Cell culture method, kit and device therefor

Abstract

A method of culturing cells is provided that includes stimulating or expanding (growing) a plurality of cells in a cell composition. In some aspects, methods and reagents are provided for expanding, e.g., stimulating or expanding (growing), a cell population, comprising binding of an agent to a molecule on the surface of the cell, thereby providing one or more signals to the cell. In some cases, the reagent is a multimerizing agent, and one or more agents are multimerized by reversibly binding to the reagent. In some aspects, the multimerized agent can provide expansion or proliferation or other stimulation of the cell population, after which such stimulatory agent can be removed by disrupting the reversible binding. Compositions, devices, and methods of use thereof are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Patent Application No. 62 / 245,249, filed October 22, 2015, entitled "Cell Cultivation Method and Kits and Apparatus Therefor," which is incorporated herein by reference in its entirety.

[0002] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING This application submits a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042002740SeqList.txt, created October 17, 2016, and is 39,887 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0003] Field In some aspects, the present disclosure relates to the incubation or culturing of cells, such as target cells, and compositions thereof, as well as the enrichment, separation, and / or sorting of cells, e.g., target cells used in such incubations, from other components, e.g., other cells in the composition. The incubation and separation steps can be performed in various orders and may overlap in time. Such methods typically involve the use of reagents, agents, and complexes (and / or complexes containing components that reversibly bind to each other) that reversibly bind to each other, with such binding generally being reversible by the addition of a substance. Thus, in some embodiments, following or during incubation and / or sorting, reagents can be dissociated by the addition of a substance to allow downstream processing, subsequent rounds of stimulation or separation, and / or to control the quality or quantity of signaling received by the cells. In some aspects, the methods involve binding of an agent to a molecule on the surface of a cell, thereby providing one or more signals to the cell. The methods generally use a reagent, e.g., a multimerization reagent, that contains multiple binding sites for agents, such that one or more agents are multimerized by reversibly binding to the reagent, e.g., thereby generating a stimulatory reagent (multimerization agent) and / or a selection reagent (multimerization selection agent) that has a stimulatory agent or selection agent multimerized thereon. In some embodiments, the incubation is performed in the presence of a support, such as a stationary phase or solid phase, in some cases in the presence of a stationary or solid phase to which a selection agent is attached. In some embodiments, the cells to be stimulated are immobilized to the stationary phase, generally indirectly, via a provided reagent. Compositions, devices, and methods of use thereof are also provided. [Background technology]

[0004] background A variety of strategies can be utilized to stimulate and / or enrich cells and cell populations in vitro.Available strategies include those for enriching and / or expanding antigen-specific T cells in vitro, and these strategies are used in adoptive immunotherapy or cancer therapy, where the infusion of these T cells has been shown to have anti-tumor reactivity in tumor hosts, or when administered to patients with viral infection.Improved strategies for culturing, enriching and selecting cells and populations in vitro are needed, for example, for research, diagnostic and therapeutic purposes.Reagents, methods, manufacturing articles and kits are provided to meet this need. Summary of the Invention

[0005] overview Methods are provided that include manipulating, culturing, and / or treating cells, such as target cells, and / or compositions thereof. Also provided are compositions, agents, reagents, and devices and articles of manufacture for use in the methods, for example, for use in any of the methods. The methods generally include a step of incubating (e.g., culturing) cells, for example, to stimulate the cells.

[0006] For example, in some embodiments, the method includes incubating a composition comprising target cells in the presence of a stimulator reversibly bound to a reagent. In some aspects, the reagent includes multiple stimulator binding sites capable of reversibly binding to the stimulator. The incubation can be performed under conditions in which the stimulator specifically binds to a molecule expressed on the surface of the target cells, thereby inducing or modulating a signal in the target cells.

[0007] In some embodiments, the method further comprises enriching or selecting cells and compositions thereof, such as target cells to be stimulated and / or target cells that have been so stimulated or stimulated. In some embodiments, the method comprises both incubation of cells (e.g., for stimulation) and enrichment or selection of cells, e.g., to select a specific population of target cells and stimulate or otherwise culture such cells after or simultaneously with selection. In some embodiments, enrichment or selection and stimulation are performed sequentially in any order, or at least partially overlap in time, or are performed simultaneously.

[0008] In some embodiments, the various steps of the method (and / or the method as a whole) are performed in a closed system, such as a sterile system; among the devices and articles provided are those for use in performing the method in such closed or sterile systems. In some embodiments, the cells are T cells.

[0009] In some aspects, the method includes incubating a composition comprising T cells in the presence of an agent (which in some cases can be referred to as a first agent, e.g., as distinct from a second, third, etc., additional agent used in the method or reagent) and / or a complex comprising the same, e.g., a multimerized agent or a reagent comprising the agent in a multimerized form. Immobilization of the agent to the reagent can be reversible. In some aspects, the multiple stimulator binding sites comprise one or more binding sites Z1 that can reversibly bind to binding partner C1; and / or the stimulator further comprises one or more binding partners C1. In some aspects, the multiple stimulator binding sites comprise two or more binding sites Z1 and / or further comprise one or more binding sites Z2 that can reversibly bind to binding partner C1; and / or the stimulator comprises two or more binding partners C1. The stimulator can further comprise binding site B2, in which case specific binding between the stimulator and a molecule on the surface of the target cell comprises an interaction between B2 and the molecule.

[0010] In some aspects, the agent is a receptor-binding substance, for example, a stimulatory substance that can deliver, induce, or modulate a signal to a target cell. The receptor-binding substance (e.g., a stimulatory substance) can be reversibly bound to a reagent, and the reagent comprises multiple binding sites that can reversibly bind to the receptor-binding substance, and thus can act as a multimerization reagent for forming a multimer of the receptor-binding substance on the oligomer reagent. In some aspects, the receptor-binding substance can specifically bind to a molecule on the surface of a cell, for example, a T cell, in a manner that induces or modulates a signal in the cell, for example, a T cell, in the composition.

[0011] In some embodiments, the reagent bound to the stimulator (which complex may in some cases be referred to as a stimulating reagent) is oligomeric in nature, but is not bound to a solid support, bead, rigid particle, spherical or substantially spherical particle, or stationary phase, and is soluble. In some aspects, the reagent has a size of less than 20 nm, less than 10 nm, less than 5 nm, or less than 1 nm. In some cases, the reagent has a density of less than 1.2 g / cm. 3 Less than or equal to 1.0 g / cm 3 has a density of less than

[0012] In some embodiments, such a stimulator (e.g., first agent) is not, is not bound to, or is associated with a solid support, stationary phase, bead, microparticle, magnetic particle, and / or matrix in said incubation, and / or is flexible, does not contain a metal or magnetic core, is composed entirely or primarily of organic multimers, is not spherical, is not substantially spherical or uniform in shape, and / or is not rigid.

[0013] In some embodiments, for example, in embodiments in which the reagent is not bound to a solid phase or beads or other elements described, stimulation is nevertheless carried out in the presence of a support, such as a stationary phase and / or a solid phase. In some aspects, at least a plurality of target cells are immobilized on such a support during at least part of the incubation. The immobilization is optionally reversible.

[0014] In some embodiments, stimulation and reversible immobilization on support are carried out by the same reagent, so one reagent is used for separation and stimulation.For example, the method can include: (a) combining the composition comprising target cells with the stimulating substance that is reversibly bound to the reagent that is immobilized on support, wherein the reagent comprises a plurality of stimulating substance binding sites that can be reversibly bound to stimulating substance and can specifically bind to the molecule that is expressed on the surface of target cells, thereby immobilizing target cells on the support; (b) separating or removing other cells of the composition from the immobilized target cells; and (c) incubating at least a portion of the immobilized target cells in the presence of the stimulating substance under the condition that the signal is induced or modulated in at least a plurality of target cells;

[0015] In some cases, the support is or includes a stationary phase; and / or the support is or includes a solid support. In some aspects, the stimulus is a first agent, and at least a portion of the incubation is performed in the presence of a second reagent immobilized on the support and a selection agent that can reversibly bind to the second reagent and also bind to a molecule on the cell, thereby promoting reversible immobilization of the cell on the support during incubation. The second reagent can include multiple selection agent binding sites, e.g., one or more (e.g., two or more) binding sites Y1, each of which can reversibly bind to the selection agent via a binding partner D1 included in the selection agent. In some embodiments, the selection agent further includes a binding site Y2 that can also bind to D1. In some embodiments, the selection agent further includes a binding site B1, e.g., such that specific binding between the selection agent and the selection marker includes an interaction between B1 and the selection marker.

[0016] In some embodiments, the second reagent and the selection agent are reversibly bound together in a complex at the time of the combining step, and the combining step is performed by combining the cells with the complex. In other embodiments, the second reagent and the selection agent are not in a complex at the time of the combining step, and the combining step is performed by adding the second reagent and the selection agent separately. Thus, these agents may or may not be pre-bound prior to combining with the cells or other compounds or compositions.

[0017] In some embodiments, the method further comprises enrichment or selection, e.g., by combining at least a plurality of target cells; (i) a selection agent capable of specifically binding to a selection marker expressed by one or more of the at least a plurality of target cells, and (ii) a selection agent that is immobilized or capable of being immobilized, directly or indirectly, to a support; and (c) the support, whereby one or more target cells of the at least a plurality are immobilized on the support via the selection agent.

[0018] The method may further comprise steps of combining, separating and / or removing other cells of the composition from the immobilized target cells, and performing washing steps, which may be performed before the start of said incubation.

[0019] The support may comprise a stationary phase and / or be or comprise a solid support.

[0020] In some aspects, the enrichment and stimulation are performed sequentially or simultaneously, or with partial overlap; in some aspects, the incubating step is performed and / or initiated prior to the combining step; or, alternatively, the incubating step is performed and / or initiated subsequent to the combining step. In some aspects, the combining step is performed during at least a portion of the incubation.

[0021] In some aspects, the reversible binding between various components in the above embodiments (e.g., between a first agent or stimulator and a reagent such as a multimerization reagent, and / or between a selection agent and a second reagent, or any or all of the reversible bindings described above) can be disrupted by the addition of a substance. In some aspects, the substance is or comprises a free binding partner; or the substance is or comprises a competitor; and / or the substance effects a change that disrupts the binding in a manner other than competing for the binding. Thus, disruption by the substance can be achieved through competition, e.g., by competing for binding with one component with another component, e.g., having more favorable binding properties. In some aspects, the substance is non-detrimental to the target cells, or to a majority of the target cells, so that the target cells can be used in a particular desired downstream process. In some cases, addition of the substance to target cells in an amount sufficient to effect said destruction does not reduce the viability and / or proliferation ability of the cells to less than or about 90%, 80%, 70%, 60%, or 50%, compared to the absence of the substance but under otherwise the same conditions. Exemplary substances are or include peptides or polypeptides, such as those that bind to streptavidin or streptavidin muteins and / or bind to the biotin-binding site thereof.

[0022] The substance may comprise a molecule selected from the group consisting of: a streptavidin-binding molecule; biotin; D-biotin; a biotin analog; or the amino acid sequence Val at a sequence position corresponding to positions 44 to 47 of streptavidin or wild-type streptavidin. 44 -Thr 45 -Ala 46 -Arg 47 Or Ile 44 -Gly 45 -Ala 46 -Arg 47and a peptide comprising or consisting of the sequence set forth in SEQ ID NOs: 1, 4, 5, and 7. In other embodiments, the agent is or comprises a metal chelator, which is optionally EDTA or EGTA.

[0023] In some aspects, agents are monomeric if they contain only one given binding site, e.g., a stimulator contains only one binding site B2 and / or a selection agent contains only one B1. For example, in some cases, a stimulator contains only a single binding site that specifically binds to the molecule; a stimulator contains only a single binding site that specifically binds to the molecule in a monovalent manner and / or specifically binds to a selection marker; and / or a selection agent specifically binds to a selection marker in a monovalent manner.

[0024] The binding sites for reversible binding of the stimulating reagent / stimulating substance (e.g., C and Z), respectively, may be the same or different, and may or may not be blockable by the same substance as the binding sites of the selecting reagent / selecting substance (e.g., D and Y). The first and second reagents may be substantially the same, and optionally, only one (e.g., the selecting substance-binding reagent) may be immobilized on a support.

[0025] Also provided are agents, reagents, complexes, and articles of manufacture, such as compositions or kits, comprising the same, e.g., for carrying out any of the method embodiments. For example, compositions are provided that include a stimulatory agent reversibly linked to a multimerization reagent, optionally a first reagent, where the stimulatory agent is capable of specifically binding to a molecule on the surface of a target cell in a manner that induces or modulates a signal in the target cell.

[0026] In some embodiments, the compositions and / or articles further comprise a support; a second reagent immobilized on the support; and a selection agent capable of reversibly binding to the second reagent and specifically binding to a selection marker on a target cell.

[0027] The composition may further comprise target cells and / or non-target cells, where the non-target cells do not express the (first) and / or second selection marker; the target cells may optionally comprise a recombinant molecule that is a chimeric receptor or a nucleic acid that expresses a recombinant molecule. The composition may further comprise a substance capable of disrupting the binding.

[0028] In some embodiments, the article of manufacture comprises a stimulating agent, a reagent that reversibly binds to the stimulating agent, an optional second reagent that binds to a selection agent, an optional selection agent, and optionally a support on which the reagent may or may not be pre-immobilized. These components can be present in separate containers, the same container, or a combination thereof. The reagent capable of reversible binding may be pre-bound in the article of manufacture, or may be separated for subsequent functioning of the reagent with an agent. In some aspects, the support and the second support are present in separate containers, where the separate containers are optionally fluidly connected to each other, allowing the cell suspension to pass through or past one of the supports to the other; the article further comprises a substance that can disrupt the reversible binding between one or more reagents and one or more agents.

[0029] In some embodiments, a second reagent is further contained in the container; and / or the (first) selection substance and / or second selection substance are further contained in the container; and / or the article further comprises a second container, optionally containing the (first) stimulus substance and / or second stimulus substance, and / or first reagent; and / or the article further comprises a third container containing a second selection substance and / or a third reagent; and / or the article further comprises a fourth container containing the substance.

[0030] Also provided are devices for carrying out the provided methods, such as any of the provided embodiments of the methods. Such devices can, in some embodiments, include any of the provided compositions or provided articles of manufacture. The devices can include, or further include, a fluid inlet fluidly connected to the composition or to one or more components of the device, and / or a fluid outlet fluidly connected to the composition or to one or more components of the device.

[0031] In some embodiments, the device includes: (a) a stimulatory substance capable of specifically binding to a molecule on the surface of a target cell in a manner that induces or modulates a signal in the target cell; (b) a first reagent capable of reversibly binding to the stimulatory substance; (c) a second reagent; (d) a support; and (e) a selection substance capable of reversibly binding to the second reagent and specifically binding to a selection marker on the target cell. In some aspects, components (a) through (e) are present in multiple containers, at least some of which are fluidly connected, optionally in a closed or sterile system, so that one or more of the components pass from one container to another within the device. In some aspects, the device further includes a sample outlet fluidly connected to at least one support, such as a stationary phase for chromatography.

[0032] In some embodiments, the article of manufacture or apparatus is a functionally closed system. The apparatus can further include one or more controllers capable of adjusting or regulating one or more characteristics of the environment in which one or more various steps are performed, such as pH, pO2, pCO2, and / or automatic temperature control of one or more vessels or components thereof and / or at least one of the at least one stationary phase for chromatography.

[0033] The article of manufacture or device can further include a fluid connection to a container containing culture medium, one or more nutrients, and / or one or more carbon sources, whereby the connection can provide such culture medium, nutrients, and / or carbon sources to cells within the device, optionally when the cells are immobilized on a stationary phase for chromatography. In some aspects, the container is an output container or a preparation container, e.g., containing a buffer or other components suitable for preparing the cells. In some aspects, at least one of the listed components and / or the container for containing or housing it is removable from the device in a sterile or aseptic manner.

[0034] In some embodiments, the method is performed in a fully or partially automated manner. Thus, in some embodiments, the device or article is capable of performing the method in an automated manner, e.g., in a manner that reduces or eliminates the need for user control or intervention during one or more steps.

[0035] In some embodiments, the incubation is carried out following the combination, and the method further comprises transferring the target cells of the composition to a different environment suitable for cell culture or expansion.In some cases, the cells are transferred to a different environment in a closed system or closed container; or the transfer comprises removing the cells to be transferred from a first container and transferring them to a second container.The different environment can be in an incubator.In some aspects, the transfer is carried out in a closed system, in which case the transfer comprises transferring a sterile sealed container containing cells to a sterile environment or to a different environment within the sealed container, and / or the transfer is carried out in a sterile environment or under sterile conditions.

[0036] In some embodiments, following transfer, the cells are separated from the stationary phase by disrupting the reversible bond, and optionally the cells are separated from the presence of the stationary phase. In some cases, the separated cells are further expanded. In some aspects, while the cells are in an environment suitable for expansion, the environment, for example, pH, pO2, pCO2 and / or temperature, is controlled, optionally in an automated manner, during at least a portion of the incubation, and / or nutrients are provided to the cells contained in at least one of the at least one stationary phase for chromatography. Transfer to a suitable environment for expansion can include separating the stationary phase from the cells, but the stationary phase is present in the device.

[0037] In some embodiments, the methods include features, such as particular steps, selection of particular agents, and / or selection of particular reagents, that allow for control or adjustment of the type, intensity, or duration of the signal received or modulated via the reagent, and / or the properties of the output composition or cell population(s) ultimately produced by the method. In some embodiments, such features are possible due to advantageous properties of the agents and reagents, such as reversibility of binding of individual components of the agents or reagents, and thus reversibility of binding of the multimerized agents to cells. Such reagent properties can be exploited to achieve control in many ways. For example, in some embodiments, the methods include providing temporal control of the signal via reversibility of binding, controlling the duration of contact between the cells and the multimerized agents, and / or controlling the duration of the signaling induced thereby.

[0038] In some embodiments, the methods include controlling, e.g., precisely controlling, the length of time that such agents are bound to the cells. For example, this can be done by actively reversing such binding at a specific time point, in some cases while still maintaining the cells for an additional period of time under other culture conditions, such as incubation at physiological temperature and / or with various nutrients. Thus, in contrast to other methods that simply involve the termination of all or substantially all signals received by the cells, the provided methods, in some aspects, allow for the specific termination or disruption of signals delivered by specific reagents.

[0039] Similarly, in some embodiments, reversibility allows a reagent to be modular in nature, permitting substitution of one or more of its components without engineering or new reagents, e.g., by simply reversing binding and combining with an additional agent or reagent under conditions that induce reversible binding. For example, by being able to reversibly bind various stimuli to the same multimerization reagent, simultaneously or at different times, users of the provided methods and compositions can substitute one or more stimuli for another one or more stimuli, thereby adjusting the nature of the particular signal delivered, e.g., by inducing a stronger, weaker, or qualitatively different signal depending on the desired outcome.

[0040] In some embodiments, temporal control and modularity are used in combination, e.g., by incubating cells in the presence of one agent for a period of time to induce reversal of binding by interference, followed by incubation in the presence of another or different agent or reagent. For example, in one embodiment, T cells are first stimulated with a reagent to deliver a signal of a particular strength or quality, and after a period of time, such signal is interfered with and replaced with a signal that is qualitatively or quantitatively different (e.g., stronger, weaker, activates a different signaling pathway, or is known to be important for a different differentiation pathway). In some embodiments, such control provides several advantages, e.g., allowing the user to maximize a desired outcome (e.g., expansion or persistence) while avoiding undesirable outcomes such as exhaustion or anergy.

[0041] In some embodiments, temporal control is achieved by disrupting the reversible binding of the reagent or agent, for example, by adding a substance.For example, in some embodiments, the reversible binding between the receptor-binding substance and the reagent is disrupted within a certain period, for example, within 5 days after the start of incubation, and / or within a certain percentage of the total length of incubation, for example, within 1 / 5, 1 / 4, 1 / 3 or 1 / 2 of the period.Therefore, in some cases, the method results in the generation of cultured T cells.

[0042] In some aspects, the incubation is carried out under conditions in which the receptor binding agent specifically binds to the molecule, thereby inducing or modulating a signal in one or more T cells in the composition.

[0043] In some embodiments, disruption of the bond between the receptor-binding substance and the reagent occurs more than 30 minutes after the start of incubation. For example, in some aspects, disruption of the bond between the receptor-binding substance and the reagent occurs 1 hour to 4 days after the start of incubation, 6 hours to 3 days after the start of incubation, 12 hours to 2 days after the start of incubation, or 1 day to 3 days after the start of incubation. In some cases, disruption of the bond between the receptor-binding substance and the reagent occurs about 1 hour to about 4 days after the start of incubation, about 6 hours to about 3 days after the start of incubation, about 12 hours to about 2 days after the start of incubation, or about 1 day to about 3 days after the start of incubation. In some aspects, the disruption occurs about 1 hour or more after the start of incubation, or within 1 day, 2 days, 3 days, or 4 days after the start of incubation.

[0044] In some embodiments, the receptor binding agent is capable of initiating a TCR / CD3 complex-associated signal in a T cell. In some aspects, the receptor binding agent specifically binds to a member of the TCR / CD3 complex. In some cases, the receptor binding agent specifically binds to CD3.

[0045] In some embodiments, the molecule (on the surface of the T cell) 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 can further specifically bind to a second molecule on one or more surfaces of the T cell. In some cases, the second molecule can induce, enhance, attenuate, or modify the signal delivered via the first molecule in the T cell.

[0046] In some aspects, the receptor-binding agent comprises a binding partner C1. In some aspects, the multiple binding sites comprised in the reagent comprise two or more binding sites Z1. In some cases, the two or more binding sites Z1 are each capable of binding to binding partner C1 to form a reversible bond between the receptor-binding agent and the reagent.

[0047] In some embodiments, disrupting the binding between the receptor binding agent and the reagent comprises introducing into the cell a composition comprising a substance capable of reversing the binding between the receptor binding agent and the reagent.

[0048] In some cases, the receptor binding substance is a first receptor binding substance, and incubation is further carried out in the presence of a second receptor binding substance.In such cases, the second receptor binding substance can specifically bind to a second molecule on the surface of one or more T cells.In some aspects, the second molecule can enhance, attenuate, or modify the signal delivered by the first molecule in T cells.

[0049] In some embodiments, the reagent comprises multiple binding sites capable of reversibly binding to a second receptor binding substance. In such cases, the second receptor binding substance is reversibly bound to the reagent. In some aspects, the multiple binding sites capable of reversibly binding to the first receptor binding substance and the multiple binding sites capable of reversibly binding to the second receptor binding substance can be the same or different.

[0050] In some aspects, the second receptor binding substance comprises a binding partner C1 or C2 that can reversibly bind to two or more binding sites Z1. In such cases, the first and second receptor binding substances are reversibly bound to the reagent via the two or more binding sites Z1. In some cases, the second receptor binding substance comprises a binding partner C2, and the reagent further comprises multiple binding sites Z2. The multiple binding sites Z2 can bind to the binding partner C2 to form reversible bonds between the second receptor binding substance and the reagent. In some aspects, C2 and C1 are the same or substantially the same, or comprise the same or substantially the same moiety. In some cases, Z1 and Z2 are the same or substantially the same, or comprise the same or substantially the same moiety.

[0051] In some cases, the reagent is a first reagent and the incubation is performed in the presence of at least a second reagent that reversibly binds to a second receptor binding agent.

[0052] In some embodiments, the incubation is performed under conditions in which the second receptor-binding agent specifically binds to the second molecule. In such aspects, binding of the second receptor-binding agent to the second molecule induces or modulates a signal that enhances, attenuates, or alters the signal delivered via the first molecule, e.g., in a T cell.

[0053] In some such embodiments, disruption of the binding between the first and second receptor binding agents and the reagent terminates or reduces the signal induced or modulated by the first receptor binding agent and terminates or reduces the signal induced or modulated by the second receptor binding agent.

[0054] In some embodiments, a method for culturing T cells is provided herein, comprising incubating a composition containing T cells in the presence of a first receptor binding substance capable of specifically binding to a first molecule expressed on the surface of the T cells. In some aspects, binding of the first receptor binding substance to the first molecule induces or modulates a TCR / CD3 complex-associated signal in the T cells. In some such embodiments, the composition is further incubated in the presence of a second receptor binding substance, the second receptor binding substance being reversibly bound to a reagent comprising multiple binding sites capable of reversibly binding thereto. In some aspects, the second receptor binding substance is capable of specifically binding to a second molecule on the surface of the T cells. In some cases, binding of the second receptor binding substance to the second molecule induces or modulates a second signal in the T cells, enhancing, attenuating, or altering the signal delivered via the first molecule. In some aspects, within 5 days after the start of incubation, the reversible binding between the second receptor binding substance and the reagent is disrupted, thereby generating cultured T cells.

[0055] In some embodiments, 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 cell.

[0056] In some embodiments, the second receptor binding agent comprises a binding partner C1. In some such embodiments, the multiple binding sites comprised in the reagent include two or more binding sites Z1, each capable of binding to binding partner C1 to form a reversible bond between the second receptor binding agent and the reagent.

[0057] In some aspects, the second signal is a signal other than a TCR / CD3 complex-associated signal. In some cases, the second signal can enhance or potentiate the TCR / CD3 complex-associated signal.

[0058] In some embodiments, the second molecule is a costimulatory molecule, an accessory molecule, a cytokine receptor, a chemokine receptor, an immune checkpoint molecule, or a member of the TNF family or TNF receptor family. In some cases, the second molecule is CD28, CD90 (Thy-1), CD95 (Apo- / Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM. In some such cases, the second molecule is CD28. In some embodiments, the modularity of the reagents allows for the substitution of one or more costimulatory and / or activating reagents, e.g., temporarily, during a given incubation. For selection purposes, modularity can also allow for sequential selection and / or stimulation when reagents are removed between steps.

[0059] In some aspects, disrupting the binding between the second receptor binding substance and the reagent (which may be the second reagent) comprises introducing into the cell a composition comprising a substance capable of reversing the binding between the second receptor binding substance and the reagent (which may be the second reagent). In some embodiments, the disruption terminates or reduces a signal induced or modulated by the second receptor binding substance; or the additional molecule is CD28 and the disruption terminates or reduces a CD28 costimulatory signal in the T cell.

[0060] In some aspects, after disruption, the composition comprising T cells is further incubated. In some such aspects, the incubation and further incubation are performed in the same container. Optionally, the further incubation is performed in the presence of the substance. In some cases, the method does not include removing the substance, receptor-binding substance, second receptor-binding substance, and / or reagent from the cell composition before the further incubation. In some embodiments, the incubation and / or further incubation is performed at about 37°C ± 2°C. Optionally, the incubation and / or further incubation is performed in the presence of an additional agent capable of delivering a signal to T cells. In some embodiments, the additional agent can enhance or induce proliferation of T cells, CD4+ T cells, and / or CD8+ T cells. In some aspects, the additional agent is a cytokine such as IL-2, IL-15, or IL-7. In some embodiments, the further incubation is carried out for a period of not more than 14 days, not more than 12 days, not more than 10 days, not more than 8 days, or not more than 6 days.

[0061] In some embodiments, a method for culturing T cells is provided herein, the method comprising incubating a composition containing T cells in the presence of a receptor-binding substance that specifically binds to a CD28 molecule on the surface of the T cells under conditions that result in CD28-mediated signaling in the cells. In some such aspects, within 5 days after the start of the incubation, binding of the receptor-binding substance to the CD28 molecule is eliminated or reduced, thereby terminating or diminishing CD28 signaling in the cells, thereby generating cultured T cells.

[0062] In some cases, the elimination or reduction is achieved within 4 days, 3 days, 2 days, or 1 day after the start of incubation. In some cases, the elimination or reduction step includes washing the cells, thereby removing or reducing receptor-binding substances that do not specifically bind to CD28 from the composition. In some aspects, the elimination step includes reversing the binding interaction between the receptor-binding substance and the CD28 molecule, and further includes washing the cells to remove or reduce the receptor-binding substance from the composition.

[0063] In some aspects, during at least a portion of the incubation and / or following the incubation, the T cells in the composition are incubated in the presence of an agent that specifically binds to a molecule of the TCR / CD3 complex, thereby inducing or modulating a TCR / CD3 complex-associated signal in the cell.

[0064] In some aspects, a method for culturing T cells is provided herein, the method comprising incubating a composition containing T cells in the presence of a receptor-binding substance. In some embodiments, the receptor-binding substance is reversibly bound to a reagent comprising multiple binding sites that can reversibly bind to the receptor-binding substance. In some cases, the receptor-binding substance can specifically bind to a molecule on the surface of T cells other than CD28 or CD3 in a manner that induces or modulates a signal in the T cells, thereby generating cultured T cells.

[0065] In some aspects, the incubation is performed under conditions in which the receptor binding agent specifically binds to the molecule, thereby inducing or modulating a signal in the T cell, which in some embodiments is not a TCR / CD3 complex-associated signal.

[0066] In some embodiments, the receptor-binding agent comprises a binding partner C1. In some such embodiments, the multiple binding sites of the reagent comprise two or more binding sites Z1, each of which can bind to a binding partner C1 to form a reversible bond between the receptor-binding agent and the reagent.

[0067] In some embodiments, the receptor-binding substance is a second receptor-binding substance, and the molecule is a second molecule. In some such embodiments, the incubation is further carried out in the presence of a first receptor-binding substance capable of specifically binding to a first molecule on the surface of one or more T cells, and the first molecule can induce or modulate a first signal in one or more T cells in the composition. In some aspects, the first receptor-binding substance is reversibly bound to a reagent, and the reagent comprises multiple binding sites for the first receptor-binding substance and the second receptor-binding substance. In some cases, the first receptor-binding substance is reversibly bound to a second reagent comprising multiple binding sites capable of reversibly binding to the first receptor-binding substance.

[0068] In some embodiments, the first receptor binding agent can initiate a TCR / CD3 complex-associated signal in a T cell. In some aspects, the first receptor binding agent specifically binds to a member of the TCR / CD3 complex. In some cases, the first receptor binding agent specifically binds to CD3.

[0069] In some embodiments, specific binding of the second receptor binding agent to the second molecule can enhance, attenuate, or modify the signal delivered via the first molecule. In some cases, specific binding of the second receptor binding agent to the second molecule can enhance or potentiate the TCR / CD3 complex-associated signal.

[0070] In some embodiments, a method for culturing T cells is provided herein, the method comprising incubating a composition containing T cells in the presence of a first receptor binding substance, the first receptor binding substance being reversibly bound to a reagent comprising multiple binding sites capable of reversibly binding thereto. In some cases, the first receptor binding substance can specifically bind to a first molecule on the surface of the T cells to induce or modulate a TCR / CD3 complex-associated signal in the T cells in the composition. In some embodiments, the method comprises incubating a composition containing T cells in the presence of a second receptor binding substance, the second receptor binding substance being reversibly bound to the reagent further comprising multiple binding sites for the second receptor binding substance, or to a second reagent comprising multiple binding sites capable of reversibly binding to the second receptor binding substance. In some aspects, the second receptor binding substance can specifically bind to a second molecule on the surface of the T cells to induce or modulate a second signal in the T cells in the composition. In some aspects, the second molecule is other than CD28. In some embodiments, the incubation is carried out under conditions in which the signal and / or a second signal is induced or modulated in the T cells in the composition, thereby generating cultured T cells.

[0071] In some embodiments, the first receptor binding substance specifically binds to a member of the TCR / CD3 complex, and / or the first receptor binding substance specifically binds to CD3. In some cases, specific binding of the second receptor binding substance to the second molecule can induce or modulate a signal other than a TCR / CD3 complex-associated signal. In some aspects, specific binding of the second receptor binding substance to the second molecule can enhance, attenuate, or modify the signal delivered via the first molecule. In some embodiments, specific binding of the second receptor binding substance to the second molecule can enhance or strengthen a TCR / CD3 complex-associated signal.

[0072] In some embodiments, the molecule, which may be a 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 may be a 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 may be a second molecule, is not CD137, and / or the receptor binding agent, which may be a second receptor binding agent, does not specifically bind to CD137.

[0073] In some aspects, the first receptor binding substance and the second receptor binding substance reversibly bind to the reagent. In some embodiments, the first receptor binding substance and the second receptor binding substance each individually comprise a binding partner C1, and the plurality of binding sites comprises two or more binding sites Z1, each capable of binding to binding partner C1 to form a reversible bond between the first and second receptor binding substances and the reagent. In other embodiments, the first receptor binding substance comprises binding partner C1, and the second receptor binding substance comprises binding partner C2, and the plurality of binding sites comprises two or more binding sites Z1, each capable of binding to binding partner C1 and binding partner C2 to form a reversible bond between the first and second receptor binding substances and the reagent. In a further embodiment, the first receptor binding agent comprises a binding partner C1, the second receptor binding agent comprises a binding partner C2, and the multiple binding sites comprise two or more binding sites Z1 and two or more binding sites Z2, each of Z1 being capable of binding to binding partner C1 to form a reversible bond between the first receptor binding agent and the reagent, and each of Z2 being capable of binding to binding partner C2 to form a reversible bond between the second receptor binding agent and the reagent.

[0074] In some embodiments, a method for culturing target cells is provided herein, the method comprising incubating a composition containing target cells in the presence of a receptor-binding substance. The receptor-binding substance can be reversibly bound to a reagent that is a streptavidin analog or streptavidin mutein that contains multiple binding sites that can reversibly bind to it. In some cases, the receptor-binding substance can specifically bind to a molecule on the surface of the target cell to induce or modulate a signal in the target cell in the composition. In some aspects, the streptavidin mutein contains a net negative charge.

[0075] In some aspects, provided herein are methods for culturing target cells, comprising incubating a composition containing target cells in the presence of a receptor-binding substance, the receptor-binding substance being reversibly bound to a reagent that is a streptavidin analog or streptavidin mutein comprising multiple binding sites capable of reversibly binding thereto. In some embodiments, the receptor-binding substance can specifically bind to a molecule on the surface of the target cells in a manner that induces or modulates a signal in the target cells in the composition. In some cases, the streptavidin analog or mutein exhibits a higher affinity for a streptavidin-binding peptide comprising the amino acid sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8) than streptavidin or a mutein comprising the amino acid sequence set forth in any of SEQ ID NOs: 1-6, thereby resulting in cultured target cells.

[0076] In some embodiments, the incubation is performed under conditions in which the receptor binding agent specifically binds to the molecule, thereby inducing or modulating a signal in one or more target cells in the composition.

[0077] In some cases, the multiple binding sites of the reagent include two or more binding sites Z1. In some cases, the receptor-binding substance includes a binding partner C1, where C1 can reversibly bind to binding site Z1, and the reversible binding between C1 and Z1 affects the reversible binding between the receptor-binding substance and the reagent. In some embodiments, the streptavidin analog or mutein includes multiple binding sites Z1, and multiple receptor-binding substances are reversibly bound to the reagent.

[0078] In some embodiments, target cells comprise blood cells, leukocytes, lymphocytes, B cells, B cell populations, T cells, T cell populations, and / or natural killer (NK) cells. In some embodiments, target cells comprise antigen-specific T cells or populations thereof, T helper cells or populations thereof, cytotoxic T cells or populations thereof, memory T cells or populations thereof, regulatory T cells or populations thereof, NK cells or populations thereof, antigen-specific B cells or populations thereof, memory B cells or populations thereof, or regulatory B cells or populations thereof. In some aspects, target cells are T cells.

[0079] In some embodiments, the molecule is present on the surface of a T cell, and the receptor binding agent can induce or modulate a signal in the T cell in the composition. In some aspects, the receptor binding agent can initiate a TCR / CD3 complex-associated signal in the T cell, and / or the receptor binding agent specifically binds to a member of the TCR / CD3 complex. In some cases, the receptor binding agent specifically binds to CD3. In some cases, the molecule is a first molecule, and the receptor binding agent can specifically bind to the first molecule and, optionally, to a second molecule on one or more surfaces of the target cell. In some embodiments, binding to the second molecule can enhance, attenuate, or modify the signal delivered via the first molecule.

[0080] In some cases, the receptor binding substance is a first receptor binding substance, and incubation is further carried out in the presence of a second receptor binding substance.The second receptor binding substance can specifically bind to a second molecule on the surface of one or more target cells.In some cases, the binding of the second receptor binding substance to the second molecule can induce or modulate the signal transmitted through the first molecule, so as to enhance, attenuate or modify the signal.

[0081] In some embodiments, the incubation is carried out under conditions in which the second receptor-binding substance specifically binds to the second molecule, thereby inducing or modulating a signal in target cells in the composition to enhance, attenuate, or modify the signal delivered via the first molecule. In some embodiments, the streptavidin mutein or analog contains multiple binding sites that can reversibly bind to the second receptor-binding substance, such that the second receptor-binding substance is reversibly bound to the streptavidin mutein or analog.

[0082] In some embodiments, the second receptor binding agent comprises a binding partner C1 or C2, which is capable of reversibly binding to two or more binding sites Z2 present on the streptavidin analog or mutein.

[0083] 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 molecules are CD40 and CD137. In some cases, the second receptor binding agent specifically binds to CD40 or CD137.

[0084] In some embodiments, the second receptor binding agent comprises a plurality of different receptor binding agents, each of which can individually bind to the same or different second molecule on the surface of a T cell in the composition and collectively induce or modulate one or more signals in the cell.

[0085] In some embodiments, the method further comprises disrupting the reversible bond between the first and / or second receptor-binding agent and the reagent, hi some aspects, the disruption occurs within 14 days, within 12 days, within 10 days, within 8 days, or within 6 days of initiating incubation.

[0086] In some embodiments, at least a portion of the incubation is performed in the presence of an additional agent capable of delivering a signal to T cells. In some cases, the additional agent can enhance or induce proliferation of T cells, CD4+ T cells and / or CD8+ T cells. In some embodiments, the additional agent is a cytokine such as IL-2, IL-15, or IL-7. In some cases, the additional agent does not specifically bind to CD28 and / or does not induce CD28 signaling.

[0087] In some embodiments, the T cells or target cells are primary cultured 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, enriched or separated CD3+ T cells, enriched or separated CD4+ T cells, or enriched or separated CD8+ T cells. In some embodiments, prior to the incubating step, the T cells are not enriched for CD62L+ cells and / or are not enriched for naive T cells. In some cases, the T cells or target cells are human cells.

[0088] In some embodiments, the reagent is not bound to a support or solid support during said incubation. In other embodiments, the reagent is bound to a support during at least a portion of said incubation, whereby the plurality of T cells or target cells are reversibly immobilized on the support during at least a portion of the incubation. In such embodiments, the support is a solid support or stationary phase.

[0089] In some embodiments, the receptor binding substance, which may be the first receptor binding substance, comprises only one binding site, e.g., B2. In some aspects, the receptor binding substance, which may be the first receptor binding substance, specifically binds to the molecule in a monovalent manner. In some embodiments, the second receptor binding substance comprises only one binding site, e.g., B4. In some cases, the second receptor binding substance specifically binds to the molecule in a monovalent manner. In some embodiments, a binding site such as B2 or B4 comprises an antibody binding site.

[0090] In some aspects, the receptor-binding substance that may be the first receptor-binding substance and / or the second receptor-binding substance are each individually an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties, an Ig domain-containing molecule, a cytokine, a chemokine, an aptamer, or an MHC molecule or binding fragment thereof. In such aspects, the receptor-binding substance that may be the first receptor-binding substance and / or the second receptor-binding substance comprises an antibody fragment, a Fab fragment, a bivalent antibody fragment, such as an (Fab)2' fragment, or a bivalent single-chain Fv (scFv) fragment. In some cases, the receptor-binding substance that may be the first receptor-binding substance and / or the second receptor-binding substance is a monovalent antibody fragment, such as an Fab fragment, an Fv fragment, or an scFv fragment. In some cases, the receptor-binding agent, which may 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, a mutein based on a lipocalin family polypeptide, a glubody, an ankyrin scaffold-based protein, a crystalline scaffold-based protein, an adnectin, or an avimer.

[0091] In some embodiments, the receptor binding agent, which may be the first receptor binding agent, includes an agent that specifically binds to CD3. The agent that specifically binds to CD3 may be an anti-CD3 antibody, a bivalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, or a proteinaceous CD3-binding molecule with antibody-like binding properties. In some embodiments, the second receptor binding agent includes an agent that specifically binds to CD28, CD90, CD95, CD137, CD154, ICOS, LAT, CD27, OX40, and / or HVEM.An agent that specifically binds to CD28, CD90, CD95, CD137, CD154, ICOS, LAT, CD27, OX40, and / or HVEM can be an anti-CD28 antibody, a bivalent 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 bivalent 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 bivalent 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 bivalent 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 bivalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment of an anti-CD137 ... a bivalent antibody fragment, a proteinaceous CD137 binding molecule with antibody-like binding properties, an anti-ICOS antibody, a bivalent 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 bivalent 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 bivalent 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 bivalent 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 bivalent 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, a 4-1BB ligand, or any mixture thereof.

[0092] In some embodiments, the reagent is or comprises streptavidin, avidin, or an analog or mutein of streptavidin that reversibly binds to biotin, a biotin analog, or a biologically active fragment thereof; an avidin or analog or mutein of streptavidin that reversibly binds to a streptavidin-binding peptide; a reagent comprising at least two chelating groups K capable of binding to a transition metal ion; an agent capable of binding to an oligohistidine affinity tag; an agent capable of binding to glutathione-S-transferase; calmodulin or an analog thereof; an agent capable of binding to calmodulin-binding peptide (CBP); an agent capable of binding to a FLAG peptide; an agent capable of binding to an HA tag; an agent capable of binding to maltose-binding protein (MBP); an agent capable of binding to an HSV epitope; an agent capable of binding to a myc epitope; or an agent capable of binding to a biotinylated carrier protein.

[0093] In some embodiments, the reagent is or comprises 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.

[0094] In some embodiments, the reagent is an oligomer or polymer of streptavidin, avidin, or an analog or mutein of streptavidin that reversibly binds to biotin or a biologically active fragment; a streptavidin or avidin analog or mutein that reversibly binds to a streptavidin-binding peptide; a reagent comprising at least two chelating groups K capable of binding to a transition metal ion; an agent capable of binding to an oligohistidine affinity tag; an agent capable of binding to glutathione-S-transferase; calmodulin or an analog thereof; an agent capable of binding to calmodulin-binding peptide (CBP); an agent capable of binding to a FLAG peptide; an agent capable of binding to an HA tag; an agent capable of binding to maltose-binding protein (MBP); an agent capable of binding to an HSV epitope; an agent capable of binding to a myc epitope; or an agent capable of binding to a biotinylated carrier protein.

[0095] In some embodiments, the reagent comprises streptavidin, avidin, a streptavidin analog or mutein, or an oligomer or polymer of an avidin analog or mutein. In some aspects, the individual molecules of the oligomer or polymer are crosslinked by a polysaccharide or a bifunctional linker.

[0096] In some embodiments, the plurality of binding sites Z comprises at least 2, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 72 or more binding sites.

[0097] In some aspects, the streptavidin-binding peptide is: TIFF0007665579000001.tif34162.

[0098] In some embodiments, the reagent comprises a streptavidin analog or mutein comprising the amino acid sequence Val44-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47 at sequence positions corresponding to positions 44 to 47 in streptavidin in the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the streptavidin analog or mutein comprises the amino acid sequence Val44-Thr45-Ala46-Arg47 at sequence positions corresponding to positions 44 to 47 in streptavidin in the amino acid sequence set forth in SEQ ID NO: 1.

[0099] In some embodiments, the streptavidin analog or mutein comprises the amino acid sequence set forth in any of SEQ ID NOs: 3-6. In some aspects, the streptavidin analog or mutein comprises an amino acid sequence 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 comprises the amino acid sequence corresponding to Val44-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 to biotin or a biologically active form thereof, a biotin analog or mutein or a biologically active fragment thereof, or a functional fragment of any of the above sequences that reversibly binds to a streptavidin-binding peptide.

[0100] In some embodiments, the streptavidin analog or mutein further comprises one or more amino acid substitutions at positions corresponding to 117, 120 and / or 121 with respect to positions in streptavidin in the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions are selected from among Glu117, Asp117, Arg117, Ser120, Ala120, Gly120, Trp121, Tyr121 or Phe121. In some embodiments, the one or more amino acid substitutions are Glu117, Gly120 or Tyr121.

[0101] In some embodiments, the streptavidin analog or mutein comprises the amino acid sequence set forth in SEQ ID NO: 27 or 28. In some aspects, the streptavidin analog or mutein comprises an amino acid sequence 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 NO: 28, and includes the amino acid sequences corresponding to Val44, 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 to biotin or a biologically active fragment, a biotin analog or mutein or a biologically active fragment thereof, or a functional fragment of any of the above sequences that reversibly binds to a streptavidin-binding peptide.

[0102] In some embodiments, binding partner C1 and / or binding partner C2 independently comprise a streptavidin-binding peptide such as: TIFF0007665579000002.tif34162.

[0103] In some embodiments, the disruption comprises introducing into cells a composition containing a substance capable of reversing the binding between the receptor-binding substance, which may be the first receptor-binding substance, and / or the second receptor-binding substance and the reagent. In some embodiments, the substance is a free binding partner and / or a competitor. In some embodiments, the substance in the composition does not cause adverse effects on T cells or 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%, respectively, compared to incubation of T cells or target cells under comparable or identical conditions without the substance. In some embodiments, the disruption terminates or reduces the signal induced or modulated by one or both of the receptor-binding substance and the second receptor-binding substance in T cells or target cells.

[0104] In some embodiments, the reagent is or comprises streptavidin, avidin, a streptavidin analog or mutein, an avidin analog or mutein, or a biologically active fragment thereof. In some embodiments, the agent comprises a streptavidin-binding peptide, biotin or a biologically active fragment, optionally D-biotin, or a biotin analog or biologically active fragment.

[0105] In some embodiments, the agent is a streptavidin-binding peptide, such as: TIFF0007665579000003.tif34164. In some embodiments, the agent is C1 or an analog thereof, or C2 or an analog thereof.

[0106] In some embodiments, the dissociation constant (K) of the reversible binding between binding site Z1 and binding partner C1 and / or the reversible binding between binding site Z2 and binding partner C2 is D ) is 10 -2 M~10 -13 It is in the range of M.

[0107] In some embodiments, prior to incubation, the cells are contacted with a selection agent that specifically binds to a marker contained on the T cells or target cells of the composition, thereby generating or obtaining a composition comprising T cells or target cells. In some embodiments, at least a portion of the incubation is performed in the presence of a selection agent that specifically binds to a marker contained on the T cells or target cells of the composition, thereby enriching the cultured T cells for T cells or target cells that contain the marker.

[0108] In some embodiments, the selection agent is reversibly bound to a reagent that further comprises multiple binding sites capable of specifically binding to the selection agent, hi some aspects, the selection agent is reversibly bound to a second reagent that comprises multiple binding sites capable of specifically binding to the selection agent.

[0109] In some embodiments, the induction or modulation of a signal results in increased expansion (proliferation) and / or activation of cultured T cells compared to incubation of T cells in the absence of the induction or modulation of the signal. In certain 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 more. In some embodiments, the induction or modulation of an additional or second signal increases the expansion (proliferation) and / or activation of T cells compared to incubation of T cells in the absence of the induction or modulation of the additional or second signal. In certain 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 more.

[0110] In some embodiments, the method increases the expansion and / or proliferation and / or survival and / or differentiation of T cells or a specific subset thereof in the composition, e.g., in the output composition produced by the method, alters the metabolic profile of such T cells or subsets in the composition, alters a subset of CD8+ T cells in the composition, and / or increases the percentage of long-lived memory T cells in the composition. In some embodiments, such an increase is compared to the starting composition, e.g., cells before incubation. In some aspects, it is compared to similar stimulation performed under different conditions in a certain way, for example, similar stimulation performed under conditions that are otherwise the same but in which binding is not disrupted until the end of incubation (e.g., without time control), and / or similar stimulation performed under conditions that are otherwise the same but in which a different combination of stimulatory substances is used, e.g., a costimulator is present in the multimerized stimulatory substance and is different from one or more or any costimulatory molecules used in the stimulatory substance in question. For example, if the stimulatory agent in question includes a costimulatory agent other than a CD28 binding molecule, a comparison or similar set of conditions may include incubation with a stimulatory agent or reagent that includes an agent that specifically binds to CD28 and / or induces or modulates CD28 signaling, and optionally includes another agent in common with the reagent in question, such as an anti-CD3 agent.

[0111] 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+ T cells is increased by 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 before incubation, in the composition after incubation but in the absence of disruption, or in the composition after incubation under similar or comparable conditions but in the presence of an agent that specifically binds to CD28 and / or induces or modulates CD28 signaling.

[0112] In some embodiments, the methods result in cultured T cells in which the proportion of CD8+ T cells or the relative or normalized proportion of CD8+ T cells in the composition is increased by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to the proportion or relative or normalized proportion of CD8+ T cells in the composition before incubation, in the composition after incubation but in the absence of disruption, or in the composition after a similar or comparative incubation, e.g., in the presence of an agent that specifically binds CD28 and / or induces or modulates CD28 signaling.

[0113] In some embodiments, the method results in an increase in the number or relative number (e.g., proportion) of cells having a particular phenotype, such as, for example, cells that exhibit characteristics of a longer-lived and / or less differentiated cell population, e.g., a long-lived memory population or a stem-like population, e.g., cells that express high levels of CD62L, CD127, CCR7, Sca1, and / or CD27, and / or cells that also express or include indicators of proliferation and indicators of a naive or resting phenotype, e.g., any of the above cells that stain low for T-bet and / or have a phenotype that is IL-7Ra+, CD95+, IL-2Rβ+, CXCR3+, and LFA-1+. In some aspects, the CD62L+ cells in the composition, optionally long-lived memory T cells or memory stem cells (T SCM ), the number or percentage of CD62L+, long-lived memory T cells or T in the composition before incubation, in the composition after incubation but in the absence of destruction, or in the composition after a similar incubation but in the presence of an agent that specifically binds to CD28 and / or induces or modulates CD28 signaling. SCMIn some embodiments, the stimulation results in an increase of 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 cell population of the same or similar method or reagent. In some embodiments, the stimulation results in an increase in the number or percentage of cells that are expanded or survive but have a less differentiated, longer-lived phenotype compared to a similar method or reagent, resulting in the production of a desired number of cells.

[0114] In some embodiments, the cultured T cells contain 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% or more of a T cell subset comprising a phenotype that is surface positive for CD62L (CD62L+) as a percentage of the total T cells in the composition or total cells in the composition.

[0115] In some embodiments, the T cell subset further comprises a phenotype comprising CD127+; and / or any one or more of CD45RA+, CD45RO-, CCR7+, CD27+ and any one or more of t-betlow, IL-7Ra+, CD95+, IL-2Rβ+, CXCR3+, LFA-1+.

[0116] In some embodiments, the T cell subset contains low levels of TCR rearrangement excisions circles (TRECs); and / or expresses a proliferation marker, which may be Ki-67; and / or exhibits the ability to proliferate in the presence of a stimuli; and / or exhibits the ability to produce cytokines, such as IFN-γ, TNF, or IL-2, in the presence of a stimuli.

[0117] In some embodiments, the stimulatory agent is an antigen, a homeostatic cytokine, such as IL-15 and / or IL-17, or an agent capable of initiating a TCR / CD3 complex-associated signal in a T cell.

[0118] In some embodiments, the T cell subset is or comprises long-lived memory T cells. In some embodiments, the T cell subset is T memory stem cells (TSCM ) or contains it.

[0119] In some embodiments, the method further comprises introducing a recombinant nucleic acid molecule into the T cells or target cells of the population. In some such aspects, the nucleic acid molecule encodes a recombinant protein, whereby the cells can express the recombinant protein. In some embodiments, the recombinant receptor is a chimeric antigen receptor or a transgenic T cell receptor (TCR). In some aspects, the method is performed in vitro or ex vivo.

[0120] In some embodiments, the method further comprises administering the cultured cells to a subject with a disease or disorder.

[0121] Provided herein, in some aspects, is a composition containing a plurality of cultured T cells or target cells produced by the methods described herein, and optionally a pharmaceutically acceptable excipient.

[0122] In some embodiments, after addition of the substance, the cells are not incubated in vitro or ex vivo at a temperature above 30° C. for more than 24 hours, more than 48 hours, more than 72 hours, or more than 96 hours.

[0123] In some aspects, provided herein is a reversible reagent, comprising a reagent comprising multiple binding sites capable of binding to a receptor-binding substance. In some embodiments, the receptor-binding substance is reversibly bound to the reagent and can specifically bind to a molecule on the surface of a T cell in a manner that induces or modulates a signal in the T cell; for example, the molecule is not CD28 or CD3.

[0124] In some embodiments, binding to the molecule induces or modulates a signal in the T cell other than a TCR / CD3 complex-associated signal; and / or binding to the molecule enhances or potentiates a TCR / CD3 complex-associated signal.

[0125] In some embodiments, the molecule is CD90 (Thy-1), CD95 (Apo- / Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM. In some cases, the molecule is not CD137.

[0126] In some embodiments, the reagents used in the methods and compositions include streptavidin analogs or muteins, typically oligomers thereof, where the muteins contain multiple binding sites capable of binding to an agent, typically capable of reversibly binding to such an agent. Such reagents are also provided. In some aspects, the streptavidin analogs or muteins contain a net negative charge and / or exhibit a higher affinity for a streptavidin-binding peptide containing the amino acid sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8) than streptavidin or muteins containing the amino acid sequence set forth in any of SEQ ID NOs: 1-6. In some embodiments, one or more agents are reversibly bound to the reagent and are capable of specifically binding to molecules on the surface of a cell. Such reagents, agents, and complexes thereof are also provided.

[0127] In some embodiments, the receptor-binding agent comprises a binding partner C1. In some aspects, the plurality of binding sites comprises two or more binding sites Z1, each capable of binding to a binding partner C1, forming a reversible bond between the receptor-binding agent or agent and the reagent.

[0128] In some embodiments, the receptor binding agent is a second receptor binding agent, the molecule is a second molecule, and the reagent further comprises c) a plurality of binding sites capable of reversibly binding to the first receptor binding agent, and d) a first receptor binding agent that is i) reversibly bound to the reagent and ii) capable of specifically binding to a first molecule on the surface of a T cell.

[0129] In some embodiments, the agent or first receptor binding agent specifically binds to a member of the TCR / CD3 complex and / or the first receptor binding agent specifically binds to CD3.

[0130] In some embodiments, the first receptor binding substance and the second receptor binding substance each individually comprise a binding partner C1, and the plurality of binding sites comprises two or more binding sites Z1, each capable of binding to binding partner C1, forming a reversible bond between the first and second receptor binding substances and the reagent. In some aspects, the first receptor binding substance comprises binding partner C1, and the second receptor binding substance comprises binding partner C2, and the plurality of binding sites comprises two or more binding sites Z1, each capable of binding to binding partner C1 and binding partner C2, forming a reversible bond between the first and second receptor binding substances and the reagent. In some embodiments, the first receptor binding substance comprises a binding partner C1, the second receptor binding substance comprises a binding partner C2, and the multiple binding sites comprise two or more binding sites Z1 and two or more binding sites Z2, each of Z1 being capable of binding to binding partner C1 to form a reversible bond between the first receptor binding substance and the reagent, and each of Z2 being capable of binding to binding partner C2 to form a reversible bond between the second receptor binding substance and the reagent.

[0131] In some embodiments, the reagent has a size of less than 20 nm, less than 10 nm, less than 5 nm, or less than 1 nm. In some embodiments, the reagent has a size of less than 1.2 g / cm 3 Less than or equal to 1.0 g / cm 3 In some embodiments, the reagent is not bound to a support or solid support. In some cases, the reagent is bound or immobilized to a support. In some cases, the support is a solid support or stationary phase. In some aspects, the support comprises a bead, particle, nanoparticle, or microsphere.

[0132] In some embodiments, the agent, the receptor-binding agent that may be the first receptor-binding agent, and / or the second receptor-binding agent are each individually an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties, a molecule containing an Ig domain, or a binding fragment thereof.

[0133] In some embodiments, the agent, the receptor binding agent which may be the second receptor binding agent, and / or the first receptor binding agent comprises an antibody fragment. In some aspects, the agent, the receptor binding agent which may be the second receptor binding agent, and / or the first receptor binding agent comprises a Fab fragment. In some cases, the agent, the receptor binding agent which may be the second receptor binding agent, and / or the first receptor binding agent is a bivalent antibody fragment, such as a (Fab)2' fragment or a bivalent single-chain Fv (scFv) fragment. In some embodiments, the agent, the receptor binding agent which may be the second receptor binding agent, and / or the first receptor binding agent is a monovalent antibody fragment selected from a Fab fragment, an Fv fragment, and an scFv fragment.

[0134] In some embodiments, the agent or first receptor-binding agent comprises an agent that specifically binds to CD3. In some embodiments, the agent that specifically binds to CD3 is an anti-CD3 antibody, a bivalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, or a proteinaceous CD3-binding molecule with antibody-like binding properties. In some embodiments, the receptor binding agent, which may be the agent or second receptor binding agent, comprises an agent that specifically binds to CD90, CD95, CD137, CD154, ICOS, LAT, CD27, OX40, and HVEM, including, for example, an anti-CD90 antibody, a bivalent 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 bivalent 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 bivalent 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 bivalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment of an anti-CD137 ... a proteinaceous CD137 binding molecule having antibody-like binding properties, an anti-ICOS antibody, a bivalent 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 bivalent 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 bivalent 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 bivalent 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 bivalent 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.

[0135] In some embodiments, the reagent is or comprises streptavidin, avidin, a streptavidin analog or mutein, and an avidin analog or mutein, hi some embodiments, the reagent comprises streptavidin, avidin, a streptavidin analog or mutein, and / or an oligomer or polymer of an avidin analog or mutein.

[0136] In some aspects, a kit is provided herein, comprising the reagent disclosed herein and, optionally, instructions for use. In some embodiments, the kit comprises a substance capable of reversing the binding between the receptor-binding substance and the reagent. In some embodiments, the kit comprises a reagent comprising multiple binding sites that can reversibly bind to the receptor-binding substance. In some aspects, the receptor-binding substance is reversibly bound to the reagent and can specifically bind to a molecule expressed on the surface of a target cell. In some cases, binding to the molecule induces or modulates a signal in the target cell. In some cases, the kit comprises a substance capable of reversing the binding between the receptor-binding substance and the reagent.

[0137] In some embodiments, the target cell is a T cell. In some embodiments, the reagent is or comprises streptavidin, avidin, a streptavidin analog or mutein, and / or an avidin analog or mutein. In some embodiments, the reagent comprises an oligomer or polymer of streptavidin, avidin, a streptavidin analog or mutein, and / or an avidin analog or mutein.

[0138] In some embodiments, the agent includes a streptavidin-binding peptide, biotin or a biologically active fragment, optionally D-biotin, or a biotin analog or biologically active fragment.

[0139] In some embodiments, the reagent has a size of less than 20 nm, less than 10 nm, less than 5 nm, or less than 1 nm. In some embodiments, the reagent has a size of less than 1.2 g / cm 3 Less than or equal to 1.0 g / cm 3 In some embodiments, the reagent is not bound to a support or solid support.

[0140] In some aspects, provided herein are compositions comprising a plurality of T cells genetically engineered to express a recombinant receptor that specifically binds to a target antigen. In some cases, 35%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the total T cells in the composition or as a percentage of the total cells in the composition comprise a T cell subset that is CD3+, CD4+, or CD8+ and CD62L+, and comprises one or more of CD127+, CD45RA+, CD45RO-, CCR7+, and CD27+, and one or more of t-betlow, IL-7Ra+, CD95+, IL-2Rβ+, CXCR3+, and LFA-1+ surface phenotypes. In some embodiments, prior to or during genetic manipulation, the plurality of T cells comprising a 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 include a GSK-P inhibitor or a recombinant homeostatic cytokine, optionally IL-7 or IL-15.

[0141] In some embodiments, the T cell subset comprises at least 5 x 10 6 Pieces, at least 1 x 10 6 pieces, or at least 2 x 10 6 Contains cells.

[0142] In some embodiments, provided herein is a composition comprising 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 comprising a T cell subset, wherein the T cell subset is CD3+, CD4+, or CD8+ and CD62L+, and comprises a surface phenotype that is one or more of CD127+, CD45RA+, CD45RO-, CCR7+, and CD27+, and one or more of t-betlow, IL-7Ra+, CD95+, IL-2Rβ+, CXCR3+, and LFA-1+. In some embodiments, the T cell subset is present at a higher percentage of total T cells in the population or at a higher number of total T cells in the population compared to a population comprising primary T cells isolated or enriched from a human subject based on the surface expression of one or more markers comprising the phenotype. In some embodiments, the T cell subset is present at a higher percentage of total T cells in the population or at a greater number of total T cells in the population compared to a population of T cells incubated in the presence of a GSK-P inhibitor. In some embodiments, the T cell subset is present at a higher percentage of total T cells in the population or at a greater number of total T cells in the population compared to a population of T cells 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 higher percentage of total T cells in the population or at a greater number of total T cells in the population compared to a population of T cells stimulated with anti-CD3 and anti-CD8, but where the stimulation or activation is for 1, 2, 3, 4, or 5 days or more, and / or where the stimulation is not interrupted with the presence of biotin or a biotin analog.

[0143] In some embodiments, the T cell subset is present in the population at about 35%, 40%, 50%, 60%, 70%, 80%, or 90% or more as a percentage of the total T cells in the population. In some embodiments, the T cell subset is present in the population at about 5×10 6 pieces, 1×10 6 pieces, 2×106 It contains one or more cells.

[0144] In some embodiments, the composition is a pharmaceutical composition.

[0145] Provided herein, in some aspects, are methods of treatment that include administering a composition, e.g., a pharmaceutical composition, described herein to a subject having a disease or condition.

[0146] In some embodiments, the cells comprise a recombinant receptor, such as a chimeric antigen receptor (CAR) or a TCR. In some aspects, the recombinant receptor, such as a CAR or transgenic TCR, specifically binds to an antigen associated with a disease or condition.

[0147] In some embodiments, the disease or condition is cancer, an autoimmune disease or disorder, or an infectious disease. [The present invention 1001] 1. A method for modulating cells, comprising incubating a composition comprising target cells in the presence of a stimulator reversibly bound to a first reagent comprising a plurality of stimulator binding sites capable of reversibly binding to the stimulator, At least a plurality of target cells are immobilized on the support during at least a portion of said incubation; and The incubation is carried out under conditions in which the stimulator specifically binds to a molecule expressed on the surface of the target cell, thereby inducing or modulating a signal in the target cell. method. [The present invention 1002] 1001. The method of claim 1001, wherein the immobilization of at least a plurality of the cells is reversible. [The present invention 1003] the plurality of stimulator binding sites include one or more binding sites Z1 capable of reversibly binding to a binding partner C1; and the stimulator further comprises one or more binding partners C1; The method of the present invention 1001 or 1002. [The present invention 1004] the plurality of stimulator binding sites comprises two or more of said binding sites Z1 and / or further comprises one or more binding sites Z2 capable of reversibly binding to a binding partner C1; and / or The stimulator comprises two or more binding partners C1; The method of the present invention 1003. [The present invention 1005] The method of any of claims 1001 to 1004, wherein the stimulator further comprises a binding site B2, and the specific binding between the stimulator and a molecule on the surface of the target cell comprises an interaction between B2 and the molecule. [The present invention 1006] the support is or comprises a stationary phase; and / or the support is or comprises a solid support; Any of the methods 1001 to 1005 of the present invention. [The present invention 1007] the reagent is a first reagent, and at least a portion of the incubation is carried out in the presence of (a) a second reagent immobilized on a support, and (b) a selection substance reversibly bound to the second reagent; specific binding by the selection agent to a selection marker expressed by at least a plurality of target cells results in reversible immobilization of the at least a plurality of target cells to the support; Any of the methods 1002 to 1005 of the present invention. [The present invention 1008] 1. A method of modulating a cell, comprising: (1) combining (a) a composition comprising target cells, (b) a selection agent (i) capable of specifically binding to a selection marker expressed by at least one or more of a plurality of target cells, and (ii) immobilized or capable of being immobilized directly or indirectly to a support, and (c) the support, whereby one or more of the at least a plurality of target cells are immobilized on the support via the selection agent; and (2) incubating at least a plurality of target cells in the presence of a stimulant reversibly bound to a reagent comprising a plurality of stimulant binding sites capable of reversibly binding to the stimulant under conditions such that the stimulant specifically binds to a molecule expressed on the surface of the target cells, thereby inducing or modulating a signal in the target cells. A method comprising: [The present invention 1009] 1008. The method of claim 10, wherein said reagent is a first reagent and said selection substance is reversibly bound to a second reagent immobilized on a support, and specific binding by said selection substance to a selection marker expressed by at least a plurality of target cells results in reversible immobilization of said at least a plurality of target cells to said support. [The present invention 1010] 1009. The method of any of claims 1007 to 1009, wherein the second reagent comprises a plurality of selection agent binding sites, each capable of reversibly binding to said selection agent. [The present invention 1011] the plurality of selectivity binding sites comprises one or more binding sites Y1 capable of reversibly binding to a binding partner D1; and the selection agent further comprises one or more binding partners D1; The method of the present invention 1010. [The present invention 1012] the plurality of selection agent binding sites comprises two or more of said binding sites Y1 and / or further comprises one or more binding sites Y2 capable of reversibly binding to a binding partner D1; and / or the selection agent comprises two or more binding partners D1; The method of the present invention 1011. [The present invention 1013] The method of any of claims 1002-1007 and 1009-1012, wherein during at least a portion of the incubation, reversible immobilization of at least a plurality of target cells is facilitated by reversible immobilization of a reagent to the support. [The present invention 1014] During the incubation, the first reagent is not bound to or associated with a solid support, stationary phase, bead, microparticle, magnetic particle and / or matrix; and / or the first reagent is flexible, does not contain a metal or magnetic core, is composed entirely or primarily of organic multimers, is not spherical, is not substantially spherical or uniform in shape, and / or is not rigid; Any of the methods 1001 to 1003 of the present invention. [The present invention 1015] (a) with at least a plurality of target cells; (b) a selection agent (i) capable of specifically binding to a selection marker expressed by one or more of the at least a plurality of target cells, and (ii) immobilized or capable of being immobilized directly or indirectly on a support; (c) the support; and further comprising the step of combining whereby one or more target cells of the at least plurality are immobilized on the support via the selection substance; Any of the methods 1001 to 1007 of the present invention. [The present invention 1016] the selection agent further comprises one or more binding partners D1 capable of reversibly binding to the binding site Z1; and / or the selection agent further comprises one or more binding partners D1 capable of reversibly binding to the binding site Y1; Any of the methods 1007 to 1015 of the present invention. [The present invention 1017] The method of any one of claims 1015 to 1016, further comprising the step of separating and / or removing other cells of said composition from the immobilized target cells after said combining step. [The present invention 1018] The method of claim 1017, further comprising performing a washing step. [The present invention 1019] 1019. The method of claim 1017 or 1018, wherein said separating step and / or said washing step is carried out before the start of said incubation. [The present invention 1020] 1020. The method of any of claims 1008 to 1019, wherein the support is or comprises a stationary phase and / or is or comprises a solid support. [The present invention 1021] the incubating step is performed and / or begins before the combining step; or the incubating step is performed and / or initiated subsequent to the combining step; Any of the methods of 1015 to 1020 of the present invention. [The present invention 1022] 1022. The method of any one of claims 1015 to 1021, wherein said combining step is carried out during at least a portion of said incubation. [The present invention 1023] 1023. The method of any one of claims 1015 to 1022, wherein the immobilization of the selected substance on the support is reversible. [The present invention 1024] the plurality of stimulator binding sites include one or more binding sites Z1 capable of reversibly binding to a binding partner C1; and the stimulator further comprises one or more binding partners C1; Any of the methods 1008 to 1023 of the present invention. [The present invention 1025] the plurality of stimulator binding sites comprises two or more of said binding sites Z1 and / or further comprises one or more binding sites Z2 capable of reversibly binding to a binding partner C1; and / or The stimulator comprises two or more binding partners C1; The method of the present invention 1024. [The present invention 1026] The method of any of claims 1013 to 1024, wherein the stimulator further comprises a binding site B2, and the specific binding between the stimulator and a molecule on the surface of the target cell comprises an interaction between B2 and the molecule. [The present invention 1027] the reagent is a first reagent; and Immobilization of the selection agent to the support is indirect, occurring via reversible binding of the selection agent to a second reagent immobilized on the support; Any of the methods of 1015 to 1026 of the present invention. [The present invention 1028] 1027. The method of claim 1027, wherein the second reagent comprises a plurality of selection agent binding sites capable of reversibly binding the selection agent. [The present invention 1029] 1029. A method according to claim 1028, wherein the plurality of selection agent binding sites comprises a binding site Y1 capable of binding to a binding partner D1, and one or more of said binding partners D1 are comprised in the selection agent. [The present invention 1030] the plurality of selection agent binding sites comprises two or more of said binding sites Y1 and / or further comprises one or more binding sites Y2 capable of reversibly binding to a binding partner D1; and / or the selection agent comprises two or more binding partners D1; The method of the present invention 1029. [The present invention 1031] Any of the methods of inventions 1027 to 1030, wherein the second reagent and the selection substance reversibly bind together to form a complex at the time of the combining step, and the combining step is carried out by combining the cells with the complex. [The present invention 1032] Any of the methods of present inventions 1027 to 1030, wherein the second reagent and the selection substance are not a complex at the time of the combining step, but are added separately to carry out the combining step. [The present invention 1033] The method of any of claims 1007 to 1012 and 1015 to 1032, wherein the selection agent further comprises a binding site B1, and the specific binding between the selection agent and the selection marker comprises an interaction between B1 and the selection marker. [The present invention 1034] The selection agent contains only one binding site B1; the selection agent contains only a single binding site that specifically binds to the selection marker; and / or The selection agent specifically binds to the selection marker in a monovalent manner; Any of the methods of 1007 to 1012 and 1015 to 1033 of the present invention. [This invention 1035] the selected agent is or comprises an agent selected from the group consisting of an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with immunoglobulin-like function, an Ig domain-containing molecule, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex; a receptor ligand; and binding fragments thereof; and / or the selection agent comprises an antibody fragment; the selection agent is or comprises a Fab fragment; The selected substance is (Fab) 2 ' selected from the group of bivalent antibody fragments consisting of fragments and bivalent single-chain Fv (scFv) fragments; the selected agent is a monovalent antibody fragment selected from the group consisting of a Fab fragment, an Fv fragment, and an scFv; and / or the selected agent is a proteinaceous binding molecule with antibody-like binding properties selected from the group consisting of aptamers, muteins based on polypeptides of the lipocalin family, glubodies, proteins based on ankyrin scaffolds, proteins based on crystalline scaffolds, adnectins, and avimers; Any of the methods of 1007 to 1012 and 1015 to 1034 of the present invention. [The present invention 1036] the selection marker is a B-cell or T-cell co-receptor; the selectable marker is or comprises a member of the T-cell or B-cell antigen receptor complex; the selection marker is or comprises a CD3 chain; the selection marker is or comprises the CD3 ζ chain; the selection marker is or includes CD8; the selection marker is or comprises CD4; and / or the specific binding between the selection agent and the selection marker does not induce a signal to the target cell or does not induce a stimulatory, activating or proliferative signal to the target cell; Any of the methods of 1007 to 1012 and 1015 to 1035 of the present invention. [This invention 1037] The stimulator contains only one binding site, B2; the stimulator contains only a single binding site that specifically binds to the molecule; or the stimulator specifically binds to the molecule in a monovalent manner; Any of the methods of 1001 to 1036 of the present invention. [The present invention 1038] the stimulatory agent is or comprises an agent selected from the group consisting of an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with immunoglobulin-like function, an Ig domain-containing molecule, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex, a receptor ligand, and binding fragments thereof; and / or the stimulator comprises an antibody fragment; the stimulator is or comprises a Fab fragment; The stimulating substance is (Fab) 2 ' selected from the group of bivalent antibody fragments consisting of fragments and bivalent single-chain Fv (scFv) fragments; the stimulator is a monovalent antibody fragment selected from the group consisting of a Fab fragment, an Fv fragment, and an scFv; and / or the stimulator is a proteinaceous binding molecule with antibody-like binding properties selected from the group consisting of aptamers, muteins based on polypeptides of the lipocalin family, glubodies, proteins based on ankyrin scaffolds, proteins based on crystalline scaffolds, adnectins, and avimers; Any of the methods of 1001 to 1037 of the present invention. [This invention 1039] the molecule expressed on the surface of the target cell is or comprises a member of the T-cell or B-cell antigen receptor complex; the molecule expressed on the surface of the target cell is or comprises a CD3 chain; the molecule expressed on the surface of the target cell is or comprises CD3ζ; the molecule expressed on the surface of the target cell is or comprises the antigen-binding portion of a T cell receptor or a B cell receptor; The molecule expressed on the surface of the target cell is a chimeric antigen receptor; Specific binding of a stimulator to the molecule can deliver a primary signal to T cells or B cells. Any of the methods of 1001 to 1038 of the present invention. [The present invention 1040] the stimulator comprises an MHC I:peptide complex or a functional portion thereof, an MHC II:peptide complex or a functional portion thereof, and / or is capable of delivering a stimulatory signal via a TCR / CD3 complex in a T cell, a CD3-containing complex in a T cell, and / or an ITAM-containing molecule in a T cell; and / or Inducing or modulating the signal results in increased expression of a cytokine in the target cell. Any of the methods of 1001 to 1039 of the present invention. [The present invention 1041] The method of claim 1040, wherein the cytokine is IL-2, IFN-γ and / or IL-4. [The present invention 1042] Any of the methods of claims 1001 to 1041, wherein the molecule expressed on the surface of the target cell is a first molecule, and the stimulatory substance is capable of further binding to a second molecule expressed on the surface of at least a plurality of the target cells. [This invention 1043] The method of any of claims 1001 to 1042, wherein the molecule expressed on the surface of the target cell is a first molecule, the stimulator is a first stimulator, and the incubation is further carried out in the presence of a second stimulator capable of binding to a second molecule expressed on the surface of at least a plurality of the target cells. [This invention 1044] The method of claim 1043, wherein the second stimulant is reversibly bound to the first reagent or is reversibly bound to the fourth reagent. [This invention 1045] the second stimulator comprises one or more binding partners C1; the second stimulator comprises one or more binding partners C2 capable of binding to the stimulator binding site; and / or the second stimulator comprises one or more binding partners C2 capable of binding to binding site Z2, and the reagent further comprises one or more binding sites Z2; The method of the present invention 1043 or 1044. [The present invention 1046] C2 and C1 are the same or substantially the same or contain the same or substantially the same moieties; Z1 and Z2 are the same or substantially the same or contain the same or substantially the same moieties; The method of the present invention 1045. [This invention 1047] The method of any of claims 1043 to 1046, wherein the second stimulator comprises one or more binding sites B4 that promote specific binding between the second stimulator and the second molecule. [This invention 1048] The method of claim 1042, wherein the stimulator further comprises one or more binding sites B4 that promote its specific binding to a second molecule. [This invention 1049] 1049. The method of any of claims 1042 to 1048, wherein specific binding of said agent or said second agent to a second molecule can enhance, attenuate, or modify the signal delivered via the first molecule. [The present invention 1050] The second molecule is a costimulatory molecule; The second molecule is an accessory molecule; The second molecule is a cytokine receptor; The second molecule is a chemokine receptor; the second molecule is an immune checkpoint molecule; or the second molecule is a member of the TNF family or the TNF receptor family; Any of methods 1042 to 1049 of the present invention. [This invention 1051] The method of claim 1050, wherein the second molecule comprises CD28, CD137, CD40 ligand, CD40, OX40, or ICOS, or a functional portion of any of the above. [This invention 1052] Any of the methods of claims 1007 to 1012 and 1015 to 1051, wherein the selection marker is a first selection marker and the selection agent is capable of further binding to a second selection marker expressed on the surface of at least a plurality of the target cells. [This invention 1053] The method of any of claims 1007 to 1012 and 1015 to 1052, wherein the selection marker is a first selection marker, the selection agent is a first selection agent, and the incubation is further performed in the presence of a second selection agent capable of binding to a second selection marker expressed on the surface of at least a plurality of the target cells. [This invention 1054] a second selected substance is reversibly bound to the second reagent; or a second selected substance is reversibly bound to a third reagent immobilized on the support or an additional support; The method of the present invention 1053. [This invention 1055] the second selection agent comprises one or more binding partners D1; ​​and / or the second selection agent comprises one or more binding partners D2 capable of binding to binding site Y1; and / or the second selected substance comprises one or more binding partners D2 capable of binding to binding site Y2, and the second reagent further comprises one or more binding sites Y2; The method of the present invention 1053 or 1054. [This invention 1056] D2 and D1 are the same or substantially the same or contain the same or substantially the same moieties; Y1 and Y2 are the same or substantially the same or contain the same or substantially the same moieties; C1 and D1 are the same or substantially the same or contain the same or substantially the same moieties; and / or Z1 and Y1 are the same or substantially the same or contain the same or substantially the same moieties; The method of the present invention 1055. [This invention 1057] 1057. The method of any of claims 1053 to 1056, wherein the second selection agent comprises one or more binding sites B3 that promote specific binding between the second selection agent and the second selection marker. [This invention 1058] The method of any of claims 1052 and 1054 to 1057, wherein said selection agent further comprises one or more binding sites B3 that promote its specific binding to the second selection marker. [This invention 1059] The second stimulator contains only one binding site, B4; the second stimulator contains only a single binding site that specifically binds to the second molecule; a second stimulatory agent specifically binds to the molecule in a monovalent manner; Any of methods 1043 to 1058 of the present invention. [The present invention 1060] the second stimulatory agent is or comprises an agent selected from the group consisting of an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with immunoglobulin-like function, an Ig domain-containing molecule, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex, a receptor ligand, and binding fragments thereof; the second stimulator comprises an antibody fragment; the second stimulatory agent is or comprises a Fab fragment; The second stimulator (Fab) 2 ' selected from the group of bivalent antibody fragments consisting of fragments and bivalent single-chain Fv (scFv) fragments; the second stimulator is a monovalent antibody fragment selected from the group consisting of a Fab fragment, an Fv fragment, and an scFv; and / or the second stimulator is a proteinaceous binding molecule with antibody-like binding properties selected from the group consisting of an aptamer, a mutein based on a lipocalin family polypeptide, a glubody, an ankyrin scaffold-based protein, a crystalline scaffold-based protein, an adnectin, and an avimer; Any of methods 1043 to 1059 of the present invention. [This invention 1061] the second selection agent contains only one binding site B3; the second selection agent contains only a single binding site that specifically binds to the second selection marker; and / or the second selection agent specifically binds to the second selection marker in a monovalent manner; Any of the methods of 1052 to 1060 of the present invention. [This invention 1062] the second selected substance is or comprises a substance selected from the group consisting of an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with immunoglobulin-like function, an Ig domain-containing molecule, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex, a receptor ligand, and binding fragments thereof; the second selected agent comprises an antibody fragment; the second selection agent is or comprises a Fab fragment; The second selected substance is (Fab) 2 ' selected from the group of bivalent antibody fragments consisting of fragments and bivalent single-chain Fv (scFv) fragments; the second selected agent is a monovalent antibody fragment selected from the group consisting of a Fab fragment, an Fv fragment, and an scFv; and / or the second selected agent is a proteinaceous binding molecule with antibody-like binding properties selected from the group consisting of an aptamer, a mutein based on a lipocalin family polypeptide, a glubody, an ankyrin scaffold-based protein, a crystalline scaffold-based protein, an adnectin, and an avimer; Any of the methods of the present invention 1052 to 1061. [This invention 1063] the second selection marker is a B-cell or T-cell co-receptor; the second selection marker is or comprises a member of the T-cell or B-cell antigen receptor complex; the second selection marker is or comprises a CD3 chain; the second selection marker is or comprises the CD3 ζ chain; the second selection marker is or comprises CD8; the second selection marker is or comprises CD4; and / or the specific binding between the second selection agent and the second selection marker does not induce a signal to the target cell, or does not induce a stimulatory, activating or proliferative signal to the target cell; Any of the methods of 1052 to 1062 of the present invention. [This invention 1064] 1. A method of modulating a cell, comprising: (a) combining a composition comprising target cells with a stimulatory substance reversibly bound to a reagent immobilized on a support, the reagent comprising a plurality of stimulatory substance binding sites each capable of reversibly binding to the stimulatory substance and capable of specifically binding to a molecule expressed on the surface of the target cells, thereby immobilizing the target cells on the support; (b) separating or removing other cells of the composition from the immobilized target cells; and (c) incubating at least a portion of the immobilized target cells in the presence of said stimulatory agent under conditions such that a signal is induced or modulated in at least a plurality of the target cells. A method comprising: [This invention 1065] 1065. The method of claim 1064, wherein the support is or comprises a solid support and / or a stationary phase. [The present invention 1066] the plurality of stimulator binding sites include one or more binding sites Z1 capable of reversibly binding to a binding partner C1; and the stimulator further comprises one or more binding partners C1; The method of claim 1064 or 1065. [This invention 1067] the plurality of stimulator binding sites comprises two or more binding sites Z1 and / or further comprises one or more binding sites Z2 capable of reversibly binding to a binding partner C1; and / or The stimulator comprises two or more binding partners C1; The method of the present invention 1066. [The present invention 1068] Any of the methods of claims 1064 to 1067, wherein the stimulator further comprises a binding site B2, and the specific binding between the stimulator and a molecule on the surface of the target cell comprises an interaction between B2 and the molecule. [The present invention 1069] The method of any of claims 1001 to 1068, wherein the bonds between one or more agents reversibly bound to the first, second and / or third reagents can be individually disrupted by addition of the agents to the cells. [The present invention 1070] a reversible bond between the stimulatory substance and the first reagent can be disrupted by the addition of said substance; and / or a reversible bond between the selection agent and the first reagent or the selection agent and the second reagent can be disrupted by the addition of said agent; and / or Each of the reversible bonds between the stimulatory substance and the first reagent and the reversible bonds between the selection substance and the second reagent and / or the first reagent can be disrupted by the addition of said substance; a reversible bond between the second stimuli and the first or fourth reagent can be disrupted by the addition of said substances; and / or a reversible bond between the second selection substance and the first reagent and / or between the second selection substance and the second reagent and / or between the second selection substance and the third reagent can be disrupted by the addition of said substance; and / or Each of the reversible bonds between the second stimulating substance and the first reagent or the fourth reagent, and the reversible bonds between the second selecting substance and the first reagent, the second reagent, and / or the third reagent can be disrupted by the addition of said substance; The method of the present invention 1069. [This invention 1071] the substance is or comprises a free binding partner; or the substance is or comprises a competitor; and / or the substance brings about a change that disrupts the binding in a way other than by competing for the binding; The method of claim 1069 or 1070. [This invention 1072] 1071. A method according to claim 1071, wherein said substance is not detrimental to the target cells or the majority of target cells and / or addition of said substance to target cells in an amount sufficient to cause said destruction does not reduce the viability and / or proliferation ability of said cells by less than 90%, 80%, 70%, 60% or 50% or less than about 90%, about 80%, about 70%, about 60% or about 50% compared to the absence of said substance but under otherwise the same conditions. [This invention 1073] The method of any of claims 1069 to 1072, wherein said substance is or comprises a peptide or polypeptide. [This invention 1074] The substance is a streptavidin-binding molecule; biotin; D-biotin; a biotin analog; streptavidin; or a substance containing the amino acid sequence Val at a sequence position corresponding to positions 44 to 47 of wild-type streptavidin. 44 -Thr 45 -Ala 46 -Arg 47 Or Ile 44 -Gly 45 -Ala 46 -Arg 47 a biotin analogue that specifically binds to a streptavidin analogue having the sequence: the agent optionally comprises a metal chelator, which is EDTA or EGTA; Any of the methods of the present invention 1069 to 1073. [This invention 1075] The support comprises a resin or matrix; the support comprises a gel filtration matrix; the support comprises a chromatography matrix; and / or The support comprises a cellulosic or organic polymer membrane; Any of the methods of 1001 to 1074 of the present invention. [This invention 1076] The method of claim 1075, wherein the chromatography matrix is ​​in a column; and / or the chromatography is column chromatography or planar chromatography. [This invention 1077] The method of any one of claims 1001 to 1076, wherein the support comprises a microparticle, a hard particle, a magnetic particle, or a bead. [This invention 1078] The method of any of claims 1001 to 1077, wherein the support is a stationary phase and is present in a vessel during all or a portion of said incubation and / or said contacting. [This invention 1079] 1078. The method of claim 1078, wherein the vessel comprises a vessel selected from the group consisting of a column, a vessel suitable for bidirectional flow, a pipette tip, a tube, and a column suitable for flow-through of a liquid sample. [The present invention 1080] Target cells include blood cells; Target cells include white blood cells; Target cells include lymphocytes; target cells include B cells; The target cells comprise a B cell population; target cells include T cells; the target cells comprise a T cell population; and / or the target cells include natural killer (NK) cells; Any of the methods of 1001 to 1079 of the present invention. [This invention 1081] The method of the present invention 1080, wherein the target cells comprise antigen-specific T cells or a population thereof, T helper cells or a population thereof, cytotoxic T cells or a population thereof, memory T cells or a population thereof, regulatory T cells or a population thereof, NK cells or a population thereof, antigen-specific B cells or a population thereof, memory B cells or a population thereof, or regulatory B cells or a population thereof. [This invention 1082] The method of any of claims 1001 to 1081, wherein the induction or modulation of the signal induces, attenuates, suppresses or enhances activation, proliferation, survival and / or growth. [This invention 1083] The method of any of claims 1001 to 1082, wherein the first reagent, the second reagent and / or the third reagent are, or comprise, independently, streptavidin, avidin, an analogue of streptavidin that reversibly binds to biotin, an analogue of avidin that reversibly binds to biotin, a reagent comprising at least two chelating groups K capable of binding to a transition metal ion, an agent capable of binding to an oligohistidine affinity tag, an agent capable of binding to glutathione-S-transferase, calmodulin or an analogue 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, and / or an agent capable of binding to a biotinylated carrier protein. [This invention 1084] The method of claim 1083, wherein the first reagent, the second reagent, and / or the third reagent are, or comprise, independently, an oligomer or a polymer. [This invention 1085] The method of any of claims 1001 to 1084, wherein the first reagent, the second reagent, or the third reagent is, independently, or comprises, streptavidin, avidin, a streptavidin analog, or an oligomer or polymer of an avidin analog, wherein the oligomer or polymer comprises monomers of the streptavidin, avidin, or analog crosslinked by a polysaccharide or a bifunctional linker. [The present invention 1086] Binding partner C1, binding partner C2, binding partner D1 and / or binding partner D2 independently comprise biotin or a biotin analog that reversibly binds to streptavidin or avidin; and / or each of binding partner C1 and binding partner C2 independently comprises biotin or a biotin analog that reversibly binds to streptavidin or avidin; and / or each of binding partner D1 and binding partner D2 independently comprises biotin or a biotin analog that reversibly binds to streptavidin or avidin; Any of the methods of the present invention 1002 to 1007, 1016 to 1063 and 1066 to 1085. [This invention 1087] the first reagent, the second reagent, and / or the third reagent independently comprise a streptavidin analog or an avidin analog that reversibly binds to biotin; the first reagent, the second reagent, and / or the third reagent independently comprise a streptavidin analog or an avidin analog that reversibly binds to a biotin analog; and / or the first reagent, the second reagent, and / or the third reagent independently comprise a streptavidin analog or an avidin analog that reversibly binds to a streptavidin-binding peptide; and / or the first reagent, the second reagent, and / or the third reagent are independently: TIFF0007665579000004.tif34169 a streptavidin or avidin analog that reversibly binds to a streptavidin-binding peptide selected from the group consisting of: Any of the methods of 1001 to 1086 of the present invention. [This invention 1088] Binding partner C1, binding partner C2, binding partner D1 and / or binding partner D2 are independently TIFF0007665579000005.tif34168 comprising a streptavidin-binding peptide selected from the group consisting of: Each of binding partner C1 and binding partner C2 independently TIFF0007665579000006.tif34164 and / or Each of binding partner D1 and binding partner D2 independently TIFF0007665579000007.tif33164 comprising a streptavidin-binding peptide selected from the group consisting of: Any of the methods of the present invention 1002 to 1007, 1016 to 1063 and 1066 to 1087. [This invention 1089] the first reagent, the second reagent, and / or the third reagent are independently: The amino acid sequence Val was added to the sequence positions corresponding to positions 44 to 47 of wild-type streptavidin. 44 -Thr 45 -Ala 46 -Arg 47 or a streptavidin analogue comprising The amino acid sequence Ile was added to the sequence positions corresponding to positions 44 to 47 of wild-type streptavidin. 44 -Gly 45 -Ala 46 -Arg 47 and a streptavidin analogue comprising Any of the methods of 1001 to 1088 of the present invention. [The present invention 1090] Any of the methods of claims 1002 to 1007, 1016 to 1063 and 1066 to 1089, wherein the binding between the binding partner C1 and / or the binding partner C2, respectively, and the binding site Z1 and / or Z2, respectively, can occur in the presence of divalent cations and / or cannot occur in the absence of divalent cations and / or is disrupted by removal of divalent cations. [This invention 1091] each of binding partners C1 and / or C2, and / or each of binding partners D1 and / or D2, independently, comprises a calmodulin-binding peptide, and the (first) reagent and / or the second reagent and / or the third reagent and / or the fourth reagent comprises calmodulin; or each of binding partners C1 and / or C2, and / or each of binding partners D1 and / or D2, independently, comprises a FLAG peptide, and the (first) reagent and / or second reagent and / or third reagent and / or fourth reagent comprises an antibody that binds to a FLAG peptide; or each of the binding partners C1 and / or C2, and / or each of the binding partners D1 and / or D2, independently, comprises an oligohistidine tag, and the (first) reagent and / or the second reagent and / or the third reagent and / or the fourth reagent comprises an antibody that binds to the oligohistidine tag; Any of the methods of 1002 to 1007, 1016 to 1063 and 1066 to 1090 of the present invention. [This invention 1092] The method of any one of claims 1002 to 1007, 1016 to 1063 and 1066 to 1091, wherein the bond between binding partner C1 and binding site Z1, and / or the bond between binding partner C2 and binding site Z2, and / or the bond between binding partner D1 and binding site Y1, and / or the bond between binding partner D2 and binding site Y2, can be disrupted by metal ion chelation, optionally achieved by the addition of EDTA or EGTA. [This invention 1093] the binding partners C1 and C2 are different and / or their interaction with said (first) reagent is disruptable by the addition of different substances or is not disruptable by the addition of the same substance; the binding partners D1 and D2 are different and / or their interaction with the second reagent is disruptable by the addition of a different substance or is not disruptable by the addition of the same substance; the binding partners C1 and / or C2 are different compared to the binding partners D1 and / or D2 and / or their interactions with the first and second reagents, respectively, can be disrupted by the addition of different substances or cannot be disrupted by the addition of the same substances, Any of the methods of 1002 to 1007, 1016 to 1063 and 1066 to 1092 of the present invention. [This invention 1094] Binding partners C1 and C2 are identical or substantially identical and / or their interaction with the first reagent is disruptable by the addition of the same substance; Binding partners D1 and D2 are identical or substantially identical and / or their interaction with a second reagent is disruptable by the addition of the same substance; the binding partners C1 and / or C2 are identical or substantially identical compared to the binding partners D1 and / or D2, and / or their interactions with the first and second reagents, respectively, are disruptable by the addition of the same substances; Any of the methods of 1002 to 1007, 1016 to 1063 and 1066 to 1093 of the present invention. [This invention 1095] The method of any of claims 1001 to 1094, further comprising the step of disrupting the bond between one or more agents reversibly bound to the first, second and / or third reagent. [This invention 1096] 1095. The method of claim 1095, wherein said disrupting step is performed after said incubation or is initiated subsequent to the initiation of said incubation. [This invention 1097] 1097. The method according to claim 1095 or 1096, wherein said disruption is carried out by introducing a substance that disrupts the interaction between binding partners C1 and / or C2 and binding sites Z1 and / or Z2. [This invention 1098] The destroying step Termination or reduction of the signal delivered by one of said stimulators; or Termination or reduction of stimulation, activation, proliferation of said cells Any of the methods of the present inventions 1095 to 1097, which cause the above. [This invention 1099] 19. The method of any one of claims 1097 to 1098, wherein said disruption comprises introducing into said cells a composition comprising said substance. [The present invention 1100] The method of any of claims 1001 to 1099, wherein said composition contacted with the second reagent further comprises non-target cells, and the method further comprises the step of separating the target cells from the non-target cells. [The present invention 1101] Any of the methods of inventions 1001 to 1100, further comprising a step of expanding the cells contained in the population of target cells prior to said incubation, or wherein the cells have been expanded in vitro prior to said incubation. [The present invention 1102] The method of any of claims 1001 to 1101, further comprising changing the medium or replenishing said substance at least once during said incubation. [The present invention 1103] Any of the methods of claims 1001 to 1102, further comprising repeating one or more steps of the method in an iterative manner, thereby successively separating and expanding one or more populations of cells for at least two cycles. [The present invention 1104] contacting target cells in the population with at least two different selection agents that specifically bind to two different selection markers in each of two different contacting steps; At least a portion of the incubation is performed between the two contacting steps with different selection agents. The method of the present invention 1103. [This invention 1105] further comprising the step of introducing the recombinant nucleic acid into target cells of the population; the nucleic acid encodes a recombinant protein, whereby the cell expresses the recombinant protein; Any of the methods 1001 to 1104 of the present invention. [The present invention 1106] 1105. A method according to claim 1105, wherein said introducing step is carried out subsequent to or during said incubation and / or while the cells are immobilized on a support. [This invention 1107] 1107. The method of any of claims 1001 to 1106, wherein said cells express the introduced recombinant protein ex vivo during at least a portion of said incubation. [This invention 1108] The method according to any one of 1105 to 1107 of the present invention, wherein a step of introducing nucleic acid is carried out between two or more of said at least two contacting steps. [This invention 1109] 1108. The method of claim 1108, wherein one of the at least two selection agents specifically binds to the recombinant protein and / or one of the stimulatory agents specifically binds to the recombinant protein. [The present invention 1110] The method of any one of claims 1001 to 1109, wherein the first reagent is not immobilized on a support. [The present invention 1111] following said incubation, further comprising transferring the target cells of the composition to a different environment; the environment is suitable for cell culture or expansion; Any of the methods 1001 to 1110 of the present invention. [The present invention 1112] The method of claim 1111, wherein the cells so transferred are transferred to a different environment in a closed system or container; or the transfer comprises removing the cells so transferred from a first container and transferring the cells to a second container. [The present invention 1113] The method of claim 1111 or 1112, wherein the different environment is within an incubator. [This invention 1114] Any of the methods of inventions 1111 to 1113, wherein the transfer is carried out in a closed system, the transfer comprises transferring a sterile sealed container containing the cells to a sterile environment or to a different environment within the sealed container, and / or the transfer is carried out in a sterile environment or under sterile conditions. [This invention 1115] 1115. The method of claim 1114, further comprising, following transfer, releasing said cells from the stationary phase by disrupting said reversible bond, and optionally removing said cells from the presence of the stationary phase. [The present invention 1116] The method of claim 1115, further comprising the step of expanding the extracted cells. [This invention 1117] Temperature, pH, pO 2 , pCO 2 and / or temperature is controlled, optionally in an automated manner, during at least a portion of said incubation. [This invention 1118] 1118. The method of any of claims 1111 to 1117, wherein the cells contained in at least one of the at least one stationary phase for chromatography are supplied with nutrients while in an environment suitable for growth. [This invention 1119] A method according to any one of claims 1111 to 1118, wherein the stationary phase is present in a device according to any one of claims 1131 to 1143, and the transfer to an appropriate environment for expansion comprises separating the stationary phase from the cells while the stationary phase is present in the device. [The present invention 1120] 1. An article of manufacture for purifying and modulating target cells, comprising: (a) a stimulatory agent capable of specifically binding to a molecule on the surface of a target cell in a manner that induces or modulates a signal in the target cell; (b) a first reagent comprising a plurality of stimulator binding sites, each capable of reversibly binding to said stimulator; (c) a second reagent; (d) a support; and (e) a selection agent capable of reversibly binding to a second reagent and specifically binding to a selection marker on a target cell. Manufactured articles, including: [This invention 1121] The method of claim 1120, wherein the support is or comprises a stationary phase and / or a solid support. [This invention 1122] 1120 or 1121, wherein the second reagent is immobilized on a support. [This invention 1123] The article of manufacture of any of claims 1120 to 1122, wherein a first reagent is reversibly bound to said stimulatory agent and / or said selection agent is reversibly bound to a second reagent. [This invention 1124] a second stimulator capable of specifically binding to a second molecule on the surface of the target cell and capable of reversibly binding to the first reagent and / or the fourth reagent; and / or a second selection agent capable of specifically binding to a second selection marker (i) contained on the target cell or (ii) contained on another target cell; The article of manufacture of any one of claims 1120 to 1123, further comprising: [This invention 1125] The method of claim 1124, wherein the other target cell expresses a molecule to which the first stimulatory agent and / or the second stimulatory agent specifically binds. [The present invention 1126] The article of manufacture of claim 1124 or 1125, wherein the second selected substance is capable of reversibly binding to the second reagent, or wherein the article further comprises a third reagent capable of reversibly binding to the second selected substance. [This invention 1127] 1127. The article of manufacture of claim 1126, wherein a third reagent is immobilized on said support or another support. [This invention 1128] 1127. An article of manufacture of claim 1127, wherein the support and second support are present in separate containers, and these different containers are optionally fluidly connected to each other to allow a cell suspension to pass through or past one of the supports to the other support. [This invention 1129] 1129. The article of manufacture of any one of claims 1120 to 1128, wherein the support is a chromatography matrix or a stationary phase comprising the same, and the article of manufacture further comprises a container containing all or part of the chromatography matrix. [The present invention 1130] 1129. The article of manufacture of claim 1129, wherein the vessel is a column. [This invention 1131] An apparatus comprising any one of the articles of manufacture of the present inventions 1120 to 1130. [This invention 1132] The device of the present invention 1131 further comprising a fluid inlet fluidly connected to one or more components of said device, and / or a fluid outlet fluidly connected to one or more components of said device. [This invention 1133] (a) a stimulatory agent capable of specifically binding to a molecule on the surface of a target cell in a manner that induces or modulates a signal in the target cell; (b) a first reagent capable of reversibly binding to the stimulator; (c) a second reagent; (d) support; (e) a selection agent capable of reversibly binding to a second reagent and specifically binding to a selection marker on a target cell. 1. An apparatus comprising: [This invention 1134] 1133. The device of claim 1133, wherein said support is or comprises a stationary phase for chromatography. [This invention 1135] The device of the present invention 1133, wherein components (a) through (e) are present in multiple containers, at least some of which are fluidly connected, whereby one or more of the components pass from one container to another within the device. [This invention 1136] Any of the devices of inventions 1131 to 1135, in a closed or sterile system. [This invention 1137] The apparatus of any of claims 1131 and 1134-1136, further comprising a sample outlet fluidly connected to one of the at least one stationary phase for chromatography. [This invention 1138] Any one of the articles of manufacture or devices of inventions 1120 to 1137, wherein the device is a functionally closed system. [This invention 1139] The method further includes one or more control units, which control the pH, pO of one or more vessels or components thereof, and / or at least one of the at least one stationary phase for chromatography. 2 , pCO 2 and / or any of the articles of manufacture or devices of the present invention 1120 to 1138, which are capable of adjusting or regulating automatic temperature control. [This invention 1140] 1120-1139. The article of manufacture or device of any of claims 1120-1139, further comprising a fluid connection to a container containing culture medium and / or one or more nutrients and / or one or more carbon sources, whereby said connection is capable of delivering such culture medium, nutrients and / or carbon sources to cells within the device, optionally when the cells are immobilized on a stationary phase for chromatography. [This invention 1141] The device of any of claims 1131 to 1140, wherein at least one of the listed components and / or the container containing it is removable from the device in a sterile or aseptic manner. [This invention 1142] Any of the apparatuses of inventions 1131 to 1141 or any of the articles of inventions 1122 to 1130, which are useful for carrying out any of the methods of inventions 1001 to 1121 or which are capable of carrying out any of the methods of inventions 1001 to 1121, optionally carried out in an automated manner. [This invention 1143] An apparatus according to any one of claims 1131 to 1141 or an article according to any one of claims 1122 to 1130, for use in a method according to any one of claims 1001 to 1121, wherein the method is carried out, optionally in an automated manner. [Brief explanation of the drawings]

[0148] FIG. 1A-E provide schematic diagrams of exemplary embodiments. FIG. 1A shows a schematic diagram of a reagent (or a representative portion thereof) having multiple binding sites for reversible binding to an agent. In this case, the reagent is shown as being capable of reversibly binding two agents, each of which can specifically bind to a molecule on a cell. The reagent has multiple binding sites, including multiple binding sites Z1, each of which can reversibly bind to an agent. In the diagram shown, the first and second agents, which may be the same in some cases, each include at least one binding partner C1. The binding partner C1 reversibly binds to binding site Z1. The first and second agents also each include binding site B2, which can specifically bind to a molecule on the surface of a cell, which may, in some cases, be on the same cell. Here, the first and second agents are shown to specifically bind to multiple molecules on the same cell. 1B shows a schematic diagram of a reagent having multiple binding sites capable of reversibly binding to a first and second agent, where the first and second agents are capable of specifically binding to molecules on a first and second cell, respectively. The reagent has multiple binding sites Z1, each capable of reversibly binding to an agent. The first and second agents may optionally be the same, and each contain a binding partner C1, which reversibly binds to binding site Z1. The first and second agents each contain a binding site B2, which may specifically bind to a molecule on the surface of a cell, where the molecule may be on the same cell or a different cell. Here, the first agent binds to a molecule on the surface of a first cell, and the second agent binds to a molecule on the surface of a second cell. FIG. 1C shows a reagent capable of reversibly binding to a first and second agent, where the first and second agents are capable of specifically binding to molecules on a first and second cell, respectively. The reagent has multiple binding sites, Z1 and Z2, which may be the same or different and each capable of reversibly binding to one or both of the agents. The first agent includes a binding partner, C1, that reversibly binds to Z1; the second agent includes a binding partner, C2, that reversibly binds to Z2. In some cases, C1 and C2 are different. In some cases, C1 and C2 are the same or substantially the same. The first agent includes a binding site, B1, that can specifically bind to a molecule on the surface of a cell, and the second agent includes at least one binding site, B3, that can specifically bind to a molecule on the surface of a cell. Binding sites B1 and B3 may bind to two different cell surface molecules, or different epitopes on a single molecule, or the same or different molecules on the surface of different cells. Here, a first agent is shown as binding to a molecule on the surface of a first cell via B1, and a second agent is bound to a molecule on the surface of a second cell. FIG. 1D shows a reagent capable of reversibly binding to a first and second agent, such as a selection agent, each capable of specifically binding to a molecule on a cell. The reagent has multiple binding sites, including Z1 and Z2, which may be the same or different and each capable of reversibly binding to an agent. The first agent includes a binding partner C1 capable of specifically binding to binding site Z1, and the second agent includes at least one binding partner C2 capable of specifically binding to binding site Z2. In some cases, C1 and C2 are different. In some cases, C1 and C2 are the same or substantially the same. The first agent includes a binding site B1 capable of specifically binding to a molecule on the surface of a cell, and the second agent includes a binding site B3 capable of specifically binding to a molecule on the surface of a cell. In some embodiments, the first agent and the second agent can be selection agents. Binding sites B1 and B3 can bind to the same or different molecules (e.g., receptors) on the surface of a single cell, the same or different epitopes on a single molecule, or the same or different molecules on the surface of different cells, where a first agent binds to a first molecule on the surface of the cell and a second agent binds to a second molecule on the surface of the same cell. FIG. 1E shows a reagent reversibly bound to a first and a second agent, each capable of specifically binding to a molecule on a cell. The reagent has multiple binding sites, including Z1 and Z2, which may be the same or different and each capable of reversibly binding to an agent. The first agent includes a binding partner C1 that can reversibly bind to Z1 of the reagent, and the second agent includes a binding partner C2 that can reversibly bind to Z2. In some cases, C1 and C2 are different. In some cases, C1 and C2 are the same or substantially the same. The first agent includes at least one binding site B2 that can specifically bind to a molecule on the surface of a cell, and the second agent includes at least one binding site B4 that can specifically bind to a molecule on the surface of a cell. In some embodiments, the first agent and the second agent can be stimulatory agents. Binding sites B2 and B4 can bind to the same or different molecules on the surface of a single cell, the same or different epitopes on a single molecule, or the same or different molecules on the surface of different cells, where a first agent binds to a first molecule on the surface of the cell and a second agent binds to a second molecule on the surface of the same cell. (FIG. 2) FIGS. 2A-E provide schematic diagrams of the exemplary embodiment shown in FIGS. 1A-E, but where the depicted reagents are shown as immobilized on a support, such as a stationary phase. (FIG. 3) FIG. 3 shows a schematic diagram of an exemplary embodiment in which an oligomeric reagent is used to multimerize a stimulator, the resulting complex is incubated with cells to deliver a signal to the cells, and then the binding is reversed. Panel A shows oligomeric reagent 1, which is shown as not bound to a support and is flexible. Stimulator 2, shown here as a Fab fragment, capable of specifically binding to a molecule on the surface of a cell is combined with the reagent. The stimulator includes a binding partner (e.g., binding partner C) that can reversibly bind to a binding site (e.g., binding site Z) on the reagent that multimerizes the stimulator. Panel B shows the binding partner reversibly bound to a binding site on the reagent. Cell 3 is added to the system. Panel C shows the multimerized stimulator (Fab fragment) specifically binding to molecule 4 on the surface of cell 3. The stimulator depicted in panel C is a stimulatory receptor-binding substance (e.g., a first receptor-binding substance and / or a second receptor-binding substance) that can induce or modulate a signal in a cell upon binding of the stimulator to a molecule on the cell. As shown in panel D, a substance 5, such as a competing reagent (e.g., biotin), is added to this composition, which can exhibit a higher binding affinity for the binding site on the reagent than for the binding partner on the stimulator, thereby disrupting the reversible binding between reagent 1 and stimulator 2. In some cases, the stimulator, e.g., a Fab fragment, can also dissociate from its interaction with molecule 4 on cell 3. In some cases, this can disrupt, reduce, and / or terminate signaling within the cell. (FIG. 4) FIG. 4 provides a schematic diagram of an exemplary embodiment of a reversible system attached to a support, such as a solid support or a surface comprising a stationary phase. Panel A shows a support 6 containing reagent 1. An agent 2, such as a Fab fragment, capable of specifically binding to a molecule on the surface of a cell is added to the system. Agent 2, e.g., a Fab fragment, comprises a binding partner (e.g., binding partner C) that can reversibly bind to a binding site (e.g., binding site Z) on the reagent. Panel B shows the binding partner reversibly bound to a binding site on the reagent. A cell 3 is added to the system. Panel C shows agent 2, e.g., a Fab fragment, bound to molecule 4 on the surface of cell 3. In some embodiments, the scFv constitutes a receptor binding agent or a selection agent. In some embodiments, the agent, e.g., a Fab fragment, can be a receptor binding agent or a selection agent. Panel C shows an exemplary receptor-binding substance (e.g., a first receptor-binding substance and / or a second receptor-binding substance) that can induce or modulate a signal in a cell upon binding of the receptor-binding substance, e.g., a Fab fragment, to a molecule on the cell. A substance 5, such as a competing reagent (e.g., biotin), is added, which may exhibit a higher binding affinity for the binding site on the reagent than for the binding partner on the agent, e.g., the Fab fragment, thereby disrupting the binding between the reagent and the agent. Panel D shows disruption of the binding between agent 2, e.g., a Fab fragment, and the reagent, resulting in dissociation of the reagent from the agent, and thus from the cell. In some cases, the agent, e.g., the Fab fragment, can also dissociate from its interaction with molecule 4 on cell 3. In some cases, this can disrupt, reduce, and / or terminate intracellular signal transduction. (FIG. 5) FIG. 5 provides a schematic diagram of an exemplary embodiment for stimulating and enriching target cells, where stimulation occurs at least in part by incubation of cells in the presence of a support 6, depicted here as a stationary phase, on which component(s) of a cell selection reagent 1 are immobilized (Panel A). Reagent 1 has a binding site for a selection agent 2 capable of binding to a molecule 4 present on some or all of the target cells. Selection agent 2 is added to the support on which reagent 1 is immobilized under conditions in which the reagent and selection agent reversibly bind, e.g., via the binding site, to generate an oligomeric complex containing the multimerized selection agent on the support (Panel B). The selection agent can include two or more selection agents. Alternatively, a reversibly bound selection agent-reagent complex can be added as a complex to the stationary phase for immobilization. As shown, cells 3, including target cells, are combined with the stationary phase and the multimerized selection agent complex, thereby reversibly immobilizing the target cells to support 6 via selection agent 2 and reagent 6 (Panel C). Optionally, unbound cells are removed before or after addition of the stimulator. A complex comprising multimerized stimulator 5 reversibly bound to oligomeric reagent 7 is added under conditions where stimulator 5 specifically binds to a molecule on the target cell, thereby inducing or modulating a signal in immobilized target cells expressing the marker (Panel D). In some embodiments, when reversible binding between different agents and reagents can be reversed by the same agent, the agent can be added to disrupt the reversible binding, detach the cells from the stationary phase, and stop stimulation, e.g., by adding a single agent. (FIG. 6A) Figure 6A shows a graph of the total cell numbers observed after (i) enriching PBMC samples for cells expressing one of the various indicated selection markers (CD3+ enrichment, CD4+ enrichment, CD8+ enrichment) and (ii) incubating the enriched cells in the presence of medium and IL-2 alone (open bars) or with a multimerization reagent reversibly bound to anti-CD3 and anti-CD28 Fab fragments (black bars). Incubation was performed on a column in the presence of a stationary phase, onto which the cells were reversibly immobilized via a selection agent specific for the relevant selection marker. (Figure 6B) Figure 6B shows the results of surface expression of CD45RA and CD45RO on CD4+ and CD8+ cells after (i) enriching PBMC samples for cells expressing one of the various indicated selection markers (CD3+ enrichment, CD4+ enrichment, CD8+ enrichment) and (ii) incubating the enriched cells in the presence of medium and IL-2 alone (unstimulated) or with multimerization reagents reversibly bound to anti-CD3 and anti-CD28 Fab fragments (a-CD3, a-CD28). Incubation was performed on a column in the presence of a stationary phase, to which cells were reversibly immobilized via a selection agent specific for the relevant selection marker. The top row shows staining before selection or incubation. (Figure 6C) Figure 6C shows the results of surface expression of CD62L and CD69 on CD4+ and CD8+ cells after (i) enriching PBMC samples for cells expressing one of the various indicated selection markers (CD3+ enrichment, CD4+ enrichment, CD8+ enrichment) and (ii) incubating the enriched cells in the presence of medium and IL-2 alone (unstimulated) or with multimerization reagents reversibly bound to anti-CD3 and anti-CD28 Fab fragments (a-CD3, a-CD28). Incubation was performed on a column in the presence of a stationary phase, to which the cells were reversibly immobilized via a selection agent specific for the relevant selection marker. (Figure 7A) Figures 7A-C show the results of an experiment in which CD3+ T responder cells were expanded after in vitro stimulation with αCD3 and αCD28 Fab fragments reversibly immobilized on streptavidin mutein Strep-tactin®-coated beads. Figure 7A is a histogram showing the size distribution (forward scatter) of stimulated cells. (Figure 7B) Figures 7A-C show the results of an experiment in which CD3+ T responder cells were expanded after in vitro stimulation with αCD3 and αCD28 Fab fragments reversibly immobilized on streptavidin mutein Strep-tactin®-coated beads. Figure 7B shows a histogram depicting the degree of proliferation as a function of the number of cells per cell division shown at the top of Figure 7B (0 represents non-dividing cells, 5 represents cells that have undergone at least five divisions). (Figure 7C) Figures 7A-C show the results of an experiment in which CD3+ T responder cells were expanded after in vitro stimulation with αCD3 and αCD28 Fab fragments reversibly immobilized on streptavidin mutein Strep-tactin®-coated beads. Figure 7C shows a photograph of the culture dish after 4 days of stimulation. (Figure 8-1) Figures 8A-E show the results of an experiment in which CD3+ T responder cells were expanded after in vitro stimulation with reversible αCD3 / αCD28 Fab fragments reversibly immobilized on a soluble oligomeric streptavidin mutein, which served as a soluble reagent. For the experiment whose results are shown in Figures 8A-E, 300,000 CD3+ responder T cells (Tresp) were labeled with 2 μM carboxyfluorescein succinimidyl ester (CFSE) and stimulated with various amounts of a soluble oligomeric streptavidin mutein preparation to which a combination of αCD3 Fab fragments and αCD28 Fab fragments (both of which possess a Strep-tag on their heavy chains as streptavidin-binding peptides) had been immobilized. ("1x" corresponds to 3 μg of oligomeric streptavidin mutein functionalized with 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 Fab; numbers indicate the "1x" amount.) Tresp cells were left unstimulated or stimulated with blank oligomeric streptavidin mutein (no Fab), used as a negative control. Tresp cells were seeded in duplicate in 48-well plates with 300,000 CD3-negative autologous feeder cells (irradiated at 30 Gy) in 1 ml of cell culture medium supplemented with 20 U / ml interleukin-2 (IL-2). Cells were incubated at 37°C without medium changes, and proliferation was analyzed after 5 days by CFSE dilution via FACS analysis (Figure 8B). Figure 8A shows the size distribution of cells after 5 days of culture. Histograms show viable CD3+ cells, while Figure 8C shows cells released from the stimulation reagent after incubation with 1 mM D-biotin and subsequent washing. Dissociation and removal of monomeric Fab fragments was analyzed by restaining with phycoerythrin-labeled oligomeric streptavidin mutein (ST-PE) as a fluorescent label, and a representative histogram is shown. Figure 8D shows the absolute number of viable (trypan blue-negative) cells after 5 days, counted using a Neubauer counting chamber, plotted against each stimulation condition. Median cell counts are shown in Figure 8D; error bars indicate standard deviation (SD).FIG. 8E shows a photograph of the culture dish after 5 days of stimulation. (Figure 8-2) See the description of Figure 8-1. (Figure 8-3) See the description of Figure 8-1. (FIG. 9) Figures 9A-B show the proliferation kinetics of purified CD4+ and CD8+ T responder cells (Tresp) stimulated in vitro with either αCD3 / αCD28 Fab fragments or αCD3 / αCD28 / αCD8 Fab reversibly immobilized to two soluble oligomeric streptavidin muteins acting as soluble reagents. The first type of oligomeric streptavidin mutein was a fraction (n≧3) of the oligomeric streptavidin mutein obtained in Example 3 (also referred to herein as the "conventional" or "smaller" oligomeric streptavidin mutein backbone and indicated by the triangle symbol with the apex downwards in Figures 9A-B). The second type of oligomeric streptavidin mutein used as a soluble reagent was an oligomer obtained by reacting the soluble oligomeric streptavidin mutein with biotinylated human serum albumin (HSA). This HSA-based soluble reagent is also referred to herein as the "larger" oligomeric streptavidin mutein backbone. In the experiments shown in Figures 9A-B, cells were expanded without medium change. Results for CD4+ T responder cells are shown in Figure 9A, and results for CD8+ T responder cells are shown in Figure 9B. In this regard, it should be noted that the soluble reagents used experimentally that are functionalized by reversibly binding to a first agent, and optionally a second and third agent, are referred to in the figures as "multimerizing agents." (Figure 10) Figures 10A-B show the proliferation kinetics of purified CD4+ and CD8+ T responder cells (Tresp) stimulated in vitro with reversibly immobilized αCD3 / αCD28 Fab fragments on two types of soluble oligomeric streptavidin muteins acting as soluble reagents. The first type of oligomeric Strep-tactin® was a fraction (n≧3) of the oligomeric streptavidin mutein obtained in Example 3 (also referred to herein as the "conventional oligomeric streptavidin mutein scaffold" and indicated by a triangular symbol with a vertex on top in Figures 10A-B). The second type of this oligomeric streptavidin mutein used as the soluble reagent was an HSA-based soluble reagent, the "large scaffold" described above. In the experiments shown in Figures 10A-B, growth was achieved by changing the medium. The results for CD4+ T responder cells are shown in Figure 10A, and the results for CD8+ T responder cells are shown in Figure 10B. (Figure 11) Figures 11A-B show combined data from the results obtained in Figures 9-10 on the expansion kinetics of purified CD4+ and CD8+ T responder cells, with Figure 11A showing the results for CD4+ T cells and Figure 11B showing the results for CD8+ T cells. The straight lines represent cultures with a medium change on day 3, while the dotted lines represent values ​​for the extent of expansion obtained without a medium change on day 3. The data shown in Figures 11A-B are normalized to the input cell number. Only data for Tresp stimulated with oligomeric streptavidin muteins (n≧3), Tresp stimulated with commercial anti-CD3 / anti-CD28 beads (positive control), and unstimulated T cells (negative control) are shown; data for the "large backbone" reagent are not shown. (FIG. 12A) FIGS. 12A-C show the activity of CD3+ central memory T cells (T) polyclonally stimulated in vitro with αCD3 / αCD28 Fab fragments reversibly immobilized to soluble oligomeric streptavidin muteins (n≧3) as described in Example 3. CM) (CD3+CD62L+CD45RA- Tcm). The graphs shown in Figures 12A-B represent the extent of proliferation as a function of the number of cells harvested at each time point, with Figure 12A showing proliferation in medium supplemented with IL-2 only. (FIG. 12B) FIGS. 12A-C show the activity of CD3+ central memory T cells (T) polyclonally stimulated in vitro with αCD3 / αCD28 Fab fragments reversibly immobilized to soluble oligomeric streptavidin muteins (n≧3) as described in Example 3. CM ) (CD3+CD62L+CD45RA- Tcm). The graphs shown in Figures 12A-B represent the extent of proliferation as a function of the number of cells harvested at each time point, and Figure 12B shows proliferation in medium supplemented with IL-2 and IL-15. (FIG. 12C) FIGS. 12A-C show the activity of CD3+ central memory T cells (T) polyclonally stimulated in vitro with αCD3 / αCD28 Fab fragments reversibly immobilized to soluble oligomeric streptavidin muteins (n≧3) as described in Example 3. CM Figure 12C shows the expansion kinetics and phenotype of CD3+CD62L+CD45RA- Tcm after 14 days of culture in these different cytokine environments. (Figure 13A) Figures 13A-B show the yield and phenotype of expanded purified CD8+ T responder cells stimulated in vitro with αCD3 / αCD28 Fab fragments reversibly immobilized to two types of soluble oligomeric streptavidin muteins, which served as soluble reagents. The first type of oligomeric streptavidin mutein was a fraction of the oligomeric streptavidin mutein obtained in Example 3 (conventional backbone), and the second type of this oligomeric streptavidin mutein used as a soluble reagent was the soluble oligomer described above, referred to herein as the "large" backbone. In these experiments, fractions of conventional oligomeric streptavidin muteins (n≧3) were also used as reagents functionalized with a single Fab fragment (third bar in Figures 13A and 13B) or with a combination of αCD3 Fab fragments and αCD28 Fab fragments. In addition to the combined stimulation with αCD3 / αCD28 Fab fragments, additional αCD8 Fab fragments (commercially available from IBA GmbH, Goettingen, Germany) were also immobilized to test whether they could selectively stimulate specific T cell subpopulations. Figure 13A shows a bar graph depicting the extent of proliferation as a function of the number of cells harvested on day 6, compared to a negative control (unstimulated purified CD8+ T responder cells) and normalized to a positive control (purified CD8+ T responder cells stimulated with commercially available anti-CD3 / anti-CD28 beads (beads to which αCD3 and αCD28 monoclonal antibodies were irreversibly immobilized)). (Figure 13B) Figures 13A-B show the yield and phenotype of expanded purified CD8+ T responder cells stimulated in vitro with αCD3 / αCD28 Fab fragments reversibly immobilized to two types of soluble oligomeric streptavidin muteins, which served as soluble reagents. The first type of oligomeric streptavidin mutein was a fraction of the oligomeric streptavidin mutein obtained in Example 3 (conventional backbone), and the second type of this oligomeric streptavidin mutein used as a soluble reagent was the soluble oligomer described above, referred to herein as the "large" backbone. In these experiments, fractions of conventional oligomeric streptavidin muteins (n≧3) were also used as reagents functionalized with a single Fab fragment (third bar in Figures 13A and 13B) or with a combination of αCD3 Fab fragments and αCD28 Fab fragments. In addition to the combined stimulation with αCD3 / αCD28 Fab fragments, an additional αCD8 Fab fragment (commercially available from IBA GmbH, Goettingen, Germany) was also immobilized to test whether it was possible to selectively stimulate specific T cell subpopulations. Figure 13B shows flow cytometry analysis of the surface expression of CD8 and the T cell surface molecule CD45RO (an indicator of T cell proliferation and activation) after cell culture. One-way ANOVA was used to compare the different stimulation conditions, and no significant differences (ns) were detected. (FIG. 14A) Figures 14A-B show the yield and phenotype of expanded purified CD8+ T responder cells stimulated in vitro with αCD3 / αCD28 Fab fragments reversibly immobilized to soluble oligomeric streptavidin muteins acting as soluble reagents functionalized with single Fab fragments or with combinations of Fab fragments (as described above). In these experiments, CD8+ T responder cells were stimulated with various amounts of soluble reagents functionalized with αCD3 and αCD28 Fab fragments (soluble oligomeric streptavidin muteins (1 mg / ml) from Example 3), optionally together with the αCD8 Fab fragments described above. The term "1x" corresponds to 1.5 µg of oligomeric streptavidin muteins functionalized with 0.5 µg of αCD3 Fab fragments alone and 1.5 µg of oligomeric streptavidin muteins functionalized with 0.5 µg of αCD28 Fab fragments alone, or to 3 µl of a preparation of oligomeric streptavidin muteins loaded with 0.5 µg of αCD3 Fab fragments and 0.5 µg of αCD28 Fab, or to 4.5 µl of a preparation of oligomeric streptavidin muteins loaded with 0.5 µg of strep-tagged αCD3, 0.5 µg of strep-tagged αCD8, and 0.5 µg of strep-tagged αCD28 Fab. Therefore, the term "2x" indicates that twice the amount of immobilized αCD3 Fab fragments was used, corresponding to 3.0 μg of oligomeric streptavidin muteins functionalized with 1 μg of αCD3 Fab fragments alone and 3.0 μg of oligomeric streptavidin muteins functionalized with 1 μg of αCD28 Fab fragments alone. Untreated Tresp cells served as a negative control, and purified CD8+ T responder cells stimulated with commercially available anti-CD3 / anti-CD28 beads (beads to which αCD3 and αCD28 monoclonal antibodies were irreversibly immobilized) served as a positive control. Figure 14A shows a bar graph depicting the extent of proliferation as a function of the number of cells harvested on day 5, compared with the negative control and normalized to the positive control. (Figure 14B) Figures 14A-B show the yield and phenotype of expanded purified CD8+ T responder cells stimulated in vitro with αCD3 / αCD28 Fab fragments reversibly immobilized to soluble oligomeric streptavidin muteins acting as soluble reagents functionalized with single Fab fragments or with combinations of Fab fragments (as described above). In these experiments, CD8+ T responder cells were stimulated with various amounts of soluble reagents functionalized with αCD3 and αCD28 Fab fragments (soluble oligomeric streptavidin muteins (1 mg / ml) from Example 3), optionally together with the αCD8 Fab fragments described above. The term "1x" corresponds to 1.5 µg of oligomeric streptavidin muteins functionalized with 0.5 µg of αCD3 Fab fragments alone and 1.5 µg of oligomeric streptavidin muteins functionalized with 0.5 µg of αCD28 Fab fragments alone, or to 3 µl of a preparation of oligomeric streptavidin muteins loaded with 0.5 µg of αCD3 Fab fragments and 0.5 µg of αCD28 Fab, or to 4.5 µl of a preparation of oligomeric streptavidin muteins loaded with 0.5 µg of strep-tagged αCD3, 0.5 µg of strep-tagged αCD8, and 0.5 µg of strep-tagged αCD28 Fab. Therefore, the term "2x" indicates that twice the amount of immobilized αCD3 Fab fragments was used, corresponding to 3.0 μg of oligomeric streptavidin muteins functionalized with 1 μg of αCD3 Fab fragments alone and 3.0 μg of oligomeric streptavidin muteins functionalized with 1 μg of αCD28 Fab fragments alone. Untreated Tresp cells served as a negative control, and purified CD8+ T responder cells stimulated with commercially available anti-CD3 / anti-CD28 beads (beads to which αCD3 and αCD28 monoclonal antibodies were irreversibly immobilized) served as a positive control. Figure 14B shows FACS analysis of CD8 and CD45RO surface expression after cell culture. (FIG. 15) Figures 15A-B show the expansion of purified CD3+ T responder cells stimulated in vitro with αCD3 / αCD28 Fab fragments reversibly immobilized on the soluble oligomeric streptavidin mutein of Example 3 used as a soluble reagent. In one experiment, in addition to the αCD3 / αCD28 Fab fragments, commercially available αCD8 Fab fragments (catalog number 6-8000-203) from IBA GmbH (Goettingen, Germany) were also immobilized on the soluble oligomer of the streptavidin mutein to test whether a reagent in which the αCD8 Fab fragment was also reversibly immobilized could be used to selectively stimulate in vitro a CD8+ T cell subpopulation within a bulk CD3+ culture. More specifically, 500,000 purified CD3+ responder T cells (Tresp) were stimulated with 3 μl of a 1 mg / ml preparation of oligomeric streptavidin muteins loaded with a combination of 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 Fab. As an alternative approach, 4.5 μl of the oligomeric streptavidin muteins were loaded with 0.5 μg of the above-mentioned αCD3 Fab, 0.5 μg of αCD8 Fab, and 0.5 μg of αCD28 Fab. Unstimulated Tresp cells served as a negative control, and Tresp cells stimulated with anti-CD3 / anti-CD28 beads (beads to which αCD3 and αCD28 monoclonal antibodies were irreversibly immobilized) served as a positive control. Figure 15A shows a graph of the cell number (degree of expansion) in cultures under each condition. FIG. 15B shows the percentage of CD4+ cells and CD8+ cells under each stimulation condition. (Fig. 16A) Figures 16A-B show the results of differential intracellular calcium mobilization in Jurkat cells labeled with the αCD3 antibody OKT3 or with Fab fragments of OKT3 multimerized with Strep-tactin® (also referred to herein as Fab multimers). In the experiment in Fig. 16A, Jurkat cells were loaded with the calcium-sensitive dye Indo-1-AM, and calcium release was triggered by injection of the αCD3 mAb OKT3 (black squares) or αCD3 OKT3 Fab multimers (derived from the parental cell line OKT3) with or without prior D-biotin blockage (dark gray triangles and light gray circles, respectively) and compared with injection of PBS (inverted open triangles). Application of ionomycin was used as a positive control. Intracellular Ca 2+ Time-resolved changes in concentration were monitored by flow cytometry based on changes in the FL6 / FL7 ratio. (Figure 16B) Figures 16A-B show the results of differential intracellular calcium mobilization in Jurkat cells labeled with the αCD3 antibody OKT3 or with Fab fragments of OKT3 multimerized with Strep-tactin® (also referred to herein as Fab multimers). In the experiment in Figure 16B, Indo-1-AM-labeled Jurkat cells were activated with different αCD3 stimuli (OKT3: upper graph, αCD3 Fab multimer: middle graph) as described in Example 11, followed by subsequent (t = 140 s) D-biotin-mediated blockade of αCD3 Fab multimer signaling. D-biotin-predissociated αCD3 Fab multimers (lower graph) and ionomycin were used as negative or positive controls. Data are representative of three separate experiments. (Figure 17) Figure 17 shows the results of reversible staining of cells with anti-CD3 OKT3 Fab multimers. Freshly isolated PBMCs were stained with either a monoclonal antibody (left dot plot, parental clone of the Fab multimer) or the cognate PE-labeled Fab multimer and analyzed before (second dot plot from the left) or after (middle dot plot) treatment with D-biotin. Remaining Fab monomers were then detected after a subsequent washing step with fresh PE-labeled Strep-Tactin® (second dot plot from the right). Secondary Fab multimer staining of reversibly stained cells served as a control (right dot plot). Only viable (PI-negative) cells are shown. Numbers within the dot plots indicate the percentage of cells within the gate. (Figure 18) Figure 18 shows the separation of cells by reversible binding of anti-CD28 Fab fragments multimerized with Strep-Tactin® labeled with phycoerythrin as a fluorescent label. CD28+ cells were sorted / isolated from freshly isolated PMBCs by Fab multimer magnetic cell sorting, as described in International Patent Application Publication No. WO 2013 / 011011. Prior to sorting, cells were counterstained with the cognate fluorescent aCD28 multimer (left dot plot) or with an antibody against the immunoglobulin kappa light chain (second dot plot from the left, αIgκ mAb). After sorting, cells were treated with D-biotin and then washed to remove magnetic beads and Fab monomers. Subsequently, released CD28+ cells were (re)stained with αCD28 Fab multimers (second dot plot from the right) or with αIgκ mAb (right dot plot) to detect potential remaining Fab monomers. Only viable (PI-negative) CD3+ cells are shown. Numbers within the dot plots indicate the percentage of cells within the gate. (FIG. 19) Figures 19A-B show the early cluster formation of T cells after activation of purified CD4+ and CD8+ T responder cells stimulated in vitro with αCD3 / αCD28 Fab fragments reversibly immobilized to soluble oligomeric streptavidin muteins (n≧3) as described in Example 3. Figure 19A shows the results for CD4+ T cells, and Figure 19B shows the results for CD8+ T cells. Data are shown for Tresp stimulated with a soluble multimerization reagent (oligomeric streptavidin mutein), Tresp stimulated with commercially available anti-CD3 / anti-CD28 beads (positive control), and unstimulated T cells (negative control). (Figure 20-1) Figures 20A-B show purified CD3+CD62L+CD45RA- T cells stimulated in vitro with both a peptide:MHC molecule complex (acting as a first agent that provides a primary activation signal to the cell) and an αCD28 Fab fragment (acting as a second agent that binds to an accessory molecule on the surface of the cell). CM The kinetics of selective antigen-specific (Ag-specific) expansion from a bulk population of responder cells are shown; unstimulated T cells (negative control) are shown. Both the antigen-specific peptide-MHC molecule complex and the αCD28 Fab fragment were reversibly immobilized to the same soluble oligomeric streptavidin muteins (n≧3) described in Example 3. The peptide used for antigen-specific expansion in Figure 20A was a peptide of amino acids 309-317 of the immediate early 1 protein restricted by HLA-C702 MHC molecules, representing the HLA-C7 / IE-1 epitope specific for cytomegalovirus (CMV): TIFF0007665579000008.tif4128 (described in Ameres et al., PLOS Pathogens, May 2013, vol. 9, issue 5, e1003383). The MHC I molecule presenting the peptide contained, at the C-terminus of its heavy chain, a streptavidin-binding peptide commercially available as "Twin-Strep-tag" from IBA GmbH (Goettingen, Germany): TIFF0007665579000009.tif4128. Figure 20A shows an exemplary flow cytometry analysis of the fraction of Ag-specific cells expanded using a peptide:MHC-I complex specific for this HLA-C7 / IE-1 epitope as the first agent to provide a primary activation signal to cells reversibly immobilized on a soluble oligomeric streptavidin mutein. The graphs in Figures 20B-20E show the kinetics of further Ag-specificity increase as a function of the number of specific peptide:MHC I multimer-positive cells, collected at the same time points as in Figure 20A, using different complexes of antigen-specific peptide and MHC I molecules as the first agent to provide a primary activation signal to cells reversibly immobilized on a soluble oligomeric streptavidin mutein. More specifically, Figure 20B shows the CMV pp65 epitope (amino acids 341-350 restricted by HLA-A2402: Figure 20C shows the expansion of Ag-specific cells expanded with peptide:MHC-I complexes specific for the CMV pp65 epitope (amino acids 265-274 restricted by HLA-B702: Figure 20D shows the expansion of Ag-specific cells grown with another peptide:MHC-I complex specific for the adenoviral hexon 5 epitope (amino acids 114-124 restricted by HLA-B702: Figure 20B shows the expansion of Ag-specific cells grown with peptide:MHC-I complexes specific for the HLA-B7 / IE-1309-317 epitope of CMV (Figure 20C). Figure 20D shows the expansion of Ag-specific cells grown with peptide:MHC-I complexes specific for the HLA-B7 / IE-1309-317 epitope of CMV (exemplary FACS data, see Figure 20A above). All peptide:MHC molecules with Twin Strep® tags are commercially available from Iba GmbH. In this context, HLA-A molecules with a "Twin-Strep® tag" at the C-terminus are * 2402, HLA-B *0702 and HLA-C * The amino acid sequences of the 0702 molecule are shown in the attached sequence listing as SEQ ID NOs: 42, 43 and 44, while the amino acid sequence of β2 microglobulin (which together with the α chain representing the HLA-encoded molecule form the respective MHC I molecule) is shown in the attached sequence listing as SEQ ID NO: 45. Also, Figure 20F shows an exemplary flow cytometry analysis of CD62L and CD127 surface expression for HLA-B7 / hexon5 114-124 stimulated / expanded cells from Figure 20D after 14 days of culture. (Figure 20-2) See the description of Figure 20-1. (Figure 20-3) See the description of Figure 20-1. (Figure 20-4) See the description of Figure 20-1. FIG. 21 shows purified CD3+CD62L+CD45RA-T cells stimulated in vitro with a) antigen-specific peptide-MHC I complexes and b) αCD28 Fab fragments reversibly immobilized to soluble oligomeric streptavidin muteins as first and second agents. CM The kinetics of selective Ag-specific expansion from responder cells are shown. For this purpose, 500,000 CD3+CD62L+CD45RA- responder T cells were cultured. CM Cells (Tresp) were incubated with 0.5 μg of peptide:MHC class I complex with streptavidin-conjugated peptide (this particular peptide is amino acids 114-124 of the hexon 5 protein of adenovirus restricted by HLA-B0702: Ag-specific stimulation was performed with 3 μl of a preparation of streptactin multimerization reagent functionalized with the peptide (representing TIFF0007665579000013.tif4128; see above) and 0.5 μg of αCD28 Fab. As an alternative stimulation, 4.5 μl of the streptactin multimerization reagent preparation was loaded with 0.5 μg of this peptide:MHC class I complex, 0.5 μg of αCD8 Fab, and 0.5 μg of αCD28 Fab. For comparison, polyclonal stimulation was performed with 3 μl of a preparation of streptactin multimerization reagent (1 mg / ml) loaded with a combination of 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 Fab. Again, as an alternative stimulation condition, 4.5 μl of a preparation of streptactin multimers loaded with 0.5 μg of αCD3 Fab, 0.5 μg of αCD8 Fab, and 0.5 μg of αCD28 Fab was used. Untreated (unstimulated) Tresp cells served as a negative control, and Tresp cells polyclonally stimulated with anti-CD3 / anti-CD28 beads served as a positive control. Tresp cells were seeded in 48-well plates in 1 ml of cell culture medium supplemented with 30 U / ml IL-2 and 5 ng / ml IL-15. Cells were incubated at 37°C with medium changes every 3 days, and cell numbers were analyzed after 7 and 14 days. The photograph shown in Figure 21 depicts the extent of cluster formation on day 5 of Ag-specific stimulation, exemplified for the adenoviral HLA-B7 / hexon 5 epitope. (Figure 22) Figures 22A-B show activation of intracellular signaling cascades in transduced Jurkat cells engineered to express an αCD19 chimeric antigen receptor (CAR) stimulated with the oligomeric Strep-tactin® of Example 3 as a soluble multimerization reagent. The specificity of CARs is typically derived from the scFv region assembled from the antigen-binding region of a monoclonal antibody (mAb), which specifically binds to a target / tumor-associated antigen, such as CD19, and links it to T cell-specific signaling (as described in Hudecek et al., Clin Cancer Res. 2013 June 15; 19(12): 3153-3164). In this experiment, the extracellular domain (ECD) of CD19, which contains the natural ligand of αCD19 CAR, as well as a polyclonal αIgG F(ab)2 fragment (donkey anti-human F(ab)2 is commercially available from Jackson Immuno Research) that recognizes the IgG4 spacer within αCD19-CAR was used as the first agent to provide a primary activation signal to Jurkat cells. Reversible immobilization to a soluble oligomeric streptavidin mutein was performed using a streptavidin-binding peptide fused to the C-terminus of the ECD of CD19: (Because streptavidin mutein "m2" binds biotin with low affinity, this binding is reversible and can be displaced, for example, by the addition of excess free biotin.) In the control experiment shown in Figure 22A, 300,000 CD3+ Jurkat responder cells (Jresp) were stimulated with various amounts of a mixture of a preparation of oligomeric streptactin (1 mg / ml) functionalized with αCD3 Fab and αCD28 Fab ("x1" corresponds to 3 μg of multimerized streptactin functionalized with 0.5 μg of aCD3 Fab and 0.5 μg of aCD28 Fab—polyclonal multimerization agent). In the experiment shown in Figure 22B, 3 μl of an oligomeric streptactin preparation was functionalized with 0.5 μg (×1) or 1 μg (×2) of the extracellular domain (ECD) of CD19; or 3 μl of an oligomeric streptactin preparation was loaded with 0.5 μg (×1) or 1 μg (×2) of αIgG, which recognizes an IgG4 spacer (both of which are CAR-specific multimerizing agents). Anti-CD3 / anti-CD28 beads (beads on which αCD3 and αCD28 monoclonal antibodies were irreversibly immobilized) or Jresp cells stimulated with PMA and ionomycin were used as positive controls. Jresp cells were seeded in 200 μl of cell culture medium supplemented with 30 U / ml IL-2 in a 1.5 ml Eppendorf tube. Cells were incubated at 37°C, placed on ice, and lysed after 0 to 20 minutes of stimulation. Detection of phosphorylated ERK indicates active MAPK signaling, and staining for housekeeper β-actin indicates equal loading of total protein per condition and time point. DETAILED DESCRIPTION OF THE INVENTION

[0149] Detailed Description The present invention provides a method for incubating cells (culturing) and / or enriching or selecting cells from a cell composition. For example, the present invention provides a method for increasing, activating, promoting the survival or differentiation (or lack thereof) of target cells, and / or inducing various other effects in target cells; and / or for separating target cells from other cells and / or other components or enriching them compared to other components. In some aspects, the cell is a T cell, for example, a bulk T cell or a cell of a specific T cell subset.

[0150] All publications, including patent documents, scientific literature, and databases, referenced in this application are incorporated by reference in their entirety for all purposes, as if each individual publication were individually incorporated by reference. If the definitions set forth herein conflict or are otherwise inconsistent with the definitions set forth in the patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein shall take precedence over the definitions incorporated herein by reference. For example, the contents of International PCT Application No. PCT / EP2015 / 058339 are incorporated by reference in their entirety.

[0151] The section headings used herein are for editorial purposes only and should not be construed as limiting the subject matter described.

[0152] I. Overview of Methods, Systems, and Reagents Provided herein are methods for incubating (culturing) cells to expand, activate, promote the survival or differentiation (or lack thereof) of target cells, and / or induce various other effects in target cells. In some embodiments, the provided methods also include one or more other processing steps, including one or more of selection, isolation, enrichment, and / or selection of cells from a composition of cells and / or transduction of cells. In some embodiments, the provided methods are performed in the presence of a multimerized agent in which one or more binding agents (e.g., antibody fragments, e.g., Fabs) are reversibly bound to a multimerization reagent, such as an oligomeric streptavidin mutein reagent.

[0153] In some embodiments, the cell culture and / or concentration is carried out in whole or in part in the presence of a support, such as a stationary phase and / or solid support, and / or when the cells are present on the support. In some embodiments, the support is a chromatography matrix, and the incubation is carried out when the cells are in a column containing a stationary phase. In some embodiments, the method is carried out when the cells are immobilized, usually indirectly immobilized on a support, such as a column. The immobilization is typically reversible so that the cells can be removed from the support after incubation.

[0154] In some embodiments, the support provides the ability to enrich a cell-containing composition or select specific cells during, before, or after incubation. For example, in some embodiments, a solid support functionalized with an agent specific for one or more target cells (e.g., a T cell marker, e.g., CD3) is used, and desired T cells are contacted with the support, bound to the support, and indirectly immobilized. In some aspects, the target cells are stimulated while immobilized on the support, for example, by adding a multimerization reagent and / or through an agent bound to the support. Thus, in some aspects, the provided methods and other embodiments advantageously allow multiple processing steps (e.g., stimulation and selection) to be performed within the same container and / or closed system or systems, thereby providing improved efficiency and safety features. An exemplary embodiment is shown in FIG. 5.

[0155] In some embodiments, the method involves a reversible reagent system capable of binding to a molecule on the surface of a target cell, e.g., a reagent comprising a receptor-binding molecule, such as a stimulator, thereby providing a signal to the cell, which in some instances may be a primary activation signal. In some embodiments, the method employs a reagent that may be a multimerization reagent that has bound to it one or more agents, e.g., a first agent that provides a signal, such as a primary activation signal and / or an accessory or costimulatory signal, to the cell, a stimulator, such as a second agent. In some embodiments, the primary activation signal may be sufficient to activate the cell to expand / proliferate. The first agent may be bound to the multimerization reagent either reversibly or irreversibly. The multimerization reagent may also be bound to a second agent that stimulates an accessory molecule on the surface of the cell. Upon binding to the accessory molecule on the surface of the cell, the second agent may stimulate the thereby activated cell to expand. The second agent may also be bound to the multimerization reagent either reversibly or irreversibly. The multimerization reagent can be either immobilized on a solid support or soluble. In some embodiments, the multimerization reagent is on a support that is a stationary phase, and at least a portion of the incubation is carried out in a column containing this stationary phase.

[0156] In one aspect, the method disclosed herein is the serial expansion of a cell population, in which an entire lymphocyte population is stimulated / expanded, and the reagents required for expansion are then removed by chromatography on a suitable stationary phase. In some embodiments, the expanded / stimulated cells, which are cultured cells, can optionally be transfected with, for example, a T cell receptor or chimeric antigen receptor (CAR), and in some aspects, can be subjected to a second stimulation expansion using a different stimulatory molecule that binds to the introduced T cell receptor or chimeric antigen receptor.

[0157] Methods for expanding T cell populations in vitro in the absence or presence of small amounts of exogenous growth factors are known in the art (see, e.g., U.S. Patent No. 6,352,694 B1 and European Patent No. EP0 700 430 B1). Generally, such methods use solid surfaces of greater than 1 μM to which various binding agents (e.g., anti-CD3 and / or anti-CD28 antibodies) are immobilized. For example, Dynabeads® CD3 / CD28 (Invitrogen) is a commercially available T cell expansion reagent that is a uniform, 4.5 μm, superparamagnetic, sterile, non-pyrogenic polystyrene bead coated with a mixture of affinity-purified monoclonal antibodies directed against CD3 and CD28 cell surface molecules on human T cells. However, in some instances, such magnetic beads are difficult to incorporate into cell expansion methods because, for example, under conditions required for clinical trials or treatment, it must be ensured that these magnetic beads are completely removed before administering the expanded T cells to a patient.

[0158] In some embodiments, the methods provided herein address these issues. In some aspects, the provided reagents are reversible, allowing the stimulatory substance to be removed from the cell composition. Also, in some aspects, the reagent, e.g., the multimerization reagent to which the stimulatory substance is bound, is not immobilized on a support, e.g., a solid support or surface. Thus, in some aspects, the reagent, e.g., the multimerization reagent, is flexible and not rigid. In some embodiments, the reagent can adapt or conform to the cell surface. In some embodiments, the reagent can be immobilized on a support, e.g., a solid support, that includes a stationary phase. In some embodiments, such methods can be used with selection agents, where similar selection agents are used that can select one or more target cells and expose them to the stimulatory substance simultaneously or sequentially. Thus, in some aspects, selection and isolation in combination with stimulation can bias the stimulation of specific cells or cell subsets.

[0159] In some embodiments, the provided methods include culturing a composition of cells, e.g., contacting the composition of cells with a reagent, e.g., a multimerization reagent coupled to one or more receptor-binding agents (e.g., stimulatory agents) (see, e.g., Figures 4A and 4B). In some embodiments, the cell composition is contacted with the multimerization reagent, and typically, after incubating the cell population with the multimerization reagent, the population of cells forms a complex / binds to the multimerization reagent through the first agent. Other cell populations contained in the starting sample that lack a particular cell surface molecule do not bind to the multimerization reagent. In this regard, it should be noted that a cell population typically has multiple copies of a cell surface molecule on its surface, and binding of these multiple copies is typically required for stimulation or activation.

[0160] Thus, a multimerization reagent typically provides two or more binding sites, e.g., Z1, to which multiple agents can be reversibly bound to present a first agent, a second agent, and / or other agents to a cell population at sufficient density. In this regard, a multimerization reagent may have multiple binding sites, e.g., Z1; for example, a streptavidin mutein (which is a homotetramer) has four such binding sites, e.g., Z1, in its native state and may further oligomerize. In some instances, a reagent may have only one binding site, e.g., Z1, for reversible binding of a binding partner such as C1. An example of such a reagent is multimeric calmodulin. Calmodulin has only one binding site for a calmodulin-binding peptide. However, calmodulin can be biotinylated and then reacted with streptavidin oligomers (see also below), thereby providing a multimerization reagent in which multiple calmodulin molecules are present at high density on a "scaffold," thereby providing multimeric calmodulin.

[0161] In some embodiments, after incubation or at other appropriate time points when it is desired to discontinue stimulation, the binding between the reversibly bound binding partner C, e.g., C1, of the agent and the binding site Z, e.g., Z1, of the multimerization reagent is disrupted by disrupting the respective reversible bonds. In some examples, disruption can be achieved by adding a competitor to an incubation / reaction mixture containing a population of cells bound to the multimerization reagent. In the case of competitive disruption of the reversible binding between the reversibly bound binding partner C, e.g., C1, of the agent and the binding site Z, e.g., Z1, of the multimerization reagent (which can also be understood as competitive elution), the incubation mixture / cell population can be contacted with free first binding partner C, e.g., C1, or with an analog of the first binding partner C that can disrupt the binding between the first binding partner and the binding site Z, e.g., Z1. In the example of binding partner C, e.g., C1, which is a streptavidin-binding peptide that binds to the biotin-binding site of streptavidin, the first free partner can be the corresponding free streptavidin-binding peptide or a competitively binding analogue, such as biotin or a biotin derivative, e.g., desthiobiotin.

[0162] In some embodiments, addition of the free partner or an analog thereof removes the binding partner C, e.g., C1, from the multimerization reagent, and because the binding partner is included in the reversibly bound agent, removal of such agent from the multimerization reagent is achieved. This agent removal then occurs particularly when the binding affinity of the binding between the first agent and the cell surface receptor is 10. -2 M~10 -13 Dissociation constants (K D ) and is also reversible, dissociating the first agent from the cell surface molecule. In some aspects, this dissociation also terminates stimulation of the cell population.

[0163] In some embodiments, the binding affinity of antibody molecules to their antigens, including, for example, cell surface receptor molecules, is typically less than K D 10 -7 M ~ about 10 -13 The affinity range is M. Thus, conventional monoclonal antibodies can be used as agents (primary or secondary receptor-binding agents, e.g., stimulatory or selective agents). In some embodiments, monoclonal antibodies can also be used in the form of their monovalent antibody fragments, e.g., Fab fragments or single-chain Fv fragments, to avoid unwanted avidity effects that result in stronger binding.

[0164] Thus, the provided methods have the advantage of allowing precise control over the duration of stimulation or expansion of a cell population, as well as precise control over the functional status of the cell population. In some aspects, brief activation of cells, e.g., T cells, by adding a substance, e.g., a competitor (e.g., biotin or an analog), within 5 days of stimulation, expands or stimulates the cells but alters one or more characteristics or features of the cells, e.g., the percentage of CD4 or CD8 T cells, the CD8 / CD4 ratio, phenotypic markers, and / or differentiation status. For example, in some aspects, the ability to temporally control signals using embodiments of the provided reagents expands less differentiated, long-lived T cell populations, e.g., long-lived memory T cells. In some instances, temporal control of signals, e.g., by disrupting binding of a multimerization reagent with a competitor, can be used to adjust or regulate the relative expansion and / or maintenance of certain subpopulations compared to others (e.g., CD4+ vs. CD8+).

[0165] In some embodiments, by dissociating the reversibly bound agent or agents from the cell surface molecule, the provided method has the additional advantage that the stimulated cell population does not contain the stimulating agent at the end of the stimulation period. Also, in some embodiments, all other reagents used in this method, i.e., the agent (e.g., the first or second receptor-binding agent, e.g., the stimulating agent or selecting agent), and the competitive reagent of binding partner C, such as C1, or its analog, can be easily removed from the stimulated cell population through a "removal cartridge" (e.g., as described in International Patent Application WO 2013 / 124474). For example, in some cases where the multimerization reagent is immobilized on a solid support, such as a bioreactor surface or magnetic beads, it is not used. Therefore, using a removal cartridge to remove the free agent and the competitive reagent can include loading the elution sample (e.g., the sample obtained after disrupting the reversible binding) into a second chromatography column.

[0166] In some embodiments, the chromatography column has a suitable stationary phase that can simultaneously function as both an affinity chromatography matrix and a gel permeation matrix. In some aspects, an affinity reagent is immobilized on the affinity chromatography matrix. In some embodiments, the affinity reagent can be, for example, streptavidin, streptavidin mutein, avidin, avidin mutein, or a mixture thereof. In some embodiments, an agent (e.g., a first or second receptor-binding agent, e.g., a stimulator or selector), a competitor of binding partner C, C1, binds to the affinity reagent and is thereby immobilized on the chromatography matrix. As a result, the eluted sample containing the isolated and expanded cell population is free of the agent (e.g., a first or second receptor-binding agent, e.g., a stimulator or selector) and the competitor. In some embodiments, the cultured composition is free of any reactants, which in some aspects is advantageous for use in connection with diagnostic applications (e.g., further FACS™ sorting) or any cell-based therapeutic application.

[0167] In some embodiments, the ability to remove reagents and other components from the composition has the added advantage of avoiding any solid support, such as magnetic beads. In some embodiments, this means that there is no or minimal risk of contaminating the activated T cells with such magnetic beads. In some embodiments, this also means that a process that meets GMP standards can be more easily constructed compared to other methods, such as the use of Dynabeads®, where additional measures must be taken to ensure that the final expanded T cell population is free of magnetic beads. Furthermore, in some embodiments, the use of a soluble multimerizing agent makes it easier to remove it from the activated cell population (T cells, B cells, or natural killer cells), since the cells can be simply sedimented by centrifugation and the supernatant containing the soluble multimerizing agent can be discarded. Alternatively, the soluble multimerizing agent can be removed from the expanded cell population in a gel-permeable matrix of a removal cartridge, such as those described above (e.g., International Patent Application Publication No. WO 2013 / 124474). In some embodiments, for example, in embodiments where a solid phase (e.g., magnetic beads) is not present, an automated closed system for cell expansion is also provided that can be incorporated into known cell expansion systems, such as the Xuri Cell Expansion System W25 and WAVE Bioreactor 2 / 10 System available from GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom) or the Quantum® Cell Expansion System available from TerumoBCT Inc. (Lakewood, CO, USA). In some embodiments, when a solid phase and / or stationary phase is used, the fact that stimulation is performed in the presence of the stationary phase or that cells can be stimulated in an immobilized state allows the entire selection and / or stimulation process to be carried out in a sterile or closed system vessel, such as a column.

[0168] In some aspects, the methods provided herein can include a cell population having at least two specific cell surface molecules. In some embodiments, the first cell surface molecule is involved in a primary activation signal for the cell population, and the second cell surface molecule is an accessory molecule on the cell surface that is involved in providing a stimulus to the cell. In some embodiments, the cell population is contacted with a multimerization reagent reversibly linked to a first agent that provides a primary activation signal to the cell and a second agent that induces or modulates an additional signal, e.g., stimulates an accessory molecule on the cell surface. The cell population can be, for example, a T cell population in which the cell surface molecule is a TCR / CD3 complex and the cell surface molecule is the accessory molecule CD28. Furthermore, as described herein, targeting other accessory molecules, such as one or more of CD90 (Thy-1), CD95 (Apo- / Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, and HVEM, is also contemplated. In some aspects, stimulation through such other accessory molecules increases less differentiated, and in some instances, longer-lived populations of T cells, e.g., long-lived memory T cells, compared to traditional stimulation through CD28. In some embodiments, both binding of the TCR / CD3 complex as a primary activation signal and binding of an accessory molecule (e.g., CD28 or other accessory molecule) may be required for T cell expansion / proliferation.

[0169] In some embodiments, the multimerization reagent comprises at least one binding site Z, e.g., Z1, for reversible binding of a first agent, and the first agent also comprises at least one binding partner C, e.g., C1, wherein the binding partner C, e.g., C1, can reversibly bind to the binding site Z, e.g., Z1, of the multimerization reagent. Thus, upon contact with or incubation with the multimerization reagent, the first agent can be reversibly bound to the multimerization reagent through a reversible bond formed between the binding partner C, e.g., C1, and the binding site Z, e.g., Z1. In addition, the second agent can comprise a binding partner C, e.g., C2, wherein the binding partner C2 can reversibly bind to the binding site Z, e.g., Z2, of the multimerization reagent. In some embodiments, upon contact with or incubation with a multimerization reagent, the second agent is reversibly bound to the multimerization reagent through a reversible bond formed between a binding partner C, e.g., C1, and a binding site Z, e.g., Z2. In some examples, C1 and C2 may be the same or substantially the same and / or may comprise the same or substantially the same moiety. In some examples, Z1 and Z2 may be the same or substantially the same and / or may comprise the same or substantially the same moiety.

[0170] In some embodiments, when used as binding partners C1 and C2, moieties that bind to the same binding site of a multimerization reagent have the advantage that the same competitive reagent (that is a competitive reagent for the first binding partner C1 and also a competitive reagent for the second binding partner C2), or an analog thereof, can be used to prevent, and in some instances, stop, the expansion of a population of target cells (e.g., T cells) and to release this population of target cells (e.g., T cells) from the multimerization reagent.

[0171] In some examples of generating a binding agent (e.g., a first or second receptor-binding agent, e.g., a stimulatory or selection agent) comprising a binding partner C, the binding partner C, e.g., C1 or C2, can be provided by the respective expression vector used for recombinant production of the agent (e.g., an antibody fragment), such that the binding partner C, e.g., C1 or C2, is part of a fusion peptide that includes the agent at either its N-terminus or C-terminus. In some embodiments, in the case of an agent that is an antibody or antigen-binding fragment, the binding partner C, e.g., C1 or C2, can be present at the C-terminus of either the light or heavy chain. This method of cloning the variable domains of a recombinant protein, e.g., an antibody molecule, and recombinantly producing the respective protein, e.g., an antibody fragment, is also known to those skilled in the art; see, e.g., Skerra, A. (1994). In some embodiments, antibody molecules are generated using, for example, well-known evolutionary methods, such as phage display (e.g., Kay, BK et al. (1996) Phage Display of Peptides and Proteins - A Laboratory Manual, 1999). st Ed., Academic Press, New York NY; Lowman, HB (1997) Annu. Rev. Biophys. Biomol. Struct. 26, 401-424 or Rodi, DJ, and Makowski, L. (1999) Curr. Opin. Biotechnol. 10, 87-93), ribosome display (reviewed in Amstutz, P. et al. (2001) Curr. Opin. Biotechnol. 12, 400-405) or mRNA display as reported in Wilson, DS et al. (2001) Proc. Natl. Acad. Sci. USA 98, 3750-3755 can be used to generate artificial binding molecules with antibody-like properties against specific targets, such as CD3 or CD28 as described, or other accessory or stimulatory molecules.

[0172] II. Reversible Reagent Systems and Related Uses In some embodiments, the method uses a reversible system in which at least one agent (e.g., a receptor-binding agent or a selection agent) capable of binding to a molecule on the surface of a cell (a cell surface molecule) is reversibly bound to a reagent. In some examples, the reagent comprises multiple binding sites capable of reversibly binding to the agent (e.g., a receptor-binding agent or a selection agent). In some examples, the reagent is a multimerization reagent. In some embodiments, at least one agent (e.g., a receptor-binding agent or a selection agent) comprises at least one binding site B capable of specifically binding to an epitope or region of the molecule, and also comprises a binding partner C that specifically binds to at least one binding site Z of the reagent. In some examples, the binding interaction between binding partner C and at least one binding site Z is a non-covalent interaction. In some embodiments, the binding interaction, e.g., a non-covalent interaction, between binding partner C and at least one binding site Z is reversible.

[0173] In some embodiments, reversible binding can be performed in the presence of a substance, such as a competing reagent (also referred to as an elution reagent), which is also a binding site or contains such a binding site capable of binding to at least one binding site Z. Typically, this substance (e.g., a competing reagent) acts as a competitor by virtue of its high binding affinity for binding site Z present in the reagent and / or by being present at a higher concentration than binding partner C, thereby separating and / or dissociating binding partner C from the reagent. In some embodiments, the affinity of the substance (e.g., competing reagent) for at least one binding site Z is higher than the affinity of binding partner C of the agent (e.g., receptor-binding agent or selection agent) for at least one binding site Z. Thus, in some examples, binding between binding site Z of the reagent and binding partner C of the agent (e.g., receptor-binding agent or selection agent) can be disrupted by the addition of a substance (e.g., competing reagent), thereby rendering binding between the agent (e.g., receptor-binding agent or selection agent) and the reagent reversible.

[0174] The reagent that can be used in such reversible system has been reported and known in the art, for example, see U.S. Patent No. 5,168,049; U.S. Patent No. 5,506,121; U.S. Patent No. 6,103,493; U.S. Patent No. 7,776,562; U.S. Patent No. 7,981,632; U.S. Patent No. 8,298,782; U.S. Patent No. 8,735,540; U.S. Patent No. 9,023,604; and International PCT Publication No. WO2013 / 124474 and WO2014 / 076277.Non-limiting examples of reagent and binding partner that can form reversible interaction and the substance (for example, competitive reagent) that can reverse such binding are described below.

[0175] A. Reagents In some embodiments, the reagent comprises one or more binding moieties Z that can reversibly bind to a binding partner C included in the agent (e.g., receptor binding agent or selection agent). In some embodiments, the reagent comprises multiple binding moieties Z, each capable of specifically binding to a binding partner C included in the agent (e.g., receptor binding agent or selection agent), such that the reagent is capable of reversibly binding to multiple agents (e.g., receptor binding agent or selection agent), e.g., a multimer-forming reagent. In some embodiments, the reagent is an oligomer or polymer of individual molecules (e.g., monomers), or a complex (e.g., tetramer) that forms individual molecules each comprising at least one binding moiety Z. In some embodiments, the reagent comprises at least two binding sites Z, at least three binding sites Z, at least four binding sites Z, e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72 or more binding sites Z. The binding sites may all be the same, or the multiple binding sites may comprise one or more different binding sites (e.g., Z1, Z2, Z3, etc.).

[0176] In some embodiments, two or more agents (e.g., receptor binding agents or selection agents) bind, e.g., reversibly bind, to a reagent, e.g., through one or more binding sites Z present on the reagent. In some instances, this positions the agents (e.g., receptor binding agents or selection agents) in close proximity to one another, such that an avidity effect can occur when a target cell having (at least two copies of) a cell surface molecule comes into contact with an agent (e.g., receptor binding agent or selection agent) having one or more binding sites B that can bind to a particular molecule.

[0177] In some embodiments, two or more different agents (e.g., receptor binding agents or selection agents) that are the same, i.e., contain the same binding site B, can be reversibly bound to the reagent. In some embodiments, at least two different (types) of agents (e.g., receptor binding agents or selection agents) can be used, and in some instances, three or four different (types) of agents, e.g., two or more different receptor binding agents and / or selection agents, can be used. For example, in some embodiments, the reagent can be reversibly bound to a first agent (e.g., receptor binding agent or selection agent) that contains binding site B1, B2, B3, or B4, etc., and a second agent (e.g., receptor binding agent or selection agent) that contains another binding site, e.g., one different from binding site B1, B2, B3, or B4. In some instances, the binding sites of the first agent and the second agent can be the same. For example, in some aspects, each of the at least two agents (e.g., receptor binding agents or selection agents) can bind to the same molecule. In some examples, the binding sites of the first agent and the second agent may be different. In some aspects, each of at least two agents (e.g., receptor binding agents or selection agents) may bind to a different molecule, e.g., the first molecule, the second molecule, etc. In some examples, the different molecules, e.g., cell surface molecules, may be present on the same target cell. In other examples, the different molecules, e.g., cell surface molecules, may be present on different target cells present within the same cell population. In some examples, a third, fourth, etc. agent (e.g., receptor binding agent or selection agent), each comprising an additional different binding site, may be bound to the same reagent.

[0178] In some embodiments, two or more different agents (e.g., receptor binding agents or selection agents) comprise the same binding partner C. In some embodiments, two or more different agents (e.g., receptor binding agents or selection agents) comprise different binding partners. In some aspects, a first agent (e.g., receptor binding agent or selection agent) may have a binding partner C1 that can specifically bind to binding site Z1 present on the reagent, and a second agent (e.g., receptor binding agent or selection agent) may have a binding partner C2 that can specifically bind to binding site Z1 or binding site Z2 present on the reagent. Thus, in some examples, multiple binding sites Z comprised in a reagent include binding sites Z1 and Z2, respectively, that can reversibly bind to binding partners C1 and C2 comprised in an agent (e.g., receptor binding agent or selection agent). In some embodiments, C1 and C2 are the same, and / or Z1 and Z2 are the same. In other aspects, one or more of the multiple binding sites Z may be different. In other examples, one or more of the multiple binding partners C may be different. It is within the skill of one of ordinary skill in the art to select any combination of different binding partners C that are compatible with a reagent comprising binding site Z, as long as each of the binding partners C is capable of interacting with, e.g., specifically binding to, one of the binding sites Z.

[0179] In some embodiments, the reagent is streptavidin, a streptavidin mutein or analog, avidin, an avidin mutein or analog (e.g., neutravidin), or a mixture thereof, and such reagent comprises one or more binding sites Z for reversible binding with a binding partner C. In some embodiments, the binding partner C can be biotin, a biotin derivative or analog, or a streptavidin-binding peptide or other molecule capable of specifically binding to streptavidin, a streptavidin mutein or analog, avidin, or an avidin mutein or analog. In some embodiments, the reagent is or comprises streptavidin, avidin, an analog or mutein of streptavidin, or an analog or mutein or avidin that reversibly binds to biotin, a biotin analog, or a biologically active fragment thereof. In some embodiments, the reagent is or comprises a streptavidin analog or mutein or an avidin analog or mutein that reversibly binds to a streptavidin-binding peptide. In some embodiments, the substance (e.g., a competitive reagent) can be biotin, a biotin derivative or analog, or a streptavidin-binding peptide that can compete with binding partner C for binding to one or more binding sites Z. In some embodiments, binding partner C and the substance (e.g., a competitive reagent) are different, and the substance (e.g., a competitive reagent) exhibits a higher binding affinity for one or more binding sites Z compared to the affinity of the binding partner.

[0180] In some embodiments, streptavidin can be wild-type streptavidin, streptavidin muteins, or analogs, such as streptavidin-like polypeptides. Similarly, avidin in some aspects includes wild-type avidin or avidin muteins or analogs, such as neutravidin, which is a deglycosylated avidin with modified arginines that typically exhibit a more neutral pi and can be used as a substitute for native avidin. Typically, deglycosylated neutral forms of avidin include commercially available forms, such as "Extravidin" available from Sigma-Aldrich or "NeutrAvidin" available from Thermo Scientific or Invitrogen.

[0181] In some embodiments, the reagent is streptavidin or a streptavidin mutein or analog. In some embodiments, wild-type streptavidin (wt-streptavidin) has the amino acid sequence (SEQ ID NO: 1) disclosed in Argarana et al., Nucleic Acids Res. 14 (1986) 1871-1882. Generally, streptavidin naturally exists as a tetramer of four identical subunits, each containing a single binding site for biotin, a biotin derivative or analog, or a biotin mimetic, i.e., a homotetramer. An exemplary sequence of a streptavidin subunit is the amino acid sequence shown in SEQ ID NO: 1, although such a sequence may also include sequences present in its homologs from other Streptomyces species. In particular, each subunit of streptavidin contains approximately 10 -14 The equilibrium dissociation constant (K D) can exhibit strong binding affinity for biotin. In some examples, streptavidin can exist as a monovalent tetramer in which only one of the four binding sites is functional (Howarth et al. (2006) Nat. Methods, 3:267-73; Zhang et al. (2015) Biochem. Biophys. Res. Commun., 463:1059-63), a bivalent tetramer in which two of the four binding sites are functional (Fairhead et al. (2013) J. Mol. Biol., 426:199-214), or in a monomeric or dimeric form (Wu et al. (2005) J. Biol. Chem., 280:23225-31; Lim et al. (2010) Biochemistry, 50:8682-91).

[0182] In some embodiments, streptavidin can be in any form, e.g., wild-type or unmodified streptavidin, e.g., streptavidin from a Streptomyces species, including at least one functional subunit comprising a binding site for biotin, a biotin derivative or analog, or a biotin mimetic, such as at least one functional subunit of wild-type streptavidin from Streptomyces avidinii, generally set forth in SEQ ID NO:1, or a functionally active fragment thereof. For example, in some embodiments, streptavidin can include fragments of wild-type streptavidin truncated at the N- and / or C-terminus. Such minimal streptavidins include any that begin at the N-terminus in the region of amino acid positions 10-16 of SEQ ID NO:1 and end at the C-terminus in the region of amino acid positions 133-142 of SEQ ID NO:1. In some embodiments, a functionally active fragment of streptavidin comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, streptavidin, such as that set forth in SEQ ID NO: 2, can further comprise an N-terminal methionine at a position corresponding to Ala13 in the numbering set forth in SEQ ID NO: 1. References to residue positions in streptavidin or streptavidin muteins refer to the residue numbering in SEQ ID NO: 1.

[0183] In some aspects, streptavidin muteins comprise polypeptides that are distinguished from the sequence of unmodified or wild-type streptavidin by one or more amino acid substitutions, deletions, or additions, but that contain at least one functional subunit containing a binding site for biotin, a biotin derivative or analog, or a streptavidin-binding peptide. In some aspects, streptavidin-like polypeptides and streptavidin muteins can be essentially immunologically equivalent to wild-type streptavidin, and in particular polypeptides that can bind to biotin, a biotin derivative, or a biotin analog with the same or different affinity as wt-streptavidin. In some examples, streptavidin-like polypeptides or streptavidin muteins can contain amino acids that are not part of wild-type streptavidin, or they can contain only a portion of wild-type streptavidin. In some embodiments, the streptavidin-like polypeptide is a polypeptide that is not identical to wild-type streptavidin because the host lacks the enzyme required to convert the polypeptide produced by the host into the structure of wild-type streptavidin. In some embodiments, streptavidin can also exist as a streptavidin tetramer and a streptavidin dimer, particularly a streptavidin homotetramer, a streptavidin homodimer, a streptavidin heterotetramer, and a streptavidin heterodimer. Generally, each subunit usually has a binding site for biotin or a biotin analog or a streptavidin-binding peptide. Examples of streptavidin or streptavidin muteins are mentioned, for example, in WO 86 / 02077, DE 19641876 A1, US Pat. No. 6,022,951, WO 98 / 40396 or WO 96 / 24606.

[0184] In some embodiments, a streptavidin mutein can include amino acids that are not part of unmodified or wild-type streptavidin or can include only a portion of wild-type or unmodified streptavidin. In some embodiments, a streptavidin mutein includes at least one subunit that can have one or more amino acid substitutions (replacements) compared to a subunit of unmodified or wild-type streptavidin, e.g., compared to the wild-type streptavidin subunit shown in SEQ ID NO:1 or a functionally active fragment thereof, e.g., shown in SEQ ID NO:2. In some embodiments, at least one subunit of the streptavidin mutein can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid differences compared to wild-type or unmodified streptavidin, and / or comprises at least one subunit comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2, and such streptavidin muteins exhibit functional activity of binding to biotin, a biotin derivative or analog, or a biotin mimetic. In some embodiments, the amino acid replacements (substitutions) are conservative or non-conservative mutations. Examples of streptavidin muteins are known in the art, see, e.g., U.S. Patent Nos. 5,168,049; 5,506,121; 6,022,951; 6,156,493; 6,165,750; 6,103,493; or 6,368,813; or International Published PCT Application No. WO2014 / 076277.

[0185] In some embodiments, streptavidin or streptavidin muteins comprise proteins comprising one or more functional subunits, e.g., two or more, three or more, four or more, and in some examples, five, six, seven, eight, nine, ten, eleven, twelve or more functional subunits, comprising one or more binding sites Z for biotin, a biotin derivative or analog, or a streptavidin-binding peptide. In some embodiments, streptavidin or streptavidin muteins may comprise monomers; dimers, including heterodimers or homodimers; tetramers, including homotetramers, heterotetramers, monovalent tetramers or divalent tetramers, or higher order multimers or oligomers thereof.

[0186] In some embodiments, the binding affinity of the streptavidin or streptavidin mutein for the peptide ligand binding partner is 1 x 10 -4 Medium, 5 x 10 -4 Medium, 1 x 10 -5 M, 5x 10 -5 Medium, 1 x 10 -6 Medium, 5 x 10 -6 M or 1 x 10 -7 Less than m, but usually 1 x 10 -13 Medium, 1 x 10 -12 M or 1 x 10 -11 For example, a peptide sequence (Strep-tag), disclosed in, for example, U.S. Pat. No. 5,506,121, functions as a biotin mimic, e.g., approximately 10 -4 M to 10 -5 K between M D The binding affinity of the streptavidin molecule can be further improved by introducing mutations into the streptavidin molecule, see, for example, U.S. Patent No. 6,103,493 or International PCT Publication No. WO2014 / 076277. In some embodiments, the binding affinity can be determined by methods known in the art, such as any of those described below.

[0187] In some embodiments, the reagent, e.g., streptavidin or streptavidin mutein, exhibits binding affinity for a peptide ligand binding partner, which may be binding partner C present in the agent (e.g., receptor binding agent or selection agent). In some embodiments, the peptide sequence comprises a sequence having the general formula set forth in SEQ ID NO:9, e.g., the sequence set forth in SEQ ID NO:10. In some embodiments, the peptide sequence has the general formula set forth in SEQ ID NO:11, e.g., the general formula set forth in SEQ ID NO:12. In one example, the peptide sequence is TIFF0007665579000015.tif4128 (also known as Strep-tag® and shown in SEQ ID NO:7). In one example, the peptide sequence is TIFF0007665579000016.tif4128 (also known as Strep-tag® II and set forth in SEQ ID NO:8). In some embodiments, the peptide ligand comprises a consecutive arrangement of at least two streptavidin binding modules, the distance between the two modules being at least 0 and no more than 50 amino acids, one binding module having 3-8 amino acids and comprising at least the sequence His-Pro-Xaa (SEQ ID NO:9), where Xaa is glutamine, asparagine, or methionine, and the other binding molecule having the same or a different streptavidin peptide ligand, e.g., set forth in SEQ ID NO:11 (see, e.g., International Publication No. WO 02 / 077018; U.S. Patent No. 7,981,632). In some embodiments, the peptide ligand comprises a sequence having a formula set forth in either SEQ ID NO:13 or 14. In some embodiments, the peptide ligand has an amino acid sequence set forth in any of SEQ ID NOs: 15-19.

[0188] In some embodiments, the reagent is or comprises a streptavidin mutein. In some embodiments, the streptavidin mutein comprises one or more mutations (e.g., amino acid substitutions) compared to wild-type streptavidin set forth in SEQ ID NO:1, or a biologically active portion thereof. For example, a biologically active portion of streptavidin may comprise an N- and / or C-terminally truncated streptavidin variant, in some instances referred to as minimal streptavidin. In some embodiments, the N-terminally truncated minimal streptavidin into which any mutations may be introduced begins at amino acid positions 10-16 at the N-terminus and ends at amino acid positions 133-142 at the C-terminus, compared to the sequence set forth in SEQ ID NO:1. In some embodiments, the N-terminally truncated streptavidin into which any mutations may be introduced comprises the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the minimal streptavidin comprises the amino acid sequence from Ala13 to Ser139, optionally with an N-terminal methionine residue in place of Ala13. For purposes of this application, the numbering of amino acid positions refers throughout to that of wt-streptavidin as shown in SEQ ID NO:1. (See Argarana et al., Nucleic Acids Res. 14 (1986), 1871-1882; see also Figure 3).

[0189] In some embodiments, the streptavidin mutein is a variant described in U.S. Patent No. 6,103,493. In some embodiments, the streptavidin mutein comprises at least one mutation within the region of amino acid positions 44-53 based on the amino acid sequence of wild-type streptavidin, e.g., as set forth in SEQ ID NO:1. In some embodiments, the streptavidin mutein comprises a mutation at one or more residues 44, 45, 46, and / or 47. In some embodiments, the streptavidin mutein comprises a substitution of Glu at position 44 of wild-type streptavidin with a hydrophobic aliphatic amino acid, e.g., Val, Ala, Ile, or Leu, any amino acid at position 45, an aliphatic amino acid, e.g., a hydrophobic aliphatic amino acid, and / or Val at position 47 with a basic amino acid, e.g., Arg or Lys, e.g., typically Arg. In some embodiments, Ala is at position 46 and / or Arg is at position 47 and / or Val or Ile is at position 44. In some embodiments, the streptavidin mutant comprises a streptavidin mutein comprising, for example, the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4, which is a sequence encoding residues Val, ... 44 -Thr 45 -Ala 46 -Arg 47 In some embodiments, the streptavidin mutein comprises residue Ile, as shown in exemplary streptavidin muteins (also known as SAM2) including, for example, the amino acid sequence set forth in SEQ ID NO:5 or 6. 44 -Gly 45 -Ala 46 -Arg 47 In some instances, such streptavidin muteins are described, for example, in U.S. Patent No. 6,103,493 and are commercially available under the trademark Strep-Tactin®.

[0190] In some embodiments, the streptavidin mutein is a variant described in International Published PCT Application No. WO 2014 / 076277. In some embodiments, the streptavidin mutein contains at least two cysteine ​​residues in the region of amino acid positions 44-53, as set forth in SEQ ID NO:1. In some embodiments, cysteine ​​residues are present at positions 45 and 52 to form disulfide bridges linking these amino acids. In such embodiments, amino acid 44 is typically glycine or alanine, amino acid 46 is typically alanine or glycine, and amino acid 47 is typically arginine. In some embodiments, the streptavidin mutein contains at least one mutation or amino acid difference in the region of amino acid residues 115-121, as set forth in SEQ ID NO:1. In some embodiments, the streptavidin mutein comprises at least one mutation at amino acid positions 117, 120, and 121 and / or a deletion of amino acid positions 118 and 119 and a substitution of at least amino acid 121.

[0191] In some embodiments, the streptavidin mutein comprises a mutation at position 117, which can be a large hydrophobic residue such as Trp, Tyr, or Phe, or a charged residue such as Glu, Asp, or Arg, or a hydrophilic residue such as Asn or Gin, or in some examples, the hydrophobic residues Leu, Met, or Ala, or the polar residues Thr, Ser, or His. In some embodiments, the mutation at position 117 is combined with a mutation at position 120, which can be a small residue such as Ser, Ala, or Gly, and a mutation at position 121, which can be a bulky hydrophobic residue such as Trp, Tyr, or Phe. In some embodiments, the mutation at position 117 is combined with a mutation at position corresponding to position 120 of wild-type streptavidin or a biologically active fragment thereof shown in SEQ ID NO:1, which may be a hydrophobic residue, e.g., Leu, Ile, Met, or Val, or usually Tyr or Phe, and a mutation at position corresponding to position 121 compared to wild-type streptavidin or a biologically active fragment thereof shown in SEQ ID NO:1, which may be a small residue such as Gly, Ala, or Ser, or a hydrophobic residue such as Gln, or Leu, Val, Ile, Trp, Tyr, Phe, or Met. In some embodiments, such muteins may also include residues Val44-Thr45-Ala46-Arg47 or residues Ile44-Gly45-Ala46-Arg47. In some embodiments, the streptavidin mutein comprises residues Val44, Thr45, Ala46, Arg47, Glu117, Gly120 and Tyr121.In some embodiments, the mutein streptavidin comprises the amino acid sequence set forth in SEQ ID NO:27 or SEQ ID NO:28, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:27 or SEQ ID NO:28, and comprises residues Val44, Thr45, Ala46, Arg47, Glu117, Gly120, and Tyr121, and exhibits functional activity of binding to biotin, a biotin analog, or a streptavidin-binding peptide.

[0192] In some embodiments, the streptavidin mutein may comprise any of the above mutations in any combination, and the resulting streptavidin mutein may be a peptide ligand ( TIFF0007665579000017.tif4128; also known as Strep-tag® and shown in SEQ ID NO: 7) -4 M and / or peptide ligands ( TIFF0007665579000018.tif4128; also known as Strep-tag® II and shown in SEQ ID NO: 8) -4 Less than 1 M and / or 1 x 10 for any of the peptide ligands shown in any of SEQ ID NOs: 7-19 -4 Medium, 5 x 10 -4 Medium, 1 x 10 -5 M, 5x 10 -5 Medium, 1 x 10 -6 Medium, 5 x 10 -6 M or 1 x 10 -7 Less than M but usually 1 x 10 -13 Medium, 1 x 10 -12 M or 1 x 10 -11 M.

[0193] In some embodiments, the streptavidin mutein exhibits an amino acid sequence set forth in any of SEQ ID NOs: 3-6, 27, or 28, or an amino acid sequence exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 3-6, 27, or 28, and is a peptide ligand ( TIFF0007665579000019.tif4128; also known as Strep-tag® and shown in SEQ ID NO: 7) -4 M and / or peptide ligands ( TIFF0007665579000020.tif4128; also known as Strep-tag® II and shown in SEQ ID NO: 8) -4 Less than 1 M and / or 1 x 10 for any of the peptide ligands shown in any of SEQ ID NOs: 7-19 -4 Medium, 5 x 10 -4 Medium, 1 x 10 -5 M, 5x 10 -5 Medium, 1 x 10 -6 Medium, 5 x 10 -6 M or 1 x 10 -7 Less than M but usually 1 x 10 -13 Medium, 1 x 10 -12 M or 1 x 10 -11 It exhibits a binding affinity greater than M.

[0194] In some embodiments, the streptavidin mutein also exhibits binding to other streptavidin ligands, such as, but not limited to, biotin, iminobiotin, lipoic acid, desthiobiotin, diaminobiotin, HABA (hydroxyazobenzenebenzoic acid), and / or dimethyl HABA. In some embodiments, the streptavidin mutein exhibits binding affinity to another streptavidin ligand, such as biotin or desthiobiotin, that is greater than the binding affinity of the streptavidin mutein to a biotin-mimetic peptide ligand, such as that set forth in any of SEQ ID NOs:7-19. Thus, in some embodiments, biotin or a biotin analog or derivative (e.g., desthiobiotin) can be used as a competitive reagent in the provided methods. For example, the interaction between a mutein streptavidin designated Strep-tactin® (e.g., comprising the sequence shown in SEQ ID NO:4) and a peptide ligand designated Strep-tag® II (e.g., shown in SEQ ID NO:8) is approximately 10 times slower than the biotin-streptavidin interaction. -13 Compared to M, it is about 10 -16 K of M D In some instances, the antibody is characterized by a binding affinity of 10 to Strep-tactin®. -10 ~10 -13 Between m or about 10 -10 ~10 -13 K between M D Biotin, which can bind with high affinity to Strep-tag® II, can compete with Strep-tag® II for this binding site.

[0195] In some examples, the reagent comprises at least two chelating groups, K, that may be capable of binding to a transition metal ion. In some embodiments, the reagent may be capable of binding to an oligohistidine affinity tag, glutathione-S-transferase, calmodulin or an analog thereof, calmodulin-binding peptide (CBP), FLAG peptide, HA tag, maltose-binding protein (MBP), an HSV epitope, a myc epitope, and / or a biotinylated carrier protein.

[0196] In some embodiments, the reagent is an oligomer or polymer. In some embodiments, the oligomer or polymer can be produced either by directly or indirectly linking individual molecules of a protein found in nature, or by directly or indirectly linking individual monomeric molecules or complexes of subunits that form individual molecules (e.g., directly or indirectly linking dimers, trimers, tetramers, etc. of a protein found in nature). For example, a streptavidin or avidin tetrameric homodimer or heterodimer can be referred to as an individual molecule or smallest building block of each oligomer or polymer. In some embodiments, the oligomer or polymer can comprise the linkage of at least two individual molecules of a protein (i.e., is a 2-mer) or can be at least a 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 25-mer, 30-mer, 35-mer, 40-mer, 45-mer, or 50-mer of individual molecules of a protein (e.g., monomer, tetramer).

[0197] Oligomers can be produced using any method known in the art, for example, as described in published U.S. patent application no. US2004 / 0082012. In some embodiments, the oligomer or polymer comprises two or more individual molecules that can be crosslinked, for example, by a polysaccharide or bifunctional linker.

[0198] In some embodiments, oligomers or polymers are obtained by crosslinking individual molecules or complexes of subunits that form individual molecules in the presence of a polysaccharide. In some embodiments, oligomers or polymers can be prepared by introducing carboxyl residues into a polysaccharide, such as dextran. In some aspects, individual molecules of a reagent (e.g., a monomer, a tetramer) can be linked to the carboxyl groups of the dextran backbone through the primary amino groups of internal lysine residues and / or the free N-terminus using conventional carbodiimide chemistry. In some embodiments, the linking reagent is used in a molar ratio of about 60 moles of individual molecules of the reagent (e.g., a monomer, a tetramer) per mole of dextran.

[0199] In some embodiments, the reagent is an oligomer or polymer of one or more streptavidin or avidin or any analog or mutein of streptavidin (e.g., Strep-Tactin® or Strep-Tactin® XT) or analog or mutein of avidin (e.g., neutravidin). In some embodiments, binding site Z is the native biotin-binding site of avidin or streptavidin, and there can be up to four binding sites in an individual molecule (e.g., a tetramer contains four binding sites Z), whereby a homotetramer can contain up to four binding sites, i.e., Z1, that are the same, and a heterotetramer can contain up to four binding sites, which can be different, e.g., comprising Z1 and Z2. In some embodiments, the oligomer is generated or produced from multiple individual molecules (e.g., multiple homotetramers) of the same streptavidin, streptavidin mutein, avidin, or avidin mutein, and in this example, each binding site Z, e.g., Z1, of the oligomer is the same. For example, in some examples, the oligomer can include multiple binding sites Z1, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, or more binding sites Z1. In some embodiments, the oligomer is generated or produced from a plurality of individual molecules, which may be heterotetramers of streptavidin, streptavidin muteins, avidin or avidin muteins, and / or from a plurality of two or more different individual molecules (e.g., different homotetramers) of streptavidin, streptavidin muteins, avidin or avidin muteins that differ with respect to their binding sites Z, e.g., Z1 and Z2; in this example, a plurality of different binding sites Z, e.g., Z1 and Z2, may be present within the oligomer.For example, in some instances, an oligomer may contain multiple binding sites Z1 and multiple binding sites Z, collectively comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50 or more total binding sites Z1 and Z2.

[0200] In some instances, each oligomer or polymer can be cross-linked by a polysaccharide. In one embodiment, oligomers or polymers of streptavidin or avidin or a streptavidin or avidin analog (e.g., neutravidin) can be prepared in a first step by introducing carboxyl residues into a polysaccharide, such as dextran, essentially as described in Noguchi, A, et al., Bioconjugate Chemistry (1992) 3,132-137. In some such aspects, streptavidin or avidin or an analog thereof can then be linked in a second step to the carboxyl groups of the dextran backbone through the primary amino groups of internal lysine residues and / or the free N-terminus using conventional carbodiimide chemistry. In some instances, cross-linked oligomers or polymers of streptavidin or avidin or any analogue of streptavidin or avidin can also be obtained by cross-linking through a bifunctional molecule that acts as a linker, such as glutaraldehyde, or by other methods reported in the art.

[0201] In some embodiments, oligomers or polymers can be obtained by crosslinking individual molecules or complexes of subunits that form individual molecules using bifunctional linkers or other chemical linkers, such as glutardialdehyde, or by other methods known in the art.In some aspects, the crosslinked oligomers or polymers of streptavidin or avidin, or any mutein or analog of streptavidin or avidin, can be obtained by crosslinking individual streptavidin or avidin molecules through a bifunctional molecule that functions as a linker, such as glutardialdehyde, or by other methods reported in the art.For example, it is possible to generate oligomers of streptavidin muteins by introducing thiol groups into streptavidin muteins (this can be done, for example, by reacting streptavidin muteins with 2-iminothiolane (Trauts' reagent) and then activating the available amino groups in the streptavidin muteins in a separate reaction). In some embodiments, this activation of the amino group can be achieved by reacting the streptavidin mutein with a commercially available heterobifunctional cross-linker, such as sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) or succinimidyl 6-[(β-maleimidopropionamido)hexanoate (SMPH). In some such embodiments, the two resulting reaction products are mixed, thereby reacting a thiol group, typically contained in one batch of modified streptavidin mutein, with an activated amino acid (e.g., by a maleimide function) in the other batch of modified streptavidin mutein. In some instances, this reaction results in the formation of a streptavidin mutein multimer / oligomer.These oligomers can have any suitable number, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50 or more individual molecules, and the degree of oligomerization can vary depending on the reaction conditions.

[0202] In some embodiments, oligomeric or polymeric reagents can be isolated through size-exclusion chromatography, and any desired fraction can be used as a reagent. For example, in some embodiments, after reacting a modified streptavidin mutein in the presence of 2-iminothiolane and a heterobifunctional crosslinker, such as sulfo-SMCC, the oligomeric or polymeric reagent can be isolated through size-exclusion chromatography, and any desired fraction can be used as a reagent. In some embodiments, oligomers do not (and need not) have a single molecular weight, but can exhibit a statistical weight distribution, such as a Gaussian distribution. In some examples, any oligomer containing four or more streptavidin or mutein tetramers, such as homotetramers or heterotetramers, can be used as a soluble reagent. For example, any oligomer containing typically 3 to 50 tetramers, such as homotetramers or heterotetramers, 10 to 40 tetramers, such as homotetramers or heterotetramers, or 25 to 35 tetramers, such as homotetramers or heterotetramers, can be used as a soluble reagent. The oligomer can have, for example, from 3 to 25 streptavidin mutein tetramers, such as homotetramers or heterotetramers. In some aspects, when the molecular weight of the streptavidin mutein is about 50 kDa, the soluble oligomer may be about 150 kDa to about 2000 kDa, about 150 kDa to about 1500 kDa, about 150 kDa to about 1250 kDa, about 150 kDa to 1000 kDa, about 150 kDa to about 500 kDa, or about 150 kDa to about 300 kDa, about 300 kDa to about 2000 kDa, about 300 kDa to about 1500 kDa, about 300 kDa to about 1250 kDa, about 300 kDa to 1000 kDa, about 300 kDa to about 500 kDa, about 500 kDa to about 2000 kDa, or about 500 kDa to about 1500 kDa. kDa, about 500 kDa to about 1250 kDa, about 500 kDa to 1000 kDa, about 1000 kDa to about 2000 kDa, about 1000 kDa to about 1500 kDa, about 1000 kDa to about 1250 kDa, about 1250 kDa to about 2000 kDa, or about 1500 kDa to about 2000 kDa.Typically, each streptavidin molecule / mutein has four biotin binding sites, so that such a reagent may provide 12 to 160 binding sites Z, for example 12 to 100 binding sites Z.

[0203] 1. Reagent Format a. Support In some embodiments, the reagent is contained in a support, e.g., a solid support or surface, e.g., a bead, or a stationary phase (chromatography matrix). In some such embodiments, the reagent is reversibly immobilized to the support. In some examples, the reagent is immobilized to the support through a covalent bond. In some aspects, the reagent is reversibly immobilized to the support by a non-covalent bond.

[0204] In some embodiments, the support is a solid support. Any solid support (surface) can be used for reversible immobilization of a reagent. Examples of solid supports onto which a reagent can be immobilized include magnetic beads, polymer beads, cell culture plates, microtiter plates, membranes, or hollow fibers. In some aspects, hollow fibers can be used as bioreactors in the Quantum® Cell Expansion System available from TerumoBCT Inc. (Lakewood, CO, USA). In some embodiments, the reagent is covalently attached to the solid support. In other embodiments, non-covalent interactions can also be used, for example, for immobilization to a plastic substrate. In some embodiments, the reagent can be, for example, streptavidin or an avidin mutein that reversibly binds to a streptavidin-binding peptide. Such streptavidin muteins can be covalently attached to any surface, for example, resins (beads) used for chromatographic purification, such as Strep-Tactin® Sepharose, Strep-Tactin® Superflow®, Strep-Tactin® Superflow® Enhanced, or Strep-Tactin® MacroPrep®, commercially available from IBA GmbH, Göttingen. Other commercially readily available examples are immobilized metal affinity chromatography (IMAC) resins, such as TALON® resins (Westburg, Leusden, The Netherlands), which can be used for the reversible immobilization of oligohistidine-tagged (his-tagged) proteins, for example, for the reversible binding of agents (e.g., receptor-binding agents or selectivities) that contain an oligohistidine tag, such as a penta- or hexa-histidine tag, as binding partner C. Other examples include calmodulin sepharose available from GE Life Sciences, which can be used with agents (e.g., receptor binding agents or selection agents) that contain calmodulin-binding peptides or sepharose as binding partner C to which glutathione binds.In some such instances, binding partner C is glutathione-S-transferase.

[0205] In some embodiments, the support comprises a stationary phase. Thus, in some embodiments, the reagent is contained in the stationary phase (also called a chromatography matrix). In some such embodiments, the reagent is reversibly immobilized to the stationary phase. In some instances, the reagent is reversibly immobilized to the stationary phase through a covalent bond. In some aspects, the reagent is reversibly immobilized to the stationary phase by a non-covalent bond.

[0206] Any material can be used as a chromatography matrix. Generally, suitable chromatography materials are essentially harmless, i.e., do not cause adverse effects on cell viability, when used under the desired conditions, for example, in a packed chromatography column. In some embodiments, the stationary phase is maintained at a predetermined location, for example, at a predetermined position, with the location of the sample moving relative to it. Thus, in some embodiments, the stationary phase is part of a chromatography system in which a mobile phase flows (either by flow-through or in batch mode), and the components contained in the liquid phase (either dissolved or dispersed) are distributed between the phases.

[0207] In some embodiments, the chromatography matrix is ​​in the form of a solid or semi-solid phase, and the sample containing the target cells to be isolated / separated is in the liquid phase. The chromatography matrix is ​​a monolithic chromatography material comprising a specific material (of any suitable size and shape) or a paper substrate or membrane. Thus, in some aspects, chromatography can be both column chromatography and planar chromatography. In some embodiments, in addition to standard chromatography columns, columns that enable bidirectional flow, such as PhyTip® columns or pipette tips available from PhyNexus, Inc., San Jose, CA, USA, can be used in column-based / flow-through mode-based methods. Thus, in some examples, pipette tips or columns that enable bidirectional flow are also included in the chromatography columns useful in the methods of the present invention. In some examples, for example, when a particulate matrix material is used, the particulate matrix material can have an average particle size of, for example, about 5 μm to about 200 μm, or about 5 μm to about 400 μm, or about 5 μm to about 600 μm. In some aspects, the chromatography matrix can be or include, for example, a polymeric resin or a metal oxide or semi-metal oxide. In some aspects, for example, when planar chromatography is used, the matrix material can be any material suitable for planar chromatography, such as a conventional cellulose-based or organic polymer-based membrane (e.g., a paper membrane, a nitrocellulose membrane, or a polyvinylidene fluoride (PVDF) membrane) or a silica-coated glass plate. In one embodiment, the chromatography matrix / stationary phase is a non-magnetic or non-magnetizable material.

[0208] In some embodiments, nonmagnetic or non-magnetizable chromatographic stationary phases suitable for the methods of the present invention include derivatized silica or cross-linked gels. In some aspects, the cross-linked gels can be based on natural polymers, such as those from classes of polymers that occur in nature. For example, natural polymers that can serve as the basis for chromatographic stationary phases are polysaccharides. In some instances, each polysaccharide is typically cross-linked. Examples of polysaccharide matrices include, but are not limited to, agarose gels (e.g., Superflow™ agarose or Sepharose® materials, such as Superflow™ Sepharose®, which are commercially available in different bead and pore sizes) or cross-linked dextran gels. A further example is a particulate cross-linked agarose matrix to which dextran is covalently attached, commercially available as Sephadex® or Superdex® (in various bead and pore sizes), both available from GE Healthcare. Another example of such a chromatographic material is Sephacryl®, also available from GE Healthcare in different bead and pore sizes.

[0209] In some embodiments, cross-linked gels can also be based on synthetic polymers, such as polymer classes that do not occur in nature. In some aspects, such synthetic polymers underlying chromatographic stationary phases are polymers that contain polar monomer units and are therefore themselves polar. Thus, in some instances, such polar polymers are hydrophilic. Hydrophilic molecules, also referred to as lipophilic, in some aspects contain moieties that can form dipole-dipole interactions with water molecules. In general, hydrophobic molecules, also referred to as lipophilic, tend to separate from water.

[0210] Examples of suitable synthetic polymers are polyacrylamide, styrene divinylbenzene gels, and copolymers of acrylates and diols or acrylamide and diols. An example is polymethacrylate gel commercially available as Fractogel®. A further example is a copolymer of ethylene glycol and methacrylate commercially available as Toyopearl®. In some embodiments, the chromatographic stationary phase can also include natural and synthetic polymer components, such as composite matrices or composites or copolymers of polysaccharides and agarose, such as polyacrylamide / agarose composites, or copolymers of polysaccharides and N,N'-methylenebisacrylamide. Examples of copolymers of dextran and N,N'-methylenebisacrylamide are the Sephacryl® series of materials mentioned above. In some embodiments, the derivatized silica can include silica particles linked to synthetic or natural polymers. Examples of such embodiments include, but are not limited to, polysaccharide-bonded silica, polyvinylpyrrolidone-bonded silica, polyethylene oxide-bonded silica, poly(2-hydroxyethylaspartamide) silica, and poly(N'-isopropylacrylamide)-bonded silica.

[0211] In some embodiments, the chromatography matrix, for example, when used in the removal cartridges described herein, is a gel filtration matrix. Generally, gel filtration can be characterized by the properties it is designed to achieve. Thus, in some aspects, a gel filtration matrix achieves separation of cells or other biological matter largely based on their size. In some such aspects, each chromatography matrix is ​​typically a granular porous material, as described above. The chromatography matrix can have a specific exclusion limit, typically defined in terms of the molecular weight above which molecules cannot enter the pores and are completely removed. In some embodiments, each molecular weight defining the size exclusion limit can be selected to be below the weight corresponding to the weight of the target cells. In such embodiments, the target cells are prevented from entering the pores of the size-exclusion chromatography matrix. Similarly, the stationary phase can have pores smaller than the size of the selected target cells. In exemplary embodiments, the chromatography matrix has an average pore size of 0 to about 500 nm.

[0212] In some embodiments, components present in a sample, such as agents (e.g., receptor-binding agents or selection agents) or competitive reagents, may have a size below the exclusion limit of the pores and thus be able to enter the pores of the chromatography matrix. In some aspects, among such components that can partially or completely enter the pore volume, larger molecules with less access to the pore volume may elute first, and the smallest molecules typically elute last. In some embodiments, the exclusion limit of the chromatography matrix is ​​selected to be below the maximum width of the target cells. Thus, in some aspects, components with access to the pore volume may remain in or on the chromatography matrix longer than target cells. Thus, in some instances, target cells may be separated from other objects / components of the sample and recovered in the eluate of the chromatography column. Thus, in some aspects, components, such as agents (e.g., receptor-binding agents or selection agents), or, if applicable, competitive reagents, may elute from the gel filtration matrix at a later time than target cells. In some embodiments, this effect is further increased, for example, when the gel permeation matrix includes a reagent comprising (e.g., covalently attached to) a binding site Z that can bind to an agent (e.g., a receptor-binding agent or a selection agent) and / or a competing reagent present in the sample. In some instances, the agent (e.g., a receptor-binding agent or a selection agent) and / or a competing reagent can be bound by the binding site Z of the reagent and thereby immobilized on the matrix. In some aspects, the method is performed in a removal cartridge.

[0213] In some embodiments, the chromatography matrix used in the methods of the present invention can also include a magnetically attractable object, such as one or more magnetically attractable particles or magnetic fluids. Each magnetically attractable particle can include a reagent having a binding site capable of binding to target cells. In some examples, the magnetically attractable particles can include diamagnetic, ferromagnetic, paramagnetic, or superparamagnetic materials. Generally, superparamagnetic materials respond to a magnetic field induced by an induced magnetic field without permanent magnetization. Iron oxide-based magnetic particles are commercially available, for example, from Dynal Biotech as Dynabeads®, Miltenyi Biotec as magnetic MagneticBeads, CPG Inc. as magnetic porous glass beads, and from various other sources, such as Roche Applied Science, BIOCLON, BioSource International Inc., micromod, AMBION, Merck, Bangs Laboratories, Polysciences, or Novagen Inc., to name a few. Magnetic nanoparticles based on superparamagnetic Co and FeCo and ferromagnetic Co nanocrystals have been reported, for example, by Hutten, A. et al. (J. Biotech. (2004), 112, 47-63). In another embodiment, the chromatography matrix used in the method of the present invention is a depression of any magnetically attractable object.

[0214] In some embodiments, a device is provided that includes at least one arrangement of first and second stationary phases, such as a chromatography column for cell selection (selection cartridge) and a second chromatography column for reagent removal (removal cartridge). The device may include multiple arrangements of first and second stationary phases (chromatography columns) fluidically connected in series. The device may include a sample inlet fluidically connected to a first stationary phase of a first arrangement of first and second stationary phases. In some embodiments, the device may also include a sample outlet for cells fluidically connected to the last second stationary phase of at least one arrangement of first and second stationary phases for chromatography. In some aspects, the device may also include a competitive reagent reservoir fluidically connected to at least one of the first stationary phases of the first and second stationary phase arrangement.

[0215] b. Soluble In some embodiments, the reagent is not bound to a solid support, i.e., it exists in a soluble form or is soluble. In principle, the same reagent can be used as a reagent immobilized on a support, for example, a solid support or a stationary phase. For example, any of the above-mentioned examples of reagents can be used without immobilizing or attaching such a reagent to a support, for example, without attaching it to a solid support or a stationary phase. In some embodiments, the reagent comprises multiple binding sites Z for reversible binding to a binding substance through interaction with a binding partner C. In some examples, the reagent is an oligomer or polymer of individual molecules, or an oligomer or polymer of a complex of subunits forming individual molecules (e.g., an oligomer or polymer of a dimeric, trimeric, or tetrameric protein). In some embodiments, the reagent can be, for example, a streptavidin mutein oligomer, a calmodulin oligomer, or a compound (oligomer) that provides at least two chelating groups K capable of binding to a transition metal ion, thereby allowing the reagent to be attached to an oligohistidine affinity tag, a multimeric glutathione-S-transferase, or a biotinylated carrier protein.

[0216] In some embodiments, the reagent is characterized by the absence of a solid support (surface) attached to the reagent. For example, in some embodiments, the reagent does not include or is not attached (directly or indirectly) to particles, beads, nanoparticles, microspheres, or other solid supports. In some embodiments, the reagent is not rigid, inflexible, or hard, or does not include or is not attached to a rigid, inflexible, or hard surface. In some embodiments, the reagent is flexible or substantially flexible. In some instances, the reagent can adjust or adapt to the shape of the surface of a cell. In some embodiments, the reagent does not include a spherical or substantially spherical shape.

[0217] In some embodiments, substantially all of the reagents, i.e., greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, are organic, consist of, or comprise organic matter. For example, in some embodiments, greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the reagents are, consist of, or comprise lipids, carbohydrates, proteins, peptides, or mixtures thereof. In some embodiments, the reagents are essentially free of, essentially free of, or essentially free of inorganic materials, inorganic cores, such as metals, e.g., iron, synthetic or inorganic polymers, e.g., styrene polymers, e.g., polystyrene, latex, silica, or magnetic cores. For example, in some embodiments, the relative percentage of inorganic matter in the reagent included as part of the reagent is less than 20%, 15%, 10%, 5% or less.

[0218] In some embodiments, the majority (i.e., greater than 50%) of the total amount of reagent in the aqueous solution, e.g., greater than 60%, 70%, 80%, 90%, 95%, 99% or more, consists of individual protein molecules, e.g., oligomers or polymers of individual molecules or complexes of subunits forming individual molecules (e.g., tetrameric molecules), that comprise the reagent. In some embodiments, the total density of the soluble reagent is greater than or equal to 1.2 g / cm 3 , 1.1 g / cm 3 , 1.0 g / cm 3 Less than or smaller than.

[0219] In some embodiments, e.g., soluble reagents that are not attached to a support or solid support (e.g., not attached to a bead) are relatively small in size, e.g., typically less than or about 20 nM in size, e.g., less than or about 15 nM, less than or about 10 nM, less than or about 5 nM, or even smaller.

[0220] In some embodiments, for example, a soluble reagent that is not attached to a support or solid support (e.g., not attached to a bead) is biologically inert, i.e., it is non-toxic to living cells. In some embodiments, the reagent can be biodegradable, e.g., it can be degraded by enzymatic activity or removed by phagocytic cells.

[0221] In some embodiments, a reagent (e.g., streptavidin or a mutein, such as a tetrameric streptavidin mutein) can be reacted with a carrier, such as an organic carrier. In some aspects, in addition to reacting with a polysaccharide, a physiologically or pharmaceutically acceptable protein, such as serum albumin (e.g., human serum albumin (HSA) or bovine serum albumin (BSA)), can be used as a carrier protein. In such examples, a reagent, such as streptavidin or a streptavidin mutein (either as an individual tetramer or in the form of an oligomer), can be linked to the carrier protein through a non-covalent interaction. In some such embodiments, biotinylated BSA (commercially available from various sources, such as ThermoFisher Scientific, Sigma Aldrich, or Vectorlabs, to name a few) can be reacted with a reagent (e.g., a streptavidin mutein). In some aspects, a portion of the reagent oligomer (e.g., a streptavidin oligomer) can be non-covalently linked to a biotinylated carrier protein through one or more binding sites Z, while leaving the majority of the binding sites Z of the oligomer available for binding to an agent (e.g., a receptor-binding agent or a selection agent) and any additional agents described herein. Thus, by such an approach, soluble reagents with multiple binding sites Z can be prepared.

[0222] In other embodiments, a reagent, such as a streptavidin mutein (either as an individual tetramer or in the form of an oligomer), can be covalently linked to a synthetic carrier, such as a polyethylene glycol (PEG) molecule. Any suitable PEG molecule can be used for this purpose, and the PEG molecule and each reagent can be soluble. Typically, PEG molecules with a molecular weight of up to 1000 Da are soluble in water or culture medium and can be used in the methods of the present invention. In some examples, such PEG-based reagents can be prepared using commercially available activated PEG molecules (e.g., PEG-NHS derivatives available from NOF North America Corporation, Irvine, California, USA, or activated PEG derivatives available from Creative PEGWorks, Chapel Hills, North Carolina, USA) and the amino group of a streptavidin mutein.

[0223] B. Agent In some embodiments, the agent (e.g., receptor-binding agent or selection agent) has one or more binding sites B for binding to a molecule on the surface of a cell, e.g., a cell surface molecule. Thus, in some examples, the agent (e.g., receptor-binding agent or selection agent) comprises one binding site B or multiple binding sites B, and specific binding between the agent (e.g., receptor-binding agent or selection agent) and the molecule on the surface of a target cell comprises an interaction between B and the molecule. In some embodiments, the agent comprises only one binding site, i.e., is monovalent. In some embodiments, the agent (e.g., receptor-binding agent or selection agent) has multiple binding sites B, including at least two, e.g., three, four, or five, binding sites B capable of binding to a cell surface molecule. In some such aspects, at least two or more binding sites B can be identical. In some embodiments, one or more of the at least two or more binding sites B can be different (e.g., B1 and B2).

[0224] In some embodiments, one or more different agents (e.g., one or more different receptor binding substances, selection substances, or other agents that bind to molecules on cells) reversibly bind to the reagent. In some embodiments, at least two, three, four, or more different agents reversibly bind to the same reagent. In some embodiments, at least two different agents reversibly bind to the same reagent, each reagent comprising one binding site B or multiple binding sites B for specific binding between the agent and the molecule. In some embodiments, at least two or more agents comprise the same binding site B, e.g., for binding to the same or substantially the same molecule. In some embodiments, at least two or more agents comprise different binding sites B, e.g., for binding to different molecules. In some embodiments, a first agent (e.g., a first receptor binding substance or a first selection substance) comprises binding site B1, B2, B3, B4, etc., and a second agent (e.g., a second receptor binding substance or a second selection substance) comprises another of binding sites B1, B2, B3, B4, etc. In some embodiments, the first agent (e.g., a first selected agent) comprises binding site B1, and the second agent (e.g., a second selected agent) comprises binding site B3. In some embodiments, the first agent (e.g., a first receptor binding substance) comprises binding site B2, and the second agent (e.g., a second receptor binding substance) comprises binding site B4. In any such embodiment, the first agent and the second agent may comprise binding partner C1 or C2. In some embodiments, C1 and C2 may be the same. In some embodiments, C1 and C2 are different. In some embodiments, the first agent and the second agent comprise the same binding partner C1.

[0225] In some instances, the dissociation constant (K) of binding between the agent (e.g., through its binding site B) and the binding site Z of the reagent is D ) is about 10 -2 M ~ about 10 -13 M or about 10 -3 M ~ about 10 -12 M or about 10 -4M ~ about 10 -11 M, or about 10 -5 M ~ about 10 -10 In some embodiments, the dissociation constant (K D ) are those with low affinity, e.g., about 10 -3 ~about 10 -7 In some embodiments, the dissociation constant (K D ) is a high affinity one, e.g., about 10 -7 ~Approx. 1 x 10 -10 It is within the range of M.

[0226] In some embodiments, dissociation of the binding of the agent to the molecule through binding site B occurs sufficiently quickly, e.g., after disruption of the reversible bond between the reagent and the agent, that the target cell is only transiently stained by or bound to the agent. off When expressed as a rate (also called the dissociation rate constant of the binding between the agent and the molecule (through binding site B), k off The speed is approximately 0.5 x 10 -4 seconds -1 or more, approximately 1 x 10 -4 seconds -1 or more, about 2 x 10 -4 seconds -1 or more, about 3 x 10 -4 seconds -1 or more, about 4 x 10 -4 seconds -1 or more, about 5 x 10 -4 seconds -1 or more, approximately 1 x 10 -3 seconds -1 or more, about 1.5 x 10 -3 seconds -1 or more, about 2 x 10 -3 seconds -1 or more, about 3 x 10 -3 seconds -1 or more, about 4 x 10 -3 seconds -1or more, about 5 x 10 -3 seconds -1 or more, approximately 1 x 10 -2 seconds or more, or about 5 x 10 -1 seconds -1 or more. The k suitable for the interaction of a particular agent with the cellular molecule. off It is within the skill of one of ordinary skill in the art to empirically determine the rate range (see, e.g., Published U.S. Application No. US2014 / 0295458). For example, a rate of, e.g., 4.0 x 10, can be used so that the majority of the agent can be removed or separated within one hour following disruption of the binding complex. -4 seconds -1 Larger, relatively high k off In another example, an agent with a rate of binding can be used, e.g., 1.0 x 10, such that the majority of the agent can be removed or separated from the cells within about 3.5 hours after disruption of the binding complex. -4 seconds -1 of low k off Agents with speed may be used.

[0227] In some embodiments, the K D and the K of the bond formed between the binding site B of the agent (e.g., receptor binding agent or selective agent) and the cell surface molecule. D , k off and k on The rate can be determined by any suitable means, for example, by fluorescence titration, equilibrium dialysis, or surface plasmon resonance.

[0228] In some aspects, a cell surface molecule is a molecule to which an agent (e.g., a receptor-binding agent or a selection agent) can be directed. In some embodiments, the cell surface molecule is a peptide or a protein, such as a receptor, e.g., a membrane receptor protein. In some embodiments, the receptor is a lipid, polysaccharide, or nucleic acid. In some embodiments, a cell surface molecule that is a protein can be a peripheral membrane protein or an integral membrane protein. In some embodiments, a cell surface molecule can have one or more membrane-spanning domains. As some illustrative examples, membrane proteins having a transmembrane domain include G protein-coupled receptors, such as olfactory receptors, rhodopsin receptors, rhodopsin pheromone receptors, peptide hormone receptors, taste receptors, GABA receptors, opiate receptors, serotonin receptors, Ca2+ receptors, melanopsin, acetylcholine, nicotinic, adrenergic, norepinephrine, catecholamine, L-DOPA-, dopamine and serotonin (biogenic amines, endorphins / enkephalins) neuropeptide receptors, neurotransmitter receptors, such as ligand-gated, voltage-gated or mechanosensory receptors, receptor kinases, such as serine / threonine kinases, tyrosine kinases, porins / channels, such as chloride channels, potassium channels, sodium channels, OMP proteins, ABC transporters (ATP-binding cassette transporters), such as amino acid transporters, Na-glucose transporters, Na / iodide transporters, ion transporters, such as light-harvesting complexes, cytochrome c oxidases, ATPases. It may be Na / K, H / K, Ca, a cell adhesion receptor, such as a metalloprotease, an integrin, or a catherin.

[0229] In some embodiments, the cell surface molecule can be an antigen that defines a desired cell population or subpopulation, for example, a population or subpopulation of blood cells, such as lymphocytes (e.g., T cells, T helper cells, e.g., CD4+ T helper cells, B cells, or natural killer cells), monocytes, or stem cells, e.g., CD34-positive peripheral stem cells or Nanog- or Oct-4-expressing stem cells. Examples of T cells include CMV-specific CD8+ T lymphocytes, cytotoxic T cells, memory T cells, and regulatory T cells (Tregs). An example of a Treg is a CD4CD25CD45RA Treg cell, and an example of a memory T cell is a CD62LCD8+-specific central memory T cell. The cell surface molecule can also be a marker for tumor cells.

[0230] As noted above, in some embodiments, the agent (e.g., receptor-binding agent or selection agent) has a binding partner C in addition to a binding site B capable of binding to a cell surface molecule. In some aspects, this binding partner C can bind to a binding site Z of a reagent, the reagent having one or more binding sites for binding partner C. In some embodiments, the non-covalent bond that can form between the binding partner C included in the agent (e.g., receptor-binding agent or selection agent) and the binding site Z of the reagent can be of any desired strength and affinity and can be disruptable or reversible under the conditions under which the method is performed. The agent (e.g., receptor-binding agent or selection agent) can include at least one additional binding partner C, including two, three, or more, and the reagent can include at least two, e.g., three, four, five, six, seven, eight, or more binding sites Z for the binding partner C included in the agent (e.g., receptor-binding agent or selection agent). As described in U.S. Pat. No. 7,776,562, U.S. Pat. No. 8,298,782 or International Patent Application WO 2002 / 054065, any combination of reagents having a binding partner C and one or more corresponding binding sites Z can be selected, for example, such that the binding partner C and the binding site Z can reversibly bind in a complex, e.g., to produce an avidity effect.

[0231] The binding partner C included in the agent (e.g., receptor-binding agent or selector) can be, for example, hydrocarbon-based (including polymeric) and can contain nitrogen, phosphorus, sulfur, carbene, halogen, or pseudohalogen groups. In some aspects, it can be an alcohol, organic acid, inorganic acid, amine, phosphine, thiol, disulfide, alkane, amino acid, peptide, oligopeptide, polypeptide, protein, nucleic acid, lipid, sugar, oligosaccharide, or polysaccharide. As a further example, it can also be a cation, anion, polycation, polyanion, polycation, electrolyte, polyelectrolyte, carbon nanotube, or carbon nanofoam. Typically, such a binding partner C has a higher affinity for the binding site of the reagent than for other entities. Examples of binding partners C include, but are not limited to, crown ethers, immunoglobulins, their fragments, and proteinaceous binding molecules with antibody-like functions.

[0232] In some embodiments, binding partner C included in the agent (e.g., receptor-binding agent or selection agent) comprises biotin, and the reagent comprises a streptavidin analog or an avidin analog that reversibly binds to biotin. In some embodiments, binding partner C included in the agent (e.g., receptor-binding agent or selection agent) comprises a biotin analog that reversibly binds to streptavidin or avidin, and the reagent comprises streptavidin, avidin, a streptavidin analog, or an avidin analog that reversibly binds to the respective biotin analog. In some embodiments, binding partner C included in the agent (e.g., receptor-binding agent or selection agent) comprises a streptavidin or an avidin-binding peptide, and the reagent comprises streptavidin, avidin, a streptavidin analog, or an avidin analog that reversibly binds to the respective streptavidin or avidin-binding peptide.

[0233] In some embodiments, the reagent is streptavidin, e.g., a streptavidin mutein, including any of the above (e.g., as set forth in SEQ ID NOs:3-6), and the binding partner C included in the agent (e.g., receptor-binding agent or selection agent) can comprise a streptavidin-binding peptide. In some embodiments, the streptavidin-binding peptide can comprise a sequence having the general formula set forth in SEQ ID NO:9, e.g., the sequence set forth in SEQ ID NO:10. In some embodiments, the peptide sequence is set forth in SEQ ID NO:11, e.g., the general formula set forth in SEQ ID NO:12. In one example, the peptide sequence is TIFF0007665579000021.tif4128 (also known as Strep-tag® and set forth in SEQ ID NO:7). In one example, the peptide sequence is TIFF0007665579000022.tif4128 (also known as Strep-tag® II and set forth in SEQ ID NO:8). In some embodiments, the peptide ligand comprises a consecutive arrangement of at least two streptavidin binding modules, the distance between the two modules being at least 0 and no more than 50 amino acids, one binding module having 3-8 amino acids and comprising at least the sequence His-Pro-Xaa (SEQ ID NO:9), where Xaa is glutamine, asparagine, or methionine, and the other binding module having the same or a different streptavidin peptide ligand (e.g., set forth in SEQ ID NO:11) (see, e.g., International Published PCT Application No. WO 02 / 077018; U.S. Patent No. 7,981,632). In some embodiments, the peptide ligand comprises a sequence having a formula set forth in either SEQ ID NO:13 or 14. In some embodiments, the peptide ligand has an amino acid sequence set forth in any of SEQ ID NOs: 15-19. In most instances, all of these streptavidin-binding peptides bind to the same binding site, i.e., the biotin-binding site of streptavidin. When one or more of such streptavidin-binding peptides are used as biotin partner C, e.g., C1 and C2, the multimerization reagent is typically a streptavidin mutein.

[0234] In some embodiments, the streptavidin-binding peptide may be further modified. In some embodiments, the streptavidin-binding peptide comprises the peptide sequence: TIFF0007665579000023.tif5128 (also known as Strep-tag® II and set forth in SEQ ID NO:8).

[0235] In some embodiments, the binding partner C of the agent (e.g., receptor-binding agent or selection agent) comprises a moiety known to those skilled in the art as an affinity tag. In such embodiments, the reagent may comprise a corresponding binding partner, e.g., an antibody or antibody fragment known to bind to the affinity tag. Some illustrative examples of known affinity tags include binding partner C included in the agent (e.g., receptor-binding agent or selection agent) include dinitrophenol or digoxigenin, oligohistidine, polyhistidine, immunoglobulin domain, maltose-binding protein, glutathione-S-transferase (GST), chitin-binding protein (CBP) or thioredoxin, calmodulin-binding peptide (CBP), FLAG peptide, TIFF0007665579000024.tif26152, Maltose-binding protein (MBP), sequence of herpes simplex virus glycoprotein D HSV epitope, sequence of TIFF0007665579000025.tif4128 TIFF0007665579000026.tif5128 "myc" epitope of transcription factor c-myc, V5 tag TIFF0007665579000027.tif5151 or glutathione-S-transferase (GST). In such embodiments, the complex formed between one or more binding sites Z of the reagent, which may be an antibody or antibody fragment, and the antigen can be competitively disrupted by adding a free antigen, i.e., a free peptide (epitope tag) or a free protein (e.g., MBP or CBP). In some embodiments, the affinity tag can also be an oligonucleotide tag. In some examples, such an oligonucleotide tag can be used, for example, to hybridize to an oligonucleotide having a complementary sequence linked to or contained in the reagent.

[0236] Further examples of suitable binding partners C include, but are not limited to, lectins, protein A, protein G, metals, metal ions, nitrilotriacetic acid derivatives (NTA), RGD motifs, dextran, polyethyleneimine (PEI), redox polymers, glycoproteins, aptamers, dyes, amylose, maltose, cellulose, chitin, glutathione, calmodulin, gelatin, polymyxin, heparin, NAD, NADP, lysine, arginine, benzamidine, polyU, or oligodT. Lectins, such as concavalin A, are known to bind to polysaccharides and glycosylated proteins. An example of a dye is a triazine dye, such as Cibacron Blue F3G-A (CB) or Red HE-3B, which specifically binds to NADH-dependent enzymes. Typically, Green A binds to CoA proteins, human serum albumin, and dehydrogenases. In some instances, the dyes 7-aminoactinomycin D and 4',6-diamidino-2-phenylindole bind to DNA. Generally, cations of metals, such as Ni, Cd, Zn, Co, or Cu, are typically linked to affinity tags, such as hexahistidine or TIFF0007665579000028.tif4143 and used to bind to an oligohistidine-containing sequence containing N-methacryloyl-(L)-cysteine ​​methyl ester.

[0237] In some embodiments, binding between a binding partner C included in the agent (e.g., receptor-binding agent or selection agent) and one or more binding sites Z of the reagent occurs in the presence of a divalent, trivalent, or tetravalent cation. In this regard, in some embodiments, the reagent comprises a divalent, trivalent, or tetravalent cation, typically held, e.g., complexed, by a suitable chelator. In some embodiments, the binding partner C included in the agent (e.g., receptor-binding agent or selection agent) can comprise a moiety that comprises, e.g., a complex with, a divalent, trivalent, or tetravalent cation. Examples of individual metal chelators include, but are not limited to, ethylenediamine, ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), N,N-bis(carboxymethyl)glycine (also known as nitrilotriacetic acid, NTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 2,3-dimercapto-1-propanol (dimercaprol), profine, and heme. For example, EDTA chelates most monovalent, divalent, trivalent, and tetravalent metal ions, such as silver (Ag). + ), calcium (Ca 2+ ), manganese (Mn 2+ ), copper (Cu 2+ ), iron (Fe 2+ ), cobalt (Co + ) and zirconium (Zr 4+ ) and BAPTA forms a complex with Ca 2+ By way of illustration, a standard method used in the art is to detect copper (Cu) presented by an oligohistidine tag and the chelator nitrilotriacetic acid (NTA). 2+ ), Nickel (Ni 2+ ), cobalt (Co 2+ ) or zinc (Zn 2+ ) ions.

[0238] In some embodiments, the binding partner C included in the agent (e.g., receptor-binding agent or selection agent) comprises a calmodulin-binding peptide, and the reagent comprises multimeric calmodulin, as described, for example, in U.S. Pat. No. 5,985,658. In some embodiments, the binding partner C included in the agent (e.g., receptor-binding agent or selection agent) comprises a FLAG peptide, and the reagent comprises an antibody that binds to the FLAG peptide, for example, the FLAG peptide that binds to the monoclonal antibody 4E11 described in U.S. Pat. No. 4,851,341. In one embodiment, the binding partner C included in the agent (e.g., receptor-binding agent or selection agent) comprises an oligohistidine tag, and the reagent comprises an antibody or transition metal ion that binds to the oligohistidine tag. In some embodiments, dissolution of all these binding complexes can be achieved by metal ion chelation, e.g., calcium chelation, for example, by adding EDTA or EGTA. In some embodiments, calmodulin, antibodies such as 4E11, or chelated metal ions or free chelators can be multimerized by conventional methods, such as biotinylation and complexation with streptavidin or avidin or oligomers thereof, or by first introducing carboxyl residues into a polysaccharide, such as dextran, essentially as described in Noguchi, A, et al. Bioconjugate Chemistry (1992) 3, 132-137, and then linking the calmodulin, antibody, or chelated metal ions or free chelators to the carboxyl groups of the polysaccharide, such as dextran, backbone via primary amino groups using conventional carbodiimide chemistry in a second step. In some such embodiments, binding between a binding partner C contained in an agent (e.g., a receptor-binding agent or a selective agent) and one or more binding sites Z of the reagent can be prevented by metal ion chelation. Metal chelation can be achieved, for example, by the addition of EGTA or EDTA.

[0239] In some embodiments, the agent (e.g., a receptor-binding agent or a selection agent) that specifically binds to a cell surface molecule can be comprised, for example, in an antibody, a fragment thereof, or a proteinaceous binding molecule with antibody-like function. In some embodiments, binding site B of the agent is an antibody binding site, for example, is or comprises one or more complementarity-determining regions (CDRs) of an antibody. Examples of (recombinant) antibody fragments include, but are not limited to, Fab fragments, Fv fragments, single-chain Fv fragments (scFv), bivalent antibody fragments such as (Fab)2' fragments, diabodies, triabodies (Iliades, P., et al., FEB S Lett (1997) 409, 437-441), decabodies (Stone, E., et al., Journal of Immunological Methods (2007) 318, 88-94), and other domain antibodies (Holt, LJ, et al., Trends Biotechnol. (2003), 21, 11, 484-490). In some embodiments, an agent (eg, a receptor binding agent or a selection agent) can include a bivalent proteinaceous artificial binding molecule, such as a dimeric lipocalin mutein, also known as a "duocalin."

[0240] In some embodiments, an agent (e.g., a receptor-binding agent or a selection agent) can have a single binding site B, i.e., can be monovalent. Examples of monovalent agents (e.g., receptor-binding agents or selection agents) include, but are not limited to, monovalent antibody fragments, proteinaceous binding molecules with antibody-like binding properties, or MHC molecules. Examples of monovalent antibody fragments include, but are not limited to, Fab fragments, Fv fragments, and single-chain Fv fragments (scFv), including bivalent single-chain Fv fragments.

[0241] In some embodiments, the agent is an antibody or an antigen-binding fragment thereof, such as a Fab fragment, an Fv fragment, a single-chain Fv fragment (scFv), or a bivalent antibody fragment, such as a F(ab')2 fragment. In some embodiments, the agent is or is derived from a parent antibody known to bind to a cellular molecule of interest. Various antibody molecules or fragments thereof against cell surface molecules are well known in the art, and any of such various antibodies can be used as an agent in the methods herein. In some embodiments, the agent is an antibody or fragment thereof containing one or more amino acid substitutions in the variable heavy chain of a parent or reference antibody, e.g., to generate an antibody with altered affinity or a sufficiently fast off-rate, as described above. For example, examples of such mutations are known in the context of mutations in the anti-CD4 antibody 13B8.2 (see, e.g., U.S. Patent No. 7,482,000, U.S. Patent Application Publication No. US2014 / 0295458, or International Patent Application No. WO2013 / 124474), and any such mutations can be made in another parent or reference antibody.

[0242] In some aspects, agents (e.g., receptor-binding agents or selection agents) that may be monovalent include, for example, monovalent antibody fragments or monovalent artificial binding molecules (proteinaceous or otherwise), such as muteins based on polypeptides of the lipocalin family (also known as "Anticalin®"), or bivalent molecules, such as antibodies or fragments in which both binding sites are maintained, e.g., F(ab')2 fragments.

[0243] Examples of proteinaceous binding molecules with antibody-like functions include muteins based on polypeptides of the lipocalin family (see, e.g., WO 03 / 029462; Beste et al., Proc. Natl. Acad. Sci. USA (1999) 96, 1898-1903). Lipocalins, such as bilin-binding protein, human neutrophil gelatinase-associated lipocalin, human apolipoprotein D, or human tear lipocalin, typically have a native ligand-binding site that can be engineered to bind to a specific target. Further examples of proteinaceous binding molecules with antibody-like binding properties that can be used as agents (e.g., receptor binders or selectors) that specifically bind to cell surface molecules include, but are not limited to, so-called glubodies (see, e.g., International Patent Application WO 96 / 23879), proteins based on ankyrin scaffolds (Mosavi, LK, et al, Protein Science (2004) 13, 6, 1435-1448) or crystalline scaffolds (e.g., International Patent Application WO 01 / 04144), the proteins described in Skerra, J. Mol. Recognit. (2000) 13, 167-187, Adnectins, tetranectins, and avimers. Avimers, including multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains, typically contain so-called A domains that occur as a series of multiple domains in various cell surface receptors (Silverman, J., et al, Nature Biotechnology (2005) 23, 1556-1561). Adnectins, usually derived from a domain of human fibronectin, typically contain three loops that can be engineered for immunoglobulin-like binding to targets (Gill, DS & Damle, NK, Current Opinion in Biotechnology (2006) 17, 653-658). Tetranectins, usually derived from a human homotrimeric protein, also typically contain loop regions within a C-type lectin domain that can be engineered for desired binding.Peptoids, which may function as protein ligands in some instances, are typically oligo(N-alkyl)glycines, which differ from peptides in that the side chains are attached to the amide nitrogen rather than the carbon atom. Peptoids are typically resistant to proteases and other modifying enzymes and can have much higher cell permeability than peptides (see, e.g., Kwon, Y.-U., and Kodadek, T., J. Am. Chem. Soc. (2007) 129, 1508-1509).

[0244] Further examples of suitable proteinaceous binding molecules include EGF-like domains, Kringle domains, fibronectin type I domains, fibronectin type II domains, fibronectin type III domains, PAN domains, Gla domains, SRCR domains, Kunitz / bovine pancreatic trypsin inhibitory domains, tendamistat, Kazal-type serine protease inhibitor domains, trefoil (P-type) domains, von Willebrand factor type C domains, anaphylatoxin-like domains, CUB domains, thyroglobulin type I repeats, LDL receptor class A domains, Sushi domains, Link domains, thrombospondin type I domains, immunoglobulin domains or immunoglobulin-like domains (e.g., domain antibodies or camelid heavy chain antibodies), C-type lectin domains, MAM domains, von Willebrand factor type A domains, somatomedin B domains, WAP-type four disulfide core domains, F5 / 8 C-type domains, hemopexin domains, SH2 domains, SH3 domains, laminin-type EGF-like domains, C2 domains, "kappa bodies" (Ill et al. Protein Eng (1997) 10, 949-57), so-called "minibodies" (Martin et al., EMBO J (1994) 13, 5303-5309), diabodies (Holliger et al., PNAS USA (1993) 90, 6444-6448), so-called "Janusis" (Traunecker et al., EMBO J (1991) 10, 3655-3659 or Traunecker et al., Int J Cancer (1992) Suppl 7, 51-52), nanobodies, microbodies, affilins, affibodies, knottins, ubiquitin, zinc finger proteins, autofluorescent proteins, or leucine-rich repeat proteins. In some embodiments, the nucleic acid molecule with antibody-like function can be an aptamer. Generally, aptamers fold into a defined three-dimensional motif and exhibit high affinity for specific target structures.

[0245] 1. Receptor-binding substances In some embodiments, the agent is a receptor-binding agent. In some embodiments, the receptor-binding agent binds to a molecule (e.g., a receptor) on the surface of a cell, and the binding between the agent and the molecule can induce or modulate a signal within the cell. In some examples, the cell surface molecule (e.g., a receptor) is a signaling molecule. In some such examples, the receptor-binding agent can specifically bind to a signaling molecule expressed by one or more cells. In some examples, the receptor-binding agent is a stimulatory agent, and the stimulatory agent can be any agent that can induce a signal within a cell (e.g., a T cell) by binding to a cell surface molecule, such as a receptor. In some embodiments, the signal can be immunostimulatory, and in this example, the receptor-binding agent or stimulatory agent can induce or modulate a signal that is involved in or stimulates an immune response by the cell (e.g., a T cell), for example, increasing immune cell proliferation or expansion, immune cell activation, immune cell differentiation, cytokine secretion, cytotoxic activity, or one or more other functional activities of an immune cell. In some embodiments, the signal may be inhibitory, in which case the receptor binding agent or stimulator may induce or modulate a signal that is involved in or inhibits an immune response in a cell (e.g., a T cell), e.g., inhibiting or reducing immune cell proliferation or expansion, immune cell activation, immune cell differentiation, cytokine secretion, cytotoxic activity, or one or more other functional activities of an immune cell.

[0246] In some embodiments, the receptor binding substance, for example, the stimulating substance, is a first receptor binding substance, for example, a first stimulating substance. In some aspects, the first receptor binding substance, for example, a first stimulating substance, binds to a receptor molecule on the surface of a cell. Thus, in some instances, the first receptor binding substance, for example, a first stimulating substance, induces or regulates a signal. In some instances, the induction or regulation of a signal by the first receptor binding substance, for example, a first stimulating substance, results in the activation, stimulation and / or proliferation (proliferation) of the cell. Thus, in some instances, the first receptor binding substance, for example, a first stimulating substance, provides a primary activation signal for the cell, thereby activating the cell.

[0247] In some embodiments, the cell population can be a lymphocyte population, including but not limited to a B cell population, a T cell population, or a natural killer cell population. An example of a cell population is a B cell bearing CD40 or CD137 (both cell populations can be expanded by binding only to a first agent that provides an activation signal, such as a 4-1BB ligand; or an αCD40 antibody molecule or an αCD137 antibody molecule (see, for example, Zhang et al., 2010, J Immunol, 184:787-795)). Other examples of agents (either first or second) that can be used to expand B cells are agents that bind to IgG, CD19, CD28, or CD14, such as αCD19, αIgG, αCD28, or αCD14 antibody molecules. It is also contemplated that the first or second agent for B cell expansion may comprise a ligand for a toll-like receptor or an interleukin, such as IL-21 (see, e.g., Dienz O, et al. 2009. J. Exp. Med. 206:69). Note that lipopolysaccharide may also be used as a first agent and may comprise the binding partner C1 as used herein, such that lipopolysaccharide-dependent activation of B cells is also encompassed within this embodiment of the invention.

[0248] Other examples of suitable cell populations include T cell populations expanded after activation by binding of a first agent to TCR / CD3 and a second agent to an accessory molecule, such as CD28, on the T cells. In this example, the first agent stimulates a TCR / CD3 complex-associated signal in the T cells, and the second agent provides a secondary stimulation by binding to CD28 as an accessory molecule. Agents that can be used to expand T cells can also include interleukins, such as IL-2, IL-7, IL-15, or IL-21 (see, e.g., Cornish et al. 2006, Blood. 108(2):600-8; Bazdar and Sieg, 2007, Journal of Virology, 2007, 81(22):12670-12674; Battalia et al., 2013, Immunology, 139(1):109-120). Other examples of agents that can be used to expand T cells are agents that bind to CD8, CD45, or CD90, such as αCD8, αCD45, or αCD90 antibodies. Examples of T cell populations include antigen-specific T cells, T helper cells, cytotoxic T cells, memory T cells (an example of a memory T cell is CD62L + CD8 + CD4 T cells are specific central memory T cells) or regulatory T cells (Tregs are examples of CD4 T cells). + CD25 + CD45RA+ Treg cells).

[0249] Another example of a suitable cell population includes natural killer cells (NK cells), which can be expanded using, for example, an agent that binds to CD16 or CD56, such as an αCD16 or αCD56 antibody. An example of such an αCD16 antibody is antibody 3G8, which has the VH sequence shown in SEQ ID NO:36 and the VL sequence shown in SEQ ID NO:37 (see, e.g., Hoshino et al., Blood. 1991 Dec 15;78(12):3232-40). Another agent that can be used to expand NK cells can be IL-15 (see, e.g., Vitale et al. 2002. The Anatomical Record. 266:87-92). Yet another example of a suitable cell population includes monocytes, which can be expanded using, for example, an agent that binds to CD14, such as an αCD14 antibody molecule.

[0250] In some embodiments, the first receptor-binding substance, e.g., the first stimulator, can stimulate a TCR / CD3 complex-associated signal in a cell, e.g., a T cell. In some aspects, the first receptor-binding substance, e.g., the first stimulator, can be a binding substance that specifically binds to CD3. In some examples, the first receptor-binding substance, e.g., the first stimulator, that specifically binds to CD3 can be selected from the group consisting of an anti-CD3 antibody, a bivalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, and a proteinaceous CD3-binding molecule with antibody-like binding properties. The bivalent antibody fragment can be a F(ab')2 fragment or a bivalent single-chain Fv fragment, and the monovalent antibody fragment can be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv). In some examples, the proteinaceous CD3 binding molecule with antibody-like binding properties can be an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, an ankyrin scaffold-based protein, a crystalline scaffold-based protein, an adnectin, or an avimer.

[0251] In some embodiments, the anti-CD3 Fab fragment may be derived from the CD3-binding monoclonal antibody produced by the hybridoma cell line OKT3 (ATCC® CRL-8001™; see also U.S. Pat. No. 4,361,549). The heavy chain variable domain and light chain variable domain of the anti-CD3 antibody OKT3 are described in Arakawa et al. J. Biochem. 120, 657-662 (1996) and comprise the amino acid sequences set forth in SEQ ID NOs: 31 and 32, respectively.

[0252] In some aspects, the receptor binding substance, e.g., the stimulator, is a second receptor binding substance, e.g., a second stimulator. In some examples, the second receptor binding substance, e.g., a second stimulator, binds to a molecule on the surface of a cell, e.g., a cell surface molecule, e.g., a receptor molecule. In some embodiments, the second receptor binding substance, e.g., a second stimulator, can enhance, attenuate, or modify the signal delivered through the first molecule. In some embodiments, the second receptor binding substance, e.g., a second stimulator, induces or modulates a signal, e.g., a second or additional signal. In some aspects, the second receptor binding substance, e.g., a second stimulator, can enhance or strengthen the signal induced by the first receptor binding substance, e.g., the first stimulator. In some embodiments, the second receptor binding substance, e.g., a second stimulator, can bind to an accessory molecule and / or stimulate or induce an accessory or secondary signal within the cell. In some aspects, the second receptor binding substance, e.g., a second stimulator, binds to a costimulatory molecule and / or provides a costimulatory signal.

[0253] In some embodiments, the receptor binding agent, e.g., stimulatory agent, which may be a second receptor binding agent, e.g., a second stimulatory agent, binds, e.g., specifically binds, to a second molecule, which may be a costimulatory molecule, an accessory molecule, a cytokine receptor, a chemokine receptor, an immune checkpoint molecule, or a member of the TNF family or the TNF receptor family.

[0254] In some embodiments, the molecule on the cell, e.g., T cell, can be CD28, and the receptor-binding agent, e.g., stimulator (which can, e.g., be a second receptor-binding agent, e.g., a second stimulator) specifically binds to CD28. In some aspects, the receptor-binding agent, e.g., stimulator (which can, e.g., be a second receptor-binding agent, e.g., a second stimulator) that specifically binds to CD28 can be selected from the group consisting of an anti-CD28 antibody, a bivalent antibody fragment of an anti-CD28 antibody, a monovalent antibody fragment of an anti-CD28 antibody, and a proteinaceous CD28-binding molecule with antibody-like binding properties. The bivalent antibody fragment can be a F(ab')2 fragment or a bivalent single-chain Fv fragment, and the monovalent antibody fragment can be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv). The proteinaceous CD28 binding molecule with antibody-like binding properties can be an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, a protein based on an ankyrin scaffold, a protein based on a crystalline scaffold, an adnectin, or an avimer.

[0255] In some embodiments, the anti-CD28 Fab fragment may be derived from the antibody CD28.3 (deposited as a synthetic single-chain Fv construct under GenBank accession number AF451974.1; see also Vanhove et al, BL...

Claims

1. 1. A method for stimulating target cells, the method comprising incubating a composition comprising target cells in the presence of a stimulating reagent in a chromatography column, At least a plurality of target cells are reversibly immobilized on a chromatography matrix present in a chromatography column during at least a portion of said incubation; The stimulating agent is in a soluble form, and (a) a first stimuli; (b) a second stimulant, and (c) an oligomer of a streptavidin molecule, an oligomer of an avidin molecule, an oligomer of a streptavidin mutein molecule, or an oligomer of an avidin mutein molecule. Including, The first and second stimuli are each (i) a streptavidin binding partner reversibly bound to the streptavidin molecule or the streptavidin mutein molecule; or (ii) an avidin binding partner reversibly bound to the avidin molecule or the avidin mutein molecule; Including, a selection agent is immobilized on the chromatography matrix, and the selection agent specifically binds to a selection marker expressed on the surface of the target cells to result in reversible immobilization of at least a plurality of target cells on the chromatography matrix; A method in which the step of incubating a composition comprising target cells in the presence of the stimulating reagent results in cellular stimulation of the target cells, and the step is performed under conditions in which a first stimulating agent specifically binds to a first molecule expressed on the surface of the target cells, thereby inducing an immunostimulatory signal within the cells of the target cells, and a second stimulating agent specifically binds to a second molecule expressed on the surface of the target cells, thereby inducing a second signal within the cells of the target cells, the second signal enhancing the immunostimulatory signal delivered through the first molecule.

2. 2. The method of claim 1, wherein the selection agent comprises an antibody, an antibody fragment, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex, or a receptor ligand that specifically binds to the selection marker.

3. 3. The method of claim 2, wherein the selection agent comprises an antibody fragment, and the antibody fragment of the selection agent is a Fab fragment.

4. The method of any one of claims 1 to 3, wherein the selection marker is a B-cell or T-cell co-receptor, or a member of the T-cell or B-cell antigen receptor complex.

5. The method of any one of claims 1 to 4, wherein the selection marker is CD8, CD4, or CD3.

6. The streptavidin binding partner or avidin binding partner comprises biotin, and the stimulating reagent comprises an oligomer of streptavidin molecules, an oligomer of avidin molecules, an oligomer of streptavidin mutein molecules, or an oligomer of avidin mutein molecules that reversibly binds to biotin, the streptavidin binding partner or avidin binding partner comprises a biotin analogue and the stimulating reagent comprises an oligomer of a streptavidin molecule, an oligomer of an avidin molecule, an oligomer of a streptavidin mutein molecule, or an oligomer of an avidin mutein molecule that reversibly binds to the biotin analogue; or the streptavidin-binding partner or avidin-binding partner comprises a streptavidin-binding peptide, and the stimulating reagent comprises an oligomer of a streptavidin molecule, an oligomer of an avidin molecule, an oligomer of a streptavidin mutein molecule, or an oligomer of an avidin mutein molecule that reversibly binds to the streptavidin-binding peptide; The method of any one of claims 1 to 5.

7. The streptavidin-binding partner or avidin-binding partner comprises a streptavidin-binding peptide comprising a sequence of amino acids set forth in any of SEQ ID NOs:7, 8, and 15-19, and the stimulating reagent comprises an oligomer of streptavidin molecules, an oligomer of avidin molecules, an oligomer of streptavidin mutein molecules, or an oligomer of avidin mutein molecules that reversibly binds to the streptavidin-binding peptide. The method according to any one of claims 1 to 6.

8. The method according to any one of claims 1 to 7, wherein the individual molecules of the oligomer are cross-linked by a polysaccharide or a bifunctional linker.

9. The method of any one of claims 1 to 8, wherein the oligomer comprises three or more tetramers of either streptavidin, avidin, a streptavidin mutein, or an avidin mutein.

10. The stimulating reagent comprises an oligomer of a streptavidin mutein molecule, the streptavidin mutein of the stimulating reagent having the amino acid sequence Val at sequence positions corresponding to positions 44 to 47 in reference to the amino acid sequence shown in SEQ ID NO:

1. 44 -Thr 45 -Ala 46 -Arg 47 or Ile 44 -Gly 45 -Ala 46 -Arg 47 The method of any one of claims 1 to 9, comprising:

11. The method of claim 10, wherein the streptavidin mutein of the stimulating reagent comprises an amino acid sequence set forth in any of SEQ ID NOs: 3-6, 27, and 28.

12. The method of any one of claims 1 to 11, wherein the first molecule expressed on the surface of the target cell is a member of a T cell or B cell antigen receptor complex or is a chimeric antigen receptor.

13. The method of any one of claims 1 to 12, wherein the first molecule expressed on the surface of the target cell is CD3.

14. 14. The method of any one of claims 1 to 13, wherein the first stimulatory agent comprises an antibody, an antibody fragment, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex, or a receptor ligand that specifically binds to the first molecule.

15. 15. The method of claim 14, wherein the first stimulatory agent comprises an antibody fragment, and the antibody fragment of the first stimulatory agent is a Fab fragment.

16. The method of any one of claims 1 to 15, wherein the second molecule is a costimulatory molecule, an accessory molecule, a cytokine receptor, a chemokine receptor, an immune checkpoint molecule, or a member of the TNF family or TNF receptor family.

17. The method of any one of claims 1 to 16, wherein the second molecule is CD28, CD137, CD40 ligand, CD40, OX40, or ICOS.

18. 18. The method of any one of claims 1 to 17, wherein the second stimulatory agent comprises an antibody, an antibody fragment, a cytokine, a chemokine, an aptamer, or a receptor ligand that specifically binds to the second molecule.

19. 20. The method of claim 18, wherein the second stimulatory agent comprises an antibody fragment, and the antibody fragment of the second stimulatory agent is a Fab fragment.

20. The method of any one of claims 1 to 19, wherein the first stimulatory agent comprises an anti-CD3 antibody or an antigen-binding fragment thereof, and the second stimulatory agent comprises an anti-CD28 antibody or an antigen-binding fragment thereof.

21. 21. The method of any one of claims 1 to 20, wherein the reversible binding of the first and / or second stimuli is disrupted by the addition of a substance comprising biotin, a biotin analogue, or a streptavidin-binding peptide.

22. 22. The method of claim 21, further comprising the step of disrupting the reversible binding of the first and / or second stimulatory agent by addition of said agent, said disruption causing a termination or reduction of stimulation of the cell.

23. 23. The method of claim 21 or 22, wherein the addition of the substance comprises the addition of D-biotin.

24. The method of any one of claims 1 to 23, wherein the chromatography matrix is ​​a non-magnetic or non-magnetizable material.

25. The method of any one of claims 1 to 24, wherein the chromatography matrix comprises a chromatography resin.

26. The method of any one of claims 1 to 25, wherein the target cells comprise lymphocytes.

27. The method of any one of claims 1 to 26, wherein the target cells comprise T cells.

28. 28. The method of any one of claims 1 to 27, wherein said incubation increases immune cell proliferation, alters immune cell differentiation, increases cytokine secretion, or increases cytotoxic activity.

29. The method according to any one of claims 1 to 28, further comprising the step of introducing a recombinant nucleic acid into the cells of the target cell, said nucleic acid encoding a recombinant protein, whereby said cells express the recombinant protein, wherein said introducing is performed while the cells are reversibly immobilized on a chromatography matrix.

30. The method of any one of claims 1 to 29, wherein the incubation is performed at a temperature of 37°C ± 5°C.

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