding to positions 44-47, e.g., with reference to the positions in the sequence of amino acids set forth in SEQ ID NO:1. 44 -Gly 45 -Ala 46 -Arg47 Alternatively, the streptavidin mutein may comprise, for example, the amino acid sequence Val at a position in streptavidin corresponding to positions 44 to 47 in the amino acid sequence shown in SEQ ID NO: 1. 44 -Thr 45 -Ala 46 -Arg 47 In some of any of the embodiments, the streptavidin mutein comprises the sequence of amino acids set forth in any of SEQ ID NOs: 3-6, 27, 28, 104, and 105. In some of any of the embodiments, the streptavidin mutein comprises the sequence of amino acids set forth in SEQ ID NO: 6.

[0057] In some embodiments, the selection agent is or comprises an agent selected from the group consisting of an antibody fragment, a proteinaceous binding molecule with immunoglobulin-like function, an Ig domain-containing molecule, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex, a receptor ligand, and a binding fragment of, for example, any of the foregoing. In some embodiments, the selection agent comprises an antibody or an antibody fragment. In some embodiments, the selection agent comprises an antibody fragment. In some embodiments, the antibody fragment is a monovalent antibody fragment.

[0058] In some embodiments, the selection marker is a T cell co-receptor; the selection marker is or comprises a member of the T cell antigen receptor complex; the selection marker is or comprises a CD3 chain; the selection marker is or comprises a CD3 zeta chain; the selection marker is or comprises CD8; the selection marker is or comprises CD4; the selection marker is or comprises CD45RA; the selection marker is or comprises CD27; the selection marker is or comprises CD28; and / or the selection marker is or comprises CCR7. In some embodiments, the selection marker is selected from the group consisting of CD3, CD4, and CD8. In some embodiments, the selection marker is CD3.

[0059] In some embodiments, the selectable marker is a member of a T cell co-receptor or a T cell antigen receptor complex. In some embodiments, the selectable marker is selected from the group consisting of CD3, CD4, CD8, CD45RA, CD27, CD28, and CCR7. In some embodiments, the selectable marker is selected from the group consisting of CD3, CD4, and CD8. In some embodiments, the selectable marker is CD3.

[0060] In some of any of the embodiments, the selection agent is directly or indirectly bound to the stationary phase, hi some of the embodiments, the selection agent is indirectly bound to the stationary phase via a selection reagent to which the selection agent reversibly binds.

[0061] In some of the optional embodiments, the stationary phase is or comprises a chromatographic matrix.

[0062] In some of any of the embodiments, the stationary phase has a binding capacity of between or about 500 million to 5 billion cells, 500 million to 4 billion cells, 500 million to 3 billion cells, 500 million to 2 billion cells, 1 billion to 5 billion cells, 1 billion to 4 billion cells, 1 billion to 3 billion cells, or 1 billion to 2 billion cells, inclusive of each boundary. In some of any of the embodiments, the stationary phase has a binding capacity of between 1 billion to 2 billion cells or between about 1 billion to 2 billion cells, inclusive of each boundary.

[0063] In some of any of the embodiments, the plurality of T cells comprises antigen-specific T cells, helper T cells, cytotoxic T cells, memory T cells, and / or regulatory T cells. In some of any of the embodiments, the T cells comprise CD3+ T cells, or CD4+ T cells and / or CD8+ T cells.

[0064] In some embodiments, the T cells are primary T cells from a human subject, or the sample comprises primary T cells from a human subject. In some embodiments, the sample is or comprises a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product. In some embodiments, the sample is an apheresis or leukapheresis product. In some embodiments, the apheresis or leukapheresis product has been previously cryo-frozen.

[0065] In some embodiments, the recombinant protein is an antigen receptor. In some embodiments, the recombinant protein is a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an extracellular antigen recognition domain that specifically binds to a target antigen and an intracellular signaling domain comprising an ITAM. In some embodiments, the intracellular signaling domain comprises the intracellular domain of the CD3 zeta (CD3ζ) chain. In some embodiments, the CAR further comprises a transmembrane domain connecting the extracellular domain and the intracellular signaling domain. In some embodiments, the transmembrane domain comprises the transmembrane portion of CD28. In some embodiments, the intracellular signaling domain further comprises the intracellular signaling domain of a T cell costimulatory molecule. In some embodiments, the T cell costimulatory molecule is selected from the group consisting of CD28 and 41BB.

[0066] In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is pseudotyped with VSV-G.

[0067] In some of any of the embodiments, the method further comprises recovering the transduced T cells after further incubation, e.g., incubating, thereby producing a transduced T cell output composition.

[0068] In some embodiments, at the time of harvest, the percentage of naive-like cells in the output composition is greater than or about 60% of the total T cells, total CD4+ T cells, total CD8+ T cells, or recombinant protein-expressing cells in the output composition, e.g., any of the foregoing. In some embodiments, the naive-like T cells comprise CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+ T cells. In some embodiments, the naive-like T cells comprise CD27+CCR7+ T cells. In some embodiments, the naive-like T cells comprise CCR7+CD45RA+ T cells.

[0069] In some embodiments, the method further comprises formulating the cells of the output composition for cryopreservation and / or administration to a subject. In some embodiments, the recovered cells are formulated in the presence of a pharmaceutically acceptable excipient or cryoprotectant.

[0070] In some of the embodiments, at least one of the method steps is performed in a closed system. In some of the embodiments, all of the method steps are performed in a closed system.

[0071] In some of any of the embodiments, at least one of the method steps is automated. In some of any of the embodiments, all of the method steps are automated.

[0072] In some embodiments, provided herein is an article of manufacture for on-column transduction of T cells, comprising: (a) a composition comprising (i) a first stimulating agent and a second stimulating agent for stimulating T cells, the first and second stimulating agents being capable of specifically binding to a first molecule and a second molecule, respectively, on the surface of the T cells, and (ii) a viral vector for transducing the T cells, the viral vector comprising a nucleic acid sequence encoding a recombinant protein; and (b) a stationary phase comprising a selection agent capable of specifically binding to a selection marker on the T cells, for immobilizing the T cells on the stationary phase.

[0073] In some of any of the embodiments, the first and second stimulating agents are reversibly bound to a T cell stimulating reagent included in the composition. In some of any of the embodiments, the selection agent is indirectly bound to the stationary phase via the selection reagent. In some of any of the embodiments, the stationary phase is or comprises a chromatography matrix. In some of any of the embodiments, the article of manufacture further comprises a container containing all or a portion of the chromatography matrix. In some of any of the embodiments, the stationary phase is a first stationary phase, the selection agent is a first selection agent, the selection marker is a first selection marker, and the article of manufacture further comprises a second stationary phase comprising a second selection agent capable of specifically binding to a second selection marker on T cells. In some of any of the embodiments, the first and second stationary phases are arranged in parallel. In some of any of the embodiments, the first and second stationary phases are arranged in series.

[0074] In some embodiments, provided herein is an apparatus that includes an article of manufacture of any of the provided embodiments.

[0075] In some of any of the embodiments, the device further comprises a fluid inlet fluidly connected to one or more components of the device and / or a fluid outlet fluidly connected to one or more components of the device. In some of any of the embodiments, the device is in a closed or sterile system.

[0076] In some of any of the embodiments, the article of manufacture or apparatus is for use in the method of any of the provided embodiments. In some of any of the embodiments, the method is performed in an automated manner.

[0077] Also provided herein, in some embodiments, are populations of T cells transduced by any of the provided methods. [Brief explanation of the drawings]

[0078] [Figure 1-1] 1A and 1B provide schematic diagrams of exemplary housing assemblies for column chromatography. Figure 1A shows an exemplary housing assembly including a temperature control member including a heating coil with an inlet and outlet for an external hot water supply and a gas supply connector for a threaded air filter. Figure 1B shows an exemplary housing assembly in an exemplary column chromatography system. [Figure 1-2] Same as above. [Figure 2]FIG. 2 provides a schematic diagram of an exemplary embodiment for stimulating and selecting target cells, where stimulation is performed, at least in part, by incubation of cells in the presence of a support 36, depicted here as a stationary phase, having immobilized thereon components of a selection reagent 31 for cell selection (Panel A), the selection reagent 31 having binding sites for a selection agent 32, which can bind to a molecule (selection marker) 34 present on some or all of the target cells. The selection agent 32 is added to the support with immobilized selection reagent 31 under conditions such that the selection agent and the selection agent reversibly bind, e.g., via the binding sites, thereby forming an oligomeric complex with the selection agent immobilized thereon (Panel B). The selection agent can include two or more agents. Alternatively, a complex of the reversibly bound selection agent and selection agent can be added to the stationary phase as a complex for immobilization. As shown in the figure, cells 33, such as target cells, are combined with a stationary phase and a multimerized selection agent complex, whereby the target cells are reversibly immobilized to a support 36 via a selection agent 32 and a reagent (selection marker) 34 (Panel C). Optionally, unbound cells are removed before or after addition of the stimulating agent. A complex comprising a multimerized stimulating agent 35 reversibly bound to an oligomeric stimulating reagent 37 is added under certain conditions, whereby the stimulating agent 35 specifically binds to a molecule on the target cells, thereby inducing or modulating a signal in the immobilized target cells expressing the marker (Panel D). [Figure 3-1]Figures 3A and 3B show the results of WST metabolic assays of T cells from three different donors incubated with anti-CD3 / anti-CD28 multimerized in different batches of oligomeric reagent. Figure 3A summarizes the WST metabolic activity, as indicated by the WST ratio, for all tested batches (pooled) compared to a reference batch containing anti-CD3 / anti-CD28 multimerized on an oligomeric scaffold with an average hydrodynamic radius of 36 nm or 101 nm. The average WST metabolic activity in T cells from different donors, as indicated by the average WST ratio, for each tested batch and the reference reagent is shown in Figure 3B. [Figure 3-2] Same as above. [Figure 4] FIG. 4 provides a schematic diagram of an exemplary on-column T cell selection and stimulation process. [Figure 5] Figure 5 shows the elution efficiency using an exemplary heat / gas column with a heating element and gas delivery element that was approximately twice that of the reference column. The estimate (gray bar) was the theoretical number of captured cells that would elute if 100% efficiency were assumed. [Figure 6] Figure 6 shows flow cytometry quantification of cells in the starting material, negative fraction, or positive fraction after on-column T cell selection and stimulation using an exemplary column with a heating element and gas delivery element. Cells were stained with antibodies recognizing surface markers including CD3, CD4, CD8, CD45, and CD14. [Figure 7-1]Figures 7A and 7B show the results of on-column selection and stimulation of T cells using an exemplary column with a heating element and gas delivery element. Cells were monitored by flow cytometry on days 1, 2, and 3 during the subsequent incubation for cell number and cell surface expression after staining the cells with antibodies recognizing CD3, CD4, CD8, and activation markers CD69 and CD25; the flow cytometry results are shown in Figure 7A. Assessment of cell number and fold expansion after the subsequent incubation indicated that selected and stimulated T cells began to increase in number on day 3, consistent with the cells' ability to proliferate, as shown in Figure 7B. [Figure 7-2] Same as above. [Figure 8-1] Figures 8A-8C provide the results of on-column T cell selection using cryopreserved apheresis samples as starting samples in an exemplary heat / gas column. Figure 8A shows that cryopreserved apheresis samples (CAPH) generally have a higher mononuclear cell content (greater than 20% as indicated by the percentage of viable CD45+ cells) compared to fresh apheresis samples (APH). Figure 8B depicts the percentage of cells positive for CD3 or CD14 in the starting material and the positive fraction. The number of T cells selected using column chromatography is shown in Figure 8C; in this case, two sequential selections were performed against CD3. [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 9] Figure 9 provides a schematic diagram of a selection and stimulation run using two identical exemplary heat / gas columns arranged in series (Run 1) and a selection and stimulation run using two identical exemplary heat / gas columns arranged in parallel (Run 2). [Figure 10-1]Figures 10A and 10B provide a comparison of the results of T cell selection and stimulation in Run 1 and Run 2. Figure 10A shows flow cytometry analysis of the starting material, negative fraction, and positive fraction, where cells were stained with antibodies recognizing surface markers including CD3, CD4, CD8, and CD14. Cells from the positive fraction were harvested and incubated, and the left panel, Figure 10B, shows the expression of activation markers CD25 and CD69 on cells on day 1 of incubation. Representative results of cell numbers in Run 1 (□) and Run 2 (●) during incubation are shown in the right panel, Figure 10B. [Figure 10-2] Same as above. [Figure 11-1] Figures 11A and 11B provide the results of on-column T cell selection using an enriched blood sample as the starting sample, with CD3 selection and stimulation on two exemplary heat / gas columns arranged in parallel. Figure 11A shows flow cytometry analysis of the starting material, the negative fraction, or the positive fraction, where cells were stained with antibodies recognizing surface markers including CD3, CD4, CD8, and CD14. Cells from the positive fraction were harvested and incubated, and the CD4 / CD8 and CD25 / CD69 expression of the incubated cells is shown in Figure 11B. [Figure 11-2] Same as above. [Figure 12] 12 provides the results of an exemplary process for selecting T cells obtained directly from whole blood using Sephadex® G-50 as the resin in an exemplary thermal / gas chromatography column. The starting material, negative fractions, and positive fractions from CD3+ T cell selection were stained with propidium iodide (PI) and CD3 antibody and quantified by flow cytometry. [Figure 13]Figure 13 shows the effect of 24-hour on-column stimulation with anti-CD3 / anti-CD28 oligomeric stimulating reagents on CD3, CD4, and CD8 surface expression (assessed as mean fluorescence intensity (MFI)) when each molecule is used as a selection marker to immobilize cells on the stationary phase of a chromatography column. Surface expression patterns are compared to control conditions without on-column stimulation with anti-CD3 / anti-CD28 oligomeric stimulating reagents. Cells were isolated from apheresis samples applied to the stationary phase. [Figure 14] Figure 14 shows exemplary kinetics of downregulation and re-expression of TCR / CD3 complexes upon on-column stimulation with anti-CD3 / anti-CD28 oligomeric stimulating reagents, where CD3 is used as a selection marker to immobilize cells on the column. Cells were isolated from apheresis samples applied to the stationary phase. Antibodies against the αβ TCR chain were used to assess CD3 / TCR complexes. [Figure 15-1] Figures 15A-15B show the phenotypic and functional characteristics of cultured T cells spontaneously detached upon on-column stimulation with anti-CD3 / anti-CD28 oligomeric stimulating reagents. Figure 15A shows, from left to right, T cell size and CD3, CD69, and CD25 expression 24 hours and 5 days after on-column stimulation. Figure 15B shows the proliferative potential of spontaneously detached cultured T cells, as indicated by cell count and magnification. Cells were isolated from apheresis samples applied to the stationary phase and collected using a washing step. [Figure 15-2] Same as above. [Figure 16-1]Figures 16A-16D show exemplary effects of incubating T cells with anti-CD3 / anti-CD28 oligomeric stimulatory reagents in the presence or absence of Compound 63 on mTor signaling and viability and proliferation kinetics. Figure 16A shows pS6 expression in viable CD8+ T cells by the memory subset. Figure 16B shows the mean fluorescence intensity (mfi) of pS6 expression in total CD8+ T cells with treatment as indicated. Figures 16C-16D show viability and total T cell numbers, respectively, over time (indicated by days; e.g., d1) in culture after the initiation of stimulation ("input"). In Figures 16C-16D, the black line corresponds to T cell compositions incubated in the presence of Compound 63, and the gray line corresponds to T cell compositions incubated in the absence of Compound 63. [Figure 16-2] Same as above. [Figure 16-3] Same as above. [Figure 16-4] Same as above. [Figure 17-1] Figures 17A-17F show exemplary functional and phenotypic characteristics of cryopreserved CAR-T cells generated using a method employing incubation with an anti-CD3 / anti-CD28 oligomeric stimulating reagent in the presence or absence of compound 63. Figure 17A shows the intracellular expression of caspases at the time of thawing. Figures 17B and 17D show the phenotypic profiles of CD8 and CD4 CAR-T cells, respectively, by subset expression of CD27 and / or CCR7. Figures 17C and 17E show intracellular IL2, IFNg, or TNF (left panels) or a combination of IL2 and / or IFNg or TNF (right panels) in CD8 and CD4 CAR-T cells, respectively, stimulated with antigen-bearing targets. Figure 17F shows the increased proliferation and viability over 12 days (left panel) and total expansion calculated by area under the growth curve (AUC) (right panel) for CAR-T cells stimulated with anti-CAR beads. [Figure 17-2] Same as above. [Figure 18-1]Figure 18A shows the yield of CD3+, CD4+, and CD8+ T cells after cell selection using either the on-column stimulation process or the alternative process described in Example 11. Figures 18B-18C show the total number of cells (Figure 18B) and the percentage of viable cells (Figure 18C) recovered after using the on-column stimulation or alternative process described in Example 11. [Figure 18-2] Same as above. [Figure 18-3] Same as above. [Figure 19-1] Figures 19A-19D show the percentage of viable cells (e.g., purity; Figure 19A) at day 5 of culture (day 8 from the start of the process), the percentage of viable cells expressing an exemplary CAR (Figure 19B), the percentage of viable cells expressing CD4 at day 8 of selection and process (Figure 19C), and the T cell phenotype distribution (percentage) for each donor (Figure 19D) for on-column stimulation or an alternative process described in Example 11. [Figure 19-2] Same as above. [Figure 19-3] Same as above. [Figure 19-4] Same as above. [Figure 20] Figure 20 shows CD19+ HEK cell lysis over time during culture with anti-CD19 CAR T cells engineered using on-column stimulation or an alternative process described in Example 11, as well as under control conditions. [Figure 21-1] Figures 21A-21C show antigen-specific CAR T cell IFNg (Figure 21A), IL-2 (Figure 21B), and TNFα (Figure 21C) production for CD4 and CD8 T cells engineered using on-column stimulation or an alternative process described in Example 11. [Figure 21-2] Same as above. [Figure 21-3] Same as above. [Figure 22-1]Figures 22A-22C show the CD4:CD8 ratio (Figure 22A), transduction efficiency of engineered T cells (CD4 and CD8 cell mix; Figure 22B), and percentage of viable cells (Figure 22C) produced using on-column stimulation or an alternative process described in Example 11. For each process, three manufacturing runs are shown. [Figure 22-2] Same as above. [Figure 22-3] Same as above. [Figure 23] Figure 23 shows tumor size by mean radiance in treatment groups 6 days after mice were injected (iv) with a B cell lymphoma cell line (Raji) and before the mice were treated with CAR-T cell compositions. Treatment groups represent CAR-T cell compositions generated by three manufacturing runs for each of the on-column stimulation or alternative processes described in Example 11. [Figure 24] Figure 24 shows the tumor burden in mice injected with a B cell lymphoma cell line (Raji) over time for each treatment group. The effect of CAR T cell therapy is shown for on-column stimulation or the alternative process described in Example 11, as well as for each of the three manufacturing runs (Figures 22A-22C). [Figure 25]Figures 25-28 provide schematic diagrams of an exemplary housing assembly for column chromatography. The housing assembly in this example includes an inlet housing member, an outlet housing member, a sidewall member, and a jacket member surrounding the sidewall member and portions of the inlet and outlet housing members. The jacket member of the exemplary housing assembly is made of two jacket components, each containing a heating coil with an inlet and an outlet for an external hot water supply. The two jacket components together form the jacket member. The exemplary housing assembly also includes a gas supply connector (not shown) for a threaded air filter, which is connected to the inlet of the inlet housing member. Figure 25 shows an enlarged view of an exemplary housing assembly. Figures 26A-26C show views of the inside (Figure 26A), side (Figure 26B), and exterior (Figure 26C) of one jacket component. Figure 27 shows a view of an exemplary housing assembly depicting the inlet for the external hot water supply and a portion of the inlet of the inlet housing member. 28 shows a diagram of an exemplary housing assembly depicting a portion of the outlet for an external hot water supply and the inlet of the outlet housing member. Optional features (not shown) for this exemplary housing assembly include a first porous member (e.g., a woven polyester mesh) configured to separate the stationary phase and the inlet of the internal cavity, a second porous member (e.g., a woven polyester mesh) configured to separate the stationary phase and the outlet of the internal cavity, and a tubing set connector. [Figure 26-1]Figures 25-28 provide schematic diagrams of an exemplary housing assembly for column chromatography. The housing assembly in this example includes an inlet housing member, an outlet housing member, a sidewall member, and a jacket member surrounding the sidewall member and portions of the inlet and outlet housing members. The jacket member of the exemplary housing assembly is made of two jacket components, each containing a heating coil with an inlet and an outlet for an external hot water supply. The two jacket components together form the jacket member. The exemplary housing assembly also includes a gas supply connector (not shown) for a threaded air filter, which is connected to the inlet of the inlet housing member. Figure 25 shows an enlarged view of an exemplary housing assembly. Figures 26A-26C show views of the inside (Figure 26A), side (Figure 26B), and exterior (Figure 26C) of one jacket component. Figure 27 shows a view of an exemplary housing assembly depicting the inlet for the external hot water supply and a portion of the inlet of the inlet housing member. 28 shows a diagram of an exemplary housing assembly depicting a portion of the outlet for an external hot water supply and the inlet of the outlet housing member. Optional features (not shown) for this exemplary housing assembly include a first porous member (e.g., a woven polyester mesh) configured to separate the stationary phase and the inlet of the internal cavity, a second porous member (e.g., a woven polyester mesh) configured to separate the stationary phase and the outlet of the internal cavity, and a tubing set connector. [Figure 26-2] Same as above. [Figure 26-3] Same as above. [Figure 27]Figures 25-28 provide schematic diagrams of an exemplary housing assembly for column chromatography. The housing assembly in this example includes an inlet housing member, an outlet housing member, a sidewall member, and a jacket member surrounding the sidewall member and portions of the inlet and outlet housing members. The jacket member of the exemplary housing assembly is made of two jacket components, each containing a heating coil with an inlet and an outlet for an external hot water supply. The two jacket components together form the jacket member. The exemplary housing assembly also includes a gas supply connector (not shown) for a threaded air filter, which is connected to the inlet of the inlet housing member. Figure 25 shows an enlarged view of an exemplary housing assembly. Figures 26A-26C show views of the inside (Figure 26A), side (Figure 26B), and exterior (Figure 26C) of one jacket component. Figure 27 shows a view of an exemplary housing assembly depicting the inlet for the external hot water supply and a portion of the inlet of the inlet housing member. 28 shows a diagram of an exemplary housing assembly depicting a portion of the outlet for an external hot water supply and the inlet of the outlet housing member. Optional features (not shown) for this exemplary housing assembly include a first porous member (e.g., a woven polyester mesh) configured to separate the stationary phase and the inlet of the internal cavity, a second porous member (e.g., a woven polyester mesh) configured to separate the stationary phase and the outlet of the internal cavity, and a tubing set connector. [Figure 28]Figures 25-28 provide schematic diagrams of an exemplary housing assembly for column chromatography. The housing assembly in this example includes an inlet housing member, an outlet housing member, a sidewall member, and a jacket member surrounding the sidewall member and portions of the inlet and outlet housing members. The jacket member of the exemplary housing assembly is made of two jacket components, each containing a heating coil with an inlet and an outlet for an external hot water supply. The two jacket components together form the jacket member. The exemplary housing assembly also includes a gas supply connector (not shown) for a threaded air filter, which is connected to the inlet of the inlet housing member. Figure 25 shows an enlarged view of an exemplary housing assembly. Figures 26A-26C show views of the inside (Figure 26A), side (Figure 26B), and exterior (Figure 26C) of one jacket component. Figure 27 shows a view of an exemplary housing assembly depicting the inlet for the external hot water supply and a portion of the inlet of the inlet housing member. 28 shows a diagram of an exemplary housing assembly depicting a portion of the outlet for an external hot water supply and the inlet of the outlet housing member. Optional features (not shown) for this exemplary housing assembly include a first porous member (e.g., a woven polyester mesh) configured to separate the stationary phase and the inlet of the internal cavity, a second porous member (e.g., a woven polyester mesh) configured to separate the stationary phase and the outlet of the internal cavity, and a tubing set connector. [Figure 29]Figures 29-31 provide schematic diagrams of an exemplary housing assembly for column chromatography. This exemplary housing assembly includes an inlet housing member, an outlet housing member, a sidewall member, and a jacket member that surrounds the sidewall member and portions of the inlet and outlet housing members. The jacket member of the exemplary housing assembly is made of three jacket components, each containing an electric heating element including a metal plate. The three jacket components together form the jacket member. The exemplary housing assembly also includes a gas supply connector for a threaded air filter (not shown), which is connected to the inlet of the inlet housing member. Figure 29 shows an enlarged view of the exemplary housing assembly. Figures 30A-30C show three views of one jacket component. Figure 30D shows the electric heating element. Figure 31 shows a view of the exemplary housing assembly, depicting the electrical connections of the electric heating element and a portion of the inlet of the outlet housing member. Optional features (not shown) for this exemplary housing assembly include a first porous member (e.g., a woven polyester mesh), a second porous member (e.g., a woven polyester mesh), and a tubing set connector. [Figure 30-1]Figures 29-31 provide schematic diagrams of an exemplary housing assembly for column chromatography. This exemplary housing assembly includes an inlet housing member, an outlet housing member, a sidewall member, and a jacket member that surrounds the sidewall member and portions of the inlet and outlet housing members. The jacket member of the exemplary housing assembly is made of three jacket components, each containing an electric heating element including a metal plate. The three jacket components together form the jacket member. The exemplary housing assembly also includes a gas supply connector for a threaded air filter (not shown), which is connected to the inlet of the inlet housing member. Figure 29 shows an enlarged view of the exemplary housing assembly. Figures 30A-30C show three views of one jacket component. Figure 30D shows the electric heating element. Figure 31 shows a view of the exemplary housing assembly, depicting the electrical connections of the electric heating element and a portion of the inlet of the outlet housing member. Optional features (not shown) for this exemplary housing assembly include a first porous member (e.g., a woven polyester mesh), a second porous member (e.g., a woven polyester mesh), and a tubing set connector. [Figure 30-2] Same as above. [Figure 30-3] Same as above. [Figure 30-4] Same as above. [Figure 31]Figures 29-31 provide schematic diagrams of an exemplary housing assembly for column chromatography. This exemplary housing assembly includes an inlet housing member, an outlet housing member, a sidewall member, and a jacket member that surrounds the sidewall member and portions of the inlet and outlet housing members. The jacket member of the exemplary housing assembly is made of three jacket components, each containing an electric heating element including a metal plate. The three jacket components together form the jacket member. The exemplary housing assembly also includes a gas supply connector for a threaded air filter (not shown), which is connected to the inlet of the inlet housing member. Figure 29 shows an enlarged view of the exemplary housing assembly. Figures 30A-30C show three views of one jacket component. Figure 30D shows the electric heating element. Figure 31 shows a view of the exemplary housing assembly, depicting the electrical connections of the electric heating element and a portion of the inlet of the outlet housing member. Optional features (not shown) for this exemplary housing assembly include a first porous member (e.g., a woven polyester mesh), a second porous member (e.g., a woven polyester mesh), and a tubing set connector. [Figure 32] Figure 32 shows CD27 surface expression on cells after immobilization on the stationary phase of a heated column using CD27 as a selection marker and on-column stimulation with an anti-CD3 / anti-CD28 oligomeric stimulating reagent. The column was heated using a jacket member containing two heating coils, each with an inlet and outlet for an external heated water supply. The heated column also had a gas supply connector for a threaded air filter. As a control, CD27-selected cells were not subjected to on-column stimulation with an anti-CD3 / anti-CD28 oligomeric stimulating reagent. Cells were isolated from an apheresis sample that had been applied to the stationary phase. [Figure 33]Figure 33 shows CD3 and CD27 surface expression in cells sequentially isolated from an apheresis sample using two separation columns. CD27 was used as the selection marker in the first column, and the positive fraction from the first column was passed through a second column carrying the CD3 selection marker. Cells immobilized on the second column were stimulated with an anti-CD3 / anti-CD28 oligomeric stimulating reagent. The second column was heated using a jacket member containing two heating coils, each with an inlet and outlet for an external heated water supply. The heated column also had a gas supply connector for a threaded air filter. [Figure 34-1] Figures 34A-34E show the CD3+ depletion (Figure 34A), CD4 and CD8 expression (Figure 34B), CD69 expression (Figure 34C), viability (Figure 34D), and viable cell count (Figure 34E) of cells after on-column stimulation in chromatography columns heated using different heating elements. The columns were heated using a jacket member containing two heating coils (water) or three metal plates (metal) as an electric heating element. The columns also had a gas supply connector for a threaded air filter. [Figure 34-2] Same as above. [Figure 34-3] Same as above. [Figure 34-4] Same as above. [Figure 34-5] Same as above. [Figure 35] Figure 35 shows CD8 and CAR expression in cells simultaneously stimulated and transduced on-column (right panel) and negative and positive control cells (left and center panels, respectively). Results shown are for cells pre-gated on live single CD45+ lymphocytes. DETAILED DESCRIPTION OF THE INVENTION

[0079] Some aspects of the present disclosure provide methods for on-column transduction of cells. In some embodiments, the cells are T cells. In some embodiments, the methods include contacting the cells or a sample containing the cells with a stimulating reagent, e.g., a T cell stimulating reagent. In some embodiments, the methods include contacting the cells or a sample containing the cells with a viral vector having a nucleic acid sequence encoding a recombinant protein, thereby producing transduced cells. In some embodiments, the cells or a sample containing the cells are contacted simultaneously with the stimulating reagent, e.g., a T cell stimulating reagent, and the viral vector. In some embodiments, the cells are immobilized on a stationary phase, e.g., a stationary phase contained in the internal cavity of a chromatography column. In some embodiments, the cells are immobilized before and at the time the cells or a sample containing the cells are contacted with the stimulating reagent, e.g., a T cell stimulating reagent, and the viral vector. In some embodiments, the cells are immobilized via a selection agent that binds to a selection marker expressed by the cells, where the selection agent is directly or indirectly immobilized on the stationary phase.

[0080] In some embodiments, the method further comprises adding cells or a sample comprising cells, e.g., T cells, to the internal cavity. In some embodiments, the method further comprises adding a composition comprising a stimulating reagent, e.g., a T cell stimulating reagent, and a viral vector, to the internal cavity. In some embodiments, the method further comprises incubating the cells or a sample comprising cells in the internal cavity in the presence of a stimulating reagent, e.g., a T cell stimulating reagent, and a viral vector. In some embodiments, the method further comprises collecting, culturing, harvesting, and / or formulating the transduced cells. Articles of manufacture and devices, including those for performing the provided methods, are also provided herein.

[0081] Methods for generating cell populations suitable for use in cell therapy, such as selected (enriched), stimulated, and engineered cell populations, often require separate selection, stimulation, and manipulation steps, which can lengthen the manufacturing process. Furthermore, selection techniques can involve mixing selected cells with selection reagents, such as selection agents such as Fab fragments, and competitive reagents and / or free-binding agents used to promote cell detachment from the stationary phase used in column chromatography, resulting in the need for additional washing and / or medium exchange steps to purify the output composition. In addition to requiring significant time to complete, multiple processing steps can result in cellular stress that can affect downstream cell processing and even cell biology. Additional methods for generating cell compositions are needed.

[0082] In some aspects, provided herein are methods for selecting cells (e.g., T cells, such as CD3+, CD4+, or CD8+ T cells) from a sample containing target cells, and stimulating and / or manipulating, e.g., transducing, the selected cells. In some embodiments, the target cells are simultaneously stimulated and transduced after selection, wherein the target cells are immobilized on the stationary phase of the chromatography column used for selection. Thus, in some aspects, the provided methods combine the steps of stimulating cells and manipulating cells, thereby reducing the time required for production. In some aspects, this combination results in improved transduction efficiency compared to other methods, where transduction is performed after cell activation and / or elution of cells from the chromatography column. In some aspects, transduction efficiency is improved by early cell transduction, e.g., as early as the onset of activation. In some embodiments, transduction efficiency is improved by transducing cells on-column because the cells are not further processed, e.g., by immobilizing and / or eluting the cells from the column.

[0083] Furthermore, in some embodiments, the provided methods also result in the natural detachment of cells from the stationary phase after activation and / or transduction. Thus, in some embodiments, the provided methods do not require the use of a competitive reagent to elute the cells and / or additional wash steps to remove the competitive reagent and selection agent after elution. In some embodiments, the methods provided herein do not require a separate step to promote detachment of cells from the stationary phase. In some embodiments, the methods provided herein do not require a separate purification step, e.g., a step to remove an agent used to promote detachment (e.g., a competitor and / or free binder). Thus, in some embodiments, the methods provided herein reduce and / or minimize cell handling, contamination, and processing time in the manufacturing process. Furthermore, the provided methods enable the use of a completely closed system that unifies on-column operations, such as cell selection, stimulation, and genetic manipulation, by performing the selection, activation, and transduction steps on a chromatography column. In some embodiments, the steps of the provided methods can be automated, or the methods can be fully automated. In some aspects, the provided methods allow for faster production with less manipulation of cells, thereby resulting in, for example, maintenance of a wider range of cell characteristics, improved cell production turnaround time, fewer hands-on defects, and reduced ultimate manufacturing costs for cell therapies. In some aspects, the provided methods and other embodiments are advantageous in that they compress multiple processing steps (e.g., selection, stimulation, and transduction) and / or eliminate processing steps (e.g., steps to remove selection reagents and / or agents used to promote detachment), and allow the compressed process to occur within the same container and / or closed system, which can provide increased efficiency and sterility.

[0084] The provided methods are capable of selecting cells, e.g., CD3+, CD4+, and CD8+ T cells, from other components, e.g., other cells in a sample, immobilizing the cells on a stationary phase of a chromatography column, stimulating and transducing the selected cells immobilized on the stationary phase, and collecting the cells, without a processing step to remove the agent used to detach the cells from the stationary phase and promote said detachment from the output composition of the selected and stimulated cells. In certain embodiments, the provided devices and methods include separate selection, stimulation, and transduction steps, and further are capable of generating a population of selected, stimulated, and transduced cells in a reduced time compared to methods that include separate selection, stimulation, and transduction steps and require additional steps to detach the cells from the stationary phase and remove the agent used to promote detachment. In certain aspects, the provided methods can generate an output population (also referred to as a composition) of selected, stimulated, and transduced cells suitable for downstream processing (e.g., culturing, expanding, and / or performing subsequent incubation, stimulation, selection, and / or transduction) within 24 hours of initiating stimulation and / or transduction on the column. In some embodiments, the methods provided herein involve the use of a stimulating agent capable of binding to a molecule on the surface of the cells, thereby delivering a stimulatory signal to the cells. In some embodiments, the stimulating agent is included in an oligomeric stimulating reagent that can be added to the stationary phase. In some embodiments, the stimulation results in the spontaneous detachment of the selected cells from the stationary phase, thus allowing for collection of the selected and stimulated cells without additional processing steps to remove the agent used to detach the cells from the stationary phase and promote said detachment from the output cell composition.In certain embodiments, the methods successfully produce a composition of contaminating (e.g., free of agents used for detachment, e.g., competitors or free binders, and / or selection agents, etc.) selected, stimulated, and transduced cells suitable for further processing, e.g., culturing, expanding, incubating, or subsequently performing stimulation, selection, and / or transduction, within 24 hours of initiating on-column stimulation and / or transduction.

[0085] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. To the extent that a definition set forth herein contradicts or otherwise conflicts with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0086] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0087] I. Methods for Selecting, Stimulating, and / or Manipulating Cells Provided herein are methods for producing an output population of cells (also referred to as an output composition), e.g., selected, stimulated, and transduced CD3+ T, CD4+ T, and / or CD8+ T cells, comprising steps for selecting, stimulating, transducing, and / or collecting the cells. In certain embodiments, the methods provided herein are used in connection with manufacturing, producing, or generating cell therapies. In some embodiments, the methods for producing or generating an output composition, e.g., selected, stimulated, and transduced T cells, comprise one or more steps for isolating cells from a subject, incubating the cells under stimulatory conditions, and genetically engineering the cells. In some embodiments, the methods comprise processing steps performed in the following order: input cells, e.g., primary CD4+ and CD8+ T cells, are isolated from a biological sample, e.g., selected or separated, incubated under stimulatory conditions, genetically engineered to introduce a recombinant polynucleotide encoding a recombinant receptor into the cells, such as by transduction or transfection, collected in a single step, and then collected, retrieved, or filled into a container, e.g., a bag or vial, as an output population. In some embodiments, the cells of the output population are reintroduced into the same subject, optionally after cryopreserving and storing the cells. In some embodiments, the output population of engineered cells is suitable for use in therapy, e.g., autologous cell therapy.

[0088] In some embodiments, the selected cells immobilized on the stationary phase are stimulated and manipulated, e.g., transduced, simultaneously, for example, by simultaneously contacting the immobilized cells with a stimulating agent or reagent and a manipulation particle, e.g., a viral vector. The term "simultaneous" or "simultaneously," as used herein, means that there is a time separation of within about 15 minutes, e.g., within about 10 minutes, about 5 minutes, or about 1 minute. For example, in connection with simultaneously initiating stimulation and transduction of cells, stimulation and transduction are initiated within 15 minutes, 10 minutes, 5 minutes, or 1 minute of each other. In connection with simultaneously contacting cells with a stimulating reagent and a viral vector, the cells are contacted with the stimulating reagent and the viral vector within 15 minutes, 10 minutes, 5 minutes, or 1 minute apart. In some embodiments, the stimulating reagent and the viral vector are contained in the same composition (e.g., a mixture containing both the stimulating reagent and the viral vector). In some embodiments, the stimulatory reagent and viral vector are contained in separate compositions (e.g., the stimulatory reagent in one composition and the viral vector in another composition) that are added to the cells with a time separation of less than about 15 minutes, about 10 minutes, about 5 minutes, or about 1 minute.

[0089] Provided herein are methods for selecting cells from a sample containing target cells (e.g., T cells, CD3+, CD4+, CD8+ T cells), immobilizing the target cells on a stationary phase of a chromatography column, stimulating and transducing the immobilized cells on the stationary phase (also referred to herein as on-column stimulation and / or on-column transduction), and collecting and / or eluting the selected, stimulated, and transduced cells that spontaneously detach from the stationary phase without the use of a competitor or free binder to facilitate detachment. Among the provided methods are methods involving selecting cells from a sample containing target cells (e.g., T cells, CD3+, CD4+, CD8+ T cells), immobilizing the target cells on a stationary phase of a chromatography column, stimulating and transducing the immobilized cells on the stationary phase, and collecting and / or eluting the selected, stimulated, and transduced cells by gravity flow. In provided embodiments, stimulation of target cells (e.g., CD3+, CD4+, or CD8+ T cells) on the stationary phase of a chromatography column promotes downregulation of a molecule used for cell selection (i.e., a selection marker), resulting in spontaneous detachment or release of the cells from the stationary phase. Cell release or detachment can occur without any additional steps or reagents. In some aspects, cells can be collected by gravity flow, for example, by adding medium or other solution to the chromatography column. In certain embodiments, the added medium or other solution does not contain a competitor or free binder to promote detachment of the cells from the stationary phase.

[0090] In certain embodiments, the provided methods are carried out to select, stimulate, and transduce T cells. In some embodiments, T cells are selected from a biological sample, e.g., an apheresis sample, by adding cells of the sample to an affinity chromatography matrix (e.g., a stationary phase) immobilized with or bound by a selection agent specific for T cells or a subset thereof, e.g., as described in Section IB-1. In provided embodiments, the method includes stimulating the cells immobilized on the stationary phase in the presence of one or more T cell stimulators. In some embodiments, the one or more stimulatory agents include an agent for delivering a stimulatory signal in the T cells. In some embodiments, the stimulatory signal is via a TCR / CD3 complex in the T cells, a CD3-containing complex in the T cells, and / or an ITAM-containing molecule in the T cells. In some embodiments, the stimulatory agent (e.g., a first stimulatory agent) is an agent that binds to CD3, e.g., an anti-CD3 antibody. In some embodiments, the one or more stimulatory agents further include a second stimulatory agent that can further stimulate or enhance a signal in the T cells. In some embodiments, the second stimulating agent is capable of specifically binding to one or more costimulatory molecules on T cells, e.g., CD28, CD90 (Thy-1), CD95 (Apo- / Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM. In some embodiments, the second stimulating agent is an agent that binds to CD28, e.g., an anti-CD28 antibody. In some embodiments, the one or more stimulating agents include an anti-CD3 antibody and an anti-CD28 antibody, e.g., an anti-CD3 Fab and an anti-CD28 Fab. In some embodiments, the one or more stimulating agents are immobilized or bound to a reagent (e.g., a stimulating reagent) that is added to the chromatography column. In certain embodiments, the stimulating reagent is a soluble polymer or oligomeric reagent. For example, the one or more stimulating agents are functionalized on an oligomeric or polymeric protein rather than on a solid surface (e.g., a bead). Exemplary oligomeric stimulating reagents for use in the provided methods are described herein, eg, in section IB-2.In some embodiments, the oligomeric stimulating reagent is an oligomeric streptavidin mutein that is functionalized or multimerized with one or more stimulating agents (eg, an anti-CD3 Fab and an anti-CD28 Fab).

[0091] In some embodiments, the method further comprises introducing a recombinant nucleic acid molecule into the immobilized T cells, wherein the nucleic acid molecule encodes a recombinant protein, thereby producing a composition comprising the transduced T cells. In some embodiments, the recombinant protein is an antigen receptor. In some embodiments, the recombinant protein is a chimeric antigen receptor. In some embodiments, the immobilized T cells are contacted with the recombinant nucleic acid molecule during stimulation of the immobilized cells. In some embodiments, transduction and stimulation of the immobilized T cells are initiated simultaneously. In some embodiments, the immobilized cells are contacted simultaneously with the recombinant nucleic acid molecule and one or more stimulating agents, e.g., stimulating agents contained in a stimulating reagent.

[0092] In some embodiments, the method includes further incubating the composition containing the transduced cells (e.g., transduced T cells) in the column. In some embodiments, the incubation is carried out at 37°C ± 2°C or about 37°C ± 2°C. In some embodiments, the incubation is carried out under conditions that do not expand or substantially expand the cells. In some embodiments, the incubation is carried out in the presence of an additional agent capable of delivering a signal to the T cells. In some embodiments, the additional agent is capable of enhancing or inducing proliferation of T cells, CD4+ T cells, and / or CD8+ T cells. In some embodiments, the additional agent is a cytokine selected from among IL-2, IL-15, and IL-7. In some embodiments, the incubation is carried out for a period of time that is within 24 hours, 12 hours, 10 hours, 8 hours, 6 hours, or 5 hours. In some embodiments, the incubation is carried out in serum-free medium.

[0093] In the provided methods, the selected, stimulated, and transduced T cells are collected by eluting or washing the selected, stimulated, and transduced cells by gravity flow.

[0094] In some embodiments, the collecting comprises washing the stationary phase with medium (e.g., serum-free medium), medium that does not contain competitor or free binder, to elute the target cells (e.g., T cells) from the stationary phase. In some embodiments, the collecting by gravity flow comprises adding medium that does not contain medium, competitor or free binder to the stationary phase to elute the T cells from the stationary phase. In some embodiments, the composition containing stimulated, transduced T cells does not contain a competitor or free binder. In some embodiments, the competitor or free binder is or contains biotin or a biotin analog, e.g., D-biotin. In some embodiments, the competitor or free binder is D-biotin. In some embodiments, the medium for washing the column and eluting the cells by gravity flow is serum-free medium that contains recombinant cytokines (e.g., IL-2, IL-15, and / or IL-7).

[0095] In some embodiments, the method includes further incubating (e.g., culturing) the composition containing the collected transduced cells (e.g., collected transduced T cells). In some embodiments, the further incubation (e.g., culturing) is carried out at 37°C ± 2°C or about 37°C ± 2°C. In some embodiments, the further incubation (e.g., culturing) is carried out under conditions that do not expand or do not substantially expand the cells. In some embodiments, the further incubation is carried out under conditions for expansion (e.g., proliferation) of the cells. In some embodiments, the further incubation (e.g., culturing) is carried out in the presence of an additional agent capable of delivering a signal to the T cells. In some embodiments, the additional agent is contained in the medium used to wash the stationary phase. In some embodiments, the additional agent is capable of enhancing or inducing proliferation of T cells, CD4+ T cells and / or CD8+ T cells. In some embodiments, the additional agent is a cytokine selected from among IL-2, IL-15, and IL-7. In some embodiments, the further incubation is carried out for a period of time that is 14 days or less, 12 days or less, 10 days or less, 8 days or less, 6 days or less, or 5 days or less.

[0096] In certain embodiments, provided herein are methods related to producing an output population of cells expressing a recombinant receptor from an initial or input population of cells. In certain embodiments, the input population is generated, produced, and / or manufactured by combining, mixing, and / or pooling cells, including those from a population of cells containing enriched T cells, enriched CD4+ T cells, and / or enriched CD8+ T cells (hereinafter also referred to as a population of enriched T cells, a population of enriched CD4+ T cells, and a population of enriched CD8+ T cells, respectively). In some embodiments, the input population of cells is a combined, mixed, and / or pooled population of CD4+ and CD8+ T cells. In certain embodiments, the methods can be used to isolate select cells from a biological sample (e.g., whole blood, apheresis), e.g., collected, collected, and / or obtained from a subject, to generate an input population of enriched T cells. In some embodiments, the provided methods may be used in connection with recovering, collecting, and / or formulating a population of enriched T cells after the cells have been stimulated, manipulated, transduced, and / or cultured.

[0097] In certain embodiments, the cells are incubated for a sufficient amount of time during or after genetically engineering the cells, for example, to allow the incorporation of a heterologous or recombinant polynucleotide encoding a recombinant protein or to allow the expression of the recombinant protein. In certain embodiments, the cells are incubated for a fixed or predetermined amount of time, for example, more than 18 hours or less than 4 days. In some embodiments, the engineering step starts or begins simultaneously with the time the cells are exposed to the stimulant.

[0098] In some embodiments, one or more process steps are carried out at least partially in serum-free medium. In some embodiments, the serum-free medium is a defined or well-defined cell culture medium. In certain embodiments, the serum-free medium is a controlled culture medium that has been processed, e.g., filtered to remove inhibitors and / or growth factors. In some embodiments, the serum-free medium contains proteins. In certain embodiments, the serum-free medium may contain serum albumin, hydrolysates, growth factors, hormones, carrier proteins, and / or attachment factors. In some embodiments, the serum-free medium comprises cytokines. In some embodiments, the serum-free medium comprises cytokines or recombinant cytokines. In some embodiments, the serum-free medium comprises recombinant IL-2, IL-15, and / or IL-7. In some embodiments, the serum-free medium comprises glutamine. In some embodiments, the serum-free medium comprises glutamine and recombinant IL-2, IL-15, and IL-7.

[0099] In some embodiments, methods are provided herein in which one or more, or all, steps in preparing cells for clinical use, e.g., adoptive cell therapy, are performed without exposing the cells to non-sterile conditions. In some embodiments, the cells are selected, stimulated, transduced, washed, and formulated, all within a closed, sterile system or device. In some embodiments, one or more of the steps are performed outside the closed system or device. In some such embodiments, the cells are transferred from the closed system or device under sterile conditions, such as by sterile transfer to a separate closed system.

[0100] In some embodiments, the methods provided herein are performed using any of the devices described in Section III.

[0101] In certain embodiments, a sample and / or an isolated portion of a sample (e.g., a buffy coat, a population of enriched T cells) may be collected, formulated for cryoprotection, frozen (e.g., cryoprotected), and / or stored at or below 0°C, -20°C, or -70°C or -80°C before, during, or after any stage or step of a method as provided herein. In some embodiments, the cells may be stored for an amount of time less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or for an amount of time less than 1, 2, 3, 4, 5, 6, 7, 8 weeks, or for an amount of time of at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or for more than 8 weeks. After storage, the sample or an isolated portion of the sample may be thawed, and processing according to the method may be resumed from the same point in the process. In certain embodiments, a cultured and / or formulated population of enriched T cells is cryoprotected and stored before being administered to a subject, for example, as an autologous cell therapy.

[0102] In certain embodiments, at any stage or step in the process, a portion of cells may be sampled or collected; for example, cells may be harvested from a population of cells (e.g., a population of T cells) while the population remains in a closed system. In certain embodiments, such cells may be analyzed for markers, characteristics, or properties, including, but not limited to, viability, apoptosis, activation, stimulation, growth, and / or exhaustion. In some embodiments, cells are sampled or collected by an automated process. In some embodiments, analysis of sampled or collected cells is automated. In certain embodiments, analysis is performed in a closed system under sterile conditions.

[0103] In some embodiments, cells or populations of cells produced and / or processed by the provided methods may be compared to cells or populations of cells processed or produced by exemplary and / or alternative processes. In certain embodiments, the alternative and / or exemplary processes may differ in one or more specific aspects, but otherwise contain similar or identical features, aspects, steps, stages, reagents, or conditions of the embodiments or aspects of the provided methods to which the exemplary or alternative processes are to be compared. For example, selected, stimulated, and transduced cells produced by the provided methods, e.g., the output composition of cells, may be compared to cells produced in a process that included separate selection, stimulation, and transduction steps, or that required the use of a competitor or free binder to detach selected cells from the stationary phase. In some embodiments, unless otherwise specified, the provided methods and the exemplary or alternative processes would otherwise be similar and / or identical, such as having similar or identical steps for selection, enrichment, stimulation, manipulation, transfection, transduction, culture, and / or formulation. In some embodiments, unless otherwise specified, the provided methods and alternative processes select and / or enrich cells from the same or similar type of biological sample and / or process cells and / or input cells of the same cell type.

[0104] In some embodiments, the selected, stimulated, and transduced cells are a composition containing stimulated, transduced T cells, where the T cells are selected from a biological sample (e.g., an apheresis or whole blood sample) containing a plurality of T cells. In some embodiments, collection and / or elution of selected, stimulated, and transduced cells that spontaneously detach from the stationary phase is achieved by gravity flow, for example, during a washing step. The methods provided herein combine cell selection, stimulation, transduction, collection, and / or elution steps, and do not require separate steps to promote detachment of selected, stimulated, and transduced cells from the stationary phase and purification steps to remove agents used to promote detachment (e.g., competitors and / or free binding agents). As such, the method reduces the number of processing steps required to produce a selected, stimulated, and transduced cell composition suitable for downstream processing (e.g., culture, expansion, subsequent incubation, stimulation, and / or selection (e.g., initial selection and / or polishing)), thereby reducing production time, minimizing potential cell stress, and reducing the possibility of contamination.

[0105] In certain embodiments, the method generates an output composition of selected, stimulated, and transduced cells suitable for downstream processing within a certain amount of time, e.g., within 24 hours. In certain embodiments, the method generates an output composition of selected, stimulated, and transduced cells suitable for downstream processing within a certain amount of time, e.g., within 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours, or within about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 hours. In certain embodiments, the method generates an output composition of selected, stimulated, and transduced cells suitable for downstream processing within 6, 5, 4, 3, or 2 hours, or within about 6, about 5, about 4, about 3, or about 2 hours. In some embodiments, the method generates an output composition of selected, stimulated, and transduced cells suitable for downstream processing within a certain amount of time, e.g., within less than 6 hours, or within less than about 6 hours. In some embodiments, the method generates an output composition of selected, stimulated, and transduced cells suitable for downstream processing within a certain amount of time, e.g., less than or about 5.5 hours. In some embodiments, the method generates an output composition of selected, stimulated, and transduced cells suitable for downstream processing within a certain amount of time, e.g., less than or about 5 hours. In some embodiments, the method generates an output composition of selected, stimulated, and transduced cells suitable for downstream processing within a certain amount of time, e.g., less than or about 4.5 hours. In some embodiments, the method generates an output composition of selected, stimulated, and transduced cells suitable for downstream processing within a certain amount of time, e.g., less than or about 4 hours. In some embodiments, the method generates an output composition of selected, stimulated, and transduced cells suitable for downstream processing within a certain amount of time, e.g., less than or about 3 hours.In some embodiments, the methods generate an output composition of selected, stimulated, and transduced cells suitable for downstream processing within a certain amount of time, e.g., less than 3-6 hours or less than about 3-6 hours. In some embodiments, the methods generate an output composition of selected, stimulated, and transduced cells suitable for downstream processing within a certain amount of time, e.g., less than 4-6 hours or less than about 4-6 hours. In some embodiments, the methods generate an output composition of selected, stimulated, and transduced cells suitable for downstream processing within a certain amount of time, e.g., less than 5-6 hours or less than about 5-6 hours. In some embodiments, the methods generate an output composition of selected, stimulated, and transduced cells suitable for downstream processing within a certain amount of time, e.g., less than 4-5 hours or less than about 4-5 hours. In some embodiments, the methods provided herein generate a composition of engineered T cells (e.g., a therapeutic cell composition) within 5 days. In some embodiments, the methods provided herein produce compositions of engineered T cells (e.g., therapeutic cell compositions) in 4-5 days or about 4-5 days. In some embodiments, the processes provided herein result in a manufacturing process that is 4 or 5 days long, or about 4 or about 5 days long. In some embodiments, the processes provided herein result in a manufacturing process that is 4 or 5 days long, or about 4 or about 5 days long. In some embodiments, the processes provided herein result in a manufacturing process that is 4 days long or 96±6 hours long, or about 4 days long or 96±6 hours long.

[0106] The provided methods include methods for selecting cells, e.g., CD3+, CD4+, and CD8+ T cells, from other components, e.g., from other cells in a sample, immobilizing the cells on a stationary phase of a chromatography column, stimulating and transducing the selected cells that are immobilized on the stationary phase, and collecting the selected, stimulated cells in the absence of a processing step to detach the cells from the stationary phase and to remove an agent (e.g., a competitor or free binder) used to facilitate said detachment of the selected, stimulated, and transduced cells from an output composition. In certain embodiments, the provided methods include methods for selecting cells, e.g., CD3+, CD4+, and CD8+ T cells, from other components, e.g., from other cells in a sample, immobilizing the cells on a stationary phase of a chromatography column, stimulating and transducing the selected cells that are immobilized on the stationary phase, and eluting and / or collecting the selected, stimulated, and transduced cells by gravity flow.

[0107] In certain aspects, the provided methods are improvements over and comparisons to numerous existing methods for generating engineered cells (e.g., T cells) for cell therapy, e.g., that include one or more additional steps after cell selection (e.g., immunoaffinity-based selection) prior to stimulating and transducing the cells. In some embodiments, the one or more additional steps present in existing methods may include an elution step or a step using a competitive reagent or free binder to recover or harvest the selected cells and / or a step to remove reagents (e.g., magnetic bead reagents or antibodies) used in the selection. In some embodiments, such additional steps may extend the process for engineering cells for cell therapy and / or may result in manipulation of the cells during the process, which may affect their differentiation state, viability, or cell number. In certain aspects, the provided methods include separate selection, stimulation, and transduction steps, generating a population of selected, stimulated, and transduced cells in a shorter amount of time compared to methods that require additional steps to detach the cells from the stationary phase and to remove agents used to promote detachment.

[0108] In certain aspects, the methods generate a selected, stimulated, and transduced cell output population (also referred to as an output composition) suitable for subsequent rounds of downstream processing (e.g., culturing, expansion and / or incubation, stimulation and / or selection (e.g., polishing)) within 24 hours of initiating on-column stimulation and transduction, also referred to herein as on-column stimulation and on-column transduction. In some embodiments, the methods generate a selected, stimulated, and transduced cell output population (e.g., an output composition) suitable for downstream processing (e.g., subsequent rounds of culturing, expansion and / or incubation, stimulation and / or selection (e.g., polishing)) within 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours or about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 hours of initiating on-column stimulation and transduction. In some embodiments, the methods generate a selected, stimulated, and transduced cell output population suitable for downstream processing (e.g., subsequent rounds of culture, expansion and / or incubation, stimulation and / or selection (e.g., polishing)) within 6, 5, 4, 3, or 2 hours or within about 6, 5, 4, 3, or 2 hours. In some embodiments, the methods generate a selected, stimulated, and transduced cell output population suitable for downstream processing (e.g., subsequent rounds of culture, expansion and / or incubation, stimulation and / or selection (e.g., polishing)) within 3-6 hours or within about 3-6 hours. In some embodiments, the methods generate a selected, stimulated, and transduced cell output population suitable for downstream processing (e.g., subsequent rounds of culture, expansion and / or incubation, stimulation and / or selection (e.g., polishing)) within 4-6 hours or within about 4-6 hours.In some embodiments, the method generates a selected, stimulated, and transduced cell output population suitable for downstream processing (e.g., subsequent rounds of culture, expansion, and / or incubation, stimulation and / or selection (e.g., polishing)) within 5-6 hours or about 5-6 hours. In some embodiments, the method generates a selected, stimulated, and transduced cell output population suitable for downstream processing (e.g., subsequent rounds of culture, expansion, and / or incubation, stimulation and / or selection (e.g., polishing)) within 4-5 hours or about 4-5 hours. In some embodiments, the method generates a selected, stimulated, and transduced cell output population suitable for downstream processing (e.g., subsequent rounds of culture, expansion, and / or incubation, stimulation and / or selection (e.g., polishing)) within 6 hours or about 6 hours. In some embodiments, within 5.5 hours or about 5.5 hours, a selected, stimulated, and transduced cell output population suitable for downstream processing (e.g., subsequent rounds of culture, expansion and / or incubation, stimulation and / or selection (e.g., polishing)) is generated. In some embodiments, within 5 hours or about 5 hours, a selected, stimulated, and transduced cell output population suitable for downstream processing (e.g., subsequent rounds of culture, expansion and / or incubation, stimulation and / or selection (e.g., polishing)) is generated. In some embodiments, within 4.5 hours or about 4.5 hours, a selected, stimulated, and transduced cell output population suitable for downstream processing (e.g., subsequent rounds of culture, expansion and / or incubation, stimulation and / or selection (e.g., polishing)) is generated. In some embodiments, within 4 hours or about 4 hours, a selected, stimulated, and transduced cell output population is produced that is suitable for downstream processing (e.g., culture, subsequent rounds of expansion and / or incubation, stimulation and / or selection (e.g., polishing)).In some embodiments, within 3 hours or about 3 hours, a selected, stimulated, and transduced cell output population is produced that is suitable for downstream processing (e.g., culture, subsequent rounds of expansion and / or incubation, stimulation and / or selection (e.g., polishing)).

[0109] In some embodiments, the method involves the use of a stimulating agent that can bind to a molecule on the surface of a cell, thereby delivering a stimulatory signal to the cell. In some embodiments, the stimulating agent is contained in an oligomeric stimulating reagent (e.g., a streptavidin mutein oligomer conjugated to anti-CD3 and anti-CD28 Fab) that can be added to the stationary phase. In some embodiments, the stimulation results in the spontaneous detachment of selected cells from the stationary phase, thereby allowing for collection and / or elution of selected, stimulated, and transduced cells in the absence of additional processing steps to remove agents used to detach cells from the stationary phase and promote said detachment from the output stimulator cell composition. In some embodiments, the stimulation results in the spontaneous detachment or release of selected cells from the stationary phase, thereby allowing for collection and / or elution of selected, stimulated, and transduced cells by gravity flow. In some embodiments, gravity flow is relied upon to collect or elute cells that have spontaneously detached from the column (e.g., the stationary phase). In some embodiments, a wash step in combination with gravity flow can be used, for example, to elute spontaneously detached cells from the column (e.g., the stationary phase). In some embodiments, the wash step may simply involve adding cell culture medium (e.g., serum-free medium), e.g., the same medium present in the cell input composition, to the column prior to adding or immobilizing the cells on the stationary phase. In certain aspects, the method successfully produces an uncontaminated (e.g., free of agents used for detachment (e.g., competitors, free binders) and / or selection agents) composition of selected, stimulated, and transduced cells suitable for further processing, e.g., culturing, expansion, incubation, or subsequent rounds of stimulation and / or selection (e.g., polishing), within 24 hours of initiating on-column stimulation and transduction.

[0110] In certain aspects, the method involves the use of an oligomeric stimulatory reagent comprising a stimulatory agent capable of delivering a stimulatory signal to a target cell (e.g., a T cell). An exemplary oligomeric reagent comprises a streptavidin mutein oligomer reversibly bound or conjugated to one or more antibodies or fragments thereof capable of delivering a stimulatory signal to a target cell, e.g., a T cell. In some embodiments, the oligomeric stimulatory reagent is a streptavidin mutein oligomer conjugated to anti-CD3 and anti-CD28 Fab. Existing reagents for use in in vitro stimulation of T cells, such as in the absence or presence of low amounts of exogenous growth factors, are known (see, e.g., U.S. Patent No. 6,352,694 B1 and European Patent EP 0 700 430 B1). Generally, such reagents can use beads, e.g., magnetic beads, with a diameter greater than 1 μm, to which various binding agents (e.g., anti-CD3 antibodies and / or anti-CD28 antibodies) are immobilized. However, in some cases, such magnetic beads are difficult to incorporate into methods for stimulating cells under conditions required, for example, for clinical trials or therapeutic purposes, because one must ensure that these magnetic beads are substantially or completely removed before administering the engineered T cells to a subject. In some embodiments, such removal, such as by exposing the cells to a magnetic field, can reduce the yield of viable cells available for cell therapy. In certain cases, such reagents, e.g., stimulatory reagents, containing magnetic beads must be incubated with cells for a minimum amount of time to allow for a sufficient amount of T cell detachment from the stimulatory reagent. Furthermore, reagents such as beads are not readily compatible with column chromatography due to physical constraints.

[0111] The provided methods utilizing oligomeric stimulating reagents (e.g., streptavidin mutein oligomers conjugated to anti-CD3 and anti-CD28 antibodies, e.g., Fabs) overcome such potential limitations. For example, in some embodiments, the provided methods include adding a soluble oligomeric reagent that is not bound to a solid support (e.g., beads) to a stationary phase to initiate stimulation. In some embodiments, the provided methods may include a step of reducing or minimizing the amount of residual oligomeric stimulating reagent that may be present at the end of the overall process of engineering cells for cell therapy. In some embodiments, the risk of residual reagent in output cells, e.g., engineered cells, produced or generated by the methods is reduced or avoided by the use of oligomeric reagents because the addition of a competing reagent or free binding agent can be used to dissociate (e.g., disrupt binding of) the oligomeric stimulating reagent from the stimulating agent in the composition containing the cells. In some embodiments, because the oligomeric stimulating reagent is soluble, it may be sufficient to reduce or remove the oligomeric stimulating reagent from the cells in the composition by one or more wash steps, for example, without the need to add a competing reagent or free binding agent. In some embodiments, this also means that a process that complies with GMP standards can be more easily established compared to other methods, such as those that require additional measures to ensure that the final population for administration is bead-free. Thus, in some aspects, removal or separation of the oligomeric stimulating reagent from the cells, such as by adding a competitor or free binder, or by one or more washing steps, results in little or no cell loss compared to removal or separation of a bead-based stimulating reagent. In some aspects, the timing of reduction, removal, or separation of the stimulating reagent or oligomeric stimulating reagent is not limited or is less limited than removal or separation of a bead-based stimulating reagent. Thus, in some aspects, the stimulating reagent or oligomeric stimulating reagent can be reduced, removed, or separated from the cells at any time or step during the provided methods.

[0112] Also provided are cells and populations prepared by the methods, including pharmaceutical populations and formulations and kits, systems and devices for carrying out the methods. Further provided are methods for the use of the cells and populations prepared by the methods, including methods for treatment, e.g., adoptive cell therapy, and pharmaceutical populations for administration to a subject.

[0113] A. Sample and Cell Preparation In certain embodiments, methods are provided herein that include selecting and / or enriching cells from a biological sample. In some embodiments, the methods provided include selecting cells or populations thereof from a biological sample, e.g., obtained from or derived from a subject, e.g., a subject with a particular disease or condition requiring or to which cell therapy is administered. In some aspects, the subject is a human, e.g., a patient requiring a particular therapeutic intervention, e.g., adoptive cell therapy, from which cells are isolated, processed, and / or manipulated. Thus, in some embodiments, the cells are primary cells, e.g., primary human cells. Samples include tissues, fluids, and other samples directly collected from a subject. Biological samples can be samples obtained directly from a biological source or samples to be processed. Biological samples include, but are not limited to, bodily fluids, e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue, and organ samples, including processed samples derived therefrom.

[0114] In some embodiments, the sample is a blood or blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsils, or other organ and / or cells derived therefrom. Samples include samples from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0115] In some examples, cells are obtained from the subject's circulating blood, for example, by apheresis or leukapheresis. The sample, in some embodiments, contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells and / or platelets, and in some embodiments, cells other than red blood cells and platelets.

[0116] In some embodiments, the sample is a sample containing T cells. 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 an apheresis sample. In some embodiments, the sample is a leukapheresis sample.

[0117] In some embodiments, blood cells collected from a subject are washed, e.g., to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or most or all divalent cations. In some aspects, the wash step is accomplished by a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 Cell Processor, Baxter) according to the manufacturer's instructions. In some aspects, the wash step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, after washing, the cells are washed in various biocompatible buffers, e.g., Ca 2+ / Mg 2+ In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

[0118] In some embodiments, a sample containing cells (e.g., an apheresis product or a leukapheresis product) is washed to remove one or more anticoagulants, such as heparin, added during apheresis or leukapheresis.

[0119] In some embodiments, a sample comprising cells (e.g., a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukapheresis product) is cryopreserved and / or cryoprotected (e.g., frozen) and then thawed prior to any steps for separation, selection, activation, stimulation, manipulation, transduction, transfection, incubation, culture, harvesting, formulation of a population of cells, and / or administration of the formulated cell population to a subject.

[0120] In certain embodiments, the apheresis product or leukapheresis product is cryopreserved and / or cryoprotected (e.g., frozen) and then thawed before being subjected to a cell selection or isolation process (e.g., a T cell selection or isolation process) as described below. In some embodiments, the thawed cell composition is subjected to dilution (e.g., with serum-free medium) and / or washing (e.g., with serum-free medium), which may, in some cases, remove or reduce unwanted or undesirable components. In some cases, dilution and / or washing removes or reduces the presence of cryoprotectants, e.g., DMSO, in the thawed sample, which, if not removed, may adversely affect cell viability, yield, or recovery upon extended room temperature exposure. In some embodiments, dilution and / or washing allows for the exchange of the medium of the thawed cryopreserved product with a serum-free medium, such as those described herein or in International Application No. PCT / US2018 / 064627, which are incorporated herein by reference.

[0121] In some embodiments, the serum-free medium comprises a basal medium supplemented with one or more supplements (e.g., OpTmizer™ T-Cell Expansion Basal Medium (ThermoFisher). In some embodiments, the one or more supplements are serum-free. In some embodiments, the serum-free medium comprises a basal medium supplemented with one or more additional components for the maintenance, proliferation, and / or activation of cells (e.g., T cells), e.g., as provided by additional supplements (e.g., OpTmizer™ T-Cell Expansion Supplement (ThermoFisher)). In some embodiments, the serum-free medium comprises a serum replacement supplement, e.g., an immune cell serum replacement, e.g., ThermoFisher, #A2596101, the CTS™ Immune Cell Serum Replacement, or a serum replacement supplement as described in Smith et al. Clin Transl Immunology. 2015 Jan;4(1): e31. In some embodiments, the serum-free medium further comprises an immune cell serum replacement as described in e31. In some embodiments, the serum-free medium further comprises a free form of an amino acid, such as, for example, L-glutamine. In some embodiments, the serum-free medium further comprises a dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine), such as, for example, the dipeptide in Glutamax™ (ThermoFisher). In some embodiments, the serum-free medium further comprises one or more recombinant cytokines, such as, for example, recombinant human IL-2, recombinant human IL-7, and / or recombinant human IL-15.

[0122] In some embodiments, after a cryopreserved and / or cryoprotected apheresis product or leukapheresis product has been subjected to a T cell selection or isolation step, no additional cryopreservation and / or cryoprotection step is performed during or between any of the subsequent steps, e.g., activation, stimulation, manipulation, transduction, transfection, incubation, culture, harvesting, formulation of a population of cells, and / or administration of a formulated cell population to a subject, etc. For example, T cells selected from a thawed cryopreserved and / or cryoprotected apheresis product or leukapheresis product are not again cryopreserved and / or cryoprotected before being thawed for downstream processing, e.g., transduction, etc.

[0123] In certain embodiments, the cryopreserved and / or cryoprotected apheresis or leukapheresis product is banked (e.g., without cell selection before freezing the sample), which, in some aspects, allows for more flexibility for subsequent manufacturing steps. In one aspect, banking cells before selection increases cell yield for downstream processes, and banking cells earlier may mean that the cells are healthier and easier to meet manufacturing success criteria. In another aspect, after thawing, the cryopreserved and / or cryoprotected apheresis or leukapheresis product may be subjected to one or more different selection methods. The advantage of this approach is, among other things, increasing the availability, efficiency, and / or other aspects of the cells of cell therapy for the treatment of a disease or condition of interest, such as in the sample donor and / or another recipient.

[0124] In some embodiments, the sample (e.g., an apheresis or leukapheresis sample) is collected at a time point after the donor is diagnosed with a disease or condition and is cryopreserved and / or cryoprotected before or without prior cell selection (e.g., without prior T cell selection, such as chromatographic selection). In some aspects, the timing of cryopreservation is also before the donor receives one or more of the following: any initial treatment for the disease or condition, any targeted therapy or any therapy designated for treatment for the disease or condition, or any therapy other than radiation therapy and / or chemotherapy. In some embodiments, the sample is collected after the first recurrence of the disease after initial treatment for the disease and before the donor or subject receives a subsequent treatment for the disease. The initial and / or subsequent treatment can be a therapy other than cell therapy. In some embodiments, the collected cells can be used in cell therapy after the initial treatment and / or subsequent treatment. In one embodiment, cryopreserved and / or cryoprotected samples without prior cell selection can help reduce initial costs, such as those associated with untreated patients in randomized clinical trials who may later require crossover therapy.

[0125] In some embodiments, the sample (e.g., an apheresis or leukapheresis sample) is collected after a second recurrence of disease after a second-line treatment for the disease and before the donor or subject receives subsequent treatment for the disease, and is cryopreserved and / or cryoprotected before or without prior cell therapy (e.g., without prior T cell selection, such as chromatographic selection). In some embodiments, the patient is identified as likely to relapse after a second-line treatment, for example, by assessing certain risk factors. In some embodiments, the risk factors are based on disease type and / or genetics, such as double-matched lymphoma, primary refractory cancer, or active B-cell lymphoma. In some embodiments, the risk factors are based on clinical findings, such as early recurrence after first-line treatment or other poor prognostic indicators after treatment (e.g., an IPI (International Prognostic Index) > 2).

[0126] In some embodiments, the sample (e.g., an apheresis or leukapheresis sample) is collected at a time before the donor or subject is diagnosed with the disease and is cryopreserved and / or cryoprotected before or without prior cell therapy (e.g., without prior T cell selection, such as chromatographic selection). In some aspects, the donor or subject may be determined to be at risk for developing the disease. In some aspects, the donor or subject may be a healthy subject. In certain cases, such as when cell therapy is needed at a later stage in life, the donor or subject may choose to bank or store cells without being considered at risk for developing the disease or diagnosed with the disease. In some embodiments, the donor or subject may be considered at risk for developing the disease based on factors such as, for example, gene mutations, genetic abnormalities, gene disruptions, family history, protein abnormalities (e.g., defects in protein production and / or protein processing), and lifestyle choices that may increase the risk of developing the disease. In some embodiments, the cells are collected as a prophylactic.

[0127] In some embodiments, cryopreserved and / or cryoprotected samples of cells (e.g., apheresis or leukapheresis samples), such as samples of cells that have not been subjected to pre-cell selection (e.g., pre-T cell selection, e.g., chromatographic selection), are stored or banked for periods of 12, 24, 36, or 48 hours or more. In some embodiments, samples are stored or banked for periods of 1, 2, 3, or 4 weeks or more. In some embodiments, samples are stored or banked long-term. In some embodiments, samples are stored for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years or more, or longer.

[0128] In some embodiments, apheresis or leukapheresis samples collected from donors are transported to a storage or processing facility in a cryogenic environment and / or cryogenically stored at a storage facility or processed at a processing facility. In some embodiments, the sample is processed before transport, e.g., by selecting T cells, e.g., CD4+ and / or CD8+ T cells. In some embodiments, such processing is performed after transport of the sample and before cryogenic storage. In some embodiments, processing is performed after thawing the sample after cryogenic storage.

[0129] By allowing a donor to store their cells at a stage when they, and therefore their cells, have not undergone extensive treatment for a disease and / or are not suffering from or diagnosed with a disease or condition, such cells may have certain advantages for use in cell therapy compared to cells harvested after one or more treatments. For example, cells harvested before one or more treatments may be healthier, exhibit higher levels of certain cellular activity, grow more rapidly, and / or be more receptive to genetic manipulation than cells that have undergone multiple treatments. Another example of an advantage of the embodiments described herein may include convenience. For example, by collecting, optionally processing, and storing a donor's cells before the donor needs cell therapy, the cells will be readily available if or when a recipient later needs them. This could increase laboratory capacity for apheresis, thereby providing a technology with greater flexibility for planning the apheresis collection process.

[0130] Exemplary methods and systems for cryogenic storage and processing of cells from a sample, such as an apheresis sample, can include those described in International Publication No. 2018170188. In some embodiments, the methods and systems involve collecting an apheresis before a patient requires cell therapy and then subjecting the apheresis sample to cryopreservation for later use in a process for engineering cells, such as T cells, with a recombinant receptor (e.g., a CAR). In some cases, such processes can include those described herein. In some embodiments, the apheresis sample is collected from a subject and cryopreserved prior to subsequent T cell selection, activation, stimulation, engineering, transduction, transfection, incubation, culture, harvesting, formulation of a population of cells, and / or administration of the formulated cell population to the subject. In such examples, the cryopreserved apheresis sample is thawed before being subjected to one or more selection steps, such as any of those described herein.

[0131] In some embodiments, a cryopreserved and / or cryoprotected sample of cells (e.g., an apheresis or leukapheresis sample), such as a sample of cells that has not been subjected to prior cell selection (e.g., has not been subjected to prior T cell selection, such as chromatographic selection), is thawed prior to its use for downstream processing for the manufacture of a cell population for cell therapy, e.g., a T cell population comprising CAR+ T cells. In some embodiments, such a cryopreserved and / or cryoprotected sample of cells (e.g., an apheresis or leukapheresis sample) is used with the processes provided herein for designing T cell therapies, such as CAR+ T cell therapies. In certain examples, no further step of cryopreservation is performed before or during the harvesting / formulation step.

[0132] B. Drug and Reagent Systems In certain embodiments, provided herein are methods that include selecting and / or enriching cells (e.g., T cells) from a biological sample using an agent (selection agent) that binds to a cell surface marker on cells present in the biological sample. In provided embodiments, the biological sample is any of those described in Section IA. In some embodiments, the biological sample is a sample containing T cells. In provided embodiments, the selection agent binds to or is immobilized on a chromatography matrix (e.g., a stationary phase) contained in a chromatography column of a device provided herein and performs specific selection of target cells of interest (e.g., T cells), as described in Section IC, thereby immobilizing the target cells (e.g., T cells) on the chromatography matrix (e.g., stationary phase). In some embodiments, the selection agent can be indirectly bound to the chromatography matrix (e.g., stationary phase) via a reagent, e.g., a selection reagent. In some embodiments, the selection reagent is covalently or non-covalently bound to the stationary phase of the column. In some embodiments, the selection reagent is a reagent that reversibly immobilizes the selection agent on the chromatography matrix (e.g., stationary phase). Exemplary selection reagents to which selection agents are attached for use in connection with the provided devices and methods are described in Section II.B.2.

[0133] In some embodiments, the selection reagent to which the selection agent binds provides a reversible system that reversibly associates the selection agent with the reagent. Exemplary reversible systems for chromatographic cell selection include those described in International Publication No. WO 2013 / 124474. In some embodiments, as described in more detail herein, the reversible system employs a reagent comprised of a streptavidin mutein molecule that reversibly binds to the selection agent via a streptavidin-binding peptide binding partner contained by the selection agent. In some embodiments, adding a free binding partner or competitor (also referred to as a competitor) disrupts the bond between the selection agent and the reagent, thereby reversing the binding of the selection agent from the reagent and releasing the immobilized cells from the selection reagent. For example, in the case of a streptavidin mutein / streptavidin-binding peptide system, an exemplary competitor is biotin (e.g., D-biotin) or a biotin analog.

[0134] In some embodiments, reversibility of binding of the selection agent on the chromatography matrix as provided herein is not required because on-column stimulation of cells immobilized on the chromatography matrix promotes downregulation of the molecule used for cell selection (i.e., the selection marker), resulting in spontaneous detachment or release of the cells from the stationary phase. Thus, cell release or detachment can occur without any additional steps or reagents. In some aspects, cells can be collected by gravity flow, e.g., by adding a medium or other solution to the chromatography column. In certain embodiments, the added medium or other solution does not contain a competitor or free binding agent to promote cell detachment from the stationary phase. For example, in the case of a streptavidin mutein / streptavidin-binding peptide system, cells can be collected by gravity flow after adding a wash or medium to the column, where the wash or medium does not contain a free binding partner or competitor, e.g., biotin (e.g., D-biotin) or a biotin analog.

[0135] In certain embodiments, provided herein are methods that include on-column stimulation of cells (e.g., T cells) immobilized on a chromatography column, such as with a selection agent or reagent. In provided embodiments, stimulation is performed using one or more agents (one or more stimulatory agents) to stimulate the cells to bind one or more receptor molecules on the cells to deliver a signal to the cells. In some embodiments, the one or more stimulatory agents are for stimulating the T cells and for delivering a primary signal to the T cells (e.g., via TCR complex signaling). and provides a costimulatory signal to the T cell (e.g., by signaling from a costimulatory receptor). In some embodiments, the selection agent and at least one of the one or more stimulatory agents are different. In some embodiments, the selection agent and each of the one or more stimulatory agents are different. In some embodiments, an agent can be used as both a selection agent and one of the one or more stimulatory agents in connection with the provided methods. In some embodiments, the one or more stimulatory agents are attached to a reagent (e.g., a stimulatory reagent) that delivers a stimulatory signal to the cell. In some embodiments, the reagent includes multiple binding sites for attaching each of the one or more stimulatory agents such that the stimulatory agents are multimerized on the agent. In certain embodiments, such stimulatory reagents are oligomeric or polymeric reagents composed of multiple individual molecules, such as multiple protein units or complexes (e.g., tetramers). Exemplary stimulatory reagents to which one or more stimulatory agents are attached, such as oligomeric stimulatory reagents for use in connection with the provided devices and methods, are described in Section IB-2. In certain embodiments, the stimulation reagent is added to the chromatography column containing the immobilized cells under suitable conditions to deliver a signal to the cells. For example, on-column stimulation is performed at a suitable temperature as described herein by heating the device described and provided herein to a suitable physiological temperature to allow signal transduction events in the cells, for example, a temperature between 30°C or about 30°C and 39°C or about 39°C, for example, about 37°C ± 2°C, such as 37°C or about 37°C.

[0136] In some embodiments, a stimulating reagent to which one or more stimulating agents are bound provides a reversible system in which one or more stimulating agents are reversibly associated with the reagent. Exemplary reversible systems for cellular stimulation include those described in International Publication No. WO 2015 / 158868, WO 2017068421, or WO 2018 / 197949. In some embodiments, the reversible system employs a reagent composed of a streptavidin mutein oligomer or polymer that reversibly binds to one or more stimulating agents via a streptavidin-binding peptide binding partner contained by the one or more stimulating agents. In some embodiments, adding a free binding partner or competitor (also called a competitor) disrupts the bond between the one or more stimulating agents and the reagent, thereby reversing the binding of the one or more stimulating agents from the reagent and terminating or preventing the stimulatory signal delivered by the one or more stimulating agents of the stimulating reagent. For example, in the case of a streptavidin mutein / streptavidin-binding peptide system, an exemplary competitor is biotin (eg, D-biotin) or a biotin analog.

[0137] In certain aspects, provided herein are methods using a reversible system in which at least one agent (e.g., a selection agent or stimulating agent) capable of binding to a molecule on the surface of a cell (a cell surface molecule) is reversibly associated with a reagent (e.g., a selection agent or stimulating agent). In some cases, the reagent comprises multiple binding sites capable of reversibly binding to the agent (e.g., a selection agent or stimulating agent). In some cases, the reagent (e.g., a selection agent or stimulating agent) is a multimerization reagent. In some embodiments, at least one agent (e.g., a selection agent or stimulating agent) comprises at least one binding site B capable of specifically binding to an epitope or region of a molecule, and also comprises a binding partner C that specifically binds to at least one binding site Z of the reagent (e.g., a selection agent or stimulating agent). In some cases, the binding interaction between binding partner C and at least one binding site Z is a non-covalent interaction. In some embodiments, the binding interaction, e.g., a non-covalent interaction, between binding partner C and at least one binding site Z is reversible.

[0138] In some embodiments, the reversible association can be mediated in the presence of a substance, such as a competitor or free binder, and thus includes a binding site that can also bind to at least one binding moiety Z. Generally, the substance (e.g., competitor or free binder) can function as a competitor by virtue of its higher binding affinity for binding site Z present in the reagent and / or by being present at a higher concentration than binding partner C, thereby displacing and / or dissociating binding partner C from the reagent. In some embodiments, the affinity of the substance (e.g., competitor or free binder) for at least one binding site Z is higher than the affinity of binding partner C of the agent (e.g., selection agent or stimulating agent) for at least one binding site Z. Thus, in some cases, the bond between binding site Z of the reagent and binding partner C of the agent (e.g., selection agent or stimulating agent) can be disrupted by the addition of a substance (e.g., competitor or free binder), thereby rendering the association between the agent (e.g., selection agent or stimulating agent) and the reagent (e.g., selection reagent or stimulating reagent) reversible.

[0139] The reagent that can be used in such reversible system is described and known in the art, for example, see U.S. Patent No. 5,168,049; U.S. Patent No. 5,506,121; U.S. Patent No. 6,103,493; U.S. Patent No. 7,776,562; U.S. Patent No. 7,981,632; U.S. Patent No. 8,298,782; U.S. Patent No. 8,735,540; U.S. Patent No. 9,023,604; and International Publication No. WO2013 / 124474 and International Publication No. WO2014 / 076277.Non-limiting examples of reagents and binding partners that can form reversible interactions, and substances that can reverse such binding (for example, competitors or free binders) are described below.

[0140] 1. Drugs In some embodiments, the agent (e.g., a selection agent or stimulating agent) has one or more binding sites B for binding to a molecule on the surface of a cell, e.g., a cell surface molecule. Thus, in some cases, the agent (e.g., a selection agent or stimulating agent) includes a binding site B or multiple binding sites B, where specific binding between the agent (e.g., a selection agent or stimulating agent) and a molecule on the surface of a target bacterium involves an interaction between B and the molecule. In some embodiments, the agent includes only a single binding site, i.e., is monovalent. In some embodiments, the agent (e.g., a selection agent or stimulating agent) has at least two, e.g., multiple binding sites B, e.g., three, four, or five binding sites B, that can bind to a cell surface molecule. In some such embodiments, at least two or more binding sites B can be identical. In some embodiments, one or more of the at least two or more binding sites B can be different (e.g., B1 and B2, etc.).

[0141] In some embodiments, one or more different agents (e.g., one or more different, e.g., selection agents or stimulating agents or other agents that bind to molecules on cells) reversibly bind to a reagent (e.g., selection agent or stimulating agent). In some embodiments, at least two, three, four, or more different agents (e.g., selection agents or stimulating agents) reversibly bind to the same reagent. In some embodiments, at least two different agents (e.g., selection agents or stimulating agents) reversibly bind to the same reagent, whereby each agent comprises a binding site B or multiple binding sites B for specific binding between the agent and the molecule. In some embodiments, at least two or more agents (e.g., selection agents or stimulating agents) comprise the same binding site B, e.g., to bind to the same or substantially the same molecule. In some embodiments, at least two or more agents (e.g., selection agents or stimulating agents) comprise different binding sites B, e.g., to bind to different molecules. In some embodiments, the first agent (e.g., a first selective agent or a first stimulating agent) comprises binding site B1, B2, B3, B4, etc., and the second agent (e.g., a second selective agent or a second stimulating agent) comprises another binding site from binding sites B1, B2, B3, B4, etc. In some embodiments, the first agent (e.g., a first selective agent) comprises binding site B1, and the second agent (e.g., a second selective agent) comprises binding site B3. In some embodiments, the first agent (e.g., a first stimulating agent) comprises binding site B2, and the second agent (e.g., a second stimulating agent) comprises binding site B4. In any of such embodiments, the first agent and the second agent can comprise binding partner C1 or C2. In some embodiments, C1 and C2 can be the same. In some embodiments, C1 and C2 are different. In some embodiments, the first agent and the second agent comprise the same binding partner C1.

[0142] In some cases, the dissociation constant (K) of binding between the drug (e.g., via binding site B) and the binding site Z of the reagent is D ) is about 10 -2 M ~ about 10 -13 M or about 10 -3M ~ about 10 -12 M or about 10 -4 M ~ about 10 -11 M or about 10 -5 M ~ about 10 -10 In some embodiments, the dissociation constant (K) for binding between the binding agent and the molecule can have a value in the range of M. D ) is, for example, about 10 -3 ~about 10 -7 K of M D In some embodiments, the dissociation constant for binding between the binding agent and the molecule (K D ) is, for example, about 10 -7 ~Approx. 1×10 -10 K of M D The dissociation constants are in the high affinity range.

[0143] In some embodiments, dissociation of the binding between the agent and the molecule via binding site B occurs sufficiently rapidly, e.g., to allow the target cell to be stained or associated with the agent only transiently after disruption of the reversible bond between the agent and the agent. In some cases, the k off When expressed in terms of rate (also called dissociation rate constant), k off The speed is about 0.5 x 10 -4 sec -1 or more, approximately 1 × 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, approximately 1 × 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, approximately 1 × 10 -2 sec -1 or more, or about 5 × 10 -1 sec -1 or higher. off It is within the skill of the art to empirically determine the rate range (see, e.g., U.S. Patent Application Publication No. 2014 / 0295458). For example, a rate of, e.g., 4.0 x 10, can be selected so that most of the drug is removed or desorbed within one hour after disruption of the binding complex. -4 sec -1 A fairly high k off In other cases, a drug concentration of, for example, 1.0×10 may be used so that most of the drug is removed or desorbed from the cells within 3.5 hours after disruption of the binding complex. -4 sec -1 Lower k such as off A rate agent may be used.

[0144] In some embodiments, the K D , and K D , k of the bond formed between the binding site B of the drug (e.g., the selective agent or stimulatory agent) and the cell surface molecule off and k on The rate can be determined by any suitable means, for example, fluorescence titration, equilibrium dialysis, or surface plasmon resonance.

[0145] In some aspects, the cell surface molecule is a molecule to which an agent (e.g., a selection agent or a stimulatory agent) can be targeted. In some embodiments, the cell surface molecule is a peptide or a protein, such as a receptor, e.g., a membrane receptor protein. In some embodiments, the receptor is a lipid, polysaccharide, or nucleic acid. In some embodiments, a cell surface molecule that is a protein can be 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, opiate receptors, serotonin receptors, Ca2+ receptors, melanopsin, neurotransmitter receptors, e.g., ligand-gated receptors, voltage-gated receptors, or mechano-gated receptors, e.g., acetylcholine, nicotine, adrenergic agonists, norepinephrine, catecholamines, L-DOPA, dopamine, and serotonin (biogenic amines, e.g., acetylcholine, nicotine, adrenergic receptors, norepinephrine, catecholamines, L-DOPA, dopamine, and serotonin). The protein may be a receptor such as a phosphodiesterase (e.g., phosphodiesterase / enkephalin), a neuropeptide receptor, a receptor kinase such as a serine / threonine kinase, a tyrosine kinase, a porin / channel such as a chloride channel, a potassium channel, a sodium channel, an OMP protein, an ABC transporter (ATP-binding cassette transporter) such as an amino acid transporter, a Na glucose transporter, a Na / iodide transporter, an ion transporter such as a light-harvesting complex, a cytochrome c oxidase, an ATPase Na / K, H / K, Ca, a cell adhesion receptor such as a metalloprotease, an integrin, or a cadherin.

[0146] In some embodiments, the cell surface molecule can be an antigen that defines a desired cell population or subpopulation, for example, a population or subpopulation of blood cells, such as lymphocytes (e.g., T cells, T helper cells, e.g., CD4+ T helper cells, B cells, or natural killer cells), monocytes, or stem cells, e.g., CD34-positive peripheral blood stem cells or Nanog- or Oct-4-expressing stem cells. Examples of T cells include CMV-specific CD8+ T lymphocytes, cytotoxic T cells, memory T cells, and regulatory T cells (Tregs). An example of a Treg is a CD4CD25CD45RA Treg cell, and an example of a memory T cell is a CD62LCD8+-specific central memory T cell. The cell surface molecule can also be a marker for tumor cells.

[0147] As explained above, in some embodiments, an agent (e.g., a selection agent or stimulating agent) has a binding partner C in addition to a binding site B that can bind to a cell surface molecule. In some aspects, this binding partner C can bind to a binding site Z of a reagent (e.g., a selection agent or stimulating agent (e.g., an oligomeric stimulating reagent)), where the reagent has one or more binding sites for binding partner C. In some embodiments, the non-covalent bond that can be formed between the binding partner C included in the agent (e.g., a selection agent or stimulating agent) and the binding site(s) Z of the reagent (e.g., a selection agent or stimulating reagent (e.g., an oligomeric stimulating reagent)) can be a bond of any desired strength and affinity, and can be disruptable or reversible under the conditions in which the method is performed. An agent (e.g., a receptor binding agent or a selection agent) can include at least one, e.g., two, three, or more, additional binding partners C, and a reagent (e.g., a selection agent or stimulating reagent (e.g., an oligomeric stimulating reagent)) can include at least two, e.g., three, four, five, six, seven, eight, or more binding sites Z for the binding partner C included in the agent (e.g., a selection agent or stimulating agent). As described in U.S. Pat. No. 7,776,562, U.S. Pat. No. 8,298,782, or WO 2002 / 054065, any combination of binding partner C and a reagent having one or more corresponding binding sites Z can be selected such that the binding partner C and binding site Z can reversibly bind in a complex, e.g., to cause an avidity effect, for example.

[0148] The binding partner C included in the agent (e.g., a selection agent or stimulator) can be, for example, hydrocarbon-based (including polymeric), and examples include nitrogen, phosphorus, sulfur, carbene, halogen, or pseudohalogen groups. In some embodiments, it can be an alcohol, organic acid, inorganic acid, amine, phosphine, thiol, disulfide, alkane, amino acid, peptide, oligopeptide, polypeptide, protein, nucleic acid, lipid, sugar, oligosaccharide, or polysaccharide. As a further example, it can be a cation, anion, polycation, polyanion, polycation, electrolyte, polyelectrolyte, carbon nanotube, or carbon nanofoam. Generally, such binding partner C has a higher affinity for the binding site of the reagent than others. Examples of respective binding partners C include, but are not limited to, crown ethers, immunoglobulins, fragments thereof, and proteinaceous binding molecules with antibody-like functions.

[0149] In some embodiments, binding partner C included in the agent (e.g., selection agent or stimulant) comprises biotin, and the reagent comprises a streptavidin analog or an avidin analog that reversibly binds to biotin. In some embodiments, binding partner C included in the agent (e.g., selection agent or stimulant) comprises a biotin analog that reversibly binds to streptavidin or avidin, and the reagent comprises streptavidin, avidin, a streptavidin analog, or an avidin analog that reversibly binds to the respective biotin analog. In some embodiments, binding partner C included in the agent (e.g., selection agent or stimulant) comprises a streptavidin or an avidin-binding peptide, and the reagent comprises a streptavidin, avidin, a streptavidin analog, or an avidin analog that reversibly binds to the respective streptavidin or avidin-binding peptide. For purposes of this specification, the term analog is used interchangeably with the term mutein in reference to mutant forms of streptavidin (eg, streptavidin analogs or streptavidin muteins) or avidin (eg, avidin analogs or avidin muteins).

[0150] In some embodiments, the reagent (e.g., the selection or stimulating reagent) is or includes streptavidin, such as a streptavidin mutein, including any of those described above (e.g., described in SEQ ID NOS: 3-6), and the binding partner C included in the agent (e.g., the selection or stimulating agent) can include a streptavidin-binding peptide. In some embodiments, the streptavidin-binding peptide can include a sequence having the general formula set forth in SEQ ID NO: 9, such as the sequence set forth in SEQ ID NO: 10. In some embodiments, the streptavidin-binding peptide sequence has the general formula set forth in SEQ ID NO: 11, such as that set forth in SEQ ID NO: 12. In one example, the streptavidin-binding peptide sequence is Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (also known as Strep-tag® and set forth in SEQ ID NO: 7). In one example, the streptavidin-binding peptide sequence is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also known as Strep-tag® II and set forth in SEQ ID NO: 8). In some embodiments, the streptavidin-binding peptide ligand comprises a consecutive arrangement of at least two streptavidin-binding modules, wherein the distance between the two modules is between 0 and 50 amino acids, wherein one link module has 3-8 amino acids and comprises at least the sequence His-Pro-Xaa (SEQ ID NO: 9), where Xaa is glutamine, asparagine, or methionine, and the other link module has the same or a different streptavidin peptide ligand, e.g., set forth in SEQ ID NO: 11 (see, e.g., WO 02 / 077018; U.S. Pat. No. 7,981,632). In some embodiments, the streptavidin-binding peptide ligand comprises a sequence having a formula set forth in any of SEQ ID NOs: 13 or 14. In some embodiments, the streptavidin-binding peptide ligand has a sequence of amino acids set forth in any of SEQ ID NOs: 15-19.In most cases, all of these streptavidin-binding peptides bind to the same binding site, i.e., the biotin-binding site of streptavidin. When one or more such streptavidin-binding peptides are used as binding partner C, e.g., C1 and C2, the multimerization reagent is typically a streptavidin mutein.

[0151] In some embodiments, the streptavidin-binding peptide may be further modified. In some embodiments, the streptavidin-binding peptide may comprise the peptide sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also known as Strep-tag® II and set forth in SEQ ID NO: 8) conjugated to nickel-charged trisNTA (also known as His-STREPPER or His / Strep-tag® II adaptor).

[0152] In some embodiments, the binding partner C of an agent (e.g., a receptor binding agent or a selection agent) comprises a moiety known to those skilled in the art as an affinity tag. In such embodiments, the reagent may comprise a corresponding binding partner, e.g., an antibody or antibody fragment, known to bind to the affinity tag. Some examples of known affinity tags include binding partner C included in an agent (e.g., a selection agent or stimulatory agent), dinitrophenol or digoxigenin, oligohistidine, polyhistidine, immunoglobulin domain, maltose binding protein, glutathione-S-transferase (GST), chitin-binding protein (CBP) or thioredoxin, calmodulin-binding peptide (CBP), FLAG'-peptide, HA tag (sequence: Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) (SEQ ID NO: 20), VSV-G tag (sequence: Tyr-Thr-Asp-Ile-Glu-Met-Asn-Arg-Leu-Gly-Lys) (SEQ ID NO: 21), HSV tag (sequence: Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp- and the like. The epitope may comprise the T7 epitope (Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly) (SEQ ID NO: 23), maltose binding protein (MBP), an HSV epitope of herpes simplex virus glycoprotein with the sequence Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp (SEQ ID NO: 24), a "myc" epitope of the transcription factor c-myc with the sequence Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu (SEQ ID NO: 25), a V5 tag (sequence: Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr) (SEQ ID NO: 26), or glutathione-S-transferase (GST). In such embodiments, the complex formed between one or more binding sites Z of the reagent, which may be an antibody or antibody fragment, and the antigen can be competitively disrupted by adding free antigen, i.e., a free peptide (epitope tag) or free protein (such as, for example, MBP or CBP).In some embodiments, the affinity tag can also be an oligonucleotide tag, which in some cases can be used to hybridize to an oligonucleotide having a complementary sequence, for example, linked to or contained in the reagent.

[0153] Further examples of suitable binding partners C include, but are not limited to, lectins, protein A, protein G, metals, metal ions, nitrilotriacetic acid derivatives (NTA), RGD motifs, dextran, polyethyleneimine (PEI), redox polymers, glycoproteins, aptamers, dyes, amylose, maltose, cellulose, chitin, glutathione, calmodulin, gelatin, polymyxin, heparin, NAD, NADP, lysine, arginine, benzamidine, polyU, or oligothymidine. Lecithins such as concanavalin A are known to bind to polysaccharides and glycosylated proteins. Illustrative examples of dyes are triazine dyes, such as cibacron blue F3G-A (CB) or red HE-3B, which specifically bind to NADH-dependent enzymes. Typically, green A binds to CoA proteins, human serum albumin, and dehydrogenases. In some cases, the dyes 7-aminoactinomycin D and 4',6-diamidino-2-phenylindole bind to DNA. Generally, metal cations such as Ni, Cd, Zn, Co, or Cu are typically used to bind affinity tags, such as oligohistidine-containing sequences, for example, hexahistidine or His-Asn-His-Arg-His-Lys-His-Gly-Gly-Gly-Cys tags (MAT tags) (SEQ ID NO: 35), and N-methacryloyl-(L)-cysteine ​​methyl ester.

[0154] In some embodiments, binding between a binding partner C included in an agent (e.g., a selection agent or stimulant) and one or more binding sites Z of a reagent occurs in the presence of a divalent, trivalent, or tetravalent cation. In this regard, in some embodiments, the reagent includes a divalent, trivalent, or tetravalent cation, typically held by, e.g., complexed with, a suitable chelator. In some embodiments, the binding partner C included in an agent (e.g., a selection agent or stimulant) can include a moiety that includes, e.g., a complex, a divalent, trivalent, or tetravalent cation, etc. Examples of respective metal chelators include, but are not limited to, 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-dimer-capto-1-propanol (dimercaprol), porphine, and heme. As an example, EDTA chelates most monovalent, divalent, trivalent, and tetravalent metal ions, such as silver (Ag + ), calcium (Ca 2+ ), manganese (Mn 2+ ), copper (Cu 2+ ), iron (Fe 2+ ), cobalt (Co + ), and zirconium (Zr 4+ ), while BAPTA forms complexes with Ca 2+ As an illustrative example, a standard method used in the art is to use an oligohistidine tag and copper (Cu 2+ ) ions, nickel (Ni 2+ ) ions, cobalt (Co 2+ ) ion, or zinc (Zn 2+ ) ions, which are presented by the chelating agent nitrilotriacetic acid (NTA).

[0155] In some embodiments, the binding partner C included in the agent (e.g., a selection agent or stimulatory agent) comprises a calmodulin-binding peptide, and the reagent comprises a multimeric calmodulin, e.g., as described in U.S. Pat. No. 5,985,658. In some embodiments, the binding partner C included in the agent (e.g., a selection agent or stimulatory agent) comprises a FLAG peptide, and the reagent comprises an antibody that binds to the FLAG peptide, e.g., the FLAG peptide that binds to the monoclonal antibody 4E11 described in U.S. Pat. No. 4,851,341. In one embodiment, the binding partner C included in the agent (e.g., a selection agent or stimulatory agent) comprises an oligohistidine tag, and the reagent comprises an antibody or a transition metal ion that binds to the oligohistidine tag. In some cases, disruption of all these binding complexes can be achieved by chelation of the metal ion, e.g., calcium chelation, by adding, for example, EDTA or EGTA. In some embodiments, calmodulin, antibodies such as 4E11, or chelating metal ions or free chelators can be multimerized by conventional methods, such as biotinylation and complexation with streptavidin or avidin or oligomers thereof, or by introducing carboxyl residues into a polysaccharide, such as dextran, in a first step, essentially as described in Noguchi, A, et al. Bioconjugate Chemistry (1992) 3, 132-137, and then linking the calmodulin, antibody, or chelating metal ions or free chelators to the carboxyl groups in the backbone of the polysaccharide, such as dextran, via primary amino groups using conventional carbodiimide chemistry in a second step. In some such embodiments, the bond between a binding partner C contained in an agent (e.g., a selection agent or stimulatory agent) and one or more binding sites Z of the reagent can be disrupted by metal ion chelation. Metal chelation can be achieved, for example, by the addition of EGTA or EDTA.

[0156] In some embodiments, an agent (e.g., a selection agent or stimulatory agent) that specifically binds to a cell surface molecule may comprise, for example, an antibody, or a fragment thereof, or a proteinaceous binding molecule with antibody-like function. In some embodiments, binding site B of the agent is an antibody binding site, e.g., is or includes one or more complementarity-determining regions of an antibody. Examples of (recombinant) antibody fragments include, but are not limited to, Fab fragments, Fv fragments, single-chain Fv fragments (scFv), bivalent antibody fragments such as (Fab)2'-fragment diabodies, triabodies (Iliades, P., et al, FEB S Lett (1997) 409, 437-441), decabodies (Stone, E., et al, Journal of Immunological Methods (2007) 318, 88-94), and other domain antibodies (Holt, LJ, et al, Trends Biotechnol. (2003), 21, 11, 484-490). In some embodiments, the agent (e.g., receptor binding agent or selection agent) may comprise a bivalent artificial proteinaceous binding molecule, such as a dimeric lipocalin mutein, also known as Duocalin.

[0157] In some embodiments, an agent (e.g., a selection agent or stimulatory agent) may have a single binding site, i.e., it may be monovalent. Examples of monovalent agents (e.g., selection agents or stimulatory agents) include, but are not limited to, monovalent antibody fragments, proteinaceous binding molecules with antibody-like binding properties, antibodies, or MHC molecules. Examples of monovalent antibody fragments include, but are not limited to, Fab fragments, Fv fragments, and single-chain Fv fragments (scFv), such as bivalent single-chain Fv fragments.

[0158] In some embodiments, the agent (e.g., a selection agent or stimulating agent) is an antibody or antigen-binding fragment thereof, such as a Fab fragment, an Fv fragment, a single-chain Fv fragment (scFv), a bivalent antibody fragment, such as an F(ab')2-fragment, etc. In some embodiments, the agent (e.g., a selection agent or stimulating agent) is or is derived from a parent antibody known to bind to a cellular molecule of interest. Various antibody molecules or fragments thereof directed against cell surface molecules are well known in the art, and any of a variety of such can be used as an agent in the methods herein. In some embodiments, the agent (e.g., a selection agent or stimulating agent) is an antibody or fragment thereof that contains one or more amino acid substitutions in the variable heavy chain of a parent or reference antibody, e.g., to generate an antibody with altered affinity, or that exhibits a sufficiently fast off-rate as described above. For example, examples of such mutations are known in the context of variants of the anti-CD4 antibody 13B8.2 (see, e.g., U.S. Patent No. 7,482,000, U.S. Patent Application Publication No. 2014 / 0295458, or WO 2013 / 124474), and any of such mutations can be generated in another parent or reference antibody.

[0159] In some embodiments, agents (e.g., selection agents or stimulatory agents) that may be monovalent include, for example, monovalent antibody fragments or monovalent artificial binding molecules (proteinaceous or otherwise), such as muteins based on polypeptides of the lipocalin family (also known as "Anticalin®"), or bivalent molecules, such as antibodies, or fragments in which both binding sites are maintained, such as F(ab')2 fragments.

[0160] Examples of proteinaceous binding molecules with antibody-like functions include muteins based on polypeptides of the lipocalin family (see, e.g., WO 03 / 029462; Beste et al., Proc. Natl. Acad. Sci. USA (1999) 96, 1898-1903). Generally, lipocalins, such as bilin-binding protein, human neutrophil gelatinase-associated lipocalin, human Apolipoprotein D, or human tear lipocalin, have a native ligand-binding site that can be engineered to bind to a predetermined target. Further examples of proteinaceous binding molecules antibodies with antibody-like binding properties that can be used as agents (e.g., selection or stimulating agents) that specifically bind to cell surface molecules include, but are not limited to, so-called glubodies (see, e.g., WO 96 / 23879), proteins based on ankyrin scaffolds (Mosavi, LK, et al, Protein Science (2004) 13, 6, 1435-1448), or crystalline scaffolds (e.g., WO 01 / 04144), the proteins described in Skerra, J. Mol. Recognit. (2000) 13, 167-187, adnectins, tetranectins, and avimers. Generally, avimers, such as multivalent avimer proteins generated by exon shuffling of a family of human receptor domains, contain so-called A-domains that occur as chains of multiple domains in cell surface receptors (Silverman, J., et al, Nature Biotechnology (2005) 23, 1556-1561). Adnectins, generally derived from domains of human fibronectin, typically contain three loops that can be engineered for immunoglobulin-like binding to targets (Gill, DS & Damle, NK, Current Opinion in Biotechnology (2006) 17, 653-658).Tetranectins, which are generally derived from the respective human homotrimeric protein, also typically contain a loop region in the C-type lectin domain that can be engineered for desired binding. Peptoids, which can function in some cases as protein ligands, are typically oligo(N-alkyl)glycines that differ from peptides in that the side chain is attached to the amide nitrogen rather than the carbon atom. Peptoids are typically resistant to proteases and other modifying enzymes and can have much higher cell permeability than peptides (see, e.g., Kwon, Y.-U., and Kodadek, T., J. Am. Chem. Soc. (2007) 129, 1508-1509).

[0161] Further examples of suitable proteinaceous binding molecules include, but are not limited to, EGF-like domains, kringle domains, fibronectin type I domains, fibronectin type II domains, fibronectin type III domains, PAN domains, Gla 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 camelid 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" (Ill et al. Protein Eng (1997) 10, 949-57), so-called "minibody" (Martin et al, EMBO J (1994) 13, 5303-5309), diabody (Holliger et al, PNAS USA (1993)90, 6444-6448), so-called "Janusis" (Traunecker et al, EMBO J (1991) 10, 3655-3659, or Traunecker et al, Int J Cancer (1992) Suppl 7, 51-52), nanobodies, microbodies, affilins, affibodies, knottins, ubiquitin, zinc finger proteins, autofluorescent proteins, or leucine-rich repeat proteins. In some embodiments, the nucleic acid molecule with antibody-like function can be an aptamer. Generally, aptamers fold into a defined three-dimensional motif and exhibit high affinity for a given target structure.

[0162] a. Selective agent In certain aspects, the methods provided herein use a selection agent. In some embodiments, an agent as described in Section IB is a selection agent. In some embodiments, the selection agent binds to a molecule on the surface of a cell, such as a cell surface molecule. In some cases, the cell surface molecule is a selection marker. In some embodiments, the selection agent can specifically bind to a selection marker expressed by one or more of the cells in the sample. In some embodiments, throughout this disclosure, reference to specific binding to a molecule, such as a cell surface molecule or cell surface receptor, does not necessarily mean that the agent binds exclusively to such molecule. For example, an agent that specifically binds to a molecule may generally bind other molecules with much lower affinity, as determined, for example, by immunoassay, BIAcore®, KinExA3000 instrument (Sapidyne Instruments, Boise, ID), or other assay. In some cases, the ability of an agent to bind to a target molecule under particular binding conditions is such that its affinity or avidity is at least 5 times greater than the average affinity or avidity of the same agent for a sampling of random peptides or polypeptides of sufficient statistical size, e.g., at least 10, 20, 30, 40, 50, 100, 250, or 500 times greater, or even at least 1000 times greater.

[0163] In some embodiments, cells, e.g., target cells (e.g., T cells), have or express a molecule, e.g., a selection marker, on their surface, such that the cells to be selected are defined by the presence of at least one common specific molecule (e.g., selection marker). In some embodiments, a sample containing target cells may also contain additional cells that lack the molecule (e.g., selection marker). For example, in some embodiments, T cells may be selected from a sample containing multiple cell types, such as red blood cells or B cells. Selection marker and receptor molecule may be used interchangeably herein to refer to cell surface molecules.

[0164] In some embodiments, the selection agent is or comprises an agent selected from the group consisting of an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with immunoglobulin-like function, a molecule containing an Ig domain, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex; a receptor ligand; and binding fragments thereof; and / or the selection agent comprises an antibody fragment; the selection agent is or comprises a Fab fragment; the selection agent is selected from the group consisting of bivalent antibody fragments consisting of (Fab)2'-fragments and bivalent single-chain Fv (scFv) fragments; the selection agent is a monovalent antibody fragment selected from the group consisting of a Fab fragment, an Fv fragment, and an scFv; and / or the selection agent is a proteinaceous binding molecule antibody with antibody-like binding properties selected from the group consisting of an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, an ankyrin scaffold-based protein, a crystalline scaffold-based protein, an adnectin, and an avimer.

[0165] In some embodiments, the selection agent further comprises a binding partner C for binding to the reagent. In some embodiments, the selection agent is selected from the group consisting of biotin, streptavidin, and a biotin analogue that reversibly binds to avidin, such as Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 15), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16), Trp-Ser -His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18), and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19), a calmodulin-binding peptide that reversibly binds to calmodulin, a FLAG peptide that reversibly binds to an antibody that binds to the FLAG peptide, and an oligohistidine tag that reversibly binds to an antibody that binds to the oligohistidine tag.

[0166] In some embodiments, the reagent is or comprises streptavidin, a streptavidin mutein, aviding, or an avidin mutein, and the selection agent comprises a binding partner C capable of binding to such a reagent, such as biotin, a biotin analog, or a streptavidin-binding peptide. In some embodiments, the selection agent is biotin, a biotin analog that reversibly binds to streptavidin or avidin, the following: Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 15), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 16), No. 17), SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18), and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19). In certain embodiments, the reagent is or comprises a streptavidin mutein (e.g., as set forth in SEQ ID NO: 6), and binding partner C is a streptavidin-binding peptide, such as any set forth in SEQ ID NO: 8 or any one of 15-19. In some embodiments, the selection agent further comprises a streptavidin-binding peptide having the sequence SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16).

[0167] In some aspects, the cell surface molecule, e.g., a selection marker, can be an antigen that defines a desired cell population or subpopulation, e.g., a population or subpopulation of blood cells, e.g., lymphocytes (e.g., T cells, T helper cells, e.g., CD4+ T helper cells, B cells, or natural killer 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 can be a marker expressed on the surface of T cells or a subset of T cells, such as CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA, and / or CD45RO. Examples of T cells include cells such as CMV-specific CD8+ T lymphocytes, cytotoxic T cells, memory T cells, and regulatory T cells (Tregs). Examples of Tregs include CD4 CD25 CD45RA Treg cells, and examples of memory T cells include CD62L CD8+ specific central memory T cells.

[0168] For example, in some aspects, specific subpopulations of T cells, e.g., cells expressing positive or high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD3+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some embodiments, such cells are selected by incubation with one or more selection agents that selectively bind to such markers. The selection agent can be any binding molecule, such as an antibody or antibody fragment, that binds to such surface markers to perform positive or negative selection of T cells or subpopulations thereof.

[0169] In some embodiments, T cells are separated from PBMC samples by negative selection of markers expressed on non-T cells, e.g., B cells, monocytes, or other leukocytes, e.g., CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed on, or relatively highly expressed in, one or more naive-like, memory, and / or effector T cell subpopulations.

[0170] In some embodiments, CD8+ cells are further enriched or depleted for naive stem cells, central memory stem cells, effector memory stem cells, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment of central memory T (TCM) cells is performed to enhance efficacy, e.g., to improve long-term survival, expansion, and / or engraftment after administration, which in some aspects is particularly robust in such subpopulations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, efficacy is further enhanced by combining TCM-enriched CD8+ T cells and CD4+ T cells.

[0171] In certain embodiments, memory T cells are present in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted for the CD62L-CD8+ and / or CD62L+CD8+ fractions, for example, by using anti-CD8 and anti-CD62L antibodies as selection agents.

[0172] In some embodiments, enrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127, and in some aspects on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is performed by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment of central memory T (TCM) cells is performed starting with a negative fraction of cells selected on the basis of CD4 expression, which is subjected to negative selection based on CD14 and CD45RA expression and positive selection based on CD62L. Such selections are performed simultaneously in some aspects and sequentially in either order in other aspects. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation is also used to generate the CD4+ cell population or subpopulation, such that both the positive and negative fractions obtained from the CD4-based separation are retained and used in subsequent steps of the method, optionally in one or more additional positive or negative selection steps below. In some embodiments, the selection of the CD4+ cell population and the selection of the CD8+ cell population are performed simultaneously. In some embodiments, the selection of the CD4+ cell population and the CD8+ cell population are performed sequentially in either order. In some embodiments, methods for selecting cells can include those described in published U.S. Patent Application Publication No. 20170037369, which is incorporated herein by reference in its entirety.

[0173] In certain embodiments, a biological sample, for example, a sample of PBMCs or other white blood cells, is subjected to selection of CD4+ T cells, and both negative and positive fractions are retained. In certain embodiments, CD8+ T cells are selected from the negative fraction. In some embodiments, a biological sample is subjected to selection of CD8+ T cells, and both negative and positive fractions are retained. In certain embodiments, CD4+ T cells are selected from the negative fraction.

[0174] In some embodiments, a selection agent that specifically binds CD4 and a selection agent that specifically binds CD8 are used to generate a population enriched for CD4+ T cells and a population enriched for CD8+ T cells, respectively.

[0175] In a specific example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, and both the negative and positive fractions are retained. The negative fraction is then subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on markers characteristic of central memory T cells, such as CD62L or CCR7, with the positive and negative selections being performed in either order.

[0176] CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, or CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO-.

[0177] In some embodiments, the selection marker is a T cell co-receptor; the selection marker is or contains a member of the T cell antigen receptor complex; the selection marker is or contains a CD3 chain; the selection marker is or contains a CD3 zeta chain; the selection marker is or contains CD8; the selection marker is or contains CD4; the selection marker is or contains CD45RA; the selection marker is or contains CD27; the selection marker is or contains CD28; and / or the selection marker is or contains CCR7. In some embodiments, the selection marker is selected from the group consisting of CD3, CD4, and CD8. In some embodiments, the selection marker is CD3.

[0178] In some embodiments, the specific binding between the selection agent and the selection marker does not induce a signal or does not induce a stimulatory, activation, or proliferative signal for the T cell. In some embodiments, the selection agent comprises a monovalent antibody fragment that binds to CD3, CD8, or CD4. In some embodiments, the selection agent is an anti-CD3 Fab, an anti-CD8 Fab, or an anti-CD4 Fab. In some embodiments, the selection agent is an anti-CD3 Fab. In some embodiments, the anti-CD3 Fab comprises an OKT3 antibody Fab fragment. In some embodiments, the anti-CD3 Fab comprises a heavy chain variable chain having the sequence set forth in SEQ ID NO: 31 and a light chain variable chain having the sequence set forth in SEQ ID NO: 32.

[0179] In some embodiments, the selection marker may be CD4, and the selection agent specifically binds to CD4. In some aspects, the selection agent that specifically binds to CD4 may be selected from the group consisting of an anti-CD4 antibody, a bivalent antibody fragment of an anti-CD4 antibody, a monovalent antibody fragment of an anti-CD4 antibody, and a proteinaceous CD4-binding molecule with antibody-like binding properties. In some embodiments, the anti-CD4 antibody, e.g., a bivalent antibody fragment or a monovalent antibody fragment (e.g., a CD4 Fab fragment), can be derived from antibody 13B8.2 or a functionally active mutant of 13B8.2 that retains specific binding to CD4. For example, exemplary mutants of antibody 13B8.2 or m13B8.2 are described in U.S. Patent No. 7,482,000, U.S. Patent Application No. US2014 / 0295458, or International Patent Application No. WO2013 / 124474, and Bes, C, et al. J Biol Chem 278, 14265-14273 (2003). The mutant Fab fragment, designated "ml3B8.2," retains the variable domain of the CD4-binding murine antibody 13B8.2 and a constant domain 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, a mutant anti-CD4 antibody, e.g., antibody 13B8.2, contains the amino acid substitution H91A in the light chain variable chain, Y92A in the light chain variable chain, H35A in the heavy chain variable chain, and / or R53A in the heavy chain variable chain, each according to Kabat numbering. In some aspects, compared to the variable domain of the 13B8.2 Fab fragment in ml3B8.2, the His residue at position 91 of the light chain (position 93 in SEQ ID NO: 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 reagent that is reversibly bound to anti-CD4 or a fragment thereof is commercially available or derived from a commercially available reagent (e.g., catalog number 6-8000-206 or 6-8000-205 or 6-8002-100; IBA GmbH, Goettingen, Germany). In some embodiments, the selection agent comprises an anti-CD4 Fab fragment.In some embodiments, the anti-CD4 Fab fragment comprises a heavy chain variable chain having the sequence set forth in SEQ ID NO: 29 and a light chain variable chain having the sequence set forth in SEQ ID NO: 30. In some embodiments, the anti-CD4 Fab fragment comprises the CDRs of the heavy chain variable chain having the sequence set forth in SEQ ID NO: 29 and the CDRs of the light chain variable chain having the sequence set forth in SEQ ID NO: 30.

[0180] In some embodiments, the selection marker may be CD8, and the selection agent specifically binds to CD8. In some aspects, the selection agent that specifically binds to CD8 may be selected from the group consisting of an anti-CD8 antibody, a bivalent antibody fragment of an anti-CD8 antibody, a monovalent antibody fragment of an anti-CD8 antibody, and a proteinaceous CD8-binding molecule with antibody-like binding properties. In some embodiments, the anti-CD8 antibody, e.g., a bivalent antibody fragment or a monovalent antibody fragment (e.g., a CD8 Fab fragment), can be derived from the antibody OKT8 (e.g., ATCC CRL-8014) or a functionally active mutant thereof that retains specific binding to CD8. In some embodiments, the reagent that reversibly binds to anti-CD8 or a fragment thereof is commercially available or derived from a commercially available reagent (e.g., catalog number 6-8003 or 6-8000-201; IBA GmbH, Goettingen, Germany). In some embodiments, the selection agent comprises an anti-CD8 Fab fragment. In some embodiments, the anti-CD8 Fab fragment comprises a heavy chain variable chain having the sequence set forth in SEQ ID NO: 36 and a light chain variable chain having the sequence set forth in SEQ ID NO: 37. In some embodiments, the anti-CD8 Fab fragment comprises the CDRs of the heavy chain variable chain having the sequence set forth in SEQ ID NO: 36 and the CDRs of the light chain variable chain having the sequence set forth in SEQ ID NO: 37.

[0181] In some embodiments, the selection marker may be CD3, and the selection agent specifically binds to CD3. In some aspects, the selection agent that specifically binds to CD3 may be selected from the group consisting of an anti-CD3 antibody, a bivalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, and a proteinaceous CD3-binding molecule with antibody-like binding properties. In some embodiments, the anti-CD3 antibody, e.g., a bivalent antibody fragment or a monovalent antibody fragment (e.g., a CD3 Fab fragment), can be derived from the antibody OKT3 (e.g., ATCC CRL-8001; see, e.g., Stemberger et al. PLoS One. 2012; 7(4): e35798) or a functionally active mutant thereof that retains specific binding to CD3. In some embodiments, the reagent that reversibly binds to anti-CD3 or a fragment thereof is commercially available or derived from a commercially available reagent (e.g., catalog number 6-8000-201, 6-8001-100; IBA GmbH, Goettingen, Germany). In some embodiments, the selection agent comprises an anti-CD3 Fab fragment. In some embodiments, the anti-CD3 Fab fragment comprises a heavy chain variable chain having the sequence set forth in SEQ ID NO: 31 and a light chain variable chain having the sequence set forth in SEQ ID NO: 32. In some embodiments, the anti-CD3 Fab fragment comprises the CDRs of the heavy chain variable chain having the sequence set forth in SEQ ID NO: 31 and the CDRs of the light chain variable chain having the sequence set forth in SEQ ID NO: 32.

[0182] In any of the above examples, the bivalent antibody fragment may be a (Fab)2'-fragment or a bivalent single-chain Fv fragment, while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv). In any of the above examples, the proteinaceous binding molecule with antibody-like binding properties may be an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, an ankyrin scaffold-based protein, a crystalline scaffold-based protein, an adnectin, and an avimer.

[0183] In some embodiments, the selection agent is directly or indirectly bound to the stationary phase, hi some embodiments, the selection agent is indirectly bound to the stationary phase via a selection reagent to which the selection agent reversibly binds. In some embodiments, the selection reagent is or comprises streptavidin, avidin, a mutein of streptavidin that reversibly binds to biotin, a biotin analog or biologically active fragment thereof, avidin or a mutein of streptavidin that reversibly binds to a streptavidin-binding peptide, a reagent containing at least two chelating groups K, wherein at least two of the chelating groups are capable of binding to a transition metal ion, an agent capable of binding to an oligohistidine affinity tag, an agent capable of binding to glutathione-S-transferase, calmodulin or an analog thereof, an agent capable of binding to calmodulin-binding peptide (CBP), an agent capable of binding to a FLAG-peptide, an agent capable of binding to an HA-tag, an agent capable of binding to maltose-binding protein (MBP), an agent capable of binding to an HSV epitope, an agent capable of binding to a myc epitope, or an agent capable of binding to a biotinylated carrier protein.

[0184] In some embodiments, the selection reagent is or contains a streptavidin or avidin mutein that reversibly binds to biotin or a biologically active fragment. In some embodiments, the selection reagent is or contains a streptavidin or avidin mutein that reversibly binds to a streptavidin-binding peptide. In some embodiments, the streptavidin or streptavidin mutein molecule reversibly binds or is capable of reversibly binding to biotin, a biotin analog, or a streptavidin-binding peptide.

[0185] b. Stimulants In certain aspects, the methods provided herein utilize a stimulatory agent. In some embodiments, an agent as described in Section IB is a stimulatory agent. In some embodiments, the stimulatory agent binds to a molecule on the surface of a cell, and the binding between the stimulatory agent and the molecule can induce, deliver, or modulate a stimulatory signal in the cell. In some cases, the cell surface molecule (e.g., a receptor) is a signaling molecule. In some such cases, the stimulatory agent is capable of specifically binding to a signaling molecule expressed by one or more target cells (e.g., T cells). In some cases, the stimulatory agent is any agent that can induce or deliver a stimulatory signal in a cell (e.g., T cell) upon binding to a cell surface molecule, e.g., a receptor. In some embodiments, the stimulatory signal may be immunostimulatory, in which case the stimulatory agent can induce, deliver, or modulate a signal in a cell (e.g., T cell) that participates in or stimulates an immune response, e.g., that increases immune cell proliferation or expansion, immune cell activation, immune cell differentiation, cytokine secretion, cytotoxic activity, or one or more other functional activities of an immune cell. In some embodiments, a stimulatory signal may be inhibitory, in which case the stimulatory agent is capable of inducing, delivering, or modulating a signal in a cell (e.g., a T cell) that is involved in or stimulates an immune response, e.g., inhibiting or reducing immune cell proliferation or expansion, immune cell activation, immune cell differentiation, cytokine secretion, cytotoxic activity, or one or more other functional activities of an immune cell.

[0186] In some embodiments, the stimulating agent is a first stimulating agent. In some embodiments, the first stimulating agent binds to receptor molecules on the surface of selected cells of the sample. Thus, in some cases, the first stimulating agent delivers, induces, or modulates a stimulating signal. In some aspects, the delivery, induction, or modulation of a stimulating signal by the first stimulating agent achieves cell stimulation. Thus, in some cases, the first stimulating agent delivers a stimulating signal to the cell, thereby stimulating the cell. In some embodiments, the first stimulating agent further induces the downregulation of a selection marker. As used herein, downregulation can include a reduction in the expression of a selection marker compared to an earlier time point.

[0187] In some embodiments, the target cell (e.g., T cell) comprises a TCR / CD3 complex and a costimulatory molecule, e.g., CD28. In this case, the first stimulatory agent binds to the TCR / CD3 complex, thereby delivering a stimulatory signal in the T cell, and the second stimulatory agent binds to the costimulatory CD28 molecule. In certain aspects, the first stimulatory agent and / or the second stimulatory agent further induce downregulation of a selection marker (e.g., a selection marker used to immobilize the target cell (e.g., T cell)).

[0188] In some embodiments, the first stimulating agent delivers a TCR / CD3 complex-associated stimulatory signal in a cell, e.g., a T cell. In some embodiments, the first stimulating agent specifically binds to a molecule containing an immunoreceptor tyrosine-based activation motif or ITAM. In some aspects, the first stimulating agent specifically binds to CD3. In some cases, the first stimulating agent that specifically binds to CD3 may be selected from the group consisting of an anti-CD3 antibody, a bivalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, and a proteinaceous CD3-binding molecule with antibody-like binding properties. The bivalent antibody fragment may be a (Fab)2'-fragment or a bivalent single-chain Fv fragment, while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv). In some cases, the proteinaceous CD3-binding molecule with antibody-like binding properties may be an aptamer, a mutein based on a lipocalin family polypeptide, a glubody, an ankyrin scaffold-based protein, a crystalline scaffold-based protein, an adnectin, or an avimer.

[0189] In some embodiments, the anti-CD3 Fab fragment can be derived from the CD3-binding monoclonal antibody produced by the hybridoma cell line OKT3 (ATCC® CRL-8001™; see also U.S. Pat. No. 4,361,549). The heavy chain variable domain and light chain variable domain of the anti-CD3 antibody OKT3 are described in Arakawa et al. J. Biochem. 120, 657-662 (1996) and comprise the amino acid sequences set forth in SEQ ID NOs: 31 and 32, respectively.

[0190] In some embodiments, the stimulating agent is a second stimulating agent. In some embodiments, the second stimulating agent binds to a molecule on the surface of a cell, e.g., a cell surface molecule, e.g., a receptor molecule. In some embodiments, the second stimulating agent can enhance, attenuate, or modify a stimulating signal delivered via a molecule bound by the first stimulating agent. In some embodiments, the second stimulating agent delivers, induces, or modulates a stimulating signal, e.g., a second or additional stimulating signal. In some aspects, the second stimulating agent enhances or strengthens a stimulating signal induced by the first stimulating agent. In some embodiments, the second stimulating agent can bind to an auxiliary molecule in the cell and / or stimulate or induce an auxiliary or secondary stimulating signal. In some aspects, the second stimulating agent binds to a costimulatory molecule and / or provides a costimulatory signal.

[0191] In some embodiments, the stimulatory agent, which may be a second stimulatory agent, binds, e.g., specifically binds, to a second molecule, which may be 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.

[0192] In some embodiments, the molecule on the cell, e.g., a T cell, can be CD28, and the stimulator (which can be, e.g., a second stimulator) specifically binds to CD28. In some aspects, the stimulator (which can be, e.g., a second stimulator) that specifically binds to CD28 can be selected from the group consisting of an anti-CD28 antibody, a bivalent antibody fragment of an anti-CD28 antibody, a monovalent antibody fragment of an anti-CD28 antibody, and a proteinaceous CD28-binding molecule with antibody-like binding properties. The bivalent antibody fragment can be a (Fab)2'-fragment or a bivalent single-chain Fv fragment, while the monovalent antibody fragment can be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv). The proteinaceous CD28-binding molecule with antibody-like binding properties can be an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, an ankyrin scaffold-based protein, a crystalline scaffold-based protein, an adnectin, and an avimer.

[0193] In some embodiments, the anti-CD28 Fab fragment can be 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) and the heavy chain variable chain and light chain comprising SEQ ID NOs: 33 and 34, respectively.

[0194] In some embodiments, the one or more stimulatory agents are anti-CD3 and anti-CD28 antibodies or antigen-binding fragments thereof. In some embodiments, the one or more stimulatory agents are anti-CD3 Fab and anti-CD28 Fab.

[0195] In some embodiments, the molecule on the cell, e.g., T cell, is CD90, and the stimulator (which may be, e.g., a second stimulator) specifically binds to CD90. In some aspects, the stimulator (which may be, e.g., a second stimulator) that specifically binds to CD90 may be selected from the group consisting of an anti-CD90 antibody, a bivalent antibody fragment of an anti-CD90 antibody, a monovalent antibody fragment of an anti-CD90 antibody, and a proteinaceous CD90-binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment may be derived from any known in the art. See, e.g., anti-CD90 antibody G7 (Biolegend, catalog number 105201).

[0196] In some embodiments, the molecule on the cell, e.g., a T cell, is CD95, and the stimulatory agent (which may be, e.g., a second stimulatory agent) specifically binds to CD95. In some aspects, the stimulatory agent (which may be, e.g., a second stimulatory agent) that specifically binds to CD95 may be selected from the group consisting of an anti-CD95 antibody, a bivalent antibody fragment of an anti-CD95 antibody, a monovalent antibody fragment of an anti-CD95 antibody, and a proteinaceous CD95-binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, in some aspects, the anti-CD90 antibody may be monoclonal mouse anti-human CD95 CH11 (Upstate Biotechnology, Lake Placid, NY), or may be anti-CD95 mAb 7C11 or anti-APO-1, as described in Paulsen et al. Cell Death & Differentiation 18.4 (2011): 619-631.

[0197] In some embodiments, the molecule on a cell, e.g., a T cell or a B cell, may be CD137, and the stimulator (e.g., may be a second stimulator) specifically binds to CD137. In some aspects, the stimulator (e.g., may be a second stimulator) that specifically binds to CD137 may be selected from the group consisting of an anti-CD137 antibody, a bivalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment of an anti-CD137 antibody, and a proteinaceous CD137-binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment may be derived from any known in the art. For example, the anti-CD137 antibody may be 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, Mittler et al. Immunol Res. 2004;29(1-3):197-208.

[0198] In some embodiments, the molecule on the cell, e.g., B cell, can be CD40, and the stimulatory agent, e.g., the stimulatory agent (which can be, e.g., a second stimulatory agent, e.g., a second stimulatory agent) specifically binds to CD40. In some aspects, the stimulatory agent (which can be, e.g., a second stimulatory agent, e.g., a second stimulatory agent) that specifically binds to CD40 can be selected from the group consisting of an anti-CD40 antibody, a bivalent antibody fragment of an anti-CD40 antibody, a monovalent antibody fragment of an anti-CD40 antibody, and a proteinaceous CD40 binding molecule with antibody-like binding properties.

[0199] In some embodiments, the molecule on the cell, e.g., a T cell, can be CD40L (CD154), and the stimulator (e.g., can be a second stimulator) specifically binds to CD40L. In some aspects, the stimulator (e.g., can be a second stimulator) that specifically binds to CD40L can be selected from the group consisting of an anti-CD40L antibody, a bivalent antibody fragment of an anti-CD40L antibody, a monovalent antibody fragment of an anti-CD40L antibody, and a proteinaceous CD40L-binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art. For example, the anti-CD40L antibody can be Hu5C8 in some aspects, as described in Blair et al. JEM vol. 191 no. 4 651-660. See also, e.g., WO1999061065, US20010026932, US7547438, and WO2001056603.

[0200] In some embodiments, the molecule on a cell, e.g., a T cell, may be an inducible T cell costimulatory molecule (ICOS), and the stimulator (e.g., a second stimulator) specifically binds to ICOS. In some aspects, the stimulator (e.g., a second stimulator) that specifically binds to ICOS may be selected from the group consisting of an anti-ICOS antibody, a bivalent antibody fragment of an anti-ICOS antibody, a monovalent antibody fragment of an anti-ICOS antibody, and a proteinaceous ICOS-binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment may be derived from any known in the art. See, for example, US20080279851 and Deng et al. Hybrid Hybridomics. 2004 Jun;23(3):176-82.

[0201] In some embodiments, the molecule on a cell, e.g., a T cell, can be a linker for activation of T cells (LAT), and the stimulator (which can be, e.g., a second stimulator) specifically binds to LAT. In some aspects, the stimulator (which can be, e.g., a second stimulator) that specifically binds to LAT can be selected from the group consisting of an anti-LAT antibody, a bivalent antibody fragment of an anti-LAT antibody, a monovalent antibody fragment of an anti-LAT antibody, and a proteinaceous LAT-binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment can be derived from any known in the art.

[0202] In some embodiments, the molecule on a cell, e.g., a T cell, may be CD27, and the stimulator (e.g., may be a second stimulator) specifically binds to CD27. In some aspects, the stimulator (e.g., may be a second stimulator) that specifically binds to CD27 may be selected from the group consisting of an anti-CD27 antibody, a bivalent antibody fragment of an anti-CD27 antibody, a monovalent antibody fragment of an anti-CD27 antibody, and a proteinaceous CD27-binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment may be derived from any known in the art. See, for example, WO2008051424.

[0203] In some embodiments, the molecule on a cell, e.g., a T cell, may be OX40, and the stimulator (e.g., may be a second stimulator) specifically binds to OX40. In some aspects, the stimulator (e.g., may be a second stimulator) that specifically binds to OX40 may be selected from the group consisting of an anti-OX40 antibody, a bivalent antibody fragment of an anti-OX40 antibody, a monovalent antibody fragment of an anti-OX40 antibody, and a proteinaceous OX40 binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment may be derived from any known in the art. See, for example, WO2013038191, Melero et al. Clin Cancer Res. 2013 Mar 1;19(5):1044-53.

[0204] In some embodiments, the molecule on a cell, e.g., a T cell, may be HVEM, and the stimulating agent (e.g., a second stimulating agent) specifically binds to HVEM. In some aspects, the stimulating agent (e.g., a second stimulating agent) that specifically binds to HVEM may be selected from the group consisting of an anti-HVEM antibody, a bivalent antibody fragment of an anti-HVEM antibody, a monovalent antibody fragment of an anti-HVEM antibody, and a proteinaceous HVEM-binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment may be derived from any known antibody in the art. See, for example, WO2006054961, WO2007001459, Park et al. Cancer Immunol Immunother. 2012 Feb;61(2):203-14.

[0205] In any of the above examples, the bivalent antibody fragment may be a (Fab)2' fragment or a bivalent single-chain Fv fragment, while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv). In any of the above examples, the proteinaceous binding molecule with antibody-like binding properties may be an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, an ankyrin scaffold-based protein, a crystalline scaffold-based protein, an adnectin, and an avimer.

[0206] In some embodiments, the stimulatory agent specifically targets a molecule expressed on the surface of a target cell, where the molecule is a TCR, a chimeric antigen receptor, or a molecule containing an immunoreceptor tyrosine-based activation motif or ITAM. For example, the molecule expressed on the surface of the target cell is selected from a T cell or B cell antigen receptor complex, a CD3 chain, CD3 zeta, an antigen-binding portion of a T cell receptor or a B cell receptor, or a chimeric antigen receptor. In some cases, the stimulatory agent targets a peptide:MHC class I complex.

[0207] In some embodiments, the stimulatory agent binds to the His-tagged extracellular domain of a molecule expressed on the surface of a target cell. In some cases, the stimulatory agent contains the peptide sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also called Strep-tag® II, set forth in SEQ ID NO: 8) conjugated with nickel-charged trisNTA (also called His-STREPPER or His / Strep-tag® II adapter). In some embodiments, the His-tagged molecule expressed on the surface of a target cell is CD19.

[0208] In some embodiments, the stimulatory agent specifically binds to the antibody portion of the recombinant receptor, e.g., the CAR. In some cases, the antibody portion of the recombinant receptor comprises at least a portion of an immunoglobulin constant region, e.g., a hinge region, e.g., an IgG4 hinge region and / or a CH1 / CL and / or an Fc region. In some embodiments, the constant region or portion is that of a human IgG, e.g., IgG4 or IgG1. In some cases, the reagent is loaded with an αIgG that recognizes the IgG4 spacer.

[0209] In some embodiments, the desired target is a T cell receptor and / or a component of a T cell receptor. In certain embodiments, the desired target is CD3. In certain embodiments, the desired target is a T cell costimulatory molecule, such as CD28, CD137 (4-1-BB), OX40, or ICOS.

[0210] In some embodiments, for example, when the stimulating agent is not bound to a stimulating agent (e.g., an oligomeric stimulating reagent) or selection reagent, the stimulating agent is an antibody, a bivalent antibody fragment, F(ab)2, or a bivalent single-chain Fv fragment.

[0211] In some embodiments, the or each of the one or more stimulating agents further comprises a binding partner C for binding to the reagent. In some embodiments, the stimulating agent, each of the one or more stimulating agents is biotin, a biotin analog that reversibly binds to streptavidin or avidin, Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 15), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17), SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16), ), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18), and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19), a calmodulin-binding peptide that reversibly binds to calmodulin, a FLAG peptide that reversibly binds to an antibody that binds to the FLAG peptide, and an oligohistidine tag that reversibly binds to an antibody that binds to the oligohistidine tag.

[0212] In some embodiments, the reagent is or contains streptavidin, a streptavidin mutein, aviding, or an avidin mutein, and the or each of the stimulating agents contains a binding partner C capable of binding to such a reagent, such as biotin, a biotin analog, or a streptavidin-binding peptide. In some embodiments, the or each of the one or more stimulating agents is biotin, a biotin analog that reversibly binds to streptavidin or avidin, Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 15), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys lu-Lys (SEQ ID NO: 17), SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19). In certain embodiments, the reagent is or contains a streptavidin mutein (e.g., as set forth in SEQ ID NO: 6), and binding partner C is a streptavidin-binding peptide, such as any set forth in SEQ ID NO: 8 or any one of 15-19. In some embodiments, the or each of the one or more stimulating agents further comprises a streptavidin-binding peptide having the sequence SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16).

[0213] 2. Reagents In some embodiments, the reagent (e.g., a selection agent or stimulating agent) contains one or more binding sites Z that are capable of reversibly binding to a binding partner C contained by the agent (e.g., a selection agent or stimulating agent). In some embodiments, the reagent contains multiple binding sites Z, each of which is capable of specifically binding to a binding partner C contained in the agent (e.g., a selection agent or stimulating agent), such that the reagent is capable of reversibly binding to multiple agents (e.g., selection agents or stimulating agents), e.g., a multimerization reagent (e.g., a selection agent or stimulating agent). In some embodiments, the reagent is an oligomer or polymer of individual molecules (e.g., monomers) or a complex (e.g., a tetramer) composed of individual molecules, each containing at least one binding site Z. In some embodiments, the reagent contains at least two binding sites Z, at least three binding sites Z, at least four binding sites Z, e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72 or more binding sites Z. The binding sites may all be identical, or multiple binding sites may contain one or more different binding sites (e.g., Z1, Z2, Z3, etc.).

[0214] In some embodiments, two or more agents (e.g., selection agents or stimulating agents) are associated with, e.g., reversibly bound to, a reagent (e.g., selection agent or stimulating agent) via one or more binding sites Z present on the reagent (e.g., selection agent or stimulating agent). In some cases, this results in the agents (e.g., selection agents or stimulating agents) being positioned in close proximity to each other, such that an avidity effect may occur when a target cell bearing (at least two copies of) a cell surface molecule is contacted with an agent (e.g., selection agent or stimulating agent) bearing one or more binding sites B that can bind to a particular molecule.

[0215] In some embodiments, two or more different agents (e.g., selection agents or stimulating agents) that are identical, i.e., contain the same binding site B, can be reversibly bound to the reagent. In some embodiments, at least two different (types) of agents (e.g., selection agents or stimulating agents), and in some cases three or four different (types) of agents, e.g., two or more different selection agents and / or stimulating agents, can be used. For example, in some embodiments, a reagent (e.g., selection agent or stimulating agent) can be reversibly bound to a first agent (e.g., selection agent or stimulating agent) containing binding site B1, B2, B3, or B4, etc., and a second agent (e.g., selection agent or stimulating agent) containing another binding site, e.g., another of binding sites B1, B2, B3, or B4. In some cases, the binding sites of the first agent and the second agent can be the same. For example, in some aspects, each of the at least two agents (e.g., selection agents or stimulating agents) can bind to the same molecule. In some cases, the binding sites of the first agent and the second agent may be different. In some embodiments, each of at least two agents (e.g., selection agents or stimulatory agents) may bind to a different molecule, e.g., the first molecule, the second molecule, etc. In some cases, the different molecules, e.g., cell surface molecules, may be present on the same target cell. In other cases, the different molecules, e.g., cell surface molecules, may be present on different target cells present in the same population of cells. In some cases, the third, fourth, etc. agents (e.g., selection agents or stimulatory agents) may be associated with the same reagent (e.g., selection reagent or stimulatory reagent), each containing an additional, different binding site.

[0216] In some embodiments, two or more different (e.g., selection or stimulating) agents contain the same binding partner C. In some embodiments, two or more different agents (e.g., selection or stimulating) contain different binding partners. In some aspects, a first agent (e.g., selection or stimulating) may have a binding partner C1 that can specifically bind to a binding site Z1 present on a reagent (e.g., selection or stimulating) and a second agent (e.g., selection or stimulating) may bind to a binding partner C2 that can specifically bind to a binding site Z1 or a binding site Z2 present on a reagent (e.g., selection or stimulating). Thus, in some cases, the multiple binding sites Z comprised by a reagent include binding sites Z1 and Z2, which are reversibly bindable to binding partners C1 and C2, respectively, comprised by an agent (e.g., selection or stimulating). In some embodiments, C1 and C2 are identical and / or Z1 and Z2 are identical. In other aspects, one or more of the multiple binding sites Z may be different. In other examples, one or more of the multiple binding partners C may be different. It is within the level of ordinary skill in the art to select any combination of different binding partners C that is compatible with a reagent containing binding site Z, as long as each of the binding partners C is capable of interacting with, e.g., specifically binding to, one of the binding sites Z.

[0217] In some embodiments, the reagent (e.g., selection reagent or stimulation reagent) is streptavidin, a streptavidin mutein or analog, avidin, an avidin mutein or analog (e.g., neutravidin), or a mixture thereof, and such reagent contains one or more binding sites Z for reversible association with a binding partner C. In some embodiments, the binding partner C can be biotin, a biotin derivative or analog, or a streptavidin-binding peptide or other molecule capable of specifically binding to streptavidin, a streptavidin mutein or analog, avidin, or an avidin mutein or analog. In some embodiments, the reagent is or contains streptavidin, avidin, a streptavidin analog or mutein, or an analog or mutein or avidin that reversibly binds to biotin, a biotin analog, or a biologically active fragment thereof. In some embodiments, the reagent (e.g., selection reagent or stimulation reagent) is or contains a streptavidin analog or mutein or an avidin analog or mutein that reversibly binds to a streptavidin-binding peptide. In some embodiments, the substance (e.g., competitor or free binder) can be biotin, a biotin derivative or analog, or a streptavidin-binding peptide that can compete for binding with binding partner C for one or more binding sites Z. In some embodiments, binding partner C and the substance (e.g., competitor or free binder) are different, and the substance (e.g., competitor or free binder) exhibits a higher binding affinity for one or more binding sites Z compared to the affinity of the binding partner.

[0218] In some embodiments, streptavidin can be wild-type streptavidin, a streptavidin mutein, or an analog, e.g., a streptavidin-like polypeptide. Similarly, avidin, in some aspects, includes wild-type avidin or avidin mutein or analog, e.g., neutravidin, a deglycosylated avidin with a modified arginine that typically exhibits a more neutral pi and can be used as a substitute for natural avidin. Generally, deglycosylated neutral forms of avidin include commercially available forms, such as "Extravidin" available through Sigma-Aldrich or "NeutrAvidin" available from Thermo Scientific or Invitrogen.

[0219] In some embodiments, the reagent (e.g., selection reagent or stimulation reagent) is streptavidin or a streptavidin mutein or analog. In some embodiments, wild-type streptavidin (wt-streptavidin) has the amino acid sequence disclosed by Argarana et al., Nucleic Acids Res. 14 (1986) 1871-1882 (SEQ ID NO: 1). Generally, streptavidin occurs naturally as a tetramer of four identical subunits, i.e., a homotetramer, with each subunit containing a single binding site for biotin, a biotin derivative or analog, or a biotin mimic. An exemplary sequence of a streptavidin subunit is the sequence of amino acids set forth in SEQ ID NO: 1, although such sequences also include sequences present in homologs from other Streptomyces species. In particular, each subunit of streptavidin may exhibit strong binding affinity for biotin, with a binding affinity of about 10 -14It has an equilibrium dissociation constant (KD) of about M. In some cases, streptavidin can exist as a monovalent tetramer in which only one of the four binding sites is functional (Howarth et al. (2006) Nat. Methods, 3:267-73; Zhang et al. (2015) Biochem. Biophys. Res. Commun., 463:1059-63), a bivalent tetramer in which two of the four binding sites are functional (Fairhead et al. (2013) J. Mol. Biol., 426:199-214), or in a monomeric or dimeric form (Wu et al. (2005) J. Biol. Chem., 280:23225-31; Lim et al. (2010) Biochemistry, 50:8682-91).

[0220] In some embodiments, streptavidin can be in any form, including at least one functional subunit containing a binding site for biotin, a biotin derivative or analog, or a biotin mimic, such as wild-type or unmodified streptavidin, e.g., streptavidin derived from a Streptomyces species, or a functionally active fragment thereof, generally containing at least one functional subunit of wild-type streptavidin from Streptomyces avidinii, as set forth in SEQ ID NO: 1, or a functionally active fragment thereof. For example, in some embodiments, streptavidin can include a fragment of wild-type streptavidin that is truncated at the N-terminus and / or C-terminus. Such minimal streptavidins include any that begin N-terminally in the region of amino acid positions 10-16 of SEQ ID NO: 1 and terminate C-terminally in the region of amino acid positions 133-142 of SEQ ID NO: 1. In some embodiments, a functionally active fragment of streptavidin contains the sequence of amino acids set forth in SEQ ID NO: 2. In some embodiments, a streptavidin such as that set forth in SEQ ID NO: 2 may further contain an N-terminal methionine at the position corresponding to Ala13 in the numbering set forth in SEQ ID NO: 1. References to residue positions in streptavidin or streptavidin muteins relate to the numbering of the residues in SEQ ID NO: 1.

[0221] In some embodiments, streptavidin muteins comprise polypeptides that are distinct from the sequence of unmodified or wild-type streptavidin by one or more amino acid substitutions, deletions, or additions, but that contain at least one functional subunit containing a binding site for biotin, a biotin derivative or analog, or a streptavidin-binding peptide. In some embodiments, streptavidin-like polypeptides and streptavidin muteins may be essentially immunologically equivalent to wild-type streptavidin, and in particular, polypeptides that can bind to biotin, a biotin derivative, or a biotin analog with the same or different affinity as wt-streptavidin. In some cases, streptavidin-like polypeptides or streptavidin muteins may contain amino acids that are not part of wild-type streptavidin, or may contain only a portion of wild-type streptavidin. In some embodiments, the streptavidin-like polypeptide is a polypeptide that is not identical to wild-type streptavidin, because the host lacks the enzyme necessary to convert the polypeptide produced by the host into the structure of wild-type streptavidin. In some embodiments, streptavidin may also exist as a streptavidin tetramer and a streptavidin dimer, particularly a streptavidin homotetramer, a streptavidin homodimer, a streptavidin heterotetramer, and a streptavidin heterodimer. Generally, each subunit usually has a binding site for biotin or a biotin analog, or a streptavidin-binding peptide. Examples of streptavidin or streptavidin muteins are described, for example, in WO86 / 02077, DE19641876A1, US6,022,951, WO98 / 40396, or WO96 / 24606.

[0222] In some embodiments, a streptavidin mutein may contain amino acids that are not part of unmodified or wild-type streptavidin, or may include only a portion of wild-type or unmodified streptavidin. In some embodiments, a streptavidin mutein contains at least one subunit that may have one more amino acid substitution (replacement) compared to a subunit of unmodified or wild-type streptavidin, e.g., compared to the wild-type streptavidin subunit set forth in SEQ ID NO: 1 or a functionally active fragment thereof set forth in SEQ ID NO: 2, for example. In some embodiments, at least one subunit of a streptavidin mutein may have 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 wild-type or unmodified streptavidin, and / or contain at least one subunit comprising 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 or 2, and such streptavidin muteins exhibit functional activity binding to biotin, a biotin derivative or analog, or a biotin mimic. In some embodiments, the amino acid replacements (substitutions) may be conservative or non-conservative mutations. Examples of streptavidin muteins are known in the art, see, e.g., U.S. Pat. Nos. 5,168,049, 5,506,121, 6,022,951, 6,156,493, 6,165,750, 6,103,493 or 6,368,813 or International Published PCT Application No. WO2014 / 076277.

[0223] In some embodiments, streptavidin or streptavidin muteins comprise proteins containing one or more functional subunits, e.g., two or more, three or more, four or more, and in some cases five, six, seven, eight, nine, ten, eleven, twelve or more functional subunits, that contain one or more binding sites Z for biotin, a biotin derivative or analog, or a streptavidin-binding peptide. In some embodiments, streptavidin or streptavidin muteins may comprise a monomer, a dimer, including a heterodimer or homodimer, a tetramer, including a homotetramer, a heterotetramer, a monovalent tetramer or a divalent tetramer, or may comprise higher order multimers or oligomers thereof.

[0224] In some embodiments, the binding affinity of the streptavidin or streptavidin mutein for the peptide ligand binding partner is greater than or equal to 1×10 -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 1×10 -7 M, but generally, 1 × 10 -13 M, 1 x 10 -12 M or 1×10 -11 For example, peptide sequences (Strep-tags), such as those disclosed in U.S. Pat. No. 5,506,121, can act as biotin mimics and demonstrate binding affinity for streptavidin, e.g., approximately 10 -4 M to 10 -5 M. In some cases, binding affinity can be further improved by making mutations in the streptavidin molecule, see, e.g., U.S. Pat. No. 6,103,493 or International Published PCT Application No. WO2014 / 076277. In some embodiments, binding affinity can be determined by methods known in the art, such as any of those described below.

[0225] In some embodiments, a reagent (e.g., a selection reagent or stimulating reagent), e.g., streptavidin or a streptavidin mutein, exhibits binding affinity to a streptavidin-binding peptide, and the streptavidin-binding peptide can be binding partner C present in an agent (e.g., a selection agent or stimulating agent). In some embodiments, the streptavidin-binding peptide contains a sequence having the general formula set forth in SEQ ID NO: 9, e.g., the sequence set forth in SEQ ID NO: 10. In some embodiments, the streptavidin-binding peptide has the general formula set forth in SEQ ID NO: 11, e.g., that set forth in SEQ ID NO: 12. In one example, the streptavidin-binding peptide is Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (also referred to as Strep-tag®, set forth in SEQ ID NO: 7). In one example, the streptavidin-binding peptide is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also referred to as Strep-tag® II, set forth in SEQ ID NO: 8). In some embodiments, the streptavidin-binding peptide contains a consecutive arrangement of at least two streptavidin-binding modules, the distance between the two modules being at least 0 and not exceeding 50 amino acids, one link module having 3-8 amino acids and containing at least the sequence His-Pro-Xaa (SEQ ID NO: 9), where Xaa is glutamine, asparagine, or methionine, and the other link module having the same or a different streptavidin peptide ligand, such as that set forth in SEQ ID NO: 11 (see, e.g., International Published PCT Application No. WO 02 / 077018; U.S. Patent No. 7,981,632). In some embodiments, the streptavidin-binding peptide contains a sequence having a formula set forth in any of SEQ ID NOs: 13 or 14. In some embodiments, the streptavidin-binding peptide has a sequence of amino acids set forth in any of SEQ ID NOs: 15-19.

[0226] In some embodiments, the reagent (e.g., selection reagent or stimulation reagent) is or contains a streptavidin mutein. In some embodiments, the streptavidin mutein contains one or more mutations (e.g., amino acid replacements) compared to wild-type streptavidin set forth in SEQ ID NO: 1 or a biologically active portion thereof. For example, the biologically active portion of streptavidin is truncated at the N-terminus and / or C-terminus, and in some cases may include a streptavidin mutant referred to as minimal streptavidin. In some embodiments, the N-terminally truncated minimal streptavidin, which may be mutated, begins at the N-terminus in the region of amino acid positions 10-16 and ends at the C-terminus in the region of amino acid positions 133-142 compared to the sequence set forth in SEQ ID NO: 1. In some embodiments, the N-terminally truncated streptavidin, which may be mutated, contains the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, minimal streptavidin contains the amino acid sequence from Ala13 to Ser139, and optionally has an N-terminal methionine residue in place of Ala13. For purposes herein, the numbering of amino acid positions refers throughout to the numbering of wt-streptavidin as shown in SEQ ID NO: 1 (see, e.g., Argarana et al., Nucleic Acids Res. 14 (1986), 1871-1882; also see FIG. 3).

[0227] In some embodiments, the streptavidin mutein is a mutant as described in U.S. Patent No. 6,103,493. In some embodiments, the streptavidin mutein contains at least one mutation within the region of amino acid positions 44-53 based on the amino acid sequence of wild-type streptavidin, such as that set forth in SEQ ID NO: 1. In some embodiments, the streptavidin mutein contains mutations at one or more residues 44, 45, 46, and / or 47. In some embodiments, the streptavidin mutein contains a substitution of Glu at position 44 of wild-type streptavidin with a hydrophobic aliphatic amino acid, e.g., Val, Ala, Ile, or Leu, any amino acid at position 45, an aliphatic amino acid, e.g., a hydrophobic aliphatic amino acid, at position 46, and / or a basic amino acid, e.g., Arg or Lys, e.g., typically Arg, for Val at position 47. In some embodiments, Ala is at position 46, and / or Arg is at position 47, and / or Val or Ile is at position 44. In some embodiments, the streptavidin mutant contains residue Val, such as that shown in the exemplary streptavidin mutein containing the sequence of amino acids set forth in SEQ ID NO:3 or SEQ ID NO:4 (also known as streptavidin mutant 1, SAM1). 44 -Thr 45 -Ala 46 -Arg 47 In some embodiments, the streptavidin mutein contains residues Ile, such as those shown in the exemplary streptavidin mutein (also known as SAM2) containing the sequence of amino acids set forth in SEQ ID NO: 5 or 6. 44 -Gly 45 -Ala 46 -Arg 47 In some cases, such streptavidin muteins are described, for example, in U.S. Patent No. 6,103,493 and are commercially available under the trademark Strep-Tactin®.

[0228] In some embodiments, the streptavidin mutein is a mutant as described in International Published PCT Application No. WO2014 / 076277. In some embodiments, the streptavidin mutein contains at least two cysteine ​​residues in the region of amino acid positions 44-53 relative to the amino acid positions 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 one such embodiment, amino acid 44 is typically glycine or alanine, amino acid 46 is typically alanine or glycine, and amino acid 47 is typically arginine. In some embodiments, the streptavidin mutein contains at least one mutation or amino acid difference in the region of amino acid residues 115-121 relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the streptavidin mutein contains at least one mutation at amino acid positions 117, 120, and 121 and / or a deletion of amino acids 118 and 119 and a substitution at least at amino acid position 121.

[0229] In some embodiments, the streptavidin mutein contains a mutation at position 117, which can be to a large hydrophobic residue such as Trp, Tyr, or Phe, or to a charged residue such as Glu, Asp, or Arg, or to a hydrophilic residue such as Asn or Gin, or in some cases 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 position 120, which can be to a small residue such as Ser, Ala, or Gly, and a mutation at position 121, which can be to a bulky hydrophobic residue such as Trp, Tyr, or Phe. In some embodiments, the mutation at position 117 is combined with a mutation at position corresponding to position 120 of wild-type streptavidin or a biologically active fragment thereof set forth in SEQ ID NO: 1, which may be a hydrophobic residue such as Leu, Ile, Met, or Val, or generally Tyr or Phe, and a mutation at position corresponding to position 121 compared to wild-type streptavidin or a biologically active fragment thereof set forth in SEQ ID NO: 1, which may 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, such muteins also contain residue Val. 44 -Thr 45 -Ala 46 -Arg 47 or residue Ile 44 -Gly 45 -Ala 46 -Arg 47 In some embodiments, the streptavidin mutein may contain residue Val. 44 , Thr 45 , Ala 46 , Arg47, Glu 117 , Gly 120 and Tyr 121In some embodiments, the mutein streptavidin comprises the sequence of amino acids set forth in SEQ ID NO:27 or SEQ ID NO:28, or a sequence of amino acids 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 of amino acids set forth in SEQ ID NO:27 or SEQ ID NO:28, and wherein the residues Val, ... 44 , Thr 45 , Ala 46 , Arg 47 , Glu 117 , Gly120 and Tyr121 and exhibits functional activity binding to biotin, biotin analogs or streptavidin-binding peptides.

[0230] In some embodiments, the streptavidin mutein may contain any of the above mutations in any combination, and the resulting streptavidin mutein has a binding affinity of 2.7×10 to the peptide ligand (Trp-Arg-His-Pro-Gln-Phe-Gly-Gly, also known as Strep-tag®, set forth in SEQ ID NO: 7). -4 M and / or 1.4 × 10 for the peptide ligand (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys; also known as Strep-tag® II, set forth in SEQ ID NO: 8). -4 M and / or 1 × 10 to any of the peptide ligands set forth in any of SEQ ID NOS: 7 to 19 -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 M, but generally, 1 × 10 -13 M, 1 x 10 -12 M or 1×10 -11 It may exhibit a binding affinity greater than M.

[0231] In some embodiments, the streptavidin mutein exhibits a sequence of amino acids set forth in any of SEQ ID NOs: 3-6, 27 or 28, or a sequence of amino acids exhibiting at least 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: 3-6, 27 or 28, and exhibits 2.7×10 -4 M and / or 1.4×10 for the peptide ligand (Trp Ser His Pro Gln Phe Glu Lys; also referred to as Strep-tag® II, set forth in SEQ ID NO: 8). -4 M and / or 1 × 10 to any of the peptide ligands set forth in any of SEQ ID NOS: 7 to 19 -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 M, but generally, 1 × 10 -13 M, 1 x 10 -12 M or 1×10 -11 It exhibits a binding affinity greater than M.

[0232] In some embodiments, the streptavidin mutein comprises the sequence of amino acids set forth in any of SEQ ID NOs: 3-6, 27, or 28, and the streptavidin-binding peptide comprises the sequence of amino acids set forth in any of SEQ ID NOs: 7-19. In some embodiments, the streptavidin mutein comprises the sequence of amino acids set forth in SEQ ID NO: 6, and the streptavidin-binding peptide comprises the sequence of amino acids set forth in any of SEQ ID NOs: 7-19. In some embodiments, the streptavidin mutein comprises the sequence of amino acids set forth in any of SEQ ID NOs: 3-6, 27, or 28, and the streptavidin-binding peptide comprises the sequence of amino acids set forth in SEQ ID NO: 16. In some embodiments, the streptavidin mutein comprises the sequence of amino acids set forth in SEQ ID NO: 6, and the streptavidin-binding peptide comprises the sequence of amino acids set forth in SEQ ID NO: 16.

[0233] In some embodiments, the streptavidin mutein also exhibits binding to other streptavidin ligands, such as, but not limited to, biotin, iminobiotin, lipoic acid, desthiobiotin, diaminobiotin, HABA (hydroxyazobenzene-benzoic acid), and / or dimethyl-HABA. In some embodiments, the streptavidin mutein exhibits binding affinity for another streptavidin ligand, such as biotin or desthiobiotin, that is greater than the binding affinity of the streptavidin mutein to a biotin-mimicking peptide ligand, such as one set forth in any of SEQ ID NOS: 7-19. Thus, in some embodiments, biotin or a biotin analog or derivative (e.g., desthiobiotin) can be utilized as a competitor in the provided methods. For example, the interaction of a mutein streptavidin designated Strep-tactin® (e.g., containing the sequence set forth in SEQ ID NO: 4) with a peptide ligand designated Strep-tag® II (e.g., set forth in SEQ ID NO: 8) is approximately 10 times faster than the biotin-streptavidin interaction. -13 Compared to M, approximately 10 -6M. In some cases, the binding affinity is characterized by a KD of about 10 -10 From 10- 13 Biotin, which can bind with high affinity to Strep-tactin® with a KD between M, can compete with Strep-tag® II for the binding site.

[0234] In some cases, the reagent or (e.g., selection or stimulating reagent) contains at least two chelating groups, K, that may be capable of binding to a transition metal ion. In some embodiments, the reagent (e.g., selection or stimulating 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, and / or a biotinylated carrier protein.

[0235] In some embodiments, the reagent (e.g., selection reagent or stimulation reagent) is an oligomer or polymer. In some embodiments, the oligomer or polymer can be generated by directly or indirectly linking individual molecules of a protein as they occur in nature, by directly or indirectly linking individual molecules of the monomer or complexes of subunits that make up the individual molecules (e.g., directly or indirectly linking dimers, trimers, tetramers, etc. of the protein as they occur in nature). For example, homodimers or heterodimers of streptavidin or avidin tetramers may be referred to as individual molecules or minimal building blocks of the respective oligomer or polymer. In some embodiments, the oligomer or polymer may contain a linkage of at least two individual molecules of a protein (e.g., is a 2-mer), or may be at least a 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 25-mer, 30-mer, 35-mer, 40-mer, 45-mer, or 50-mer of individual molecules of a protein (e.g., monomer, tetramer).

[0236] Oligomers can be produced using any method known in the art, such as any of those described in published U.S. patent application no. US2004 / 0082012. In some embodiments, the oligomer or polymer contains two or more individual molecules that may be crosslinked, such as by a polysaccharide or bifunctional linker.

[0237] In some embodiments, oligomers or polymers are obtained by crosslinking individual molecules or complexes of subunits constituting individual molecules in the presence of a polysaccharide. In some embodiments, oligomers or polymers can be prepared by introducing carboxyl residues into a polysaccharide, such as dextran. In some aspects, individual molecules (e.g., monomers, tetramers) of a reagent can be coupled to carboxyl groups in the dextran backbone via the primary amino groups of lysine residues and / or the free N-terminus using conventional carbodiimide chemistry. In some embodiments, the coupling reaction is carried out at a molar ratio of about 60 moles of individual molecules (e.g., monomers, tetramers) of the reagent per mole of dextran.

[0238] In some embodiments, the reagent (e.g., selection reagent or stimulation reagent) is an oligomer or polymer of one or more streptavidin or avidin, or any analog or mutein of streptavidin (e.g., Strep-Tactin® or Strep-Tactin® XT) or analog or mutein of avidin (e.g., neutravidin). In some embodiments, binding site Z is the natural biotin-binding site of avidin or streptavidin, and there may be up to four binding sites in an individual molecule (e.g., a tetramer contains four binding sites Z), such that a homotetramer may contain up to four binding sites that are identical, i.e., Z1, while a heterotetramer may contain up to four binding sites that may be different, e.g., containing Z1 and Z2. In some embodiments, an oligomer is made or generated from multiple individual molecules (e.g., multiple homotetramers) of the same streptavidin, streptavidin mutein, avidin, or avidin mutein, in which case each binding site Z, e.g., Z1, of the oligomer is identical. For example, in some cases, an oligomer may contain multiple binding sites Z1, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, or more binding sites Z1. In some embodiments, the oligomer is made or generated from a plurality of individual molecules, which may be heterotetramers of streptavidin, streptavidin muteins, avidin or avidin muteins, and / or from a plurality of two or more different individual molecules (e.g., different homotetramers) of streptavidin, streptavidin muteins, avidin or avidin muteins, whose binding sites Z are different, e.g., Z1 and Z2, in which case multiple different binding sites Z, e.g., Z1 and Z2, may be present in the oligomer.For example, in some cases, an oligomer may contain multiple binding sites Z1 and multiple binding sites Z, which in combination may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50 or more combined binding sites Z1 and Z2.

[0239] In some cases, each oligomer or polymer can be cross-linked by a polysaccharide. In one embodiment, oligomers or polymers of streptavidin, avidin, streptavidin analogs, or avidin analogs (e.g., neutravidin) can be prepared essentially by introducing carboxyl residues into a polysaccharide, such as dextran, in a first step, as described in Noguchi, A, et al., Bioconjugate Chemistry (1992) 3,132-137. In some such embodiments, streptavidin or avidin, or its analogs, can then be linked to carboxyl groups in the dextran backbone via the primary amino groups and / or free N-terminus of internal lysine residues using conventional carbodiimide chemistry in a second step. In some cases, crosslinked oligomers or polymers of streptavidin or avidin, or of any analogue of streptavidin or avidin, may also be obtained by crosslinking via a bifunctional molecule that acts as a linker, e.g., glutardialdehyde, or by other methods described in the art.

[0240] In some embodiments, oligomers or polymers are obtained by crosslinking individual molecules or complexes of subunits that make up individual molecules using bifunctional linkers or other chemical linkers, such as glutardialdehyde, or by other methods known in the art. In some aspects, crosslinked oligomers or polymers of streptavidin or avidin, or of any mutein or analog of streptavidin or avidin, may be obtained by crosslinking individual streptavidin or avidin molecules via bifunctional molecules that act as linkers, such as glutardialdehyde, or by other methods described in the art. For example, it is possible to create oligomers of streptavidin muteins by introducing thiol groups into the streptavidin mutein (this can be done, for example, by reacting the streptavidin mutein with 2-iminothiolane (Trauts' reagent) and, for example, by activating, in a separate reaction, available amino groups in the streptavidin mutein. In some embodiments, this activation of the amino groups is achieved by cross-linking the streptavidin mutein with commercially available heterobifunctional cross-linkers, such as sulfosuccinimidyl 4-(N-maleimidomethyl) thiol. This can be achieved by reaction with sulfohexane-1-carboxylate (sulfoSMCC) or succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH). In some such embodiments, the two reaction products so obtained are mixed together, typically leading to reaction of a thiol group contained in one batch of modified streptavidin mutein with an activated amino acid (such as by a maleimide function) of another batch of modified streptavidin mutein. In some cases, this reaction results in the formation of multimers / oligomers of streptavidin muteins.These oligomers may have any suitable number of individual molecules, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50 or more, and the degree of oligomerization may vary depending on the reaction conditions.

[0241] In some embodiments, oligomeric or polymeric reagents (e.g., selection or stimulation reagents) can be isolated via size-exclusion chromatography, and any desired fraction can be used as a reagent. For example, in some embodiments, after reacting a modified streptavidin mutein in the presence of 2-iminothiolane and a heterobifunctional crosslinker, e.g., sulfo-SMCC, the oligomeric or polymeric reagent can be isolated via size-exclusion chromatography, and any desired fraction can be used as a reagent. In some embodiments, the oligomer does not (and need not) have a single molecular weight, but a statistical weight distribution, such as a Gaussian distribution, can be observed. In some cases, any oligomer having more than three streptavidin or mutein tetramers, e.g., homotetramers or heterotetramers, can be used as a soluble reagent, e.g., generally 3-50 tetramers, e.g., homotetramers or heterotetramers, 10-40 tetramers, e.g., homotetramers or heterotetramers, or 25-35 tetramers, e.g., homotetramers or heterotetramers. The oligomer may have, for example, 3 to 25 streptavidin mutein tetramers, e.g., homotetramers or heterotetramers. In some embodiments, when the streptavidin mutein has a molecular weight of about 50 kDa, the soluble oligomer may be about 150 kDa to about 2000 kDa, about 150 kDa to about 1500 kDa, about 150 kDa to about 1250 kDa, about 150 kDa to about 1000 kDa, about 150 kDa to about 500 kDa, or about 150 kDa to about 300 kDa, about 300 kDa to about 2000 kDa, about 300 kDa to about 1500 kDa, or about 300 kDa to about 1250 kDa. a, about 300 kDa to 1000 kDa, about 300 kDa to about 500 kDa, about 500 kDa to about 2000 kDa, about 500 kDa to about 1500 kDa, about 500 kDa to about 1250 kDa, about 500 kDa to 1000 kDa, about 1000 kDa to about 2000 kDa, about 1000 kDa to about 1500 kDa, about 1000 kDa to about 1250 kDa, about 1250 kDa to about 2000 kDa, or about 1500 kDa to about 2000 kDa.Generally, each streptavidin molecule / mutein has four biotin binding sites, so that such a reagent may provide 12 to 160 binding sites Z, for example 12 to 100 binding sites Z.

[0242] a. Oligomer Stimulation Reagent In certain embodiments, the stimulating reagent contains an oligomeric stimulating reagent, e.g., a streptavidin mutein reagent, conjugated to, linked to, or attached to one or more stimulating agents. As noted above, in some embodiments, one or more stimulating agents have attached binding domains or binding partners (e.g., binding partner C) that can bind to the oligomeric stimulating reagent at a specific binding site (e.g., binding site Z). In some embodiments, multiple stimulating agents are reversibly bound to the oligomeric stimulating reagent. In various embodiments, the oligomeric stimulating reagent has multiple specific binding sites, Z, which in certain embodiments are reversibly bound to multiple stimulating agents at binding domains (e.g., binding partner C). In some embodiments, the amount of bound agent is reduced or decreased in the presence of a competitor, e.g., an agent that is also capable of binding to a specific binding site (e.g., binding site Z).

[0243] In some embodiments, the stimulating reagent is or includes a reversible system in which at least one stimulating agent (e.g., a stimulating agent capable of generating a signal in a cell, e.g., a T cell) is associated, e.g., reversibly associated, with the oligomeric stimulating reagent. In some embodiments, the reagent contains multiple binding sites capable of binding, e.g., reversibly binding, to the stimulating agent. In some cases, the reagent is an oligomeric stimulating reagent having at least one agent attached that is capable of generating a signal (e.g., a stimulating signal) in a cell, e.g., a T cell. In some embodiments, the stimulating agent contains at least one binding site, e.g., binding site B, that can specifically bind to an epitope or region of a molecule, and also contains a binding partner, also referred to herein as binding partner C, that specifically binds to at least one binding site of the oligomeric stimulating reagent, binding site Z of the reagent. In some embodiments, the binding interaction between binding partner C and at least one binding site Z is a non-covalent interaction. In some cases, the binding interaction between binding partner C and at least one binding site Z is a covalent interaction. In some embodiments, the binding interaction, eg, the non-covalent interaction, between binding partner C and at least one binding site Z is reversible.

[0244] Substances that can be used as oligomer stimulating reagents in such reversible systems are known, for example, see 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, 9,023,604 and International Published PCT Application Nos. WO2013 / 124474 and WO2014 / 076277. Non-limiting examples of reagents and binding partners that can form reversible interactions and substances (e.g., competitors) that can reverse such binding are described below.

[0245] In some embodiments, the oligomeric stimulating reagent is an oligomer of streptavidin, a streptavidin mutein or analog, avidin, an avidin mutein or analog (e.g., neutravidin), or a mixture thereof, and such oligomeric stimulating reagent contains one or more binding sites for reversible association with a binding domain of a stimulating agent (e.g., binding partner C). In some embodiments, the binding domain of a stimulating agent can be biotin, a biotin derivative or analog, or a streptavidin-binding peptide or other molecule capable of specifically binding to streptavidin, a streptavidin mutein or analog, avidin, or an avidin mutein or analog.

[0246] In certain embodiments, one or more stimulatory agents (e.g., agents capable of generating a signal in a cell, e.g., a T cell) associate with, e.g., reversibly bind to, the oligomeric stimulatory reagent, such as through multiple specific binding sites (e.g., binding site Z) present on the oligomeric stimulatory reagent. In some cases, this results in the stimulatory agents being positioned in close proximity to each other, such that an avidity effect can occur when a target cell bearing (at least two copies of) a cell surface molecule bound or recognized by the stimulatory agent is contacted with the agent.

[0247] In some embodiments, the oligomeric stimulation reagent is an oligomer or mixture thereof composed of streptavidin oligomers, streptavidin mutein oligomers, streptavidin analog oligomers, avidin oligomers, avidin muteins, or avidin analogs (e.g., neutravidin). In certain embodiments, the oligomeric stimulation reagent contains a specific binding site capable of binding to the binding domain (e.g., binding partner C) of the stimulator. In some embodiments, the binding domain can be biotin, a biotin derivative or analog, or a streptavidin-binding peptide or other molecule capable of specifically binding to streptavidin, a streptavidin mutein or analog, avidin, or an avidin mutein or analog. Examples of streptavidin, streptavidin muteins, streptavidin analogs, avidin, avidin muteins, or avidin analogs (e.g., neutravidin) and binding domain molecules, e.g., biotin, biotin derivatives or analogs, or streptavidin-binding peptides or other molecules capable of specifically binding to streptavidin, streptavidin muteins or analogs, avidin, or avidin muteins or analogs, that are contemplated to comprise the oligomeric stimulating reagent system are described in Section IB. The methods provided herein further contemplate that the oligomeric stimulating reagent can include molecules 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, and / or a biotinylated carrier protein (see Section IB).

[0248] In certain embodiments, the present disclosure provides oligomeric stimulating reagents that are composed of and / or contain multiple streptavidin or streptavidin mutein tetramers.In certain embodiments, the present disclosure provides oligomeric stimulating reagents that contain multiple binding sites that can reversibly bind or reversibly bind to one or more stimulating agents.In some embodiments, the oligomeric stimulating reagents have a radius between 70nm and 125nm, including the boundary, for example, an average radius of 1x10, including the boundary. 7 g / mol to 1 × 10 9The oligomeric stimulatory reagent has a molecular weight between 1,000 and 5,000 g / mol and / or a streptavidin or streptavidin mutein tetramer between 1,000 and 5,000 g / mol, inclusive. In some embodiments, the oligomeric stimulatory reagent is conjugated, e.g., reversibly conjugated, to one or more stimulatory agents, e.g., agents that bind to molecules, e.g., receptors, on the surface of cells. In certain embodiments, the one or more stimulatory agents are agents described herein, e.g., in Section IB. In some embodiments, the one or more stimulatory agents contain a monovalent binding site (e.g., binding site B). In some embodiments, the monovalent binding site binds to CD3. In some embodiments, the monovalent binding site binds to a costimulatory molecule, e.g., as described herein. In some embodiments, the monovalent binding site binds to CD28. In some embodiments, the one or more stimulatory agents contain a monovalent binding site capable of binding to CD3 and / or CD28. In some embodiments, the stimulating agent is an anti-CD3 and / or anti-CD28 antibody or antigen-binding fragment thereof, e.g., an antibody or antigen-binding fragment thereof containing a binding partner, e.g., a streptavidin-binding peptide, e.g., Strep-tag® II. In certain embodiments, one or more agents are anti-CD3 and / or anti-CD28 Fabs containing a binding partner, e.g., a streptavidin-binding peptide, e.g., Strep-tag® II. In certain embodiments, one or more agents comprise streptavidin-based oligomers, e.g., streptavidin mutein oligomers, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fabs. In some embodiments, the oligomeric stimulating reagent is any of those described in WO2015 / 158868 or WO2018 / 197949.

[0249] In some embodiments, the present disclosure provides oligomeric stimulating reagents that are composed of and / or contain multiple streptavidin or streptavidin mutein tetramers.In certain embodiments, the present disclosure provides oligomeric stimulating reagents that contain multiple binding sites that can reversibly bind or reversibly bind one or more stimulating agents.In some embodiments, the oligomeric particles have a radius of between 80nm and 120nm, including the boundary, for example, an average radius of 7.5x10, including the boundary. 6 g / mol to 2 × 10 8 The oligomeric stimulating reagent has a molecular weight between 500 and 10,000 g / mol and / or a streptavidin or streptavidin mutein tetramer between 500 and 10,000 g / mol, inclusive. In some embodiments, the oligomeric stimulating reagent is conjugated, e.g., reversibly conjugated, to one or more stimulating agents, e.g., agents that bind to molecules, e.g., receptors, on the surface of cells. In certain embodiments, the one or more stimulating agents are agents described herein, e.g., in Section IB. In some embodiments, the stimulating agent is an anti-CD3 and / or anti-CD28 antibody or antigen-binding fragment thereof, e.g., an antibody or antigen-binding fragment thereof containing a binding partner, C, e.g., a streptavidin-binding peptide, e.g., Strep-tag® II. In certain embodiments, the one or more agents is a binding partner, e.g., an anti-CD3 and / or anti-CD28 Fab containing a streptavidin-binding peptide, e.g., Twin-Strep-tag (e.g., SEQ ID NO: 16).

[0250] In some embodiments, the oligomeric stimulation reagent used in the provided methods is any of the oligomeric stimulation reagents described herein.

[0251] In some embodiments, the cells are 6per individual cell 0.01μg, 0.02μg, 0.03μg, 0.04μg, 0.05μg, 0.1μg, 0.2μg, 0.3μg, 0. 4μg, 0.5μg, 0.75μg, 1μg, 2μg, 2.2μg, 2.4μg, 2.6μg, 2.8μg, 3μg, 4μg, 5μg, 6 μg, 7μg, 8μg, 9μg or 10μg, approximately 0.01μg, 0.02μg, 0.03μg, 0.04μg, 0.05μg, 0 .1μg, 0.2μg, 0.3μg, 0.4μg, 0.5μg, 0.75μg, 1μg, 2μg, 2.2μg, 2.4μg, 2.6μg, 2.8 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, or 10 μg of, or at least 0.01 μg, 0.02 μg, 0.03 μg, 0.04 μg, 0.05 μg, 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.75 μg, 1 μg, 2 μg, 2.2 μg, 2.4 μg, 2.6 μg, 2.8 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, or 10 μg of oligomeric stimulating reagent (e.g., Strep-tagged anti-CD3 and Strep-tagged anti-CD28 In some embodiments, cells are stimulated in the presence of a streptavidin-based oligomer (e.g., a streptavidin mutein oligomer) conjugated to a Fab. 6 In certain embodiments, cells are stimulated in the presence of, or about, 4 μg per cell. 6 Stimulation is performed in the presence of, or about, 0.8 μg per cell. In certain embodiments, 4 μg of oligomeric stimulating reagent is or comprises 3 μg of oligomeric particles and 1 μg of attached agent, e.g., 0.5 μg of anti-CD3 Fab and 0.5 μg of anti-CD28 Fab.

[0252] In some embodiments, the cells are 6In some embodiments, cells are stimulated or subjected to stimulation in the presence of 3 μg or about 3 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers, e.g., streptavidin mutein oligomers, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fab) per cell. 6 In some embodiments, cells are stimulated or subjected to stimulation in the presence of 2.75 μg or about 2.75 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers, e.g., streptavidin mutein oligomers, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fab) per cell. 6 In some embodiments, cells are stimulated or subjected to stimulation in the presence of 2.5 μg or about 2.5 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers, e.g., streptavidin mutein oligomers, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fab) per cell. 6 In some embodiments, cells are stimulated or subjected to stimulation in the presence of 2.25 μg or about 2.25 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers, e.g., streptavidin mutein oligomers, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fab) per cell. 6 The cells are stimulated or subjected to stimulation in the presence of 2 μg or about 2 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers, e.g., streptavidin mutein oligomers, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fab) per cell. In certain embodiments, the cells are stimulated or subjected to stimulation in the presence of 10 μg or about 2 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers, e.g., streptavidin mutein oligomers, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fab). 6The cells are stimulated or subjected to stimulation in the presence of, or about, 1.8 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers, e.g., streptavidin mutein oligomers, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fab) per cell. In certain embodiments, the cells are stimulated or subjected to stimulation in the presence of, or about, 1.8 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers, e.g., streptavidin mutein oligomers, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fab) per cell. 6 The cells are stimulated or subjected to stimulation in the presence of, or about, 1.6 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers, e.g., streptavidin mutein oligomers, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fab) per cell. In certain embodiments, the cells are stimulated or subjected to stimulation in the presence of, or about, 1.6 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers, e.g., streptavidin mutein oligomers, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fab) per cell. 6 The cells are stimulated or subjected to stimulation in the presence of, or about, 1.4 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers, e.g., streptavidin mutein oligomers, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fab) per cell. In certain embodiments, the cells are stimulated or subjected to stimulation in the presence of, or about, 1.4 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers, e.g., streptavidin mutein oligomers, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fab) per cell. 6 The cells are stimulated or subjected to stimulation in the presence of, or about, 1.2 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers, e.g., streptavidin mutein oligomers, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fab) per cell. In certain embodiments, the cells are stimulated or subjected to stimulation in the presence of, or about, 1.2 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers, e.g., streptavidin mutein oligomers, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fab) per cell. 6 The cells are stimulated or subjected to stimulation in the presence of 1 μg or about 1 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers, e.g., streptavidin mutein oligomers, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fab) per cell. In certain embodiments, the cells are stimulated or subjected to stimulation in the presence of 1 μg or about 1 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers, e.g., streptavidin mutein oligomers, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fab) per cell. 6In some embodiments, cells are stimulated or subjected to stimulation in the presence of 0.8 μg or about 0.8 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers, e.g., streptavidin mutein oligomers, conjugated to Strep-tagged anti-CD3 and Strep-tagged anti-CD28 Fab) per cell. 8 , 9×10 8 , 8×10 8 , 7×10 8 , 6×10 8 , 5×10 8 , 4×10 8 , 3×10 8 , 2 × 10 8 , 1×10 8 of or about 10 x 10 8 , about 9×10 8 , about 8×10 8 , about 7×10 8 , about 6×10 8 , about 5×10 8 , about 4×10 8 , about 3×10 8 , about 2×10 8 , about 1×10 8 In some embodiments, the cells are stimulated or subjected to stimulation in the presence of 7×10 oligomeric stimulation reagent. 8 , 6×10 8 , 5×10 8 , 4×10 8 , 3×10 8 or about 7 x 10 8 , about 6×10 8 , about 5×10 8 , about 4×10 8 , about 3×10 8 In some embodiments, the cells are stimulated or subjected to stimulation in the presence of 7×10 oligomeric stimulation reagent. 8 ~3×10 8 or about 7 x 10 8 ~3×10 8 In some embodiments, the cells are stimulated or subjected to stimulation in the presence of 6×10 oligomeric stimulation reagent. 8 ~4×10 8 or about 6 x 108 ~4×10 8 In some embodiments, the cells are stimulated or subjected to stimulation in the presence of 6×10 oligomeric stimulation reagent. 8 ~5×10 8 or about 6 x 10 8 ~5×10 8 In some embodiments, the cells are stimulated or subjected to stimulation in the presence of 5×10 oligomeric stimulation reagent. 8 or about 5 x 10 8 The oligomers are stimulated or subjected to stimulation in the presence of a stimulating agent.

[0253] In some embodiments, the cells, e.g., selected cells of a sample, are chromatin-depleted at or near 3:1, 2.5:1, 2:1, 1.5:1, 1.25:1, 1.2:1, 1.1:1, 1:1, 0.9:1, 0.8:1, 0.75:1, 0.67:1, 0.5:1, 0.3:1, or 0.2:1 or near 3:1, about 2.5:1, about 2:1, about 1.5:1, about 1.25:1, about 1.2:1, about 1.1:1, about 1:1, about 0.9:1, about 0.8:1, about In certain embodiments, the cells are stimulated or subjected to stimulation in the presence of an oligomeric stimulation reagent to cell ratio of 0.75:1, about 0.67:1, about 0.5:1, about 0.3:1, or about 0.2:1, the oligomeric stimulation reagent to cell ratio is between 2.5:1 and 0.2:1, between 2:1 and 0.5:1, between 1.5:1 and 0.75:1, between 1.25:1 and 0.8:1, or between 1.1:1 and 0.9:1. In certain embodiments, the oligomeric stimulation reagent to cell ratio is about 1:1 or is 1:1. In certain embodiments, the oligomeric stimulation reagent to cell ratio is about 0.3:1 or is 0.3:1. In certain embodiments, the oligomeric stimulation reagent to cell ratio is about 0.2:1 or is 0.2:1.

[0254] C. Cell Selection, Stimulation and Manipulation Methods are provided herein that include combining cell selection by a column chromatography step with stimulation and / or manipulation, e.g., transduction. In certain embodiments, cells of a sample are selected using any of the exemplary selection agents described in Section IB-1. Thus, in certain aspects, when cells are immobilized on a column (e.g., by a selection agent), stimulation and transduction are performed during the selection step. In some embodiments, the stimulatory conditions include conditions that stimulate and / or deliver stimulatory signals to cells, e.g., CD3+, CD4+, or CD8+ T cells. For example, the selection is to enrich or select for T cells or a certain subset thereof, and the stimulatory conditions include conditions that stimulate signals arising from components of the TCR complex (e.g., CD3) and / or costimulatory molecules (e.g., CD28). In some embodiments, the stimulatory conditions are or include incubating target cells (e.g., T cells) immobilized on a chromatographic matrix (e.g., a stationary phase) with a stimulatory agent, e.g., an agent that delivers a stimulatory signal or is capable of delivering a stimulatory signal, thereby stimulating the selected cells. In some embodiments, the selected cells are T cells or a subset thereof, and the stimulatory agent binds to, stimulates, and / or activates components of the TCR complex (e.g., CD3) and / or costimulatory molecules (e.g., CD28). In certain embodiments, stimulating a population of cells under stimulatory conditions creates or generates a population of selected, stimulated cells (also referred to herein as a population of stimulated cells).

[0255] In certain embodiments, a heterologous or recombinant polynucleotide is introduced into the cell upon stimulation. In certain embodiments, a heterologous or recombinant polynucleotide is introduced into the cell at the onset of stimulation.

[0256] 1. Cell Selection by Chromatography Provided herein are methods for selecting cells, e.g., T cells, from a sample by chromatographic isolation, e.g., by column chromatography, including affinity chromatography or gel permeation chromatography. In some embodiments, the method utilizes a selection agent that binds to a selection marker located on the surface of target cells, e.g., cells to be isolated, selected, or enriched. Such methods may also be described as (traceless) cell affinity chromatography technology (CATCH), and may include any of the methods or techniques described in PCT application numbers WO2013124474 and WO2015164675, which are hereby incorporated by reference in their entirety. Exemplary selection agents are described in Section IB-1.

[0257] In any of the foregoing embodiments, the sample may be or may include a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product. In some embodiments, the apheresis or leukapheresis product is freshly isolated from a subject. In other embodiments, the apheresis or leukapheresis product is thawed from a cryopreserved apheresis or leukapheresis product. In some embodiments, the target cells are T cells.

[0258] In some embodiments, cells, e.g., target cells, have or express a selection marker as described herein on the cell surface, such that the cells to be isolated, selected, or enriched are defined by the presence of at least one common specific receptor molecule. In some embodiments, a sample containing target cells may also contain additional cells that lack the selection marker. For example, in some embodiments, T cells are selected, isolated, or enriched from a sample containing multiple cell types, e.g., red blood cells or B cells.

[0259] In some embodiments, the selection agent is included in a chromatography column, e.g., directly or indirectly bound to a chromatography matrix (e.g., stationary phase). In some embodiments, the selection agent is present on the chromatography matrix (e.g., stationary phase) at the time the sample is added to the column. In some embodiments, the selection agent can be indirectly bound to the chromatography matrix (e.g., stationary phase) via a reagent, e.g., a selection reagent. In some embodiments, the selection reagent is covalently or non-covalently bound to the stationary phase of the column. In some embodiments, the selection reagent is reversibly immobilized on the chromatography matrix (e.g., stationary phase). In some cases, the selection reagent is immobilized on the chromatography matrix (e.g., stationary phase) via a covalent bond. In some aspects, the selection reagent is reversibly immobilized on the chromatography matrix (e.g., stationary phase) non-covalently.

[0260] In some embodiments, the selection agent may be present on the chromatography matrix (e.g., stationary phase) at the time the sample is added to the chromatography column (e.g., stationary phase), e.g., it may be bound to the chromatography matrix (e.g., stationary phase) directly (e.g., covalently or non-covalently) or indirectly via a selection reagent. Thus, upon addition of the sample, the target cells may be bound by the selection agent and immobilized on the chromatography matrix (e.g., stationary phase) of the column. Alternatively, in some embodiments, the selection agent may be added to the sample. In this case, the selection agent binds to target cells (e.g., T cells) in the sample, and the sample may then be added to a chromatography matrix (e.g., stationary phase) containing the selection reagent, where the selection agent already bound to the target cells binds to the selection reagent, thereby immobilizing the target cells on the chromatography matrix (e.g., stationary phase). In some embodiments, the selection agent binds to a selection reagent described herein via a binding partner C, as described herein, contained in the selection agent.

[0261] In some embodiments, two or more selection agents are associated with, e.g., reversibly or irreversibly bound to, the selection reagent, e.g., via one or more binding sites Z present on the selection reagent. In some cases, this results in the selection agents being positioned in close proximity to each other, such that an avidity effect may occur when a target cell bearing (at least two copies of) a cell surface molecule (e.g., a selection marker) is contacted with a selection agent that can bind to a particular molecule (e.g., a selection marker).

[0262] In some embodiments, two or more different selection agents that are identical, i.e., have the same selection marker binding specificity, can be reversibly bound to the selection reagent. In some embodiments, it is possible to use at least two different selection agents, and in some cases, three or four different selection agents, that bind to different selection markers. In some aspects, each of the at least two selection agents can bind to a different molecule (e.g., selection marker), e.g., a first molecule, a second molecule, etc. In some cases, the different molecules (e.g., selection agents), e.g., cell surface molecules, can be present on the same target cell. In other cases, the different molecules (e.g., selection markers), e.g., cell surface molecules, can be present on different target cells present in the same population of cells. In some cases, a third, fourth, etc. selection agent can be associated with the same reagent, each containing an additional different binding site.

[0263] In some embodiments, two or more different selection agents contain the same binding partner C. In some embodiments, two or more different selection agents contain different binding partners. In some aspects, a first selection agent may have a binding partner C1 that can specifically bind to a binding site Z1 present on the selection reagent, and a second selection agent may have a binding partner C2 that can specifically bind to a binding site Z1 or to a binding site Z2 present on the selection reagent. Thus, in some cases, the multiple binding sites Z contained by the selection reagent include binding sites Z1 and Z2, which are reversibly bindable to binding partners C1 and C2 contained by the selection agent, respectively. In some embodiments, C1 and C2 are identical and / or Z1 and Z2 are identical. In other aspects, one or more of the multiple binding sites Z may be different. In other examples, one or more of the multiple binding partners C may be different. It is within the level of one skilled in the art to select any combination of different binding partners C that are compatible with a selection reagent containing binding site Z, as long as each of the binding partners C is capable of interacting with, e.g., specifically binding to, one of the binding sites Z.

[0264] In some embodiments, the reversible bond formed between binding partner C and binding site Z can be disrupted by a competitor and / or free binder. In some embodiments, the competitor and / or free binder can be biotin, a biotin derivative or analog, or a streptavidin-binding peptide that can compete for binding of one or more binding sites Z to binding partner C. In some embodiments, the binding partner C and the competitor and / or free binder are different, and the competitor and / or free binder exhibits a higher binding affinity for one or more binding sites Z compared to the affinity of the binding partner. In certain aspects of any of the methods provided herein, the addition of a competitor and / or free binder to the stationary phase of the chromatography column to disrupt binding of the selection agent to the selection reagent is not necessary to desorb target cells (e.g., T cells) from the chromatography matrix (e.g., stationary phase).

[0265] In some embodiments, cells of the sample, e.g., target cells, can be depleted from the sample, such as by rinsing, releasing, or washing the remaining sample from the chromatography matrix (e.g., stationary phase). In some embodiments, one or more (e.g., 2, 3, 4, 5, 6) washing steps are used to remove unbound cells and debris from the chromatography matrix (e.g., stationary phase). In some embodiments, the sample is allowed to permeate the matrix for at least 5, 10, 16, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, or 120 minutes, or about 5, 10, 16, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, or 120 minutes, after which one or more washing steps are performed.

[0266] Any material can be utilized as a chromatography matrix (e.g., stationary phase). Generally, suitable chromatography materials are essentially non-toxic, i.e., not detrimental to cell viability, such as when used in a packed chromatography column under desired conditions. In some embodiments, the stationary phase remains in a predetermined position, such as a predetermined configuration, while the sample position is changed. Thus, in some embodiments, the stationary phase is part of a chromatography system through which a mobile phase flows (by flow-through or in a batch mode), resulting in the distribution of components (dissolved or dispersed) contained in the liquid phase between the phases.

[0267] In some embodiments, the chromatography matrix has a solid or semi-solid phase, while the sample containing the target cells to be isolated / separated is in a fluid phase. The chromatography matrix can be a particulate material (of any size and shape) or an integrated chromatography material including paper or membranes. Thus, in some aspects, the chromatography can be both column chromatography and planar chromatography. In some embodiments, in addition to standard chromatography columns, columns that allow bidirectional flow, such as PhyTip® columns or pipette tips available from PhyNexus, Inc., San Jose, California, USA, can be used for column-based / flow-through mode-based methods. Thus, in some cases, pipette tips or columns that allow bidirectional flow are also included by the chromatography columns useful in the present methods. In some cases, for example, when a particulate matrix material is used, the particulate matrix material can have an average particle size of, for example, about 5 μm to about 200 μm, about 5 μm to about 400 μm, or about 5 μm to about 600 μm. In some aspects, the chromatography matrix can be or include, for example, a polymeric resin or a metal or metalloid oxide. In some aspects, when planar chromatography is used, the matrix material can be any material suitable for planar chromatography, such as a conventional cellulose-based or organic polymer-based membrane (e.g., a paper membrane, a nitrocellulose membrane, or a polyvinylidene difluoride (PVDF) membrane) or a silica-coated glass plate. In one embodiment, the chromatography matrix / stationary phase is a non-magnetic or non-magnetizable material.

[0268] In some embodiments, non-magnetic or non-magnetizable chromatographic stationary phases suitable for the present methods include derivatized silica or cross-linked gels. In some aspects, the cross-linked gels can be based on natural polymers, e.g., naturally occurring polymer classes. For example, the natural polymers on which the chromatographic stationary phase is based are polysaccharides. In some cases, each polysaccharide is typically cross-linked. Examples of polysaccharide matrices include, but are not limited to, agarose gels (e.g., Superflow™ agarose or Sepharose® materials, e.g., Superflow™ Sepharose®, which are commercially available in different bead and pore sizes) or gels of cross-linked dextran(s). A further illustrative example is a particulate cross-linked agarose matrix to which dextran is covalently attached, which is commercially available as Sephadex® or Superdex® (in various bead sizes and with various pore sizes), both available from GE Healthcare. Another illustrative example of such a chromatographic material is Sephacryl®, also available from GE Healthcare in different bead and pore sizes.

[0269] In some embodiments, the cross-linked gel can also be based on synthetic polymers, for example, polymer classes that do not occur in nature. In some aspects, such synthetic polymers on which the chromatographic stationary phase is based have polar monomer units and are therefore themselves polar polymers. Thus, in some cases, such polar polymers are hydrophilic. Hydrophilic molecules are also called lipophilic and, in some aspects, contain moieties that can form dipole-dipole interactions with water molecules. In general, hydrophobic molecules are also called lipophilic and tend to separate from water.

[0270] Generally, the chromatography method is fluid chromatography, usually liquid chromatography. In some embodiments, chromatography can be carried out in a flow-through mode, where a fluid sample containing cells, for example, target cells, is applied to one end of a column containing a chromatography matrix, for example, by gravity flow or by a pump, and the fluid sample is present in the column at the other end of the column. Furthermore, chromatography can be carried out in a "up-and-down" mode, where a fluid sample containing cells to be isolated is applied to one end of a column containing a chromatography matrix packed in a pipette tip, for example, by a pipette, and the fluid sample enters and presents in the chromatography matrix / pipette tip at the other end of the column. Alternatively, chromatography can also be carried out in a batch mode, where a chromatography material (stationary phase) is incubated with a sample containing cells, for example, under repeated contact and removal of the fluid sample by means of shaking, rotation, or, for example, a pipette.

[0271] In some aspects, any material can be utilized as a chromatography matrix in connection with the provided embodiments, so long as the material is suitable for chromatographic isolation, e.g., cell selection. In certain aspects, a suitable chromatography material is at least harmless or essentially harmless, e.g., not detrimental to cell viability, when used in a packed chromatography column under the desired conditions for cell isolation and / or cell separation. In some aspects, the chromatography matrix typically remains in a predetermined position in a predetermined configuration, but the positions of the sample to be separated and of the components contained therein are altered. Thus, in some aspects, the chromatography matrix is ​​a "stationary phase."

[0272] Typically, each chromatography matrix has a solid or semi-solid phase, while the sample containing the target cells to be isolated / separated is in a fluid phase. The mobile phase used to achieve chromatographic separation is also a fluid phase. The chromatography matrix can be a particulate material (of any suitable size and shape) or an integrated chromatography material including paper material or a membrane. Thus, chromatography can be both column chromatography and planar chromatography. In addition to standard chromatography columns, columns or pipette tips that allow bidirectional flow can be used for column-based / flow-through mode-based chromatographic separation of cells as described herein. In some embodiments, a particulate matrix material is used, which can have an average particle size of, for example, about 5 μm to about 200 μm, about 5 μm to about 400 μm, or about 5 μm to about 600 μm. In some embodiments, planar chromatography is used, and the matrix material can be any material suitable for planar chromatography, such as a conventional cellulose-based or organic polymer-based membrane (e.g., a paper membrane, a nitrocellulose membrane, or a polyvinylidene difluoride (PVDF) membrane) or a silica-coated glass plate.

[0273] In some embodiments, the chromatography matrix / stationary phase is a non-magnetic or non-magnetizable material. Such materials can include derivatized silica or cross-linked gels. Cross-linked gels (usually manufactured in bead form) can be based on natural polymers, such as cross-linked polysaccharides. Suitable examples include, but are not limited to, agarose gels or cross-linked dextran(s) gels. Cross-linked gels can also be based on synthetic polymers, i.e., polymer classes that do not occur in nature. Typically, such synthetic polymers on which chromatographic stationary phases for cell separation are based are polymers that have polar monomer units and are therefore themselves polar.

[0274] Illustrative examples of suitable synthetic polymers include polyacrylamide(s), styrene-divinylbenzene gels, and copolymers of acrylates and diols or acrylamide and diols. An illustrative example is polymethacrylate gel commercially available as Fractogel®. A further example is copolymers of ethylene glycol and methacrylate commercially available as Toyopearl®. In some embodiments, the chromatographic stationary phase can also include natural and synthetic polymer components, such as composite matrices or conjugates or copolymers of polysaccharides and agarose, e.g., polyacrylamide / agarose conjugates or conjugates or copolymers of polysaccharides and N,N'-methylenebisacylamide. Illustrative examples of copolymers of dextran and N,N'-methylenebisacylamide include the Sephacryl® series of materials mentioned above. Derivatized silica can include silica particles coupled to synthetic or natural polymers. Examples of such embodiments include, but are not limited to, polysaccharide-grafted silica, polyvinylpyrrolidone-grafted silica, polyethylene oxide-grafted silica, poly(2-hydroxyethylaspartamide) silica, and poly(N-isopropylacrylamide)-grafted silica.

[0275] Other components present in the sample, such as stimulating agents and / or stimulating reagents (e.g., oligomeric stimulating reagents), may have a size below the exclusion limit of the pores, allowing them to enter the pores of the size-exclusion chromatography matrix. Among such components that can partially or completely enter the pore volume, larger molecules with less access to the pore volume typically elute first, while the smallest molecules typically elute last. In some embodiments, the exclusion limit of the size-exclusion chromatography matrix is ​​selected to be less than the maximum width of the target cells. Therefore, components that access the pore volume typically remain in / on the size-exclusion chromatography matrix longer than the target cells. Thus, the target cells can be collected separately from other components / components of the sample in the eluate of the chromatography column. Therefore, components such as stimulating reagents elute from the gel filtration matrix at a later time than the target cells.

[0276] The chromatography matrix utilized in the provided embodiments also includes a magnetically attractable substance, such as one or more magnetically attractable particles or ferrofluids. Each magnetically attractable particle can include a selection reagent having a binding site (e.g., a selection agent) capable of binding to and immobilizing target cells on the chromatography matrix. The magnetically attractable particles can include diamagnetic, ferromagnetic, paramagnetic, or superparamagnetic materials. Superparamagnetic materials respond to magnetic fields, and the induced magnetic field does 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 various other sources, such as Roche Applied Science, BIOCLON, BioSource International Inc., micromod, AMBION, Merck, Bangs Laboratories, Polysciences, or Novagen Inc., to name just a few. Superparamagnetic Co and FeCo-based magnetic nanoparticles and ferromagnetic Co nanocrystals have been described, for example, by Huetten, A. et al. (J. Biotech. (2004), 112, 47-63). However, in some embodiments, the chromatography matrix utilized in the provided embodiments is completely devoid of magnetically attractable materials.

[0277] Consistent with co-pending International Patent Application PCT / EP2012 / 063969, published as WO2013 / 011011 (the entire disclosure of which is incorporated herein by reference for all purposes), the strength of binding between a selection agent and a selection marker on a target cell may not be critical to the reversibility of binding of the target cell to the selection agent via the selection agent. Rather, the dissociation constant (KD) of binding between the selection agent and the selection marker via binding site B may be of low affinity, e.g., about 10 -3 ~about 10-7 M or of high affinity, e.g., about 10 -7 ~Approx. 1×10 -10 Regardless of the strength of binding, meaning whether it is within the KD range of M, target cells can be reversibly stained as long as the dissociation of the binding between the selective agent and the receptor molecule via binding site B occurs sufficiently rapidly. In this regard, the dissociation rate constant (k off ) is approximately 3 × 10 -5 seconds -1 or greater (this dissociation rate constant is constant and characterizes the dissociation reaction of the complex formed between binding site B of the receptor-binding reagent and the receptor molecule on the surface of the target cell). The dissociation rate constant (k on ) can have any value. To ensure sufficiently reversible binding between the selection marker and the selection agent, the k of the binding equilibrium off The value is approximately 3 x 10 -5 seconds -1 More than 5×10-5 seconds -1 More than, for example, 1 x 10 -4 seconds -1 That's it, 5 x 10 -4 seconds -1 That's it, 1×10 -3 seconds -1 Over 5×10-3 seconds -1 That's it, 1×10 -2 seconds -1 That's it, 1×10 -1 seconds -1 or more or 5 x 10 -1 seconds -1 More than or about 1 x 10 -4 seconds -1 That's about 5 x 10 -4 seconds -1 More than 1 × 10 -3 seconds -1 Above, approximately 5×10-3 seconds -1 More than 1 × 10 -2 seconds -1 More than 1 × 10 -1 seconds -1 or more or about 5 x 10 -1 seconds -1It is advantageous to select the kinetic and thermodynamic constants to have values ​​equal to or greater than 1.013 bar. It should be noted here that the values ​​of the kinetic and thermodynamic constants as used herein refer to conditions of atmospheric pressure, i.e., 1.013 bar, and room temperature, i.e., 25°C.

[0278] In some embodiments, multiple rounds of cell selection steps are performed, and the positively or negatively selected fraction from one step is subjected to another selection step, e.g., subsequent positive or negative selection. In certain embodiments, methods, techniques, and reagents for selection, isolation, and enrichment are described, for example, in PCT Application No. WO2015164675, which is hereby incorporated by reference in its entirety.

[0279] In some embodiments, a single selection step can be used to isolate target cells (e.g., CD3+ T cells) from a sample. In some embodiments, the single selection step can be performed on a single chromatography column. In some examples, the single selection step can deplete cells expressing multiple markers simultaneously. Similarly, multiple cell types can be positively selected simultaneously. In certain embodiments, the selection step is repeated or performed more than once, and the positively or negatively selected fraction from a step is subjected to the same selection step, e.g., repeated positive or negative selection. In some examples, the single selection step is repeated and / or performed more than once, e.g., to increase the purity of the selected cells and / or to further remove and / or deplete negatively selected cells from the negatively selected fraction. In certain embodiments, one or more selection steps are performed 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times. In certain embodiments, one or more selection steps are performed and / or repeated between 1 and 10 times, between 1 and 5 times, or between 3 and 5 times. In some embodiments, two selection steps are performed.

[0280] Cell selection can be performed using one or more chromatography columns. In some embodiments, the one or more chromatography columns are included in a closed system. In some embodiments, the closed system is, for example, an automated closed system requiring minimal or no user (e.g., human) input. In some embodiments, cell selection is performed continuously (e.g., continuous selection techniques). In some embodiments, one or more chromatography columns are arranged in series. For example, a first column can be oriented so that the output (e.g., eluent) of the column can be fed to a second chromatography column, for example, via connected tubing. In some embodiments, multiple chromatography columns can be arranged in series. In some embodiments, cell selection can be achieved by performing sequential positive and negative selection steps, with the next step subjecting the negative and / or positive fractions from the previous step to further selection, and the entire process being performed in the same tube or set of tubes. In some embodiments, a sample containing target cells is subjected to sequential selections, where a first selection is accomplished to enrich for one of the CD4+ or CD8+ populations, and unselected cells from the first selection are used as a source of cells for a second selection to enrich for the other of the CD4+ or CD8+ populations. In some embodiments, further selection(s) are accomplished to enrich for subpopulations of one or both of the CD4+ or CD8+ populations, e.g., central memory T (T CM) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+. In some embodiments, a sample containing target cells is subjected to sequential selection, in which a first selection is achieved to enrich for the CD3+ population, and the selected cells are used as a source of cells for a second selection to enrich for the CD3+ population. In some embodiments, a sample containing target cells is subjected to sequential selection, in which a first selection is achieved on a first stationary phase (e.g., in a first chromatography column) to enrich for the CD3+ population, and the flow-through containing unbound cells is used as a source of cells for a second selection to enrich for the CD3+ population, and the first and second stationary phases are arranged sequentially. In some embodiments, further selection(s) can be achieved to identify subpopulations of the CD3+ population, such as central memory T (T CM ) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+. In some embodiments, a sample containing target cells is subjected to sequential selections, where a first selection is achieved to enrich for a CD3+ population, and the selected cells are used as a source of cells for a second selection to enrich for a CD4+ population. In some embodiments, further selection(s) are achieved to enrich for subpopulations of the CD3+CD4+ population, e.g., central memory (T CM) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+. In some embodiments, a sample containing target cells is subjected to sequential selections, where a first selection is achieved to enrich for a CD3+ population, and the selected cells are used as a source of cells for a second selection to enrich for a CD8+ population. In some embodiments, further selection(s) are achieved to enrich for subpopulations of the CD3+CD8+ population, e.g., central memory T (T CM ) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+. In some embodiments, it is contemplated that specific subpopulations of T cells (e.g., CD3+ cells), e.g., cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are selected by positive or negative sequential selection techniques. The sequential selection methods can be performed in either order.

[0281] In some embodiments, a sample containing target cells is subjected to sequential selection, in which a first selection is achieved on a first stationary phase (e.g., in a first chromatography column) to enrich for the CD3+ population, and the selected cells are used as a source of cells for a second selection on a second stationary phase (e.g., in a second chromatography column) to enrich for a subpopulation of the CD3+ population, with the first and second stationary phases being arranged sequentially. In some embodiments, further selection(s) are achieved to enrich for a subpopulation of the CD3+ population, e.g., central memory T (T CM ) cells, naive T cells and / or cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+ and / or CD45RO+, can be enriched.

[0282] In some embodiments, the sample containing the target cells is subjected to a first selection step in which the target cells are ... CM ) cells, naive T cells and / or cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+ and / or CD45RO+ markers, is achieved, and the selected cells are used as a source of cells for a second selection to enrich for a subpopulation of the CD3+ population on a second stationary phase (e.g., in a second chromatography column), and subjected to sequential selection in which the first and second stationary phases are arranged sequentially.

[0283] In some embodiments, cell selection is performed in parallel (e.g., parallel selection techniques). In some embodiments, one or more chromatography columns are arranged in parallel. For example, two or more columns can be arranged so that a sample is loaded onto two or more columns simultaneously, e.g., via tubing, which allows the sample to be added to each column without having to pass through the first column. For example, using parallel selection techniques, cell selection can be achieved by simultaneously performing positive and / or negative selection steps in a closed system, e.g., in which the entire process is performed in the same tube or set of tubes. In some embodiments, a sample containing target cells is loaded onto two or more chromatography columns, and each column undergoes parallel selection to achieve selection of a cell population. In some embodiments, two or more chromatography columns independently achieve selection of CD3+, CD4+, or CD8+ populations. In some embodiments, two or more chromatography columns, including affinity chromatography or gel permeation chromatography, independently achieve selection of the same cell population. For example, two or more chromatography columns can achieve selection of CD3+ cells. In some embodiments, two or more chromatography columns, including affinity chromatography or gel permeation chromatography, independently achieve the selection of different cell populations. For example, two or more chromatography columns may independently achieve the selection of CD3+ cells, CD4+ cells, and CD8+ cells. In some embodiments, further selection(s) using, for example, sequential selection techniques, can be achieved to enrich for subpopulations of one or all of the cell populations selected by parallel selection. For example, the selected cells may be selected from central memory (T CM) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+. In some embodiments, a sample containing target cells is subjected to parallel selection, where parallel selection is accomplished on two or more columns to enrich for the CD3+ population. In some embodiments, further selection(s) are accomplished to enrich for subpopulations of the CD3+ population, e.g., central memory (T CM) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+. In some embodiments, a sample containing target cells is subjected to parallel selection, where selection is accomplished independently on two or more columns to enrich for CD3+ and CD4+ populations. In some embodiments, further selection(s) can be accomplished to enrich for subpopulations of the CD3+ and CD4+ populations, e.g., central memory (TCM) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+. In some embodiments, a sample containing target cells is subjected to parallel selections, in which parallel selections are accomplished to enrich for the CD3+ and CD8+ populations. In some embodiments, further selection(s) can be achieved to enrich for subpopulations of the CD3+ and CD8+ populations, e.g., central memory (TCM) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+. In some embodiments, a sample containing target cells is subjected to parallel selections that are achieved to enrich for CD4+ and CD8+ populations.In some embodiments, further selection(s) can be achieved to enrich for subpopulations of the CD4+ and CD8+ populations, e.g., central memory (TCM) cells, naive T cells, and / or cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+. In some aspects, it is contemplated that specific subpopulations of T cells (e.g., CD3+, CD4+, CD8+ T cells), e.g., cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are selected by positive or negative parallel selection techniques. In some embodiments, serial and parallel selection techniques may be used in combination.

[0284] In some embodiments, two columns are used for parallel selection. In some embodiments, the two columns select for the same cell type (e.g., the same selection marker). In some embodiments, the two columns each select for CD3+ T cells.

[0285] In some embodiments, cell selection is performed by positive or negative selection to deplete CD57+ cells and enrich for T cells. An exemplary method for depleting CD57+ cells is described in WO2020 / 097132. In some embodiments, specific subpopulations of T cells, such as cells positive for one or more surface markers or cells expressing high levels of one or more surface markers, e.g., CD3+, CD4+, CD8+, or CD57+ T cells, are isolated by positive or negative selection techniques. In some embodiments, such cells are selected by incubation with one or more selection agents, e.g., antibodies or antibody fragments that specifically bind to such markers. In certain embodiments, CD57+ cells are depleted from a sample, e.g., a PBMC sample, by negative selection of cells positive for CD57 expression, and non-selected cells (CD57- cells) are used as a source of cells for a second selection to enrich for T cells on a second stationary phase (e.g., in a second chromatography column), where the first and second stationary phases are arranged sequentially. For example, in some embodiments, CD57+ cells are depleted from a sample, e.g., a PBMC sample, by negative selection of cells positive for CD57 expression, and the unselected cells (CD57- cells) are used as a source of cells for a second selection to enrich for the CD3+ population on a second stationary phase (e.g., in a second chromatography column), where the first and second stationary phases are arranged sequentially.

[0286] For example, in embodiments using multiple columns in sequential selection, e.g., a first chromatography column and a second chromatography column, the provided methods are performed such that one or more stimulating agents or reagents are added to the last chromatography column (e.g., the second chromatography column) used in the final step of selecting or enriching a subpopulation of cells. In certain embodiments, the chromatography column to which the one or more stimulating agents or reagents are added is subjected to heating using the devices provided herein. For examp...

Claims

1. 1. A method for on-column transduction of T cells comprising: (a) simultaneously contacting a plurality of T cells with a T cell stimulatory reagent and a viral vector comprising a nucleic acid sequence encoding a recombinant protein, wherein the plurality of T cells are immobilized on a stationary phase contained within the 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, and specific binding of the selection agent to the selection marker achieves immobilization of the plurality of T cells on the stationary phase; contacting, wherein the T cell stimulating reagent comprises a first stimulating agent that specifically binds CD3 and a second stimulating agent that specifically binds CD28; (b) incubating a plurality of T cells in the presence of a T cell stimulatory reagent and a viral vector, wherein at least a portion of the incubating is performed at a temperature between 35° C. and 39° C.; and (c) collecting a plurality of T cells from the chromatography column within 24 hours of contacting, thereby generating a composition comprising T cells transduced with the recombinant protein; A method comprising:

2. 1. A method for on-column transduction of T cells comprising: (a) adding a sample comprising a plurality of T cells to an internal cavity of a chromatography column, the internal cavity comprising a stationary phase comprising a selection agent that specifically binds to a selection marker expressed on the surface of the plurality of T cells, thereby immobilizing the plurality of T cells on the stationary phase; (b) simultaneously contacting the plurality of T cells immobilized on the chromatography column with a T cell stimulatory reagent and a viral vector comprising a nucleic acid sequence encoding a recombinant protein, the T cell stimulatory reagent comprising a first stimulatory agent that specifically binds CD3, and a second stimulatory agent that specifically binds CD28; (c) incubating the plurality of T cells in the presence of a T cell stimulatory reagent and a viral vector, wherein at least a portion of the incubating is performed at a temperature between 35° C. and 39° C.; and (d) collecting a plurality of the T cells from the chromatography column within 24 hours of contacting, thereby generating a composition comprising the T cells transduced with the recombinant protein; A method comprising:

3. The method of claim 1 or claim 2, wherein the T cell stimulating reagent and the viral vector are contacted with the T cells as a mixture in the same composition.

4. 1. A method for on-column transduction of T cells comprising: (a) preparing a mixture comprising a T cell stimulatory reagent and a viral vector preparation; (b) contacting a plurality of T cells with the mixture in a chromatography column, the plurality of T cells being immobilized on a stationary phase contained within an interior cavity of the 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, and specific binding of the selection agent to the selection marker achieves immobilization of the plurality of T cells on the stationary phase; contacting, wherein the T cell stimulating reagent comprises a first stimulating agent that specifically binds CD3 and a second stimulating agent that specifically binds CD28; (c) incubating the plurality of T cells in the presence of a T cell stimulatory reagent and a viral vector, wherein at least a portion of the incubating is performed at a temperature between 35° C. and 39° C.; and (d) collecting a plurality of the T cells from the chromatography column within 24 hours of contacting, thereby generating a composition comprising the T cells transduced with the recombinant protein; A method comprising:

5. 3. The method of claim 2, wherein the contacting is initiated within 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, or 120 minutes after adding the sample to the internal cavity.

6. (a) at least a portion of the incubating is carried out at a temperature of or about 37°C; (b) the temperature of the stationary phase is regulated by one or more heating elements configured to provide heat to the stationary phase; (c) the incubation is carried out in serum-free medium; and / or (d) the T cell stimulatory reagent and viral vector are contacted with a plurality of T cells in serum-free medium, and incubation is performed in serum-free medium, optionally the serum-free medium comprises one or more recombinant T cell stimulatory cytokines, and optionally further comprising one or more recombinant cytokines selected from: (i) selected from IL-2, IL-15 and IL-7; and / or (ii) IL-2, IL-15 and IL-7; 5. The method according to any one of claims 1, 2 and 4.

7. (a) T cell stimulating reagents, each at 10 ６ contacting the T cells in an amount of between 0.1 μg and 20 μg, inclusive, between 0.4 μg and 8 μg, inclusive, or between 0.8 μg and 4 μg, inclusive, per T cell immobilized on the stationary phase of cells or per putative T cell immobilized on the stationary phase; (b) The T cell stimulating reagent is 10 ６ contacted with a plurality of T cells in an amount between 1 μg and 2 μg, inclusive, per plurality of T cells immobilized on a stationary phase of cells or per estimated plurality of T cells immobilized on a stationary phase; (c) the viral vector is administered to a plurality of T cells; ６ and / or contacted with a volume of each viral vector preparation of between 0.1 μL and 100 μL, inclusive, between 0.5 μL and 50 μL, inclusive, or between 1 μL and 25 μL, inclusive, per T cell population immobilized on the stationary phase of cells or per estimated T cell population immobilized on the stationary phase; and / or (d) the viral vector is administered to a plurality of T cells; ６ A volume of viral vector preparation between 2 μL and 10 μL, inclusive, per T cell population immobilized on a stationary phase of 10 cells or per estimated T cell population immobilized on a stationary phase. ６ A volume of 6 μL is contacted per T cell immobilized on the stationary phase of cells or per estimated T cell immobilized on the stationary phase.

5. The method according to any one of claims 1, 2 and 4.

8. The viral vector preparation was 1×10 ６ TU / mL to 1 x 10 ９ Between TU / mL, 1 x 10 ６ TU / mL to 1 x 10 ８ Between TU / mL, 1 x 10 ６ TU / mL to 1 x 10 ７ Between TU / mL, 1 x 10 ７ TU / mL to 1 x 10 ９ Between TU / mL, 1 x 10 ７ TU / mL to 1 x 10 ８ Between TU / mL or 1 x 10 ８ TU / mL to 1 x 10 ９ The method of claim 4, wherein the antibody has a titer of between 100 and 150 μg / mL.

9. To be collected: (a) performed within 22 hours, 20 hours, 18 hours, 16 hours, 16 hours, 14 hours, 12 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, or 5 hours after contacting; (b) performed after between 2 hours and 24 hours, 2 hours and 22 hours, 2 hours and 20 hours, 2 hours and 18 hours, 2 hours and 16 hours, 2 hours and 14 hours, 2 hours and 12 hours, 2 hours and 10 hours, 2 hours and 9 hours, 2 hours and 8 hours, 2 hours and 7 hours, 2 hours and 6 hours, 2 hours and 5 hours, 3 hours and 6 hours, 3 hours and 5 hours, 4 hours and 6 hours, or 4 hours and 5 hours, inclusive, of contacting; (c) performed 4.5 hours after the contacting; and / or (d) adding a wash buffer to the column to collect the one or more cells released from immobilization on the stationary phase during incubation, optionally the wash buffer being a cell culture medium, and further optionally comprising: (i) the cell culture medium is a serum-free medium; and / or (ii) the cell culture medium does not contain a competitor for eluting the T cells from the stationary phase; 5. The method according to any one of claims 1, 2 and 4.

10. (a) the collecting does not include adding to the stationary phase a medium that includes a competitor for eluting the plurality of T cells from the stationary phase; Here, optionally: (i) the competitor comprises biotin or a biotin analog, optionally wherein the biotin analog is desthiobiotin; and / or (ii) the competitor comprises D-biotin; and / or (b) the composition comprising the recombinant protein-transduced T cells does not comprise a competitor; Optionally: (i) the competitor comprises biotin or a biotin analog, optionally wherein the biotin analog is desthiobiotin; and / or (ii) the competitor comprises D-biotin; 5. The method according to any one of claims 1, 2 and 4.

11. (a) the first stimulatory agent comprises a monovalent antibody fragment that binds CD3 and the second stimulatory agent comprises a monovalent antibody fragment that binds CD28, optionally the monovalent antibody fragment is selected from the group consisting of a Fab fragment, an Fv fragment, and a single chain Fv fragment (scFv); (b) the T cell stimulating reagent comprises a first stimulating agent that is an anti-CD3 Fab and a second stimulating agent that is an anti-CD28 Fab; and / or (c) the first stimulant and the second stimulant are immobilized on a solid surface, optionally on a bead, or are reversibly bound to a soluble oligomeric reagent, optionally: (i) the soluble oligomeric reagent is an oligomer comprising a plurality of streptavidin or streptavidin mutein tetramers; (ii) the soluble oligomeric reagent comprises between 1000 and 3000 streptavidin or streptavidin mutein tetramers, inclusive; and / or (iii) both the first stimulatory agent and the second stimulatory agent comprise a binding partner that reversibly binds to the soluble oligomeric reagent, optionally the binding partner reversibly binds to the biotin binding site of streptavidin or a streptavidin mutein tetramer, further optionally the binding partner is biotin, a biotin analog, or a streptavidin binding peptide; 5. The method according to any one of claims 1, 2 and 4.

12. (a) the selection agent comprises an agent selected from the group consisting of an antibody, an antibody fragment, a proteinaceous binding molecule with immunoglobulin-like function, a molecule containing an Ig domain, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex, a receptor ligand and a binding fragment of any of the foregoing, optionally comprising an antibody or an antibody fragment, and optionally wherein the antibody fragment is a monovalent antibody fragment; (b) the selectable marker is a member of a T cell co-receptor or T cell antigen receptor complex; (c) the selection marker is selected from the group consisting of CD3, CD4, CD8, CD45RA, CD27, CD28 and CCR7; and / or (d) the selection marker is CD3; 5. The method according to any one of claims 1, 2 and 4.

13. (a) the stationary phase comprises a chromatographic matrix; (b) the stationary phase has a binding capacity of between 500 million and 5 billion cells, 500 million and 4 billion cells, 500 million and 3 billion cells, 500 million and 2 billion cells, 1 billion and 5 billion cells, 1 billion and 4 billion cells, 1 billion and 3 billion cells, or 1 billion and 2 billion cells, inclusive of each boundary; (c) the stationary phase has a binding capacity of 1 to 2 billion cells, including the interface; (d) the plurality of T cells comprises antigen-specific T cells, helper T cells, cytotoxic T cells, memory T cells and / or regulatory T cells; (e) the T cells comprise CD3+ T cells, or comprise CD4+ T cells and / or CD8+ T cells; and / or (f) the T cells are primary T cells from a human subject; 5. The method according to any one of claims 1, 2 and 4.

14. The recombinant protein is (i) an antigen receptor and / or (ii) a chimeric antigen receptor (CAR), optionally wherein the CAR comprises an extracellular antigen recognition domain that specifically binds to a target antigen and an intracellular signaling domain comprising an ITAM, and further optionally: (a) the intracellular signaling domain comprises the intracellular domain of the CD3 zeta (CD3ζ) chain; (b) the CAR further comprises a transmembrane domain linking the extracellular domain and the intracellular signaling domain, optionally the transmembrane domain comprises the transmembrane portion of CD28; and / or (c) the intracellular signaling domain further comprises an intracellular signaling domain of a T cell costimulatory molecule, optionally the T cell costimulatory molecule is selected from the group consisting of CD28 and 41BB; 5. The method according to any one of claims 1, 2 and 4.

15. (a) a viral vector comprising: (i) is a retroviral vector; (ii) is a lentiviral vector; and / or (iii) is pseudotyped with VSV-G; (b) at least one of the method steps is performed in a closed system; (c) all of the method steps are performed in a closed system; (d) at least one of the steps of the method is automated; and / or (e) all of the method steps are automated; 5. The method according to any one of claims 1, 2 and 4.

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