Methods, kits, and devices for expanding a population of cells

Abstract

To provide in vitro methods of expanding a population of cells such as lymphocytes.SOLUTION: A method comprises contacting a sample comprising a population of cells with a multimerization reagent. The multimerization reagent has reversibly immobilized thereon a first agent that provides a primary activation signal to the cells and optionally a second agent that provides a co-stimulatory signal. The present invention also provides multimerization reagents, kits, arrangements and an apparatus for expanding cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This invention claims the benefit of priority to U.S. Provisional Patent Application No. 61 / 980,506, entitled "Methods, Kits And Apparatus For Expanding A Population Of Cells," filed in the U.S. Patent and Trademark Office on April 16, 2014, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] FIELD OF THE INVENTION The present invention relates to the expansion (proliferation) of populations of cells, such as populations of lymphocytes. The present invention generally provides novel methods and reagents for expanding (proliferating) cell populations, which require binding of a receptor-binding molecule (e.g., a first agent described herein) to a receptor molecule on the surface of the cells to provide a primary activation signal to the cells. The present invention uses a multimerization reagent to which is immobilized (bound) a first agent that provides a primary activation signal to the cells. This primary activation signal may itself be sufficient to activate the cells to expand / proliferate. This first agent may bind reversibly or irreversibly to the multimerization reagent. A second agent that stimulates an accessory molecule on the surface of the cells may also be immobilized (bound) on the multimerization reagent. When the second agent binds to the accessory molecule on the surface of the cells, it can stimulate the activated cells to expand. This second agent may also bind reversibly or irreversibly to the multimerization reagent. The multimerizing agent may be immobilized on a solid support or may be soluble. In one embodiment, the method disclosed herein is the continuous expansion of a population of cells by stimulating / expanding an entire population of lymphocytes, then removing the reagents required for expansion by chromatography on a suitable stationary phase, and optionally transfecting the expanded / stimulated cells with, for example, a T cell receptor or chimeric antigen receptor (CAR) and subjecting them to a second stimulation expansion with a different stimulatory molecule that binds to the introduced T cell receptor or chimeric antigen receptor. The present invention also relates to an apparatus for expanding a selected cell population.

[0003] Background of the Invention The development of techniques for expanding T cell populations in vitro has been essential for many advances in our understanding of T cell recognition of antigens and T cell activation. The development of culture methods for generating human antigen-specific T cell clones is useful for identifying pathogen- and tumor-expressed antigens recognized by T cells in order to establish immunotherapeutic approaches to treat various human diseases. Antigen-specific T cells can be expanded in vitro for use in adoptive cellular immunotherapy or cancer therapy, where infusion of such T cells has been shown to have antitumor reactivity in tumor-bearing hosts. Furthermore, adoptive immunotherapy has also been used to treat viral infections in immunocompromised individuals.

[0004] Recently established methods for expanding human T cells in vitro in the absence of exogenous growth factors and accessory molecules are described in U.S. Pat. No. 6,352,694 B1 and European Patent No. 0 700 430 B1. These patents disclose in vitro methods for inducing and expanding a population of T cells. The methods involve contacting a population of T cells with a solid surface onto which are directly immobilized (a) a first agent that provides a primary activation signal to the T cells to activate the T cells, and (b) a second agent that stimulates accessory molecules on the surface of the T cells to stimulate the activated T cells. Binding of the first and second agents to the T cells induces proliferation / expansion of the T cells. The preferred first agent described in U.S. Pat. No. 6,352,694 B1 and European Patent No. 0 700 430 B1 is a monoclonal anti-CD3 antibody that binds to the TCR / CD3 (TCR = T cell receptor) complex and stimulates TCR / CD3 complex-associated signals in T cells. According to these two patents, the preferred second agent is a monoclonal anti-CD28 antibody that binds to the accessory molecule CD28 present on T cells. Binding of this second agent to the CD28 accessory molecule provides the essential costimulation required for the expansion / proliferation of activated T cells. Alternatively, Dynabeads® CD3 / CD28 (Invitrogen) are commercially available for the expansion of T cells. Dynabeads® CD3 / CD28 CTS™ are uniform 4.5 μm superparamagnetic, sterile, non-pyrogenic polystyrene beads coated with a mixture of affinity-purified monoclonal antibodies against the CD3 and CD28 cell surface molecules on human T cells.

[0005] However, such magnetic beads are difficult to incorporate into methods for expanding cells under conditions required for, for example, clinical trials or therapeutic purposes, because it must be ensured that the magnetic beads are completely removed before administering the expanded T cells to a patient. Therefore, the present invention aims to provide an alternative method for expanding cell populations, such as regulatory T cells or central memory T cells, for research, diagnostic, and, particularly, therapeutic purposes. Ideally, the novel method should also be amenable to incorporation into automated processes that can be used to rapidly and easily expand desired cell populations for therapeutic applications.

[0006] This object is solved by the subject matter of the independent claims, in particular by the methods, kits, arrangements and devices described in the independent claims.

[0007] Summary of the Invention The present invention provides methods, kits, arrangements, and devices for the in vitro expansion of a population of desired cells that have receptor molecules on their surface that, when bound by a suitable agent, can provide a primary activation signal to the population of cells, thereby activating said population of cells for expansion (proliferation). Thus, the methods of the present invention can also be used to induce a population of cells to proliferate.

[0008] According to a first aspect, the present invention provides an in vitro method of expanding a population of cells, comprising contacting a sample comprising said population of cells with a multimerization reagent, a first agent that provides a primary activation signal to the cell is reversibly immobilized (bound) to the multimerization reagent; the multimerization reagent comprises at least one binding site Z1 for reversibly binding of the first agent; the first agent comprises at least one binding partner C1, wherein the binding partner C1 can reversibly bind to the binding site Z1 of the multimerization reagent, and the first agent binds to the multimerization reagent via the reversible bond formed between the binding partner C1 and the binding site Z1; and The first agent binds to a receptor molecule on the surface of the cell to provide a primary activation signal to the cell, thereby activating the cell.

[0009] According to a second aspect, the present invention provides an in vitro method of expanding a population of cells, comprising contacting a sample comprising said population of cells with a multimerization reagent; the multimerization reagent is in a soluble form, and a first agent that provides a primary activation signal to the cell is immobilized (bound) to the multimerization reagent; the multimerization reagent comprises at least one binding site Z1 for binding of the first agent; the first agent comprises at least one binding partner C1, wherein the binding partner C1 is capable of binding to the binding site Z1 of the multimerization reagent, and the first agent binds to the multimerization reagent via a bond formed between the binding partner C1 and the binding site Z1; and The first agent binds to a receptor molecule on the surface of the cell to provide a primary activation signal to the cell, thereby activating the cell.

[0010] According to a third aspect, the present invention provides a reagent kit for expanding a population of cells, comprising: (i) a multimerization reagent comprising at least one binding site Z for reversibly binding a first agent; (ii) a first agent that binds to a receptor molecule on the surface of the cell to provide a primary activation signal to the cell, thereby activating the cell; and wherein the first agent comprises at least one binding partner C1, which can reversibly bind to the binding site Z1 of the multimerization reagent, and the first agent binds to the multimerization reagent via a reversible bond formed between the binding partner C1 and the binding site Z1; (iii) a second agent that stimulates an accessory molecule on the surface of the cell; and wherein the second agent comprises a binding partner C2, which can reversibly bind to binding site Z2 of the multimerization reagent, and the second agent binds to the multimerization reagent via a bond formed between the binding partner C2 and binding site Z2, and the second agent binds to an accessory molecule on the surface of the cell, thereby stimulating the activated cell. A reagent kit comprising:

[0011] According to a fourth aspect, the present invention provides a reagent kit for expanding a population of cells, comprising: (i) a multimerization reagent in soluble form and comprising at least one binding site Z for reversibly binding a first agent; (ii) a first agent that binds to a receptor molecule on the surface of the cell to provide a primary activation signal to the cell, thereby activating the cell; and wherein the first agent comprises at least one binding partner C1, the binding partner C1 being capable of binding to the binding site Z1 of the multimerization reagent, and the first agent binds to the multimerization reagent via a reversible bond formed between the binding partner C1 and the binding site Z1. A reagent kit comprising:

[0012] According to a fifth aspect, the present invention provides an in vitro method for serially expanding a population of lymphocytes, said population of lymphocytes comprising T cells, said method comprising: contacting a sample containing T cells comprising said population of lymphocytes with a multimerization reagent; the multimerization reagent is in a soluble form, and (i) a first agent that provides a primary activation signal to the T cell and (ii) a second agent that stimulates an accessory molecule on the surface of the T cell are reversibly immobilized on the multimerization reagent; the multimerization reagent comprises at least one binding site Z1 for reversibly binding of the first agent; the first agent comprises at least one binding partner C1, which can reversibly bind to the binding site Z1 of the multimerization reagent, and the first agent binds to the multimerization reagent via the reversible bond formed between the binding partner C1 and the binding site Z1; the multimerization reagent comprises at least one binding site Z2 for reversibly binding of the second agent; the second agent comprises at least one binding partner C2, which can reversibly bind to the binding site Z2 of the multimerization reagent; and the first agent binds to the multimerization reagent via a reversible bond formed between the binding partner C2 and the binding site Z2; the first agent binds to a receptor molecule on the surface of the T cell to provide a primary activation signal to the cell, thereby activating the T cell; The second agent binds to the accessory molecule on the surface of the T cell and stimulates the activated cell, whereby the first agent and the second agent together induce the T cell to expand.

[0013] According to a sixth aspect, the present invention provides a bioreactor and a stationary phase arrangement for chromatography, comprising: the bioreactor is suitable for cell expansion; the stationary phase is suitable for cell separation and reagent removal, the stationary phase being a gel filtration matrix and / or an affinity chromatography matrix, the gel filtration and / or affinity chromatography matrix comprising an affinity reagent, the affinity reagent comprising a binding site Z1 that specifically binds to a binding partner C1 comprised in a first agent, and / or the affinity reagent comprising a binding site Z2 that specifically binds to a binding partner C2 comprised in a second agent, thereby being suitable for immobilizing the first agent and / or the second agent, the first binding partner C1 and / or the free second binding partner C2 on the stationary phase, The bioreactor and the stationary phase are provided in a fluid connection arrangement.

[0014] According to a seventh aspect, the present invention provides an apparatus for purifying and expanding a population of cells, the apparatus comprising a bioreactor according to the sixth aspect and at least one arrangement of a stationary phase for chromatography.

[0015] According to an eighth aspect, the present invention provides a multimerization reagent capable of expanding a population of cells, comprising: in soluble form and comprising at least one binding site Z1 for reversibly binding a first agent that provides a primary activation signal to said cell; the first agent that provides a primary activation signal to the cell is reversibly immobilized (bound) to the multimerization reagent; the first agent comprises at least one binding partner C1, wherein the binding partner C1 is capable of reversibly binding to the at least one binding site Z1 of the multimerization reagent; the first agent binds to the multimerization reagent via a reversible bond that can be formed between the binding partner C1 and the binding site Z1; A multimerization reagent is provided.

[0016] According to a ninth aspect, the present invention provides a composition capable of expanding a population of cells, comprising: (i) a first multimerization reagent in soluble form and comprising at least one binding site Z1 for reversibly binding a first agent that provides a primary activation signal to said cell, the first agent that provides a primary activation signal to the cell is reversibly immobilized (bound) to the first multimerization reagent; a first multimerization reagent, wherein the first agent comprises at least one binding partner C1, which can reversibly bind to the at least one binding site Z1 of the multimerization reagent, and the first agent binds to the multimerization reagent via the reversible bond formed between the binding partner C1 and the binding site Z1; (ii) a second multimerization reagent in soluble form and comprising at least one binding site Z2 for reversible binding of a second agent that stimulates an accessory molecule on the surface of said cell, the second agent, which stimulates an accessory molecule on the surface of the cell, is reversibly immobilized (bound) to the multimerization reagent; the second agent comprises a binding partner C2, which is capable of binding to the at least one binding site Z2 of the multimerization reagent; and a second multimerization reagent, which binds to the multimerization reagent via a bond formed between the binding partner C2 and the binding site Z2. [Brief explanation of the drawings]

[0017] The present invention will be better understood by reference to the detailed description when considered in conjunction with the non-limiting examples and accompanying drawings. The drawings illustrate embodiments of the methods of the present invention. Without being bound by theory, the drawings include conclusions regarding the underlying expansion mechanism. The conclusions are provided for illustrative purposes only and merely serve to visualize that the expansion method can be achieved at the molecular level. [Figure 1A]Figure 1 illustrates an embodiment of an in vitro expansion method for expanding a population of cells bearing a cell surface receptor to which a first agent can bind, thereby providing an activation signal for cell expansion. As shown in Figure 1a, a sample containing a population of cells (2) bearing a surface receptor molecule (30) is contacted with a multimerization reagent (4). The population of cells (2) is present in a mixture with another population of cells (22) that does not possess the surface receptor molecule (30). A first agent (6) that provides a primary activation signal to the cells is reversibly immobilized (bound) to the multimerization reagent (4). The multimerization reagent (4) contains at least one binding site Z1 (42) for reversible binding of the first agent (6), which in turn contains at least one binding partner C1 (6a), capable of reversibly binding to the binding site Z1 (44) of the multimerization reagent. Thus, for immobilization, the first agent (6) binds to the multimerization reagent (4) via a reversible bond formed between the binding partner C1 (6a) and the binding site Z1 (42). In the example shown in Figure 1, the multimerization reagent (4) has a second binding site Z2 (44), which is not used in this example. The multimerization reagent (4) is itself immobilized on a solid support (10), such as magnetic beads, polymer beads, or the surface of a cell culture plate or reactor. Population of cells (2) can be, for example, a lymphocyte cell population, such as a population of B cells that can be activated via the CD40 receptor (see, e.g., Carpenter et al., Journal of Translational Medicine 2009, 7:93 "Activation of human B cells by the agonist CD40 antibody CP-870,893 and augmentation with simultaneous toll-like receptor 9 stimulation"). In this case, cell surface receptor (30) is CD40, and first agent (6) can be any CD40-binding molecule that provides the desired activation signal, e.g., monoclonal antibody CP-870,893, or an antibody-binding fragment thereof, such as a monovalent Fab fragment.The binding partner C1 of the first agent (6) can be, for example, any affinity peptide fused or conjugated to the C-terminus of one of the two polypeptide chains (heavy or light) of an antibody molecule. The binding partner C1 (6a) can be, for example, a streptavidin-binding peptide, such as the peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 01) (also known as "Strep-tag®") described in U.S. Pat. No. 5,506,121, or a streptavidin-binding peptide having a sequential arrangement of two or more individual binding molecules as described in WO 02 / 077018 or U.S. Pat. No. 7,981,632. When a streptavidin-binding peptide is used as binding partner C1, the multimerization reagent (4) can be any streptavidin mutein to which the streptavidin peptide (=first binding partner C1 (6a)) reversibly binds via its (biotin) binding site Z1 (42), as shown diagrammatically in Figure 1. Such a multimerization reagent may be a streptavidin mutein (analog) comprising the amino acid sequence Va144-Thr45-Ala46-Arg47 (SEQ ID NO: 02) at positions 44-47 of wild-type streptavidin, or a streptavidin mutein (analog) comprising the amino acid sequence Ile44-Gly45-Ala46-Arg47 (SEQ ID NO: 03) at positions 44-47 of wild-type streptavidin, both of which are described, for example, in U.S. Patent No. 6,103,493 and are commercially available under the trademark Strep-Tactin®. In the example of Figure 1, the multimerization reagent (4) may further comprise a multimeric calmodulin or glutathione-S-transferase, either of which forms a reversible bond with a calmodulin-binding peptide or glutathione. Thus, binding site Z2 (44) can be formed by calmodulin or glutathione-S-transferase. Such protein conjugates, for example, of calmodulin and streptavidin muteins, can be prepared by standard protein chemistry, for example, by using bifunctional linkers.As shown in Figure 1b, cell population (2) is contacted with multimerization reagent (4), and typically after incubation with the multimerization reagent (4), cell population (2) complexes / binds with the multimerization reagent via first agent (6). The first agent specifically binds to a cell surface receptor molecule, such as C40 in this example, and provides an activation signal for cell expansion, e.g., of B cells. Other cell populations (22) contained in the initial sample that do not have the specific cell surface molecule (30) do not bind to the multimerization reagent. In this regard, it is noted that cell population (2) typically has multiple copies of cell surface molecule (30) on its surface, and binding of these multiple copies is typically required for activation. Thus, the multimerizing agent (4) typically provides more than one binding site Z1 (not shown in the scheme of FIG. 1 ) so that multiple first agents (6) can reversibly bind to achieve "multimerization" of the first agents, i.e., present a sufficient density of first agents to the cell population (2). In this regard, as used herein, it should be noted that the multimerizing agent itself may have multiple binding sites Z1; for example, a streptavidin mutein (which is a homotetramer) in its native state has four such binding sites Z1. However, the multimerizing reagent may also be based on a compound that itself has only one binding site Z1 for the reversible binding of the binding partner C1. An example of such a compound is multimeric calmodulin. Calmodulin itself has only one binding site for a calmodulin-binding peptide. However, calmodulin can be biotinylated and then reacted with streptavidin-oligomers (see also below) to provide a multimerization reagent in which multiple calmodulin molecules are densely displayed on a "scaffold," thereby providing multimeric calmodulin. As shown in Figure 1c, after incubation (typically for a period of time suitable for expanding the desired cell population), the binding between the binding partner C1 (6a) of the first agent (6) and the binding site Z1 of the multimerization reagent (4) is separated by dissociating their respective reversible bonds.This separation can be achieved by adding a competitor to the incubation / reaction mixture containing the population of cells (2) bound to the multimerization reagent. For competitive separation of the reversible binding between the binding partner C1 (6a) of the first agent and the binding moiety Z1 (22) of the multimerization reagent (which can be understood as competitive elution), the incubation mixture / population of cells can be contacted with the free first binding partner C1 (20) or an analog of the first binding partner C that can separate the binding between the first binding partner C1 (6a) and the binding moiety Z1 (22). In the example of binding partner C1 being a streptavidin-binding peptide that binds to the biotin-binding site of streptavidin, the free first partner C1 (20) can be the corresponding free streptavidin-binding peptide or a competitively binding analog. Such an analog can be, for example, biotin or a biotin derivative such as desthiobiotin. As shown in Figure 1d, addition of the first free partner 20 or its analog displaces the binding partner C1 (6a) from the multimerization reagent 4, which, in turn, displaces the first agent 6 from the multimerization reagent 4, since the binding partner C1 is contained in the first agent 6. This displacement of the first agent 6 then dissociates the first agent 6 from the cell surface receptor 30, particularly if the binding affinity between the first agent and the cell surface receptor 30 has a dissociation constant (Kd) in the range of 10-2 M to 10-13 M and is therefore reversible. This dissociation also terminates stimulation of the cell population 2. Thus, the present invention offers the advantage of precisely controlling the duration of stimulation or expansion of the cell population, thereby tightly controlling the functional state of the cell population. It is noted in this context that the binding affinity of an antibody molecule for its antigen (which in this example includes a cell surface receptor molecule such as CD40) typically falls within an affinity range of Kd of 10 M to 10 M. Thus, conventional monoclonal antibodies can be used as first agents in the present invention (and, of course, as second agents, as described below).To avoid any undesirable avidity effects that lead to stronger binding, monoclonal antibodies can also be used in the form of monovalent antibody fragments, such as Fab fragments or single-chain Fv fragments. Furthermore, due to the dissociation of the first agent from the cell surface molecule (30), the present invention has the additional advantage that the stimulated cell population (2) is free of the stimulating agent at the end of the stimulation period, and all other reagents used in the method, i.e., the first agent (6) and the free first partner (20) of binding partner C1 or its analog, can be easily removed from the stimulated cell population (2) via the "removal cartridge" described in WO 2013 / 124474, while retaining the multimerization reagent (4) immobilized on a solid support, such as a bioreactor surface or magnetic beads. Returning to the removal of the free agent (6) and the free first partner (20), the eluate sample obtained in FIG. 1d of the present application may be loaded onto the second chromatography column of WO 2013 / 124474, as described in the "removal cartridge" in WO 2013 / 124474 (see, for example, FIG. 4 therein). This chromatography column has a suitable stationary phase that can act as both an affinity chromatography matrix and a gel permeation matrix. An affinity reagent is immobilized on this affinity chromatography matrix. In this example, the affinity reagent may be, for example, streptavidin, streptavidin mutein, avidin, avidin mutein, or a mixture thereof. The free first agent (6), the first partner (20) of binding partner C1 (also referred to herein as the "competitor reagent"), binds to the affinity reagent and is immobilized on the chromatography matrix. As a result, the eluate sample containing the isolated and expanded cell population 2 is free of the first agent 6 and the competing reagent 20. The expanded cell population 2, free of any reactants, is ready for further use, for example, for diagnostic applications (e.g., further FACS™ sorting) or any cell-based therapeutic application. [Figure 1B]Figure 1 illustrates an embodiment of an in vitro expansion method for expanding a population of cells bearing a cell surface receptor to which a first agent can bind, thereby providing an activation signal for cell expansion. As shown in Figure 1a, a sample containing a population of cells (2) bearing a surface receptor molecule (30) is contacted with a multimerization reagent (4). The population of cells (2) is present in a mixture with another population of cells (22) that does not possess the surface receptor molecule (30). A first agent (6) that provides a primary activation signal to the cells is reversibly immobilized (bound) to the multimerization reagent (4). The multimerization reagent (4) contains at least one binding site Z1 (42) for reversible binding of the first agent (6), which in turn contains at least one binding partner C1 (6a), capable of reversibly binding to the binding site Z1 (44) of the multimerization reagent. Thus, for immobilization, the first agent (6) binds to the multimerization reagent (4) via a reversible bond formed between the binding partner C1 (6a) and the binding site Z1 (42). In the example shown in Figure 1, the multimerization reagent (4) has a second binding site Z2 (44), which is not used in this example. The multimerization reagent (4) is itself immobilized on a solid support (10), such as magnetic beads, polymer beads, or the surface of a cell culture plate or reactor. Population of cells (2) can be, for example, a lymphocyte cell population, such as a population of B cells that can be activated via the CD40 receptor (see, e.g., Carpenter et al., Journal of Translational Medicine 2009, 7:93 "Activation of human B cells by the agonist CD40 antibody CP-870,893 and augmentation with simultaneous toll-like receptor 9 stimulation"). In this case, cell surface receptor (30) is CD40, and first agent (6) can be any CD40-binding molecule that provides the desired activation signal, e.g., monoclonal antibody CP-870,893, or an antibody-binding fragment thereof, such as a monovalent Fab fragment.The binding partner C1 of the first agent (6) can be, for example, any affinity peptide fused or conjugated to the C-terminus of one of the two polypeptide chains (heavy or light) of an antibody molecule. The binding partner C1 (6a) can be, for example, a streptavidin-binding peptide, such as the peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 01) (also known as "Strep-tag®") described in U.S. Pat. No. 5,506,121, or a streptavidin-binding peptide having a sequential arrangement of two or more individual binding molecules as described in WO 02 / 077018 or U.S. Pat. No. 7,981,632. When a streptavidin-binding peptide is used as binding partner C1, the multimerization reagent (4) can be any streptavidin mutein to which the streptavidin peptide (=first binding partner C1 (6a)) reversibly binds via its (biotin) binding site Z1 (42), as shown diagrammatically in Figure 1. Such a multimerization reagent may be a streptavidin mutein (analog) comprising the amino acid sequence Va144-Thr45-Ala46-Arg47 (SEQ ID NO: 02) at positions 44-47 of wild-type streptavidin, or a streptavidin mutein (analog) comprising the amino acid sequence Ile44-Gly45-Ala46-Arg47 (SEQ ID NO: 03) at positions 44-47 of wild-type streptavidin, both of which are described, for example, in U.S. Patent No. 6,103,493 and are commercially available under the trademark Strep-Tactin®. In the example of Figure 1, the multimerization reagent (4) may further comprise a multimeric calmodulin or glutathione-S-transferase, either of which forms a reversible bond with a calmodulin-binding peptide or glutathione. Thus, binding site Z2 (44) can be formed by calmodulin or glutathione-S-transferase. Such protein conjugates, for example, of calmodulin and streptavidin muteins, can be prepared by standard protein chemistry, for example, by using bifunctional linkers.As shown in Figure 1b, cell population (2) is contacted with multimerization reagent (4), and typically after incubation with the multimerization reagent (4), cell population (2) complexes / binds with the multimerization reagent via first agent (6). The first agent specifically binds to a cell surface receptor molecule, such as C40 in this example, and provides an activation signal for cell expansion, e.g., of B cells. Other cell populations (22) contained in the initial sample that do not have the specific cell surface molecule (30) do not bind to the multimerization reagent. In this regard, it is noted that cell population (2) typically has multiple copies of cell surface molecule (30) on its surface, and binding of these multiple copies is typically required for activation. Thus, the multimerizing agent (4) typically provides more than one binding site Z1 (not shown in the scheme of FIG. 1 ) so that multiple first agents (6) can reversibly bind to achieve "multimerization" of the first agents, i.e., present a sufficient density of first agents to the cell population (2). In this regard, as used herein, it should be noted that the multimerizing agent itself may have multiple binding sites Z1; for example, a streptavidin mutein (which is a homotetramer) in its native state has four such binding sites Z1. However, the multimerizing reagent may also be based on a compound that itself has only one binding site Z1 for the reversible binding of the binding partner C1. An example of such a compound is multimeric calmodulin. Calmodulin itself has only one binding site for a calmodulin-binding peptide. However, calmodulin can be biotinylated and then reacted with streptavidin-oligomers (see also below) to provide a multimerization reagent in which multiple calmodulin molecules are densely displayed on a "scaffold," thereby providing multimeric calmodulin. As shown in Figure 1c, after incubation (typically for a period of time suitable for expanding the desired cell population), the binding between the binding partner C1 (6a) of the first agent (6) and the binding site Z1 of the multimerization reagent (4) is separated by dissociating their respective reversible bonds.This separation can be achieved by adding a competitor to the incubation / reaction mixture containing the population of cells (2) bound to the multimerization reagent. For competitive separation of the reversible binding between the binding partner C1 (6a) of the first agent and the binding moiety Z1 (22) of the multimerization reagent (which can be understood as competitive elution), the incubation mixture / population of cells can be contacted with the free first binding partner C1 (20) or an analog of the first binding partner C that can separate the binding between the first binding partner C1 (6a) and the binding moiety Z1 (22). In the example of binding partner C1 being a streptavidin-binding peptide that binds to the biotin-binding site of streptavidin, the free first partner C1 (20) can be the corresponding free streptavidin-binding peptide or a competitively binding analog. Such an analog can be, for example, biotin or a biotin derivative such as desthiobiotin. As shown in Figure 1d, addition of the first free partner 20 or its analog displaces the binding partner C1 (6a) from the multimerization reagent 4, which, in turn, displaces the first agent 6 from the multimerization reagent 4, since the binding partner C1 is contained in the first agent 6. This displacement of the first agent 6 then dissociates the first agent 6 from the cell surface receptor 30, particularly if the binding affinity between the first agent and the cell surface receptor 30 has a dissociation constant (Kd) in the range of 10-2 M to 10-13 M and is therefore reversible. This dissociation also terminates stimulation of the cell population 2. Thus, the present invention offers the advantage of precisely controlling the duration of stimulation or expansion of the cell population, thereby tightly controlling the functional state of the cell population. It is noted in this context that the binding affinity of an antibody molecule for its antigen (which in this example includes a cell surface receptor molecule such as CD40) typically falls within an affinity range of Kd of 10 M to 10 M. Thus, conventional monoclonal antibodies can be used as first agents in the present invention (and, of course, as second agents, as described below).To avoid any undesirable avidity effects that lead to stronger binding, monoclonal antibodies can also be used in the form of monovalent antibody fragments, such as Fab fragments or single-chain Fv fragments. Furthermore, due to the dissociation of the first agent from the cell surface molecule (30), the present invention has the additional advantage that the stimulated cell population (2) is free of the stimulating agent at the end of the stimulation period, and all other reagents used in the method, i.e., the first agent (6) and the free first partner (20) of binding partner C1 or its analog, can be easily removed from the stimulated cell population (2) via the "removal cartridge" described in WO 2013 / 124474, while retaining the multimerization reagent (4) immobilized on a solid support, such as a bioreactor surface or magnetic beads. Returning to the removal of the free agent (6) and the free first partner (20), the eluate sample obtained in FIG. 1d of the present application may be loaded onto the second chromatography column of WO 2013 / 124474, as described in the "removal cartridge" in WO 2013 / 124474 (see, for example, FIG. 4 therein). This chromatography column has a suitable stationary phase that can act as both an affinity chromatography matrix and a gel permeation matrix. An affinity reagent is immobilized on this affinity chromatography matrix. In this example, the affinity reagent may be, for example, streptavidin, streptavidin mutein, avidin, avidin mutein, or a mixture thereof. The free first agent (6), the first partner (20) of binding partner C1 (also referred to herein as the "competitor reagent"), binds to the affinity reagent and is immobilized on the chromatography matrix. As a result, the eluate sample containing the isolated and expanded cell population 2 is free of the first agent 6 and the competing reagent 20. The expanded cell population 2, free of any reactants, is ready for further use, for example, for diagnostic applications (e.g., further FACS™ sorting) or any cell-based therapeutic application. [Figure 1C]Figure 1 illustrates an embodiment of an in vitro expansion method for expanding a population of cells bearing a cell surface receptor to which a first agent can bind, thereby providing an activation signal for cell expansion. As shown in Figure 1a, a sample containing a population of cells (2) bearing a surface receptor molecule (30) is contacted with a multimerization reagent (4). The population of cells (2) is present in a mixture with another population of cells (22) that does not possess the surface receptor molecule (30). A first agent (6) that provides a primary activation signal to the cells is reversibly immobilized (bound) to the multimerization reagent (4). The multimerization reagent (4) contains at least one binding site Z1 (42) for reversible binding of the first agent (6), which in turn contains at least one binding partner C1 (6a), capable of reversibly binding to the binding site Z1 (44) of the multimerization reagent. Thus, for immobilization, the first agent (6) binds to the multimerization reagent (4) via a reversible bond formed between the binding partner C1 (6a) and the binding site Z1 (42). In the example shown in Figure 1, the multimerization reagent (4) has a second binding site Z2 (44), which is not used in this example. The multimerization reagent (4) is itself immobilized on a solid support (10), such as magnetic beads, polymer beads, or the surface of a cell culture plate or reactor. Population of cells (2) can be, for example, a lymphocyte cell population, such as a population of B cells that can be activated via the CD40 receptor (see, e.g., Carpenter et al., Journal of Translational Medicine 2009, 7:93 "Activation of human B cells by the agonist CD40 antibody CP-870,893 and augmentation with simultaneous toll-like receptor 9 stimulation"). In this case, cell surface receptor (30) is CD40, and first agent (6) can be any CD40-binding molecule that provides the desired activation signal, e.g., monoclonal antibody CP-870,893, or an antibody-binding fragment thereof, such as a monovalent Fab fragment.The binding partner C1 of the first agent (6) can be, for example, any affinity peptide fused or conjugated to the C-terminus of one of the two polypeptide chains (heavy or light) of an antibody molecule. The binding partner C1 (6a) can be, for example, a streptavidin-binding peptide, such as the peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 01) (also known as "Strep-tag®") described in U.S. Pat. No. 5,506,121, or a streptavidin-binding peptide having a sequential arrangement of two or more individual binding molecules as described in WO 02 / 077018 or U.S. Pat. No. 7,981,632. When a streptavidin-binding peptide is used as binding partner C1, the multimerization reagent (4) can be any streptavidin mutein to which the streptavidin peptide (=first binding partner C1 (6a)) reversibly binds via its (biotin) binding site Z1 (42), as shown diagrammatically in Figure 1. Such a multimerization reagent may be a streptavidin mutein (analog) comprising the amino acid sequence Va144-Thr45-Ala46-Arg47 (SEQ ID NO: 02) at positions 44-47 of wild-type streptavidin, or a streptavidin mutein (analog) comprising the amino acid sequence Ile44-Gly45-Ala46-Arg47 (SEQ ID NO: 03) at positions 44-47 of wild-type streptavidin, both of which are described, for example, in U.S. Patent No. 6,103,493 and are commercially available under the trademark Strep-Tactin®. In the example of Figure 1, the multimerization reagent (4) may further comprise a multimeric calmodulin or glutathione-S-transferase, either of which forms a reversible bond with a calmodulin-binding peptide or glutathione. Thus, binding site Z2 (44) can be formed by calmodulin or glutathione-S-transferase. Such protein conjugates, for example, of calmodulin and streptavidin muteins, can be prepared by standard protein chemistry, for example, by using bifunctional linkers.As shown in Figure 1b, cell population (2) is contacted with multimerization reagent (4), and typically after incubation with the multimerization reagent (4), cell population (2) complexes / binds with the multimerization reagent via first agent (6). The first agent specifically binds to a cell surface receptor molecule, such as C40 in this example, and provides an activation signal for cell expansion, e.g., of B cells. Other cell populations (22) contained in the initial sample that do not have the specific cell surface molecule (30) do not bind to the multimerization reagent. In this regard, it is noted that cell population (2) typically has multiple copies of cell surface molecule (30) on its surface, and binding of these multiple copies is typically required for activation. Thus, the multimerizing agent (4) typically provides more than one binding site Z1 (not shown in the scheme of FIG. 1 ) so that multiple first agents (6) can reversibly bind to achieve "multimerization" of the first agents, i.e., present a sufficient density of first agents to the cell population (2). In this regard, as used herein, it should be noted that the multimerizing agent itself may have multiple binding sites Z1; for example, a streptavidin mutein (which is a homotetramer) in its native state has four such binding sites Z1. However, the multimerizing reagent may also be based on a compound that itself has only one binding site Z1 for the reversible binding of the binding partner C1. An example of such a compound is multimeric calmodulin. Calmodulin itself has only one binding site for a calmodulin-binding peptide. However, calmodulin can be biotinylated and then reacted with streptavidin-oligomers (see also below) to provide a multimerization reagent in which multiple calmodulin molecules are densely displayed on a "scaffold," thereby providing multimeric calmodulin. As shown in Figure 1c, after incubation (typically for a period of time suitable for expanding the desired cell population), the binding between the binding partner C1 (6a) of the first agent (6) and the binding site Z1 of the multimerization reagent (4) is separated by dissociating their respective reversible bonds.This separation can be achieved by adding a competitor to the incubation / reaction mixture containing the population of cells (2) bound to the multimerization reagent. For competitive separation of the reversible binding between the binding partner C1 (6a) of the first agent and the binding moiety Z1 (22) of the multimerization reagent (which can be understood as competitive elution), the incubation mixture / population of cells can be contacted with the free first binding partner C1 (20) or an analog of the first binding partner C that can separate the binding between the first binding partner C1 (6a) and the binding moiety Z1 (22). In the example of binding partner C1 being a streptavidin-binding peptide that binds to the biotin-binding site of streptavidin, the free first partner C1 (20) can be the corresponding free streptavidin-binding peptide or a competitively binding analog. Such an analog can be, for example, biotin or a biotin derivative such as desthiobiotin. As shown in Figure 1d, addition of the first free partner 20 or its analog displaces the binding partner C1 (6a) from the multimerization reagent 4, which, in turn, displaces the first agent 6 from the multimerization reagent 4, since the binding partner C1 is contained in the first agent 6. This displacement of the first agent 6 then dissociates the first agent 6 from the cell surface receptor 30, particularly if the binding affinity between the first agent and the cell surface receptor 30 has a dissociation constant (Kd) in the range of 10-2 M to 10-13 M and is therefore reversible. This dissociation also terminates stimulation of the cell population 2. Thus, the present invention offers the advantage of precisely controlling the duration of stimulation or expansion of the cell population, thereby tightly controlling the functional state of the cell population. It is noted in this context that the binding affinity of an antibody molecule for its antigen (which in this example includes a cell surface receptor molecule such as CD40) typically falls within an affinity range of Kd of 10 M to 10 M. Thus, conventional monoclonal antibodies can be used as first agents in the present invention (and, of course, as second agents, as described below).To avoid any undesirable avidity effects that lead to stronger binding, monoclonal antibodies can also be used in the form of monovalent antibody fragments, such as Fab fragments or single-chain Fv fragments. Furthermore, due to the dissociation of the first agent from the cell surface molecule (30), the present invention has the additional advantage that the stimulated cell population (2) is free of the stimulating agent at the end of the stimulation period, and all other reagents used in the method, i.e., the first agent (6) and the free first partner (20) of binding partner C1 or its analog, can be easily removed from the stimulated cell population (2) via the "removal cartridge" described in WO 2013 / 124474, while retaining the multimerization reagent (4) immobilized on a solid support, such as a bioreactor surface or magnetic beads. Returning to the removal of the free agent (6) and the free first partner (20), the eluate sample obtained in FIG. 1d of the present application may be loaded onto the second chromatography column of WO 2013 / 124474, as described in the "removal cartridge" in WO 2013 / 124474 (see, for example, FIG. 4 therein). This chromatography column has a suitable stationary phase that can act as both an affinity chromatography matrix and a gel permeation matrix. An affinity reagent is immobilized on this affinity chromatography matrix. In this example, the affinity reagent may be, for example, streptavidin, streptavidin mutein, avidin, avidin mutein, or a mixture thereof. The free first agent (6), the first partner (20) of binding partner C1 (also referred to herein as the "competitor reagent"), binds to the affinity reagent and is immobilized on the chromatography matrix. As a result, the eluate sample containing the isolated and expanded cell population 2 is free of the first agent 6 and the competing reagent 20. The expanded cell population 2, free of any reactants, is ready for further use, for example, for diagnostic applications (e.g., further FACS™ sorting) or any cell-based therapeutic application. [Figure 1D]Figure 1 illustrates an embodiment of an in vitro expansion method for expanding a population of cells bearing a cell surface receptor to which a first agent can bind, thereby providing an activation signal for cell expansion. As shown in Figure 1a, a sample containing a population of cells (2) bearing a surface receptor molecule (30) is contacted with a multimerization reagent (4). The population of cells (2) is present in a mixture with another population of cells (22) that does not possess the surface receptor molecule (30). A first agent (6) that provides a primary activation signal to the cells is reversibly immobilized (bound) to the multimerization reagent (4). The multimerization reagent (4) contains at least one binding site Z1 (42) for reversible binding of the first agent (6), which in turn contains at least one binding partner C1 (6a), capable of reversibly binding to the binding site Z1 (44) of the multimerization reagent. Thus, for immobilization, the first agent (6) binds to the multimerization reagent (4) via a reversible bond formed between the binding partner C1 (6a) and the binding site Z1 (42). In the example shown in Figure 1, the multimerization reagent (4) has a second binding site Z2 (44), which is not used in this example. The multimerization reagent (4) is itself immobilized on a solid support (10), such as magnetic beads, polymer beads, or the surface of a cell culture plate or reactor. Population of cells (2) can be, for example, a lymphocyte cell population, such as a population of B cells that can be activated via the CD40 receptor (see, e.g., Carpenter et al., Journal of Translational Medicine 2009, 7:93 "Activation of human B cells by the agonist CD40 antibody CP-870,893 and augmentation with simultaneous toll-like receptor 9 stimulation"). In this case, cell surface receptor (30) is CD40, and first agent (6) can be any CD40-binding molecule that provides the desired activation signal, e.g., monoclonal antibody CP-870,893, or an antibody-binding fragment thereof, such as a monovalent Fab fragment.The binding partner C1 of the first agent (6) can be, for example, any affinity peptide fused or conjugated to the C-terminus of one of the two polypeptide chains (heavy or light) of an antibody molecule. The binding partner C1 (6a) can be, for example, a streptavidin-binding peptide, such as the peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 01) (also known as "Strep-tag®") described in U.S. Pat. No. 5,506,121, or a streptavidin-binding peptide having a sequential arrangement of two or more individual binding molecules as described in WO 02 / 077018 or U.S. Pat. No. 7,981,632. When a streptavidin-binding peptide is used as binding partner C1, the multimerization reagent (4) can be any streptavidin mutein to which the streptavidin peptide (=first binding partner C1 (6a)) reversibly binds via its (biotin) binding site Z1 (42), as shown diagrammatically in Figure 1. Such a multimerization reagent may be a streptavidin mutein (analog) comprising the amino acid sequence Va144-Thr45-Ala46-Arg47 (SEQ ID NO: 02) at positions 44-47 of wild-type streptavidin, or a streptavidin mutein (analog) comprising the amino acid sequence Ile44-Gly45-Ala46-Arg47 (SEQ ID NO: 03) at positions 44-47 of wild-type streptavidin, both of which are described, for example, in U.S. Patent No. 6,103,493 and are commercially available under the trademark Strep-Tactin®. In the example of Figure 1, the multimerization reagent (4) may further comprise a multimeric calmodulin or glutathione-S-transferase, either of which forms a reversible bond with a calmodulin-binding peptide or glutathione. Thus, binding site Z2 (44) can be formed by calmodulin or glutathione-S-transferase. Such protein conjugates, for example, of calmodulin and streptavidin muteins, can be prepared by standard protein chemistry, for example, by using bifunctional linkers.As shown in Figure 1b, cell population (2) is contacted with multimerization reagent (4), and typically after incubation with the multimerization reagent (4), cell population (2) complexes / binds with the multimerization reagent via first agent (6). The first agent specifically binds to a cell surface receptor molecule, such as C40 in this example, and provides an activation signal for cell expansion, e.g., of B cells. Other cell populations (22) contained in the initial sample that do not have the specific cell surface molecule (30) do not bind to the multimerization reagent. In this regard, it is noted that cell population (2) typically has multiple copies of cell surface molecule (30) on its surface, and binding of these multiple copies is typically required for activation. Thus, the multimerizing agent (4) typically provides more than one binding site Z1 (not shown in the scheme of FIG. 1 ) so that multiple first agents (6) can reversibly bind to achieve "multimerization" of the first agents, i.e., present a sufficient density of first agents to the cell population (2). In this regard, as used herein, it should be noted that the multimerizing agent itself may have multiple binding sites Z1; for example, a streptavidin mutein (which is a homotetramer) in its native state has four such binding sites Z1. However, the multimerizing reagent may also be based on a compound that itself has only one binding site Z1 for the reversible binding of the binding partner C1. An example of such a compound is multimeric calmodulin. Calmodulin itself has only one binding site for a calmodulin-binding peptide. However, calmodulin can be biotinylated and then reacted with streptavidin-oligomers (see also below) to provide a multimerization reagent in which multiple calmodulin molecules are densely displayed on a "scaffold," thereby providing multimeric calmodulin. As shown in Figure 1c, after incubation (typically for a period of time suitable for expanding the desired cell population), the binding between the binding partner C1 (6a) of the first agent (6) and the binding site Z1 of the multimerization reagent (4) is separated by dissociating their respective reversible bonds.This separation can be achieved by adding a competitor to the incubation / reaction mixture containing the population of cells (2) bound to the multimerization reagent. For competitive separation of the reversible binding between the binding partner C1 (6a) of the first agent and the binding moiety Z1 (22) of the multimerization reagent (which can be understood as competitive elution), the incubation mixture / population of cells can be contacted with the free first binding partner C1 (20) or an analog of the first binding partner C that can separate the binding between the first binding partner C1 (6a) and the binding moiety Z1 (22). In the example of binding partner C1 being a streptavidin-binding peptide that binds to the biotin-binding site of streptavidin, the free first partner C1 (20) can be the corresponding free streptavidin-binding peptide or a competitively binding analog. Such an analog can be, for example, biotin or a biotin derivative such as desthiobiotin. As shown in Figure 1d, addition of the first free partner 20 or its analog displaces the binding partner C1 (6a) from the multimerization reagent 4, which, in turn, displaces the first agent 6 from the multimerization reagent 4, since the binding partner C1 is contained in the first agent 6. This displacement of the first agent 6 then dissociates the first agent 6 from the cell surface receptor 30, particularly if the binding affinity between the first agent and the cell surface receptor 30 has a dissociation constant (Kd) in the range of 10-2 M to 10-13 M and is therefore reversible. This dissociation also terminates stimulation of the cell population 2. Thus, the present invention offers the advantage of precisely controlling the duration of stimulation or expansion of the cell population, thereby tightly controlling the functional state of the cell population. It is noted in this context that the binding affinity of an antibody molecule for its antigen (which in this example includes a cell surface receptor molecule such as CD40) typically falls within an affinity range of Kd of 10 M to 10 M. Thus, conventional monoclonal antibodies can be used as first agents in the present invention (and, of course, as second agents, as described below).To avoid any undesirable avidity effects that lead to stronger binding, monoclonal antibodies can also be used in the form of monovalent antibody fragments, such as Fab fragments or single-chain Fv fragments. Furthermore, due to the dissociation of the first agent from the cell surface molecule (30), the present invention has the additional advantage that the stimulated cell population (2) is free of the stimulating agent at the end of the stimulation period, and all other reagents used in the method, i.e., the first agent (6) and the free first partner (20) of binding partner C1 or its analog, can be easily removed from the stimulated cell population (2) via the "removal cartridge" described in WO 2013 / 124474, while retaining the multimerization reagent (4) immobilized on a solid support, such as a bioreactor surface or magnetic beads. Returning to the removal of the free agent (6) and the free first partner (20), the eluate sample obtained in FIG. 1d of the present application may be loaded onto the second chromatography column of WO 2013 / 124474, as described in the "removal cartridge" in WO 2013 / 124474 (see, for example, FIG. 4 therein). This chromatography column has a suitable stationary phase that can act as both an affinity chromatography matrix and a gel permeation matrix. An affinity reagent is immobilized on this affinity chromatography matrix. In this example, the affinity reagent may be, for example, streptavidin, streptavidin mutein, avidin, avidin mutein, or a mixture thereof. The free first agent (6), the first partner (20) of binding partner C1 (also referred to herein as the "competitor reagent"), binds to the affinity reagent and is immobilized on the chromatography matrix. As a result, the eluate sample containing the isolated and expanded cell population 2 is free of the first agent 6 and the competing reagent 20. The expanded cell population 2, free of any reactants, is ready for further use, for example, for diagnostic applications (e.g., further FACS™ sorting) or any cell-based therapeutic application. [Figure 2A]Figure 2 illustrates a further embodiment of the expansion method of the present invention. As shown in Figure 2a, a sample contains a population of cells (2) bearing two specific cell surface molecules (30) and (32). Cell surface molecule (30) is responsible for the primary activation signal for the cell population, while cell surface molecule (32) is an accessory molecule on the cell surface that is involved in providing stimulation to the cells. The population of cells may be, for example, a T cell population in which cell surface molecule (30) is the TCR / CD3 complex and cell surface molecule (32) is the accessory molecule CD28. T cell expansion / proliferation requires the binding of both the TCR / CD3 complex as a primary activation signal and CD28 as a costimulator. T cell population (2) is present in a mixture with another cell population (22) that does not possess surface receptor molecules (30) and (32). In this embodiment, cell population (2) is also contacted with a multimerization reagent (4). A first agent (6) that provides a primary activation signal to cells is reversibly immobilized (bound) on the multimerization reagent (4). Furthermore, a second agent (8) that stimulates CD28 as an accessory molecule on the cell surface is reversibly immobilized (bound) on the multimerization reagent. The multimerization reagent (4) includes at least one binding site Z1 (42) for reversibly binding the first agent (6), and the first agent (6) includes at least one binding partner C1 (6a), which can reversibly bind to the binding site Z1 (44) of the multimerization reagent. Thus, due to immobilization, the first agent (6) binds to the multimerization reagent (4) via the reversible bond formed between the binding partner C1 (6a) and the binding site Z1 (42). Further, in the embodiment shown in Figure 2, the second agent (8) comprises a binding partner C2 (8a), which can reversibly bind to binding site Z2 (44) of the multimerization reagent (4). The second agent (8) binds to the multimerization reagent (4) via a reversible bond formed between binding partner C2 (8a) and binding site Z2 (44). In this embodiment, the first agent (6) may be a monoclonal anti-CD3 antibody or an antigen-binding fragment thereof, such as a Fab fragment. The second agent (8) may be a monoclonal anti-CD28 antibody or an antigen-binding fragment thereof, such as a Fab fragment.The first binding partner (6a) can be a streptavidin-binding peptide (6a) fused or conjugated to an anti-CD3 antibody or anti-CD3 antibody fragment. The second binding partner (8a) can be a calmodulin-binding peptide conjugated or fused to a CD28 antibody or CD28-binding antibody fragment. In this context, it is noted that monoclonal antibodies against CD3 or CD28 are well known (see, e.g., U.S. Pat. No. 6,352,694 B or European Patent No. 0 700 430 B1, discussed above) and are commercially available from a number of sources, including Santa Cruz Biotechnology (Santa Cruz, CA, USA), Life Technologies (Carlsbad, CA, USA), BD Biosciences (San Jose, CA, USA), Biolegend (San Diego, CA, USA), or Miltenyi Biotec (Bergisch Gladbach, Germany), to name just a few. Such monoclonal antibodies can therefore be used as first and second agents and can be chemically linked (conjugated) to, for example, binding partners C1 and C2. Alternatively, the variable domain genes from a hybridoma cell line can be cloned or an antibody whose amino acid sequence is known can be used and the respective antibody fragments, such as Fab fragments or Fv, can be produced recombinantly.When using approaches such as those described in the Examples section for both the hybridoma cell line OKT3 (ATCC® CRL-8001™, described in U.S. Pat. No. 4,361,549) producing the monoclonal anti-CD3 antibody and the anti-CD28 antibody 28.3 described in Vanhove et al., BLOOD, 15 July 2003, Vol. 102, No. 2, pages 564-570 and GenBank accession number AF451974.1, the binding partners C1 and C2 are conveniently provided by the respective expression vectors used for recombinant production, such that the antibody fragment has the binding partner C1 or C2 as a fusion peptide at the C-terminus of either the light or heavy chain (in this situation, the same procedure as described in Arakawa et al., J. Biochem. 120, 657-662). (1996) the amino acid sequences of the heavy and light chain variable domains of the antibody OKT3 are shown for illustrative purposes as SEQ ID NOS: 17 and 18 and in the attached sequence listing, while the amino acid sequences of the variable domains of the anti-CD28 antibody 28.3 described by Vanhove et al. (supra) are shown in the attached sequence listing as SEQ ID NOS: 19 (VH) and 20 (VL). Furthermore, this methodology for cloning the variable domains of antibody molecules and recombinantly producing the respective antibody fragments is well known to those skilled in the art (see, for example, Skerra, A. (1994) A general vector, pASK84, for cloning, bacterial production, and single-step purification of antibody Fab fragments. Gene 141, 79-84, or Skerra, A. (1993) Bacterial expression of immunoglobulin fragments. Curr Opin Immunol. 5, 256-562).Finally, methods such as phage display (reviewed, for example, in Kay, BK et al. (1996) Phage Display of Peptides and Proteins - A Laboratory Manual, 1st 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, for example, in Amstutz, P. et al. (2001) Curr. Opin. Biotechnol. 12, 400-405), or mRNA display (Wilson, DS et al. (2001) Proc. Natl. Acad. Sci. USA 98, It is also possible to generate artificial binding molecules, such as antibody molecules, with antibody-like properties for a given target, such as CD3 or CD28, by well-known evolutionary methods, such as those reported in [Publication ID No. 3750-3755], as shown in the example of Figure 2. In the example shown in Figure 2, the multimerization reagent (4) has two distinct binding sites, Z1 (42) and Z2 (44). Using the binding partner C1 (6a), which is a streptavidin-binding peptide, the binding site Z1 (42) of the multimerization reagent (4) is provided by a suitable streptavidin mutein to which the streptavidin peptide (6a) reversibly binds. Since the binding partner C2 is a calmodulin-binding peptide, the binding site Z2 (44) of the multimerization reagent (4) is provided by the multimeric calmodulin. The multimerization reagent (4) may be a single molecule, e.g., a conjugate of multimeric calmodulin and streptavidin (this alternative is typically used in the case of soluble multimerization), or it may consist of two independent molecules. The latter option is preferred when the multimerization reagent (44) is immobilized on a solid support, as shown in Figure 2. In this case, a mixture of streptavidin mutein and calmodulin may be coated (immobilized) on the solid support, e.g., in a 1:1 molar ratio relative to the binding sites Z1 and Z2.Note that in this situation, the immobilization of calmodulin on the surface of the solid support obviates the need to prepare multimeric calmodulin as described above, but the immobilization of calmodulin on the surface is sufficient to present calmodulin (which, as described above, has only a single binding site for the calmodulin-binding peptide) at a density high enough to ensure binding of cell population (2). For example, in this case, a bivalent antibody fragment having two binding sites for CD28, or an intact antibody itself having two identical binding sites, can be used as second reagent (8). As shown in Figure 2b, T cell population (2) is contacted with multimerization reagent (4), and typically after incubation of the cell population with multimerization reagent (4), T cell population (2) forms a complex with / binds to the multimerization reagent via first agent (6) and second agent (8). The first agent (6) and the second agent (8) specifically bind to the TCR / CD3 complex and the accessory molecule CD28 to induce T cell proliferation / expansion. As shown in Figure 2c, after incubation (typically for a period of time suitable for expanding the desired cell population), the binding partner C1 (6a) of the first agent (6) and the binding site Z1 of the multimerization reagent (4) are separated by dissociating their respective reversible bonds. Similarly, the binding partner C2 (8a) of the second agent (8) and the binding site Z2 of the multimerization reagent (4) are separated by dissociating their respective reversible bonds. The reversible binding between the binding partner C1 (6a) of the first agent 6 and the binding site Z1 can be disrupted by biotin (acting as a free analog of the first partner 20), while the reversible binding between the binding partner C2 (8a) of the first agent 8 and the binding site Z2 can be disrupted by adding a metal chelator (calcium chelator) such as EDTA or EGTA (acting as a free analog of the second partner 20), since the binding of calmodulin to the calmodulin-binding peptide is calcium ion (Ca2+) dependent. This, of course, means that the contacting of the cell population 2 is carried out in a Ca2+-containing buffer.As shown in Figure 2d, addition of the first free partner and second free partner analogs 20 displaces binding partners C1 (6a) and C2 (8a), respectively, from multimerization reagent 4, thereby displacing first agent 6 and second agent 8 from multimerization reagent 4. This displacement of first agent 6 and second agent 8 then dissociates first agent 6 and second agent 8 from the TCR / CD3 complex and accessory molecule CD28, thereby terminating the stimulation / expansion of cell population 2. Thus, as described above, the present invention provides the advantage of being able to precisely control the duration of stimulation or expansion of a T cell population, and therefore the functional state of the T cell population. After elution of the cells as shown in Figure 1d, the first agent (6), the second reagent (8), and the analogs (20) of the free first and second binding partners of binding partner C1 and C2 can be easily removed from the stimulated cell population (2) via a "removal cartridge" as described in WO 2013 / 124474. Furthermore, and importantly, if the initial sample was a population of lymphocytes, e.g., in the form of PMBCs obtained from a Ficoll gradient, the T cell population (2) is available for continuous expansion, as defined herein. Since the expanded cell population (e.g., by initial stimulation via CD3 / CD28) can be transfected with, e.g., a T cell receptor (TCR) or a chimeric antigen receptor (CAR, also known as an artificial T cell receptor) during expansion, the genetically modified cells can then be released from the initial stimulus and subsequently stimulated with a second type of stimulus, e.g., via a de novo introduced receptor. These secondary stimuli may include antigenic stimulation in the form of peptide / MHC molecules, cognate (bridged) ligands of the genetically introduced receptor (e.g., the natural ligand of the CAR), or any ligand (such as an antibody) that binds directly within the framework of the new receptor (e.g., by recognizing a constant region within the receptor). The T cell population obtained from this serial expansion can then be used for adoptive cell transfer. [Figure 2B]Figure 2 illustrates a further embodiment of the expansion method of the present invention. As shown in Figure 2a, a sample contains a population of cells (2) bearing two specific cell surface molecules (30) and (32). Cell surface molecule (30) is responsible for the primary activation signal for the cell population, while cell surface molecule (32) is an accessory molecule on the cell surface that is involved in providing stimulation to the cells. The population of cells may be, for example, a T cell population in which cell surface molecule (30) is the TCR / CD3 complex and cell surface molecule (32) is the accessory molecule CD28. T cell expansion / proliferation requires the binding of both the TCR / CD3 complex as a primary activation signal and CD28 as a costimulator. T cell population (2) is present in a mixture with another cell population (22) that does not possess surface receptor molecules (30) and (32). In this embodiment, cell population (2) is also contacted with a multimerization reagent (4). A first agent (6) that provides a primary activation signal to cells is reversibly immobilized (bound) on the multimerization reagent (4). Furthermore, a second agent (8) that stimulates CD28 as an accessory molecule on the cell surface is reversibly immobilized (bound) on the multimerization reagent. The multimerization reagent (4) includes at least one binding site Z1 (42) for reversibly binding the first agent (6), and the first agent (6) includes at least one binding partner C1 (6a), which can reversibly bind to the binding site Z1 (44) of the multimerization reagent. Thus, due to immobilization, the first agent (6) binds to the multimerization reagent (4) via the reversible bond formed between the binding partner C1 (6a) and the binding site Z1 (42). Further, in the embodiment shown in Figure 2, the second agent (8) comprises a binding partner C2 (8a), which can reversibly bind to binding site Z2 (44) of the multimerization reagent (4). The second agent (8) binds to the multimerization reagent (4) via a reversible bond formed between binding partner C2 (8a) and binding site Z2 (44). In this embodiment, the first agent (6) may be a monoclonal anti-CD3 antibody or an antigen-binding fragment thereof, such as a Fab fragment. The second agent (8) may be a monoclonal anti-CD28 antibody or an antigen-binding fragment thereof, such as a Fab fragment.The first binding partner (6a) can be a streptavidin-binding peptide (6a) fused or conjugated to an anti-CD3 antibody or anti-CD3 antibody fragment. The second binding partner (8a) can be a calmodulin-binding peptide conjugated or fused to a CD28 antibody or CD28-binding antibody fragment. In this context, it is noted that monoclonal antibodies against CD3 or CD28 are well known (see, e.g., U.S. Pat. No. 6,352,694 B or European Patent No. 0 700 430 B1, discussed above) and are commercially available from a number of sources, including Santa Cruz Biotechnology (Santa Cruz, CA, USA), Life Technologies (Carlsbad, CA, USA), BD Biosciences (San Jose, CA, USA), Biolegend (San Diego, CA, USA), or Miltenyi Biotec (Bergisch Gladbach, Germany), to name just a few. Such monoclonal antibodies can therefore be used as first and second agents and can be chemically linked (conjugated) to, for example, binding partners C1 and C2. Alternatively, the variable domain genes from a hybridoma cell line can be cloned or an antibody whose amino acid sequence is known can be used and the respective antibody fragments, such as Fab fragments or Fv, can be produced recombinantly.When using approaches such as those described in the Examples section for both the hybridoma cell line OKT3 (ATCC® CRL-8001™, described in U.S. Pat. No. 4,361,549) producing the monoclonal anti-CD3 antibody and the anti-CD28 antibody 28.3 described in Vanhove et al., BLOOD, 15 July 2003, Vol. 102, No. 2, pages 564-570 and GenBank accession number AF451974.1, the binding partners C1 and C2 are conveniently provided by the respective expression vectors used for recombinant production, such that the antibody fragment has the binding partner C1 or C2 as a fusion peptide at the C-terminus of either the light or heavy chain (in this situation, the same procedure as described in Arakawa et al., J. Biochem. 120, 657-662). (1996) the amino acid sequences of the heavy and light chain variable domains of the antibody OKT3 are shown for illustrative purposes as SEQ ID NOS: 17 and 18 and in the attached sequence listing, while the amino acid sequences of the variable domains of the anti-CD28 antibody 28.3 described by Vanhove et al. (supra) are shown in the attached sequence listing as SEQ ID NOS: 19 (VH) and 20 (VL). Furthermore, this methodology for cloning the variable domains of antibody molecules and recombinantly producing the respective antibody fragments is well known to those skilled in the art (see, for example, Skerra, A. (1994) A general vector, pASK84, for cloning, bacterial production, and single-step purification of antibody Fab fragments. Gene 141, 79-84, or Skerra, A. (1993) Bacterial expression of immunoglobulin fragments. Curr Opin Immunol. 5, 256-562).Finally, methods such as phage display (reviewed, for example, in Kay, BK et al. (1996) Phage Display of Peptides and Proteins - A Laboratory Manual, 1st 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, for example, in Amstutz, P. et al. (2001) Curr. Opin. Biotechnol. 12, 400-405), or mRNA display (Wilson, DS et al. (2001) Proc. Natl. Acad. Sci. USA 98, It is also possible to generate artificial binding molecules, such as antibody molecules, with antibody-like properties for a given target, such as CD3 or CD28, by well-known evolutionary methods, such as those reported in [Publication ID No. 3750-3755], as shown in the example of Figure 2. In the example shown in Figure 2, the multimerization reagent (4) has two distinct binding sites, Z1 (42) and Z2 (44). Using the binding partner C1 (6a), which is a streptavidin-binding peptide, the binding site Z1 (42) of the multimerization reagent (4) is provided by a suitable streptavidin mutein to which the streptavidin peptide (6a) reversibly binds. Since the binding partner C2 is a calmodulin-binding peptide, the binding site Z2 (44) of the multimerization reagent (4) is provided by the multimeric calmodulin. The multimerization reagent (4) may be a single molecule, e.g., a conjugate of multimeric calmodulin and streptavidin (this alternative is typically used in the case of soluble multimerization), or it may consist of two independent molecules. The latter option is preferred when the multimerization reagent (44) is immobilized on a solid support, as shown in Figure 2. In this case, a mixture of streptavidin mutein and calmodulin may be coated (immobilized) on the solid support, e.g., in a 1:1 molar ratio relative to the binding sites Z1 and Z2.Note that in this situation, the immobilization of calmodulin on the surface of the solid support obviates the need to prepare multimeric calmodulin as described above, but the immobilization of calmodulin on the surface is sufficient to present calmodulin (which, as described above, has only a single binding site for the calmodulin-binding peptide) at a density high enough to ensure binding of cell population (2). For example, in this case, a bivalent antibody fragment having two binding sites for CD28, or an intact antibody itself having two identical binding sites, can be used as second reagent (8). As shown in Figure 2b, T cell population (2) is contacted with multimerization reagent (4), and typically after incubation of the cell population with multimerization reagent (4), T cell population (2) forms a complex with / binds to the multimerization reagent via first agent (6) and second agent (8). The first agent (6) and the second agent (8) specifically bind to the TCR / CD3 complex and the accessory molecule CD28 to induce T cell proliferation / expansion. As shown in Figure 2c, after incubation (typically for a period of time suitable for expanding the desired cell population), the binding partner C1 (6a) of the first agent (6) and the binding site Z1 of the multimerization reagent (4) are separated by dissociating their respective reversible bonds. Similarly, the binding partner C2 (8a) of the second agent (8) and the binding site Z2 of the multimerization reagent (4) are separated by dissociating their respective reversible bonds. The reversible binding between the binding partner C1 (6a) of the first agent 6 and the binding site Z1 can be disrupted by biotin (acting as a free analog of the first partner 20), while the reversible binding between the binding partner C2 (8a) of the first agent 8 and the binding site Z2 can be disrupted by adding a metal chelator (calcium chelator) such as EDTA or EGTA (acting as a free analog of the second partner 20), since the binding of calmodulin to the calmodulin-binding peptide is calcium ion (Ca2+) dependent. This, of course, means that the contacting of the cell population 2 is carried out in a Ca2+-containing buffer.As shown in Figure 2d, addition of the first free partner and second free partner analogs 20 displaces binding partners C1 (6a) and C2 (8a), respectively, from multimerization reagent 4, thereby displacing first agent 6 and second agent 8 from multimerization reagent 4. This displacement of first agent 6 and second agent 8 then dissociates first agent 6 and second agent 8 from the TCR / CD3 complex and accessory molecule CD28, thereby terminating the stimulation / expansion of cell population 2. Thus, as described above, the present invention provides the advantage of being able to precisely control the duration of stimulation or expansion of a T cell population, and therefore the functional state of the T cell population. After elution of the cells as shown in Figure 1d, the first agent (6), the second reagent (8), and the analogs (20) of the free first and second binding partners of binding partner C1 and C2 can be easily removed from the stimulated cell population (2) via a "removal cartridge" as described in WO 2013 / 124474. Furthermore, and importantly, if the initial sample was a population of lymphocytes, e.g., in the form of PMBCs obtained from a Ficoll gradient, the T cell population (2) is available for continuous expansion, as defined herein. Since the expanded cell population (e.g., by initial stimulation via CD3 / CD28) can be transfected with, e.g., a T cell receptor (TCR) or a chimeric antigen receptor (CAR, also known as an artificial T cell receptor) during expansion, the genetically modified cells can then be released from the initial stimulus and subsequently stimulated with a second type of stimulus, e.g., via a de novo introduced receptor. These secondary stimuli may include antigenic stimulation in the form of peptide / MHC molecules, cognate (bridged) ligands of the genetically introduced receptor (e.g., the natural ligand of the CAR), or any ligand (such as an antibody) that binds directly within the framework of the new receptor (e.g., by recognizing a constant region within the receptor). The T cell population obtained from this serial expansion can then be used for adoptive cell transfer. [Figure 2C]Figure 2 illustrates a further embodiment of the expansion method of the present invention. As shown in Figure 2a, a sample contains a population of cells (2) bearing two specific cell surface molecules (30) and (32). Cell surface molecule (30) is responsible for the primary activation signal for the cell population, while cell surface molecule (32) is an accessory molecule on the cell surface that is involved in providing stimulation to the cells. The population of cells may be, for example, a T cell population in which cell surface molecule (30) is the TCR / CD3 complex and cell surface molecule (32) is the accessory molecule CD28. T cell expansion / proliferation requires the binding of both the TCR / CD3 complex as a primary activation signal and CD28 as a costimulator. T cell population (2) is present in a mixture with another cell population (22) that does not possess surface receptor molecules (30) and (32). In this embodiment, cell population (2) is also contacted with a multimerization reagent (4). A first agent (6) that provides a primary activation signal to cells is reversibly immobilized (bound) on the multimerization reagent (4). Furthermore, a second agent (8) that stimulates CD28 as an accessory molecule on the cell surface is reversibly immobilized (bound) on the multimerization reagent. The multimerization reagent (4) includes at least one binding site Z1 (42) for reversibly binding the first agent (6), and the first agent (6) includes at least one binding partner C1 (6a), which can reversibly bind to the binding site Z1 (44) of the multimerization reagent. Thus, due to immobilization, the first agent (6) binds to the multimerization reagent (4) via the reversible bond formed between the binding partner C1 (6a) and the binding site Z1 (42). Further, in the embodiment shown in Figure 2, the second agent (8) comprises a binding partner C2 (8a), which can reversibly bind to binding site Z2 (44) of the multimerization reagent (4). The second agent (8) binds to the multimerization reagent (4) via a reversible bond formed between binding partner C2 (8a) and binding site Z2 (44). In this embodiment, the first agent (6) may be a monoclonal anti-CD3 antibody or an antigen-binding fragment thereof, such as a Fab fragment. The second agent (8) may be a monoclonal anti-CD28 antibody or an antigen-binding fragment thereof, such as a Fab fragment.The first binding partner (6a) can be a streptavidin-binding peptide (6a) fused or conjugated to an anti-CD3 antibody or anti-CD3 antibody fragment. The second binding partner (8a) can be a calmodulin-binding peptide conjugated or fused to a CD28 antibody or CD28-binding antibody fragment. In this context, it is noted that monoclonal antibodies against CD3 or CD28 are well known (see, e.g., U.S. Pat. No. 6,352,694 B or European Patent No. 0 700 430 B1, discussed above) and are commercially available from a number of sources, including Santa Cruz Biotechnology (Santa Cruz, CA, USA), Life Technologies (Carlsbad, CA, USA), BD Biosciences (San Jose, CA, USA), Biolegend (San Diego, CA, USA), or Miltenyi Biotec (Bergisch Gladbach, Germany), to name just a few. Such monoclonal antibodies can therefore be used as first and second agents and can be chemically linked (conjugated) to, for example, binding partners C1 and C2. Alternatively, the variable domain genes from a hybridoma cell line can be cloned or an antibody whose amino acid sequence is known can be used and the respective antibody fragments, such as Fab fragments or Fv, can be produced recombinantly.When using approaches such as those described in the Examples section for both the hybridoma cell line OKT3 (ATCC® CRL-8001™, described in U.S. Pat. No. 4,361,549) producing the monoclonal anti-CD3 antibody and the anti-CD28 antibody 28.3 described in Vanhove et al., BLOOD, 15 July 2003, Vol. 102, No. 2, pages 564-570 and GenBank accession number AF451974.1, the binding partners C1 and C2 are conveniently provided by the respective expression vectors used for recombinant production, such that the antibody fragment has the binding partner C1 or C2 as a fusion peptide at the C-terminus of either the light or heavy chain (in this situation, the same procedure as described in Arakawa et al., J. Biochem. 120, 657-662). (1996) the amino acid sequences of the heavy and light chain variable domains of the antibody OKT3 are shown for illustrative purposes as SEQ ID NOS: 17 and 18 and in the attached sequence listing, while the amino acid sequences of the variable domains of the anti-CD28 antibody 28.3 described by Vanhove et al. (supra) are shown in the attached sequence listing as SEQ ID NOS: 19 (VH) and 20 (VL). Furthermore, this methodology for cloning the variable domains of antibody molecules and recombinantly producing the respective antibody fragments is well known to those skilled in the art (see, for example, Skerra, A. (1994) A general vector, pASK84, for cloning, bacterial production, and single-step purification of antibody Fab fragments. Gene 141, 79-84, or Skerra, A. (1993) Bacterial expression of immunoglobulin fragments. Curr Opin Immunol. 5, 256-562).Finally, methods such as phage display (reviewed, for example, in Kay, BK et al. (1996) Phage Display of Peptides and Proteins - A Laboratory Manual, 1st 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, for example, in Amstutz, P. et al. (2001) Curr. Opin. Biotechnol. 12, 400-405), or mRNA display (Wilson, DS et al. (2001) Proc. Natl. Acad. Sci. USA 98, It is also possible to generate artificial binding molecules, such as antibody molecules, with antibody-like properties for a given target, such as CD3 or CD28, by well-known evolutionary methods, such as those reported in [Publication ID No. 3750-3755], as shown in the example of Figure 2. In the example shown in Figure 2, the multimerization reagent (4) has two distinct binding sites, Z1 (42) and Z2 (44). Using the binding partner C1 (6a), which is a streptavidin-binding peptide, the binding site Z1 (42) of the multimerization reagent (4) is provided by a suitable streptavidin mutein to which the streptavidin peptide (6a) reversibly binds. Since the binding partner C2 is a calmodulin-binding peptide, the binding site Z2 (44) of the multimerization reagent (4) is provided by the multimeric calmodulin. The multimerization reagent (4) may be a single molecule, e.g., a conjugate of multimeric calmodulin and streptavidin (this alternative is typically used in the case of soluble multimerization), or it may consist of two independent molecules. The latter option is preferred when the multimerization reagent (44) is immobilized on a solid support, as shown in Figure 2. In this case, a mixture of streptavidin mutein and calmodulin may be coated (immobilized) on the solid support, e.g., in a 1:1 molar ratio relative to the binding sites Z1 and Z2.Note that in this situation, the immobilization of calmodulin on the surface of the solid support obviates the need to prepare multimeric calmodulin as described above, but the immobilization of calmodulin on the surface is sufficient to present calmodulin (which, as described above, has only a single binding site for the calmodulin-binding peptide) at a density high enough to ensure binding of cell population (2). For example, in this case, a bivalent antibody fragment having two binding sites for CD28, or an intact antibody itself having two identical binding sites, can be used as second reagent (8). As shown in Figure 2b, T cell population (2) is contacted with multimerization reagent (4), and typically after incubation of the cell population with multimerization reagent (4), T cell population (2) forms a complex with / binds to the multimerization reagent via first agent (6) and second agent (8). The first agent (6) and the second agent (8) specifically bind to the TCR / CD3 complex and the accessory molecule CD28 to induce T cell proliferation / expansion. As shown in Figure 2c, after incubation (typically for a period of time suitable for expanding the desired cell population), the binding partner C1 (6a) of the first agent (6) and the binding site Z1 of the multimerization reagent (4) are separated by dissociating their respective reversible bonds. Similarly, the binding partner C2 (8a) of the second agent (8) and the binding site Z2 of the multimerization reagent (4) are separated by dissociating their respective reversible bonds. The reversible binding between the binding partner C1 (6a) of the first agent 6 and the binding site Z1 can be disrupted by biotin (acting as a free analog of the first partner 20), while the reversible binding between the binding partner C2 (8a) of the first agent 8 and the binding site Z2 can be disrupted by adding a metal chelator (calcium chelator) such as EDTA or EGTA (acting as a free analog of the second partner 20), since the binding of calmodulin to the calmodulin-binding peptide is calcium ion (Ca2+) dependent. This, of course, means that the contacting of the cell population 2 is carried out in a Ca2+-containing buffer.As shown in Figure 2d, addition of the first free partner and second free partner analogs 20 displaces binding partners C1 (6a) and C2 (8a), respectively, from multimerization reagent 4, thereby displacing first agent 6 and second agent 8 from multimerization reagent 4. This displacement of first agent 6 and second agent 8 then dissociates first agent 6 and second agent 8 from the TCR / CD3 complex and accessory molecule CD28, thereby terminating the stimulation / expansion of cell population 2. Thus, as described above, the present invention provides the advantage of being able to precisely control the duration of stimulation or expansion of a T cell population, and therefore the functional state of the T cell population. After elution of the cells as shown in Figure 1d, the first agent (6), the second reagent (8), and the analogs (20) of the free first and second binding partners of binding partner C1 and C2 can be easily removed from the stimulated cell population (2) via a "removal cartridge" as described in WO 2013 / 124474. Furthermore, and importantly, if the initial sample was a population of lymphocytes, e.g., in the form of PMBCs obtained from a Ficoll gradient, the T cell population (2) is available for continuous expansion, as defined herein. Since the expanded cell population (e.g., by initial stimulation via CD3 / CD28) can be transfected with, e.g., a T cell receptor (TCR) or a chimeric antigen receptor (CAR, also known as an artificial T cell receptor) during expansion, the genetically modified cells can then be released from the initial stimulus and subsequently stimulated with a second type of stimulus, e.g., via a de novo introduced receptor. These secondary stimuli may include antigenic stimulation in the form of peptide / MHC molecules, cognate (bridged) ligands of the genetically introduced receptor (e.g., the natural ligand of the CAR), or any ligand (such as an antibody) that binds directly within the framework of the new receptor (e.g., by recognizing a constant region within the receptor). The T cell population obtained from this serial expansion can then be used for adoptive cell transfer. [Figure 2D]Figure 2 illustrates a further embodiment of the expansion method of the present invention. As shown in Figure 2a, a sample contains a population of cells (2) bearing two specific cell surface molecules (30) and (32). Cell surface molecule (30) is responsible for the primary activation signal for the cell population, while cell surface molecule (32) is an accessory molecule on the cell surface that is involved in providing stimulation to the cells. The population of cells may be, for example, a T cell population in which cell surface molecule (30) is the TCR / CD3 complex and cell surface molecule (32) is the accessory molecule CD28. T cell expansion / proliferation requires the binding of both the TCR / CD3 complex as a primary activation signal and CD28 as a costimulator. T cell population (2) is present in a mixture with another cell population (22) that does not possess surface receptor molecules (30) and (32). In this embodiment, cell population (2) is also contacted with a multimerization reagent (4). A first agent (6) that provides a primary activation signal to cells is reversibly immobilized (bound) on the multimerization reagent (4). Furthermore, a second agent (8) that stimulates CD28 as an accessory molecule on the cell surface is reversibly immobilized (bound) on the multimerization reagent. The multimerization reagent (4) includes at least one binding site Z1 (42) for reversibly binding the first agent (6), and the first agent (6) includes at least one binding partner C1 (6a), which can reversibly bind to the binding site Z1 (44) of the multimerization reagent. Thus, due to immobilization, the first agent (6) binds to the multimerization reagent (4) via the reversible bond formed between the binding partner C1 (6a) and the binding site Z1 (42). Further, in the embodiment shown in Figure 2, the second agent (8) comprises a binding partner C2 (8a), which can reversibly bind to binding site Z2 (44) of the multimerization reagent (4). The second agent (8) binds to the multimerization reagent (4) via a reversible bond formed between binding partner C2 (8a) and binding site Z2 (44). In this embodiment, the first agent (6) may be a monoclonal anti-CD3 antibody or an antigen-binding fragment thereof, such as a Fab fragment. The second agent (8) may be a monoclonal anti-CD28 antibody or an antigen-binding fragment thereof, such as a Fab fragment.The first binding partner (6a) can be a streptavidin-binding peptide (6a) fused or conjugated to an anti-CD3 antibody or anti-CD3 antibody fragment. The second binding partner (8a) can be a calmodulin-binding peptide conjugated or fused to a CD28 antibody or CD28-binding antibody fragment. In this context, it is noted that monoclonal antibodies against CD3 or CD28 are well known (see, e.g., U.S. Pat. No. 6,352,694 B or European Patent No. 0 700 430 B1, discussed above) and are commercially available from a number of sources, including Santa Cruz Biotechnology (Santa Cruz, CA, USA), Life Technologies (Carlsbad, CA, USA), BD Biosciences (San Jose, CA, USA), Biolegend (San Diego, CA, USA), or Miltenyi Biotec (Bergisch Gladbach, Germany), to name just a few. Such monoclonal antibodies can therefore be used as first and second agents and can be chemically linked (conjugated) to, for example, binding partners C1 and C2. Alternatively, the variable domain genes from a hybridoma cell line can be cloned or an antibody whose amino acid sequence is known can be used and the respective antibody fragments, such as Fab fragments or Fv, can be produced recombinantly.When using approaches such as those described in the Examples section for both the hybridoma cell line OKT3 (ATCC® CRL-8001™, described in U.S. Pat. No. 4,361,549) producing the monoclonal anti-CD3 antibody and the anti-CD28 antibody 28.3 described in Vanhove et al., BLOOD, 15 July 2003, Vol. 102, No. 2, pages 564-570 and GenBank accession number AF451974.1, the binding partners C1 and C2 are conveniently provided by the respective expression vectors used for recombinant production, such that the antibody fragment has the binding partner C1 or C2 as a fusion peptide at the C-terminus of either the light or heavy chain (in this situation, the same procedure as described in Arakawa et al., J. Biochem. 120, 657-662). (1996) the amino acid sequences of the heavy and light chain variable domains of the antibody OKT3 are shown for illustrative purposes as SEQ ID NOS: 17 and 18 and in the attached sequence listing, while the amino acid sequences of the variable domains of the anti-CD28 antibody 28.3 described by Vanhove et al. (supra) are shown in the attached sequence listing as SEQ ID NOS: 19 (VH) and 20 (VL). Furthermore, this methodology for cloning the variable domains of antibody molecules and recombinantly producing the respective antibody fragments is well known to those skilled in the art (see, for example, Skerra, A. (1994) A general vector, pASK84, for cloning, bacterial production, and single-step purification of antibody Fab fragments. Gene 141, 79-84, or Skerra, A. (1993) Bacterial expression of immunoglobulin fragments. Curr Opin Immunol. 5, 256-562).Finally, methods such as phage display (reviewed, for example, in Kay, BK et al. (1996) Phage Display of Peptides and Proteins - A Laboratory Manual, 1st 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, for example, in Amstutz, P. et al. (2001) Curr. Opin. Biotechnol. 12, 400-405), or mRNA display (Wilson, DS et al. (2001) Proc. Natl. Acad. Sci. USA 98, It is also possible to generate artificial binding molecules, such as antibody molecules, with antibody-like properties for a given target, such as CD3 or CD28, by well-known evolutionary methods, such as those reported in [Publication ID No. 3750-3755], as shown in the example of Figure 2. In the example shown in Figure 2, the multimerization reagent (4) has two distinct binding sites, Z1 (42) and Z2 (44). Using the binding partner C1 (6a), which is a streptavidin-binding peptide, the binding site Z1 (42) of the multimerization reagent (4) is provided by a suitable streptavidin mutein to which the streptavidin peptide (6a) reversibly binds. Since the binding partner C2 is a calmodulin-binding peptide, the binding site Z2 (44) of the multimerization reagent (4) is provided by the multimeric calmodulin. The multimerization reagent (4) may be a single molecule, e.g., a conjugate of multimeric calmodulin and streptavidin (this alternative is typically used in the case of soluble multimerization), or it may consist of two independent molecules. The latter option is preferred when the multimerization reagent (44) is immobilized on a solid support, as shown in Figure 2. In this case, a mixture of streptavidin mutein and calmodulin may be coated (immobilized) on the solid support, e.g., in a 1:1 molar ratio relative to the binding sites Z1 and Z2.Note that in this situation, the immobilization of calmodulin on the surface of the solid support obviates the need to prepare multimeric calmodulin as described above, but the immobilization of calmodulin on the surface is sufficient to present calmodulin (which, as described above, has only a single binding site for the calmodulin-binding peptide) at a density high enough to ensure binding of cell population (2). For example, in this case, a bivalent antibody fragment having two binding sites for CD28, or an intact antibody itself having two identical binding sites, can be used as second reagent (8). As shown in Figure 2b, T cell population (2) is contacted with multimerization reagent (4), and typically after incubation of the cell population with multimerization reagent (4), T cell population (2) forms a complex with / binds to the multimerization reagent via first agent (6) and second agent (8). The first agent (6) and the second agent (8) specifically bind to the TCR / CD3 complex and the accessory molecule CD28 to induce T cell proliferation / expansion. As shown in Figure 2c, after incubation (typically for a period of time suitable for expanding the desired cell population), the binding partner C1 (6a) of the first agent (6) and the binding site Z1 of the multimerization reagent (4) are separated by dissociating their respective reversible bonds. Similarly, the binding partner C2 (8a) of the second agent (8) and the binding site Z2 of the multimerization reagent (4) are separated by dissociating their respective reversible bonds. The reversible binding between the binding partner C1 (6a) of the first agent 6 and the binding site Z1 can be disrupted by biotin (acting as a free analog of the first partner 20), while the reversible binding between the binding partner C2 (8a) of the first agent 8 and the binding site Z2 can be disrupted by adding a metal chelator (calcium chelator) such as EDTA or EGTA (acting as a free analog of the second partner 20), since the binding of calmodulin to the calmodulin-binding peptide is calcium ion (Ca2+) dependent. This, of course, means that the contacting of the cell population 2 is carried out in a Ca2+-containing buffer.As shown in Figure 2d, addition of the first free partner and second free partner analogs 20 displaces binding partners C1 (6a) and C2 (8a), respectively, from multimerization reagent 4, thereby displacing first agent 6 and second agent 8 from multimerization reagent 4. This displacement of first agent 6 and second agent 8 then dissociates first agent 6 and second agent 8 from the TCR / CD3 complex and accessory molecule CD28, thereby terminating the stimulation / expansion of cell population 2. Thus, as described above, the present invention provides the advantage of being able to precisely control the duration of stimulation or expansion of a T cell population, and therefore the functional state of the T cell population. After elution of the cells as shown in Figure 1d, the first agent (6), the second reagent (8), and the analogs (20) of the free first and second binding partners of binding partner C1 and C2 can be easily removed from the stimulated cell population (2) via a "removal cartridge" as described in WO 2013 / 124474. Furthermore, and importantly, if the initial sample was a population of lymphocytes, e.g., in the form of PMBCs obtained from a Ficoll gradient, the T cell population (2) is available for continuous expansion, as defined herein. Since the expanded cell population (e.g., by initial stimulation via CD3 / CD28) can be transfected with, e.g., a T cell receptor (TCR) or a chimeric antigen receptor (CAR, also known as an artificial T cell receptor) during expansion, the genetically modified cells can then be released from the initial stimulus and subsequently stimulated with a second type of stimulus, e.g., via a de novo introduced receptor. These secondary stimuli may include antigenic stimulation in the form of peptide / MHC molecules, cognate (bridged) ligands of the genetically introduced receptor (e.g., the natural ligand of the CAR), or any ligand (such as an antibody) that binds directly within the framework of the new receptor (e.g., by recognizing a constant region within the receptor). The T cell population obtained from this serial expansion can then be used for adoptive cell transfer. [Figure 3A]Figure 3 shows a further embodiment of the expansion method of the present invention. The sample used in this example also contains a population of T cells (2) bearing two specific cell surface molecules (30) and (32), where the cell surface molecule (30) is a TCR / CD3 complex and the cell surface molecule (32) is the accessory molecule CD28. Figure 3a shows the population of T cells (2) after contact with a multimerization reagent (4). Also in this example, a first agent (6), an anti-CD3 antibody or antigen-binding fragment thereof that provides a primary activation signal to T cells, and a second agent (8), an anti-CD28 antibody or antigen-binding fragment thereof that stimulates CD28 as an accessory molecule, are reversibly immobilized (bound) to the multimerization reagent (4). The multimerization reagent (4) shown in the example of Figure 3 contains only one binding site, Z1 (42), for reversible binding of both the first agent (6) and the second agent (8). Both the first agent (6) and the second agent (8) comprise at least one binding partner C1 (6a, 8a), and both the binding partner C1 (6a) and the binding partner (8a) can reversibly bind to the binding site Z1 (44) of the multimerization reagent. Thus, for immobilization, the first agent (6) and the second agent (8) bind to the multimerization reagent (4) via reversible bonds formed between the binding partner C1 (6a) and the binding partner C2, respectively, and the binding site Z1 (42). The binding partners C1 and C2 can be different or identical.For example, binding partner C1 can be a streptavidin-binding peptide of the sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 01), "Strep-tag®"), while binding partner C2 can be a streptavidin-binding peptide of the sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 04), also known as "di-tag3") or the sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 05), also known as "di-tag2"), Junttila et al., Proteomics 5 (2005), The multimerization reagent (4) may be a streptavidin-binding peptide (described in U.S. Pat. Nos. 1199-1203 or U.S. Pat. No. 7,981,632). 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 these streptavidin-binding peptides are used as binding partners C1 and C2, the multimerization reagent (4) is a streptavidin mutein. As shown in FIG. 3, a soluble multimerization reagent (4) is used. In the case of a streptavidin mutein, the soluble multimerization reagent may be, for example, an oligomer or polymer of streptavidin or avidin, or any mutein (analog) of streptavidin or avidin. The oligomer may contain three or more monomers of streptavidin, avidin, or muteins thereof. The oligomer or polymer may be cross-linked by a polysaccharide.Such oligomers or polymers of streptavidin or avidin or streptavidin or avidin muteins can be prepared in a first step by introducing carboxyl residues into a polysaccharide, such as dextran, essentially as described in "Noguchi, A., Takahashi, T., Yamaguchi, T., Kitamura, K., Takakura, Y., Hashida, M. & Sezaki, H. (1992). Preparation and properties of the immunoconjugate composed of anti-human colon cancer monoclonal antibody and mitomycin C dextran conjugate. Bioconjugate Chemistry 3, 132-137." In a second step, streptavidin or avidin or muteins thereof are linked to carboxyl groups on the dextran backbone via internal lysine residues and / or the free N-terminal primary amino group using conventional carbodiimide chemistry. Alternatively, cross-linked oligomers or polymers of streptavidin or avidin, or any muteins of streptavidin or avidin, can be obtained by cross-linking via a bifunctional linker, such as glutaraldehyde, or by other methods described in the literature. Using binding partners C1 and C2 that bind to the same binding site (42) of the multimerizing agent has the advantage that the same free partner (for the first binding partner C1 and for the second binding partner C2) or its analog can be used to terminate the expansion of T cell population (2) and release it from the multimerizing agent, as shown in Figure 3b. In the example of Figure 3, analogs of the first and second partners C1 and C2, such as biotin or biotin derivatives (iminobiotin or desthiobiotin), can be conveniently used to terminate the expansion and release T cell population (2).As shown in Figure 3c, after elution of the cells as shown in Figure 1d, the first agent (6), the second reagent (8), and biotin as the analog of the free first partner of binding partner C1 and the free second partner of binding partner C2 (20) can be easily removed from the stimulated cell population (2) via the "removal cartridge" described in WO 2013 / 124474. Furthermore, the embodiment using soluble multimerization reagent (4) has the additional advantage of avoiding any solid support, such as magnetic beads. This means that there is no risk of contamination of activated T cells with such magnetic beads. This also means that a process that complies with GMP standards can be established much more easily than known methods, such as the use of Dynabeads®, which require additional measurements to ensure that the final expanded T cell population is free of magnetic beads. Furthermore, the use of a soluble multimerizer makes it much easier to remove it from activated cell populations (T cells, B cells, or even natural killer cells) because the cells can be simply sedimented by centrifugation and the supernatant containing the soluble multimerizer can be discarded. Alternatively, the soluble multimerizer may be removed from the expanded cell population in the gel permeation matrix of the removal cartridge of WO 2013 / 124474. Because no solid phase (e.g., magnetic beads) is present, the present invention also provides an automated, closed system for expanding cells 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). [Figure 3B]Figure 3 shows a further embodiment of the expansion method of the present invention. The sample used in this example also contains a population of T cells (2) bearing two specific cell surface molecules (30) and (32), where the cell surface molecule (30) is a TCR / CD3 complex and the cell surface molecule (32) is the accessory molecule CD28. Figure 3a shows the population of T cells (2) after contact with a multimerization reagent (4). Also in this example, a first agent (6), an anti-CD3 antibody or antigen-binding fragment thereof that provides a primary activation signal to T cells, and a second agent (8), an anti-CD28 antibody or antigen-binding fragment thereof that stimulates CD28 as an accessory molecule, are reversibly immobilized (bound) to the multimerization reagent (4). The multimerization reagent (4) shown in the example of Figure 3 contains only one binding site, Z1 (42), for reversible binding of both the first agent (6) and the second agent (8). Both the first agent (6) and the second agent (8) comprise at least one binding partner C1 (6a, 8a), and both the binding partner C1 (6a) and the binding partner (8a) can reversibly bind to the binding site Z1 (44) of the multimerization reagent. Thus, for immobilization, the first agent (6) and the second agent (8) bind to the multimerization reagent (4) via reversible bonds formed between the binding partner C1 (6a) and the binding partner C2, respectively, and the binding site Z1 (42). The binding partners C1 and C2 can be different or identical.For example, binding partner C1 can be a streptavidin-binding peptide of the sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 01), "Strep-tag®"), while binding partner C2 can be a streptavidin-binding peptide of the sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 04), also known as "di-tag3") or the sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 05), also known as "di-tag2"), Junttila et al., Proteomics 5 (2005), The multimerization reagent (4) may be a streptavidin-binding peptide (described in U.S. Pat. Nos. 1199-1203 or U.S. Pat. No. 7,981,632). 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 these streptavidin-binding peptides are used as binding partners C1 and C2, the multimerization reagent (4) is a streptavidin mutein. As shown in FIG. 3, a soluble multimerization reagent (4) is used. In the case of a streptavidin mutein, the soluble multimerization reagent may be, for example, an oligomer or polymer of streptavidin or avidin, or any mutein (analog) of streptavidin or avidin. The oligomer may contain three or more monomers of streptavidin, avidin, or muteins thereof. The oligomer or polymer may be cross-linked by a polysaccharide.Such oligomers or polymers of streptavidin or avidin or streptavidin or avidin muteins can be prepared in a first step by introducing carboxyl residues into a polysaccharide, such as dextran, essentially as described in "Noguchi, A., Takahashi, T., Yamaguchi, T., Kitamura, K., Takakura, Y., Hashida, M. & Sezaki, H. (1992). Preparation and properties of the immunoconjugate composed of anti-human colon cancer monoclonal antibody and mitomycin C dextran conjugate. Bioconjugate Chemistry 3, 132-137." In a second step, streptavidin or avidin or muteins thereof are linked to carboxyl groups on the dextran backbone via internal lysine residues and / or the free N-terminal primary amino group using conventional carbodiimide chemistry. Alternatively, cross-linked oligomers or polymers of streptavidin or avidin, or any muteins of streptavidin or avidin, can be obtained by cross-linking via a bifunctional linker, such as glutaraldehyde, or by other methods described in the literature. Using binding partners C1 and C2 that bind to the same binding site (42) of the multimerizing agent has the advantage that the same free partner (for the first binding partner C1 and for the second binding partner C2) or its analog can be used to terminate the expansion of T cell population (2) and release it from the multimerizing agent, as shown in Figure 3b. In the example of Figure 3, analogs of the first and second partners C1 and C2, such as biotin or biotin derivatives (iminobiotin or desthiobiotin), can be conveniently used to terminate the expansion and release T cell population (2).As shown in Figure 3c, after elution of the cells as shown in Figure 1d, the first agent (6), the second reagent (8), and biotin as the analog of the free first partner of binding partner C1 and the free second partner of binding partner C2 (20) can be easily removed from the stimulated cell population (2) via the "removal cartridge" described in WO 2013 / 124474. Furthermore, the embodiment using soluble multimerization reagent (4) has the additional advantage of avoiding any solid support, such as magnetic beads. This means that there is no risk of contamination of activated T cells with such magnetic beads. This also means that a process that complies with GMP standards can be established much more easily than known methods, such as the use of Dynabeads®, which require additional measurements to ensure that the final expanded T cell population is free of magnetic beads. Furthermore, the use of a soluble multimerizer makes it much easier to remove it from activated cell populations (T cells, B cells, or even natural killer cells) because the cells can be simply sedimented by centrifugation and the supernatant containing the soluble multimerizer can be discarded. Alternatively, the soluble multimerizer may be removed from the expanded cell population in the gel permeation matrix of the removal cartridge of WO 2013 / 124474. Because no solid phase (e.g., magnetic beads) is present, the present invention also provides an automated, closed system for expanding cells 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). [Figure 3C]Figure 3 shows a further embodiment of the expansion method of the present invention. The sample used in this example also contains a population of T cells (2) bearing two specific cell surface molecules (30) and (32), where the cell surface molecule (30) is a TCR / CD3 complex and the cell surface molecule (32) is the accessory molecule CD28. Figure 3a shows the population of T cells (2) after contact with a multimerization reagent (4). Also in this example, a first agent (6), an anti-CD3 antibody or antigen-binding fragment thereof that provides a primary activation signal to T cells, and a second agent (8), an anti-CD28 antibody or antigen-binding fragment thereof that stimulates CD28 as an accessory molecule, are reversibly immobilized (bound) to the multimerization reagent (4). The multimerization reagent (4) shown in the example of Figure 3 contains only one binding site, Z1 (42), for reversible binding of both the first agent (6) and the second agent (8). Both the first agent (6) and the second agent (8) comprise at least one binding partner C1 (6a, 8a), and both the binding partner C1 (6a) and the binding partner (8a) can reversibly bind to the binding site Z1 (44) of the multimerization reagent. Thus, for immobilization, the first agent (6) and the second agent (8) bind to the multimerization reagent (4) via reversible bonds formed between the binding partner C1 (6a) and the binding partner C2, respectively, and the binding site Z1 (42). The binding partners C1 and C2 can be different or identical.For example, binding partner C1 can be a streptavidin-binding peptide of the sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 01), "Strep-tag®"), while binding partner C2 can be a streptavidin-binding peptide of the sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)3-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 04), also known as "di-tag3") or the sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)2-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 05), also known as "di-tag2"), Junttila et al., Proteomics 5 (2005), The multimerization reagent (4) may be a streptavidin-binding peptide (described in U.S. Pat. Nos. 1199-1203 or U.S. Pat. No. 7,981,632). 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 these streptavidin-binding peptides are used as binding partners C1 and C2, the multimerization reagent (4) is a streptavidin mutein. As shown in FIG. 3, a soluble multimerization reagent (4) is used. In the case of a streptavidin mutein, the soluble multimerization reagent may be, for example, an oligomer or polymer of streptavidin or avidin, or any mutein (analog) of streptavidin or avidin. The oligomer may contain three or more monomers of streptavidin, avidin, or muteins thereof. The oligomer or polymer may be cross-linked by a polysaccharide.Such oligomers or polymers of streptavidin or avidin or streptavidin or avidin muteins can be prepared in a first step by introducing carboxyl residues into a polysaccharide, such as dextran, essentially as described in "Noguchi, A., Takahashi, T., Yamaguchi, T., Kitamura, K., Takakura, Y., Hashida, M. & Sezaki, H. (1992). Preparation and properties of the immunoconjugate composed of anti-human colon cancer monoclonal antibody and mitomycin C dextran conjugate. Bioconjugate Chemistry 3, 132-137." In a second step, streptavidin or avidin or muteins thereof are linked to carboxyl groups on the dextran backbone via internal lysine residues and / or the free N-terminal primary amino group using conventional carbodiimide chemistry. Alternatively, cross-linked oligomers or polymers of streptavidin or avidin, or any muteins of streptavidin or avidin, can be obtained by cross-linking via a bifunctional linker, such as glutaraldehyde, or by other methods described in the literature. Using binding partners C1 and C2 that bind to the same binding site (42) of the multimerizing agent has the advantage that the same free partner (for the first binding partner C1 and for the second binding partner C2) or its analog can be used to terminate the expansion of T cell population (2) and release it from the multimerizing agent, as shown in Figure 3b. In the example of Figure 3, analogs of the first and second partners C1 and C2, such as biotin or biotin derivatives (iminobiotin or desthiobiotin), can be conveniently used to terminate the expansion and release T cell population (2).As shown in Figure 3c, after elution of the cells as shown in Figure 1d, the first agent (6), the second reagent (8), and biotin as the analog of the free first partner of binding partner C1 and the free second partner of binding partner C2 (20) can be easily removed from the stimulated cell population (2) via the "removal cartridge" described in WO 2013 / 124474. Furthermore, the embodiment using soluble multimerization reagent (4) has the additional advantage of avoiding any solid support, such as magnetic beads. This means that there is no risk of contamination of activated T cells with such magnetic beads. This also means that a process that complies with GMP standards can be established much more easily than known methods, such as the use of Dynabeads®, which require additional measurements to ensure that the final expanded T cell population is free of magnetic beads. Furthermore, the use of a soluble multimerizer makes it much easier to remove it from activated cell populations (T cells, B cells, or even natural killer cells) because the cells can be simply sedimented by centrifugation and the supernatant containing the soluble multimerizer can be discarded. Alternatively, the soluble multimerizer may be removed from the expanded cell population in the gel permeation matrix of the removal cartridge of WO 2013 / 124474. Because no solid phase (e.g., magnetic beads) is present, the present invention also provides an automated, closed system for expanding cells 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). [Figure 4A]Figure 4 shows the results of an experiment in which CD3+ responder T cells expanded after in vitro stimulation with αCD3 and αCD28 Fab fragments reversibly immobilized on beads coated with the streptavidin mutein Strep-tactin®. Figure 4A is a histogram showing the size distribution (forward scatter) of stimulated cells, Figure 4B is a histogram showing the extent of proliferation according to the number of cells per cell division indicated on Figure 4B (0 represents cells that have not divided; 5 represents cells that have divided at least five times), and Figure 4C shows a photograph of the culture dish after 4 days of stimulation. [Figure 4B] Figure 4 shows the results of an experiment in which CD3+ responder T cells expanded after in vitro stimulation with αCD3 and αCD28 Fab fragments reversibly immobilized on beads coated with the streptavidin mutein Strep-tactin®. Figure 4A is a histogram showing the size distribution (forward scatter) of stimulated cells, Figure 4B is a histogram showing the extent of proliferation according to the number of cells per cell division indicated on Figure 4B (0 represents cells that have not divided; 5 represents cells that have divided at least five times), and Figure 4C shows a photograph of the culture dish after 4 days of stimulation. [Figure 4C] Figure 4 shows the results of an experiment in which CD3+ responder T cells expanded after in vitro stimulation with αCD3 and αCD28 Fab fragments reversibly immobilized on beads coated with the streptavidin mutein Strep-tactin®. Figure 4A is a histogram showing the size distribution (forward scatter) of stimulated cells, Figure 4B is a histogram showing the extent of proliferation according to the number of cells per cell division indicated on Figure 4B (0 represents cells that have not divided; 5 represents cells that have divided at least five times), and Figure 4C shows a photograph of the culture dish after 4 days of stimulation. [Figure 5A]Figure 5 shows the results of differential intracellular calcium mobilization in Jurkat cells labeled with either the αCD3 antibody OKT3 or the Fab fragment of OKT3 multimerized with Strep-tactin® (also referred to herein as Fab multimer). Figure 5A: Jurkat cells were loaded with the calcium-sensitive dye Indo-1-AM, and calcium release was triggered by injection of αCD3 mAb (black squares) or αCD3 OKT3 Fab multimers (derived from the parental cell line OKT3) with or without prior cleavage with D-biotin (dark gray triangles and light gray circles, respectively), compared with injection of PBS (white inverted triangles). Application of ionomycin served as a positive control. Time-resolved changes in intracellular Ca2+ concentration were monitored by flow cytometry based on changes in the FL6 / FL7 ratio. Figure 5B: Indo-1-AM-labeled Jurkat cells were activated with various αCD3 stimuli as described in Figure 4a (OKT3: upper graph and αCD3Fab multimer: middle graph), followed by D-biotin-mediated disruption of αCD3Fab multimer signaling (t = 140 s). Injection of PBS (lower graph) and ionomycin served as negative or positive controls. Data are representative of three separate experiments. [Figure 5B]Figure 5 shows the results of differential intracellular calcium mobilization in Jurkat cells labeled with either the αCD3 antibody OKT3 or the Fab fragment of OKT3 multimerized with Strep-tactin® (also referred to herein as Fab multimer). Figure 5A: Jurkat cells were loaded with the calcium-sensitive dye Indo-1-AM, and calcium release was triggered by injection of αCD3 mAb (black squares) or αCD3 OKT3 Fab multimers (derived from the parental cell line OKT3) with or without prior cleavage with D-biotin (dark gray triangles and light gray circles, respectively), compared with injection of PBS (white inverted triangles). Application of ionomycin served as a positive control. Time-resolved changes in intracellular Ca2+ concentration were monitored by flow cytometry based on changes in the FL6 / FL7 ratio. Figure 5B: Indo-1-AM-labeled Jurkat cells were activated with various αCD3 stimuli as described in Figure 4a (OKT3: upper graph and αCD3Fab multimer: middle graph), followed by D-biotin-mediated disruption of αCD3Fab multimer signaling (t = 140 s). Injection of PBS (lower graph) and ionomycin served as negative or positive controls. Data are representative of three separate experiments. [Figure 6] Figure 6 shows the results of reversible staining of cells with anti-CD3 OKT3 Fab multimers. Freshly isolated PBMCs were stained with either monoclonal antibodies (left dot plot, parental clone of the Fab multimer) or cognate PE-labeled Fab multimers and analyzed before (second column from the left) or after (middle column) treatment with D-biotin. After a subsequent washing step with fresh PE-labeled Strep-Tactin®, remaining Fab monomers were then detected (second column from the right). Secondary Fab multimer staining of reversibly stained cells served as a control (right column). Only viable (PI-negative) cells are shown. Numbers in the dot plots indicate the percentage of cells within the gate. [Figure 7]Figure 7 shows the isolation of cells by reversible binding of anti-CD28 Fab fragments multimerized with Strep-Tactin® labeled with phycoerythrin as a fluorescent label. CD28+ cells were selected / isolated from freshly isolated PMBCs by Fab multimer magnetic cell selection, as described in WO 2013 / 011011. Prior to selection, cells were control stained with either cognate fluorescent aCD28 multimers (left dot plot) or an antibody against the immunoglobulin kappa light chain (second dot plot from the left, α-Ig kappa mAb). After selection, cells were treated with D-biotin and then washed to remove magnetic beads and Fab monomers. Released CD28+ cells were then (re)stained with either CD28 Fab multimers (second dot plot from the right) or α-Ig kappa mAb (right dot plot) to detect any remaining Fab monomers. Only viable (PI-negative) CD3+ cells are shown. Numbers in the dot plots indicate the percentage of cells within the gate. [Figure 8-1]Figure 8 shows the results of an experiment in which CD3+ responder T cells were expanded after in vitro stimulation with reversibly immobilized αCD3 / αCD28 Fab fragments on soluble oligomeric Strep-tactin®, which served as a soluble multimerization reagent. In the experiment whose results are shown in Figure 8, 300,000 CD3+ responder T cells (Tresp) were labeled with 2 μM carboxyfluorescein succinimidyl ester (CFSE) and stimulated with various amounts of a preparation of soluble Streptactin oligomers onto which combinations of αCD3 Fab fragments and αCD28 Fab fragments, both of which have a Strep-tag on the heavy chain as a streptavidin-binding peptide, were immobilized. ("1×" corresponds to 3 μg of multimerized Strep-tactin functionalized with 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 Fab; numbers indicate multiples of "1×"). Tresp cells were left unstimulated or stimulated with blank Strep-Tactin multimers (no Fab) to serve as a negative control. Tresp cells were seeded in duplicate 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) in 48-well plates. Cells were incubated at 37°C without medium changes, and proliferation was analyzed according to CFSE dilution after 5 days by FACS analysis (Figure 8B). Figure 8A shows the size distribution of cells after 5 days of culture. The histogram shows viable CD3+ cells, while Figure 8C shows cells after culture treated with 1 mM D-biotin, washed, and then released with stimulatory reagent. Dissociation and removal of monomeric Fab fragments were analyzed by restaining with phycoerythrin-labeled Strep-Tactin® (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 and plotted against each stimulation condition. Median cell counts are shown in Figure 8D; error bars indicate standard deviation (SD). Figure 8E shows a photograph of the culture dish after 5 days of stimulation. [Figure 8-2]Figure 8 shows the results of an experiment in which CD3+ responder T cells were expanded after in vitro stimulation with reversibly immobilized αCD3 / αCD28 Fab fragments on soluble oligomeric Strep-tactin®, which served as a soluble multimerization reagent. In the experiment whose results are shown in Figure 8, 300,000 CD3+ responder T cells (Tresp) were labeled with 2 μM carboxyfluorescein succinimidyl ester (CFSE) and stimulated with various amounts of a preparation of soluble Streptactin oligomers onto which combinations of αCD3 Fab fragments and αCD28 Fab fragments, both of which have a Strep-tag on the heavy chain as a streptavidin-binding peptide, were immobilized. ("1×" corresponds to 3 μg of multimerized Strep-tactin functionalized with 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 Fab; numbers indicate multiples of "1×"). Tresp cells were left unstimulated or stimulated with blank Strep-Tactin multimers (no Fab) to serve as a negative control. Tresp cells were seeded in duplicate 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) in 48-well plates. Cells were incubated at 37°C without medium changes, and proliferation was analyzed according to CFSE dilution after 5 days by FACS analysis (Figure 8B). Figure 8A shows the size distribution of cells after 5 days of culture. The histogram shows viable CD3+ cells, while Figure 8C shows cells after culture treated with 1 mM D-biotin, washed, and then released with stimulatory reagent. Dissociation and removal of monomeric Fab fragments were analyzed by restaining with phycoerythrin-labeled Strep-Tactin® (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 and plotted against each stimulation condition. Median cell counts are shown in Figure 8D; error bars indicate standard deviation (SD). Figure 8E shows a photograph of the culture dish after 5 days of stimulation. [Figure 8-3]Figure 8 shows the results of an experiment in which CD3+ responder T cells were expanded after in vitro stimulation with reversibly immobilized αCD3 / αCD28 Fab fragments on soluble oligomeric Strep-tactin®, which served as a soluble multimerization reagent. In the experiment whose results are shown in Figure 8, 300,000 CD3+ responder T cells (Tresp) were labeled with 2 μM carboxyfluorescein succinimidyl ester (CFSE) and stimulated with various amounts of a preparation of soluble Streptactin oligomers onto which combinations of αCD3 Fab fragments and αCD28 Fab fragments, both of which have a Strep-tag on the heavy chain as a streptavidin-binding peptide, were immobilized. ("1×" corresponds to 3 μg of multimerized Strep-tactin functionalized with 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 Fab; numbers indicate multiples of "1×"). Tresp cells were left unstimulated or stimulated with blank Strep-Tactin multimers (no Fab) to serve as a negative control. Tresp cells were seeded in duplicate 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) in 48-well plates. Cells were incubated at 37°C without medium changes, and proliferation was analyzed according to CFSE dilution after 5 days by FACS analysis (Figure 8B). Figure 8A shows the size distribution of cells after 5 days of culture. The histogram shows viable CD3+ cells, while Figure 8C shows cells after culture treated with 1 mM D-biotin, washed, and then released with stimulatory reagent. Dissociation and removal of monomeric Fab fragments were analyzed by restaining with phycoerythrin-labeled Strep-Tactin® (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 and plotted against each stimulation condition. Median cell counts are shown in Figure 8D; error bars indicate standard deviation (SD). Figure 8E shows a photograph of the culture dish after 5 days of stimulation. [Figure 9]FIG. 9 shows a description of the continuous expansion method of the present invention (FIG. 9a), while FIG. 9b briefly describes some features and advantages of continuous expansion. [Figure 10]FIG. 10 illustrates an arrangement of the present invention that can be used with the expansion methods of the present invention. The arrangement (100) includes a bioreactor (50), a first "removal cartridge" (70), and a second "removal cartridge" (90). The bioreactor (50) is fluidly connected to the first removal cartridge, which in turn is fluidly connected to the second removal cartridge (90). The arrangement (100) can be part of an apparatus for automated cell expansion and purification as described herein. The bioreactor (50) performs an expansion method described herein, such as the expansion method illustrated in FIG. 3 utilizing a soluble multimerization reagent. In this case, after terminating activation / expansion of cell population (2) by adding competitor (20) (the free partner of binding partner C1 or an analog thereof), the reaction mixture discharged from the bioreactor contains the expanded cell population (2), the first agent (6), the second agent (8), and the soluble multimerization reagent (4). In this example, the first agent (6) is a CD3-binding antibody fragment containing a streptavidin-binding peptide as the binding partner C1, the second agent (8) is a CD28-binding antibody fragment containing a streptavidin-binding peptide as the binding partner C1, and the competitor (20) (a free analog of the binding partner C1) is biotin. The reaction mixture is applied to a first removal cartridge (70). This first removal cartridge (70) is the removal cartridge described in WO 2013 / 124474, which includes a chromatography column having a suitable stationary phase. The stationary phase can function as an affinity chromatography matrix and also as a gel permeation matrix. An affinity reagent is immobilized on this affinity chromatography matrix. In this example, the affinity reagent can be, for example, streptavidin, a streptavidin mutein, avidin, an avidin mutein, or a mixture thereof. Therefore, the first agent (6) and the second agent (8) bind to the affinity reagent via these streptavidin-binding peptides, and biotin as a competitor (20) also binds to the affinity reagent.Thus, all three reagents are immobilized on the chromatography matrix of the first removal cartridge, while the expanded cell population (2) and soluble multimerization reagent (4) pass through the stationary phase. This "flow-through" is then applied to a second removal cartridge (90), which also contains a stationary phase. This stationary phase contains a second affinity reagent capable of binding to binding site Z1 (42) of multimerization reagent (4). This affinity reagent may be, for example, biotin covalently bound to the stationary phase. Such a stationary phase may be, for example, d-biotin Sepharose™, available from Affiland SA (Ans-Liege, Belgium). Thus, the soluble multimerization reagent (4) binds to (is retained by) the stationary phase of the second removal cartridge (90), while the expanded cell population (2) passes through the stationary phase and is free of any reactants. Population of cells (2) is now ready for any further use, such as diagnostic applications (e.g., further FACS™ sorting) or any cell-based therapeutic application. Of course, it is possible to change the order of the first "removal cartridge" (70) and the second "removal cartridge" (90) in configuration (100), such that bioreactor (50) is (directly) fluidically connected to second removal cartridge (90), and first removal cartridge (70) is subsequently positioned and fluidly connected to second removal cartridge (90). In this configuration, multimerization reagent (4) is first removed from population of cells (2), followed by first agent (6), second agent (8), and, for example, competitor (20). Such configurations are also encompassed by the present invention and may be part of an apparatus for automated cell expansion and purification as described herein. [Figure 11]11 shows a further embodiment of an arrangement of the present invention that can be used with the expansion methods of the present invention. This arrangement (110) includes a bioreactor (50), a first "removal cartridge" (70), and a second "removal cartridge" (90). The bioreactor (50) is fluidly connected to the first removal cartridge (70), which in turn is fluidly connected to the second removal cartridge (90). The second removal cartridge (110) is further fluidly connected to the bioreactor (50). This arrangement (110) can also be part of an apparatus for automated cell expansion and purification as described herein. For example, when used with an expansion method using a soluble multimerization reagent (4), a purified, expanded population of cells (2) is obtained as the eluate of the second removal cartridge (90). Since removal cartridge (90) is fluidly connected to bioreactor (50), population of cells (2) may be returned to bioreactor (50) for serial clonal expansion, e.g., as described above, by transfecting the population of cells with a gene for a T cell receptor and then further (second) expanding the population of cells using the expansion method of the present invention. [Figure 12]Figure 12 shows a further embodiment of an arrangement of the present invention that can be used with the expansion method of the present invention. This arrangement (120) includes a bioreactor (50), a first "removal cartridge" (70), and a second "removal cartridge" (90). The bioreactor (50) is fluidly connected to the first removal cartridge (70), which in turn is fluidly connected to the second removal cartridge (90). Similar to the embodiment shown in Figure 11, a second removal cartridge (110) is fluidly connected to the bioreactor (50). However, a "selection cartridge" (92) described in WO 2013 / 124474 is positioned between the second removal cartridge (90) and the bioreactor (50). Thus, as described in WO 2013 / 124474, a subpopulation (2a) of cells contained in the population of cells (2) can be selected / enriched via this "selection cartridge" (92). This subpopulation of cells (2a) may be transferred to a bioreactor (50), for example, for continuous expansion as described herein. Alternatively (not shown), this subpopulation of cells (2a) may be used for cell-based therapy. It is also noted that the use of the soluble multimerization reagents described herein allows for the design of automated cell purification and expansion devices that are functionally closed and therefore less prone to contamination. Furthermore, because the soluble multimerization reagents eliminate the need for solid phase materials such as magnetic beads, such cell purification devices can also be designed as continuous-flow devices. [Figure 13]Figure 13 shows the expansion kinetics of purified CD4+ and CD8+ responder T cells (Tresp) stimulated in vitro with either the αCD3 / αCD28 Fab fragment or the αCD3 / αCD28 / αCD8 reversibly immobilized on two types of soluble oligomeric Strep-tactin® muteins, which act as soluble multimerization reagents. The first type of oligomeric Strep-tactin® was a fraction of the oligomeric streptavidin muteins (n≧3) obtained in Example 5 (also referred to herein as the "conventional Strep-tactin® scaffold" and indicated by a downward-pointing triangle symbol in Figure 13), and the second type of this oligomeric streptavidin mutein used as a soluble multimerization reagent was an oligomer obtained by reacting the soluble oligomeric streptavidin mutein with biotinylated human serum albumin (HSA). This HSA-based soluble multimerization reagent is also referred to herein as the "large Streptactin® scaffold." In the experiment of Figure 13, expansion was performed without changing the medium. Results for CD4+ responder T cells are shown in Figure 13A, and results for CD8+ responder T cells are shown in Figure 13B. Note that in this context, the soluble multimerization reagent used in the experiment, functionalized by reversibly binding a first agent, and optionally a second and third agent, is referred to in the figures as the "Streptamer® multimer." [Figure 14]Figure 14 shows the expansion kinetics of purified CD4+ and CD8+ responder T cells (Tresp) stimulated in vitro with the reversibly immobilized αCD3 / αCD28 Fab fragment, which was reversibly immobilized with two types of soluble oligomeric Strep-tactin® acting as soluble multimerization reagents. The first type of oligomeric Strep-tactin® was a fraction of the oligomeric streptavidin muteins (n≧3) obtained in Example 5 (also referred to herein as the "conventional Streptactin® scaffold" and indicated by an upturned triangle symbol in Figure 14), while the second type of this oligomeric streptavidin mutein used as the soluble multimerization reagent was the HSA-based soluble multimerizer described above (the "large Streptactin® scaffold"). In the experiments shown in Figure 14, expansion was performed by changing the medium. The results for CD4+ responder T cells are shown in Figure 14, and the results for CD8+ responder T cells are shown in Figure 14B. [Figure 15] Figure 15 shows the combined data from the results obtained in Figures 13 and 14 for the expansion kinetics of purified CD4+ and CD8+ responder T cells, with Figure 15A showing the results for CD4+ T cells and Figure 15B showing the results for CD8+ T cells. A straight line is used for cultures in which the medium was changed on day 3, while the dashed line indicates the value obtained for the extent of expansion when the medium was not changed on day 3. The data shown in Figure 15 are normalized to the input cell number. Only data for Tresp stimulated with oligomeric streptavidin muteins (n≧3), Tresp stimulated with commercially available Dynabeads (positive control), and unstimulated T cells (negative control) are shown; data for the multimerization reagent with the "large Streptactin® backbone" are not shown. [Figure 16]Figure 16 shows the early cluster formation of T cells after activation of purified CD4+ and CD8+ responder T cells stimulated in vitro with αCD3 / αCD28 Fab fragments reversibly immobilized on soluble oligomeric streptavidin muteins (n≧3) as described in Example 5. Figure 16A shows the results for CD4+ T cells, and Figure 16B shows the results for CD8+ T cells. Data are shown for Tresp stimulated with soluble multimerization reagent (oligomeric streptavidin muteins), Tresp stimulated with commercially available Dynabeads (positive control), and unstimulated T cells (negative control). [Figure 17-1] Figure 17 shows the expansion kinetics and phenotype of CD3+ central memory T cells (Tcm) (CD3+CD62L+CD45RA-Tcm) polyclonally stimulated in vitro with αCD3 / αCD28 Fab fragments reversibly immobilized on soluble oligomeric streptavidin muteins (n≧3) as described in Example 5. The graphs shown in Figure 17 show the extent of proliferation according to the number of cells harvested at each time point: Figure 17A shows proliferation in medium supplemented with IL-2 alone, and Figure 17B shows proliferation in medium supplemented with IL-2 and IL-15. Figure 17C shows flow cytometry analysis of CD62L and CD127 surface expression after 14 days of culture in these variable cytokine environments. [Figure 17-2] Figure 17 shows the expansion kinetics and phenotype of CD3+ central memory T cells (Tcm) (CD3+CD62L+CD45RA-Tcm) polyclonally stimulated in vitro with αCD3 / αCD28 Fab fragments reversibly immobilized on soluble oligomeric streptavidin muteins (n≧3) as described in Example 5. The graphs shown in Figure 17 show the extent of proliferation according to the number of cells harvested at each time point: Figure 17A shows proliferation in medium supplemented with IL-2 alone, and Figure 17B shows proliferation in medium supplemented with IL-2 and IL-15. Figure 17C shows flow cytometry analysis of CD62L and CD127 surface expression after 14 days of culture in these variable cytokine environments. [Figure 18-1]Figure 18 shows the kinetics of selective antigen-specific (Ag-specific) expansion from a bulk population of purified CD3+CD62L+CD45RA- responder Tcm cells stimulated in vitro with both peptide:MHC molecule complexes (acting as a first agent providing a primary activation signal to the cells) and αCD28Fab fragments (acting as a second agent binding to accessory molecules on the surface of the cells), and unstimulated T cells (negative control). Both the antigen-specific peptide:MHC molecule complexes and αCD28Fab fragments were reversibly immobilized on the same soluble oligomeric streptavidin muteins (n≧3) as described in Example 5. The peptide used for antigen-specific amplification in Figure 18A was the peptide CRVLCCYVL (SEQ ID NO: 06), which is amino acids 309-317 of the immediate early 1 protein restricted by the HLA-C702 MHC molecule (described in Ameres et al., PLOS Pathogens, May 2013, vol. 9, issue 5, e1003383), representing the HLA-C7 / IE-1 epitope specific for cytomegalovirus (CMV). The MHC I molecule presenting this peptide carries a streptavidin-binding peptide (SAWSHPQFEK(GGGS)2GGSAWSHPQFEK (SEQ ID NO: 07), commercially available as "Twin-Strep-tag®" from IBA GmbH, Göttingen, Germany) at the C-terminus of the heavy chain. Figure 18A shows an exemplary flow cytometry analysis of the fraction of Ag-specific cells expanded using peptide:MHC-I complexes specific for this HLA-C7 / IE-1 epitope as the first agent to provide a primary activation signal to cells reversibly immobilized on soluble oligomeric streptavidin muteins. The graphs in Figures 18B-18E show the kinetics of further Ag-specific expansion according to the number of specific peptide:MHC-I multimer-positive cells harvested per time point, similar to Figure 18A, using different complexes of antigen-specific peptides and MHC I molecules as the first agent to provide a primary activation signal to cells reversibly immobilized on soluble oligomeric streptavidin muteins.More specifically, Figure 18B shows the expansion of Ag-specific cells expanded using a peptide:MHC-I complex specific for the CMV pp65 epitope (amino acids 341-350 (QYDPVAALF (SEQ ID NO: 08)) restricted by HLA-A2402), and Figure 18C shows the expansion of Ag-specific cells expanded using another peptide:MHC-I complex specific for the CMV pp65 epitope (amino acids 265-274 (RPHERNGFTV (SEQ ID NO: 09)) restricted by HLA-B702). Figure 18D shows the expansion of Ag-specific cells grown with peptide:MHC-I complexes specific for the hexon 5 epitope of adenovirus (amino acids 114-124 (CPYSGTAYNSL (SEQ ID NO: 10)) restricted by HLA-B702), and Figure 18E shows the expansion of Ag-specific cells grown with peptide:MHC-I complexes specific for the HLA-B7 / IE-1309-317 epitope of CMV (see Figure 18A above for exemplary FACS data). All peptide:MHC molecules with Strep® tags are commercially available from Iba GmbH. In this context, the amino acid sequences of HLA-A*2402, HLA-B*0702, and HLA-C*0702 molecules with a "Twin-Strep®" tag at their C-terminus are shown in the attached sequence listing as SEQ ID NOs:21, 22, and 23, respectively, while the amino acid sequence of β2-microglobulin (which forms the α chain, i.e., the respective MHC I molecule together with the HLA-encoding molecule) is shown in the attached sequence listing as SEQ ID NO:24. Furthermore, Figure 18F shows an exemplary flow cytometry analysis of the surface expression of C62L and CD127 for HLA-B7 / Hexon5114-124 stimulated / expanded cells from Figure 18D after 14 days of culture. [Figure 18-2]Figure 18 shows the kinetics of selective antigen-specific (Ag-specific) expansion from a bulk population of purified CD3+CD62L+CD45RA- responder Tcm cells stimulated in vitro with both peptide:MHC molecule complexes (acting as a first agent providing a primary activation signal to the cells) and αCD28Fab fragments (acting as a second agent binding to accessory molecules on the surface of the cells), and unstimulated T cells (negative control). Both the antigen-specific peptide:MHC molecule complexes and αCD28Fab fragments were reversibly immobilized on the same soluble oligomeric streptavidin muteins (n≧3) as described in Example 5. The peptide used for antigen-specific amplification in Figure 18A was the peptide CRVLCCYVL (SEQ ID NO: 06), which is amino acids 309-317 of the immediate early 1 protein restricted by the HLA-C702 MHC molecule (described in Ameres et al., PLOS Pathogens, May 2013, vol. 9, issue 5, e1003383), representing the HLA-C7 / IE-1 epitope specific for cytomegalovirus (CMV). The MHC I molecule presenting this peptide carries a streptavidin-binding peptide (SAWSHPQFEK(GGGS)2GGSAWSHPQFEK (SEQ ID NO: 07), commercially available as "Twin-Strep-tag®" from IBA GmbH, Göttingen, Germany) at the C-terminus of the heavy chain. Figure 18A shows an exemplary flow cytometry analysis of the fraction of Ag-specific cells expanded using peptide:MHC-I complexes specific for this HLA-C7 / IE-1 epitope as the first agent to provide a primary activation signal to cells reversibly immobilized on soluble oligomeric streptavidin muteins. The graphs in Figures 18B-18E show the kinetics of further Ag-specific expansion according to the number of specific peptide:MHC-I multimer-positive cells harvested per time point, similar to Figure 18A, using different complexes of antigen-specific peptides and MHC I molecules as the first agent to provide a primary activation signal to cells reversibly immobilized on soluble oligomeric streptavidin muteins.More specifically, Figure 18B shows the expansion of Ag-specific cells expanded using a peptide:MHC-I complex specific for the CMV pp65 epitope (amino acids 341-350 (QYDPVAALF (SEQ ID NO: 08)) restricted by HLA-A2402), and Figure 18C shows the expansion of Ag-specific cells expanded using another peptide:MHC-I complex specific for the CMV pp65 epitope (amino acids 265-274 (RPHERNGFTV (SEQ ID NO: 09)) restricted by HLA-B702). Figure 18D shows the expansion of Ag-specific cells grown with peptide:MHC-I complexes specific for the hexon 5 epitope of adenovirus (amino acids 114-124 (CPYSGTAYNSL (SEQ ID NO: 10)) restricted by HLA-B702), and Figure 18E shows the expansion of Ag-specific cells grown with peptide:MHC-I complexes specific for the HLA-B7 / IE-1309-317 epitope of CMV (see Figure 18A above for exemplary FACS data). All peptide:MHC molecules with Strep® tags are commercially available from Iba GmbH. In this context, the amino acid sequences of HLA-A*2402, HLA-B*0702, and HLA-C*0702 molecules with a "Twin-Strep®" tag at their C-terminus are shown in the attached sequence listing as SEQ ID NOs:21, 22, and 23, respectively, while the amino acid sequence of β2-microglobulin (which forms the α chain, i.e., the respective MHC I molecule together with the HLA-encoding molecule) is shown in the attached sequence listing as SEQ ID NO:24. Furthermore, Figure 18F shows an exemplary flow cytometry analysis of the surface expression of C62L and CD127 for HLA-B7 / Hexon5114-124 stimulated / expanded cells from Figure 18D after 14 days of culture. [Figure 18-3]Figure 18 shows the kinetics of selective antigen-specific (Ag-specific) expansion from a bulk population of purified CD3+CD62L+CD45RA- responder Tcm cells stimulated in vitro with both peptide:MHC molecule complexes (acting as a first agent providing a primary activation signal to the cells) and αCD28Fab fragments (acting as a second agent binding to accessory molecules on the surface of the cells), and unstimulated T cells (negative control). Both the antigen-specific peptide:MHC molecule complexes and αCD28Fab fragments were reversibly immobilized on the same soluble oligomeric streptavidin muteins (n≧3) as described in Example 5. The peptide used for antigen-specific amplification in Figure 18A was the peptide CRVLCCYVL (SEQ ID NO: 06), which is amino acids 309-317 of the immediate early 1 protein restricted by the HLA-C702 MHC molecule (described in Ameres et al., PLOS Pathogens, May 2013, vol. 9, issue 5, e1003383), representing the HLA-C7 / IE-1 epitope specific for cytomegalovirus (CMV). The MHC I molecule presenting this peptide carries a streptavidin-binding peptide (SAWSHPQFEK(GGGS)2GGSAWSHPQFEK (SEQ ID NO: 07), commercially available as "Twin-Strep-tag®" from IBA GmbH, Göttingen, Germany) at the C-terminus of the heavy chain. Figure 18A shows an exemplary flow cytometry analysis of the fraction of Ag-specific cells expanded using peptide:MHC-I complexes specific for this HLA-C7 / IE-1 epitope as the first agent to provide a primary activation signal to cells reversibly immobilized on soluble oligomeric streptavidin muteins. The graphs in Figures 18B-18E show the kinetics of further Ag-specific expansion according to the number of specific peptide:MHC-I multimer-positive cells harvested per time point, similar to Figure 18A, using different complexes of antigen-specific peptides and MHC I molecules as the first agent to provide a primary activation signal to cells reversibly immobilized on soluble oligomeric streptavidin muteins.More specifically, Figure 18B shows the expansion of Ag-specific cells expanded using a peptide:MHC-I complex specific for the CMV pp65 epitope (amino acids 341-350 (QYDPVAALF (SEQ ID NO: 08)) restricted by HLA-A2402), and Figure 18C shows the expansion of Ag-specific cells expanded using another peptide:MHC-I complex specific for the CMV pp65 epitope (amino acids 265-274 (RPHERNGFTV (SEQ ID NO: 09)) restricted by HLA-B702). Figure 18D shows the expansion of Ag-specific cells grown with peptide:MHC-I complexes specific for the hexon 5 epitope of adenovirus (amino acids 114-124 (CPYSGTAYNSL (SEQ ID NO: 10)) restricted by HLA-B702), and Figure 18E shows the expansion of Ag-specific cells grown with peptide:MHC-I complexes specific for the HLA-B7 / IE-1309-317 epitope of CMV (see Figure 18A above for exemplary FACS data). All peptide:MHC molecules with Strep® tags are commercially available from Iba GmbH. In this context, the amino acid sequences of HLA-A*2402, HLA-B*0702, and HLA-C*0702 molecules with a "Twin-Strep®" tag at their C-terminus are shown in the attached sequence listing as SEQ ID NOs:21, 22, and 23, respectively, while the amino acid sequence of β2-microglobulin (which forms the α chain, i.e., the respective MHC I molecule together with the HLA-encoding molecule) is shown in the attached sequence listing as SEQ ID NO:24. Furthermore, Figure 18F shows an exemplary flow cytometry analysis of the surface expression of C62L and CD127 for HLA-B7 / Hexon5114-124 stimulated / expanded cells from Figure 18D after 14 days of culture. [Figure 19]Figure 19 shows the kinetics of selective Ag-specific expansion from purified CD3+CD62L+CD45RA- responder Tcm cells stimulated in vitro with a) antigen-specific peptide-MHC class I complexes and b) αCD28Fab fragments reversibly immobilized on soluble oligomeric streptavidin muteins as primary and secondary agents. To this end, 500,000 CD3+CD62L+CD45RA- responder Tcm cells (Tresp) were stimulated in an Ag-specific manner with 3 μL of a preparation of Streptactin multimerization reagent functionalized with 0.5 μg of peptide:MHC class I complexes and 0.5 μg of αCD28Fab, each containing a streptavidin-binding peptide (the specific peptide represents amino acids 114-124 of the Hexon 5 protein of adenovirus restricted by HLA-B0702 (CPYSGTAYNSL, SEQ ID NO: 10), see above). Alternatively, 4.5 μL of a streptactin multimerization reagent preparation was loaded with 0.5 μg of this peptide:MHC class I complex, 0.5 μg of αCD8Fab, and 0.5 μg of αCD28Fab. For comparison, polyclonal stimulation was performed using 3 μL of a 1 mg / mL streptactin multimerization reagent preparation loaded with a combination of 0.5 μg of αCD3Fab and 0.5 μg of αCD28Fab. Furthermore, as an alternative to the above stimulation conditions, 4.5 μL of a streptactin multimer preparation loaded with 0.5 μg of αCD3Fab, 0.5 μg of αCD8Fab, and 0.5 μg of αCD28Fab was used. Untreated (unstimulated) Tresp cells served as a negative control, and Tresp cells polyclonally stimulated with Dynabeads served as a positive control. Tresp cells were seeded in 1 mL of cell culture medium supplemented with 30 U / mL IL-2 and 5 ng / mL IL-15 in a 48-well plate. The cells were incubated at 37°C with medium changes every 3 days, and cell numbers were analyzed after 7 and 14 days. The photograph in Figure 19 shows the extent of cluster formation on day 5 for Ag-specific stimulation, exemplified by the adenoviral HLA-B7 / Hexon5 epitope. [Figure 20]Figure 20 shows the yield and phenotype of purified CD8+ responder T cell expansion stimulated in vitro with αCD3 / αCD28 Fab fragments reversibly immobilized on two types of soluble oligomeric Strep-tactin®, which act as soluble multimerization reagents. The first type of oligomeric Strep-tactin® is a fraction of the oligomeric streptavidin mutein obtained in Example 5 (conventional backbone). The second type of this oligomeric streptavidin mutein used as a soluble multimerization reagent is the soluble oligomer described above and is referred to herein as the "large" Streptactin® backbone. In these experiments, fractions of conventional oligomeric streptavidin muteins (n≧3) were also used as multimerization reagents functionalized with a single Fab fragment (third bar in Figures 20A and 20B) or a combination of αCD3 and αCD28 Fab fragments. To further test whether combined stimulation with the αCD3 / αCD28 Fab fragments could preferentially stimulate specific T cell subpopulations, we also immobilized an additional αCD8 Fab fragment (commercially available from IBA GmbH, Göttingen, Germany). Figure 20A shows a bar graph representing the degree of proliferation according to the number of cells harvested on day 6, compared with a negative control (unstimulated purified CD8 responder T cells) and normalized to a positive control (purified CD8 T responders stimulated with commercially available Dynabeads (beads to which αCD3 and αCD28 monoclonal antibodies are irreversibly immobilized)). Figure 20B 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 various stimulation conditions, and no significant differences (ns) were detected. [Figure 21]Figure 21 shows the yield and phenotype of the expansion of purified CD8+ responder T cells stimulated in vitro with αCD3 / αCD28 Fab fragments reversibly immobilized on soluble oligomeric Strep-tactin® acting as a soluble multimerization reagent functionalized with single or combinations of Fab fragments (as already described above). In these experiments, CD8+ responder T cells were stimulated with various amounts of soluble multimerization reagent functionalized with αCD3 and αCD28 Fab fragments (soluble oligomeric Strep-tactin® (1 mg / mL) from Example 5), optionally together with the above αCD8 Fab fragments. The term "1x" corresponds to 1.5 µg of multimerized streptactin functionalized with 0.5 µg of αCD3Fab fragments only and 1.5 µg of multimerized streptactin functionalized with 0.5 µg of αCD28Fab only, or 3 µL of a preparation of oligomeric streptactin loaded with 0.5 µg of αCD3Fab fragments and 0.5 µg of αCD28Fab, or 4.5 µL of a preparation of streptactin multimers loaded with 0.5 µg of streptactin-tagged, 0.5 µg of streptactin-tagged, 0.5 µg of streptactin-tagged, and 0.5 µg of streptactin-tagged αCD28Fab. Therefore, the term "2x" means that twice the amount of immobilized αCD3Fab fragments was used, corresponding to 3.0 μg of multimerized streptactin functionalized with only 1 μg of αCD3Fab fragments and 3.0 μg of multimerized streptactin functionalized with only 1 μg of αCD28Fab. Untreated Tresp cells served as a negative control, and purified CD8+ T responders stimulated with commercially available Dynabeads (beads to which αCD3 and αCD28 monoclonal antibodies were irreversibly immobilized) served as a positive control. Figure 21A shows a bar graph representing the degree of proliferation according to the cell number harvested on day 5 compared with the negative control and normalized to the positive control. Figure 21B shows FACS analysis of surface expression of CD8 and CD45RO after cell culture. [Figure 22]Figure 22 shows the activation of intracellular signaling cascades in transduced Jurkat cells engineered to express an αCD19 chimeric antigen receptor (CAR) and stimulated with the oligomeric Strep-tactin® of Example 5 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) that specifically binds to a target / tumor-associated antigen, such as CD19, and couples 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, a polyclonal αIgG F(ab)2 fragment (donkey anti-human F(ab)2 is commercially available from Jackson Immuno Research) recognizing the IgG4 spacer within αCD19-CAR, in addition to the extracellular domain (ECD) of CD19 containing the natural ligand of αCD19CAR, was also used as the first agent to provide a primary activation signal to Jurkat cells. Reversible immobilization to a soluble oligomeric streptavidin mutein was provided by the streptavidin peptide SAWSHPQFEK(GGGS)2GGSAWSHPQFEK (SEQ ID NO: 07) fused to the C-terminus of the ECD of CD19, or by a biotinylated (Fab)2 fragment of αIgG (streptavidin mutein "m2" binds biotin with low affinity, so 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 a mixture of various amounts of a preparation (1 mg / mL) of oligomeric streptactin functionalized with αCD3Fab and αCD28Fab ("x1" corresponds to 3 μg of multimerized streptactin functionalized with 0.5 μg of aCD3Fab polyclonal streptamer multimer and 0.5 μg of aCD28Fab polyclonal streptamer multimer).In the experiment shown in Figure 22B, 3 μL of oligomeric streptactin preparation was functionalized with 3 μL of oligomeric streptactin preparation loaded with 0.5 μg (×1) or 1 μg (×2) of the extracellular domain (ECD) of CD19 or 0.5 μg (×1) or 1 μg (×2) of αIgG recognizing an IgG4 spacer (both of which are CAR-specific Streptamer® multimers). Jresp cells were stimulated with Dynabeads (beads on which αCD3 and αCD28 monoclonal antibodies were irreversibly immobilized) or with PMA and ionomycin, which served 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 and stimulated for 0 or 20 minutes, then placed on ice and lysed. [Figure 23]23 shows the expansion of purified CD3+ responder T cells stimulated in vitro with αCD3 / αCD28 Fab fragments reversibly immobilized on the soluble oligomeric Strep-tactin® of Example 5, which serves as a soluble multimerization 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, Göttingen, Germany were immobilized on soluble oligomers of streptavidin muteins to test whether it was possible to preferentially stimulate in vitro a CD8+ T cell subpopulation within a bulk CD3+ culture using a multimerization reagent of the invention in which the αCD8 Fab fragment was also reversibly immobilized. More specifically, 500,000 purified CD3+ responder T cells (Tresp) were stimulated with 3 μL of a 1 mg / mL preparation of oligomeric streptavidin loaded with a combination of 0.5 μg of αCD3 and 0.5 μg of αCD28Fab. As an alternative approach, 4.5 μL of streptactin oligomers were loaded with 0.5 μg of the above αCD3, 0.5 μg of αCD8Fab, and 0.5 μg of αCD28Fab. Unstimulated Tresp cells served as a negative control, and Tresp cells stimulated with Dynabeads (beads to which αCD3 and αCD28 monoclonal antibodies were irreversibly immobilized) served as a positive control. [Figure 24]Figure 24 shows an exemplary strategy for generating oligomeric streptavidin muteins that can be used as soluble multimerization reagents of the present invention. Figure 24A shows the use of streptavidin mutein "m2" (SAm2), which contains the amino acid sequence 11e44-Gly45-Ala46-Arg47 (SEQ ID NO: 03) at sequence positions 44-47 of wild-type streptavidin, in a first step to generate oligomeric streptavidin muteins with a "conventional backbone." In a second step, oligomeric soluble streptavidin muteins with a "large backbone" can be generated by coupling the streptavidin mutein to a biotinylated carrier protein such as human serum albumin (HSA) or by coupling the streptavidin mutein to a synthetic carrier such as PEG. Figure 24B: Biotinylation of human serum albumin (HSA).

[0018] Detailed Description of the Invention The present invention provides methods, kits, and devices for expanding a population of cells or inducing and expanding a population of T cells.

[0019] The term "population of cells," as used herein, encompasses all cells that can be expanded by binding of a first agent that provides a primary activation signal to the cells to a cell surface receptor. It is also possible that expansion of a population of cells may require binding of a second agent to a second cell surface receptor (accessory molecule) to generate a costimulatory signal necessary for cell expansion. In some embodiments, the cell population may be a lymphocyte population, including, but not limited to, a population of B cells, a population of T cells, or a population of natural killer cells. An illustrative example of a cell population is CD40- or CD137-bearing B cells (both cell populations can proliferate upon binding a first agent that provides an activation signal, e.g., 4-1BB ligand; or αCD40 or αCD137 antibody molecules alone (see, e.g., Zhang et al., 2010, J Immunol, 184:787-795)). Other illustrative examples of agents (either first or second) that can be used to expand B cells include 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 expanding B cells may include 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). It is noted that lipopolysaccharide-dependent activation of B cells is also encompassed by the present invention, since, as used herein, lipopolysaccharide can also be used as a first agent and can comprise the binding partner C1. Other illustrative examples of suitable cell populations include T cell populations that are expanded following activation by a first agent binding to TCR / CD3 and a second agent binding to an accessory molecule on the T cells, such as CD28. In this case, the first agent stimulates a TCR / CD3 complex-associated signal in the T cell, and the second agent provides a secondary stimulus by binding to CD28 as an accessory molecule.Agents that can be used to expand T cells may 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 illustrative examples of agents that can be used to expand T cells are agents that bind to CD8, CD45, or CD90, e.g., antibodies to αCD8, αCD45, or αCD90. Illustrative examples of T cell populations include antigen-specific T cells, helper T cells, cytotoxic T cells, memory T cells (an illustrative example of a memory T cell is CD62L). + CD8 + specific central memory T cells), or regulatory T cells (an illustrative example of a Treg is a CD4 + CD25 +The term "T cell (population)," as used herein, also includes T cells comprising a chimeric antigen receptor (CAR), also known as an artificial T cell receptor or a chimeric T cell receptor. Thus, T cell populations comprising a chimeric antigen receptor can also be expanded using the methods, reagents, and devices of the present invention. In this regard, see also Example 15, in which Jurkat cells expressing a chimeric CD19-specific antigen receptor (CAR) were stimulated with a soluble multimerization reagent of the present invention. Another illustrative 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. In an illustrative example, such an αCD16 antibody is antibody 3G8, having the VH sequence set forth in SEQ ID NO: 25 and the VL sequence set forth in SEQ ID NO: 26 (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 illustrative 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. The cell population can be of any mammalian origin, including, but not limited to, human, rabbit, guinea pig, squirrel, hamster, cat, dog, lemur, goat, pig, horse, rhesus monkey, macaque, or chimpanzee.

[0020] Therefore, in light of the above, the present invention relates to methods for selectively inducing the ex vivo expansion of a population of cells, such as B cells, T cells, or natural killer cells, in the absence of exogenous growth factors and accessory molecules, such as lymphokines. Furthermore, proliferation of these cells, such as B cells or T cells, can be induced without the need for antigen, thus providing an expanded cell population, such as a T cell population, that is polyclonal in terms of antigen reactivity. The methods disclosed herein provide for sustained expansion of a selected population of T cells, such as CD4+ or CD8+ T cells, over an extended period of time, allowing for a multi-fold increase in the number of these cells relative to the original T cell population. Generally, in the case of (clonal) expansion of a lymphocyte population, as described herein, all progeny can share the same antigen specificity as the cell population selected for expansion.

[0021] In light of the above, the present invention also provides a method for expanding a population of antigen-specific T cells. To produce a population of antigen-specific T cells, T cells are contacted with an antigen in a form suitable for triggering a primary activation signal in the T cells, i.e., the antigen is presented to the T cells so that a signal is triggered in the T cells via the TCR / CD3 complex. For example, the antigen can be presented to the T cells by antigen-presenting cells in association with MHC molecules. Antigen-presenting cells, such as B cells, macrophages, monocytes, dendritic cells, Langerhans cells, or other cells capable of presenting antigen to T cells, can be incubated with T cells in the presence of antigen (e.g., soluble antigen) so that the antigen-presenting cells present the antigen to the T cells. Alternatively, cells expressing an antigen of interest can be incubated with T cells. For example, tumor cells expressing a tumor-associated antigen can be incubated with T cells to induce a tumor-specific response. Similarly, cells infected with a pathogen (e.g., a virus) that present an antigen of the pathogen can be incubated with T cells. Following antigen-specific activation of the population of T cells, the cells can be expanded according to the methods of the present invention. For example, after antigen specificity is established, T cells can be expanded by culturing them with an anti-CD3 antibody (used as a first agent) and an anti-CD28 antibody (used as a second agent) according to the methods described herein. In another embodiment, the first agent can be an MHC I:peptide complex that binds to the antigen-specific T cell population. In such an embodiment, any antigen-specific peptide known and capable of being complexed with the respective MHC I molecule can be used. See, in this regard, Examples 11 and 12, which illustrate the selective antigen-specific expansion of Tcm responder cells from bulk CD3+ central memory T cells for four different antigen-specific cells. Alternatively, the natural ligand of the receptor that triggers cell expansion can be used as the first agent. See, in this regard, Example 15, in which the extracellular domain of CD19 triggered activation of an intracellular signaling cascade in transduced Jurkat cells engineered to express a chimeric CD19-binding antigen receptor (CAR).

[0022] The cell population sample can be from any suitable source, typically a sample of body tissue or a bodily fluid such as blood. In the latter case, the sample can be, for example, a population of peripheral blood mononuclear cells (PBMCs), which can be obtained by standard isolation methods such as Ficoll gradients of blood cells. However, the cell population to be expanded can also be in a purified form and isolated using the reversible cell staining / isolation techniques described in U.S. Patent No. 7,776,562, U.S. Patent No. 8,298,782, WO 02 / 054065, or WO 2013 / 011011. Alternatively, the cell population can be obtained by negative magnetic immunoadhesion-mediated cell sorting, as described in U.S. Patent No. 6,352,694 B1 or European Patent No. 0 700 430 B1. When the isolation methods described herein are used in basic research, the sample can be cells from an in vitro cell culture experiment. The sample is typically prepared in a fluid form such as a solution or dispersion.

[0023] In light of the above, in one embodiment, the present invention provides an in vitro method for expanding a population of cells, the method comprising contacting a sample containing the population of cells with a multimerization reagent. A first agent that provides a primary activation signal to the cells is reversibly immobilized (bound) to the multimerization reagent, and the multimerization reagent comprises at least one binding site Z1 for reversible binding of the first agent. The first agent comprises at least one binding partner C1, which is capable of reversibly binding to binding site Z1 of the multimerization reagent, and the first agent binds to the multimerization reagent via the reversible bond formed between the binding partner C1 and the binding site Z1. The first agent binds to a receptor molecule on the surface of the cells to provide a primary activation signal to the cells, thereby activating the cells.

[0024] In another embodiment, the present invention provides a method in which a second agent that stimulates an accessory molecule on the surface of a cell is reversibly immobilized (bound) to a multimerization agent. The second agent comprises binding partner C2, which can reversibly bind to binding site Z2 of the multimerization reagent, and the second agent binds to the multimerization reagent via a reversible bond formed between binding partner C2 and binding site Z2. The second agent binds to the accessory molecule on the surface of the cell and stimulates the activated cell. In this embodiment, the first agent can stimulate a TCR / CD3 complex-associated signal in T cells and can be a binding agent that specifically binds to CD3. In this embodiment, the accessory molecule on the T cell can be CD28, and the second agent that binds to the accessory molecule is a binding agent that specifically binds to CD28. In this case, the first agent that specifically binds to CD3 may be selected from the group consisting of an anti-CD3 antibody, a bivalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, and a proteinaceous CD3-binding molecule with antibody-like binding properties. The second agent that specifically binds to CD28 may 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 may be a (Fab)2' fragment or a bivalent single-chain Fv fragment, while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv). The proteinaceous CD3- or CD28-binding molecule with antibody-like binding properties may be an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, an ankyrin scaffold-based protein, a crystalline scaffold-based protein, an adnectin, or an avimer.

[0025] In general, the first and second agents used in the present invention may be, for example, antibodies, fragments thereof, and proteinaceous binding molecules with antibody-like functions. Examples of (recombinant) antibody fragments are Fab fragments, Fv fragments, single-chain Fv fragments (scFv), bivalent antibody fragments such as (Fab)2' fragments, diabodies, triabodies (Iliades, P., et al., FEBS Lett (1997) 409, 437-441), decabodies (Stone, E., et al., Journal of Immunological Methods (2007) 318, 88-94), and other domain antibodies (Holt, LJ, et al., Trends Biotechnol. (2003), 21, 11, 484-490). In some embodiments, one or more binding sites of the first or second agent may be a bivalent artificial proteinaceous binding molecule, such as a dimeric lipocalin mutein, also known as a "duocalin." In some embodiments, the receptor-binding reagent may have a single second binding site, i.e., be monovalent. Examples of monovalent first or second 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.

[0026] As mentioned above, examples of proteinaceous binding molecules with antibody-like functions are 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, have natural ligand-binding sites that can be engineered to bind to a given target. Further examples of proteinaceous binding molecules with antibody-like binding properties that can be used as receptor binding reagents that specifically bind to receptor molecules include, but are not limited to, so-called glubodies (see, e.g., WO 96 / 23879), proteins based on ankyrin scaffolds (Mosavi, LK, et al., Protein Science (2004) 13, 6, 1435-1448), or proteins based on crystalline scaffolds (e.g., 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, contain so-called A domains that occur as strings of multiple domains in some cell surface receptors (Silverman, J., et al., Nature Biotechnology (2005) 23, 1556-1561). Adnectins, derived from a domain of human fibronectin, 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, derived from the respective human homotrimeric protein, similarly contain loop regions in a C-type lectin domain that can be engineered for desired binding (ibid.).Peptoids, which can act as protein ligands, are oligo(N-alkyl)glycines that differ from peptides in that the side chain is attached to the amide nitrogen rather than the α-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).Further examples of suitable proteinaceous binding molecules include EGF-like domains, kringle domains, fibronectin type I domains, fibronectin type II domains, fibronectin type III domains, PAN domains, G1a domains, SRCR domains, Kunitz / bovine pancreatic trypsin inhibitor domains, tendamistat, Kazal-type serine protease inhibitor domains, trefoil (P-type) domains, von Willebrand factor type C domains, anaphylatoxin-like domains, CUB domains, thyroglobulin type I repeats, LDL receptor class A domain, Sushi domain, Link domain, thrombospondin type I domain, immunoglobulin domain or immunoglobulin-like domain (e.g., domain antibody or camel heavy chain antibody), C-type lectin domain, MAM domain, von Willebrand factor type A domain, somatomedin B domain, WAP-type 4 disulfide core domain, F5 / 8 C-type domain, hemopexin domain, SH2 domain, SH3 domain, laminin-type EGF-like domain, C2 domain, "kappa body" (Ill. et al., Protein Eng (1997) 10, 949-57, so-called "minibodies" (Martin et al., EMBO J (1994) 13, 5303-5309), bispecific antibodies (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, ubiquitins, zinc finger proteins, autofluorescent proteins, or leucine-rich repeat proteins. Examples of nucleic acid molecules with antibody-like functions are aptamers. Aptamers fold into defined three-dimensional motifs and exhibit high affinity for a given target structure.

[0027] Turning now to multimerization reagents, the binding sites Z1 and Z2 of the multimerization reagent may be identical (see also the example in Figure 3), in which case a single multimerization agent may be used.

[0028] In embodiments using reversibly binding first and, optionally, second agents, the multimerization reagent may be immobilized on a solid surface. Any solid surface (support) can be used to immobilize the multimerization reagent. Illustrative examples of solid surfaces onto which the multimerization reagent can be immobilized include magnetic beads, polymer beads, cell culture plates, microtiter plates, membranes, or hollow fibers. Hollow fibers are used as bioreactors in, for example, the Quantum® Cell Expansion System available from TerumoBCT Inc. (Lakewood, CO, USA). The multimerization reagent is typically covalently bound to the solid support; however, noncovalent interactions can be used, if desired, for example, to immobilize the reagent on a plastic substrate. As described in more detail below, the multimerization reagent can be, for example, a streptavidin or avidin mutein that reversibly binds to a streptavidin-binding peptide. Such streptavidin muteins can be covalently bound to any surface, for example, resins (beads) used for chromatographic purification, and are commercially available from IBA GmbH, Göttingen, e.g., in the form of Strep-Tactin® Sepharose, Strep-Tactin® Superflow®, Strep-Tactin® Superflow® high capacity, or Strep-Tactin® MacroPrep®. Other illustrative examples of readily commercially available multimerization reagents are immobilized metal affinity chromatography (IMAC) resins, such as TALON® resin (Westburg, Leusden, The Netherlands), which can generally be used for the reversible immobilization of oligohistidine-tagged (his-tagged) proteins, i.e., for the reversible binding of first or second agents bearing an oligohistidine tag, such as a penta- or hexa-histidine tag, as the first binding partner C1 or second binding partner C2, respectively.Another example of a multimerization reagent is calmodulin sepharose, available from GE Life Sciences, or glutathione-conjugated sepharose, which can be used with a first or second agent comprising a calmodulin-binding peptide as binding partner C1 or C2, in which case the binding partner C1 or C2 is glutathione-S-transferase.

[0029] In another embodiment of the method of the present invention, the multimerization reagent may be in a soluble form. In principle, the same multimerization agent may be used as for a multimerization reagent immobilized on a solid support. The multimerization reagent may be in a soluble form, for example, a streptavidin mutein oligomer, a calmodulin oligomer, or a compound (oligomer) that provides at least two chelating groups K, which bind to a transition metal ion and allow the binding of moiety A to an oligohistidine affinity tag, a multimeric glutathione-S-transferase, or a biotinylated carrier protein.

[0030] As explained above, the first and second agents, in addition to their respective binding sites capable of binding to cell surface receptor molecules, have a binding partner C1 or C2 (hereinafter referred to as "binding partner C" for ease of reference). This binding partner C can bind to a binding site Z of the multimerization reagent (Z meaning either binding site Z1 or binding site Z2 of the multimerization reagent). The non-covalent bond formed between the binding partner C contained in the first or second agent and the binding site Z of the multimerization reagent can be of any desired strength and affinity, as long as it is separable or reversible under the conditions under which the method of the present invention is carried out. The dissociation constant (K D ) is about 10 -2 M~about 10 -13 M may have a value in the range of, for example, about 10 -2 M~about 10 -13 M, or about 10 -3 M~about 10 -12M, or about 10 -4 M~about 10 -11 M, or about 10 -5 M~about 10 -10 K of M D The K of this bond D , and the K of the bond formed between the binding site B of the receptor-binding reagent and the receptor molecule. D , k off , and k on The rate can be determined by any suitable means, such as, for example, fluorescence titration, equilibrium dialysis, or surface plasmon resonance. The receptor molecule binding reagent may contain at least one (including two, three, or more) second binding partner C, and the affinity reagent may contain at least two (e.g., three, four, five, six, seven, eight, or more) binding sites for the binding partners contained in the receptor molecule binding reagent. As described in U.S. Pat. No. 7,776,562, U.S. Pat. No. 8,298,782, or WO 2002 / 054065, any combination of binding partner C and affinity agent having one or more corresponding binding sites Z may be selected, which typically corresponds to the avidity effect, as long as the binding partner C and the binding site Z of the affinity agent can reversibly bind to or multimerize in a (multivalent) complex.

[0031] The binding partner contained in the first or second agent may be an oligopeptide, polypeptide, protein, nucleic acid, lipid, sugar, oligosaccharide, or polysaccharide. Such binding partners have a higher affinity for the binding site of the multimerization reagent than other substances. Examples of binding partners include, but are not limited to, immunoglobulin molecules, fragments thereof, and proteinaceous binding molecules with antibody-like functions.

[0032] In some embodiments, binding partner C included in the first or second agent comprises biotin, and the affinity reagent comprises a streptavidin analog or an avidin analog that reversibly binds to biotin.

[0033] In some embodiments, binding partner C included in the first or second agent comprises a biotin analog that reversibly binds to streptavidin or avidin, and the affinity reagent comprises a streptavidin analog or an avidin analog that reversibly binds to streptavidin, avidin, or the respective biotin analog.

[0034] In some further embodiments, the binding partner C included in the first or second agent comprises streptavidin or an avidin-binding peptide, and the affinity reagent comprises streptavidin, avidin, a streptavidin analogue or an avidin analogue that reversibly binds to the respective streptavidin or avidin-binding peptide.

[0035] In some embodiments, the binding partner included in the first or second agent may comprise the streptavidin-binding peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 01), and the affinity reagent may comprise the amino acid sequence Va1 at sequence positions 44-47 of wild-type streptavidin. 44 -Thr 45 -Ala 46 -Arg 47 Streptavidin mutein (analog) containing (SEQ ID NO: 02) or wild-type streptavidin containing the amino acid sequence lle at positions 44 to 47 44 -Gly45-Ala 46 -Arg 47 (SEQ ID NO: 03), both of which are described, for example, in U.S. Patent No. 6,103,493 and are commercially available under the trademark Strep-Tactin®. The streptavidin-binding peptide may be a single peptide, such as the "Strep-tag®" described, for example, in U.S. Patent No. 5,506,121, or a streptavidin-binding peptide having a sequential arrangement of two or more individual binding molecules, as described in WO 02 / 077018 or U.S. Patent No. 7,981,632.

[0036] In some embodiments, the binding partner C of the first or second agent comprises a moiety known to those of skill in the art as an affinity tag. In such embodiments, the affinity reagent comprises the 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 partners comprised in the first or second agent such as oligohistidine, immunoglobulin domains, maltose binding protein, glutathione-S-transferase (GST), chitin-binding protein (CBP) or thioredoxin, calmodulin-binding peptide (CBP), FLAG' peptide, HA tag (sequence: Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala (SEQ ID NO: 11)), VSV-G tag (sequence: Tyr-Thr-Asp-Ile-Glu-Met-Asn-Arg-Leu-Gly-Lys (SEQ ID NO: 12)), HSV tag (sequence: Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp (SEQ ID NO: 13)), and the like. 13)), the T7 epitope (Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly (SEQ ID NO: 14)), maltose binding protein (MBP), an HSV epitope of herpes simplex virus glycoprotein D with the sequence Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp (SEQ ID NO: 13), a "myc" epitope of the transcription factor c-myc with the sequence Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu (SEQ ID NO: 15), a V5 tag (sequence Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr, SEQ ID NO: 16), or glutathione S-transferase (GST). In such embodiments, the complex formed between one or more binding sites of the multimerization reagent, in this case, between an antibody or antibody fragment and an antigen, can be competitively separated by adding free antigen, i.e., a free peptide (epitope tag) or free protein (e.g., MBP or CBP). The affinity tag may also be an oligonucleotide tag.Such oligonucleotide tags can be used, for example, to hybridize the oligonucleotide to a complementary sequence attached to or contained in an affinity reagent.

[0037] In some embodiments, binding between the binding partner C in the first or second agent and one or more binding sites of the multimerization reagent occurs in the presence of a divalent, trivalent, or tetravalent cation. In this regard, in some embodiments, the multimerization reagent typically comprises a divalent, trivalent, or tetravalent cation that is retained, e.g., complexed, using a suitable chelator. The binding partner in the receptor-binding reagent, in such embodiments, may comprise, e.g., a complexing moiety that comprises, e.g., a divalent, trivalent, or tetravalent cation. Examples of respective metal chelators include, but are not limited to, ethylenediamine, ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), N,N-bis(carboxymethyl)glycine (also known as nitrilotriacetic acid, NTA), or 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). For example, EDTA dissolves most monovalent, divalent, trivalent, and tetravalent metal ions, such as calcium (Ca 2+ ), manganese (Mn 2 +), copper (Cu 2+ ), iron (Fe 2+ ), cobalt (Co 3+ ), and zirconium (Zr 4+ ), while BAPTA forms a complex with Ca 2+ As an illustrative example, a standard method used in the art is to use an oligohistidine tag and copper (Cu 2+ ), Nickel (Ni 2+ ), cobalt (Co 3+ ), or zinc (Zn 2+ ) ions, which is presented using the chelating agent nitrilotriacetic acid (NTA).

[0038] In some embodiments, the binding partner C in the first or second agent comprises a calmodulin-binding peptide, and the affinity reagent comprises multimeric calmodulin, e.g., as described in U.S. Pat. No. 5,985,658 or as described herein with reference to FIG. 2. In some embodiments, the binding partner C in the first or second agent comprises a FLAG peptide, and the affinity reagent comprises an antibody that binds to the FLAG peptide, e.g., monoclonal antibody 4E11, as described in U.S. Pat. No. 4,851,341. In one embodiment, the binding partner C in the first or second agent comprises an oligohistidine tag, and the affinity reagent comprises an antibody or transition metal ion that binds to the oligohistidine tag. Separation of all these binding complexes can be achieved by chelation of the metal ion, e.g., calcium, by adding, for example, EDTA or EGTA (see above). Calmodulin, antibodies such as 4E11, or chelating metal ions or free chelators can be multimerized by conventional methods, e.g., biotinylation and complexation with streptavidin or avidin or multimers thereof, or by introducing carboxyl residues into a polysaccharide, e.g., dextran, in a first step essentially as described in Noguchi, A, et al. Bioconjugate Chemistry (1992) 3, 132-137, and then linking calmodulin, antibodies, chelating metal ions, or free chelators to the carboxyl groups of the polysaccharide, e.g., dextran backbone via primary amino groups using conventional carbodiimide chemistry in a second step. In such embodiments, the bond between the binding partner C contained in the first or second agent and one or more binding sites Z of the multimerization reagent can be separated by chelation of the metal ion. Metal chelation can be achieved, for example, by the addition of EGTA or EDTA.

[0039] In some embodiments, particularly when the multimerization reagent is in a soluble form and based on streptavidin or avidin, it is an oligomer or polymer of streptavidin or avidin, or any mutein (analog) of streptavidin or avidin. The binding site Z is the natural biotin bond of avidin or streptavidin. Each oligomer or polymer can be cross-linked by a polysaccharide. In one embodiment, an oligomer or polymer of streptavidin or avidin, or a mutein (analog) of streptavidin or avidin, is prepared in a first step by introducing a carboxyl residue into a polysaccharide, such as dextran, essentially as described in Noguchi, A, et al., Bioconjugate Chemistry (1992) 3, 132-137. In a second step, streptavidin or avidin or analogs thereof may then be linked to the carboxyl groups on the dextran backbone via internal lysine residues and / or the free N-terminal primary amino group using conventional carbodiimide chemistry. Furthermore, crosslinked oligomers or polymers of streptavidin or avidin, or any mutein (analog) of streptavidin or avidin, can also be obtained by crosslinking individual streptavidin or avidin molecules (tetrameric homodimers of streptavidin or avidin are referred to herein as "individual molecules," i.e., the smallest building blocks of the respective oligomers or polymers) via a bifunctional molecule, such as glutaric dialdehyde, which acts as a linker, or by other methods described in the art. For example, in a first step, it is possible to prepare oligomers of streptavidin muteins by introducing thiol groups into the streptavidin mutein (this can be done, for example, by reaction of the streptavidin mutein with 2-iminothiolane (Trauts' reagent) and then activating available amino groups on the streptavidin mutein in a separate reaction).This activation of the amino groups can be achieved by reacting the streptavidin mutein with commercially available heterobifunctional cross-linkers, such as sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) or succinimidyl-6-[(β-maleimidopropionamido)hexanoate (SMPH). In a second step, the two reaction products thus obtained are mixed to allow the thiol groups contained in one batch of modified streptavidin muteins to react with the activated amino acids (via maleimide functional groups) of the other batch of modified streptavidin muteins. This reaction results in the formation of streptavidin mutein multimers / oligomers. These oligomers can have any suitable number of "individual molecules" or "streptavidin components" greater than three, and the degree of oligomerization can vary depending on the reaction conditions (see Figure 24). After reacting these two batches of modified streptavidin muteins, the oligomeric soluble multimerization reagent is typically isolated via size exclusion chromatography, and any desired fraction can be used as a multimerization reagent. Typically, oligomers do not (and need not) have a single molecular weight; they usually exhibit a statistical weight distribution, such as a Gaussian distribution. Any oligomer containing more than three streptavidin homotetramers (building blocks; (n≧3)) can be used as a soluble multimerization reagent. The oligomer can have, for example, 3 to 25 streptavidin mutein homotetramers. Where a streptavidin mutein, such as mutein "m1" or "m2," described in more detail below, has a molecular weight of approximately 50 kDa, these soluble oligomers have a molecular weight of approximately 150 kDa to approximately 1250 kDa. Since each streptavidin molecule / mutein has four biotin binding sites, such a multimerization reagent, as described herein, provides 12 to 100 binding sites Z1 (and Z2).

[0040] According to the above disclosure, in addition to such oligomeric multimerization reagents containing only cross-linked streptavidin homotetramers, tetrameric streptavidin muteins can be reacted with carriers to obtain multimerization reagents for use in the present invention. In addition to the reaction with polysaccharides described above, physiologically or pharmaceutically acceptable proteins such as serum albumin (e.g., human serum albumin (HSA) or bovine serum albumin (BSA)) can also be used as carrier proteins. In such cases, streptavidin muteins (either as individual homotetramers or in the form of oligomers with n≧3) can be linked to carrier proteins via non-covalent interactions. For this purpose, biotinylated BSA (commercially available from various sources, such as ThermoFisher Scientific, Sigma-Aldrich, or Vectorlabs, to name a few) can be reacted with streptavidin muteins. In this manner, a portion of the streptavidin oligomer is noncovalently bound to the biotinylated carrier protein via one or more biotin-binding sites (Z1, Z2), leaving the majority of the oligomer's binding sites (Z1, Z2) available for binding to agents, such as the first agent and optionally the second agent, as well as any additional agents described herein. Thus, this approach allows for convenient preparation of soluble multimerization reagents with multiple binding sites Z1 (see Figure 24). Alternatively, streptavidin muteins (either as individual homotetramers or in the form of oligomers with n > 3) can be covalently bound to a synthetic carrier, such as a polyethylene glycol (PEG) molecule. Any suitable PEG molecule can be used for this purpose, as long as the PEG molecule and the respective multimerization reagent are soluble. Typically, all PEG molecules with a molecular weight of 1000 Da or less are soluble in water or the culture medium that can be used in the present invention.Such PEG-based multimerization reagents can be easily prepared using commercially available activated PEG molecules bearing the amino groups of streptavidin muteins (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).

[0041] For streptavidin or wild-type streptavidin (wt-streptavidin), reference is made to the amino acid sequence disclosed by Argarana et al., Nucleic Acids Res. 14 (1986) 1871-1882. Streptavidin muteins are polypeptides that differ from the wild-type streptavidin sequence by one or more amino acid substitutions, deletions, or additions and retain the binding properties of wt-streptavidin. Streptavidin-like polypeptides and streptavidin muteins are essentially polypeptides that are immunologically equivalent to wild-type streptavidin and, specifically, can bind to biotin, biotin derivatives, or biotin analogs with the same or different affinity as wt-streptavidin. Streptavidin-like polypeptides or streptavidin muteins may contain amino acids that are not part of wild-type streptavidin, or may contain only a portion of wild-type streptavidin. Streptavidin-like polypeptides are not identical to wild-type streptavidin because the host lacks the enzymes necessary to convert the polypeptide they produce into the structure of wild-type streptavidin. The term "streptavidin" also includes streptavidin tetramers and streptavidin dimers, specifically streptavidin homotetramers, streptavidin homodimers, streptavidin heterotetramers, and streptavidin heterodimers. Each subunit typically 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.

[0042] In a preferred embodiment, the streptavidin mutein used as the multimerization reagent is a streptavidin mutein also described in U.S. Pat. No. 6,103,493 and German Patent No. 196 41 876.3. These streptavidin muteins have at least one mutation in the region of amino acid positions 44 to 53 based on the amino acid sequence of wild-type streptavidin. A minimal streptavidin mutein whose N-terminus begins in the region of amino acids 10 to 16 of wild-type streptavidin and whose C-terminus ends in the region of amino acids 133 to 142 of wild-type streptavidin is preferred. Examples of such preferred streptavidin muteins have a hydrophobic aliphatic amino acid in place of Glu at position 44, any amino acid at position 45, a hydrophobic aliphatic amino acid at position 46, and / or a basic amino acid in place of Val at position 47. The streptavidin mutein contains the amino acid sequence Va1 at sequence positions 44-47. 44 -Thr 45 -Ala 46 -Arg 47 (SEQ ID NO: 02), or a mutein containing the amino acid sequence IIe at positions 44 to 47 of wild-type streptavidin 44 -Gly 45 -Ala 46 -Arg 47 (SEQ ID NO: 03). Such muteins are described, for example, in U.S. Patent No. 6,103,493 and are commercially available from IBA GmbH in the form of mutein "m1" and mutein "m2" under the trade name Strep-Tactin®.

[0043] In some embodiments, the methods of the present invention may be used to deplete a sample of reagents previously used in cell expansion. For example, the first or second agent and its respective free partner (competitor) may be present in the eluate of the expansion method. Using the methods of the present invention, such reagents can be at least essentially (including completely) removed from a sample, e.g., from a cell population. As an illustrative example, the first or second agent defined above can be depleted from a sample to a level below the detection limit of, e.g., FACS or Western blot. A competing reagent (free first or second binding partner, or an analog thereof) may be used to terminate and control expansion and release the cell population from the multimerizing agent. This competing reagent may have a binding site capable of specifically binding to binding site Z of the affinity reagent in the "removal cartridge" of WO 2013 / 124474. In such embodiments, each method of the present invention may also function to deplete (including remove) the first and second agents, as well as the competing reagent.

[0044] The methods of the present invention may be performed at any temperature that does not at least essentially impair the viability of the cell population. References herein to conditions that are at least essentially not harmful, detrimental, or at least essentially not impair viability refer to conditions that result in at least 70% (including at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5%) of the population of cells being expanded with sufficient viability. In some embodiments, the methods of the present invention are performed at a temperature of about 20°C or higher. Depending on the cell population being expanded, a suitable temperature range may be, for example, about 20°C to about 45°C (including about 25°C to about 40°C or about 32°C to 37°C). In some embodiments, the methods of the present invention are performed at a constant temperature value or a selected temperature value ± about 5°C, ± about 4°C, ± about 3°C, ± about 2°C, ± about 1°C, or ± about 0.5°C. Those skilled in the art can experimentally determine the appropriate temperature, taking into account the properties of the cells and the expansion conditions. Typically, human cells expand at temperatures such as 37°C.

[0045] In a further embodiment, the present invention provides an in vitro method for expanding a population of cells, comprising contacting a sample containing the population of cells with a multimerization reagent, wherein the multimerization reagent is in a soluble form, and a first agent that provides a primary activation signal to the cells is immobilized (bound) to the multimerization reagent. The multimerization reagent comprises at least one binding site Z1 for binding of the first agent, and the first agent comprises at least one binding partner C1, which is capable of binding to the binding site Z1 of the multimerization reagent. The first agent binds to the multimerization reagent via the bond formed between the binding partner C1 and the binding site Z1, and the first agent binds to a receptor molecule on the surface of the cells to provide a primary activation signal to the cells and activate the cells. It is expressly noted herein that when a soluble multimerization agent is used, the bond between the binding partner C1 and the binding site Z1 does not need to be reversible.

[0046] In this second embodiment, a second agent that stimulates an accessory molecule on the surface of a cell is immobilized (bound) on the multimerization reagent, and the second agent comprises a binding partner C2 that can bind to binding site Z2 of the multimerization reagent. The second agent binds to the multimerization reagent via the bond formed between binding partner C2 and binding site Z2, and the second agent binds to an accessory molecule on the surface of the cell and stimulates the activated cell.

[0047] In one embodiment of this second method, the bond formed between binding partner C1 and binding site Z1 may be irreversible and / or the bond formed between binding partner C2 and binding site Z2 may also be irreversible.

[0048] In a different embodiment of this second method, the bond formed between binding partner C1 and binding site Z1 may be irreversible. Also, the bond formed between binding partner C2 and binding site Z2 may be irreversible. In this case, the dissociation constant (K d ) is 10 -2 M~10 -13 M may be in the range.

[0049] In this second method based on a soluble multimerization reagent, the multimerization reagent and all other reagents, as well as the first and second agents, and the cell population may otherwise be used in the same manner as disclosed above for methods using reversibility between the first or second agent and the multimerization reagent.

[0050] The present invention further provides a reagent kit for expanding a population of cells, comprising: (i) a multimerization reagent comprising at least one binding site Z for reversibly binding a first agent; (ii) a first agent that binds to a receptor molecule on the surface of the cell to provide a primary activation signal to the cell, thereby activating the cell, the first agent comprising at least one binding partner C1, the binding partner C1 being capable of reversibly binding to a binding site Z1 of the multimerization reagent, the first agent binding to the multimerization reagent via a reversible bond formed between the binding partner C1 and the binding site Z1; (iii) a second agent that stimulates an accessory molecule on the surface of the cell, the second agent comprising a binding partner C2, the binding partner C2 being capable of reversibly binding to binding site Z2 of the multimerization reagent, the second agent binding to the multimerization reagent via a bond formed between the binding partner C2 and the binding site Z2, the second agent binding to an accessory molecule on the surface of the cell, thereby stimulating the activated cell; A reagent kit comprising:

[0051] The present invention also provides a reagent kit for expanding a population of cells, comprising: (i) a multimerization reagent in soluble form and comprising at least one binding site Z for reversibly binding a first agent; (ii) a first agent that binds to a receptor molecule on the surface of the cell to provide a primary activation signal to the cell, thereby activating the cell, the first agent comprising at least one binding partner C1, the binding partner C1 being capable of binding to a binding site Z1 of the multimerization reagent, the first agent binding to the multimerization reagent via a reversible bond formed between the binding partner C1 and the binding site Z1; A reagent kit comprising:

[0052] This second reagent kit may further include (iii) a second agent that stimulates an accessory molecule on the surface of the cell, the second agent comprising a binding partner C2, the binding partner C2 being capable of binding to binding site Z2 of the multimerization reagent, and the second agent binding to the multimerization reagent via a bond formed between the binding partner C2 and the binding site Z2.

[0053] The kits disclosed herein are particularly useful when the population of cells is a lymphocyte population.

[0054] In accordance with the above disclosure, the present invention also provides novel multimerization reagents and novel compositions comprising such multimerization reagents capable of expanding a cell population. Such multimerization reagents capable of expanding a cell population are in a soluble form and include at least one binding site Z1 for reversibly binding a first agent that provides a primary activation signal to the cells, where the first agent that provides a primary activation signal to the cells is reversibly immobilized (bound) to the multimerization reagent; the first agent includes at least one binding partner C1, which can reversibly bind to at least one binding site Z1 of the multimerization reagent, and the first agent binds to the multimerization reagent via the reversible bond formed between the binding partner C1 and the binding site Z1. It should be noted herein that any of the first agents described herein may be immobilized on such multimerization agents.

[0055] The multimerization reagents of the present invention may further comprise at least one binding site Z2 for reversible binding of a second agent that stimulates an accessory molecule on the surface of a cell, wherein the second agent that stimulates an accessory molecule on the surface of a cell is reversibly immobilized (bound) to the multimerization reagent, and the second agent comprises a binding partner C2 that can bind to at least one binding site Z2 of the multimerization reagent. In this embodiment, the second agent binds to the multimerization reagent via a bond formed between binding partner C2 and binding site Z2.

[0056] Furthermore, in light of the above disclosure, such multimerization reagents can expand lymphocyte populations or subpopulations contained within the lymphocyte populations. The lymphocyte population to be expanded may be any suitable population, for example, a B cell population, a T cell population, or a natural killer cell population. The T cell population may be an antigen-specific T cell population, a helper T cell population, a cytotoxic T cell population, a memory T cell, a regulatory T cell, or a natural killer T cell population. Thus, in such an embodiment of the multimerization reagent, the first agent is capable of stimulating a TCR / CD3 complex-associated signal in T cells. Thus, the first agent present in the multimerization reagent may be a binding agent that specifically binds to CD3, while the second agent that binds to the accessory molecule may be a binding agent that specifically binds to CD28 or CD137.

[0057] In embodiments of the multimerization reagent, the first agent that specifically binds CD3 can be an anti-CD3 antibody, a bivalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, and / or a proteinaceous CD3 binding molecule with antibody-like binding properties. In these embodiments, the second agent that specifically binds CD28 or CD137 can be an anti-CD28 antibody, a bivalent antibody fragment of an anti-CD28 antibody, a monovalent antibody fragment of an anti-CD28 antibody, a proteinaceous CD28 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 antibody, a proteinaceous CD137 binding molecule with antibody-like binding properties, a 4-1BB ligand, and any mixture thereof. Therefore, the multimerization reagent of the present invention may generally have immobilized thereon a mixture of one type of first agent and one type of second agent, for example, an anti-CD3 antibody as the first agent and, for example, an anti-CD28 antibody and a 4-1BB ligand as the (binding moiety) second agent.

[0058] When the multimerization reagent is used to expand B cells, the first agent immobilized on the multimerization reagent may be a binding reagent that specifically binds CD40 or CD137. In accordance with the disclosure provided herein, in such embodiments, the first binding reagent that specifically binds CD40 or CD137 may be selected from an anti-CD40 antibody, a bivalent antibody fragment of an anti-CD40 antibody, a monovalent antibody fragment of an anti-CD40 antibody, and a proteinaceous CD40 binding molecule with antibody-like binding properties, or an anti-CD137 antibody, a bivalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment of an anti-CD137 antibody, a proteinaceous CD137 binding molecule with antibody-like binding properties, and a CD40 ligand (CD154).

[0059] Also, in accordance with the general disclosure of the present invention, in the multimerization reagents described herein, the binding sites Z1 and Z2 of the multimerization reagent may be the same. As noted above, such multimerization reagents may include streptavidin oligomers or polymers, avidin oligomers or polymers, streptavidin analog oligomers or polymers that reversibly bind to biotin, avidin analog oligomers or polymers that reversibly bind to biotin, and reagents containing at least two chelating groups K, which are capable of binding to transition metal ions, thereby enabling the reagents to bind to oligohistidine affinity tags, polymeric glutathione-S-transferases, polymeric calmodulins, and biotinylated carrier proteins.

[0060] The novel compositions provided herein are capable of expanding a population of cells, (i) a first multimerization reagent in soluble form and comprising at least one binding site Z1 for reversibly binding of a first agent that provides a primary activation signal to the cell, wherein the first agent that provides a primary activation signal to the cell is reversibly immobilized (bound) on the first multimerization reagent, the first agent comprising at least one binding partner C1 that can reversibly bind to the at least one binding site Z1 of the multimerization reagent, and the first agent binds to the multimerization reagent via the reversible bond formed between the binding partner C1 and the binding site Z1; (ii) a second multimerization reagent in soluble form and comprising at least one binding site Z2 for reversible binding of a second agent that stimulates an accessory molecule on the surface of the cell, wherein the second agent that stimulates an accessory molecule on the surface of the cell is reversibly immobilized (bound) to the multimerization reagent, the second agent comprising a binding partner C2 that is capable of binding to the at least one binding site Z2 of the multimerization reagent, and the second agent binds to the multimerization reagent via a bond formed between the binding partner C2 and the binding site Z2; may include:

[0061] Such novel compositions are, for example, the reaction mixtures used in Example 13, in which two separate multimerization reagents were functionalized with only αCD3Fab fragments or only αCD28Fab fragments. In this context, it is noted that such compositions were shown in Example 13 to have the same expansion efficiency as a single multimerization reagent in which both the first and second agents are co-immobilized. Thus, the combined use of two or more multimerization reagents individually functionalized with only one agent (e.g., one first agent or one second agent) is functionally equivalent to using a single binding site multimerization reagent in which both the first and second agents are immobilized for expansion. It is also noted that in this context, the multimerization reagents of the present invention may be functionalized with multiple agents (e.g., one, two, three, four, or even more agents) intended for use in expanding a selected cell population. A third or fourth agent may, for example, provide a stimulus for expanding a desired subpopulation of cells. In this context, see, for example, Example 13, in which a soluble multimerization reagent was reversibly functionalized with three reagents—αCD3 Fab fragments as the first reagent, αCD28 Fab fragments as the second reagent, and αCD8 Fab fragments as the third reagent—to enrich for a subpopulation of CD8+ T cells in a sample of a population of CD3+ T cells (lymphocytes). By using a combination of such agents, all of which can be reversibly immobilized on the same multimerization reagent, the present invention makes it possible, for example, to preferentially expand or selectively enrich any desired cell (sub)population from a sample containing a variety of different subpopulations. However, it should be noted that in this context, it is also possible to use three different multimerization reagents for this purpose, e.g., a first multimerization reagent functionalized only with αCD3 Fab fragments, a second multimerization reagent functionalized with αCD28 Fab fragments, and a third multimerization reagent functionalized with αCD8 Fab fragments. Similarly, it is also possible to use only two different multimerization reagents: a first multimerization reagent functionalized only with the αCD3 Fab fragment and a second multimerization reagent functionalized with both the αCD28 Fab fragment and the αCD8 Fab fragment. Thus, the present invention allows the modular design of any type of desired expansion reagent.

[0062] The present invention also provides an in vitro method for serially expanding a population of lymphocytes, wherein the population of lymphocytes comprises T cells, the method comprising: contacting a sample containing said T cells, said sample comprising a population of lymphocytes, with a multimerization reagent; the multimerization reagent is in a soluble form, and (i) a first agent that provides a primary activation signal to the T cell and (ii) a second agent that stimulates an accessory molecule on the surface of the T cell are reversibly immobilized on the multimerization reagent; the multimerization reagent comprises at least one binding site Z1 for reversibly binding of the first agent; the first agent comprises at least one binding partner C1, which can reversibly bind to the binding site Z1 of the multimerization reagent, and the first agent binds to the multimerization reagent via a reversible bond formed between the binding partner C1 and the binding site Z1; the multimerization reagent comprises at least one binding site Z2 for reversibly binding of the second agent; the second agent comprises at least one binding partner C2, which is capable of reversibly binding to the binding site Z2 of the multimerization reagent; and the first agent binds to the multimerization reagent via a reversible bond formed between the binding partner C2 and the binding site Z2; the first agent binds to a receptor molecule on the surface of the T cell to provide a primary activation signal to the cell, thereby activating the T cell; The second agent binds to the accessory molecule on the surface of the T cell and stimulates the activated cell, whereby the first agent and the second agent together induce the T cell to expand.

[0063] In this method, a sample containing a population of lymphocytes, including a population of T cells, is then contacted with a soluble multimerization reagent to which the first and second agents are immobilized, and the T cells specifically bind to the multimerization reagent.

[0064] Contacting the sample containing T cells, including the population of lymphocytes, with the multimerization reagent may be carried out in a bioreactor, such as a hollow fiber bioreactor (e.g., the hollow fiber bioreactor of the Quantum® cell expansion system) or a plastic bag bioreactor (e.g., the Cellbag® used in the Xuri Cell Expansion System W25 manufactured by GE Healthcare).

[0065] The method further comprises contacting the lymphocyte population (a reaction mixture containing T cells bound to the multimerization reagent via the first and second agents) with (i) a free form of the first binding partner C1 or an analog thereof capable of dissociating the binding between the first binding partner C1 and binding site Z1, and (ii) a free form of the second binding partner C2 or an analog thereof capable of dissociating the binding between the second binding partner C2 and binding site Z2. By doing so, the reversible binding between the binding partner C2 of the second agent and binding site Z2 of the multimerization reagent is dissociated in addition to the reversible binding between the binding partner C1 of the first agent and binding site Z1, thereby releasing the T cells bound to the multimerization reagent via the first and second agents into the eluate and halting T cell expansion.

[0066] In this method, the eluate containing the expanded T cell population (the reaction mixture in which the expansion reaction has already been terminated by the addition of the free form of the first partner or its analog) can be subjected to chromatography on a suitable (first) stationary phase. The (first) stationary phase can be a gel filtration matrix and / or affinity chromatography matrix, as described in WO 2013 / 124474. The gel filtration and / or affinity chromatography matrix contains an affinity reagent, which contains binding moieties Z1 and / or Z2 that specifically bind to binding partners C1 and / or C2 contained in the first agent or second agent. In this way, the first agent, the second agent, the first binding partner C1, and / or the free form of the second binding partner C2 are immobilized on the stationary phase. In this method, the first stationary phase is fluidly connected to the bioreactor.

[0067] In one embodiment of this continuous expansion, the binding sites Z1 and Z2 of the multimerizing agent are identical. Furthermore, a single multimerizing agent may be used. When a soluble multimerizing agent is used, the T cell population (or generally, the expanded cell population) is separated from the soluble multimerizing agent. The separation / removal may be performed using a second stationary phase. For this purpose, the mixture containing the T cells and the soluble multimerizing agent is subjected to chromatography on a suitable second stationary phase before or after application to the first stationary phase. This second stationary phase may be a gel filtration matrix and / or an affinity chromatography matrix, which contains an affinity reagent. The affinity reagent contained in the chromatography resin, if present, contains a binding partner D that (specifically) binds to binding site Z1 and / or binding site Z2 of the multimerizing agent, thereby immobilizing the multimerizing agent on the stationary phase. When a streptavidin-based multimerizing agent is used and both the first and second agents have streptavidin-binding peptides as binding partners C1 and C2, the binding partner D contained in this second stationary phase affinity reagent can be biotin. Soluble oligomers of streptavidin or streptavidin muteins used as multimerizing agents then bind to biotin that is covalently bound to a chromatography matrix, such as commercially available biotin-Sepharose™.

[0068] In this method of serial expansion, the first agent can stimulate a TCR / CD3 complex-associated signal in T cells, and thus the first agent can be a binding reagent that specifically binds to CD3. Furthermore, the accessory molecule on the T cell can be CD28. In this case, the second agent that binds to the accessory molecule is a binding reagent that specifically binds to CD28.

[0069] In this method of serial expansion, for example, T cells may be transfected with a T cell receptor (TCR) or a chimeric antigen receptor (CAR, also known as an artificial T cell receptor) during or after expansion. This transfection to introduce the gene for the desired receptor may be performed, for example, using any suitable retroviral vector. The genetically modified cell population may then be released from the initial stimulus (e.g., CD3 / CD28 stimulation) and then stimulated with a second type of stimulus, for example, via the de novo introduced receptor. This second type of stimulus may include antigen stimulation in the form of a peptide / MHC molecule, a cognate (cross-linking) ligand of the genetically introduced receptor (e.g., a natural ligand of the CAR), or any ligand (e.g., an antibody) that binds directly within the framework of the new receptor (e.g., by recognizing a constant region within the receptor). See, for example, Cheadle et al., "Chimeric antigen receptors for T-cell based therapy" Methods Mol Biol. 2012; 907:645-66 or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine Vol. 65: 333-347 (2014).

[0070] In this method, the population of lymphocytes comprising T cells can be a population of peripheral blood mononuclear cells (PBMCs) or a population of enriched or purified T cells. The population of lymphocytes can be derived, for example, from whole blood, or from a non-mobilized apheresis product, or from a frozen tissue specimen.

[0071] In this method of serial expansion based on a soluble multimerization reagent, the multimerization reagent and all other reagents, as well as the first and second agents, and the cell population may otherwise be used in the same manner as disclosed above for methods using reversibility between the first or second agent and the multimerization reagent.

[0072] The present invention further relates to a bioreactor and a first stationary phase for chromatography. The bioreactor is suitable for cell expansion, and the stationary phase is suitable for cell separation and reagent removal. The first stationary phase is a gel filtration and / or affinity chromatography matrix, which comprises an affinity reagent, the affinity reagent comprising a binding site Z1 that specifically binds to a binding partner C1 contained in a first agent, and / or the affinity reagent comprising a binding site Z2 that specifically binds to a binding partner C2 contained in a second agent. The first stationary phase is thereby suitable for immobilizing the first and / or second agent, the first binding partner C1, and / or the free form of the second binding partner C2 thereon. The bioreactor and the stationary phase are further fluidically connected. This configuration can be used in the serial expansion described above and can be incorporated into known cell expansion systems such as the Quantum® cell expansion system or the Xuri Cell Expansion System W25.

[0073] In this arrangement, the first stationary phase is comprised in a chromatography column or is a planar stationary phase. The arrangement may further comprise a second stationary phase fluidly connected to the first stationary phase. The second stationary phase may be a gel filtration matrix and / or an affinity chromatography matrix, and the gel filtration and / or affinity chromatography matrix comprises an affinity reagent. This affinity reagent may comprise a binding partner D that (specifically) binds to the binding site Z1 of the multimerization reagent, thereby making it suitable for immobilizing the multimerization reagent on the stationary phase.

[0074] The present invention is further directed to an apparatus for purifying and expanding a population of cells, comprising a bioreactor as defined above and at least one arrangement of a first or second stationary phase for chromatography.

[0075] The device may further include multiple arrangements of bioreactors and stationary phases fluidly connected in series.

[0076] The device may include a sample inlet fluidly connected to the bioreactor and the bioreactor of the chromatographic stationary phase arrangement, and may include a sample outlet for purified and expanded target cells, the sample outlet fluidly connected to the last stationary phase of at least one of the bioreactor and chromatographic stationary phase arrangements.

[0077] Finally, the device may be designed as a functionally closed system.

[0078] As one of ordinary skill in the art will readily appreciate from the present disclosure, other compositions of matter, means, uses, methods, or steps, now existing or later developed, that perform substantially the same function or achieve substantially the same results as the corresponding exemplary embodiments described herein, can likewise be utilized in accordance with the present invention.

[0079] Experimental Example Example 1: Stimulation / expansion of CD3+ responder T cells by αCD3 / αCD28 Fab fragments reversibly immobilized on beads coated with the streptavidin mutein Strep-tactin® 300,000 CD3+CD62L responder T cells (Tresp, isolated by sequential magnetic enrichment from unmobilized donor apheresis products) were labeled with 3 μM CFSE and stimulated with 5 μL of a 15 μL preparation of Streptactin® beads (10 mg magnetic particles per mL, 35 μg Streptactin® loaded per mg beads) loaded with 0.5 μg αCD3Fab fragment alone, 0.5 μg αCD28Fab fragment alone, or a mixture of 0.5 μg αCD3Fab fragment and 0.5 μg αCD28Fab.

[0080] The αCD3 Fab fragment used was derived from a CD3-binding monoclonal antibody produced by the hybridoma cell line OKT3. The hybridoma cell line OKT3 and the OKT3 antibody are described in U.S. Patent No. 4,361,549, and the cell line is deposited under accession number ATCC® CRL-8001™. The CD28 Fab used was derived from the monoclonal anti-human CD28 antibody CD28.3 (Vanhove et al., BLOOD, 15 July 2003, Vol. 102, No. 2, pages 564-570). The nucleotide sequence of the variable domain of this antibody CD28.3 has been deposited in GenBank in the form of a synthetic single-chain Fv construct, anti-human CD28 antibody scFv28.3, under GenBank accession number AF451974.1.

[0081] Both Fab fragments were recombinantly produced in E. coli as described in WO 2013 / 011011 and WO 2013 / 124474, with IgG1 consensus sequences as constant regions (CH1 and Ckappa). The heavy chains of both Fab fragments were fused at their carboxy termini to a consecutive arrangement of two streptavidin-binding modules (SAWSHPQFEK(GGGS)GGSAWSHPQFEK (SEQ ID NO: 07)), commercially available as "Twin-Strep-tag®" from IBA GmbH, Göttingen, Germany. The αCD3 Fab fragment was used as the first agent, with the streptavidin-binding peptide serving as binding partner C1, and the αCD28 Fab fragment was used as the second agent, with the streptavidin-binding peptide serving as binding partner C2. The (tetrameric) streptavidin mutein "Strep-tactin®" served as a multimerization reagent to which both Fab fragments were reversibly immobilized.

[0082] In expansion experiments, Tresp cells stimulated with blank beads (no Fab) served as a negative control. Tresp cells were seeded in triplicates with 300,000 CD3+ autologous feeder cells (irradiated at 30 Gy) in 3 mL of complete cell culture medium (RPMI (Gibco) supplemented with 10% (v / v) fetal bovine serum, L-glutamine, b-mercaptoethanol, HEPES, penicillin, streptomycin, and gentamicin) supplemented with 10 U / mL interleukin-2 (IL-2) in 48-well plates. The cells were incubated at 37°C without medium changes and analyzed by FACS analysis after 4 days. FACS staining and analysis were performed after 10 minutes of incubation with 100 μM D-biotin. One representative plot for each condition is shown in Figure 4. The plot shows viable CD3+ cells stained with propidium iodide (PI) to distinguish live / dead. Figure 4a is a histogram showing the size distribution (forward scatter) of stimulated cells. Figure 4a shows that after in vitro stimulation with αCD3 / αCD28 Fab fragments reversibly immobilized on beads coated with the streptavidin mutein Strep-tactin®, specific cell populations of Tresp cells were stimulated and expanded (increased in size / number compared to the unstimulated "beads only" control) when incubated in the presence of beads immobilized with a mixture of 0.5 μg of αCD3 Fab fragments and 0.5 μg of αCD28 Fab. Figure 4B shows a histogram of the dilution of the proliferation dye CFSE, which represents the degree of proliferation according to the number of cells per cell division (0, shown at the top of Figure 4B, represents cells that have not divided; 5 represents cells that have divided at least five times). Figure 4B shows that most of the T cell population stimulated with beads immobilized with a mixture of 0.5 μg of αCD3Fab fragment and 0.5 μg of αCD28Fab underwent three cell divisions and exhibited a more uniform proliferation pattern (fewer undivided cells at the "0" peak) than that observed with a single stimulation alone.Furthermore, the absolute increase in proliferation (more cells uniformly proliferated after 4 days of stimulation with αCD3- and αCD28-functionalized beads) was reflected by a more significant consumption of medium, as shown by the indicator color change to yellow in Figure 4C.

[0083] Example 2: Analysis of differential intracellular calcium mobilization in Jurkat cells Here, we investigated real-time flow cytometric analysis of differential intracellular calcium mobilization induced in Jurkat cells labeled with either the αCD3 antibody clone OKT3 or the Fab fragment of OKT3 multimerized with Strep-tactin®.

[0084] To this end, Jurkat cells were loaded with the calcium-sensitive dye Indo-1-AM and calcium release was triggered by injection of either the αCD3 monoclonal antibody OKT3 (produced by the hybridoma cell line OKT3, see above, black squares) or αCD3 Fab fragments (derived from the parental cell line OKT3) multimerized by reversible binding of its streptavidin-binding peptide to soluble Strep-Tactin fluorescently conjugated with phycoerythrin. The intact multimeric OKT3 Fab-Strep-Tactin complex triggered calcium release over the same period as the parental antibody clone (dark gray triangles). Cell activation was completely avoided by injection of D-biotin-treated, predissociated Fab-Strep-Tactin complexes (light gray circles), as well as injection of a PBS negative control (white inverted triangles). Application of ionomycin served as a positive control for calcium influx. Intracellular Ca 2+The time-resolved changes in OKT3 concentration were monitored by flow cytometry based on the change in the FL6 / FL7 ratio. Figure 5A shows that both the parent antibody OKT3 and the monovalent OKT3 multimerized Fab fragments affect calcium release; that is, the monovalent OKT3 multimerized Fab fragments are essentially as functional as the parent antibody. Furthermore, when biotin was added to Strep-tactin on which the OKT3 Fab fragments were immobilized before the addition of Streptactin-OKT3 Fab fragments, the multimeric OKT3 Fab fragments failed to trigger calcium release. In this case, biotin disrupted the reversible bond formed between Strep-tactin as a multimerizing agent and the OKT3 Fab fragments. Therefore, the monovalent Fab fragments were displaced from the multimerizing agent and, after dissociation, were unable to trigger calcium release by binding to CD3 on Jurkat cells.

[0085] In the experiment shown in Figure 5B, Indo-1-AM-labeled Jurkat cells were activated with OKT3-derived αCD3Fab-Strep-Tactin complexes as described in Figure 5A. Injection of intact (top graph) or predissociated complexes (bottom graph) served as positive and negative controls, respectively. Furthermore, stimulation of cells with intact Fab-Strep-Tactin complexes followed by injection of D-biotin (at t = 140 s, near the activation peak) rapidly disrupted αCD3Fab multimer signaling (middle graph). Injection of ionomycin into the predissociated Fab complexes served as a positive control. Data are representative of three separate experiments. Importantly, Figure 5B shows that adding D-biotin to the samples rapidly displaces the Fab fragments from the Strep-Tactin multimerizer, thereby effectively terminating calcium release even during ongoing calcium stimulation, demonstrating that the dissociated OKT3 Fab fragments are no longer biologically active. Similarly, multimeric OKT3 Fab fragments were unable to trigger calcium release when biotin was added to the Strep-Tactin-OKT3 Fab fragment multimers before adding the Streptactin-OKT3 Fab sample to Jurkat cells.

[0086] Example 3: Reversible staining of cells with CD3Fab multimers In this example, we investigate the reversible staining of cells with CD3 Fab multimers. Freshly isolated PBMCs were stained with either the αCD3 monoclonal antibody clone OKT3 (left dot plot, parent clone of the Fab multimer) or the cognate phycoerythrin (PE)-labeled OKT3 Fab multimer and analyzed before (second column from the left) or after (middle column) treatment with D-biotin. After a subsequent washing step with new PE-labeled Strep-Tactin®, remaining Fab monomers were detected (second column from the right). Secondary Fab multimer staining of reversibly stained cells served as a control (right column). Only viable CD3 cells, negative for propidium iodide (PI) staining for live / dead discrimination, are shown in Figure 6. Numbers in the dot plots indicate the percentage of cells within the gate. This experiment demonstrates that staining of CD3+ PBMCs with anti-CD3 Fab fragments multimerized with streptactin as a multimerization reagent is completely reversible by the addition of D-biotin, and that monovalent Fab fragments alone do not bind to CD3 molecules present on PBMCs.

[0087] Example 4: Reversible isolation of cells with CD28Fab multimers This example demonstrates the isolation of cells by reversible binding of anti-CD28 Fab fragments multimerized with Strep-Tactin® magnetic particles (these magnetic particles are available from IBA GmbH, Gottingen, Germany). Fab fragments derived from the antibody CD28.3, described in Example 1 above, were used for this purpose. CD28+ cells were selected / isolated from freshly isolated PMBCs by Fab multimer magnetic cell selection essentially as described in WO 2013 / 011011. Prior to selection, cells were control stained with either a cognate fluorescent αCD28 multimer (left dot plot) or an antibody against the immunoglobulin kappa light chain (second dot plot from the left, α-Ig kappa mAb) as a control stain. After selection, CD28+ cells were treated with D-biotin and then washed to remove the magnetic beads and Fab monomers. Released CD28+ cells were then (re)stained with either CD28Fab multimers (second dot plot from the right) or α-Igkappa mAb (right dot plot) to detect any potential remaining Fab monomers. 陰性 ) Only CD3+ cells are shown. Numbers in the dot plots indicate the percentage of cells within the gate. Figure 7 shows that CD28+ cells can be isolated from PMBCs using such multimerized anti-CD28 Fab fragments, and that after selection, all isolation reagents, including anti-CD28 Fab monomers, can be removed.

[0088] Example 5: Stimulation / expansion of CD3+ responder T cells by αCD3 / αCD28 Fab fragments reversibly immobilized on soluble Strep-tactin In this example, CD3+ responder T cells (isolated by magnetic selection from a sample of fresh PBMCs obtained from a Ficoll gradient) were stimulated in vitro with αCD3 / αCD28 Fab fragments reversibly immobilized on soluble oligomeric Strep-tactin®, which served as a soluble multimerization reagent, and then expanded. Oligomeric Strep-tactin® was obtained by polymerizing Strep-tactin® with sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, product number 22122 Thermo Scientific) and iminothiolane (product number 26101 Thermo Scientific) according to the manufacturer's protocol. Oligomeric streptavidin was separated from monomeric (unreacted) and dimeric streptavidin muteins by size exclusion chromatography, and the fraction of oligomeric streptavidin muteins (n≧3) thus obtained was used as a soluble multimerization reagent.

[0089] For in vitro expansion, 300,000 CD3+ responder T cells (Tresp) were labeled with 2 μM carboxyfluorescein succinimidyl ester (CFSE) and stimulated with various amounts of a soluble Strep-tactin® oligomer containing the αCD3 OKT3 Fab fragment and the αCD28 Fab fragment of antibody 28.3 (both of which have the Twin-Strep-tag® heavy chain as a streptavidin-binding peptide). ("1×" corresponds to 3 μg of multimerized Streptactin functionalized with 0.5 μg of the αCD3 monomeric Fab fragment and 0.5 μg of the αCD28 monomeric Fab fragment; "0.5×," "2×," and "5×" indicate n-fold increases from "1×.") Tresp cells were left unstimulated or stimulated with blank Strep-tactin multimers (no Fab), which served as a negative control. Tresp cells were seeded in duplicate with 300,000 CD3-negative autologous feeder cells (irradiated at 30 Gy) in 1 mL of cell culture medium supplemented with 20 U / mL IL-2 in 48-well plates. Cells were incubated at 37°C without medium changes, and proliferation was analyzed according to CFSE dilution after 5 days by FACS analysis. Figure 8A shows the increased size distribution of proliferating cells after 5 days of culture compared to the negative control. Figure 8B shows that CD3+ Tresp cells were appropriately stimulated and proliferated vigorously when a mixture of αCD3Fab and αCD28Fab fragments was incubated with immobilized soluble oligomeric Strep-tactin® (compared to solid Streptactin magnetic particles in Figure 4). The results in Figures 8a and 8b show that under these in vitro conditions, the majority of CD3+ responder T cells divided (2-5 cell divisions) after engagement of surface CD28 and TCR / CD3 complexes with αCD3 and αCD28 Fab fragments reversibly immobilized on soluble Strep-Tactin® oligomers. After in vitro expansion, the soluble Fab-Strep-Tactin stimulatory reagent was dissociated and removed after D-biotin treatment.The dissociation and removal of monomeric Fab fragments was analyzed by flow cytometry by restaining the cells with phycoerythrin-labeled Strep-Tactin® (ST-PE). Representative histograms (dark gray histograms) are shown compared with the appropriate ST-PE-only negative control (light gray histogram). Figure 8C shows that both Fab fragments were completely dissociated and removed from the expanded cells. Figure 8D shows the absolute number of viable (trypan blue-negative) cells after 5 days. Counts were counted using a Neubauer counting chamber and plotted for each stimulation condition. Median cell counts are shown in Figure 8D, and error bars indicate standard deviation (SD). Figure 8D also shows that mixtures of αCD3 and αCD28 Fab fragments immobilized on soluble Strep-Tactin multimerization reagent were all equally effective in expanding CD3+ cells, resulting in an approximately four-fold increase in absolute cell counts.

[0090] Example 6: Proliferation kinetics of purified CD4+ and CD8+ responder T cells stimulated in vitro with reversible aCD3 / aCD28 Fab-Streptamer multimers without medium exchange In this example, we investigated the expansion kinetics of purified CD4+ and CD8+ responder T cells (Tresp) stimulated in vitro with reversibly immobilized soluble oligomeric streptavidin muteins, the αCD3 / αCD28 Fab fragments. For this purpose, two different sizes of soluble oligomeric Strep-tactin® muteins served as soluble multimerization reagents. The first type of oligomeric Strep-tactin® was a fraction of the oligomeric streptavidin muteins (n≧3) obtained in Example 5 (also referred to herein as the "conventional Streptactin® scaffold" and indicated by an upturned triangle symbol in Figure 13). The second type of oligomeric streptavidin mutein used as a soluble multimerization reagent was an oligomeric streptavidin mutein (n≧3) reacted with biotinylated human serum albumin (also referred to herein as the "large Streptactin® backbone").

[0091] In this example, 500,000 purified CD4+ or CD8+ responder T cells (Tresp) were stimulated separately with these two different streptamer multimers as described above: either the Streptactin backbone of Example 5 (using a solution concentration of 1 mg of oligomeric streptavidin mutein per mL) or the large Streptactin backbone (0.1 mg / mL). 3 μL of both different backbones were loaded with a combination of 0.5 μg of αCD3 used in the above example and 0.5 μg of αCD28Fab, which has the streptavidin-binding peptide SAWSHPQFEK(GGGS)GGSAWSHPQFEK (SEQ ID NO: 07) at the C-terminus of the heavy chain of the Fab fragment. Additionally, 4.5 μL of conventional Streptactin scaffold was loaded with 0.5 μg of αCD3 Fab fragment, 0.5 μg of αCD8 Fab fragment (IBA GmbH Göttingen, also carrying the streptavidin-binding peptide SAWSHPQFEK(GGGS)2GGSAWSHPQFEK (SEQ ID NO: 07) at the C-terminus of the Fab fragment), and 0.5 μg of αCD28 Fab fragment. Untreated (unstimulated) Tresp cells served as a negative control, and Tresp cells stimulated with commercially available Dynabeads (beads onto which αCD3 and αCD28 monoclonal antibodies were irreversibly immobilized) served as a positive control. Tresp cells were seeded in duplicate in 1 mL of cell culture medium (RPMI 1640 (Gibco) supplemented with 10% (v / v) fetal bovine serum, 0.025% (w / v) L-glutamine, 0.025% (w / v) L-arginine, 0.1% (w / v) HEPES, 0.001% (w / v) gentamicin, 0.002% (w / v) streptomycin, and 0.002% (w / v) penicillin) in 48-well plates. Cells were incubated at 37°C without medium changes, and cell numbers were analyzed after 1, 3, and 6 days. In the experiment shown in Figure 13, expansion was performed without medium changes.Results for CD4+ responder T cells are shown in Figure 13A and results for CD8+ responder T cells are shown in Figure 13B, with graphs representing the extent of proliferation according to the number of cells harvested per time point for CD4+ Tresp (Figure 13A) and CD8+ Tresp in Figure 13B.

[0092] As can be seen in Figure 13A, the "small" soluble multimerization reagent with reversibly immobilized αCD3 and αCD28 Fab fragments resulted in the same amount of CD4+ T cell expansion as Dynabeads (the current standard reagent for T cell expansion), while the "large" oligomeric soluble streptactin resulted in even better expansion compared to Dynabeads. This improvement may be caused by the soluble "large oligomeric multimerization reagent" being able to simultaneously bind more T cells than the "small" soluble oligomers and, consequently, stimulate more CD4+ T cells than the "small" oligomers.

[0093] As can be seen from Figure 13B, the soluble multimerization reagent of the present invention enabled CD8+ T cell expansion at least as efficiently as Dynabeads within the first 3 days. Furthermore, during this period, expansion experiments using a soluble multimerization reagent to which αCD8 Fab fragments were reversibly immobilized in addition to αCD3 and αCD28 Fab fragments (as the first and second agents) showed the highest degree of expansion under these culture conditions. This indicates that the selectivity of expansion can be increased or regulated by using stimuli (here, αCD8 Fab fragments) specific to particular subpopulations of cells, resulting in the acquisition of greater amounts of the desired cell (sub)population.

[0094] Thus, to summarize the above, Example 6 demonstrates that the functionality of the soluble multimerization reagent used in the present invention in terms of triggering T cell expansion is comparable to the current standard methodology using Dynabeads for this purpose. However, because the streptavidin-based reversible interaction between the first and second agents and the multimerization reagent allows stimulation to be controlled (and, if necessary, terminated) by adding a competitor such as biotin, the present invention offers a significant advantage over Dynabeads technology because expansion conditions can be optimized (e.g., stimulation could be stopped in the experiment of Figure 13B after 3 days). Furthermore, because the soluble multimerization reagent can be easily removed from the reaction (e.g., by immobilizing the reagent on a biotinylated column after the expansion reaction), the expansion method of the present invention can be performed and automated in the closed system required for GMP production of cells for treatment purposes, for example, without having to deal with the removal of beads such as Dynabeads.

[0095] Example 7: Growth kinetics of purified CD4+ and CD8+ responder T cells stimulated in vitro with reversible aCD3 / aCD28 Fab-Streptamer multimers following medium exchange In this example, we also investigated the expansion kinetics of purified CD4+ and CD8+ responder T cells (Tresp) stimulated in vitro with αCD3 / αCD28 Fab fragments reversibly immobilized on soluble oligomeric streptavidin muteins. For this purpose, two different sizes of soluble oligomeric Strep-tactin® muteins served as soluble multimerization reagents. The first type of oligomeric Strep-tactin® was a fraction of the oligomeric streptavidin muteins (n≧3) obtained in Example 5 (also referred to herein as the "conventional Streptactin® scaffold" and indicated by a downward-pointing triangle symbol in Figure 13). This second type of oligomeric streptavidin mutein used as a soluble multimerization reagent was obtained by reacting the oligomeric Strep-tactin (n≧3) obtained in Example 5 with biotinylated human serum albumin. This soluble oligomeric multimerization reagent is also referred to herein as the "large Streptactin® backbone."

[0096] In this example, 400,000 purified CD4+ or CD8+ responder T cells (Tresp) were stimulated separately with either of the two different streptamer multimers described above: the Streptactin scaffold (1.0 mg / mL) of Example 5 or the large Streptactin scaffold (0.1 mg / mL). 3 μL of both different scaffolds was loaded with a combination of 0.5 μg of the αCD3 Fab fragment and 0.5 μg of the αCD28 Fab fragment. Additionally, 4.5 μL of the Streptactin scaffold of Example 5 was loaded with 0.5 μg of the αCD3, 0.5 μg of the αCD8 Fab, and 0.5 μg of the αCD28 Fab fragments. Untreated (unstimulated) Tresp cells served as a negative control, and Tresp cells stimulated with Dynabeads (to which αCD3 and αCD28 monoclonal antibodies were irreversibly immobilized) served as a positive control. Tresp cells were seeded in duplicate in 1 mL of cell culture medium supplemented with 30 U / mL IL-2 in 48-well plates. Cells were incubated at 37°C with a medium change on day 3, and cell numbers were analyzed after 1, 3, and 6 days. Results for CD4+ responder T cells are shown in Figure 14A, and results for CD8+ responder T cells are shown in Figure 14B. Graphs represent the extent of proliferation according to the number of cells harvested per time point for CD4+ Tresp (Figure 14A) and CD8+ Tresp in Figure 14B.

[0097] As can be seen in Figure 14A, the soluble multimerization reagent of the present invention, in which αCD3 and αCD28 Fab fragments were reversibly immobilized, resulted in superior CD4+ T cell expansion compared to Dynabeads.

[0098] As is evident from Figure 14B, the soluble multimerization reagent of the present invention was able to expand CD8+ T cells at least as efficiently as Dynabeads within the first 6 days. Note that during this period, expansion experiments using large soluble multimerization reagents bearing αCD3 and αCD28 Fab fragments (as the first and second agents) showed the highest degree of expansion under these culture conditions. This may also be due to the soluble "large oligomeric multimerization reagents" being able to simultaneously bind more T cells than "small" soluble oligomers and, consequently, stimulate more CD4+ T cells than "small" oligomers.

[0099] Example 8: Expansion kinetics of purified CD4+ and CD8+ T cell cultures with or without medium changes In this example, the combined data from Examples 6 and 7 were normalized to the input cell number for the "small" soluble multimerization reagents and the positive and negative controls. Normalized data were not available for the "large" multimerization reagents. As described in Examples 6 and 7, 400,000 to 500,000 CD4+ or CD8+ responder T cells (Tresp) were stimulated with 3 μL of a streptactin multimer preparation (1 mg / mL; immobilized with 0.5 μg of αCD3 Fab fragment and 0.5 μg of αCD28 Fab fragment). Untreated (unstimulated) Tresp cells served as a negative control, and Tresp cells stimulated with Dynabeads served as a positive control. Tresp cells were seeded in duplicate in 1 mL of cell culture medium supplemented with 30 U / mL IL-2 in a 48-well plate. Tresp cells were seeded in duplicate in 1 mL of cell culture medium supplemented with 30 U / mL IL-2 in 48-well plates. Cells were incubated at 37°C with or without a medium change on day 3 (solid line in Figure 15 ) and cell counts were analyzed after 1, 3, and 6 days. As is evident from the normalized data in Figure 15A, the "small" soluble multimerization reagent, in which αCD3Fab and αCD28Fab fragments were reversibly immobilized, resulted in an approximately 2.5-fold expansion of CD4+ T cells, whereas expansion using Dynabeads resulted in an approximately 1.8-fold expansion. Thus, the use of the soluble multimerization reagent of the present invention further improved CD4+ T cell expansion over Dynabeads. Similarly, Figure 15B confirms that the soluble multimerization reagent of the present invention was able to expand CD8+ T cells at least as efficiently as Dynabeads within the first 3 days.

[0100] Example 9: Early cluster formation after activation of purified CD4+ and CD8+ responder T cells stimulated in vitro with reversible aCD3 / aCD28 Fab-Streptamer multimers In this example, 400,000 CD4+ or CD8+ responder T cells (Tresp) were stimulated with 3 μL of a 1 mg / mL preparation of oligomeric streptactin multimerization reagent loaded with a combination of 0.5 μg of αCD3Fab and 0.5 μg of αCD28Fab. Untreated (unstimulated) Tresp cells served as a negative control, and Tresp cells stimulated with Dynabeads served as a positive control. Tresp cells were seeded in duplicate in 1 mL of cell culture medium supplemented with 30 U / mL IL-2 in a 48-well plate. Cells were incubated at 37°C and analyzed microscopically after 1 and 2 days. Figure 16A shows stimulation of CD4+ Tresp (Figure 16A) and CD8+ Tresp (Figure 16B) with Dynabeads (middle row) and Streptamers multimers (bottom row), respectively. The photographs represent the extent of cluster formation: for clarity, exemplary clusters for stimulation with soluble streptavidin mutein oligomers are shown as circles in Figures 16A and 16B. Clusters for Dynabead stimulation are easily visualized by the accumulation of darkly colored stimulation particles. As is evident, early cluster formation occurred in both CD4+ and CD8+ T cells when the expansion method of the present invention using soluble oligomer multimerization reagents was used.

[0101] Example 10: Expansion kinetics and phenotype of polyclonally activated / expanded bulk CD3+ central memory T cells (Tcm) In this example, 500,000 CD3+CD62L+CD45RA- responder Tcm cells (Tresp) were stimulated with 3 μL of the soluble oligomeric streptactin preparation (1 mg / mL) described in Example 5 loaded with a combination of 0.5 μg of αCD3 and 0.5 μg of αCD28Fab. An additional stimulation condition was 4.5 μL of a streptactin multimer preparation loaded with 0.5 μg of αCD3, 0.5 μg of αCD8Fab, and 0.5 μg of αCD28Fab. Untreated (unstimulated) Tresp cells served as a negative control, and Tresp cells stimulated with Dynabeads (to which αCD3 and αCD28 monoclonal antibodies were irreversibly immobilized) served as a positive control. Tresp cells were seeded in 1 mL of cell culture medium supplemented with 30 U / mL IL-2 alone or 30 U / mL IL-2 and 5 ng / mL IL-15 in 48-well plates. Cells were incubated at 37°C with medium changes every 3 days, and cell numbers were analyzed after 7 and 14 days. Graphs show the extent of proliferation according to the number of cells harvested at each time point in medium supplemented with IL-2 alone (Figure 17A) and medium supplemented with IL-2 and IL-15 (Figure 17B). As can be seen in both Figures 17A and 17B, soluble multimerization reagents with reversibly immobilized CD3 Fab and αCD28 Fab fragments resulted in superior cell expansion compared to Dynabeads. As further shown by flow cytometry analysis of CD62L and CD127 surface expression after 14 days of culture in varying cytokine environments in Figure 17C, the experimental approach using the soluble multimerization reagents of the present invention preserves a higher content of long-lived memory T cells expressing CD127 than expansion using Dynabeads under both conditions selected herein, demonstrating a further advantage of the method of the present invention.

[0102] Example 11: Selective antigen-specific expansion of responder Tcm cells from bulk CD3+ central memory T cells (kinetics and phenotype) In this example, the kinetics and phenotype of selective antigen-specific (Ag-specific) expansion from purified CD3+CD62L+CD45RA- responder Tcm cells were investigated.

[0103] More specifically, CD3+CD62L+CD45RA- responder Tcm cells were stimulated in vitro with both peptide:MHC molecule complexes (acting as the first agent providing the primary activation signal to the cells) and αCD28Fab fragments (acting as the second reagent stimulating accessory molecules on the cell surface). Both the antigen-specific peptide-MHC molecule complexes and the αCD28Fab fragments were reversibly immobilized on soluble oligomeric streptavidin muteins (n≧3) as described in Example 5. The peptide used for antigen-specific amplification was the peptide CRVLCCYVL (SEQ ID NO: 06), which represents amino acids 309-317 of the immediate early 1 protein (described in Ameres et al., PLOS Pathogens, May 2013, vol. 9, issue 5, e1003383), representing the cytomegalovirus (CMV)-specific HLA-C7 / IE-1 epitope. The MHC I molecule presenting this peptide has a streptavidin-binding peptide (SAWSHPQFEK(GGGS)2GGSAWSHPQFEK (SEQ ID NO: 07), commercially available as "Twin-Strep-tag®" from IBA GmbH, Gottingen, Germany) at the C-terminus of the alpha chain (heavy chain).

[0104] For this purpose, 500,000 CD3+CD62L+CD45RA- responder Tcm cells (Tresp) were Ag-specifically stimulated with 3 μL of a soluble oligomeric streptactin multimerization reagent preparation functionalized with 0.5 μg of peptide:MHC class I complexes containing streptavidin-binding peptides and 0.5 μg of the above-described αCD28 Fab. Alternatively, 4.5 μL of the streptactin multimerization reagent preparation was loaded with 0.5 μg of these peptide:MHC class I complexes, 0.5 μg of CD8α Fab, and 0.5 μg of αCD28 Fab. For comparison, polyclonal stimulation was performed with 3 μL of a 1 mg / mL streptactin multimerization reagent preparation loaded with a combination of 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 Fab. As an alternative to the above stimulation conditions, 4.5 μL of a streptactin multimerization reagent preparation reversibly loaded with 0.5 μg of αCD3Fab, 0.5 μg of αCD8Fab, and 0.5 μg of αCD28Fab was used. Untreated (unstimulated) Tresp cells served as a negative control, and Tresp cells polyclonally stimulated with Dynabeads (beads to which αCD3 and αCD28 monoclonal antibodies were irreversibly immobilized) served as a positive control. Tresp cells were seeded in 1 mL of cell culture medium supplemented with 30 U / mL IL-2 and 5 ng / mL IL-15 in a 48-well plate. The cells were incubated at 37°C with medium changes every 3 days, and cell numbers were analyzed after 7 and 14 days. An exemplary flow cytometry analysis of the fraction of Ag-specific cells stimulated / expanded via soluble strept-tactin oligomers immobilized with peptide:MHC-I complexes for the HLA-C7 / IE-1 epitope (of CMV) (Figure 18A) shows that these antigen-specific T cells were specifically expanded.(Similar to the expansion experiment shown in Figure 18A, the extent of expansion of different Ag specificities is represented according to the number of peptide:MHC1 multimer-positive cells collected per time point.) The graphs in Figures 18B-18E show that multimerization reagents using the respective complexes of Ag-specific peptide and MHC1 molecule provide the greatest number of expanded cells (from a 20-fold increase (see Figure 18B) in the number of Ag-specific cells recognizing the pp65 epitope of CMV (amino acids 341-350 (QYDPVAALF (SEQ ID NO: 08)) restricted by HLA-A2402 to HLA-B7 / IE-1 of CMV). 309-317 The results show up to a 98-fold increase (see Figure 18E) in the number of Ag-specific cells that recognize the epitope (CRVLCCYVL (SEQ ID NO: 06)), thereby demonstrating that the expansion method of the present invention is fully applicable to the expansion of Ag-specific cells. Finally, the exemplary flow cytometry analysis of CD62L and CD127 surface expression after 14 days of culture with the (adenoviral) HLA-B7 / Hexon5 epitope shown in Figure 18F further confirms that the experimental approach using the soluble multimerization reagents of the present invention preserves a higher content of long-lived memory T cells expressing CD127 under polyclonal and Ag-specific stimulation conditions.

[0105] Example 12: Selective Ag-specific expansion kinetics and phenotype of bulk central memory T cells This example examines the kinetics of selective Ag-specific expansion from purified CD3+CD62L+CD45RA- responder Tcm cells stimulated in vitro with a) antigen-specific peptide-MHC I complexes and b) αCD28 Fab fragments reversibly immobilized as first and second agents on soluble oligomeric streptavidin muteins.

[0106] For this purpose, 500,000 CD3+CD62L+CD45RA- responder Tcm cells (Tresp) were stimulated specifically with 3 μL of a streptactin multimerization reagent preparation functionalized with 0.5 μg of a peptide:MHC class I complex containing a streptavidin-conjugated peptide (the specific peptide represents amino acids 114-124 of the adenoviral Hexon 5 protein restricted by HLA-B07 (CPYSGTAYNSL, SEQ ID NO: 10)) and 0.5 μg of αCD28 Fab. Alternatively, 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 using 3 μL of a 1 mg / mL streptactin multimerization reagent preparation loaded with a combination of 0.5 μg of αCD3Fab and 0.5 μg of αCD28Fab. Furthermore, as an alternative to the above stimulation conditions, 4.5 μL of a Streptactin multimer preparation loaded with 0.5 μg of αCD3Fab, 0.5 μg of αCD8Fab, and 0.5 μg of αCD28Fab was used. Untreated (unstimulated) Tresp cells served as a negative control, and Tresp cells polyclonally stimulated with Dynabeads served as a positive control. Tresp cells were seeded in 1 mL of cell culture medium supplemented with 30 U / mL IL-2 and 5 ng / mL IL-15 in a 48-well plate. Cells were incubated at 37°C with medium changes every 3 days, and cell numbers were analyzed after 7 and 14 days. The photographs shown in Figure 19 depict the extent of cluster formation on day 5, demonstrating exemplary Ag-specific stimulation for the adenoviral HLA-B7 / Hexon5 epitope. As can be seen in Figure 19, we were able to specifically expand such adenovirus antigen-specific cells from the original CD3+CD62L+CD45RA- responder Tcm population.

[0107] Example 13: Yield and phenotype of expanded CD8+ T cells - Size variation of soluble multimerization reagents and addition of αCD8-Fab additive for stimulation In this example, we investigated the expansion of purified CD8+ responder T cells stimulated in vitro with αCD3 / αCD28 Fab fragments, which were reversibly immobilized soluble oligomeric streptavidin muteins. Furthermore, we investigated the effect of adding αCD8 Fab to the multimerization reagent to increase the specificity of expansion for CD8+ T cells.

[0108] To this end, 300,000 purified CD8+ responder T cells (Tresp) were stimulated separately with two different soluble streptactin-based multimerization reagents: the small oligomeric streptactin multimerization reagent (1 mg / mL) from Example 5 or the large streptactin oligomer (0.1 mg / mL). 3 μL of each of the different multimerization reagents (backbone) was loaded with a combination of 0.5 μg of the αCD3 and 0.5 μg of the αCD28 Fab fragment. Additionally, 4.5 μL of the small streptactin multimerization reagent (backbone) was loaded with 0.5 μg of the αCD3, 0.5 μg of the αCD8 Fab, and 0.5 μg of the αCD28 Fab fragment. Additionally, only 3 μL of the "small" Streptactin multimerization reagent (backbone) functionalized with only 0.5 μg of the αCD3 Fab fragment or only 0.5 μg of the αCD28 Fab fragment was used. Unstimulated Tresp cells served as a negative control, and Tresp cells stimulated with Dynabeads served as a positive control. Tresp cells were seeded in duplicate in 1 mL of cell culture medium supplemented with 30 U / mL IL-2 in a 48-well plate. Cells were incubated at 37°C for 3 days, with the medium replaced, and analyzed after 6 days. Figure 20A shows the extent of proliferation according to the cell number harvested on day 6, compared with the negative control and normalized to the positive control. Figure 20A shows that expansion of CD8+ T cells using the soluble multimerization reagent of the present invention results in a higher yield of CD8+ T cells than expansion using Dynabeads. FACS analysis of CD8 surface expression (Figure 20B) and CD45RO surface expression (Figure 20C) after cell culture shows that the same phenotype of CD8+ T cells was expanded by either the multimerization reagent of the present invention or Dynabeads (no significant differences (ns) were detected when comparing various stimulation conditions using one-way ANOVA). The improved yield of CD8+ cells using the expansion method of the present invention compared to Dynabeads may be due to the fact that soluble multimerization reagents may be more accessible to target receptors on the cell surface than antibodies immobilized on Dynabeads.This improved yield can be highly advantageous when expanding populations of rare cells from initial samples.

[0109] Furthermore, when the expansion yield obtained using a multimerization reagent on which both 0.5 μg of the αCD3Fab fragment and 0.5 μg of the αCD28Fab fragment were co-immobilized (second column from the left in Figure 20B) was compared to the yield using two multimerization reagents functionalized with only the αCD3Fab fragment or only the αCD28Fab fragment (third column from the left in Figure 20B), it was found that both experiments had the same expansion efficiency. Thus, these experiments demonstrate that the use of a single multimerization reagent on which both the first and second agents are co-immobilized is functionally equivalent for expansion to the use of two separate multimerization reagents loaded with only the first and second agents, respectively.

[0110] Example 14: Yield and phenotype of expanded CD8+ T cells - Titration of different soluble multimerization reagents immobilizing various ratios of αCD3Fab and αCD28Fab fragments In this example, we investigated the yield and phenotype of expanded CD8+ responder T cells (Tresp) stimulated in vitro with various amounts of αCD3 / αCD28 Fab fragments reversibly immobilized on a soluble oligomeric streptavidin mutein.

[0111] For this purpose, 300,000 CD8+ responder T cells (Tresp) were stimulated with various amounts of a mixture of "small" oligomeric streptactin multimerization reagent preparations (1 mg / mL) functionalized with only αCD3Fab and only αCD28Fab ("1x" corresponds to 1.5 μg of streptactin multimerization reagent functionalized with only 0.5 μg of αCD3 and 1.5 μg of multimerized streptactin functionalized with only 0.5 μg of the αCD28Fab fragment), or with 3 μL of a streptactin multimerization reagent preparation loaded with 0.5 μg of αCD3 and 0.5 μg of CD28Fab, or with 4.5 μL of a streptactin multimerization reagent preparation loaded with 0.5 μg of αCD3, 0.5 μg of strep-tagged αCD8, and 0.5 μg of αCD28Fab. Untreated Tresp cells served as a negative control, and Tresp stimulated with Dynabeads served as a positive control. Tresp cells were seeded in 1 mL of cell culture medium supplemented with 30 U / mL IL-2 in 48-well plates. Cells were incubated at 37°C without changing the medium and analyzed after 5 days. Figure 21A depicts the extent of proliferation according to the cell number harvested on day 5, compared to the negative control and normalized to the positive control. Figure 21A shows that expansion of CD8+ T cells using various soluble multimerization reagents of the present invention resulted in higher yields of CD8+ T cells than expansion using dynabeads (particularly, the 5x cumulative total reagent volume condition resulted in an optimal expansion / initiated cell division, resulting in an increase in total cells over time). FACS analysis of CD8 (Figure 21B) and CD45RO (Figure 21C) surface expression after cell culture shows that the same phenotype of CD8+ T cells was expanded by either the various multimerization reagents of the present invention or commercially available Dynabeads.

[0112] Example 15: Activation of intracellular signaling cascades following stimulation of aCD19-CAR-transduced Jurkat cells with Streptamers multimers In this example, we investigated the activation of intracellular signaling cascades in transduced Jurkat cells engineered to express a tumor-specific chimeric antigen receptor (CAR), here CD19, and stimulated with the oligomeric Strep-tactin® of Example 5 as a soluble multimerization reagent.

[0113] For this purpose, 300,000 Jurkat responder cells (Jresp) were incubated with (A) a mixture of various amounts of a streptactin multimerization reagent preparation (1 mg / mL) functionalized with αCD3Fab and αCD28Fab fragments described herein ("x1" corresponds to 3 μg of streptactin multimerization reagent functionalized with 0.5 μg of αCD3Fab and 0.5 μg of αCD28Fab, providing a "polyclonal streptactin-based multimerization reagent"), or (B) 3 μL of a streptactin multimerization reagent preparation functionalized with 0.5 μg (x1) or 1 μg (x2) of the extracellular domain (ECD) of CD19 (the natural ligand of αCD19-CAR, providing a "CAR-specific streptactin-based soluble multimerization reagent"), or αIgG4 recognizing the IgG4 spacer within αCD19-CAR. The cells were stimulated with 3 μL of a 0.5 μg (×1) or 1 μg (×2) loaded streptactin multimerization reagent preparation (also providing a "CAR-specific streptavidin mutein-based multimerization reagent"). The CD19 ECD with a hexahistidine tag (SEQ ID NO: 27) was obtained from Sino Biological / Life technologies and functionalized to bind to the streptavidin-based multimerization reagent by mixing the CD19 ECD with the adapter molecule His-STREPPER (IBA GmbH, Germany, order number 2-0920-005) at a 1:1 molecular ratio and incubating at room temperature for 15 minutes. The His-STREPPER adapter molecule contains a hexahistidine tag and a chelating moiety that binds to a streptavidin-binding peptide, thereby providing the CD19 ECD with a streptavidin-binding peptide that can temporarily bind to a target molecule, here a multimerization reagent based on a streptavidin mutein. JResp was stimulated with Dynabeads (beads on which αCD3 and αCD28 monoclonal antibodies are irreversibly immobilized) or PMA and ionomycin, which served as a positive control.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 and stimulated for 0–20 minutes before being placed on ice and lysed. Detection of phosphorylated ERK indicates active MAPK signaling, and staining for housekeeper β-actin indicates equal amounts of total protein loaded per condition and time. Jurkat cells can be activated / expanded via binding of the CD19 extracellular domain to a CD19-specific chimeric antigen receptor, as shown by comparing Figure 22A, which shows Jurkat cell activation via a polyclonal streptactin multimerization reagent, with Figure 22B, which shows Jurkat cell activation via two CAR-specific streptactin-based multimerization reagents. Since genetic downstream processing of T cells is mostly performed only on preselected cell populations, global activation via cross-linking of CARs introduced via an IgG4 spacer domain (which is conserved within various CARs with different specificities) broadens the scope of application for reversible cell stimulation / expansion in these in vitro cell processing situations.

[0114] Thus, this experiment demonstrates that, in principle, any cell population that is activated by binding of an agent (ligand) that provides a primary activation signal to the cell population can be expanded using a first agent that is reversibly immobilized on a multimerization reagent as described above.

[0115] Example 16: Yield and subset composition of expanded CD3+ T cells with the addition of αCD8-Fab for stimulation This experiment was performed on purified CD3 / αCD28 Fab fragments stimulated in vitro with reversibly immobilized αCD3 / αCD28 Fab fragments on the soluble oligomeric Strep-tactin® of Example 5, which served as a soluble multimerization reagent. +Expansion of responder T cells is shown. In one experiment, to test whether it is possible to preferentially stimulate specific T cell subpopulations in vitro with reversible aCD3 / aCD28Fab-Streptamer multimers, in addition to the αCD3 / αCD28Fab fragments, the αCD8Fab fragment (catalog number 6-8000-203) commercially available from IBA GmbH, Göttingen, Germany, was immobilized on soluble oligomers of streptavidin muteins. More specifically, 500,000 purified CD3 + Responder T cells (Tresp) were stimulated with 3 μL of a 1 mg / mL preparation of oligomeric streptavidin loaded with a combination of 0.5 μg of αCD3 and 0.5 μg of αCD28 Fab. As an alternative approach, 4.5 μL of streptactin oligomers were loaded with 0.5 μg of αCD3, 0.5 μg of streptactin-tagged αCD8 Fab, and 0.5 μg of streptactin-tagged αCD28 Fab. Unstimulated Tresp cells served as a negative control, and Tresp cells stimulated with Dynabeads (beads to which αCD3 and αCD28 monoclonal antibodies were irreversibly immobilized) served as a positive control. As can be seen in Figure 23A, the multimerization reagent reversibly loaded with αCD3 Fab fragments, αCD28 Fab fragments, and αCD8 Fab fragments provided the greatest number of expanded CD3+ T cells. The expanded cell count is 1 × 1 × 10 6, yields approximately 30% higher than T cell expansion using commercially available Dynabeads. Furthermore, and more importantly, as shown in Figure 23B, the use of this multimerization reagent containing αCD3Fab, αCD28Fab, and αCD8Fab fragments resulted in the highest amount of CD8+ T cells compared to both expansion using Dynabeads or the soluble multimerization reagent of the present invention containing only αCD3Fab and αCD28Fab fragments as the first and second agents described herein. Thus, this experiment also demonstrates the advantage of the present invention, that in addition to a first agent providing a primary activation signal to the desired cell population and, optionally, a second agent providing a costimulatory signal, additional agents specific for activation of the desired cell population can be immobilized on the multimerization reagent. Thus, by doing so, the present invention offers the possibility, for example, of preferentially expanding or selectively enriching any desired cell (sub)population from a sample containing a variety of different subpopulations.

[0116] Example 17: Parallel antigen-specific expansion of responder Tcm cells from a single pool In this example, we examine the kinetics of parallel antigen-specific (Ag-specific) expansion from a single pool of responder T cells stimulated in vitro with multiple reversible peptide:MHC / αCH28 Fab-Streptamer multimers.

[0117] 500,000 CD3+CD62L+CD45RA- responder Tcm cells (Tresp) are simultaneously stimulated with multiple Ag specificities using 3 μL of streptactin multimers functionalized with 0.5 μg of each peptide:MHC class I complex bearing a streptavidin-binding peptide and 0.5 μg of αCD28Fab bearing a streptavidin-binding peptide, respectively. Alternatively, 4.5 μL of streptactin-based multimerization reagent functionalized with 0.5 μg of the peptide:MHC class I complex bearing a streptavidin-binding peptide, 0.5 μg of αCD8Fab, and 0.5 μg of αCD28Fab, respectively, is used for each specificity. For comparison, polyclonal stimulation was performed using 3 μL of a streptactin-based multimerization reagent preparation (1 mg / mL) reversibly loaded with a combination of 0.5 μg of αCD3Fab and 0.5 μg of αCD28Fab. Similarly, as an alternative to the above stimulation conditions, 4.5 μL of a streptactin-based multimerization reagent preparation reversibly loaded with 0.5 μg of αCD3Fab, 0.5 μg of αCD8Fab, and 0.5 μg of αCD28Fab (each containing a streptavidin-binding peptide) could be used. Untreated (unstimulated) Tresp cells served as a negative control, and Tresp cells polyclonally stimulated with Dynabeads (beads coated with αCD3mAb and αCD28mAb) served as a positive control. Tresp cells are seeded in 1 mL of cell culture medium supplemented with 30 U / mL IL-2 and 5 ng / mL IL-15 in 48-well plates. Cells are incubated at 37°C with medium changes every 3 days, and cell numbers are analyzed after 7 and 14 days.

[0118] Example 18: Preferential proliferation of CD8+ T cells among CD3+ responder T cells stimulated in vitro with a streptavidin-based multimerization reagent reversibly functionalized with αCD3 / αCD8 / αCD28 Fab fragments 300,000 CD3+ responder T cells (Tresp) are stimulated with 3 μL of a streptactin multimerization preparation (1 mg / mL) or multimerization reagent preparation using a large streptactin scaffold (0.1 mg / mL) loaded with a combination of 0.5 μg of αCD3 and 0.5 μg of αCD28Fab, or 4.5 μL of a streptactin-based multimerization reagent preparation loaded with 0.5 μg of αCD3, 0.5 μg of αCD8Fab, and 0.5 μg of αCD28Fab, or a mixture of 3 μL of a streptactin-based multimerization reagent preparation containing only 0.5 μg of αCD3Fab and only 0.5 μg of αCD28Fab (each Fab fragment also containing a streptavidin-binding peptide). Untreated Tresp cells served as a negative control, and Tresp stimulated with Dynabeads (beads coated with αCD3 mAb and αCD28 mAb) served as a positive control. Tresp cells were seeded in duplicate in 1 mL of cell culture medium supplemented with 30 U / mL IL-2 in 48-well plates. Cells were incubated at 37°C for 3 days, after which the medium was changed, and analyzed 6 days later.

[0119] Example 19: Preferential proliferation of CD8+ T cells among CD3+ responder T cells stimulated in vitro with a streptavidin-based multimerization reagent reversibly functionalized with αCD3 and αCD28 Fab fragments 300,000 CD3+ responder T cells (Tresp) were incubated with various amounts of a mixture of streptactin-based multimerization reagent preparations (1 mg / mL) functionalized with only αCD3Fab fragments and only αCD28Fab fragments (1.5 μg of streptactin-based multimerization reagent functionalized with only 0.5 μg of αCD3Fab fragments and 1.5 μg of streptactin-based multimerization reagent functionalized with only 0.5 μg of αCD28Fab fragments), or various amounts of a mixture of a streptactin-based multimerization reagent preparation functionalized with αCD3Fab fragments and αCD28Fab fragments with or without αCD8Fab fragments (each Fab fragment also possesses a streptavidin-binding peptide) (0.5 μg of streptactin-based multimerization reagent functionalized with 0.5 μg of αCD3Fab fragments and 0.5 μg of αCD28Fab fragments without αCD8Fab fragments). The cells were stimulated with 4.5 μL of a streptactin multimerization reagent preparation loaded with 3 μg of αCD3 Fab fragments, or 0.5 μg of αCD3 Fab fragments, 0.5 μg of αCD8 Fab fragments, and 0.5 μg of αCD28 Fab fragments (the Fab fragments also carry streptavidin-binding peptides). Untreated Tresp cells served as a negative control, and Tresp stimulated with Dynabeads (beads coated with αCD3 mAb and αCD28 mAb) served as a positive control. Tresp cells were seeded in 1 mL of cell culture medium supplemented with 30 U / mL IL-2 in a 48-well plate. The cells were incubated at 37°C for 3 days, after which the medium was changed, and analyzed 6 days later.

[0120] The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0121] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitations, not specifically disclosed herein. Thus, for example, the terms "comprise," "include," "contain," and the like, shall be read expansively and without limitation. Furthermore, the terms and phrases used herein are used as terms of description and not of limitation, and the use of such terms and phrases is not intended to exclude any equivalents of the features shown and described or portions thereof, recognizing that various modifications are possible within the scope of the invention as claimed. Thus, while the invention has been specifically disclosed by exemplary embodiments and optional features, it should be understood that modifications and variations of the invention disclosed and embodied herein may be reclassified by those skilled in the art, and that such modifications and variations are considered to be within the scope of the invention.

[0122] The invention has been described broadly and generically herein. Each narrower species and subgeneric grouping within the generic disclosure also forms part of the invention. This includes the generic description of the invention with a condition or negative limitation that removes any inventive subject matter from the genus, regardless of whether the omitted material is specifically recited herein.

[0123] Other embodiments are within the scope of the following claims. Furthermore, when features or aspects of the invention are described as a Markush group, those skilled in the art will recognize that the invention also can be described as any individual member or subgroup of members of the Markush group.

Claims

1. 1. An in vitro method of stimulating a population of T cells, comprising contacting a sample containing the population of T cells with a multimerization reagent, the multimerization reagent is in a soluble form and reversibly binds to a first agent and a second agent; the first agent comprises (i) an anti-CD3 Fab and (ii) a first streptavidin binding partner reversibly bound to a first binding site of the multimerization reagent, the first streptavidin binding partner being fused to the C-terminus of a heavy chain of the first agent; the second agent comprises (i) an anti-CD28 Fab and (ii) a second streptavidin binding partner reversibly bound to a second binding site of the multimerization reagent, the second streptavidin binding partner being fused to the C-terminus of a heavy chain of the second agent; the multimerization reagent comprises an oligomer of a streptavidin mutein reversibly bound to a first and a second streptavidin binding partner; the first agent binds to CD3 receptor molecules on the surface of T cells in the population and provides a primary activation signal to the T cells; and The in vitro method wherein the second agent binds to a CD28 receptor molecule on the surface of the T cells and provides a costimulatory signal to the T cells, thereby stimulating the T cells in the population.

2. The method of claim 1 , wherein the first agent stimulates a TCR / CD3 complex-associated signal in a T cell.

3. 3. The method of claim 1 or 2, wherein the oligomer comprises three or more tetramers of the streptavidin mutein.

4. The method of any one of claims 1 to 3, wherein the oligomers are cross-linked by polysaccharides or via bifunctional linkers.

5. (a) the first and second streptavidin binding partners each comprise biotin, and the streptavidin mutein reversibly binds to biotin; (b) the first and second streptavidin binding partners each comprise a biotin analog that reversibly binds to streptavidin, and the streptavidin mutein reversibly binds to the biotin analog; or (c) the first and second streptavidin binding partners each comprise a streptavidin-binding peptide, and the streptavidin mutein reversibly binds to the streptavidin-binding peptide; The method according to any one of claims 1 to 4.

6. the first and second streptavidin binding partners each comprise a streptavidin-binding peptide, and the streptavidin mutein reversibly binds to the streptavidin-binding peptide; The method according to any one of claims 1 to 5.

7. The first streptavidin-binding partner and the second streptavidin-binding partner are, respectively, the streptavidin-binding peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 1) or the streptavidin-binding peptide SAWSHPQFEK(GGGS). ２ GGSAWSHPQFEK (SEQ ID NO: 7), The method according to any one of claims 1 to 6.

8. The streptavidin mutein has the amino acid sequence Va1 at positions 44 to 47 of the wild-type streptavidin. ４４ -Thr ４５ -Ala ４６ -Arg ４７ or the amino acid sequence lle at sequence positions 44-47 of wild-type streptavidin ４４ -Gly ４５ -Ala ４６ -Arg ４７ Including, The method according to any one of claims 1 to 7.

9. the N-terminal amino acid residue of the streptavidin mutein is within the region of amino acids 10 to 16 of the wild-type streptavidin amino acid sequence, and the C-terminal amino acid residue of the streptavidin mutein is within the region of amino acids 133 to 142 of the wild-type streptavidin amino acid sequence; 9. The method of claim 8.

10. Disrupting the binding between the first streptavidin binding partner of the first agent and the first binding site of the multimerization reagent; and / or Dissociating the binding between the second streptavidin binding partner of the second agent and the second binding site of the multimerization reagent. Also includes The method according to any one of claims 1 to 9.

11. Dissociating the binding between the first streptavidin binding partner of the first agent and the first binding site of the multimerization reagent and / or dissociating the binding between the second streptavidin binding partner of the second agent and the second binding site of the multimerization reagent results in termination of stimulation of the T cell. The method of claim 10.

12. The dissociation constant for reversible binding between the first streptavidin binding partner and the first binding site (K d ), and / or the dissociation constant for reversible binding between the second streptavidin binding partner and the second binding site (K ｄ ) but 10 －２ M to 10 －１３ M or 10 －５ M to 10 －１０ M is in the range The method according to any one of claims 1 to 11.

13. Disrupting the reversible binding between the first streptavidin binding partner and the first binding site of the multimerization reagent by contacting the population of T cells with a free first streptavidin binding partner or an analogue of the first streptavidin binding partner that is capable of disrupting the binding between the first streptavidin binding partner and the first binding site; and / or dissociating the reversible binding between the second streptavidin binding partner and the second binding site of the multimerization reagent by contacting the population of T cells with a free second streptavidin binding partner or an analog of the second streptavidin binding partner that is capable of dissociating the binding between the second streptavidin binding partner and the second binding site; The method according to any one of claims 1 to 12.

14. The method of claim 13, wherein the reversible binding between the first streptavidin binding partner and the first binding site and / or the reversible binding between the second streptavidin binding partner and the second binding site is separated by contacting the T cell with biotin or a biotin analogue; (a) the first and second streptavidin binding partners each contain biotin, and the streptavidin mutein reversibly binds to biotin; (b) the first and second streptavidin binding partners each comprise a biotin analog that reversibly binds to streptavidin, and the streptavidin mutein reversibly binds to the biotin analog; or (c) the first and second streptavidin binding partners each comprise a streptavidin-binding peptide, and the streptavidin mutein reversibly binds to the streptavidin-binding peptide; The method according to any one of claims 1 to 13.

15. contacting the sample with a multimerization reagent results in specific binding of T cells to the multimerization reagent, and the method further comprises separating the T cells bound to the multimerization reagent from a population of unbound cells. The method according to any one of claims 1 to 14.

16. Either after or during stimulation, a T cell receptor or a chimeric antigen receptor is introduced into the T cells of the population. The method according to any one of claims 1 to 15.

17. T cells are stimulated with a third agent that binds to the introduced T cell receptor or chimeric antigen receptor.

17. The method of claim 16.

18. (i) a multimerization reagent, the multimerization reagent being in a disposable form and comprising a first binding site for reversible binding of a first agent and a second binding site for reversible binding of a second agent; (ii) a first agent comprising (1) an anti-CD3 Fab and (2) a first streptavidin binding partner capable of reversibly binding to a first binding site of the multimerization reagent, wherein the first streptavidin binding partner is fused to the C-terminus of the heavy chain of the first agent, and wherein the first agent binds to CD3 receptor molecules on the surface of T cells in the population and provides a primary activation signal to the T cells; (iii) (1) a second agent comprising an anti-CD28 Fab and a second streptavidin binding partner capable of reversibly binding to a second binding site of the multimerization reagent, wherein the second streptavidin binding partner is fused to the C-terminus of the heavy chain of the second agent, and wherein the second agent binds to a CD28 receptor molecule on the surface of a T cell and provides costimulation to the cell, thereby stimulating the T cells in the population; 1. A reagent kit for stimulating a population of T cells, comprising: A reagent kit, wherein the multimerization reagent comprises oligomers of streptavidin muteins that reversibly bind to first and second streptavidin binding partners.

19. A composition for stimulating a population of T cells, comprising a multimerization reagent reversibly bound to a first agent and a second agent, the multimerization reagent is in a soluble form; the first agent comprises (i) an anti-CD3 Fab and (ii) a first streptavidin binding partner reversibly bound to a first binding site of the multimerization reagent, the first streptavidin binding partner being fused to the C-terminus of a heavy chain of the first agent; the second agent comprises (i) an anti-CD28 Fab and (ii) a second streptavidin binding partner reversibly bound to a second binding site of the multimerization reagent, the second streptavidin binding partner being fused to the C-terminus of a heavy chain of the second agent; the multimerization reagent comprises an oligomer of a streptavidin mutein, the streptavidin mutein comprising a first binding site and a second binding site that form reversible bonds to a first and a second streptavidin binding partner, respectively; a first agent binds to CD3 receptor molecules on the surface of T cells in the population and provides a primary activation signal to the T cells; and The composition, wherein the second agent binds to the CD28 receptor molecule on the surface of the T cells and provides a costimulatory signal to the T cells, thereby stimulating the T cells in the population.

20. the first agent stimulates a TCR / CD3 complex-associated signal in a T cell; 20. The composition of claim 19.

21. The oligomer comprising three or more tetramers of a streptavidin mutein.

21. The composition of claim 19 or 20.

22. The oligomer is cross-linked by a polysaccharide or via a bifunctional linker. The composition according to any one of claims 19 to 21.

23. (a) the first and second streptavidin binding partners each comprise biotin, and the streptavidin mutein reversibly binds to the biotin; (b) the first and second streptavidin binding partners each comprise a biotin analog that reversibly binds to streptavidin, and the streptavidin mutein reversibly binds to the biotin analog; or (c) the first and second streptavidin binding partners each comprise a streptavidin-binding peptide, and the streptavidin mutein reversibly binds to the streptavidin-binding peptide. The composition according to any one of claims 19 to 22.

24. the first and second streptavidin binding partners each comprise a streptavidin-binding peptide, and the streptavidin mutein reversibly binds to the streptavidin-binding peptide; The composition according to any one of claims 19 to 23.

25. The first streptavidin-binding partner and the second streptavidin-binding partner are, respectively, the streptavidin-binding peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 1) or the streptavidin-binding peptide SAWSHPQFEK(GGGS). ２ GGSAWSHPQFEK (SEQ ID NO: 7), The composition according to any one of claims 19 to 24.

26. The streptavidin mutein has the amino acid sequence Va1 at positions 44-47 of the wild-type streptavidin sequence. ４４ -Thr ４５ -Ala ４６ -Arg ４７ or the amino acid sequence lle at sequence positions 44-47 of wild-type streptavidin ４４ -Gly ４５ -Ala ４６ -Arg ４７ Including, The composition according to any one of claims 19 to 25.

27. the N-terminal amino acid residue of the streptavidin mutein is within the region of amino acids 10 to 16 of the wild-type streptavidin amino acid sequence, and the C-terminal amino acid residue of the streptavidin mutein is within the region of amino acids 133 to 142 of the wild-type streptavidin amino acid sequence; 27. The composition of claim 26.

28. The dissociation constant for reversible binding between the first streptavidin binding partner and the first binding site (K d ), and / or the dissociation constant for reversible binding between the second streptavidin binding partner and the second binding site (K ｄ ) but 10 －２ M to 10 －１３ M or 10 －５ M to 10 －１０ M is in the range The composition according to any one of claims 19 to 27.

29. the reversible binding between the first streptavidin-binding partner and the first binding site and / or between the second streptavidin-binding partner and the second binding site can be dissociated in the presence of biotin, desthiobiotin or iminobiotin; The composition according to any one of claims 19 to 28.

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