Provided herein are methods that relate, in some aspects, to the incubation or culturing, such as to induce stimulation of expansion (proliferation), activation, costimulation and/or survival, of a composition of cells, such as a population of lymphocytes. In some aspects, provided are methods and reagents for the stimulation, e.g., of expansion (proliferation), survival or persistence, activation, costimulation, or other effect, of cell populations that involve binding of agents to a molecule on the surface of the cells, thereby providing one or more signals to the cells. In some cases, the reagents are reagents containing a plurality of binding sites for agents, such as multimerization reagents, and thus the one or more agents are multimerized by reversibly binding to the reagent, e.g., thereby creating a stimulatory reagent (multimerized agent), having stimulatory agents multimerized thereon. In some aspects, the multimerized agent can provide for expansion or proliferation or other stimulation of a population of cells, and then such stimulatory agents can be removed by disruption of the reversible bond. Also provided are compositions, apparatus and methods of use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser. No. 15/770,179 filed Apr. 20, 2018, which is a U.S. National Stage of International Application No. PCT/IB2016/001618 filed Oct. 20, 2016, which claims priority from U.S. Provisional Application No. 62/245,252 filed Oct. 22, 2015, entitled “Methods for Culturing Cells and Kits and Apparatus for Same,” and U.S. Provisional Application No. 62/245,261 filed Oct. 22, 2015, entitled “Methods for Culturing Cells and Kits and Apparatus for Same,” the contents of each of which is incorporated by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042003610SeqList.xml, created on Oct. 5, 2022, which is 124,420 bytes in size. The information in electronic format of the Sequence Listing is incorporated by reference in its entirety.

FIELD

[0003] The present disclosure relates in some aspects to the incubation or culturing, such as to induce stimulation of expansion (proliferation), activation, costimulation and/or survival, of a composition of cells such as a population of lymphocytes. In some aspects, the disclosure provides methods and reagents for the stimulation, e.g., of expansion (proliferation), survival or persistence, activation, costimulation, or other effect, of cell populations that involve binding of agents to a molecule on the surface of the cells, thereby providing one or more signals to the cells. In some cases, the reagents are reagents containing a plurality of binding sites for agents, such as multimerization reagents, and thus the one or more agents are multimerized by reversibly binding to the reagent, e.g., thereby creating a stimulatory reagent (multimerized agent), having stimulatory agents multimerized thereon. In some aspects, the multimerized agent can provide for expansion or proliferation or other stimulation of a population of cells, and then such stimulatory agents can be removed by disruption of the reversible bond. Also provided are compositions, apparatus and methods of use thereof.

BACKGROUND

[0004] Various strategies are available for stimulating T cell populations in vitro, including for expanding antigen-specific T cells in vitro for use in adoptive cellular immunotherapy or cancer therapy in which infusions of such T cells have been shown to have anti-tumor reactivity in a tumor-bearing host or for use to treat viral infections. Improved strategies are needed for expanding cell populations in vitro, including for research, diagnostic and therapeutic purposes. Provided are reagents, methods, articles of manufacture and kits that meet such needs.

SUMMARY

[0005] Provided herein are methods for incubating cells and compositions thereof, such as for culturing such cells, using reversible reagents for inducing or modulating a signal in a target cell. In some embodiments, the cells are T cells.

[0006] In some aspects the method involves incubating a composition containing T cells in the presence of an agent, such as a receptor-binding agent, e.g., a stimulatory agent. The receptor-binding agent (e.g., stimulatory agent) may be reversibly bound to a reagent containing a plurality of binding sites capable of reversibly binding to the receptor-binding agent. In some aspects, the receptor-binding agent is capable of specifically binding to a molecule on the surface of the cells, e.g., T cells, such as in a manner that induces or modulates a signal in the cells, e.g., the T cells in the composition. In some embodiments, the methods involve features, such as particular steps, selection of particular agents and/or selection of particular reagents, which allow the control or adjustment of the type or strength or duration of the signal received or modulated via the reagent, and/or of properties of the output composition or cell population(s) ultimately generated by the methods. In some embodiments, such features are possible due to advantageous properties of the agents and reagents, such as the reversibility of binding of the individual components of the agents or reagents, and thus, the reversibility of the binding of the multimerized agents and the cells. Such properties of the reagents can be exploited to achieve control in a number of ways. For example, in some embodiments, the methods, via reversibility of binding, include exerting temporal control of the signal, controlling the duration of the period in which the cells are in contact with the multimerized agents, and/or the duration of the signaling induced thereby.

[0007] In some embodiments, the methods involve control, e.g., precise control, of the length of the time period under which such agents are bound to cells. For example, this may be done by actively reversing such binding at a particular timepoint, and in some cases while still maintaining the cells for an additional time period other culture conditions, such as incubation at a physiological temperature and/or with various nutrients. Thus, as opposed to other methods which simply involve the termination of all or substantially all signals received by the cells, the provided methods in some aspects allow the specific termination or disruption of signals delivered by particular reagents.

[0008] Likewise, in some embodiments, the reversibility allows the reagents to be modular in nature, permitting the substitution of one or more components thereof without engineering or new reagents, e.g., by simply reversing binding and combining with additional agents or reagents, under conditions where reversible binding is induced. For example, by being able to reversibly bind various stimulatory agents to the same multimerization reagent, either at the same time or at different times, the user of the provided methods and compositions may adjust the nature of the particular signal being delivered, e.g., by substituting one or more stimulatory agent for another one or more stimulatory agent, such as to induce a stronger or weaker or qualitatively different signal, depending on the desired outcome.

[0009] In some embodiments, temporal control and modularity are used in combination, e.g., by incubating cells in the presence of one agent for a certain period of time, inducing reversal of binding by disruption, followed by incubation in the presence of other or more different agents or reagents. For example, in one embodiment, T cells are initially stimulated with a reagent to deliver a particular strength or quality of signal, and after a certain period of time, such signal is disrupted and a qualitatively or quantitatively different (e.g., stronger or weaker or activating different signaling pathways or known to be important for different differentiation pathways) signal is substituted. In some embodiments, such control provides advantages, for example, allowing the user to maximize desired outcomes (e.g., expansion or persistence) while avoiding undesirable outcomes such as exhaustion or anergy.

[0010] In some embodiments, temporal control is achieved by disrupting the reversible binding of the reagents or agents, such as by the addition of a substance. For example, in some embodiments, within a period of time, e.g., within 5 days after initiation of the incubation, and/or within a certain percentage of the total length of the incubation, such as within ⅕, ¼ ⅓, or ½ of the time, the reversible binding between the receptor-binding agent and the reagent is disrupted. Thus, in some cases the method results in the generation of cultured T cells.

[0011] In some embodiments, the incubation is performed under conditions in which the receptor-binding agent specifically binds to the molecule, thereby inducing or modulating the signal in one or more T cells in the composition.

[0012] In some embodiments, the disruption of the binding between the receptor-binding agent and the reagent is effected more than 30 minutes after the initiation of the incubation. For example, in some aspects, the disruption of the binding between the receptor-binding agent and the reagent is effected between 1 hour and 4 days after initiation of the incubation, between 6 hours and 3 days after initiation of the incubation, between 12 hours and 2 days after initiation of the incubation, or between 1 day and 3 days after initiation of the incubation. In some cases, the disruption of the binding between the receptor-binding agent and the reagent is effected between about 1 hour and about 4 days after initiation of the incubation, between about 6 hours and about 3 days after initiation of the incubation, between about 12 hours and about 2 days after initiation of the incubation, or between about 1 day and about 3 days after initiation of the incubation. In some aspects, the disruption is effected greater than or equal to about 1 hour after initiation of said incubation and within 1 day, 2 days, 3 days or 4 days after initiation of the incubation. In some embodiments, binding of the receptor-binding agent is capable of initiatingor does initiates a TCR/CD3 complex-associated signal in the T cells. In some aspects, the receptor-binding agent specifically binds to a member of a TCR/CD3 complex. In some instances, the receptor-binding agent specifically binds to CD3.

[0013] In some embodiments, the molecule (the molecule on the surface of the T cells) is a component of the TCR/CD3 complex or is CD3. In some aspects, the molecule is a first molecule and the receptor-binding agent is further capable of specifically binding to a second molecule on the surface of one or more of the T cells. In some cases the second molecule is capable of inducing or enhancing, dampening, or modifying a signal delivered through the first molecule in the T cells.

[0014] In some aspects, the receptor-binding agent includes a binding partner C1. In some aspects, the plurality of binding sites contained by the reagent includes two or more binding sites, Z1. In some instances, the two or binding sites Z1 each are capable of binding to the binding partner C1 to form the reversible bond between the receptor-binding agent and the reagent. In some embodiments, the disruption of the binding between the receptor-binding agent and the reagent includes introducing to the cells a composition containing a substance capable of reversing the bond between the receptor-binding agent and the reagent.

[0015] In some embodiments, the receptor-binding agent is a first receptor-binding agent and the incubation is further carried out in the presence of a second receptor-binding agent, which is capable of specifically binding to a second molecule on the surface of one or more of the T cells. In some embodiments, the binding of the second receptor-binding agent to the second molecule or does enhance, dampen, or modify a signal delivered through the first molecule in the T cells.

[0016] In some embodiments, the reagent contains a plurality of binding sites capable of reversibly binding to the second receptor-binding agent. In some such cases, the second receptor-binding agent is reversibly bound to the reagent. In some aspects, the plurality of binding sites capable of reversibly binding to the first receptor-binding agent and the plurality of binding sites capable of reversibly binding to the second receptor-binding agent can be the same or can be different.

[0017] In some ects, the second receptor-binding agent includes a binding partner C1 or C2, which is capable of reversibly binding to the two or more binding sites Z1. In some such instances, the first and second receptor-binding agents are reversibly bound to the reagent via the two or more binding sites Z1. In some cases, the second receptor-binding agent contains a binding partner C2 and the reagent further contains a plurality of binding sites Z2. The plurality of binding sites Z2 may be capable of binding to the binding partner C2 to form the reversible bond between the second receptor-binding agent and the reagent. In some aspects, C2 and C1 are the same or substantially the same, or contain the same or substantially the same moiety. In some instances, Z1 and Z2 are the same or substantially the same or contain the same or substantially the same moiety.

[0018] In some cases, the reagent is a first reagent and the incubation is carried out in the presence of at least a second reagent which is reversibly bound to the second receptor-binding agent. In some embodiments, the incubation is performed under conditions in which the second receptor-binding agent specifically binds to the second molecule. In some such aspects, the bind of the second receptor-binding agent to the second molecule induces or modulates a signal, e.g., that enhances, dampens, or modifies a signal delivered through the first molecule in the T cells.

[0019] In some embodiments, said disruption includes introducing to the cells a composition that contains a substance capable of reversing the bond between the first receptor-binding agent and the reagent and/or the second receptor-binding agent and the reagent. In some embodiments, said disruption terminates or lessens the signal induced or modulated by the first receptor-binding agent and terminates or lessens the signal induced or modulated by the second receptor-binding agent.

[0020] Provided herein in some embodiments is a method for culturing T cells that includes incubating a composition containing T cells in the presence of a first receptor-binding agent that is capable of specifically binding to a first molecule expressed on the surface of the T cells. In some aspects, the binding of the first receptor-binding agent to the first molecule induces or modulates a TCR/CD3 complex-associated signal in the T cell. In some such embodiments, the composition further is incubated in the presence of a second receptor-binding agent that is reversibly bound to a reagent containing a plurality of binding sites capable of reversibly binding to the second receptor-binding agent. In some aspects, the second receptor-binding agent is capable of specifically binding to a second molecule on the surface of the T cells. In some cases, the binding of the second receptor-binding agent to the second molecule induces or modulates a second signal in the T cell, such as to enhance, dampen or modify a signal delivered through the first molecule. In some aspects, within 5 days after initiation of the incubation, the reversible binding between the second receptor-binding agent and the reagent is disrupted, thereby generating cultured T cells.

[0021] In some embodiments, the second signal enhances, dampens or modifies a signal delivered through the first molecule in the T cells.

[0022] In some embodiments of any of the methods provided herein, the incubation is performed under conditions in which the first receptor-binding agent specifically binds to the first molecule and/or the second receptor-binding agent specifically binds to the second molecule, thereby inducing or modulating one or more signals in the T cells. In some embodiments, said disruption is carried out greater than 30 minutes after initiation of said incubation. In some embodiments, said disruption is carried out between 1 hour and 4 days after initiation of said incubation, between 6 hours and 3 days after initiation of said incubation, between 12 hours and 2 days after initiation of said incubation or between 1 day and 3 days after initiation of said incubation; or said disruption is carried out between about 1 hour and about 4 days after initiation of said incubation, between about 6 hours and about 3 days after initiation of said incubation, between about 12 hours and about 2 days after initiation of said incubation or between about 1 day and about 3 days after initiation of said incubation; or said disruption is carried out greater than or equal to about 1 hour after initiation of said incubation and within 1 day, 2 days, 3 days or 4 days after initiation of said incubation.

[0023] In some embodiments, the second receptor-binding agent includes a binding partner C1. In some such embodiments, the plurality of binding sites contained by the reagent includes two or more binding sites, Z1, which each are capable of binding to the binding partner C1 to form the reversible bond between the second receptor-binding agent and the reagent.

[0024] In some embodiments of any of the methods provided herein, the second signal is a signal other than a TCR/CD3 complex-associated signal; the second signal is capable of enhancing or potentiating a TCR/CD3 complex-associated signal; or the second molecule is a costimulatory molecule, accessory molecule, cytokine receptor, chemokine receptor, immune checkpoint molecule or is a member of the TNF family or the TNF receptor family. In some embodiments, the second 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 molecule is CD28.

[0025] In some embodiments, the second molecule is or includes an adhesion molecule or is a factor that induces cytokine production, chemokine production and/or expression of an adhesion molecule.

[0026] In some embodiments, the second receptor-binding agent specifically binds to a cytokine receptor selected from among IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, Type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2. In some embodiments, the second receptor-binding agent is a ligand that specifically binds to a cytokine receptor selected from among IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, Type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2; and/or the second receptor-binding agent is a ligand selected from among IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-1β, IL-12, IL-15, IL-17 and TNF, or is a biologically active fragment thereof. In some embodiments, the second receptor-binding agent specifically binds to a cytokine receptor selected from among IL-12R, IFN-gammaR, IL-4R and IL-17R; or the second receptor-binding agent is a ligand selected from among IL-2, IL-4, IL-7, IL-1β, IL-15 and IL-17 or is a biologically active fragment thereof.

[0027] In some embodiments, the second receptor-binding agent specifically binds to a cytokine receptor selected from among IL-7R, IL-21R and CD132 (IL receptor common gamma chain). In some embodiments, the second receptor-binding agent is a ligand that specifically binds to a cytokine receptor selected from among IL-7R, IL-21R and CD132 (IL receptor common gamma chain); and/or the second receptor-binding agent is a ligand selected from among IL-7, IL-21, IL-2, IL-4, IL-9 and IL-15, or is a biologically active fragment thereof. In some embodiments, the second receptor-binding agent specifically binds to a cytokine receptor selected from among IL-7R, IL-21R and CD132 (IL receptor common gamma chain); or the second receptor-binding agent is a ligand selected from among IL-7, IL-21, IL-2, IL-4, IL-9 and IL-15, or is a biologically active fragment thereof.

[0028] In some embodiments, the second receptor-binding agent specifically binds to a chemokine receptor selected from among CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4. In some embodiments, the second receptor-binding agent is a ligand that specifically binds to a cytokine receptor selected from among CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4; or the second receptor-binding agent is a ligand selected from among CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof. In some embodiments, the second receptor-binding agent specifically binds to a chemokine receptor selected from among CXCR3, CCR7, CXCR1 and CXCR4; or the second receptor-binding agent is a ligand selected from among CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof.

[0029] In some embodiments, the adhesion molecule is selected from among CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin), and CD29/CD49d (VLA-4), CD106 (VCAM-1) or is a biologically active fragment thereof. In some embodiments, the adhesion molecule is selected from among LFA-1, L-selectin, VCAM-1 and VLA-4 or is a biologically active fragment thereof.

[0030] In some embodiments, the factor is a nuclear factor. In some embodiments, the factor is a retinoic acid receptor-related orphan receptor gamma (RORgamma) or RORalpha.

[0031] In some embodiments, said disruption includes introducing to the cells a composition that contains a substance capable of reversing the bond between the second receptor-binding agent and the reagent. In some embodiments, said disruption terminates or lessens the signal induced or modulated by the second receptor-binding agent. For example, in some embodiments, the second molecule is CD28 and the disruption terminates or lessens the CD28 costimulatory signal in the T cells.

[0032] In some embodiments, the incubation is further carried out in the presence of a third receptor-binding agent, which is capable of specifically binding to a third molecule on the surface of one or more of the T cells.

[0033] In some embodiments of any of the methods provided herein, the first receptor-binding agent specifically binds to a first molecule expressed on the surface of the T cells in a manner that induces or modulates a TCR/CD3 complex-associated signal in the T cell; the second receptor-binding agent specifically binds to the second molecule, thereby inducing or modulating a second signal in the T cells; and the third receptor-binding agent specifically binds to a third molecule on the surface of the T cells that induces or modulates a further signal in the cell.

[0034] In some embodiments, the first molecule is CD3.

[0035] In some embodiments, the second signal enhances, dampens or modifies a signal delivered through the first molecule. In some embodiments, the second molecule is a costimulatory molecule, accessory molecule, cytokine receptor, chemokine receptor, immune checkpoint molecule or is a member of the TNF family or the TNF receptor family. In some embodiments, the second 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 molecule is CD28.

[0036] In some embodiments, the third molecule is a cytokine receptor, a chemokine receptor, or is or includes an adhesion molecule or is a factor that induces cytokine production, chemokine production and/or expression of an adhesion molecule. In some embodiments, the third receptor-binding agent specifically binds to a cytokine receptor selected from among IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, Type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2. In some embodiments, the third receptor-binding agent is a ligand that specifically binds to a cytokine receptor selected from among IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, Type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2; and/or the third receptor-binding agent is a ligand selected from among IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-1β, IL-12, IL-15, IL-17 and TNF, or is a biologically active fragment thereof.

[0037] In some embodiments, the third receptor-binding agent specifically binds to a cytokine receptor selected from among IL-12R, IFN-gammaR, IL-4R and IL-17R; or the third receptor-binding agent is a ligand selected from among IL-2, IL-4, IL-7, IL-1β, IL-15 and IL-17 or is a biologically active fragment thereof. In some embodiments, the third receptor-binding agent specifically binds to a cytokine receptor selected from among IL-7R, IL-21R and CD132 (IL receptor common gamma chain). In some embodiments, the third receptor-binding agent is a ligand that specifically binds to a cytokine receptor selected from among IL-7R, IL-21R and CD132 (IL receptor common gamma chain); and/or the third receptor-binding agent is a ligand selected from among IL-7, IL-21, IL-2, IL-4, IL-9 and IL-15, or is a biologically active fragment thereof. In some embodiments, the third receptor-binding agent specifically binds to a cytokine receptor selected from among IL-7R, IL-21R and CD132 (IL receptor common gamma chain); or the third receptor-binding agent is a ligand selected from among IL-7, IL-21, IL-2, IL-4, IL-9 and IL-15, or is a biologically active fragment thereof.

[0038] In some embodiments, the third receptor-binding agent specifically binds to a chemokine receptor selected from among CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4. In some embodiments: the third receptor-binding agent is a ligand that specifically binds to a cytokine receptor selected from among CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4; or the third receptor-binding agent is a ligand selected from among CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof. In some embodiments, the third receptor-binding agent specifically binds to a chemokine receptor selected from among CXCR3, CCR7, CXCR1 and CXCR4; or the third receptor-binding agent is a ligand selected from among CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof.

[0039] In some embodiments, the adhesion molecule is selected from among CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin), and CD29/CD49d (VLA-4), CD106 (VCAM-1) or is a biologically active fragment thereof. In some embodiments, the adhesion molecule is selected from among LFA-1, L-selectin, VCAM-1 and VLA-4 or is a biologically active fragment thereof.

[0040] In some embodiments, the factor is a nuclear factor. In some embodiments, the factor is a retinoic acid receptor-related orphan receptor gamma (RORgamma) or RORalpha.

[0041] In some embodiments, the third receptor-binding agent is reversibly bound to the first reagent or the second reagent; or the incubation is carried out in the presence of a further reagent that is reversibly bound to the third receptor-binding agent.

[0042] In some embodiments of any of the methods provided herein, the method also includes, after said disruption, further incubating the composition that contains the T cells. In some embodiments, the incubation and further incubation are carried out in the same vessel; and/or the further incubation is carried out in the presence of the substance; and/or the method does not comprise removing the substance, receptor-binding agent, second receptor-binding agent and/or reagent from the cell composition prior to the further incubation.

[0043] In some embodiments, the incubation and/or further incubation is carried out at or about 37° C.±2° C.; and/or the incubation and/or further incubation is carried out in the presence of a further agent that is capable of delivering a signal to T cells. In some embodiments, the further agent is capable of enhancing or inducing proliferation of T cells, CD4+ T cells and/or CD8+ T cells. In some embodiments, the further 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 time that is no more than 14 days, no more than 12 days, no more than 10 days, no more than 8 days or no more than 6 days.

[0044] Provided herein are methods for culturing T cells that include: incubating a composition that contains T cells in the presence of a receptor-binding agent that specifically binds to a CD28 molecule on the surface of T cells under conditions to effect signaling through CD28 in the cells; and within 5 days after initiation of said incubation, eliminating or reducing the binding of the receptor-binding agent and the CD28 molecule, whereby the CD28 signaling is terminated or lessened in the cells, thereby generating cultured T cells. In some embodiments, the elimination or reduction is carried out within 4 days, within 3 days, within 2 days or within 1 day after initiation of the incubation.

[0045] In some instances, the elimination or reduction is effected within 4 days, within 3 days, within 2 days or within 1 day after initiation of the incubation. In some cases, the eliminating or reducing includes washing the cells, whereby any receptor-binding agent that is not specifically bound to CD28 is removed or reduced from the composition. In some aspects, the eliminating includes reversing the binding interaction between the receptor-binding agent and the CD28 molecule, further including washing the cells to remove or reduce the receptor-binding agent from the composition.

[0046] In some aspects, during at least a portion of the incubation and/or subsequent to the incubation, T cells in the composition are incubated in the presence of an agent that specifically binds a molecule of the TCR/CD3 complex, whereby a TCR/CD3 complex-associated signal is induced or modulated in the cells.

[0047] Provided herein in some aspects is a method for culturing T cells including incubating a composition containing T cells in the presence of a receptor-binding agent. In some embodiments, the receptor-binding agent is reversibly bound to a reagent containing a plurality of binding sites capable of reversibly binding to the receptor-binding agent. In some cases, the receptor-binding agent is capable of specifically binding to a molecule on the surface of the target cells other than CD28, CD3 or CD40 in a manner that induces or modulates a signal in the target cells and/or alters a function of the target cells, thereby generating cultured target cells. In some embodiments, the molecule is not CD137 and/or the receptor-binding agent does not specifically bind to CD137.

[0048] In some aspects, the incubation is performed under conditions in which the receptor-binding agent specifically binds to the molecule, thereby inducing or modulating the signal in the T cells. In some embodiments, the signal is not a TCR/CD3 complex-associated signal.

[0049] In some embodiments, the signal is not a TCR/CD3 complex-associated signal.

[0050] In some embodiments, the receptor-binding agent includes a binding partner C1. In some such embodiments, the plurality of binding sites of the reagent includes two or more binding sites, Z1, which each are capable of binding to the binding partner C1 to form the reversible bond between the receptor-binding agent and the reagent.

[0051] In some embodiments, the molecule is selected from among CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 and HVEM; and/or the receptor-binding agent specifically binds to CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 or HVEM.

[0052] In some embodiments, the receptor-binding agent is a second receptor-binding agent and the molecule is a second molecule, and the incubation is further carried out in the presence of a first receptor-binding agent, which is capable of specifically binding to a first molecule on the surface of one or more of the T cells, which first molecule is optionally capable of inducing or modulating a first signal in one or more T cells in the composition. In some embodiments, the first receptor-binding agent is reversibly bound to the reagent, said reagent that includes a plurality of binding sites for the first receptor-binding agent and the second receptor-binding agent; or the first receptor-binding agent is reversibly bound to a second reagent that includes a plurality of binding sites capable of reversibly binding to the first receptor-binding agent.

[0053] In some embodiments, the first receptor-binding agent is capable of initiating a TCR/CD3 complex-associated signal in the T cells. In some aspects, the first receptor-binding agent specifically binds to a member of a TCR/CD3 complex. In some cases, the first receptor-binding agent specifically binds to CD3.

[0054] In some embodiments, the specific binding of the second receptor-binding agent to the second molecule is capable of enhancing, dampening or modifying a signal delivered through the first molecule. In some cases, the specific binding of the second receptor-binding agent to the second molecule is capable of enhancing or potentiating a TCR/CD3 complex-associated signal.

[0055] Provided herein in some embodiments is a method for culturing T cells, which method includes incubating a composition containing T cells in the presence of a first receptor-binding agent which is reversibly bound to a reagent containing a plurality of binding sites capable of reversibly binding to the first receptor-binding agent. In some cases, first receptor-binding agent is capable of specifically binding to a first molecule on the surface of the T cells, such as to induce or modulate a TCR/CD3 complex-associated signal in T cells in the composition. In some embodiments, the method includes incubating the composition containing the T cells in the presence of a second receptor-binding agent which may be reversibly bound to the reagent further containing a plurality of binding sites for the second receptor-binding agent or to a second reagent containing a plurality of binding sites capable of reversibly binding to the second receptor-binding agent. In some aspects, the second receptor-binding agent is capable of specifically binding to a second molecule on the surface of T cells such as to induce or modulate a second signal in T cells in the composition. In some aspects, the second molecule is other than CD28. In some embodiments, the incubation is performed under conditions in which the signal and/or second signal are induced or modulated in T cells in the composition, thereby generating cultured T cells.

[0056] In some embodiments, the first receptor-binding agent specifically binds to a member of a TCR/CD3 complex and/or the first receptor-binding agent specifically binds to CD3.

[0057] In some instances, the specific binding of the second receptor-binding agent to the second molecule is capable of inducing or modulating a signal other than a TCR/CD3 complex-associated signal. In some aspects, the specific binding of the second receptor-binding agent to the second molecule is capable of enhancing, dampening or modifying a signal delivered through the first molecule. In some embodiments, the specific binding of the second receptor-binding agent to the second molecule is capable of enhancing or potentiating a TCR/CD3 complex-associated signal.

[0058] In some embodiments, the molecule, which can be the second molecule, is CD90 (Thy-1), CD95 (Apo-/Fas), CD137, (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 or HVEM. In some aspects, the receptor-binding agent, which can be the second receptor-binding agent, specifically binds to CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 or HVEM. In some embodiments, the molecule, which can be a second molecule, is not CD137 and/or the receptor-binding agent, which can be the second receptor-binding agent, does not specifically bind to CD137.

[0059] In some aspects, the first receptor-binding agent and the second receptor-binding agent reversibly bind to the reagent. In some embodiments, the first receptor-binding agent and second receptor-binding agent each individually include a binding partner C1, and the plurality of binding sites includes two or more binding site, Z1, which each are capable of binding to the binding partner C1 to form the reversible bond between the first and second receptor-binding agent and the reagent. In other embodiments, the first receptor-binding agent includes a binding partner C1, the second receptor-binding agent includes a binding partner C2, and the plurality of binding sites includes two or more binding sites, Z1, which each are capable of binding to the binding partner C1 and the binding partner C2 to form the reversible bond between the first and second receptor-binding agent and the reagent. In still further embodiments, the first receptor-binding agent includes a binding partner C1, the second receptor binding agent includes a binding partner C2, and the plurality of binding sites includes two or more binding site, Z1, which each are capable of binding to the binding partner C1 to form the reversible bond between the first receptor-binding agent and the reagent and two or more binding site, Z2, which each are capable of binding to the binding partner C2 to form the reversible bond between the second receptor-binding agent and the reagent.

[0060] In some embodiments, the receptor-binding agent specifically binds to a cytokine receptor selected from among IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, Type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2. In some embodiments, the receptor-binding agent is a ligand that specifically binds to a cytokine receptor selected from among IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, Type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2; and/or the receptor-binding agent is a ligand selected from among IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-1β, IL-12, IL-15, IL-17 and TNF, or is a biologically active fragment thereof. In some embodiments, the receptor-binding agent specifically binds to a cytokine receptor selected from among IL-12R, IFN-gammaR, IL-4R and IL-17R; or the receptor-binding agent is a ligand selected from among IL-2, IL-4, IL-7, IL-1β, IL-15 and IL-17 or is a biologically active fragment thereof.

[0061] In some embodiments, the receptor-binding agent specifically binds to a cytokine receptor selected from among IL-7R, IL-21R and CD132 (IL receptor common gamma chain). In some embodiments, the receptor-binding agent is a ligand that specifically binds to a cytokine receptor selected from among IL-7R, IL-21R and CD132 (IL receptor common gamma chain); and/or the receptor-binding agent is a ligand selected from among IL-7, IL-21, IL-2, IL-4, IL-9 and IL-15, or is a biologically active fragment thereof. In some embodiments, the receptor-binding agent specifically binds to a cytokine receptor selected from among IL-7R, IL-21R and CD132 (IL receptor common gamma chain); or the receptor-binding agent is a ligand selected from among IL-7, IL-21, IL-2, IL-4, IL-9 and IL-15, or is a biologically active fragment thereof.

[0062] In some embodiments, the receptor-binding agent specifically binds to a chemokine receptor selected from among CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4. In some embodiments, the receptor-binding agent is a ligand that specifically binds to a cytokine receptor selected from among CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4; or the receptor-binding agent is a ligand selected from among CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof. In some embodiments, the receptor-binding agent specifically binds to a chemokine receptor selected from among CXCR3, CCR7, CXCR1 and CXCR4; or the receptor-binding agent is a ligand selected from among CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof.

[0063] In some embodiments, the adhesion molecule is selected from among CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin), and CD29/CD49d (VLA-4), CD106 (VCAM-1) or is a biologically active fragment thereof. In some embodiments, the adhesion molecule is selected from among LFA-1, L-selectin, VCAM-1 and VLA-4 or is a biologically active fragment thereof.

[0064] In some embodiments, the factor is a nuclear factor. In some embodiments, the factor is a retinoic acid receptor-related orphan receptor gamma (RORgamma) or RORalpha.

[0065] In some embodiments of any of the methods provided herein, the incubation is performed under conditions in which the receptor-binding agent specifically binds to the molecule, thereby inducing or modulating the signal in the target cells and/or altering a function in the target cells. In some embodiments, the receptor-binding agent includes a binding partner C1, and the plurality of binding sites includes two or more binding site, Z1, which each are capable of binding to the binding partner C1 to form the reversible bond between the receptor-binding agent and the reagent. In some embodiments, the receptor-binding agent is an additional receptor-binding agent and the molecule is an additional molecule, and the incubation is further carried out in the presence of a first receptor-binding agent, which is capable of specifically binding to a first molecule on the surface of one or more of the T cells, which first molecule is optionally capable of inducing or modulating a first signal in one or more T cells in the composition.

[0066] In some embodiments, the first receptor-binding agent is reversibly bound to the reagent, said reagent that includes a plurality of binding sites for the first receptor-binding agent and the additional receptor-binding agent; or the first receptor-binding agent is reversibly bound to a second reagent that includes a plurality of binding sites capable of reversibly binding to the first receptor-binding agent. In some embodiments, the first receptor-binding agent is capable of initiating a TCR/CD3 complex-associated signal in the T cells; and/or the first receptor-binding agent specifically binds to a member of a TCR/CD3 complex; and/or the first receptor-binding agent specifically binds to CD3.

[0067] In some embodiments, the first receptor-binding agent includes a binding partner C2, and the plurality of binding sites includes two or more binding site, Z2, which each are capable of binding to the binding partner C2 to form the reversible bond between the first receptor-binding agent and the reagent.

[0068] In some embodiments, the incubation is further carried out in the presence of a second receptor-binding agent, which is capable of specifically binding to a second molecule on the surface of one or more of the T cells, which second molecule is optionally capable of inducing or modulating a signal in target cells in the composition to enhance, dampen or modify a signal delivered through the first molecule.

[0069] In some embodiments, the second signal is a signal other than a TCR/CD3 complex-associated signal; the second signal is capable of enhancing or potentiating a TCR/CD3 complex-associated signal; or the second molecule is a costimulatory molecule, accessory molecule, cytokine receptor, chemokine receptor, immune checkpoint molecule or is a member of the TNF family or the TNF receptor family. In some embodiments, the second molecule is selected from among CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 and HVEM; and/or the second receptor-binding agent specifically binds to CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 or HVEM. In some embodiments, the second molecule is selected from among CD28 and CD137. In some embodiments, the second molecule is CD28.

[0070] Provided herein are methods for culturing target cells, which method includes incubating a composition containing target cells in the presence of a receptor-binding agent. The receptor-binding agent may be reversibly bound to a reagent that is a streptavidin analog or mutein containing a plurality of binding sites capable of reversibly binding to the receptor-binding agent. In some cases, the receptor-binding agent is capable of specifically binding to a molecule on the surface of the target cells, such as to induce or modulate a signal in target cells in the composition. In some aspects, the mutein streptavidin includes a net negative charge or exhibits an isoelectric point less than the streptavidin mutein comprising the sequence set forth in SEQ ID NO:4 or 6, thereby generating cultured target cells.

[0071] Provided herein in some aspects is a method for culturing target cells, the method including incubating a composition containing target cells in the presence of a receptor-binding agent that is reversibly bound to a reagent that is a streptavidin analog or mutein containing a plurality of binding sites capable of reversibly binding to the receptor-binding agent. In some embodiments, the receptor-binding agent is capable of specifically binding to a molecule on the surface of the target cells in a manner that induces or modulates a signal in target cells in the composition. In some cases, the streptavidin analog or mutein exhibits a higher affinity for a streptavidin-binding peptide containing the sequence of amino acids Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8) than a streptavidin or mutein containing the sequence of amino acids set forth in any of SEQ ID NOS: 1-6, thereby generating cultured target cells.

[0072] In some embodiments, the incubation is performed under conditions in which the receptor-binding agent specifically binds to the molecule, thereby inducing or modulating the signal in one or more target cells in the composition.

[0073] In some instances, the plurality of binding sites of the reagent includes two or more binding sites, Z1. In some cases, the receptor-binding agent includes a binding partner C1, which is capable of reversibly binding to the binding site Z1, wherein the reversible binding between C1 and Z1 effects the reversible binding between the receptor-binding agent and the reagent. In some embodiments, the streptavidin analog or mutein includes a plurality of binding sites Z1, and a plurality of receptor-binding agents are reversibly bound to the reagent.

[0074] In some embodiments, the target cells comprise immune cells. For example, in some embodiments, the target cells comprise blood cells; the target cells comprise leukocytes; the target cells comprise lymphocytes; the target cells comprise B cells; the target cells comprise a B cell population; the target cells comprise T cells; the target cells comprise a T cell population; and/or the target cells comprise natural killer (NK) cells. In some embodiments, the target cells comprise antigen-specific T cells or a population thereof, a T helper cell or population thereof, a cytotoxic T cell or population thereof, a memory T cell or population thereof, a regulatory T cell or population thereof, an NK cell or population thereof, antigen-specific B cells or a population thereof, a memory B cell or population thereof, or a regulatory B cell or population thereof. In some embodiments, the target cells are T cells.

[0075] In some embodiments, the molecule is present on the surface of T cells, and the receptor-binding agent is capable of inducing or modulating a signal in T cells in the composition. In some aspects, the receptor-binding agent is capable of initiating a TCR/CD3 complex-associated signal in the T cells and/or the receptor-binding agent specifically binds to a member of a TCR/CD3 complex. In some cases, the receptor-binding agent, specifically binds to CD3. In some instances, the molecule is a first molecule and the receptor-binding agent is capable of specifically binding to the first molecule and, in some cases, a second molecule on the surface of one or more of the target cells. In some embodiments, binding to the second molecule is capable of enhancing, dampening, or modifying a signal delivered through the first molecule.

[0076] In some embodiments, the molecule is a first molecule and the receptor-binding agent is capable of specifically binding to the first molecule and a second molecule on the surface of one or more of the target cells, which binding to the second molecule induces or modulates a signal in the target cells. In some embodiments, binding to the second molecule is optionally capable of enhancing, dampening, or modifying a signal delivered through the first molecule. [0077], the receptor-binding agent is a first receptor-binding agent and the incubation is further carried out in the presence of a second receptor-binding agent. The second receptor-binding agent may be capable of specifically binding to a second molecule on the surface of one or more of the target cells. In some cases, binding of the second receptor-binding agent to the second molecule is capable of inducing or modulating a signal to enhance, dampen, or modify a signal delivered through the first molecule.

[0077] In some embodiments, the incubation is performed under conditions in which the second receptor-binding agent specifically binds to the second molecule, thereby inducing or modulating a signal in target cells in the composition to enhance, dampen or modify a signal delivered through the first molecule. In some embodiments, the streptavidin mutein or analog includes a plurality of binding sites capable of reversibly binding to the second receptor-binding agent, whereby the second receptor-binding agent is reversibly bound to the streptavidin mutein or analog.

[0078] In some embodiments, the second receptor-binding agent includes a binding partner C1 or C2, which is capable of reversibly binding to the two or more binding sites Z2 present in the streptavidin analog or mutein.

[0079] In some cases, the additional molecule is CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 or HVEM. In some embodiments, the second receptor-binding agent specifically binds to CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 or HVEM. In some aspects, the additional molecule is CD40 and CD137. In some instances, the second receptor-binding agent specifically binds to CD40 or CD137.

[0080] In some embodiments, the second molecule is or includes an adhesion molecule or is a factor that induces cytokine production, chemokine production and/or expression of an adhesion molecule.

[0081] In some embodiments, the second receptor-binding agent specifically binds to a cytokine receptor selected from among IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, Type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2. In some embodiments, the second receptor-binding agent is a ligand that specifically binds to a cytokine receptor selected from among IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, Type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2; and/or the second receptor-binding agent is a ligand selected from among IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-1β, IL-12, IL-15, IL-17 and TNF, or is a biologically active fragment thereof. In some embodiments, the second receptor-binding agent specifically binds to a cytokine receptor selected from among IL-12R, IFN-gammaR, IL-4R and IL-17R; or the second receptor-binding agent is a ligand selected from among IL-2, IL-4, IL-7, IL-1β, IL-15 and IL-17 or is a biologically active fragment thereof.

[0082] In some embodiments, the second receptor-binding agent specifically binds to a cytokine receptor selected from among IL-7R, IL-21R and CD132 (IL receptor common gamma chain). In some embodiments, the second receptor-binding agent is a ligand that specifically binds to a cytokine receptor selected from among IL-7R, IL-21R and CD132 (IL receptor common gamma chain); and/or the second receptor-binding agent is a ligand selected from among IL-7, IL-21, IL-2, IL-4, IL-9 and IL-15, or is a biologically active fragment thereof. In some embodiments, the second receptor-binding agent specifically binds to a cytokine receptor selected from among IL-7R, IL-21R and CD132 (IL receptor common gamma chain); or the second receptor-binding agent is a ligand selected from among IL-7, IL-21, IL-2, IL-4, IL-9 and IL-15, or is a biologically active fragment thereof.

[0083] In some embodiments, the second receptor-binding agent specifically binds to a chemokine receptor selected from among CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4. In some embodiments, the second receptor-binding agent is a ligand that specifically binds to a cytokine receptor selected from among CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4; or the second receptor-binding agent is a ligand selected from among CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof. In some embodiments, the second receptor-binding agent specifically binds to a chemokine receptor selected from among CXCR3, CCR7, CXCR1 and CXCR4; or the second receptor-binding agent is a ligand selected from among CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof.

[0084] In some embodiments, the adhesion molecule is selected from among CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin), and CD29/CD49d (VLA-4), CD106 (VCAM-1) or is a biologically active fragment thereof. In some embodiments, the adhesion molecule is selected from among LFA-1, L-selectin, VCAM-1 and VLA-4 or is a biologically active fragment thereof.

[0085] In some embodiments, the factor is a nuclear factor. In some embodiments, the factor is a retinoic acid receptor-related orphan receptor gamma (RORgamma) or RORalpha. [0087], the second receptor-binding agent includes a plurality of different receptor-binding agents, each of which is capable of individually binding to the same or different second molecule on the surface of T cells in the composition to collectively induce or modulate one or more signals in the cells.

[0086] In some embodiments, the method further includes disrupting the reversible binding between the first and/or second receptor-binding agent and the reagent. In some aspects, the disruption is effected within 14 days after initiation of the incubation, within 12 days after initiation of the incubation, within 10 days after initiation of the incubation within 8 days after initiation of the incubation or within 6 days after initiation of the incubation.

[0087] In some embodiments, at least a portion of the incubation is carried out in the presence of a further agent that is capable of delivering a signal to T cells. In some cases, the further agent is capable of enhancing or inducing proliferation of T cells, CD4+ T cells and/or CD8+ T cells. In some embodiments, the further agent is a cytokine such as IL-2, IL-15 or IL-7. In some instances, the further agent does not specifically bind to CD28 and/or induce CD28 signaling.

[0088] In some embodiments, the T cells or target cells are primary cells from a subject. In some cases, the T cells or target cells are directly isolated from a subject. In some embodiments, the T cells are unfractionated T cells, are enriched or isolated CD3+ T cells, are enriched or isolated CD4+ T cells, or are enriched or isolated CD8+ T cells. In some embodiments, prior to the incubating the T cells are not enriched for CD62L+ cells and/or are not enriched for naïve T cells. In some cases, the T cells or target cells are human cells.

[0089] In some aspects, the reagent has a size that is less than 20 nm, less than 10 nm, less than 5 nm or less than 1 nm. In some instances, the reagent has a density of less than 1.2 g/cm3 or less than 1.0 g/cm3.

[0090] In some embodiments, the reagent is not, and is not bound to or associated with, a solid support, stationary phase, a bead, a microparticle, a magnetic particle, and/or a matrix during said incubation; and/or the reagent is flexible, does not contain a metal or magnetic core, is comprised entirely or primarily of organic multimer, is not spherical, is not substantially spherical or uniform in shape and/or is not rigid.

[0091] In some embodiments, the reagent is not bound to a support or a solid support during said incubation. In some embodiments, the reagent is bound to a support during at least a portion of the incubation, whereby a plurality of the T cells or target cells are reversibly immobilized on the support during at least a portion of the incubation. In some embodiments, the support is or includes a stationary phase; and/or the support is or includes a solid support.

[0092] In some embodiments, the receptor-binding agent, which can be the first receptor-binding agent, contains only one binding site, such as B2. In some aspects, the receptor-binding agent, which can be the first receptor-binding agent, specifically binds to the molecule in a monovalent manner. In some embodiments, the second receptor-binding agent contains only one binding site, such as B4. In some cases, the second receptor-binding agent specifically binds to the molecule in a monovalent manner. In some embodiments, the binding site, such as B2 or B4, contains an antibody combining site.

[0093] In some aspects, the receptor-binding agent, which can be a first receptor-binding agent, and/or the second receptor-binding agent each individually is an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties, a molecule containing Ig domains, a cytokine, a chemokine, an aptamer, or MHC molecule or binding fragments thereof. In some such aspects, the receptor-binding agent, which can be the first receptor-binding agent, and/or the second receptor-binding agent include an antibody fragment, a Fab fragment, a divalent antibody fragment such as a F(ab′).sub.2-fragment or a divalent single-chain Fv (scFv) fragment. In some cases, the receptor-binding agent, which can be the first receptor-binding agent, and/or the second receptor-binding agent is a monovalent antibody fragment such as a Fab fragment, an Fv fragment or an scFv fragment. In some instances, the receptor-binding agent, which can be the first receptor-binding agent, and/or the second receptor-binding agent is a proteinaceous binding molecule with antibody-like binding properties, such as an aptamer, mutein based on a polypeptide of the lipocalin family, glubody, protein based on the ankyrin scaffold, protein based on the crystalline scaffold, adnectin or an avimer.

[0094] In some embodiments, the receptor-binding agent, which can be the first receptor-binding agent, includes an agent that specifically binds to CD3. The agent that specifically binds CD3 can be an anti-CD3-antibody, a divalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3-antibody, or a proteinaceous CD3 binding molecule with antibody-like binding properties. In some embodiments, the second receptor-binding agent includes an agent that specifically binds to CD28, CD90, CD95, CD137, CD154, ICOS, LAT, CD27, OX40 and/or HVEM. The agent that specifically binds to CD28, CD90, CD95, CD137, CD154, ICOS, LAT, CD27, OX40 and/or HVEM can be an anti-CD28-antibody, a divalent antibody fragment of an anti-CD28 antibody, an antibody fragment of an anti-CD28-antibody, a proteinaceous CD28 binding molecule with antibody-like binding properties, an anti-CD90-antibody, a divalent antibody fragment of an anti-CD90 antibody, an antibody fragment of an anti-CD90-antibody, a proteinaceous CD90 binding molecule with antibody-like binding properties, an anti-CD95-antibody, a divalent antibody fragment of an anti-CD95 antibody, an antibody fragment of an anti-CD95-antibody, a proteinaceous CD95 binding molecule with antibody-like binding properties, an anti-CD154-antibody, a divalent antibody fragment of an anti-CD154 antibody, a monovalent antibody fragment of an anti-CD154-antibody, a proteinaceous CD154 binding molecule with antibody-like binding properties, an anti-CD137-antibody, a divalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment of an anti-CD137-antibody, a proteinaceous CD137 binding molecule with antibody-like binding properties, an anti-ICOS-antibody, a divalent antibody fragment of an anti-ICOS antibody, an antibody fragment of an anti-ICOS-antibody, a proteinaceous ICOS binding molecule with antibody-like binding properties, an anti-LAT-antibody, a divalent antibody fragment of an anti-LAT antibody, an antibody fragment of an anti-LAT-antibody, a proteinaceous LAT binding molecule with antibody-like binding properties, an anti-CD27-antibody, a divalent antibody fragment of an anti-CD27 antibody, an antibody fragment of an anti-CD27-antibody, a proteinaceous CD27 binding molecule with antibody-like binding properties, an anti-OX40-antibody, a divalent antibody fragment of an anti-OX40 antibody, an antibody fragment of an anti-OX40-antibody, a proteinaceous OX40 binding molecule with antibody-like binding properties, an anti-HVEM-antibody, a divalent antibody fragment of an anti-HVEM antibody, an antibody fragment of an anti-HVEM-antibody, a proteinaceous HVEM binding molecule with antibody-like binding properties, 4-1BB ligand, or any mixture thereof.

[0095] In some embodiments of any of the methods provided herein, the receptor-binding agent comprise an antibody fragment; the receptor-binding agent comprise a Fab fragment; the receptor-binding agent is a divalent antibody fragment selected from among a F(ab′).sub.2-fragment and a divalent single-chain Fv (scFv) fragment; the receptor-binding agent is a monovalent antibody fragment selected from among a Fab fragment, an Fv fragment and an scFv fragment; and/or the receptor-binding agent is a proteinaceous binding molecule with antibody-like binding properties selected from among aptamers, muteins based on a polypeptide of the lipocalin family, glubodies, proteins based on the ankyrin scaffold, proteins based on the crystalline scaffold, adnectins and avimers.

[0096] In some embodiments of any of the methods provided herein, the reagent is or contains streptavidin, avidin, an analog or mutein of streptavidin that reversibly binds biotin, a biotin analog or a biologically active fragment thereof; an analog or mutein of avidin or streptavidin that reversibly binds a streptavidin-binding peptide; a reagent that includes at least two chelating groups K, wherein the at least two 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 a 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.

[0097] In some embodiments, the reagent is or contains a streptavidin analog or mutein or an avidin analog or mutein that reversibly binds to biotin or a biologically active fragment; a streptavidin analog or mutein or an avidin analog or mutein that reversibly binds to a biotin analog or a biologically active fragment; and/or a streptavidin analog or mutein or an avidin analog or mutein that reversibly binds to a streptavidin-binding peptide.

[0098] In some embodiments, the reagent is an oligomer or polymer of streptavidin, avidin, an analog or mutein of streptavidin that reversibly binds biotin or a biologically active fragment; a streptavidin or avidin analog or mutein that reversibly binds a streptavidin-binding peptide; a reagent that includes at least two chelating groups K, wherein the at least two 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 a 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. In some embodiments, the reagent includes an oligomer or polymer of streptavidin, avidin, a streptavidin analog or mutein or and an avidin analog or mutein. In some aspects, individual molecules of the oligomer or polymer are crosslinked by a polysaccharide or a bifunctional linker.

[0099] In some embodiments, the plurality of binding sites Z include at least 2, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 72 or more binding sites.

[0100] In some aspects, the streptavidin-binding peptide is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 17), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) or 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).

[0101] In some embodiments, the reagent includes a streptavidin analog or mutein containing the amino acid sequence Va144-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47 at sequence positions corresponding to positions 44 to 47 with reference to positions in streptavidin in the sequence of amino acids set forth in SEQ ID NO: 1. In some embodiments, the streptavidin analog or mutein includes the amino acid sequence Va144-Thr45-Ala46-Arg47 at sequence positions corresponding to positions 44 to 47 with reference to positions in streptavidin in the sequence of amino acids set forth in SEQ ID NO:1.

[0102] In some embodiments, the streptavidin analog or mutein includes the sequence of amino acids set forth in any of SEQ ID NOS: 3-6. In some aspects, the streptavidin analog or mutein includes a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS:3-6 and contains the amino acid sequence corresponding to Va144-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47. In some embodiments, the streptavidin analog or mutein reversibly binds to biotin or a biologically active form thereof, a biotin analog or mutein or a biologically active fragment thereof or a streptavidin-binding peptide. In some embodiments, the streptavidin analog or mutein binds a functional fragment of any of the above sequences that reversibly binds to biotin or a biologically active form thereof, a biotin analog or mutein or a biologically active fragment thereof or a streptavidin-binding peptide.

[0103] In some embodiments, the streptavidin analog or mutein further contains an amino acid replacement or replacements at a position corresponding to 117, 120 and/or 121 with reference to positions in streptavidin in the sequence of amino acids set forth in SEQ ID NO: 1. In some embodiments, the amino acid replacement or replacements are selected from among Glu117, Asp117, Arg117, Ser120, Ala120, Gly120, Trp121, Tyr121 or Phe121. In some embodiments, the amino acid replacement or replacements are Glu117, Gly120 or Tyr121.

[0104] In some embodiments, the streptavidin analog or mutein contains the sequence of amino acids set forth in SEQ ID NO: 27 or 28. In some aspects, the streptavidin analog or mutein contains a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOS:28 and contains the amino acid sequence corresponding to Va144, Thr45, Ala46, Arg47, Glu117, Gly120 and Tyr121. In some embodiments, the streptavidin analog or mutein reversibly binds to biotin or a biologically active fragment, a biotin analog or mutein or a biologically active fragment thereof or a streptavidin-binding peptide. In some embodiments, the streptavidin analog or mutein reversibly binds a functional fragment any of the above sequences that reversibly binds to biotin or a biologically active fragment, a biotin analog or mutein or a biologically active fragment thereof or a streptavidin-binding peptide.

[0105] In some embodiments, the binding partner C1 and/or the binding partner C2, independently, includes a streptavidin-binding peptide such as Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 17), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) or 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);

[0106] In some embodiments, the disruption includes introducing to the cells a composition containing a substance capable of reversing the bond between the receptor-binding agent, which can be the first receptor-binding agent, and/or the second-receptor-binding agent and the reagent. In some embodiments, the substance is a free binding partner and/or is a competition agent. In some embodiments, the substance in the composition is not detrimental to the T cells or to the target cells. In some aspects, the addition of the substance does not reduce the percentage of surviving T cells or target cells to less than 90%, 80%, 70%, 60%, or 50%, as compared to incubation of the T cells or target cells, respectively, under comparable or the same conditions, without the substance. In some embodiments, the disruption terminates or lessens the signal induced or modulated by one or both of the receptor-binding agent or second receptor-binding agent in the T cells or the target cells.

[0107] In some embodiments, the reagent is or contains a streptavidin, avidin, a streptavidin analog or mutein or and an avidin analog or mutein or biologically active fragments thereof. In some embodiments, the substance contains a streptavidin-binding peptide, biotin or a biologically active fragment, optionally a D-biotin, or a biotin analog or biologically active fragment.

[0108] In some embodiments, the substance is a streptavidin-binding peptide such as Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 17), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) or 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 embodiments, the substance is C1 or an analog thereof or is C2 or an analog thereof.

[0109] In some embodiments, the dissociation constant (K.sub.D) for the reversible binding between the binding site Z1 and the binding partner C1 and/or for the reversible binding between the binding site Z2 and the binding partner C2 is in the range of 10-2 M to 10-13 M.

[0110] In some embodiments, prior to the incubation, cells are contacted with a selection agent that specifically binds to a marker contained by T cells or target cells of the composition, thereby generating or obtaining the composition containing the T cells or target cells. In some embodiments, at least a portion of the incubation is carried out in the presence of a selection agent that specifically binds to a marker comprised by T cells or target cells of the composition, and the cultured T cells are enriched for T cells or target cells containing the marker.

[0111] In some embodiments, the selection agent is reversibly bound to the reagent, and the reagent further contains a plurality of binding sites capable of specifically binding the selection agent. In some aspects, the selection agent is reversibly bound to a second reagent containing a plurality of binding sites capable of specifically binding to the selection agent.

[0112] In some embodiments, the induction or modulation of the signal effects an increase in expansion (proliferation) and/or activation of the cultured T cells compared to incubation of T cells in the absence of the induction or modulation of the signal. In some embodiments, the increase is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater. In some embodiments, the induction or modulation of the additional or second signal increases expansion (proliferation) and/or activation of the T cells compared to incubation of T cells in the absence of the induction or modulation of the additional or second signal. In some embodiments, the increase is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater.

[0113] In some embodiments, the method increases expansion and/or proliferation of T cells in the composition, alters the metabolic profile of T cells in the composition, alters the subset of CD8+ T cells in the composition; and/or increases the percentage of long-lived memory T cells in the composition, each compared to the T cells in the composition prior to the incubation, subsequent to the incubation but in the absence of the disruption or subsequent to the incubation but in the presence of an agent that specifically binds CD28 and/or induces or modulates CD28 signaling.

[0114] In some embodiments, the methods result in cultured T cells in which the number or percentage of CD3+ T cells, CD4+ T cells or CD8+ cells is increased at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold compared to the number or percentage of CD3+ T cells, CD4+ T cells or CD8+ T cells, respectively, in the composition prior to the incubation, subsequent to the incubation but in the absence of the disruption, or subsequent to an analogous incubation but performed in the presence of an agent that specifically binds CD28 and/or induces or modulates CD28 signaling. In some embodiments, the methods result in cultured T cells in which the ratio of CD8+ T cells or the relative or normalized ratio of CD8+ T cells in the composition is increased at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold compared to the ratio or the relative or normalized ratio of CD8+ T cells in the composition prior to the incubation, subsequent to the incubation but in the absence of the disruption, or subsequent to an analogous incubation but in the presence of an agent that specifically binds CD28 and/or induces or modulates CD28 signaling. In some embodiments, the methods result in cultured T cells in which the number or percentage of CD62L+, optionally long-lived memory T cells or memory stem cells (T.sub.SCM), in the composition is increased at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold compared to the number or percentage of the corresponding population of cells, either CD62L+, long-lived memory T cells or T.sub.SCM, in the composition prior to the incubation, subsequent to the incubation but in the absence of the disruption, or subsequent to an analogous incubation but in the presence of an agent that specifically binds CD28 and/or induces or modulates CD28 signaling.

[0115] In some embodiments, the cultured T cells contain greater than 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90% of a T cell subset containing a phenotype that is surface positive for CD62L (CD62L+) as a percentage of the total T cells in the composition or the total cells in the composition.

[0116] In some embodiments, the T cell subset further includes a phenotype including CD127+; and/or any one or more of CD45RA+, CD45RO−, CCR7+ and CD27+ and any one or more of t-bet.sup.low, IL-7Ra+, CD95+, IL-2Rβ+, CXCR3+ and LFA-1+.

[0117] In some embodiments, the T cell subset includes a low level of TCR rearrangement excisions circles (TREC); and/or expresses a proliferation marker, which can be Ki-67; and/or exhibits the capacity to proliferate in the presence of a stimulatory agent; and/or exhibits the capacity to produce a cytokine such as IFN-gamma, TNF or IL-2 in the presence of a stimulatory agent.

[0118] In some embodiments, the stimulatory agent is an antigen, a homeostatic cytokine, such as IL-15 and/or IL-17, or is an agent that is capable of initiating a TCR/CD3 complex-associated signal in the T cells.

[0119] In some embodiments, the T cell subset is or contains long-lived memory T cells. In some embodiments, the T cell subset is or includes T memory stem cells (T.sub.SCM).

[0120] In some embodiments, the method further includes introducing a recombinant nucleic acid molecule into T cells or target cells of the population. In some such aspects, the nucleic acid molecule may encode a recombinant protein, whereby cells express the recombinant protein. In some embodiments, the recombinant receptor is a chimeric antigen receptor or transgenic T cell receptor (TCR). In some aspects, the method is performed in vitro or ex vivo.

[0121] In some embodiments, the method further includes administering the cultured cells to a subject having a disease or condition.

[0122] Provided herein in some aspects is a composition containing a plurality of cultured T cells or target cells produced by the methods provided herein, and optionally a pharmaceutically acceptable excipient.

[0123] In some embodiments, after addition of the substance, the cells have not been incubated in vitro or ex vivo at a temperature greater than 30° C. for more than 24 hours, more than 48 hours, more than 72 hours or more than 96 hours.

[0124] Also provided herein are articles of manufacture that include: a) a reagent that includes a plurality of binding sites capable of binding to a receptor-binding agent; and b) the receptor-binding agent which i) is reversibly bound to the reagent and ii) is capable of specifically binding to a molecule on the surface of T cells in a manner that induces or modulates a signal in T cells. In some embodiments the molecule is not CD28, CD3, or CD40.

[0125] In some embodiments, binding the molecule induces or modulates a signal in a T cell other than a TCR/CD3 complex-associated signal; and/or binding the molecule enhances or potentiates a TCR/CD3 complex-associated signal. In some embodiments, the molecule is selected from among CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 and HVEM. In some embodiments, the molecule is not CD137.

[0126] In some embodiments, the receptor-binding agent specifically binds to a cytokine receptor, specifically binds to a chemokine receptor, is or includes an adhesion molecule or is a factor that induces cytokine production, chemokine production and/or expression of an adhesion molecule.

[0127] In some embodiments, the receptor-binding agent specifically binds to a cytokine receptor selected from among IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, Type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2. In some embodiments, the receptor-binding agent is a ligand that specifically binds to a cytokine receptor selected from among IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, Type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2; and/or the receptor-binding agent is a ligand selected from among IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-1β, IL-12, IL-15, IL-17 and TNF, or is a biologically active fragment thereof. In some embodiments, the receptor-binding agent specifically binds to a cytokine selected from among IL-12R, IFN-gammaR, IL-4R and IL-17R; or the receptor-binding agent is a ligand selected from among IL-2, IL-4, IL-7, IL-1β, IL-15 and IL-17 or is a biologically active fragment thereof.

[0128] In some embodiments, the second receptor-binding agent specifically binds to a cytokine receptor selected from among IL-7R, IL-21R and CD132 (IL receptor common gamma chain). In some embodiments, the second receptor-binding agent is a ligand that specifically binds to a cytokine receptor selected from among IL-7R, IL-21R and CD132 (IL receptor common gamma chain); and/or the second receptor-binding agent is a ligand selected from among IL-7, IL-21, IL-2, IL-4, IL-9 and IL-15, or is a biologically active fragment thereof. In some embodiments, the second receptor-binding agent specifically binds to a cytokine receptor selected from among IL-7R, IL-21R and CD132 (IL receptor common gamma chain); or the second receptor-binding agent is a ligand selected from among IL-7, IL-21, IL-2, IL-4, IL-9 and IL-15, or is a biologically active fragment thereof.

[0129] In some embodiments, the receptor-binding agent specifically binds to a chemokine receptor selected from among CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4. In some embodiments, the receptor-binding agent is a ligand that specifically binds to a cytokine receptor selected from among CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4; or the receptor-binding agent is a ligand selected from among CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof. In some embodiments, the receptor-binding agent specifically binds to a cytokine selected from among CXCR3, CCR7, CXCR1 and CXCR4; or the receptor-binding agent is a ligand selected from among CXCL9, CXCL10, CCL19, CCL21 and CCL25 or is a biologically active fragment thereof.

[0130] In some embodiments, the adhesion molecule is selected from among CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin), and CD29/CD49d (VLA-4), CD106 (VCAM-1) or is a biologically active fragment thereof. In some embodiments, the adhesion molecule is selected from among LFA-1, L-selectin, VCAM-1 and VLA-4 or is a biologically active fragment thereof.

[0131] In some embodiments, the factor is a nuclear factor. In some embodiments, the factor is a retinoic acid receptor-related orphan receptor gamma (RORgamma) or RORalpha.

[0132] Provided herein are methods for culturing target cells that include incubating a composition that contains target cells in the presence of one or more receptor-binding agent, wherein the receptor-binding agent reversibly bound to a reagent that is a streptavidin analog or mutein containing a plurality of binding sites capable of binding to an agent. In some aspects, the streptavidin analog or mutein includes a net negative charge or exhibits an isoelectric point less than the streptavidin mutein comprising the sequence of amino acids set forth in SEQ ID NO:4 or 6 and/or exhibits a higher affinity for a streptavidin-binding peptide containing the sequence of amino acids Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8) than a streptavidin or mutein containing the sequence of amino acids set forth in any of SEQ ID NOS: 1-6. In some embodiments, the agent is reversibly bound to the reagent and is capable of specifically binding to a molecule on the surface of a cell.

[0133] In some embodiments, the receptor-binding agent includes a binding partner C1. In some aspects, the plurality of binding sites includes two or more binding sites, Z1, which each are capable of binding to the binding partner C1 to form the reversible bond between the receptor-binding agent or agent and the reagent.

[0134] In some embodiments, the agent is a selection agent or a receptor-binding agent.

[0135] In some embodiments, the agent is a receptor-binding agent that specifically binds to a molecule that is or includes a member of a TCR/CD3 complex, CD3, a costimulatory molecule, accessory molecule, cytokine receptor, chemokine receptor, immune checkpoint molecule or is a member of the TNF family or the TNF receptor family or is or includes an adhesion molecule or is a factor that induces cytokine production, chemokine production and/or expression of an adhesion molecule.

[0136] In some embodiments, the receptor-binding agent is a second receptor-binding agent and the molecule is a second molecule, and the reagent further includes: c) a plurality of binding sites capable of reversibly binding to a first receptor-binding agent and d) the first receptor-binding agent which i) is reversibly bound to the reagent and ii) is capable of specifically binding to a first molecule on the surface of a T cell.

[0137] In some embodiments, the agent or the first receptor-binding agent specifically binds to a member of a TCR/CD3 complex and/or the first receptor-binding agent specifically binds to CD3.

[0138] In some embodiments, the first receptor-binding agent and second receptor-binding agent each individually contains a binding partner C1, and the plurality of binding sites includes two or more binding site, Z1, which each are capable of binding to the binding partner C1 to form the reversible bond between the first and second receptor-binding agent and the reagent. In some aspects, the first receptor-binding agent contains a binding partner C1, the second receptor-binding agent contains a binding partner C2, and the plurality of binding sites include two or more binding sites, Z1, which each are capable of binding to the binding partner C1 and the binding partner C2 to form the reversible bond between the first and second receptor-binding agent and the reagent. In some embodiments, the first receptor-binding agent contains a binding partner C1, the second receptor binding agent contains a binding partner C2, and the plurality of binding sites includes two or more binding sites, Z1, which each are capable of binding to the binding partner C1 to form the reversible bond between the first receptor-binding agent and the reagent and two or more binding sites, Z2, which each are capable of binding to the binding partner C2 to form the reversible bond between the second receptor-binding agent and the reagent.

[0139] In some embodiments, the reagent has a size that is less than 20 nm, less than 10 nm, less than 5 nm or less than 1 nm. In some embodiments, the reagent has a density of less than 1.2 g/cm3 or less than 1.0 g/cm3. In some embodiments, the reagent is not bound to a support or solid support. In some instances, the reagent is bound or immobilized to a support. In some cases, the support is a solid support or a stationary phase. In some aspects, the support includes a bead, a particle, a nanoparticle or a microsphere.

[0140] In some embodiments, the agent individually is selected from among an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties, a molecule containing Ig domains, and binding fragments thereof.

[0141] In some embodiments, the agent, the receptor-binding agent, which can be the second receptor-binding agent, and/or the first receptor-binding agent contains an antibody fragment. In some aspects, the agent, the receptor-binding agent, which can be the second receptor-binding agent, and/or the first receptor-binding agent contains a Fab fragment. In some cases, the agent, the receptor-binding agent, which can be the second receptor-binding agent, and/or the first receptor-binding agent is a divalent antibody fragment such as a F(ab′).sub.2-fragment or a divalent single-chain Fv (scFv) fragment. In some embodiments, the agent, the receptor-binding agent, which can be the second receptor-binding agent, and/or the first receptor-binding agent is a monovalent antibody fragment selected from among a Fab fragment, an Fv fragment and an scFv fragment.

[0142] In some embodiments, the agent or first receptor-binding agent includes an agent that specifically binds to CD3. In some embodiments, the agent that specifically binds CD3 is an anti-CD3-antibody, a divalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3-antibody, or a proteinaceous CD3 binding molecule with antibody-like binding properties. In some embodiments, the agent or receptor-binding agent, which can be the second receptor-binding agent, includes an agent that specifically binds to CD90, CD95, CD137, CD154, ICOS, LAT, CD27, OX40 and HVEM, such as an anti-CD90-antibody, a divalent antibody fragment of an anti-CD90 antibody, an antibody fragment of an anti-CD90-antibody, a proteinaceous CD90 binding molecule with antibody-like binding properties, an anti-CD95-antibody, a divalent antibody fragment of an anti-CD95 antibody, an antibody fragment of an anti-CD95-antibody, a proteinaceous CD95 binding molecule with antibody-like binding properties, an anti-CD154-antibody, a divalent antibody fragment of an anti-CD154 antibody, a monovalent antibody fragment of an anti-CD154-antibody, a proteinaceous CD154 binding molecule with antibody-like binding properties, an anti-CD137-antibody, a divalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment of an anti-CD137-antibody, a proteinaceous CD137 binding molecule with antibody-like binding properties, an anti-ICOS-antibody, a divalent antibody fragment of an anti-ICOS antibody, a monovalent antibody fragment of an anti-ICOS-antibody, a proteinaceous ICOS binding molecule with antibody-like binding properties, an anti-LAT-antibody, a divalent antibody fragment of an anti-LAT antibody, a monovalent antibody fragment of an anti-LAT-antibody, a proteinaceous LAT binding molecule with antibody-like binding properties, an anti-CD27-antibody, a divalent antibody fragment of an anti-CD27 antibody, a monovalent antibody fragment of an anti-CD27-antibody, a proteinaceous CD27 binding molecule with antibody-like binding properties, an anti-OX40-antibody, a divalent antibody fragment of an anti-OX40 antibody, a monovalent antibody fragment of an anti-OX40-antibody, a proteinaceous OX40 binding molecule with antibody-like binding properties, an anti-HVEM-antibody, a divalent antibody fragment of an anti-HVEM antibody, a monovalent antibody fragment of an anti-HVEM-antibody, a proteinaceous HVEM binding molecule with antibody-like binding properties, or any mixture thereof.

[0143] In some embodiments, the reagent is or contains streptavidin, avidin, a streptavidin analog or mutein or and an avidin analog or mutein. In some embodiments, the reagent includes an oligomer or polymer of streptavidin, avidin, a streptavidin analog or mutein or and an avidin analog or mutein.

[0144] Provided herein in some aspects is a kit, including the reagent disclosed herein and optionally instructions for use. In some embodiments, the kit contains a substance capable of reversing the bond between the receptor-binding agent and the reagent. In some embodiments, the kit contains a reagent containing a plurality of binding sites capable of reversibly binding to a receptor-binding agent. In some aspects, the receptor-binding agent is reversibly bound to the reagent and is capable of specifically binding to a molecule expressed on the surface of target cells. In some instances, binding to the molecule induces or modulates a signal in the target cells. In some cases the kit contains a substance capable of reversing the bond between the receptor-binding agent and the reagent.

[0145] In some embodiments, the target cells are T cells. In some embodiments, the reagent is or includes streptavidin, avidin, a streptavidin analog or mutein or and an avidin analog or mutein. In some embodiments, the reagent includes an oligomer or polymer of streptavidin, avidin, a streptavidin analog or mutein or and an avidin analog or mutein.

[0146] In some embodiments, the substance includes a streptavidin-binding peptide, biotin or a biologically active fragment, optionally a D-biotin, or a biotin analog or biologically active fragment.

[0147] In some embodiments, the reagent has a size that is less than 20 nm, less than 10 nm, less than 5 nm or less than 1 nm. In some embodiments, the reagent has a density of less than 1.2 g/cm3 or less than 1.0 g/cm3. In some embodiments, the reagent is not bound to a support or solid support.

[0148] Provided herein in some aspects is a composition including a plurality of T cells genetically engineered to express a recombinant receptor that specifically binds to a target antigen. In some cases, greater than 35%, 40%, 50%, 60%, 70%, 80% or 90% of the cells include a T cell subset containing a surface phenotype that is CD3+, CD4+ or CD8+ and CD62L+ and one or more of CD127+, CD45RA+, CD45RO−, CCR7+ and CD27+ and one or more of t-bet.sup.low, IL-7Ra+, CD95+, IL-2Rβ+, CXCR3+ and LFA-1+ as a percentage of the total T cells in the composition or the total cells in the composition. In some embodiments, prior to or during the genetic engineering, the plurality of T cells containing the T cell subset were not incubated in the presence of a GSK-P inhibitor; were not incubated in the presence of a recombinant homeostatic cytokine, optionally IL-7 or IL-15; or were not enriched for CD62L+ cells. In some embodiments, the composition does not contain a GSK-P inhibitor or a recombinant homeostatic cytokine, optionally IL-7 or IL-15.

[0149] In some embodiments, the T cell subset includes at least 5×10.sup.6, at least 1×10.sup.6 or at least 2×10.sup.6 cells.

[0150] Provided herein in some embodiments is a composition including a plurality of T cells genetically engineered to express a recombinant receptor that specifically binds to a target antigen. In some aspects, the genetically engineered T cells are derived from transducing a population of T cells containing a T cell subset containing a surface phenotype that is CD3+, CD4+ or CD8+ and CD62L+ and one or more of CD127+, CD45RA+, CD45RO−, CCR7+ and CD27+ and one or more of t-bet.sup.low, IL-7Ra+, CD95+, IL-2Rβ+, CXCR3+ and LFA-1+. In some embodiments, the T cell subset is present at a greater percentage of the total T cells in the population or a greater number of total T cells in the population compared to a population containing primary T cells that were isolated or enriched from a human subject based on surface expression of one or markers containing the phenotype. In some embodiments, the T cell subset is present at a greater percentage of the total T cells in the population or a greater number of total T cells in the population compared to a population of T cells that were incubated in the presence of a GSK-P inhibitor. In some embodiments, the T cell subset is present at a greater percentage of the total T cells in the population or a greater number of total T cells in the population compared to a population of T cells that were incubated in the presence of a recombinant homeostatic cytokine, optionally IL-7 or IL-15. In some embodiments, the T cell subset is present at a greater percentage of the total T cells in the population or a greater number of total T cells in the population compared to a population of T cells that were stimulated by anti-CD3 and anti-CD8, but in which the stimulation or activation was for greater than 1 day, 2 days, 3 days, 4 days or 5 days and/or the stimulation was not disrupted in the presence of biotin or a biotin analog.

[0151] In some embodiments, the T cell subset is present in the population at or about greater than 35%, 40%, 50%, 60%, 70%, 80% or 90% as a percentage of the total T cells in the population. In some embodiments, the T cell subset includes at least 5×10.sup.6 cells, 1×10.sup.6 cells, 2×10.sup.6 cells or more.

[0152] In some embodiments, the composition is a pharmaceutical composition.

[0153] Provided herein in some aspects is a method of treatment including administering to a subject having a disease or condition a composition, e.g., pharmaceutical composition, as described herein.

[0154] In some embodiments, the cells include a recombinant receptor, such as a chimeric antigen receptor (CAR) or TCR. In some aspects, the recombinant receptor, such as CAR or transgenic TCR specifically binds to an antigen associated with the disease or condition.

[0155] In some embodiments, the disease or condition is a cancer, and autoimmune disease or disorder, or an infectious disease