Methods, kits and apparatus for expanding population of cells

Abstract

To provide novel methods for expanding cell populations, such as regulatory T cells or central memory T cells, for research, diagnostic and, in particular, therapeutic purposes.SOLUTION: The present invention relates to in vitro-methods of expanding a population of cells such as lymphocytes, comprising contacting a sample comprising a population of cells with a multimerization reagent. The multimerization reagent has reversibly immobilized thereon (bound thereto) a first agent that provides a primary activation signal to the cells and optionally, a second agent that provides a co-stimulatory signal. The 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 THEINVENTION The present invention relates to the expansion (proliferation) of a population of cells, such as a population of lymphocytes. The present invention provides novel methods and reagents for expanding (proliferating) a population of cells, which generally require binding of a receptor binding molecule (such as a first agent described herein) to a receptor molecule on the surface of the cell to provide a primary activation signal to the cell. The present invention uses a multimerization reagent to which is immobilized (bound) a first agent that provides a primary activation signal to the cell. This primary activation signal may be sufficient in itself to activate the cell to expand / proliferate. This first agent may be reversibly bound or irreversibly bound to the multimerization reagent. Also immobilized (bound) on the multimerization reagent may be a second agent that stimulates an accessory molecule on the surface of the cell. When the second agent binds to an accessory molecule on the surface of the cell, it may stimulate the activated cell to expand. Also, this second agent may be reversibly bound or irreversibly bound 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 sequential expansion of a population of cells, where a complete population of lymphocytes is stimulated / expanded, then the reagents required for expansion are removed by chromatography on a suitable stationary phase, and the expanded / stimulated cells are optionally transfected, for example, with a T cell receptor or a chimeric antigen receptor (CAR) and subjected 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] 2. Background of the Invention The development of techniques for expanding T cell populations in vitro has been essential to many of the advances in understanding 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 pathogens and tumor-expressed antigens that are recognized by T cells to establish immunotherapeutic methods for treating 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. In addition, 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 US Pat. No. 6,352,694 B1 and EP Pat. No. 0 700 430 B1. These patents disclose in vitro methods for inducing a population of T cells to proliferate. The methods include contacting a population of T cells with a solid surface on 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. The binding of the first and second agents to the T cells induces the T cells to proliferate / expand. The preferred first agent described in US Pat. No. 6,352,694 B1 and EP Pat. No. 0 700 430 B1 is a monoclonal anti-CD3 antibody that binds to the TCR / CD3 (TCR=T cell receptor) complex and stimulates a TCR / CD3 complex-associated signal in the 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. Meanwhile, 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 integrate into methods for expanding cells under the conditions required for e.g. clinical trials or therapeutic purposes, since it must be ensured that these 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, in particular, therapeutic purposes. Ideally, the novel method should also be amenable to integration into an automated process that can be used to rapidly and easily expand the desired cell population 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 according to 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 multimerisation reagent, a first agent that provides a primary activation signal to the cell is reversibly immobilized (bound) on the multimerization reagent; the multimerization reagent comprises at least one binding site Z1 for reversibly binding the first agent; The first agent comprises at least one binding partner C1, the binding partner C1 being capable of reversibly binding to the binding site Z1 of the multimerization reagent, the first agent binds to the multimerization reagent via a reversible bond formed between the binding partner C1 and the binding site Z1, and A method is provided in which 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 multimerisation 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) on 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, the binding partner C1 being capable of binding to the binding site Z1 of the multimerization reagent, the first agent being bound to the multimerization reagent via a bond formed between the binding partner C1 and the binding site Z1, and A method is provided in which 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 the 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 a 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 of serially expanding a population of lymphocytes, said population of lymphocytes comprising T cells, said method comprising: contacting a sample comprising 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 the first agent; the first agent comprises at least one binding partner C1, the binding partner C1 being capable of reversibly 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; the multimerization reagent comprises at least one binding site Z2 for reversibly binding 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 relates to an arrangement of a bioreactor and a stationary phase for chromatography, comprising: the bioreactor is suitable for expanding cells; the stationary phase is suitable for the separation of cells and removal of reagents, 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 which specifically binds to a binding partner C1 comprised in a first agent and / or the affinity reagent comprising a binding site Z2 which 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, Provided is an arrangement in which the bioreactor and the stationary phase are in fluid connection.

[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: is in soluble form and comprises 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) on the multimerization reagent; the first agent comprises at least one binding partner C1, said binding partner C1 being capable of reversibly binding to said 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 reversible binding of 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) on the first multimerization reagent; a first multimerization reagent, the first agent comprising at least one binding partner C1, the binding partner C1 being capable of reversibly binding to the at least one 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; (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, the second agent, which stimulates an accessory molecule on the surface of the cell, is reversibly immobilized (bound) onto the multimerization reagent; The second agent comprises a binding partner C2, the binding partner C2 being capable of binding to the at least one binding site Z2 of the multimerization reagent, and the second agent comprises a second multimerization reagent that binds to the multimerization reagent via a bond formed between the binding partner C2 and the binding site Z2. [Brief description of the drawings]

[0017] The invention will be better understood by reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, which illustrate embodiments of the method of the 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 a molecular level. [Figure 1A]FIG. 1 shows an embodiment of an in vitro expansion method for expanding a population of cells having a cell surface receptor to which a first agent can bind to provide an activation signal for the cells to expand. As shown in FIG. 1a, a sample containing a population of cells (2) having a surface receptor molecule (30) is contacted with a multimerization reagent (4). The population of cells (2) is in a mixture with another population of cells (22) that does not have the surface receptor molecule (30). A first agent (6) that provides a primary activation signal to the cells is reversibly immobilized (bound) on 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), which can reversibly bind 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. The 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, the cell surface receptor (30) is CD40, and the first agent (6) can be any CD40 binding molecule that provides the desired activation signal, for example, 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) may 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) may 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 US 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 US Pat. No. 7,981,632. When a streptavidin-binding peptide is used as binding partner C1, the multimerization reagent (4) may 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 FIG. 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 lle44-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 trade name Strep-Tactin®. In the embodiment of FIG. 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, the 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 made by standard protein chemistry, for example by using bifunctional linkers.As shown in FIG. 1b, after contacting cell population (2) with multimerization reagent (4) and typically incubating the cell population with the multimerization reagent (4), the cell population (2) complexes / binds with the multimerization reagent via a 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, for example, 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 that binding of these multiple copies is typically necessary 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 population of cells (2). In this regard, it is noted that as used herein, the multimerizing agent may itself have multiple binding sites Z1, for example, streptavidin mutein in its native state (which is a homotetramer) has four such binding sites Z1. However, it is also possible that the multimerizing reagent is based on a compound that itself has only one binding site Z1 for the binding partner C1 to reversibly bind. Such an example is multimeric calmodulin. Calmodulin itself has only one binding site for calmodulin-binding peptides. 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 (usually performed for a period suitable for expanding the desired cell population), the bond between the binding partner C1 (6a) of the first agent (6) and the binding site Z1 of the multimerization reagent (4) is separated by separating the respective reversible bonds.The separation can be achieved by adding a competitor to the incubation / reaction mixture containing the population of cells bound to the multimerization reagent (2). For competitive separation of the reversible binding between the binding partner C1 (6a) of the first agent and the binding site 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 analogue of said first binding partner C, which is capable of separating the binding between the first binding partner C1 (6a) and the binding site Z1 (22). In the example of a binding partner C1 that is 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 analogue. Such an analogue can be, for example, biotin or a biotin derivative such as desthiobiotin. As shown in FIG. 1d, the addition of the first free partner (20) or an analogue thereof displaces the binding partner C1 (6a) from the multimerization reagent (4) and thus 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 of the binding between the first agent and the cell surface receptor (30) has a dissociation constant (Kd) in the range of 10-2M to 10-13M and is therefore also reversible. Due to this dissociation, the stimulation of the cell population (2) also ends. Thus, the present invention provides the advantage that the duration of the stimulation or expansion of the cell population can be precisely controlled and therefore the functional state of the cell population can also be precisely controlled. 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) is typically in the affinity range of Kd of 10-7 M to 10-13 M. Thus, conventional monoclonal antibodies can be used as the first agent in the present invention (and, of course, as the second agent, as described below).To avoid any undesired avidity effects leading to tighter 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 invention has the additional advantage that the stimulated cell population is free of the stimulating agent at the end of the stimulation period and that all other reagents used in the method, i.e. the first agent (6) and the free first partner (20) of the binding partner C1 or its analogue, 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 the bioreactor surface or on a solid support such as magnetic beads. Returning therefore 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 an affinity chromatography matrix and at the same time as a gel permeation matrix. On this affinity chromatography matrix, an affinity reagent is immobilized. The affinity reagent may, in this embodiment, be, for example, streptavidin, streptavidin mutein, avidin, avidin mutein, or a mixture thereof. The first agent (6), the free first partner (20) of the binding partner C1 (also referred to herein as the "competitive 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]FIG. 1 shows an embodiment of an in vitro expansion method for expanding a population of cells having a cell surface receptor to which a first agent can bind to provide an activation signal for the cells to expand. As shown in FIG. 1a, a sample containing a population of cells (2) having a surface receptor molecule (30) is contacted with a multimerization reagent (4). The population of cells (2) is in a mixture with another population of cells (22) that does not have the surface receptor molecule (30). A first agent (6) that provides a primary activation signal to the cells is reversibly immobilized (bound) on 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), which can reversibly bind 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. The 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, the cell surface receptor (30) is CD40, and the first agent (6) can be any CD40 binding molecule that provides the desired activation signal, for example, 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) may 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) may 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 US 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 US Pat. No. 7,981,632. When a streptavidin-binding peptide is used as binding partner C1, the multimerization reagent (4) may 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 FIG. 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 lle44-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 trade name Strep-Tactin®. In the embodiment of FIG. 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, the 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 made by standard protein chemistry, for example by using bifunctional linkers.As shown in FIG. 1b, after contacting cell population (2) with multimerization reagent (4) and typically incubating the cell population with the multimerization reagent (4), the cell population (2) complexes / binds with the multimerization reagent via a 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, for example, 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 that binding of these multiple copies is typically necessary 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 population of cells (2). In this regard, it is noted that as used herein, the multimerizing agent may itself have multiple binding sites Z1, for example, streptavidin mutein in its native state (which is a homotetramer) has four such binding sites Z1. However, it is also possible that the multimerizing reagent is based on a compound that itself has only one binding site Z1 for the binding partner C1 to reversibly bind. Such an example is multimeric calmodulin. Calmodulin itself has only one binding site for calmodulin-binding peptides. 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 (usually performed for a period suitable for expanding the desired cell population), the bond between the binding partner C1 (6a) of the first agent (6) and the binding site Z1 of the multimerization reagent (4) is separated by separating the respective reversible bonds.The separation can be achieved by adding a competitor to the incubation / reaction mixture containing the population of cells bound to the multimerization reagent (2). For competitive separation of the reversible binding between the binding partner C1 (6a) of the first agent and the binding site 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 analogue of said first binding partner C, which is capable of separating the binding between the first binding partner C1 (6a) and the binding site Z1 (22). In the example of a binding partner C1 that is 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 analogue. Such an analogue can be, for example, biotin or a biotin derivative such as desthiobiotin. As shown in FIG. 1d, the addition of the first free partner (20) or an analogue thereof displaces the binding partner C1 (6a) from the multimerization reagent (4) and thus 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 of the binding between the first agent and the cell surface receptor (30) has a dissociation constant (Kd) in the range of 10-2M to 10-13M and is therefore also reversible. Due to this dissociation, the stimulation of the cell population (2) also ends. Thus, the present invention provides the advantage that the duration of the stimulation or expansion of the cell population can be precisely controlled and therefore the functional state of the cell population can also be precisely controlled. 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) is typically in the affinity range of Kd of 10-7 M to 10-13 M. Thus, conventional monoclonal antibodies can be used as the first agent in the present invention (and, of course, as the second agent, as described below).To avoid any undesired avidity effects leading to tighter 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 invention has the additional advantage that the stimulated cell population is free of the stimulating agent at the end of the stimulation period and that all other reagents used in the method, i.e. the first agent (6) and the free first partner (20) of the binding partner C1 or its analogue, 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 the bioreactor surface or on a solid support such as magnetic beads. Returning therefore 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 an affinity chromatography matrix and at the same time as a gel permeation matrix. On this affinity chromatography matrix, an affinity reagent is immobilized. The affinity reagent may, in this embodiment, be, for example, streptavidin, streptavidin mutein, avidin, avidin mutein, or a mixture thereof. The first agent (6), the free first partner (20) of the binding partner C1 (also referred to herein as the "competitive 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]FIG. 1 shows an embodiment of an in vitro expansion method for expanding a population of cells having a cell surface receptor to which a first agent can bind to provide an activation signal for the cells to expand. As shown in FIG. 1a, a sample containing a population of cells (2) having a surface receptor molecule (30) is contacted with a multimerization reagent (4). The population of cells (2) is in a mixture with another population of cells (22) that does not have the surface receptor molecule (30). A first agent (6) that provides a primary activation signal to the cells is reversibly immobilized (bound) on 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), which can reversibly bind 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. The 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, the cell surface receptor (30) is CD40, and the first agent (6) can be any CD40 binding molecule that provides the desired activation signal, for example, 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) may 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) may 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 US 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 US Pat. No. 7,981,632. When a streptavidin-binding peptide is used as binding partner C1, the multimerization reagent (4) may 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 FIG. 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 lle44-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 trade name Strep-Tactin®. In the embodiment of FIG. 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, the 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 made by standard protein chemistry, for example by using bifunctional linkers.As shown in FIG. 1b, after contacting cell population (2) with multimerization reagent (4) and typically incubating the cell population with the multimerization reagent (4), the cell population (2) complexes / binds with the multimerization reagent via a 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, for example, 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 that binding of these multiple copies is typically necessary 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 population of cells (2). In this regard, it is noted that as used herein, the multimerizing agent may itself have multiple binding sites Z1, for example, streptavidin mutein in its native state (which is a homotetramer) has four such binding sites Z1. However, it is also possible that the multimerizing reagent is based on a compound that itself has only one binding site Z1 for the binding partner C1 to reversibly bind. Such an example is multimeric calmodulin. Calmodulin itself has only one binding site for calmodulin-binding peptides. 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 (usually performed for a period suitable for expanding the desired cell population), the bond between the binding partner C1 (6a) of the first agent (6) and the binding site Z1 of the multimerization reagent (4) is separated by separating the respective reversible bonds.The separation can be achieved by adding a competitor to the incubation / reaction mixture containing the population of cells bound to the multimerization reagent (2). For competitive separation of the reversible binding between the binding partner C1 (6a) of the first agent and the binding site 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 analogue of said first binding partner C, which is capable of separating the binding between the first binding partner C1 (6a) and the binding site Z1 (22). In the example of a binding partner C1 that is 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 analogue. Such an analogue can be, for example, biotin or a biotin derivative such as desthiobiotin. As shown in FIG. 1d, the addition of the first free partner (20) or an analogue thereof displaces the binding partner C1 (6a) from the multimerization reagent (4) and thus 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 of the binding between the first agent and the cell surface receptor (30) has a dissociation constant (Kd) in the range of 10-2M to 10-13M and is therefore also reversible. Due to this dissociation, the stimulation of the cell population (2) also ends. Thus, the present invention provides the advantage that the duration of the stimulation or expansion of the cell population can be precisely controlled and therefore the functional state of the cell population can also be precisely controlled. 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) is typically in the affinity range of Kd of 10-7 M to 10-13 M. Thus, conventional monoclonal antibodies can be used as the first agent in the present invention (and, of course, as the second agent, as described below).To avoid any undesired avidity effects leading to tighter 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 invention has the additional advantage that the stimulated cell population is free of the stimulating agent at the end of the stimulation period and that all other reagents used in the method, i.e. the first agent (6) and the free first partner (20) of the binding partner C1 or its analogue, 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 the bioreactor surface or on a solid support such as magnetic beads. Returning therefore 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 an affinity chromatography matrix and at the same time as a gel permeation matrix. On this affinity chromatography matrix, an affinity reagent is immobilized. The affinity reagent may, in this embodiment, be, for example, streptavidin, streptavidin mutein, avidin, avidin mutein, or a mixture thereof. The first agent (6), the free first partner (20) of the binding partner C1 (also referred to herein as the "competitive 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]FIG. 1 shows an embodiment of an in vitro expansion method for expanding a population of cells having a cell surface receptor to which a first agent can bind to provide an activation signal for the cells to expand. As shown in FIG. 1a, a sample containing a population of cells (2) having a surface receptor molecule (30) is contacted with a multimerization reagent (4). The population of cells (2) is in a mixture with another population of cells (22) that does not have the surface receptor molecule (30). A first agent (6) that provides a primary activation signal to the cells is reversibly immobilized (bound) on 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), which can reversibly bind 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. The 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, the cell surface receptor (30) is CD40, and the first agent (6) can be any CD40 binding molecule that provides the desired activation signal, for example, 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) may 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) may 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 US 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 US Pat. No. 7,981,632. When a streptavidin-binding peptide is used as binding partner C1, the multimerization reagent (4) may 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 FIG. 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 lle44-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 trade name Strep-Tactin®. In the embodiment of FIG. 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, the 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 made by standard protein chemistry, for example by using bifunctional linkers.As shown in FIG. 1b, after contacting cell population (2) with multimerization reagent (4) and typically incubating the cell population with the multimerization reagent (4), the cell population (2) complexes / binds with the multimerization reagent via a 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, for example, 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 that binding of these multiple copies is typically necessary 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 population of cells (2). In this regard, it is noted that as used herein, the multimerizing agent may itself have multiple binding sites Z1, for example, streptavidin mutein in its native state (which is a homotetramer) has four such binding sites Z1. However, it is also possible that the multimerizing reagent is based on a compound that itself has only one binding site Z1 for the binding partner C1 to reversibly bind. Such an example is multimeric calmodulin. Calmodulin itself has only one binding site for calmodulin-binding peptides. 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 (usually performed for a period suitable for expanding the desired cell population), the bond between the binding partner C1 (6a) of the first agent (6) and the binding site Z1 of the multimerization reagent (4) is separated by separating the respective reversible bonds.The separation can be achieved by adding a competitor to the incubation / reaction mixture containing the population of cells bound to the multimerization reagent (2). For competitive separation of the reversible binding between the binding partner C1 (6a) of the first agent and the binding site 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 analogue of said first binding partner C, which is capable of separating the binding between the first binding partner C1 (6a) and the binding site Z1 (22). In the example of a binding partner C1 that is 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 analogue. Such an analogue can be, for example, biotin or a biotin derivative such as desthiobiotin. As shown in FIG. 1d, the addition of the first free partner (20) or an analogue thereof displaces the binding partner C1 (6a) from the multimerization reagent (4) and thus 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 of the binding between the first agent and the cell surface receptor (30) has a dissociation constant (Kd) in the range of 10-2M to 10-13M and is therefore also reversible. Due to this dissociation, the stimulation of the cell population (2) also ends. Thus, the present invention provides the advantage that the duration of the stimulation or expansion of the cell population can be precisely controlled and therefore the functional state of the cell population can also be precisely controlled. 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) is typically in the affinity range of Kd of 10-7 M to 10-13 M. Thus, conventional monoclonal antibodies can be used as the first agent in the present invention (and, of course, as the second agent, as described below).To avoid any undesired avidity effects leading to tighter 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 invention has the additional advantage that the stimulated cell population is free of the stimulating agent at the end of the stimulation period and that all other reagents used in the method, i.e. the first agent (6) and the free first partner (20) of the binding partner C1 or its analogue, 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 the bioreactor surface or on a solid support such as magnetic beads. Returning therefore 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 an affinity chromatography matrix and at the same time as a gel permeation matrix. On this affinity chromatography matrix, an affinity reagent is immobilized. The affinity reagent may, in this embodiment, be, for example, streptavidin, streptavidin mutein, avidin, avidin mutein, or a mixture thereof. The first agent (6), the free first partner (20) of the binding partner C1 (also referred to herein as the "competitive 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]FIG. 2 shows a further embodiment of the expansion method of the present invention. As shown in FIG. 2a, the sample contains a population of cells (2) that has two specific cell surface molecules (30) and (32). The cell surface molecule (30) is responsible for the primary activation signal to the cell population, while the cell surface molecule (32) is an accessory molecule on the cell surface that is responsible for providing a stimulus to the cells. The population of cells can be, for example, a T cell population, where the cell surface molecule (30) is the TCR / CD3 complex and the cell surface molecule (32) is the accessory molecule CD28. The expansion / proliferation of T cells requires the binding of both the TCR / CD3 complex as a primary activation signal and CD28 as a costimulator. The population of T cells (2) is in a mixture with another cell population (22) that does not have the surface receptor molecules (30) and (32). Also in this embodiment, the cell population (2) is contacted with a multimerization reagent (4). A first agent (6) that provides a primary activation signal to a cell is reversibly immobilized (bound) on the multimerization reagent (4). In addition, a second agent (8) that stimulates CD28 as an accessory molecule on the surface of a cell is reversibly immobilized (bound) on the multimerization reagent. The multimerization reagent (4) comprises at least one binding site Z1 (42) for reversibly binding the first agent (6), and the first agent (6) comprises at least one binding partner C1 (6a), which can reversibly bind 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). Further, in the embodiment shown in Figure 2, the second agent (8) comprises a binding partner C2 (8a), which can reversibly bind to the 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 the binding partner C2 (8a) and the binding site Z2 (44). In this embodiment, the first agent (6) can be a monoclonal anti-CD3 antibody, or an antigen-binding fragment thereof, such as a Fab fragment. The second agent (8) can be a monoclonal anti-CD28 antibody, or an antigen-binding fragment thereof, such as a Fab fragment.The first binding partner (6a) may be a streptavidin-binding peptide (6a) fused or conjugated to an anti-CD3 antibody or an anti-CD3 antibody fragment. The second binding partner (8a) may be a calmodulin-binding peptide conjugated or fused to a CD28 antibody or a CD28-binding antibody fragment. In this context, it is noted that, for example, monoclonal antibodies against CD3 or CD28 are well known (see, for example, 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, such as 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 the first and second agents and can be, for example, chemically linked (conjugated) to the binding partners C1 and C2. Alternatively, it is possible to clone the genes of the variable domains from a hybridoma cell line or to use an antibody whose amino acid sequence is known and recombinantly produce the respective antibody fragments, such as Fab fragments or Fv.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 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). The amino acid sequences of the heavy and light chain variable domains of the antibody OKT3 described in (1996) 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). This methodology of cloning the variable domains of antibody molecules and recombinantly producing the respective antibody fragments is also 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, 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, as in the example of FIG. 2, by well-known evolutionary methods, such as those reported in Ref. 3750-3755. In the example shown in FIG. 2, the multimerization reagent (4) has two different binding sites Z1 (42) and Z2 (44). With 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 a multimeric calmodulin. The multimerization reagent (4) may be a single molecule, e.g. a conjugate of multimeric calmodulin and streptavidin (this alternative is usually 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 muteins 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.It is noted that in this situation, the immobilization of calmodulin on the surface of the solid support does not require the preparation of multimeric calmodulin as explained above, but the immobilization of calmodulin on the surface is sufficient to present calmodulin (which, as mentioned above, has only a single binding site for the calmodulin-binding peptide at a sufficiently high density to ensure the binding of the cell population (2)). For example, in this case, a bivalent antibody fragment with two binding sites for CD28 or an intact antibody, itself with two identical binding sites, can be used as the second reagent (8). As shown in FIG. 2b, after contacting the T cell population (2) with the multimerization reagent (4) and, typically, incubating the cell population with the multimerization reagent (4), the T cell population (2) forms a complex / binds to the multimerization reagent via the first agent (6) and the 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 FIG. 2c, after incubation (usually 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. Similarly, the binding between the binding partner C2 (8a) of the second agent (8) and the binding site Z2 of the multimerization reagent (4) is 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 analogue of the first partner (20)), whereas 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 analogue 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, the addition of the first free partner and second free partner analogs (20) displaces the binding partners C1 (6a) and C2 (8a), respectively, from the multimerization reagent (4), which in turn displaces the first agent (6) and the second agent (8) from the multimerization reagent (4). This displacement of the first agent (6) and the second agent (8) then dissociates the first agent (6) and the second agent (8) from the TCR / CD3 complex and the accessory molecule CD28, thereby terminating the stimulation / expansion of the cell population (2). Thus, as discussed above, the present invention provides the advantage that the duration of stimulation or expansion of a T cell population can be precisely controlled, and therefore the functional state of the T cell population can also be tightly controlled. After elution of the cells as shown in FIG. 1d, the first agent (6), the second reagent (8) and the analogue of the free first partner of the binding partner C1 and the free second partner of the binding partner C2 (20) can be easily removed from the stimulated cell population (2) via the “removal cartridge” described in WO 2013 / 124474. Furthermore, and importantly, if the initial sample was a population of lymphocytes, for example 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 during expansion with, for example, a T cell receptor (TCR) or a chimeric antigen receptor (CAR, also known as an artificial T cell receptor), the genetically modified cells can then be released from the initial stimulation and then stimulated with a second type of stimulation, for example via a de novo introduced receptor. These second stimuli may include antigenic stimuli in the form of peptide / MHC molecules, cognate (cross-linking) 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]FIG. 2 shows a further embodiment of the expansion method of the present invention. As shown in FIG. 2a, the sample contains a population of cells (2) that has two specific cell surface molecules (30) and (32). The cell surface molecule (30) is responsible for the primary activation signal to the cell population, while the cell surface molecule (32) is an accessory molecule on the cell surface that is responsible for providing a stimulus to the cells. The population of cells can be, for example, a T cell population, where the cell surface molecule (30) is the TCR / CD3 complex and the cell surface molecule (32) is the accessory molecule CD28. The expansion / proliferation of T cells requires the binding of both the TCR / CD3 complex as a primary activation signal and CD28 as a costimulator. The population of T cells (2) is in a mixture with another cell population (22) that does not have the surface receptor molecules (30) and (32). Also in this embodiment, the cell population (2) is contacted with a multimerization reagent (4). A first agent (6) that provides a primary activation signal to a cell is reversibly immobilized (bound) on the multimerization reagent (4). In addition, a second agent (8) that stimulates CD28 as an accessory molecule on the surface of a cell is reversibly immobilized (bound) on the multimerization reagent. The multimerization reagent (4) comprises at least one binding site Z1 (42) for reversibly binding the first agent (6), and the first agent (6) comprises at least one binding partner C1 (6a), which can reversibly bind 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). Further, in the embodiment shown in Figure 2, the second agent (8) comprises a binding partner C2 (8a), which can reversibly bind to the 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 the binding partner C2 (8a) and the binding site Z2 (44). In this embodiment, the first agent (6) can be a monoclonal anti-CD3 antibody, or an antigen-binding fragment thereof, such as a Fab fragment. The second agent (8) can be a monoclonal anti-CD28 antibody, or an antigen-binding fragment thereof, such as a Fab fragment.The first binding partner (6a) may be a streptavidin-binding peptide (6a) fused or conjugated to an anti-CD3 antibody or an anti-CD3 antibody fragment. The second binding partner (8a) may be a calmodulin-binding peptide conjugated or fused to a CD28 antibody or a CD28-binding antibody fragment. In this context, it is noted that, for example, monoclonal antibodies against CD3 or CD28 are well known (see, for example, 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, such as 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 the first and second agents and can be, for example, chemically linked (conjugated) to the binding partners C1 and C2. Alternatively, it is possible to clone the genes of the variable domains from a hybridoma cell line or to use an antibody whose amino acid sequence is known and recombinantly produce the respective antibody fragments, such as Fab fragments or Fv.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 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). The amino acid sequences of the heavy and light chain variable domains of the antibody OKT3 described in (1996) 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). This methodology of cloning the variable domains of antibody molecules and recombinantly producing the respective antibody fragments is also 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, 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, as in the example of FIG. 2, by well-known evolutionary methods, such as those reported in Ref. 3750-3755. In the example shown in FIG. 2, the multimerization reagent (4) has two different binding sites Z1 (42) and Z2 (44). With 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 a multimeric calmodulin. The multimerization reagent (4) may be a single molecule, e.g. a conjugate of multimeric calmodulin and streptavidin (this alternative is usually 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 muteins 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.It is noted that in this situation, the immobilization of calmodulin on the surface of the solid support does not require the preparation of multimeric calmodulin as explained above, but the immobilization of calmodulin on the surface is sufficient to present calmodulin (which, as mentioned above, has only a single binding site for the calmodulin-binding peptide at a sufficiently high density to ensure the binding of the cell population (2)). For example, in this case, a bivalent antibody fragment with two binding sites for CD28 or an intact antibody, itself with two identical binding sites, can be used as the second reagent (8). As shown in FIG. 2b, after contacting the T cell population (2) with the multimerization reagent (4) and, typically, incubating the cell population with the multimerization reagent (4), the T cell population (2) forms a complex / binds to the multimerization reagent via the first agent (6) and the 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 FIG. 2c, after incubation (usually 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. Similarly, the binding between the binding partner C2 (8a) of the second agent (8) and the binding site Z2 of the multimerization reagent (4) is 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 analogue of the first partner (20)), whereas 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 analogue 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, the addition of the first free partner and second free partner analogs (20) displaces the binding partners C1 (6a) and C2 (8a), respectively, from the multimerization reagent (4), which in turn displaces the first agent (6) and the second agent (8) from the multimerization reagent (4). This displacement of the first agent (6) and the second agent (8) then dissociates the first agent (6) and the second agent (8) from the TCR / CD3 complex and the accessory molecule CD28, thereby terminating the stimulation / expansion of the cell population (2). Thus, as discussed above, the present invention provides the advantage that the duration of stimulation or expansion of a T cell population can be precisely controlled, and therefore the functional state of the T cell population can also be tightly controlled. After elution of the cells as shown in FIG. 1d, the first agent (6), the second reagent (8) and the analogue of the free first partner of the binding partner C1 and the free second partner of the binding partner C2 (20) can be easily removed from the stimulated cell population (2) via the “removal cartridge” described in WO 2013 / 124474. Furthermore, and importantly, if the initial sample was a population of lymphocytes, for example 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 during expansion with, for example, a T cell receptor (TCR) or a chimeric antigen receptor (CAR, also known as an artificial T cell receptor), the genetically modified cells can then be released from the initial stimulation and then stimulated with a second type of stimulation, for example via a de novo introduced receptor. These second stimuli may include antigenic stimuli in the form of peptide / MHC molecules, cognate (cross-linking) 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]FIG. 2 shows a further embodiment of the expansion method of the present invention. As shown in FIG. 2a, the sample contains a population of cells (2) that has two specific cell surface molecules (30) and (32). The cell surface molecule (30) is responsible for the primary activation signal to the cell population, while the cell surface molecule (32) is an accessory molecule on the cell surface that is responsible for providing a stimulus to the cells. The population of cells can be, for example, a T cell population, where the cell surface molecule (30) is the TCR / CD3 complex and the cell surface molecule (32) is the accessory molecule CD28. The expansion / proliferation of T cells requires the binding of both the TCR / CD3 complex as a primary activation signal and CD28 as a costimulator. The population of T cells (2) is in a mixture with another cell population (22) that does not have the surface receptor molecules (30) and (32). Also in this embodiment, the cell population (2) is contacted with a multimerization reagent (4). A first agent (6) that provides a primary activation signal to a cell is reversibly immobilized (bound) on the multimerization reagent (4). In addition, a second agent (8) that stimulates CD28 as an accessory molecule on the surface of a cell is reversibly immobilized (bound) on the multimerization reagent. The multimerization reagent (4) comprises at least one binding site Z1 (42) for reversibly binding the first agent (6), and the first agent (6) comprises at least one binding partner C1 (6a), which can reversibly bind 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). Further, in the embodiment shown in Figure 2, the second agent (8) comprises a binding partner C2 (8a), which can reversibly bind to the 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 the binding partner C2 (8a) and the binding site Z2 (44). In this embodiment, the first agent (6) can be a monoclonal anti-CD3 antibody, or an antigen-binding fragment thereof, such as a Fab fragment. The second agent (8) can be a monoclonal anti-CD28 antibody, or an antigen-binding fragment thereof, such as a Fab fragment.The first binding partner (6a) may be a streptavidin-binding peptide (6a) fused or conjugated to an anti-CD3 antibody or an anti-CD3 antibody fragment. The second binding partner (8a) may be a calmodulin-binding peptide conjugated or fused to a CD28 antibody or a CD28-binding antibody fragment. In this context, it is noted that, for example, monoclonal antibodies against CD3 or CD28 are well known (see, for example, 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, such as 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 the first and second agents and can be, for example, chemically linked (conjugated) to the binding partners C1 and C2. Alternatively, it is possible to clone the genes of the variable domains from a hybridoma cell line or to use an antibody whose amino acid sequence is known and recombinantly produce the respective antibody fragments, such as Fab fragments or Fv.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 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). The amino acid sequences of the heavy and light chain variable domains of the antibody OKT3 described in (1996) 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). This methodology of cloning the variable domains of antibody molecules and recombinantly producing the respective antibody fragments is also 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, 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, as in the example of FIG. 2, by well-known evolutionary methods, such as those reported in Ref. 3750-3755. In the example shown in FIG. 2, the multimerization reagent (4) has two different binding sites Z1 (42) and Z2 (44). With 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 a multimeric calmodulin. The multimerization reagent (4) may be a single molecule, e.g. a conjugate of multimeric calmodulin and streptavidin (this alternative is usually 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 muteins 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.It is noted that in this situation, the immobilization of calmodulin on the surface of the solid support does not require the preparation of multimeric calmodulin as explained above, but the immobilization of calmodulin on the surface is sufficient to present calmodulin (which, as mentioned above, has only a single binding site for the calmodulin-binding peptide at a sufficiently high density to ensure the binding of the cell population (2)). For example, in this case, a bivalent antibody fragment with two binding sites for CD28 or an intact antibody, itself with two identical binding sites, can be used as the second reagent (8). As shown in FIG. 2b, after contacting the T cell population (2) with the multimerization reagent (4) and, typically, incubating the cell population with the multimerization reagent (4), the T cell population (2) forms a complex / binds to the multimerization reagent via the first agent (6) and the 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 FIG. 2c, after incubation (usually 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. Similarly, the binding between the binding partner C2 (8a) of the second agent (8) and the binding site Z2 of the multimerization reagent (4) is 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 analogue of the first partner (20)), whereas 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 analogue 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, the addition of the first free partner and second free partner analogs (20) displaces the binding partners C1 (6a) and C2 (8a), respectively, from the multimerization reagent (4), which in turn displaces the first agent (6) and the second agent (8) from the multimerization reagent (4). This displacement of the first agent (6) and the second agent (8) then dissociates the first agent (6) and the second agent (8) from the TCR / CD3 complex and the accessory molecule CD28, thereby terminating the stimulation / expansion of the cell population (2). Thus, as discussed above, the present invention provides the advantage that the duration of stimulation or expansion of a T cell population can be precisely controlled, and therefore the functional state of the T cell population can also be tightly controlled. After elution of the cells as shown in FIG. 1d, the first agent (6), the second reagent (8) and the analogue of the free first partner of the binding partner C1 and the free second partner of the binding partner C2 (20) can be easily removed from the stimulated cell population (2) via the “removal cartridge” described in WO 2013 / 124474. Furthermore, and importantly, if the initial sample was a population of lymphocytes, for example 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 during expansion with, for example, a T cell receptor (TCR) or a chimeric antigen receptor (CAR, also known as an artificial T cell receptor), the genetically modified cells can then be released from the initial stimulation and then stimulated with a second type of stimulation, for example via a de novo introduced receptor. These second stimuli may include antigenic stimuli in the form of peptide / MHC molecules, cognate (cross-linking) 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]FIG. 2 shows a further embodiment of the expansion method of the present invention. As shown in FIG. 2a, the sample contains a population of cells (2) that has two specific cell surface molecules (30) and (32). The cell surface molecule (30) is responsible for the primary activation signal to the cell population, while the cell surface molecule (32) is an accessory molecule on the cell surface that is responsible for providing a stimulus to the cells. The population of cells can be, for example, a T cell population, where the cell surface molecule (30) is the TCR / CD3 complex and the cell surface molecule (32) is the accessory molecule CD28. The expansion / proliferation of T cells requires the binding of both the TCR / CD3 complex as a primary activation signal and CD28 as a costimulator. The population of T cells (2) is in a mixture with another cell population (22) that does not have the surface receptor molecules (30) and (32). Also in this embodiment, the cell population (2) is contacted with a multimerization reagent (4). A first agent (6) that provides a primary activation signal to a cell is reversibly immobilized (bound) on the multimerization reagent (4). In addition, a second agent (8) that stimulates CD28 as an accessory molecule on the surface of a cell is reversibly immobilized (bound) on the multimerization reagent. The multimerization reagent (4) comprises at least one binding site Z1 (42) for reversibly binding the first agent (6), and the first agent (6) comprises at least one binding partner C1 (6a), which can reversibly bind 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). Further, in the embodiment shown in Figure 2, the second agent (8) comprises a binding partner C2 (8a), which can reversibly bind to the 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 the binding partner C2 (8a) and the binding site Z2 (44). In this embodiment, the first agent (6) can be a monoclonal anti-CD3 antibody, or an antigen-binding fragment thereof, such as a Fab fragment. The second agent (8) can be a monoclonal anti-CD28 antibody, or an antigen-binding fragment thereof, such as a Fab fragment.The first binding partner (6a) may be a streptavidin-binding peptide (6a) fused or conjugated to an anti-CD3 antibody or an anti-CD3 antibody fragment. The second binding partner (8a) may be a calmodulin-binding peptide conjugated or fused to a CD28 antibody or a CD28-binding antibody fragment. In this context, it is noted that, for example, monoclonal antibodies against CD3 or CD28 are well known (see, for example, 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, such as 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 the first and second agents and can be, for example, chemically linked (conjugated) to the binding partners C1 and C2. Alternatively, it is possible to clone the genes of the variable domains from a hybridoma cell line or to use an antibody whose amino acid sequence is known and recombinantly produce the respective antibody fragments, such as Fab fragments or Fv.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 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). The amino acid sequences of the heavy and light chain variable domains of the antibody OKT3 described in (1996) 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). This methodology of cloning the variable domains of antibody molecules and recombinantly producing the respective antibody fragments is also 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, 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, as in the example of FIG. 2, by well-known evolutionary methods, such as those reported in Ref. 3750-3755. In the example shown in FIG. 2, the multimerization reagent (4) has two different binding sites Z1 (42) and Z2 (44). With 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 a multimeric calmodulin. The multimerization reagent (4) may be a single molecule, e.g. a conjugate of multimeric calmodulin and streptavidin (this alternative is usually 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 muteins 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.It is noted that in this situation, the immobilization of calmodulin on the surface of the solid support does not require the preparation of multimeric calmodulin as explained above, but the immobilization of calmodulin on the surface is sufficient to present calmodulin (which, as mentioned above, has only a single binding site for the calmodulin-binding peptide at a sufficiently high density to ensure the binding of the cell population (2)). For example, in this case, a bivalent antibody fragment with two binding sites for CD28 or an intact antibody, itself with two identical binding sites, can be used as the second reagent (8). As shown in FIG. 2b, after contacting the T cell population (2) with the multimerization reagent (4) and, typically, incubating the cell population with the multimerization reagent (4), the T cell population (2) forms a complex / binds to the multimerization reagent via the first agent (6) and the 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 FIG. 2c, after incubation (usually 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. Similarly, the binding between the binding partner C2 (8a) of the second agent (8) and the binding site Z2 of the multimerization reagent (4) is 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 analogue of the first partner (20)), whereas 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 analogue 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, the addition of the first free partner and second free partner analogs (20) displaces the binding partners C1 (6a) and C2 (8a), respectively, from the multimerization reagent (4), which in turn displaces the first agent (6) and the second agent (8) from the multimerization reagent (4). This displacement of the first agent (6) and the second agent (8) then dissociates the first agent (6) and the second agent (8) from the TCR / CD3 complex and the accessory molecule CD28, thereby terminating the stimulation / expansion of the cell population (2). Thus, as discussed above, the present invention provides the advantage that the duration of stimulation or expansion of a T cell population can be precisely controlled, and therefore the functional state of the T cell population can also be tightly controlled. After elution of the cells as shown in FIG. 1d, the first agent (6), the second reagent (8) and the analogue of the free first partner of the binding partner C1 and the free second partner of the binding partner C2 (20) can be easily removed from the stimulated cell population (2) via the “removal cartridge” described in WO 2013 / 124474. Furthermore, and importantly, if the initial sample was a population of lymphocytes, for example 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 during expansion with, for example, a T cell receptor (TCR) or a chimeric antigen receptor (CAR, also known as an artificial T cell receptor), the genetically modified cells can then be released from the initial stimulation and then stimulated with a second type of stimulation, for example via a de novo introduced receptor. These second stimuli may include antigenic stimuli in the form of peptide / MHC molecules, cognate (cross-linking) 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]FIG. 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) with 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. FIG. 3a shows the population of T cells (2) after contact with a multimerization reagent (4). Also in this example, a first agent (6) of an anti-CD3 antibody or an antigen-binding fragment thereof that provides a primary activation signal to T cells and a second agent (8) of an anti-CD28 antibody or an antigen-binding fragment thereof that stimulates CD28 as an accessory molecule are reversibly immobilized (bound) on the multimerization reagent (4). The multimerization reagent (4) shown in the example of FIG. 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), both of which 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 may 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 may 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), 1199-1203 or US 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 such 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, this 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 muteins of streptavidin or avidin can be prepared in a first step by introducing carboxyl residues into a polysaccharide, e.g., 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 in the dextran backbone via internal lysine residues and / or primary amino groups at the free N-terminus using conventional carbodiimide chemistry. Alternatively, cross-linked oligomers or polymers of streptavidin or avidin or any mutein of streptavidin or avidin can be obtained by cross-linking via a bifunctional linker such as glutardialdehyde or by other methods described in the literature. The use of moieties that bind to the same binding site (42) of the multimerizing agent as binding partners C1 and C2 has the advantage that the same free partner (of the first binding partner C1 and also of the second binding partner C2) or its analogue can be used to terminate the expansion of the T cell population (2) and to release this T cell population (2) from the multimerizing agent, as shown in Figure 3b. In the example of Figure 3, analogues of the first and second partners C1 and C2, such as biotin or biotin derivatives (iminobiotin or desthiobiotin), can be conveniently used for the termination of the expansion and elution of the T cell population (2).As shown in FIG. 3c, after elution of the cells as shown in FIG. 1d, the first agent (6), the second reagent (8) and biotin as an analogue (20) of the free first partner of binding partner C1 and the free second partner of binding partner C2 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 that any solid support such as magnetic beads can be avoided. This means that there is no risk of contamination of the activated T cells with such magnetic beads. This also means that a process compliant with GMP standards can be established much easier compared to known methods such as the use of Dynabeads®, where additional measurements must be performed to ensure that the final expanded T cell population is free of magnetic beads. Furthermore, the use of a soluble multimerizing agent makes it much easier to remove it from the activated cell population (T cells, B cells, or even natural killer cells), since the cells can be simply sedimented by centrifugation and the supernatant containing the soluble multimerizing agent can be discarded. Alternatively, the soluble multimerizing agent may be removed from the expanded cell population in the gel permeation matrix of the removal cartridge of WO 2013 / 124474. Since there is no solid phase (e.g., magnetic beads), the present invention also provides an automated closed system for expanding cells that can be integrated 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]FIG. 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) with 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. FIG. 3a shows the population of T cells (2) after contact with a multimerization reagent (4). Also in this example, a first agent (6) of an anti-CD3 antibody or an antigen-binding fragment thereof that provides a primary activation signal to T cells and a second agent (8) of an anti-CD28 antibody or an antigen-binding fragment thereof that stimulates CD28 as an accessory molecule are reversibly immobilized (bound) on the multimerization reagent (4). The multimerization reagent (4) shown in the example of FIG. 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), both of which 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 may 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 may 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), 1199-1203 or US 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 such 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, this 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 muteins of streptavidin or avidin can be prepared in a first step by introducing carboxyl residues into a polysaccharide, e.g., 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 in the dextran backbone via internal lysine residues and / or primary amino groups at the free N-terminus using conventional carbodiimide chemistry. Alternatively, cross-linked oligomers or polymers of streptavidin or avidin or any mutein of streptavidin or avidin can be obtained by cross-linking via a bifunctional linker such as glutardialdehyde or by other methods described in the literature. The use of moieties that bind to the same binding site (42) of the multimerizing agent as binding partners C1 and C2 has the advantage that the same free partner (of the first binding partner C1 and also of the second binding partner C2) or its analogue can be used to terminate the expansion of the T cell population (2) and to release this T cell population (2) from the multimerizing agent, as shown in Figure 3b. In the example of Figure 3, analogues of the first and second partners C1 and C2, such as biotin or biotin derivatives (iminobiotin or desthiobiotin), can be conveniently used for the termination of the expansion and elution of the T cell population (2).As shown in FIG. 3c, after elution of the cells as shown in FIG. 1d, the first agent (6), the second reagent (8) and biotin as an analogue (20) of the free first partner of binding partner C1 and the free second partner of binding partner C2 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 that any solid support such as magnetic beads can be avoided. This means that there is no risk of contamination of the activated T cells with such magnetic beads. This also means that a process compliant with GMP standards can be established much easier compared to known methods such as the use of Dynabeads®, where additional measurements must be performed to ensure that the final expanded T cell population is free of magnetic beads. Furthermore, the use of a soluble multimerizing agent makes it much easier to remove it from the activated cell population (T cells, B cells, or even natural killer cells), since the cells can be simply sedimented by centrifugation and the supernatant containing the soluble multimerizing agent can be discarded. Alternatively, the soluble multimerizing agent may be removed from the expanded cell population in the gel permeation matrix of the removal cartridge of WO 2013 / 124474. Since there is no solid phase (e.g., magnetic beads), the present invention also provides an automated closed system for expanding cells that can be integrated 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]FIG. 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) with 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. FIG. 3a shows the population of T cells (2) after contact with a multimerization reagent (4). Also in this example, a first agent (6) of an anti-CD3 antibody or an antigen-binding fragment thereof that provides a primary activation signal to T cells and a second agent (8) of an anti-CD28 antibody or an antigen-binding fragment thereof that stimulates CD28 as an accessory molecule are reversibly immobilized (bound) on the multimerization reagent (4). The multimerization reagent (4) shown in the example of FIG. 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), both of which 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 may 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 may 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), 1199-1203 or US 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 such 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, this 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 muteins of streptavidin or avidin can be prepared in a first step by introducing carboxyl residues into a polysaccharide, e.g., 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 in the dextran backbone via internal lysine residues and / or primary amino groups at the free N-terminus using conventional carbodiimide chemistry. Alternatively, cross-linked oligomers or polymers of streptavidin or avidin or any mutein of streptavidin or avidin can be obtained by cross-linking via a bifunctional linker such as glutardialdehyde or by other methods described in the literature. The use of moieties that bind to the same binding site (42) of the multimerizing agent as binding partners C1 and C2 has the advantage that the same free partner (of the first binding partner C1 and also of the second binding partner C2) or its analogue can be used to terminate the expansion of the T cell population (2) and to release this T cell population (2) from the multimerizing agent, as shown in Figure 3b. In the example of Figure 3, analogues of the first and second partners C1 and C2, such as biotin or biotin derivatives (iminobiotin or desthiobiotin), can be conveniently used for the termination of the expansion and elution of the T cell population (2).As shown in FIG. 3c, after elution of the cells as shown in FIG. 1d, the first agent (6), the second reagent (8) and biotin as an analogue (20) of the free first partner of binding partner C1 and the free second partner of binding partner C2 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 that any solid support such as magnetic beads can be avoided. This means that there is no risk of contamination of the activated T cells with such magnetic beads. This also means that a process compliant with GMP standards can be established much easier compared to known methods such as the use of Dynabeads®, where additional measurements must be performed to ensure that the final expanded T cell population is free of magnetic beads. Furthermore, the use of a soluble multimerizing agent makes it much easier to remove it from the activated cell population (T cells, B cells, or even natural killer cells), since the cells can be simply sedimented by centrifugation and the supernatant containing the soluble multimerizing agent can be discarded. Alternatively, the soluble multimerizing agent may be removed from the expanded cell population in the gel permeation matrix of the removal cartridge of WO 2013 / 124474. Since there is no solid phase (e.g., magnetic beads), the present invention also provides an automated closed system for expanding cells that can be integrated 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 were 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 shown on Figure 4B (0 represents cells that have not divided; 5 represents cells that have divided at least 5 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 were 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 shown on Figure 4B (0 represents cells that have not divided; 5 represents cells that have divided at least 5 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 were 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 shown on Figure 4B (0 represents cells that have not divided; 5 represents cells that have divided at least 5 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 grey triangles and light grey circles, respectively) and compared to 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: Jurkat cells labeled with Indo-1-AM were activated by various αCD3 stimuli as described in Figure 4a (OKT3: top graph and αCD3Fab multimer: middle graph), followed by D-biotin-mediated disruption of αCD3Fab multimer signaling (t = 140 s). Injection of PBS (bottom graph) and ionomycin served as negative or positive controls. Data are representative of three different 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 grey triangles and light grey circles, respectively) and compared to 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: Jurkat cells labeled with Indo-1-AM were activated by various αCD3 stimuli as described in Figure 4a (OKT3: top graph and αCD3Fab multimer: middle graph), followed by D-biotin-mediated disruption of αCD3Fab multimer signaling (t = 140 s). Injection of PBS (bottom graph) and ionomycin served as negative or positive controls. Data are representative of three different 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, parent clone of Fab multimers) or cognate PE-labeled Fab multimers and analyzed before (second row from the left) or after (middle row) treatment with D-biotin. Remaining Fab monomers were then detected after a subsequent washing step with fresh PE-labeled Strep-Tactin® (second row from the right). Secondary Fab multimer staining of reversibly stained cells served as a control (right row). 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 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 possible 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 reversible αCD3 / αCD28 Fab fragments reversibly immobilized on soluble oligomeric Strep-tactin® acting 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 on which a combination of αCD3 Fab fragments and αCD28 Fab, both of which have a Strep-tag on the heavy chain as streptavidin binding peptides, was immobilized. ("1x" 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 "1x"). Tresp cells were left unstimulated or stimulated with blank Strep-tactin multimers (without Fab) serving 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 cell culture medium supplemented with 20 U / mL interleukin 2 (IL-2) in 48-well plates. Cells were incubated at 37°C without medium change 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 and released by stimulating reagent after washing. Dissociation and removal of monomeric Fab fragments was analyzed by re-staining with phycoerythrin-labeled Strep-Tactin® (ST-PE) as a fluorescent label and representative histograms are 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 numbers 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 reversible αCD3 / αCD28 Fab fragments reversibly immobilized on soluble oligomeric Strep-tactin® acting 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 on which a combination of αCD3 Fab fragments and αCD28 Fab, both of which have a Strep-tag on the heavy chain as streptavidin binding peptides, was immobilized. ("1x" 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 "1x"). Tresp cells were left unstimulated or stimulated with blank Strep-tactin multimers (without Fab) serving 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 cell culture medium supplemented with 20 U / mL interleukin 2 (IL-2) in 48-well plates. Cells were incubated at 37°C without medium change 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 and released by stimulating reagent after washing. Dissociation and removal of monomeric Fab fragments was analyzed by re-staining with phycoerythrin-labeled Strep-Tactin® (ST-PE) as a fluorescent label and representative histograms are 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 numbers 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 reversible αCD3 / αCD28 Fab fragments reversibly immobilized on soluble oligomeric Strep-tactin® acting 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 on which a combination of αCD3 Fab fragments and αCD28 Fab, both of which have a Strep-tag on the heavy chain as streptavidin binding peptides, was immobilized. ("1x" 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 "1x"). Tresp cells were left unstimulated or stimulated with blank Strep-tactin multimers (without Fab) serving 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 cell culture medium supplemented with 20 U / mL interleukin 2 (IL-2) in 48-well plates. Cells were incubated at 37°C without medium change 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 and released by stimulating reagent after washing. Dissociation and removal of monomeric Fab fragments was analyzed by re-staining with phycoerythrin-labeled Strep-Tactin® (ST-PE) as a fluorescent label and representative histograms are 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 numbers 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 an illustration 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 shows an arrangement of the invention that can be used with the expansion method of the invention. The arrangement (100) comprises 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 expansion and purification of cells as described herein. In the bioreactor (50), an expansion method as described herein is carried out, for example the expansion method shown in FIG. 3 utilizing a soluble multimerization reagent. In this case, after terminating the activation / expansion of the cell population (2) by adding a competitor (20) (the free partner of the 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 embodiment, the first agent (6) is a CD3-binding antibody fragment comprising a streptavidin-binding peptide as binding partner C1, the second agent (8) is a CD28-binding antibody fragment comprising a streptavidin-binding peptide as binding partner C1, and the competitor (20) (free analogue of binding partner C1) is biotin. This reaction mixture is applied to a first removal cartridge (70). This first removal cartridge (70) is a removal cartridge as described in WO 2013 / 124474, comprising a chromatography column with a suitable stationary phase. The stationary phase may function as an affinity chromatography matrix and at the same time as a gel permeation matrix. An affinity reagent is immobilized on this affinity chromatography matrix. The affinity reagent may in this embodiment be, for example, streptavidin, streptavidin mutein, avidin, avidin mutein, or a mixture thereof. Thus, the first agent (6) and the second agent (8) bind to the affinity reagent via their streptavidin-binding peptides. Biotin as a competitor (20) also binds to the affinity reagent.Thus, all three reagents are immobilized on the chromatographic matrix of the first removal cartridge, while the expanded cell population (2) and the 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. On this stationary phase is included a second affinity reagent that can bind to the binding site Z1 (42) of the multimerization reagent (4). This affinity reagent can be, for example, biotin covalently bound to the stationary phase. Such a stationary phase can be, for example, d-biotin Sepharose™ available from Affiland SA (Ans-Liege, Belgium). Thus, the soluble multimerization reagent (4) binds (is retained) to 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. The population of cells (2) is now ready for any further use, for example for diagnostic applications (e.g. further FACS™ sorting) or any cell-based therapeutic application. Of course, it is also possible to change the order of the first "removal cartridge" (70) and the second "removal cartridge" (90) in the arrangement (100), so that the bioreactor (50) is (directly) fluidically connected to the second removal cartridge (90), and the first removal cartridge (70) is subsequently arranged and fluidically connected to the second removal cartridge (90). In this arrangement, first the multimerization reagent (4) is removed from the population of cells (2), followed by the first agent (6), the second (8) and, for example, the competitor (20). Such an arrangement is also encompassed by the present invention and may be part of an apparatus for automated cell expansion and purification as described herein. [Figure 11]FIG. 11 shows a further embodiment of an arrangement of the invention that can be used with the expansion method of the invention. The arrangement (110) comprises 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 is in turn fluidly connected to the second removal cartridge (90). The second removal cartridge (110) is further fluidly connected to the bioreactor (50). The arrangement (110) may 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 and expanded population of cells (2) is obtained as an eluate of the second removal cartridge (90). The removal cartridge (90) is fluidly connected to the bioreactor (50) so that the population of cells (2) may be returned to the bioreactor (50) for continued 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 methods of the present invention. [Figure 12]Figure 12 shows a further embodiment of an arrangement of the invention that can be used with the expansion method of the invention. This arrangement (120) comprises 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). As in the embodiment shown in figure 11, a second removal cartridge (110) is fluidly connected to the bioreactor (50). However, a "selection cartridge" (92) as described in WO 2013 / 124474 is placed between the second removal cartridge (90) and the bioreactor (50). Thus, via this "selection cartridge" (92) a subpopulation of cells (2a) contained in the population of cells (2) can be selected / enriched, as described in WO 2013 / 124474. 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 susceptible to contamination. Furthermore, such cell purification devices can also be designed as continuous flow devices, since the soluble multimerization reagents eliminate the need for solid phase materials such as magnetic beads. [Figure 13]Figure 13 shows the expansion kinetics of the proliferation of purified CD4+ and CD8+ responder T cells (Tresp) stimulated in vitro with either αCD3 / αCD28 Fab fragments or αCD3 / αCD28 / αCD8 reversibly immobilized on two types of soluble oligomeric Strep-tactin® muteins 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 "conventional Streptactin® scaffolds" and indicated in Figure 13 by a triangle symbol with a downward pointing tip), and the second type of this oligomeric streptavidin mutein used as soluble multimerization reagent was an oligomer obtained by reacting the soluble oligomeric streptavidin muteins 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 FIG. 13, expansion was performed without changing the medium. Results for CD4+ responder T cells are shown in FIG. 13A, and results for CD8+ responder T cells are shown in FIG. 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 the proliferation of purified CD4+ and CD8+ responder T cells (Tresp) stimulated in vitro with the reversibly immobilized fragment αCD3 / αCD28 Fab fragment, 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 oligomeric streptavidin muteins (n≧3) obtained in Example 5 (also referred to herein as "conventional Streptactin® scaffold" and indicated in Figure 14 by an upturned triangle symbol), and the second type of this oligomeric streptavidin mutein used as soluble multimerization reagent was the HSA-based soluble multimerizer described above ("large Streptactin® scaffold"). In the experiment of Figure 14, the expansion was performed by changing the medium. The results for CD4+ responder T cells are shown in FIG. 14, and the results for CD8+ responder T cells are shown in FIG. 14B. [Figure 15] Figure 15 shows the combined data of the results obtained in Figures 13 and 14 for the expansion kinetics of the proliferation 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 shows 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, and not for the multimerization reagent with the "large Streptactin® backbone". [Figure 16]Figure 16 shows 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 per time point, Figure 17A shows proliferation in medium supplemented with IL-2 only, and Figure 17B shows proliferation in medium supplemented with IL-2 and IL-15. Figure 17C shows flow cytometric analysis of surface expression of CD62L and CD127 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 per time point, Figure 17A shows proliferation in medium supplemented with IL-2 only, and Figure 17B shows proliferation in medium supplemented with IL-2 and IL-15. Figure 17C shows flow cytometric analysis of surface expression of CD62L and CD127 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) are shown. Both the antigen-specific peptide-MHC molecule complexes and the 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 expansion 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), which represents the HLA-C7 / IE-1 epitope specific for cytomegalovirus (CMV). 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 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 providing 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 providing a primary activation signal to cells reversibly immobilized on soluble oligomeric streptavidin muteins.More specifically, FIG. 18B shows the expansion of Ag-specific cells expanded using a peptide:MHC-I complex specific for the pp65 epitope of CMV (amino acids 341-350 (QYDPVAALF (SEQ ID NO: 08)) restricted by HLA-A2402), and FIG. 18C shows the expansion of Ag-specific cells expanded using another peptide:MHC-I complex specific for the pp65 epitope of CMV (amino acids 265-274 (RPHERNGFTV (SEQ ID NO: 09)) restricted by HLA-B702). FIG. 18D shows the expansion of Ag-specific cells grown with peptide:MHC-I complexes specific for the hexon5 epitope of adenovirus (amino acids 114-124 (CPYSGTAYNSL (SEQ ID NO: 10) restricted by HLA-B702)), and FIG. 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 FIG. 18A above for exemplary FACS data). All peptide:MHC molecules with Strep®-Tag are commercially available from IbaGmbH. In this context, the amino acid sequences of HLA-A*2402, HLA-B*0702 and HLA-C*0702 molecules with "Twin-Strep-tag®" as C-terminus are shown in the attached sequence listing as SEQ ID NO:21, 22 and 23, while the amino acid sequence of β2-microglobulin (which forms the respective MHC I molecule together with the α-chain, i.e. the molecule encoding HLA) is shown in the attached sequence listing as SEQ ID NO:24. Furthermore, FIG. 18F shows an exemplary flow cytometry analysis of the surface expression of C62L and CD127 after 14 days of culture for HLA-B7 / Hexon5114-124 stimulated / expanded cells from FIG. 18D. [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) are shown. Both the antigen-specific peptide-MHC molecule complexes and the 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 expansion 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), which represents the HLA-C7 / IE-1 epitope specific for cytomegalovirus (CMV). 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 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 providing 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 providing a primary activation signal to cells reversibly immobilized on soluble oligomeric streptavidin muteins.More specifically, FIG. 18B shows the expansion of Ag-specific cells expanded using a peptide:MHC-I complex specific for the pp65 epitope of CMV (amino acids 341-350 (QYDPVAALF (SEQ ID NO: 08)) restricted by HLA-A2402), and FIG. 18C shows the expansion of Ag-specific cells expanded using another peptide:MHC-I complex specific for the pp65 epitope of CMV (amino acids 265-274 (RPHERNGFTV (SEQ ID NO: 09)) restricted by HLA-B702). FIG. 18D shows the expansion of Ag-specific cells grown with peptide:MHC-I complexes specific for the hexon5 epitope of adenovirus (amino acids 114-124 (CPYSGTAYNSL (SEQ ID NO: 10) restricted by HLA-B702)), and FIG. 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 FIG. 18A above for exemplary FACS data). All peptide:MHC molecules with Strep®-Tag are commercially available from IbaGmbH. In this context, the amino acid sequences of HLA-A*2402, HLA-B*0702 and HLA-C*0702 molecules with "Twin-Strep-tag®" as C-terminus are shown in the attached sequence listing as SEQ ID NO:21, 22 and 23, while the amino acid sequence of β2-microglobulin (which forms the respective MHC I molecule together with the α-chain, i.e. the molecule encoding HLA) is shown in the attached sequence listing as SEQ ID NO:24. Furthermore, FIG. 18F shows an exemplary flow cytometry analysis of the surface expression of C62L and CD127 after 14 days of culture for HLA-B7 / Hexon5114-124 stimulated / expanded cells from FIG. 18D. [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) are shown. Both the antigen-specific peptide-MHC molecule complexes and the 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 expansion 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), which represents the HLA-C7 / IE-1 epitope specific for cytomegalovirus (CMV). 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 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 providing 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 providing a primary activation signal to cells reversibly immobilized on soluble oligomeric streptavidin muteins.More specifically, FIG. 18B shows the expansion of Ag-specific cells expanded using a peptide:MHC-I complex specific for the pp65 epitope of CMV (amino acids 341-350 (QYDPVAALF (SEQ ID NO: 08)) restricted by HLA-A2402), and FIG. 18C shows the expansion of Ag-specific cells expanded using another peptide:MHC-I complex specific for the pp65 epitope of CMV (amino acids 265-274 (RPHERNGFTV (SEQ ID NO: 09)) restricted by HLA-B702). FIG. 18D shows the expansion of Ag-specific cells grown with peptide:MHC-I complexes specific for the hexon5 epitope of adenovirus (amino acids 114-124 (CPYSGTAYNSL (SEQ ID NO: 10) restricted by HLA-B702)), and FIG. 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 FIG. 18A above for exemplary FACS data). All peptide:MHC molecules with Strep®-Tag are commercially available from IbaGmbH. In this context, the amino acid sequences of HLA-A*2402, HLA-B*0702 and HLA-C*0702 molecules with "Twin-Strep-tag®" as C-terminus are shown in the attached sequence listing as SEQ ID NO:21, 22 and 23, while the amino acid sequence of β2-microglobulin (which forms the respective MHC I molecule together with the α-chain, i.e. the molecule encoding HLA) is shown in the attached sequence listing as SEQ ID NO:24. Furthermore, FIG. 18F shows an exemplary flow cytometry analysis of the surface expression of C62L and CD127 after 14 days of culture for HLA-B7 / Hexon5114-124 stimulated / expanded cells from FIG. 18D. [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 I complex and b) αCD28Fab fragments reversibly immobilized on soluble oligomeric streptavidin muteins as first and second agents. For this purpose, 500.000 CD3+CD62L+CD45RA- responder Tcm cells (Tresp) were Ag-specifically stimulated with 3 μL of a preparation of Streptactin multimerization reagent functionalized with 0.5 μg peptide:MHC class I complex and 0.5 μg αCD28Fab with a streptavidin-binding peptide (the specific peptide represents amino acids 114-124 of the Hexon5 protein of adenovirus restricted by HLA-B0702 (CPYSGTAYNSL, SEQ ID NO: 10), see above). 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 αCD8Fab, and 0.5 μg of αCD28Fab. For comparison, polyclonal stimulation was performed with 3 μL of a streptactin multimerization reagent preparation (1 mg / mL) 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 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. The photographs shown in FIG. 19 show the extent of cluster formation on day 5 for Ag-specific stimulation exemplified for the HLA-B7 / Hexon5 epitope of adenovirus. [Figure 20]Figure 20 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 two types of soluble oligomeric Strep-tactin® acting 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 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 Figure 20A and Figure 20B) or a combination of αCD3 and αCD28 Fab fragments. Furthermore, to test whether it is possible to preferentially stimulate certain T cell subpopulations upon combined stimulation with αCD3 / αCD28 Fab fragments, an additional αCD8 Fab fragment (commercially available from IBA GmbH, Göttingen, Germany) was also immobilized. Figure 20A shows a bar graph representing the degree of proliferation according to the number of cells harvested on day 6, compared to the negative control (unstimulated purified CD8+ responder T cells) and normalized to the positive control (purified CD8+ T responders stimulated with commercially available Dynabeads (beads on which αCD3 and αCD28 monoclonal antibodies are irreversibly immobilized)). Figure 20B shows a flow cytometric 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. No significant differences (ns) were detected when comparing the various stimulation conditions using one-way ANOVA. [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 Fab fragments 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 αCD8 Fab fragments described above. The term "1x" corresponds to 1.5 µg of multimerized streptactin functionalized with only 0.5 µg of αCD3Fab fragments and 1.5 µg of multimerized streptactin functionalized with only 0.5 µg of αCD28Fab, 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. Thus, 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 on which αCD3 and αCD28 monoclonal antibodies are irreversibly immobilized) served as a positive control. Figure 21A shows a bar graph representing the degree of proliferation according to the number of cells harvested on day 5, compared to the negative control and normalized to the positive control. Figure 21B shows the FACS analysis of the 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 αCD19 chimeric antigen receptor (CAR) and stimulated with oligomeric Strep-tactin® of Example 5 as a soluble multimerization reagent. The specificity of CARs typically comes 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 links it to T cell-specific signaling (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) that recognizes the IgG4 spacer in αCD19-CAR, in addition to the extracellular domain (ECD) of CD19 that contains 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 of FIG. 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 of FIG. 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 CD19 extracellular domain (ECD) or 0.5 μg (×1) or 1 μg (×2) of αIgG recognizing IgG4 spacer, both of which are CAR-specific Streptamer® multimers. Jresp were stimulated with Dynabeads (beads on which αCD3 and αCD28 monoclonal antibodies are 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 1.5 mL Eppendorf tubes. Cells were incubated at 37° C. and stimulated for 0 or 20 minutes before being placed on ice and lysed. [Figure 23]Figure 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, the αCD8 Fab fragments commercially available from IBA GmbH, Göttingen, Germany (catalog number 6-8000-203) were immobilized on the soluble oligomers of streptavidin muteins to test whether it is 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 fragments are also reversibly immobilized. More specifically, 500.000 purified CD3+ responder T cells (Tresp) were stimulated with 3 μL of a preparation of oligomeric streptavidin (1 mg / mL) loaded with a combination of 0.5 μg αCD3 and 0.5 μg αCD28Fab. As an alternative approach, 4.5 μL of Streptactin oligomers were loaded with 0.5 μg αCD3, 0.5 μg αCD8Fab, and 0.5 μg αCD28Fab. Unstimulated Tresp cells served as a negative control, and Tresp stimulated with Dynabeads (beads on which αCD3 and αCD28 monoclonal antibodies are irreversibly immobilized) served as a positive control. [Figure 24]FIG. 24 shows an exemplary strategy for generating oligomeric streptavidin muteins that can be used as soluble multimerization reagents of the present invention. FIG. 24A shows that in a first step, streptavidin mutein "m2" (SAm2) containing the amino acid sequence lle44-Gly45-Ala46-Arg47 (SEQ ID NO: 03) at sequence positions 44-47 of wild type streptavidin is used 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. FIG. 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 the proliferation of a population of T cells.

[0019] The term "population of cells" as used herein includes all cells that can be expanded by binding a first agent to a cell surface receptor that provides a primary activation signal to the cells. 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 population of lymphocytes, 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 B cells that have CD40 or CD137 (both cell populations can expand when bound to a first agent that provides an activation signal, e.g., 4-1BB ligand; or only αCD40 or αCD137 antibody molecules (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 are agents that bind IgG, CD19, CD28, or CD14, such as αCD19, αIgG, αCD28, or αCD14 antibody molecules. It is also envisioned that the first or second agents for expanding B cells may include ligands for toll-like receptors or interleukins, 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 lipopolysaccharide, as used herein, can also be used as a first agent and can include binding partner C1. Other illustrative examples of suitable cell populations include T cell populations that are expanded after 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, a first agent stimulates a TCR / CD3 complex-associated signal in the T cell, and a second agent provides a second stimulus by binding to CD28 as an accessory molecule.Agents that can be used to expand T cells can 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 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, a T cell population comprising a chimeric antigen receptor can also be expanded using the methods, reagents, and devices of the 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 invention. Another illustrative example of a suitable cell population includes natural killer cells (NK cells), which can be expanded, for example, with an agent that binds to CD16 or CD56, such as an antibody of αCD16 or αCD56. In an illustrative example, such an αCD16 antibody is the 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, for example, 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, for example, with 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 a method 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, the 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. In general, 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 generate a population of antigen-specific T cells, the 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 through the TCR / CD3 complex. For example, the antigen can be presented to the T cells by an antigen-presenting cell in conjunction with an MHC molecule. An antigen-presenting cell, such as a B cell, macrophage, monocyte, dendritic cell, Langerhans cell, or other cell capable of presenting an antigen to a T cell, may be incubated with the T cells in the presence of an antigen (e.g., a soluble antigen) so that the antigen-presenting cell presents the antigen to the T cells. Alternatively, a cell expressing an antigen of interest may be incubated with the T cells. For example, a tumor cell expressing a tumor-associated antigen may be incubated with the T cells to induce a tumor-specific response. Similarly, a cell infected with a pathogen (e.g., a virus) that presents an antigen of the pathogen may be incubated with the T cells. Following antigen-specific activation of the population of T cells, the cells may be expanded according to the method of the present invention. For example, after antigen specificity is established, T cells can be expanded by culturing with anti-CD3 antibody (used as a first agent) and 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 that is known and can be complexed with the respective MHC I molecule can be used. In this regard, see 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 the expansion of the cells can be used as the first agent. In this regard, see Example 15, where the extracellular domain of CD19 caused the activation of an intracellular signaling cascade in transduced Jurkat cells engineered to express a chimeric CD19-binding antigen receptor (CAR).

[0022] The sample of the cell population may come from any suitable source, typically any sample of body tissue or body fluid such as blood. In the latter case, the sample may be, for example, a population of peripheral blood mononuclear cells (PBMCs) that can be obtained by standard isolation methods such as Ficoll gradients of blood cells. However, the cell population to be expanded may be in purified form and may have been isolated using the reversible cell staining / isolation techniques described in US Pat. No. 7,776,562, US Pat. No. 8,298,782, WO 02 / 054065, or WO 2013 / 011011. Alternatively, the population of cells may be obtained by cell sorting via negative magnetic immunoadhesion as described in US Pat. No. 6,352,694 B1 or EP 0 700 430 B1. When the isolation methods described herein are used in basic research, the sample may 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 view of the above, in one embodiment, the present invention provides an in vitro method for expanding a population of cells, comprising contacting a sample containing a population of cells with a multimerization reagent. A first agent that provides a primary activation signal to cells is reversibly immobilized (bound) on the multimerization reagent, and the multimerization reagent comprises at least one binding site Z1 for reversibly binding 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 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.

[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) on a multimerization agent. The second agent comprises a binding partner C2 that can reversibly bind to the binding site Z2 of the multimerization reagent, and the second agent binds to the multimerization reagent via a reversible bond formed between the binding partner C2 and the binding site Z2. The second agent binds to an accessory molecule on the surface of a cell to stimulate the activated cell. In this embodiment, the first agent can stimulate a TCR / CD3 complex-associated signal in a T cell and can be a binding agent that specifically binds to CD3. In this embodiment, the accessory molecule on a 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, a protein based on ankyrin scaffold, a protein based on a crystalline scaffold, an adnectin, and 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, bispecific antibodies, trispecific antibodies (Iliades, P., et al., FEBS Lett (1997) 409, 437-441), decaspecific antibodies (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 proteinaceous artificial 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., may 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, for example, 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, the so-called glubodies (see, e.g., WO 96 / 23879), proteins based on ankyrin scaffolds (Mosavi, LK, et al., Protein Science (2004) 13, 6, 1435-1448) or crystalline scaffolds (see, 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 arise as strings of multiple domains in several 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 a 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 ligands for proteins, 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, the so-called "minibodies" (Martin et al., EMBO J (1994) 13, 5303-5309), bispecific antibodies (Holliger et al., PNAS USA (1993) 90, 6444-6448), the 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 show 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 FIG. 3), in which case a single multimerization agent may be used.

[0028] In embodiments using a first and, optionally, second agent that binds reversibly, the multimerization reagent may be immobilized on a solid surface. Any solid surface (support) may be used to immobilize the multimerization reagent. Illustrative examples of solid surfaces to which the multimerization reagent may 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 usually covalently bound to the solid support, although non-covalent interactions can be used, for example, to immobilize on a plastic substrate, if desired. As will also be described in more detail below, the multimerization reagent may be, for example, a streptavidin or avidin mutein that reversibly binds to a streptavidin-binding peptide. Such streptavidin muteins can be covalently attached to any surface, for example resins (beads) used for chromatographic purification, and are commercially available from IBA GmbH, Göttingen, for example 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 a first or second agent bearing an oligohistidine tag, such as a penta- or hexa-histidine tag, as the first binding partner C1 or the second binding partner C2, respectively.Another example of a multimerization reagent is calmodulin sepharose available from GE Life Sciences, which can be used with a first or second agent that includes a calmodulin-binding peptide as binding partner C1 or C2, or a glutathione-conjugated sepharose, in which case the binding partner C1 or C2 is glutathione-S-transferase.

[0029] In another embodiment of the method of the invention, the multimerization reagent may be in soluble form. In principle, the same multimerization agent may be used as for the multimerization reagent immobilized on a solid support. The multimerization reagent may be in soluble form, for example, a streptavidin mutein oligomer, a calmodulin oligomer, a compound (oligomer) that provides at least two chelating groups K, which are capable of binding to a transition metal ion and allowing the binding of the 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 the binding sites capable of binding to the respective cell surface receptor molecules, have a binding partner C1 or C2 (which for ease of reference will be referred to hereinafter as "binding partner C"). This binding partner C is capable of binding 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 may be of any desired strength and affinity, as long as it is separable or reversible under the conditions in which the method of the 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 may be D , and the K of the bond formed between the binding site B of the receptor-binding agent 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 comprise at least one (including two, three, or more) second binding partner C, and the affinity reagent may comprise 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 US Pat. No. 7,776,562, US Pat. No. 8,298,782, or WO 2002 / 054065, any combination of binding partner C and affinity agent with one or more corresponding binding sites Z may be selected, which typically aligns with 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, a polypeptide, a protein, a nucleic acid, a lipid, a sugar, an oligosaccharide, or a polysaccharide. Such a binding partner has a higher affinity for the binding site of the multimerization reagent than other substances. Examples of the respective binding partners include, but are not limited to, immunoglobulin molecules, fragments thereof, and proteinaceous binding molecules with antibody-like functions.

[0032] In some embodiments, the 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 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 having 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 a corresponding binding partner, such as an antibody or antibody fragment, that is known to bind to the affinity tag. As some illustrative examples of known affinity tags, the binding partner included in the first or second agent may be an oligohistidine, an immunoglobulin domain, a maltose binding protein, glutathione-S-transferase (GST), chitin binding protein (CBP) or thioredoxin, calmodulin binding peptide (CBP), a FLAG' peptide, an HA tag (sequence: Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala (SEQ ID NO: 11)), a VSV-G tag (sequence: Tyr-Thr-Asp-Ile-Glu-Met-Asn-Arg-Leu-Gly-Lys (SEQ ID NO: 12)), an HSV tag (sequence: Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp (SEQ ID NO: 13)), a glycerol-binding domain (Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp (SEQ ID NO: 14)), a glycerol-binding domain (Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp (SEQ ID NO: 15)), a glycerol-binding domain (Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp (SEQ ID NO: 16)), a glycerol-binding domain (Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp (SEQ ID NO: 17)), a glycerol-binding domain (Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp (SEQ ID NO: 18)), a glycerol-binding domain (Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp (SEQ ID NO: 19), 13)), the T7 epitope (Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly (SEQ ID NO: 14)), maltose binding protein (MBP), the 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), the "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), the 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 an embodiment, the complex formed between one or more binding sites of a multimerization reagent, in this case an antibody or antibody fragment and an antigen, can be competitively dissociated by adding free antigen, i.e., a free peptide (epitope tag) or a 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 with a complementary sequence that is linked to or contained in an affinity reagent.

[0037] In some embodiments, binding between the binding partner C of 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 of the receptor binding reagent may, in such embodiments, 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 called nitrilotriacetic acid, NTA), or 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). For example, EDTA is capable of binding 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+ ), whereas 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 of the first or second agent comprises a calmodulin-binding peptide and the affinity reagent comprises a 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 of the first or second agent comprises a FLAG peptide and the affinity reagent comprises an antibody that binds to the FLAG peptide, e.g., the FLAG peptide that binds to the monoclonal antibody 4E11, as described in U.S. Pat. No. 4,851,341. In one embodiment, the binding partner C of the first or second agent comprises an oligohistidine tag and the affinity reagent comprises an antibody or a transition metal ion that binds to the oligohistidine tag. The separation of all these binding complexes can be achieved by chelation of the metal ion, e.g., chelation of calcium, e.g., by adding EDTA or EGTA (see above). Calmodulin, antibodies such as 4E11, or chelating metal ions or free chelating agents can be multimerized by conventional methods, for example by biotinylation and complexation with streptavidin or avidin or multimers thereof, or by introducing carboxyl residues into a polysaccharide, for example dextran, in a first step essentially as described in Noguchi, A, et al. Bioconjugate Chemistry (1992) 3, 132-137, and then linking calmodulin or antibodies or chelating metal ions or free chelating agents to the carboxyl groups of the polysaccharide, for example dextran backbone via primary amino groups using conventional carbodiimide chemistry. In such an embodiment, 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. Chelation of the metal can be achieved, for example, by addition of EGTA or EDTA.

[0039] In some embodiments, specifically when the multimerization reagent is in soluble form and based on streptavidin or avidin, it is an oligomer or polymer of streptavidin or avidin, or of 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, the oligomer or polymer of streptavidin or avidin, or of a mutein (analog) of streptavidin or avidin, is prepared in a first step by introducing a carboxyl residue into a polysaccharide, for example, dextran, essentially as described in Noguchi, A, et al., Bioconjugate Chemistry (1992) 3,132-137. In a second step, streptavidin or avidin or analogues thereof may then be linked to the carboxyl groups in the dextran backbone via internal lysine residues and / or the primary amino groups of the free N-terminus using conventional carbodiimide chemistry.Furthermore, crosslinked oligomers or polymers of streptavidin or avidin, or of 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 acting as a linker, e.g., glutardialdehyde, 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 a thiol group into the streptavidin mutein (this can be done, for example, by reaction of the streptavidin mutein with 2-iminothiolane (Trauts' reagent) and activating an available amino group 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 react the thiol groups contained in one batch of modified streptavidin muteins with the activated amino acids (by maleimide functional groups) of the other batch of modified streptavidin muteins. This reaction results in the formation of multimers / oligomers of streptavidin muteins. 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 FIG. 24). After reacting the 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, the oligomer does not (and need not) have a single molecular weight, but usually has a statistical weight distribution, such as a Gaussian distribution. Any oligomer containing more than three streptavidin homotetramers (building blocks; (n≧3)) may be used as a soluble multimerization reagent. The oligomer may 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 about 50 kDa, these soluble oligomers have a molecular weight of about 150 kDa to about 1250 kDa. Since each streptavidin molecule / mutein has four biotin binding sites, such a multimerization reagent provides between 12 and 100 binding sites Z1 (and Z2) as described herein.

[0040] In accordance with 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 used in the present invention. In addition to the reaction with polysaccharides described above, it is also possible to use physiologically or pharma- ceutical acceptable proteins such as serum albumin (e.g., human serum albumin (HSA) or bovine serum albumin (BSA)) as carrier proteins. In such cases, streptavidin muteins (as individual homotetramers or even 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 just a few) can be reacted with streptavidin muteins. In this way, a portion of the streptavidin oligomer is non-covalently bound to the biotinylated carrier protein via one or more biotin binding sites (Z1, Z2), leaving the majority of the binding sites (Z1, Z2) of the oligomer available for binding to agents, such as the first agent and optionally the second agent, as well as any further agents described herein. Thus, such an approach allows for convenient preparation of soluble multimerization reagents with multiple binding sites Z1 (see FIG. 24). Alternatively, the streptavidin muteins (as individual homotetramers or even 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 in 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 are distinguished from the sequence of wild-type streptavidin by the substitution, deletion or addition of one or more amino acids and that retain the binding properties of wt-streptavidin. Streptavidin-like polypeptides and streptavidin muteins are polypeptides that are essentially immunologically equivalent to wild-type streptavidin and, in particular, can bind biotin, biotin derivatives or biotin analogs with the same or different affinity as wt-streptavidin. A streptavidin-like polypeptide or streptavidin mutein may contain amino acids that are not part of wild-type streptavidin or may contain only a portion of wild-type streptavidin. A streptavidin-like polypeptide is also a polypeptide that is not identical to wild-type streptavidin because the host does not have the enzymes required to convert the polypeptide it produces 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 usually has a binding site for biotin or a biotin analog, or a streptavidin-binding peptide. Examples of streptavidins 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 US 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 44-53 based on the amino acid sequence of wild-type streptavidin. A minimal streptavidin mutein whose N-terminus starts in the region of amino acids 10-16 of wild-type streptavidin and whose C-terminus ends in the region of amino acids 133-142 of wild-type streptavidin is preferred. Examples of such preferred streptavidin muteins have a hydrophobic aliphatic amino acid instead of Glu at position 44, any amino acid at position 45, a hydrophobic aliphatic amino acid at position 46, or / and a basic amino acid instead of Val at position 47. The streptavidin mutein has the amino acid sequence Va1 at sequence positions 44-47. 44 -Thr 45 -Ala 46 -Arg 47 (SEQ ID NO: 02), or a mutein comprising the amino acid sequence lle at positions 44 to 47 of wild-type streptavidin. 44 -Gly 45 -Ala 46 -Arg 47 (SEQ ID NO: 03). Such muteins are, for example, described 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] The method according to the invention may in some embodiments be used to deplete a sample of reagents already used in cell expansion. For example, the first or second agent and their respective free partners (competitors) may be present in the eluate of the expansion method. Using the method according to the invention, such reagents may be at least essentially (including completely) removed from the sample, for example from the cell population. As an illustrative example, the first or second agent as defined above may be depleted from the sample to a level below the detection limit of, for example, FACS or Western blot. A competing reagent (free first or second binding partner, or an analogue thereof) may be used to terminate and control the expansion and release the cell population from the multimerization agent. This competing reagent may have a binding site capable of specifically binding to the binding site Z of the affinity reagent in the "removal cartridge" of WO 2013 / 124474. In such an embodiment, the respective method according to the invention may also function for the depletion (including removal) of the first and second agents, as well as the competing reagent.

[0044] The methods of the invention may be carried out at any temperature at which the viability of the cell population is at least essentially unimpaired. When referring herein to conditions that are at least essentially unharmful, undetrimental, and at least essentially unimpairing to viability, reference is made to conditions at which the percentage of the population of cells that are expanded with sufficient viability is 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%). In some embodiments, the methods of the invention are carried out at a temperature of about 20° C. or higher. Depending on the cell population to be 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 invention are carried out at a constant temperature value, or at 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 are expanded 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 a population of cells with a multimerization reagent, the multimerization reagent being in a soluble form, and a first agent providing a primary activation signal to the cells being immobilized (bound) on the multimerization reagent. The multimerization reagent comprises at least one binding site Z1 for the first agent to bind, the first agent comprising at least one binding partner C1, the binding partner C1 being capable of binding to the binding site Z1 of the multimerization reagent. 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 and activate the cell. 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 have 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 agent, the second agent comprising a binding partner C2 that can bind to binding site Z2 of the multimerization reagent. The second agent binds to the multimerization reagent via a 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, in addition to the first and second agents, the multimerization reagent and all other reagents, and cell populations may otherwise be used in the same manner as disclosed above for the method 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 a 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 comprise (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 the 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.

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

[0054] According to the above disclosure, the present invention also provides a novel multimerization reagent and a novel composition comprising a multimerization reagent capable of expanding a population of cells. Such a multimerization reagent capable of expanding a population of cells is a multimerization reagent in a soluble form and comprises at least one binding site Z1 for reversibly binding a first agent that provides a primary activation signal to the cells, and the first agent that provides a primary activation signal to the cells is reversibly immobilized (bound) on the multimerization reagent; the first agent comprises 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 a multimerization agent.

[0055] The multimerization reagents of the invention may further comprise at least one binding site Z2 for reversibly binding a second agent that stimulates an accessory molecule on the surface of a cell, the second agent being reversibly immobilized (bound) on the multimerization reagent, the second agent comprising a binding partner C2 that is capable of binding 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 the binding partner C2 and the binding site Z2.

[0056] Also, in light of the above disclosure, such a multimerization reagent can expand a lymphocyte population or a subpopulation contained in said lymphocyte population. The lymphocyte population to be expanded can be any suitable population, for example, a B cell population, a T cell population, or a natural killer cell population. The T cell population can be an antigen-specific T cell population, a helper T cell population, a cytotoxic T cell, 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 can stimulate a TCR / CD3 complex-associated signal in T cells. Thus, the first agent present in the multimerization reagent can be a binding agent that specifically binds to CD3, while the second agent that binds to the accessory molecule can be a binding agent that specifically binds to CD28 or CD137.

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

[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 identical. As mentioned above, such multimerization reagents may include oligomers or polymers of streptavidin, oligomers or polymers of avidin, oligomers or polymers of analogs of streptavidin that bind reversibly to biotin, oligomers or polymers of analogs of avidin that bind reversibly to biotin, reagents that contain at least two chelating groups K that 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 reversible binding of a first agent that provides a primary activation signal to the cell, said first agent being reversibly immobilized (bound) on said first multimerization reagent, said first agent comprising at least one binding partner C1 capable of reversibly binding to said at least one binding site Z1 of said multimerization reagent, said first agent binding to said multimerization reagent via a reversible bond formed between said binding partner C1 and said binding site Z1; (ii) a second multimerization reagent in soluble form and comprising at least one binding site Z2 for reversibly binding a second agent that stimulates an accessory molecule on the surface of the cell, the second agent that stimulates an accessory molecule on the surface of the cell being reversibly immobilized (bound) to the multimerization reagent, the second agent comprising a binding partner C2 capable of binding to the at least one 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; may include:

[0061] Such novel compositions are, for example, the reaction mixtures used in Example 13, where two separate multimerization reagents are 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 immobilized together. 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 for expansion a multimerization reagent of one binding site in which both the first and second agents are immobilized. In this context, it is also noted that the multimerization reagents of the invention are functionalized with many agents (e.g., one, two, three, four, or even more agents) intended to be used to expand 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, where a soluble multimerization reagent was reversibly functionalized with three reagents, namely αCD3Fab fragments as the first reagent, αCD28Fab fragments as the second reagent, and αCD8Fab fragments as the third reagent, in order to enrich a subpopulation of CD8+ T cells in a sample of a population of CD3+ T cells (lymphocytes). By using such a combination of 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 is noted that in this context it is also possible to use three different multimerization reagents for this purpose, for example a first multimerization reagent functionalized only with αCD3Fab fragments, a second multimerization reagent functionalized with αCD28Fab fragments, and a third multimerization reagent functionalized with αCD8Fab fragments. Similarly, it is also possible to use only two different multimerization reagents, a first multimerization reagent functionalized only with αCD3 Fab fragments and a second multimerization reagent functionalized with both αCD28 Fab fragments and αCD8 Fab fragments. Thus, the present invention allows the design of any kind of desired expansion reagent in a modular manner.

[0062] The present invention also provides an in vitro method for serially expanding a population of lymphocytes, the population of lymphocytes comprising 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 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 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.

[0063] In this method, a sample containing a population of lymphocytes, which contains 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 the T cells, including the population of lymphocytes, with the multimerization reagent may be performed 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 from GE Healthcare).

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

[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 an analogue thereof) may be subjected to chromatography on a suitable (first) stationary phase. The (first) stationary phase may be a gel filtration and / or affinity chromatography matrix as described in WO 2013 / 124474. This gel filtration and / or affinity chromatography matrix comprises an affinity reagent, which comprises binding sites Z1 and / or Z2 that specifically bind to the binding partners C1 and / or C2 contained in the first agent or the 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 successive expansion, the binding sites Z1 and Z2 of the multimerizing agent are identical. Moreover, a single multimerizing agent may be used. When a soluble multimerizing agent is used, the T cell population (or in general 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 comprising the T cells and the soluble multimerizing agent is subjected to chromatography on a suitable second stationary phase before or after application to said first stationary phase. This second stationary phase may be a gel filtration matrix and / or an affinity chromatography matrix, which comprises an affinity reagent. The affinity reagent contained in the chromatography resin, if present, comprises a binding partner D that (specifically) binds to the binding site Z1 and / or the 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 a streptavidin-binding peptide as binding partner C1 or 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 reagents then bind to biotin which is usually covalently bound to a chromatographic 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 the T cell, 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, during or after expansion, the T cells may for example be transfected with a T cell receptor (TCR) or a chimeric antigen receptor (CAR, also known as an artificial T cell receptor). This transfection to introduce the gene of the desired receptor may be carried out, for example, with 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 antigenic stimulation in the form of a peptide / MHC molecule, a cognate (crosslinking) ligand of the genetically introduced receptor (e.g., the 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 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 a non-mobilized apheresis product, or a frozen tissue specimen.

[0071] In this method of continuous expansion based on a soluble multimerization reagent, in addition to the first and second agents, the multimerization reagent and all other reagents, and cell populations 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 invention further aims at the arrangement of a bioreactor and a first stationary phase for chromatography. The bioreactor is suitable for the expansion of cells and the stationary phase is suitable for the separation of cells and the removal of reagents. The first stationary phase is a gel filtration and / or affinity chromatography matrix, which comprises an affinity reagent, which comprises a binding site Z1 that specifically binds to a binding partner C1 contained in a first agent and / or which comprises a binding site Z2 that specifically binds to a binding partner C2 contained in a second agent. Thereby, the first stationary phase is suitable for immobilizing thereon the first agent and / or the second agent, the first binding partner C1 and / or the second binding partner C2 in free form. Furthermore, the bioreactor and the stationary phase are fluidically connected. This configuration can be used in the continuous 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 in fluid connection with the first stationary phase. The second stationary phase may be 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 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 stationary phase or a second stationary phase for chromatography.

[0075] The apparatus may further include multiple arrangements of bioreactors and stationary phases in fluid connection in series.

[0076] The device may include a sample inlet fluidly connected to the bioreactor of the bioreactor and chromatographic stationary phase arrangement, and the device 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 developed in the future, that perform substantially the same function or achieve substantially the same results as the corresponding exemplary embodiments described herein, can similarly 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 non-mobilized 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 fragments alone, 0.5 μg αCD28Fab fragments alone, or a mixture of 0.5 μg αCD3Fab fragments and 0.5 μg αCD28Fab.

[0080] The αCD3Fab 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 US Patent No. 4,361,549, which cell line is deposited under the accession number ATCC® CRL-8001™. The CD28Fab 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 the synthetic single-chain Fv construct anti-human CD28 antibody scFv28.3 under the 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 the constant regions (CH1 and Ckappa). The heavy chains of both Fab fragments were fused at the carboxy terminus to a consecutive arrangement of two streptavidin binding modules (SAWSHPQFEK(GGGS)2GGSAWSHPQFEK (SEQ ID NO: 07), commercially available as "Twin-Strep-tag" from IBA GmbH, Gottingen, 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" serves as a multimerization reagent where both Fab fragments are reversibly immobilized.

[0082] In the expansion experiments, Tresp cells stimulated with blank beads (no Fab) served as a negative control. Tresp cells were seeded in triplets with 300.000 CD3 cells 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 change and analyzed by FACS analysis after 4 days. FACS staining and analysis were performed after incubation with 100 μM D-biotin for 10 min. One representative plot for each condition is shown in Figure 4. The plots show viable CD3+ cells stained with propidium iodide (PI) for live / dead discrimination. Figure 4a shows 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®, a specific cell population of Tresp cells was 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 αCD3 Fab fragments and 0.5 μg 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 5 times). FIG. 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 displayed a more uniform proliferation pattern (fewer undivided peak "0" cells) than with a single stimulation alone.Moreover, 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 FIG. 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 a Fab fragment of OKT3 multimerized with Strep-tactin®.

[0084] For this purpose, Jurkat cells were loaded with the calcium-sensitive dye Indo-1-AM and calcium release was triggered by injecting either the αCD3 monoclonal antibody OKT3 (produced by the hybridoma cell line OKT3, see above, black squares) or the αCD3 Fab fragment (derived from the parental cell line OKT3) multimerized by reversible binding of its streptavidin-binding peptide to soluble Strep-Tactin fluorescently conjugated with phycoerythrin. In the case of the intact multimeric OKT3 Fab-Strep-Tactin complex, calcium release was triggered over the same period as the parental antibody clone (dark grey triangles). Cell activation could be completely avoided by injection of Fab-Strep-Tactin complexes treated with D-biotin and previously dissociated (light grey 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 concentration were monitored by flow cytometry based on the change in the FL6 / FL7 ratio. From FIG. 5A, it can be seen that both the parent antibody OKT3 and the multimerized monovalent Fab fragments of OKT3 affect calcium release, i.e., the multimerized monovalent Fab fragments of OKT3 are essentially as functional as the parent antibody. Note that when biotin was added to Strep-tactin on which the OKT3 Fab fragments were immobilized before the addition of the Streptactin-OKT3 Fab fragments, the multimeric OKT3 Fab fragments could not trigger calcium release. In this case, biotin dissociated the reversible bond formed between Strep-tactin as a multimerizing agent and the OKT3 Fab fragments. Thus, the monovalent Fab fragments were displaced from the multimerizing agent and, after dissociation, could not trigger calcium release by binding to CD3 of Jurkat cells.

[0085] In the experiment shown in Figure 5B, Indo-1-AM-labeled Jurkat cells were activated by OKT3-derived αCD3Fab-Strep-Tactin complexes as described in Figure 5A. Injection of intact (top graph) or pre-dissociated complexes (bottom graph) served as positive or 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) led to a rapid collapse of αCD3Fab multimer signaling (middle graph). Injection of ionomycin into the pre-dissociated Fab complexes served as a positive control. Data are representative of three different experiments. Importantly, Figure 5B shows that the addition of D-biotin to the samples rapidly displaced the Fab fragments from the Strep-Tactin multimerizer, thereby effectively terminating calcium release even during ongoing calcium stimulation, and demonstrates that the dissociated OKT3 Fab fragments are no longer biologically active. Similarly, the multimeric OKT3 Fab fragments were unable to trigger calcium release when biotin was added to the Strep-tactin-OKT3 Fab fragment multimers before the addition of the Streptactin-OKT3 Fab samples 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 multimers and analyzed before (second row from the left) or after (middle row) treatment with D-biotin. The remaining Fab monomers were then detected after a subsequent washing step with fresh PE-labeled Strep-Tactin® (second row from the right). Secondary Fab multimer staining of reversibly stained cells served as a control (right row). Only viable CD3 cells, negative for staining with propidium iodide (PI) for live / dead discrimination, are shown in FIG. 6. The numbers in the dot plots indicate the percentage of cells within the gate. This experiment shows 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 shows the isolation of cells by reversible binding of anti-CD28 Fab fragments multimerized with Strep-Tactin® magnetic particles (the 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 by Fab multimer magnetic cell selection from freshly isolated PMBCs essentially as described in WO 2013 / 011011. Before selection, cells were control stained with either cognate fluorescent αCD28 multimers (left dot plot) or an antibody against 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 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 possible remaining Fab monomers. 陰性 ) Only CD3+ cells are shown. Numbers in dot plots indicate percentage of cells within the gate. Figure 7 shows that such multimerized anti-CD28 Fab fragments can be used to isolate CD28+ cells from PMBCs, 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 expanded after in vitro stimulation with αCD3 / αCD28 Fab fragments reversibly immobilized on soluble oligomeric Strep-tactin®, which acts as a soluble multimerization reagent. 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 (Thermo Scientific). 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 preparation of soluble Strep-tactin® oligomers immobilized with a combination of the αCD3 OKT3 Fab fragment and the αCD28 Fab fragment of antibody 28.3 (both of which have the above-mentioned Twin-Strep-tag® as streptavidin-binding peptide in the heavy chain). ("1×" corresponds to 3 μg of multimerized Streptactin functionalized with 0.5 μg of αCD3 monomeric Fab fragment and 0.5 μg of αCD28 monomeric Fab fragment, and the numbers "0.5×", "2×" and "5×" indicate n-fold amounts of "1×", respectively). Tresp cells were left unstimulated or stimulated with blank Strep-tactin multimers (without Fab) serving 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 changing the medium and proliferation was analyzed according to CFSE dilution after 5 days by FACS analysis. Figure 8A shows the increase in 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 association 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.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 grey histograms) are shown compared to the appropriate ST-PE only negative control (light grey histograms). Figure 8C shows that both Fab fragments were completely dissociated and completely removed from the expanded cells. Figure 8D shows the absolute number of viable (trypan blue negative) cells after 5 days. Numbers were counted using a Neubauer counting chamber and plotted for each stimulation condition. Median cell numbers are shown in Figure 8D, error bars indicate standard deviation (SD). Figure 8D shows that mixtures of αCD3 and αCD28 Fab fragments immobilized on soluble Strep-tactin multimerization reagent were all equally effective in expanding CD3+ cells, and absolute cell numbers increased by about 4-fold.

[0090] Example 6: Kinetics of proliferation 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 αCD3 / αCD28 Fab fragments that were reversibly immobilized 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", indicated in FIG. 13 by a triangular symbol with an upturned tip). 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 Strepactin® scaffold").

[0091] In this example, 500.000 purified CD4+ or CD8+ responder T cells (Tresp) were stimulated separately with these two different Streptamers multimers as described above, i.e. either with the Streptactin scaffold of Example 5 (using a solution concentration of 1 mg oligomeric streptavidin mutein per mL) or with the large Streptactin scaffold (0.1 mg / mL). 3 μL of both different scaffolds were loaded with a combination of 0.5 μg αCD3 used in the above example and 0.5 μg αCD28Fab carrying the streptavidin binding peptide SAWSHPQFEK(GGGS)2GGSAWSHPQFEK (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 αCD3 Fab fragment, 0.5 μg 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 αCD28 Fab fragment. Untreated (unstimulated) Tresp cells served as a negative control and Tresp cells stimulated with commercially available Dynabeads (beads on which αCD3 and αCD28 monoclonal antibodies are 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, 0.002% (w / v) penicillin) supplemented with 30 U / mL IL-2 in 48-well plates. Cells were incubated at 37°C without changing the medium and cell numbers were analyzed after 1, 3, and 6 days. In the experiment in Figure 13, expansion was performed without changing the medium.The results for CD4+ responder T cells are shown in FIG. 13A and for CD8+ responder T cells in FIG. 13B, with graphs representing the extent of proliferation according to the number of cells harvested per time point for CD4+Tresp (FIG. 13A) and CD8+Tresp in FIG. 13B.

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

[0093] As is evident from Figure 13B, the soluble multimerization reagent of the present invention allowed CD8+ T cell expansion at least as efficiently as Dynabeads within the first 3 days. It should be noted that during this period, expansion experiments using a soluble multimerization reagent with reversibly immobilized αCD8 Fab fragments in addition to αCD3 and αCD28 Fab fragments (as first and second agents) showed the highest expansion under these culture conditions. This indicates that it is possible to increase or modulate the selectivity of expansion by using a stimulus (herein αCD8 Fab fragments) specific for a particular subpopulation of cells, resulting in a higher amount of the desired cell (sub)population.

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

[0095] Example 7: Kinetics of proliferation of purified CD4+ and CD8+ responder T cells stimulated in vitro with reversible aCD3 / aCD28 Fab-Streptamer multimers with 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 "conventional Streptactin® scaffold", indicated in FIG. 13 by a triangle symbol with a tip pointing down). The 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 Strepactin® backbone."

[0096] In this example, 400.000 purified CD4+ or CD8+ responder T cells (Tresp) were stimulated separately with these two different Streptamers multimers as described above, i.e., either the Streptactin scaffold of Example 5 (1.0 mg / mL) or the large Streptactin scaffold (0.1 mg / mL). 3 μL of both different scaffolds were 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 (on 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., medium was changed on day 3, and cell numbers were analyzed after 1, 3, and 6 days. Results for CD4+ responder T cells are shown in FIG. 14A and for CD8+ responder T cells in FIG. 14B, with graphs representing the extent of proliferation according to the number of cells harvested per time point for CD4+Tresp (FIG. 14A) and CD8+Tresp in FIG. 14B.

[0097] As can be seen in FIG. 14A, the soluble multimerization reagent of the present invention, in which αCD3 and αCD28 Fab fragments are reversibly immobilized, led to 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. However, during this period, the expansion experiment using the large soluble multimerization reagent with αCD3 and αCD28 Fab fragments (as first and second agents) showed the highest expansion under these culture conditions. This could also be caused by the soluble "large oligomer multimerization reagent" being able to simultaneously bind more T cells than the "small" soluble oligomers, and therefore stimulate more CD4+ T cells than the "small" oligomers.

[0099] Example 8: Expansion kinetics of purified CD4+ and CD8+ T cell cultures with or without medium replacement 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. No normalized data was obtained for the "large" multimerization reagents. 400.000-500.000 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 αCD3Fab fragments and 0.5 μg of αCD28Fab fragments) as described in Examples 6 and 7. 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 48-well plates. 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 FIG. 15 ) and cell counts were analyzed after 1, 3, and 6 days. As is evident from the normalized data in FIG. 15A, the “small” soluble multimerization reagent with reversibly immobilized αCD3Fab and αCD28Fab fragments resulted in an expansion of CD4+ T cells by about 2.5-fold, whereas expansion with Dynabeads resulted in an expansion rate of about 1.8-fold. Thus, the use of the soluble multimerization reagent of the invention further improves the expansion of CD4+ T cells over Dynabeads. Similarly, FIG. 15B confirms that the soluble multimerization reagent of the invention could 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 preparation (1 mg / mL) of oligomeric Streptactin multimerization reagent loaded with a combination of 0.5 μg αCD3Fab and 0.5 μg 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 48-well plates. Cells were incubated at 37° C. and analyzed microscopically after 1 and 2 days. Stimulation of CD4+Tresp (FIG. 16A) and CD8+Tresp (FIG. 16B) is shown with Dynabeads (middle row) and Streptamers multimers (bottom row), respectively. The photographs represent the extent of cluster formation: for ease of visualization, exemplary clusters for stimulation with soluble streptavidin mutein oligomers are shown as circles in Figures 16A and 16B. Clusters for Dynabead stimulation are easily visible by the accumulation of darkly colored stimulation particles. As is evident, early cluster formation occurred in both CD4+ and CD8+ T cells when using the expansion method of the present invention using soluble oligomer multimerization reagents.

[0101] Example 10: Expansion kinetics and phenotype of polyclonal 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) of Example 5 loaded with a combination of 0.5 μg αCD3 and 0.5 μg αCD28Fab. In addition, 4.5 μL of a streptactin multimer preparation loaded with 0.5 μg αCD3, 0.5 μg αCD8Fab, and 0.5 μg αCD28Fab were used as an additional stimulation condition. Untreated (unstimulated) Tresp cells served as a negative control, and Tresp cells stimulated with Dynabeads (on 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. The graphs show the extent of proliferation according to the number of cells harvested per time point in medium supplemented with IL-2 alone in FIG. 17A and in medium supplemented with IL-2 and IL-15 in FIG. 17B. As can be seen in both FIG. 17A and FIG. 17B, soluble multimerization reagents with reversibly immobilized CD3Fab and αCD28Fab fragments resulted in better cell expansion than Dynabeads. As further shown by flow cytometric 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 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 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 was investigated.

[0103] More specifically, CD3+CD62L+CD45RA- responder Tcm cells were stimulated in vitro with both peptide:MHC molecule complexes (acting as a first agent providing the primary activation signal to the cells) and αCD28Fab fragments (acting as a second reagent stimulating accessory molecules on the surface of the cells). Both antigen-specific peptide-MHC molecule complexes and 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), amino acids 309-317 of the immediate early 1 protein (described in Ameres et al, PLOS Pathogens, May 2013, vol. 9, issue 5, e1003383) that represents the HLA-C7 / IE-1 epitope specific for cytomegalovirus (CMV). 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 preparation of soluble oligomeric Streptactin multimerization reagent functionalized with 0.5 μg of peptide:MHC class I complexes with streptavidin-binding peptides and 0.5 μg of αCD28Fab described above. Alternatively, 4.5 μL of a preparation of Streptactin multimerization reagent was loaded with 0.5 μg of these peptide:MHC class I complexes, 0.5 μg of CD8αFab and 0.5 μg of αCD28Fab. For comparison, polyclonal stimulation was performed with 3 μL of a preparation of Streptactin multimerization reagent (1 mg / mL) loaded with a combination of 0.5 μg of αCD3Fab and 0.5 μg of αCD28Fab. Additionally, as an alternative to the above stimulation conditions, 4.5 μL of a preparation of Streptactin multimerization reagent 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 on which αCD3 and αCD28 monoclonal antibodies are 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 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. 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 (CMV) HLA-C7 / IE-1 epitope (FIG. 18A) shows that these antigen-specific T cells were specifically expanded.(Similar to the expansion experiment shown in FIG. 18A, the extent of expansion of different Ag specificities is represented according to the number of peptide:MHCI multimer positive cells harvested per time point.) The graphs in FIG. 18B-E 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 FIG. 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 Up to a 98-fold increase (see FIG. 18E) in the number of Ag-specific cells recognizing 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 surface expression of CD62L and CD127 after 14 days of culture with the (adenoviral) HLA-B7 / Hexon5 epitope shown in FIG. 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 in 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) αCD28Fab fragments reversibly immobilized on soluble oligomeric streptavidin muteins as first and second agents.

[0106] For this purpose, 500.000 CD3+CD62L+CD45RA- responder Tcm cells (Tresp) were Ag-specifically stimulated with 3 μL of a preparation of Streptactin multimerization reagent functionalized with 0.5 μg of peptide:MHC class I complex with a streptavidin-binding peptide (the specific peptide represents amino acids 114-124 of the Hexon5 protein of the adenovirus restricted by HLA-B07 (CPYSGTAYNSL, SEQ ID NO: 10)) and 0.5 μg of αCD28Fab. Alternatively, 4.5 μL of a preparation of Streptactin multimerization reagent 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 with 3 μL of a Streptactin multimerization reagent preparation (1 mg / mL) loaded with a combination of 0.5 μg αCD3Fab and 0.5 μg αCD28Fab. Additionally, as an alternative to the above stimulation conditions, 4.5 μL of a Streptactin multimer preparation loaded with 0.5 μg αCD3Fab, 0.5 μg αCD8Fab, and 0.5 μg 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 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. The photographs shown in Figure 19 depict the extent of cluster formation on day 5, showing an exemplary Ag-specific stimulation for the adenovirus 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 that 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] For this purpose, 300.000 purified CD8+ responder T cells (Tresp) were stimulated separately with two different Streptactin-based soluble multimerization reagents, namely the small oligomeric Streptactin multimerization reagent of Example 5 (1 mg / mL) or the large Streptactin oligomer (0.1 mg / mL). 3 μL of both different multimerization reagents (backbone) were loaded with a combination of 0.5 μg of the αCD3 and 0.5 μg of the αCD28 Fab fragment. Furthermore, 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. Furthermore, only 3 μL of the "small" Streptactin multimerization reagent (backbone) functionalized with only 0.5 μg of αCD3 Fab fragment or only 0.5 μg of αCD28 Fab fragment was used. Unstimulated Tresp cells served as a negative control and Tresp 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 48-well plates. Cells were incubated at 37° C. for 3 days, medium was changed, and analyzed after 6 days. FIG. 20A represents the extent of proliferation according to cell number harvested on day 6, compared to the negative control and normalized to the positive control. FIG. 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 surface expression of CD8 (FIG. 20B) and CD45RO (FIG. 20C) after cell culture shows that the same phenotype of CD8+ T cells was expanded by either the multimerization reagent of the invention or Dynabeads (no significant differences (ns) were detected when comparing the various stimulation conditions using one-way ANOVA). The improved yield of CD8+ cells using the expansion method of the 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, the amplification yield obtained using a multimerization agent in which both 0.5 μg of αCD3Fab fragments and 0.5 μg of αCD28Fab fragments are immobilized together (second column from the left in FIG. 20B) is compared with the yield using two multimerization reagents functionalized with only αCD3Fab fragments or only αCD28Fab fragments (third column from the left in FIG. 20B), showing that both experiments had the same amplification efficiency. Thus, these experiments show that the use of one multimerization reagent in which both the first and second agents are immobilized together is functionally equivalent for amplification 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 with immobilized αCD3Fab and αCD28Fab fragments at different ratios 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 (1 mg / mL) of a "small" oligomeric Streptactin multimerization reagent preparation functionalized only with αCD3Fab and only with αCD28Fab ("1x" corresponds to 1.5 μg of Streptactin multimerization reagent functionalized only with 0.5 μg of αCD3 and 1.5 μg of multimerized Streptactin functionalized only with 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. FIG. 21A represents the extent of proliferation according to cell numbers harvested on day 5 compared to the negative control and normalized to the positive control. FIG. 21A shows that expansion of CD8+ T cells using various soluble multimerization reagents of the invention results in higher yields of CD8+ T cells than expansion using dynabeads (especially the cumulative total reagent volume of 5× conditions results in optimal expansion of cells over time / initiating cell division, resulting in increased total cells). FACS analysis of surface expression of CD8 (Figure 21B) and CD45RO (Figure 21C) 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 by 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 preparation (1 mg / mL) of the Streptactin multimerization reagent 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 preparation of Streptactin multimerization reagent functionalized with 0.5 μg (x1) or 1 μg (x2) of the extracellular domain (ECD) of CD19 (the natural ligand of the αCD19-CAR, providing a "CAR-specific soluble Streptactin-based multimerization reagent"), which recognizes the IgG4 spacer within the αCD19-CAR. Stimulation was performed with 3 μL of a preparation of Streptactin multimerization reagent (also providing a "CAR-specific streptavidin mutein-based multimerization reagent") loaded with 0.5 μg (×1) or 1 μg (×2). The ECD of CD19 with a hexahistidine tag was obtained from Sino Biological / Life technologies (SEQ ID NO: 27) and functionalized to bind to the streptavidin-based multimerization reagent by mixing the ECD of CD19 with the adapter molecule His-STREPPER (IBA GmbH, Germany, order number 2-0920-005) in 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 an ECD of CD19 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 with 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 min before being placed on ice and lysed. Detection of phosphorylated ERK indicates active MAPK signaling, and staining of housekeeper β-actin indicates that equal amounts of total protein were 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 can be seen from the comparison of Figure 22A, which shows activation of Jurkat cells via a "polyclonal Streptactin multimerization reagent," and Figure 22B, which shows activation of Jurkat cells 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) widens the scope of application for reversible cell stimulation / expansion in these in vitro cell processing situations.

[0114] Thus, this experiment shows 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 onto 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 carried out to investigate the effect of αCD3 / αCD28 Fab fragments stimulated in vitro with αCD3 / αCD28 Fab fragments reversibly immobilized on the soluble oligomeric Strep-tactin® of Example 5, which served as a soluble multimerization reagent. +Expansion of responder T cells. 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 oligomeric streptavidin preparation (1 mg / mL) loaded with a combination of 0.5 μg αCD3 and 0.5 μg αCD28Fab. As an alternative approach, 4.5 μL of Streptactin oligomer was loaded with 0.5 μg αCD3, 0.5 μg strep-tagged αCD8Fab, and 0.5 μg strep-tagged αCD28Fab. Unstimulated Tresp cells served as a negative control, and Tresp stimulated with Dynabeads (beads on which αCD3 and αCD28 monoclonal antibodies are irreversibly immobilized) served as a positive control. As can be seen in Figure 23A, the multimerization reagent reversibly loaded with αCD3Fab fragments, αCD28Fab fragments, and also αCD8Fab fragments provided the highest number of expanded CD3+ T cells. The expanded cell count is 1 × 1 × 10 6and the yield was about 30% higher than the expansion of T cells using commercially available Dynabeads. Moreover, and more importantly, as shown in FIG. 23B, the amount of CD8+ T cells was highest using this multimerization reagent with αCD3Fab fragments, αCD28Fab fragments, and αCD8Fab fragments compared to both expansion using Dynabeads or the soluble multimerization reagent of the invention with only αCD3Fab fragments and αCD28Fab fragments as the first and second agents described herein. Thus, this experiment also shows the advantage of the present invention that in addition to the first agent providing a primary activation signal to the desired cell population and, optionally, the second agent providing a costimulatory signal, a further agent specific for the 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, to preferentially expand or selectively enrich 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, 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 is examined.

[0117] 500.000 CD3+CD62L+CD45RA- responder Tcm cells (Tresp) are stimulated simultaneously for multiple Ag specificities with 3 μL of Streptactin multimers functionalized with 0.5 μg of each peptide:MHC class I complex with streptavidin binding peptide and 0.5 μg of αCD28Fab also with streptavidin binding peptide for each specificity. As an alternative approach, 4.5 μL of Streptactin-based multimerization reagent functionalized with 0.5 μg of peptide:MHC class I complex with streptavidin binding peptide, 0.5 μg of αCD8Fab, and 0.5 μg of αCD28Fab, as described herein, for each specificity, are used. For comparison, polyclonal stimulation is performed with 3 μL of a Streptactin-based multimerization reagent preparation (1 mg / mL) reversibly loaded with a combination of 0.5 μg αCD3Fab and 0.5 μg 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 αCD3Fab, 0.5 μg αCD8Fab, and 0.5 μg αCD28Fab (each with a streptavidin-binding peptide) may be used. Untreated (unstimulated) Tresp cells serve as a negative control, and Tresp cells polyclonally stimulated with Dynabeads (beads coated with αCD3mAb and αCD28mAb) serve as a positive control. Tresp cells are seeded in 48-well plates in 1 mL of cell culture medium supplemented with 30 U / mL IL-2 and 5 ng / mL IL-15. Cells 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 αCD3 and 0.5 μg αCD28Fab, or with 4.5 μL of a Streptactin-based multimerization reagent preparation loaded with 0.5 μg αCD3, 0.5 μg αCD8Fab and 0.5 μg αCD28Fab, or with 3 μL of a mixture of a Streptactin-based multimerization reagent preparation with only 0.5 μg αCD3Fab and only 0.5 μg αCD28Fab (each Fab fragment also carrying a streptavidin binding peptide). Untreated Tresp cells serve as a negative control and Tresp stimulated with Dynabeads (beads coated with αCD3mAb and αCD28mAb) serve as a positive control. Tresp cells are seeded in duplicate in 48-well plates in 1 mL of cell culture medium supplemented with 30 U / mL IL-2. Cells are incubated at 37° C. for 3 days before medium change and analyzed after 6 days.

[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 Streptactin-based multimerization reagent functionalized with only 0.5 μg αCD3Fab fragments and 1.5 μg Streptactin-based multimerization reagent functionalized with only 0.5 μg αCD28Fab fragments) or various amounts of a mixture of Streptactin-based multimerization reagent preparations functionalized with αCD3Fab fragments and αCD28Fab fragments with or without αCD8Fab fragments (each Fab fragment also has a streptavidin binding peptide) (Streptactin-based multimerization reagent functionalized with 0.5 μg αCD3Fab fragments and 0.5 μg αCD28Fab fragments without αCD8Fab fragments). The cells are stimulated with 4.5 μL of a preparation of Streptactin multimerization reagent loaded with 3 μg of αCD3 Fab fragments, or 0.5 μg of αCD8 Fab fragments, 0.5 μg of αCD28 Fab fragments (the Fab fragments also carry streptavidin, a binding peptide). Untreated Tresp cells serve as a negative control, and Tresp stimulated with Dynabeads (beads coated with αCD3 mAb and αCD28 mAb) serve as a positive control. Tresp cells are seeded in 1 mL of cell culture medium supplemented with 30 U / mL IL-2 in 48-well plates. Cells are incubated at 37° C. for 3 days before changing the medium and analyzing after 6 days.

[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 commonly known general knowledge.

[0121] The invention illustratively described herein may be suitably practiced in the absence of any element or limitation not specifically disclosed herein. Thus, for example, the terms "comprise", "include", "contain", etc. shall be read expansively and without limitation. Furthermore, the terms and expressions used herein are used as descriptive terms and not limiting, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, although the present invention has been specifically disclosed by exemplary embodiments and optional features, it should be understood that modifications and variations of the invention embodied herein disclosed herein may be reclassified by those skilled in the art, and such modifications and variations are considered to be within the scope of the present 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 of skill in the art will recognize that the invention can also be described as any individual member or subgroup of members of the Markush group.

Claims

1. 1. An in vitro method of expanding a population of cells, comprising contacting a sample containing the population of cells with a multimerization reagent; a first agent that provides a primary activation signal to the cell is reversibly immobilized (bound) on the multimerization reagent; the multimerization reagent comprises at least one binding site Z1 for reversibly binding the first agent; the first agent comprises at least one binding partner C1, the binding partner C1 being capable of reversibly binding to the 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; and The method wherein 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.

2. a second agent that stimulates accessory molecules on the surface of the cell is reversibly immobilized (bound) on the multimerizing agent; the second agent comprises a binding partner C2, the binding partner C2 being capable of reversibly binding to a binding site Z2 of the multimerization reagent, and the second agent binds to the multimerization reagent via a reversible bond formed between the binding partner C2 and the binding site Z2; The method of claim 1 , wherein the second agent binds to an accessory molecule on the surface of the cell, thereby stimulating the cell to be activated.

3. The method of claim 1 or 2, wherein the population of cells is a lymphocyte population.

4. The method of claim 3 , wherein the lymphocyte population is a B cell population, a T cell population, a natural killer cell population, or a mixture thereof.

5. 5. The method of claim 4, wherein the T cell population is an antigen-specific T cell population, a helper T cell population, a cytotoxic T cell, a memory T cell, a regulatory T cell, or a natural killer T cell population.

6. The method of claim 5 , wherein the first agent comprises an MHC I:peptide complex or the first agent stimulates a TCR / CD3 complex-associated signal in the T cell.

7. The method of claim 6 , wherein the first agent is a binding reagent that specifically binds to CD3.

8. The method of claim 6 or 7, wherein the accessory molecule on the T cell is CD28 or CD137.

9. The method of claim 8, wherein the second agent that binds to the accessory molecule is a binding reagent that specifically binds to CD28 or CD137, or the second agent is a mixture of two different types of binding reagents, both of which specifically bind to CD28 or CD137.

10. the first agent that specifically binds to CD3 is 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; and / or The method of any of claims 7 to 9, wherein the second agent that specifically binds to CD28 is 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, 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.

11. The bivalent antibody fragment is (Fab) ２ 11. The method of claim 10, wherein the Fv fragment is a single chain Fv fragment or a bivalent single chain Fv fragment.

12. The method of claim 10 , wherein the monovalent antibody fragment is selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv).

13. The method of claim 10, wherein the proteinaceous CD3, CD28, or CD137 binding molecule with antibody-like binding properties is selected from the group consisting of aptamers, muteins based on polypeptides of the lipocalin family, glubodies, proteins based on ankyrin scaffolds, proteins based on crystalline scaffolds, adnectins, and avimers.

14. The method of claim 4, wherein the cell population is a B cell population and the first agent is a binding agent that specifically binds to CD40 or CD137.

15. The method of claim 14, wherein the first binding reagent that specifically binds to CD40 or CD137 is selected from the group consisting of an anti-CD40 antibody, a bivalent antibody fragment of an anti-CD40 antibody, a monovalent antibody fragment of an anti-CD40 antibody, and a proteinaceous CD40 binding molecule with antibody-like binding properties, 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).

16. The method of claim 15, wherein the bivalent antibody fragment is a (Fab)2' fragment or a bivalent single chain Fv fragment.

17. 16. The method of claim 15, wherein the monovalent antibody fragment is selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv).

18. The method of claim 15, wherein the proteinaceous CD40-binding molecule or proteinaceous CD137-binding molecule with antibody-like binding properties is selected from the group consisting of aptamers, muteins based on polypeptides of the lipocalin family, glubodies, proteins based on ankyrin scaffolds, proteins based on crystalline scaffolds, adnectins, and avimers.

19. The method of any one of claims 3 and 14 to 18, wherein the accessory molecule on the B cell is CD40.

20. 20. The method of claim 19, wherein the second agent is CD40 ligand (CD154) or CD137.

21. The method according to any one of claims 2 to 20, wherein the binding sites Z1 and Z2 of the multimerization reagent are identical.

22. The method of claim 21 , wherein a single multimerization reagent is used.

23. The method according to any one of claims 1 to 22, wherein the multimerization reagent is immobilized on a solid surface.

24. 24. The method of claim 23, wherein the solid surface is selected from the group consisting of magnetic beads, polymer beads, cell culture plates, microtiter plates, membranes, hollow fibers, and combinations thereof.

25. 25. The method of claim 24, wherein the multimerization reagent is selected from the group consisting of streptavidin, avidin, analogs of streptavidin that bind reversibly to biotin, analogs of avidin that bind reversibly to biotin, and reagents comprising at least two chelating groups K, said at least two chelating groups capable of binding to a transition metal ion, thereby enabling moiety A to bind to an oligohistidine affinity tag, a polymeric glutathione-S-transferase, a polymeric calmodulin, and a biotinylated carrier protein.

26. The method of any one of claims 1 to 22, wherein the multimerization reagent is in a soluble form.

27. 27. The method of claim 26, wherein the multimerization reagent comprises an oligomer or polymer of streptavidin, an oligomer or polymer of avidin, an oligomer or polymer of a streptavidin analogue that binds reversibly to biotin, an oligomer or polymer of an avidin analogue that binds reversibly to biotin, a reagent comprising at least two chelating groups K, said at least two chelating groups capable of binding to a transition metal ion, thereby enabling the reagent to bind to an oligohistidine affinity tag, a polymeric glutathione-S-transferase, a polymeric calmodulin, and a biotinylated carrier protein.

28. 28. The method of claim 27, wherein the streptavidin, avidin, or streptavidin analog or avidin analog that reversibly binds to biotin is cross-linked by a polysaccharide or the cross-linked oligomer or polymer of streptavidin, avidin, or of streptavidin or avidin analog that reversibly binds to biotin is obtained by cross-linking via a bifunctional linker.

29. 29. The method of claim 27 or 28, wherein the cross-linked oligomer or polymer of streptavidin, avidin, or of an analogue of streptavidin or avidin that reversibly binds to biotin, comprises three or more streptavidin monomers.

30. (a) the binding partners C1 and C2 each independently comprise biotin, and the multimerization reagent comprises a streptavidin analog or an avidin analog that reversibly binds to biotin; or (b) the binding partners C1 and C2, independently of one another, comprise a biotin analogue that reversibly binds to streptavidin or avidin, and the multimerization reagent comprises streptavidin, avidin, or a streptavidin analogue or an avidin analogue that reversibly binds to the biotin analogue; or 30. The method of any one of claims 1 to 29, wherein (c) the binding partners C1 and C2 each independently comprise a streptavidin or avidin-binding peptide, and the multimerization reagent comprises streptavidin, avidin, or a streptavidin analogue or an avidin analogue that reversibly binds to the streptavidin or avidin-binding peptide.

31. The binding partners C1 and C2 each independently comprise the streptavidin-binding peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys, and the multimerization reagent comprises the amino acid sequence Va1 at sequence positions 44-47 of wild-type streptavidin. 44 -Thr 45 -Ala 46 -Arg 47 or a streptavidin analogue comprising the amino acid sequence lle at sequence positions 44-47 of wild-type streptavidin. 44 -Gly 45 -Ala 46 -Arg 47 31. The method of claim 30, comprising a streptavidin analog comprising:

32. 32. The method according to any one of claims 1 to 31, wherein the binding between the binding partner C1 and the binding site Z1 of the multimerization reagent and the binding between the binding partner C2 and the binding site Z2 of the multimerization reagent occurs in the presence of a divalent cation.

33. the binding partner C1 and the binding partner C2, independently of each other, comprise a calmodulin-binding peptide, and the multimerization reagent comprises calmodulin; or said binding partner C comprises a FLAG peptide and said multimerisation reagent comprises an antibody that binds to said FLAG peptide; or 33. The method of claim 32, wherein the first linkage C comprises an oligohistidine tag and the multimerization reagent comprises an antibody that binds to the oligohistidine tag.

13. The method according to claim 11 or 12, wherein the bond between the binding partner C and the binding site Z of the multimerization reagent is separated by chelation of a metal ion.

34. 34. The method of claim 33, wherein chelation of the metal is achieved by the addition of EDTA or EGTA.

35. The method according to any one of claims 2 to 30, wherein said binding partners C1 and C2 are different.

36. The method according to any one of claims 2 to 30, wherein said binding partners C1 and C2 are identical.

37. 37. The method of any one of claims 1 to 36, further comprising separating the binding between the binding partner C1 of the first agent and / or the binding partner C2 of the second agent and the binding site Z1 and / or Z2 of the multimerization reagent.

38. The method of claim 37, wherein stimulation of the cell is terminated by dissociating the binding between the binding partner C1 of the first agent and / or the binding partner C2 of the second agent and the binding site Z1 and / or Z2 of the multimerization reagent.

39. The dissociation constant (K ｄ ) but 10 －２ M to 10 －１３ The method of any one of claims 1 to 38, wherein M is in the range of

40. 40. The method according to any of claims 1 to 39, wherein the reversible binding between the binding partner C1 of the first agent and / or the binding partner C2 of the second agent and the binding site Z1 and / or binding site Z2 of the multimerization reagent is separated by contacting the population of cells with a free first binding partner C1 or an analogue of the first binding partner C1 capable of separating the binding between the first binding partner C1 and the binding site Z1, and / or a free partner of the second binding partner C2 or an analogue of the second binding partner C2 capable of separating the binding between the second binding partner C2 and the binding site Z2.

41. 41. The method of claim 40, wherein the first free partner and / or the second free partner is a compound that is not harmful to the cell.

42. (a) the binding partners C1 and C2 each independently comprise biotin, and the multimerization reagent comprises a streptavidin analogue or an avidin analogue that reversibly binds to biotin; (b) the binding partners C1 and C2, independently of each other, comprise a biotin analogue that reversibly binds to streptavidin or avidin, and the multimerization reagent comprises streptavidin, avidin, or a streptavidin analogue or an avidin analogue that reversibly binds to the biotin analogue; or (c) the binding partners C1 and C2 each independently comprise a streptavidin or avidin-binding peptide, and the multimerization reagent comprises streptavidin, or avidin, or a streptavidin analog, or an avidin analog that reversibly binds to the streptavidin or avidin-binding peptide; The method of claims 25 to 41, wherein the reversible bond is disrupted by contacting the cells with biotin or a biotin analogue.

43. 40. The method of claims 25 to 39, wherein when the binding between the binding partner C1 and the binding site Z1 of the multimerization reagent and the binding between the binding partner C2 and the binding site Z2 of the multimerization reagent occurs in the presence of a divalent cation, the reversible binding is disrupted by contacting the cells with a metal chelator.

44. 1. An in vitro method of expanding a population of cells, comprising contacting a sample containing the 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) on 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, the binding partner C1 being capable of binding to the binding site Z1 of the multimerization reagent, the first agent being bound to the multimerization reagent via a bond formed between the binding partner C1 and the binding site Z1; and The method wherein 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.

45. a second agent that stimulates an accessory molecule on the surface of the cell is immobilized (bound) on the multimerization agent; the second agent comprises a binding partner C2, the binding partner C2 being capable of binding to a 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; 45. The method of claim 44, wherein the second agent binds to an accessory molecule on the surface of the cell, thereby stimulating the cell to be activated.

46. 46. ​​The method of claim 44 or 45, wherein the bond formed between the binding partner C1 and the binding site Z1 is irreversible and / or the bond formed between the binding partner C2 and the binding site Z2 is irreversible.

47. 46. ​​The method of claim 44 or 45, wherein the bond formed between the binding partner C1 and the binding site Z1 is reversible and / or the bond formed between the binding partner C2 and the binding site Z2 is reversible.

48. The dissociation constant (K ｄ ) but 10 －２ M to 10 －１３ 48. The method of claim 47, wherein the range of M is

49. The method of any one of claims 44 to 48, wherein the population of cells is a lymphocyte population.

50. 50. The method of claim 49, wherein the lymphocyte population is a B cell population, a T cell population, or a natural killer cell population.

51. 51. The method of claim 50, wherein the T cell population is an antigen-specific T cell population, a helper T cell population, cytotoxic T cells, memory T cells, regulatory T cells, or natural killer T cells.

52. 52. The method of claim 51, wherein the first agent comprises an MHC I:peptide complex or the first agent stimulates a TCR / CD3 complex-associated signal in the T cell.

53. 53. The method of claim 52, wherein the first agent is a binding reagent that specifically binds to CD3.

54. 54. The method of claim 52 or 53, wherein the accessory molecule on the T cell is CD28.

55. 55. The method of claim 54, wherein the second agent that binds to the accessory molecule is a binding reagent that specifically binds to CD28.

56. said first binding reagent that specifically binds to CD3 is 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; and / or The method of any one of claims 53 to 55, wherein the second agent that specifically binds to CD28 is 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.

57. The bivalent antibody fragment is (Fab) ２ 57. The method of claim 56, wherein the Fv fragment is a single chain Fv fragment or a bivalent single chain Fv fragment.

58. 57. The method of claim 56, wherein the monovalent antibody fragment is selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv).

59. 59. The method of claim 58, wherein the proteinaceous CD3 or CD28 binding molecule with antibody-like binding properties is selected from the group consisting of aptamers, muteins based on polypeptides of the lipocalin family, glubodies, proteins based on ankyrin scaffolds, proteins based on crystalline scaffolds, adnectins, and avimers.

60. 51. The method of claim 50, wherein the first agent is a binding reagent that specifically binds to CD40 or CD137.

61. The method of claim 60, wherein the first binding reagent that specifically binds to CD40 or CD137 is selected from the group consisting of an anti-CD40 antibody, a bivalent antibody fragment of an anti-CD40 antibody, a monovalent antibody fragment of an anti-CD40 antibody, and a proteinaceous CD40 binding molecule with antibody-like binding properties, or an anti-CD137 antibody, a bivalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment of an anti-CD137 antibody, and a proteinaceous CD137 binding molecule with antibody-like binding properties.

62. 62. The method of claim 61, wherein the bivalent antibody fragment is a (Fab)2' fragment or a bivalent single chain Fv fragment.

63. 62. The method of claim 61 , wherein the monovalent antibody fragment is selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv).

64. The method of claim 61, wherein the proteinaceous CD40 binding molecule or CD137 binding molecule with antibody-like binding properties is selected from the group consisting of aptamers, muteins based on polypeptides of the lipocalin family, glubodies, proteins based on ankyrin scaffolds, proteins based on crystalline scaffolds, adnectins, and avimers.

65. The method of any one of claims 50 and 60 to 64, wherein the accessory molecule on the B cell is CD40.

66. 66. The method of claim 65, wherein the second agent is CD40 ligand (CD154) or CD137.

67. 1. 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 a 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:

68. 1. 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:

69. The reagent kit of claim 68, further comprising: (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, the second agent binding to the multimerization reagent via a bond formed between the binding partner C2 and the binding site Z2.

70. The kit of any one of claims 67 to 69, wherein the population of cells is a lymphocyte population.

71. 71. The kit of claim 70, wherein the lymphocyte population is a B cell population, a T cell population, or a natural killer cell population.

72. 72. The kit of claim 71, wherein the T cell population is an antigen-specific T cell population, a helper T cell population, cytotoxic T cells, memory T cells, regulatory T cells, or natural killer T cells.

73. The kit of claims 67-72, wherein the first agent comprises an MHC I:peptide complex or the first agent stimulates a TCR / CD3 complex-associated signal in the T cell.

74. 74. The kit of claim 73, wherein the first agent is a binding reagent that specifically binds to CD3.

75. The kit of any one of claims 72 to 74, wherein the accessory molecule on the T cell is CD28.

76. 76. The kit of claim 75, wherein the second agent that binds to the accessory molecule is a binding reagent that specifically binds to CD28.

77. said first binding reagent that specifically binds to CD3 is 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; and / or The kit of any of claims 74 to 76, wherein the second agent that specifically binds to CD28 is 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.

78. 68. The kit of claim 67, wherein the multimerizing agent is immobilized on a solid surface.

79. 79. The kit of claim 78, wherein the solid surface is selected from the group consisting of magnetic beads, polymeric beads, cell culture plates, microtiter plates, membranes, hollow fibers, and combinations thereof.

80. 80. The kit of claim 79, wherein the multimerization reagent is selected from the group consisting of streptavidin, avidin, analogs of streptavidin that bind reversibly to biotin, analogs of avidin that bind reversibly to biotin, and reagents comprising at least two chelating groups K, said at least two chelating groups capable of binding to a transition metal ion, thereby enabling moiety A to bind to an oligohistidine affinity tag, a polymeric glutathione-S-transferase, a polymeric calmodulin, and a biotinylated carrier protein.

81. 68. The kit of claim 67, wherein the multimerization reagent is in a soluble form.

82. 82. The kit of any of claims 68 to 78 or 81, wherein the multimerization reagent is selected from the group consisting of streptavidin, avidin, an analogue of streptavidin that reversibly binds to biotin, an analogue of avidin that reversibly binds to biotin, a reagent comprising at least two chelating groups K, said at least two chelating groups being capable of binding to a transition metal ion, thereby enabling the reagent to bind to an oligohistidine affinity tag, a polymeric glutathione-S-transferase, a polymeric calmodulin, and a biotinylated carrier protein.

83. 83. The kit of claim 82, wherein the streptavidin, avidin, or streptavidin analog or avidin analog that reversibly binds to biotin is cross-linked by a polysaccharide or a cross-linked oligomer or polymer of streptavidin, avidin, or of streptavidin or avidin analog that reversibly binds to biotin, is obtained by cross-linking via a bifunctional linker.

84. 1. 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 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 the first agent; the first agent comprises at least one binding partner C1, the binding partner C1 being capable of reversibly 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; the multimerization reagent comprises at least one binding site Z2 for reversibly binding 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 method wherein 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.

85. 85. The method of claim 84, wherein the T cells specifically bind to the multimerization reagent by contacting a sample containing the T cells, the sample comprising a population of lymphocytes, with the multimerization reagent to which the first and second agents are immobilized.

86. 86. The method of claim 84 or 85, further comprising separating said T cells that are bound to said multimerizing agent from non-bound cells of said lymphocyte population.

87. 87. The method of any of claims 84-86, wherein contacting the sample comprising the T cells comprising a population of lymphocytes with the multimerization reagent to which the first and second agents are immobilized is performed in a bioreactor, such as a culture plate, a hollow fiber bioreactor, or a plastic bag bioreactor.

88. The method according to any one of claims 84 to 88, further comprising contacting the population of lymphocytes (a reaction mixture containing the T cells bound to the multimerization reagent via the first agent and the second agent) with (i) a first binding partner C1 or an analogue thereof in free form capable of dissociating the binding between the first binding partner C1 and the binding site Z1, and (ii) a second binding partner C2 or an analogue thereof in free form capable of dissociating the binding between the second binding partner C2 and the binding site Z2, The reversible bond between the binding partner C1 and the binding site Z1 of the first agent, as well as the reversible bond between the binding partner C2 of the second agent and the binding site Z2 of the multimerization reagent, are separated, thereby releasing the T cells from the soluble multimerization reagent and from the first agent and the second agent, and stopping the expansion of the T cells.

89. 89. The method of claim 88, comprising recovering an eluate containing the released T cells, the soluble multimerization reagent, the first agent and / or the second agent, the free form of binding partner C1 or an analog thereof, and the free form of binding partner C2 or an analog thereof.

90. The method of any one of claims 84 to 89, wherein the binding sites Z1 and Z2 of the multimerization reagent are identical.

91. 91. The method of claim 90, wherein a single multimerization reagent is used.

92. 92. The method of any of claims 89 to 91, further comprising subjecting the eluate to chromatography on a suitable stationary phase, wherein the stationary phase is a gel filtration and / or affinity chromatography matrix, the gel filtration and / or affinity chromatography matrix comprising an affinity reagent comprising binding moieties Z1 and / or Z2 that specifically bind to the binding partner C1 and / or C2 comprised in the first agent or the second agent, thereby immobilizing the first agent, the second agent, the first binding partner C1 or an analogue thereof, and / or the free form of the second binding partner C2 or an analogue thereof to the stationary phase.

93. 93. The method of any of claims 88-92, wherein the stationary phase is fluidly connected to the bioreactor.

94. 94. The method of claim 93, wherein the T cells are separated from the soluble multimerization reagent.

95. The method of claim 93, wherein the mixture comprising the T cells and the multimerization reagent is subjected to chromatography on a suitable stationary phase, the stationary phase being a gel filtration and / or affinity chromatography matrix, the gel filtration and / or affinity chromatography matrix comprising an affinity reagent, the affinity reagent comprising a binding partner D that (specifically) binds to the binding site Z1 of the multimerization reagent, thereby immobilizing the multimerization reagent on the stationary phase.

96. The method of any one of claims 84 to 95, wherein the first agent stimulates a TCR / CD3 complex-associated signal in the T cell.

97. 97. The method of claim 96, wherein the first agent is a binding reagent that specifically binds to CD3.

98. The method of any one of claims 84 to 97, wherein the accessory molecule on the T cell is CD28.

99. 99. The method of claim 98, wherein the second agent that binds to the accessory molecule is a binding reagent that specifically binds to CD28.

100. the first agent that specifically binds to CD3 is 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; and / or The method of any of claims 97 to 99, wherein the second agent that specifically binds to CD28 is 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.

101. The bivalent antibody fragment is (Fab) ２ 101. The method of claim 100, wherein the Fv fragment is a single chain Fv fragment or a bivalent single chain Fv fragment.

102. 102. The method of claim 101, wherein the monovalent antibody fragment is selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv).

103. The method of any one of claims 84 to 102, wherein the multimerization reagent comprises a streptavidin oligomer or polymer, an avidin oligomer or polymer, an oligomer or polymer of a streptavidin analog that reversibly binds to biotin, or an oligomer or polymer of an avidin analog that reversibly binds to biotin.

104. The method of claim 103, wherein the streptavidin, avidin, or streptavidin analog or avidin analog that reversibly binds to biotin is cross-linked by a polysaccharide or the cross-linked oligomer or polymer of streptavidin, avidin, or of streptavidin or avidin analog that reversibly binds to biotin is obtained by cross-linking via a bifunctional linker.

105. 105. The method of claim 103 or 104, wherein the cross-linked oligomer or polymer of streptavidin, avidin, or of an analogue of streptavidin or avidin that reversibly binds to biotin, comprises three or more streptavidin monomers.

106. (a) the binding partners C1 and C2 each independently comprise biotin, and the multimerization reagent comprises a streptavidin analog or an avidin analog that reversibly binds to biotin; or (b) the binding partners C1 and C2, independently of one another, comprise a biotin analogue that reversibly binds to streptavidin or avidin, and the multimerization reagent comprises streptavidin, avidin, or a streptavidin analogue or an avidin analogue that reversibly binds to the biotin analogue; or (c) the binding partners C1 and C2 each independently comprise a streptavidin or avidin-binding peptide, and the multimerization reagent comprises streptavidin, or avidin, or a streptavidin analogue, or an avidin analogue, that reversibly binds to the streptavidin or avidin-binding peptide.

107. The binding partner C comprises the streptavidin-binding peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys, and the multimerization reagent ligates the amino acid sequence Va1 at sequence positions 44-47 of wild type streptavidin. 44 -Thr 45 -Ala 46 -Arg 47 or a streptavidin analogue comprising the amino acid sequence lle at sequence positions 44-47 of wild-type streptavidin. 44 -Gly 45 -Ala 46 -Arg 47 The method of claim 106, comprising a streptavidin analog comprising:

108. The dissociation constant (K ｄ ) but 10 －２ M to 10 －１３ The method of any one of claims 84 to 107, wherein M is in the range of

109. The method according to claims 89 to 108, wherein the first free partner and / or the second free partner is a compound that is not harmful to the cell.

110. 110. The method of claim 109, wherein the reversible binding is separated by contacting the cells with biotin or a biotin analogue.

111. 111. The method of any of claims 92 to 110, wherein a T cell receptor or a chimeric antigen receptor is introduced into the T cells after or during expansion.

112. The method of claim 111, wherein the T cells are stimulated with a third agent that binds to the introduced T cell receptor or the chimeric antigen receptor.

113. The method of any of claims 84 to 112, wherein the population of lymphocytes comprising T cells is a population of peripheral blood mononuclear cells (PBMCs) or a population of enriched or purified T cells.

114. An arrangement of a bioreactor and a (first) stationary phase for chromatography, comprising: the bioreactor is suitable for expanding cells; said (first) stationary phase is suitable for the separation of cells and the removal of reagents, the stationary phase is a gel filtration and / or affinity chromatography matrix, the gel filtration and / or affinity chromatography matrix comprising an affinity reagent, the affinity reagent comprising a binding site Z1 which specifically binds to a binding partner C1 contained in a first agent and / or the affinity reagent comprising a binding site Z2 which specifically binds to a binding partner C2 contained 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 form of the second binding partner C2 on the stationary phase, An arrangement in which the bioreactor and the stationary phase are in fluid connection.

115. 115. The arrangement of claim 114, wherein the first stationary phase is comprised in a chromatography column or is a planar stationary phase.

116. 116. The arrangement of claim 115, further comprising a second stationary phase, said second stationary phase in fluid connection with said first stationary phase.

117. The arrangement of claim 116, wherein the second stationary phase is 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 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.

118. 118. The arrangement of claim 117, wherein the second stationary phase and the bioreactor are fluidly connected to each other.

119. An apparatus for purifying and expanding target cells, comprising a bioreactor as defined in claims 114 to 118 and at least one arrangement of at least one chromatographic stationary phase.

120. 120. The apparatus of claim 119, comprising a bioreactor and a sample inlet fluidly connected to said bioreactor of said arrangement of at least one chromatographic stationary phase.

121. 121. The device of claim 119 or 120, comprising a sample outlet for purified and expanded target cells, said sample outlet being fluidly connected to a bioreactor and said stationary phase of said arrangement of at least one stationary phase for chromatography.

122. The method of any one of claims 119 to 121, wherein the device is a functionally closed system.

123. A multimerization reagent capable of expanding a population of cells, comprising: is in soluble form and comprises 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) on the multimerization reagent; A multimerization reagent, wherein the first agent comprises at least one binding partner C1, the binding partner C1 being capable of reversibly binding to the at least one 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.

124. and further comprising at least one binding site Z2 for reversibly binding a second agent that stimulates an accessory molecule on the surface of the cell; the second agent, which stimulates an accessory molecule on the surface of the cell, is reversibly immobilized (bound) onto the multimerization reagent; The multimerization reagent of claim 123, wherein the second agent comprises a binding partner C2, the binding partner C2 being 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.

125. 125. The multimerization reagent of claim 123 or 124, capable of expanding a lymphocyte population.

126. 126. The multimerization reagent of claim 125, wherein the lymphocyte population to be expanded is a B cell population, a T cell population, or a natural killer cell population.

127. The multimerization reagent of claim 126, wherein the T cell population is an antigen-specific T cell population, a helper T cell population, a cytotoxic T cell, a memory T cell, a regulatory T cell, or a natural reactivator T cell population.

128. 128. The multimerization reagent of claim 127, wherein the first agent comprises an MHC I:peptide complex or stimulates a TCR / CD3 complex-associated signal in the T cell.

129. 129. The multimerization reagent of claim 128, wherein the first agent is a binding reagent that specifically binds to CD3.

130. 130. The multimerization reagent of any of claims 124-129, wherein the second agent that binds to the accessory molecule is a binding reagent that specifically binds to CD28 or CD137.

131. the first agent that specifically binds to CD3 is 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; and The multimerization reagent according to any of claims 128 to 130, wherein the second agent that specifically binds to CD28 or CD137 is 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, 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.

132. The bivalent antibody fragment is (Fab) ２ 132. The multimerizing reagent of claim 131, which is a Fv fragment or a bivalent single chain Fv fragment.

133. 132. The multimerization reagent of claim 131, wherein the monovalent antibody fragment is selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv).

134. The multimerization reagent of claim 133, wherein the proteinaceous CD3, CD28, or CD137 binding molecule with antibody-like binding properties is selected from the group consisting of aptamers, muteins based on polypeptides of the lipocalin family, glubodies, proteins based on ankyrin scaffolds, proteins based on crystalline scaffolds, adnectins, and avimers.

135. 127. The multimerization reagent of claim 126, wherein the first agent is a binding reagent that specifically binds to CD40 or CD137, and the cell population is a B cell population.

136. The multimerization reagent of claim 135, wherein the first binding reagent that specifically binds to CD40 or CD137 is selected from the group consisting of an anti-CD40 antibody, a bivalent antibody fragment of an anti-CD40 antibody, a monovalent antibody fragment of an anti-CD40 antibody, and a proteinaceous CD40 binding molecule with antibody-like binding properties, 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).

137. The multimerization reagent of claim 136, wherein the bivalent antibody fragment is a (Fab)2' fragment or a bivalent single chain Fv fragment.

138. 137. The multimerization reagent of claim 136, wherein the monovalent antibody fragment is selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv).

139. The multimerization reagent of claim 136, wherein the proteinaceous CD40-binding molecule or proteinaceous CD137-binding molecule with antibody-like binding properties is selected from the group consisting of aptamers, muteins based on polypeptides of the lipocalin family, glubodies, proteins based on ankyrin scaffolds, proteins based on crystalline scaffolds, adnectins, and avimers.

140. The multimerization reagent of claim 126, wherein the accessory molecule on the B cell is CD40 or CD137 or a mixture thereof.

141. 141. The multimerization reagent of claim 140, wherein the second agent is CD40 ligand (CD154), CD137, or a mixture thereof, and the cell population is a B cell population.

142. The multimerization reagent according to any one of claims 124 to 141, wherein the binding sites Z1 and Z2 of the multimerization reagent are identical.

143. 143. The multimerization reagent of any of claims 124 to 142, wherein the multimerization reagent comprises an oligomer or polymer of streptavidin, an oligomer or polymer of avidin, an oligomer or polymer of an analog of streptavidin that reversibly binds to biotin, an oligomer or polymer of an analog avidin that reversibly binds to biotin, a reagent comprising at least two chelating groups K, said at least two chelating groups capable of binding to a transition metal ion, thereby enabling the reagent to bind to an oligohistidine affinity tag, a polymeric glutathione-S-transferase, a polymeric calmodulin, and a biotinylated carrier protein.

144. The multimerization reagent of claim 143, wherein the streptavidin, avidin, or streptavidin analog or avidin analog that reversibly binds to biotin is cross-linked by a polysaccharide or a cross-linked oligomer or polymer of streptavidin, avidin, or of streptavidin or avidin analog that reversibly binds to biotin is obtained by cross-linking via a bifunctional linker.

145. 145. The multimerization reagent of claim 143 or 144, wherein the cross-linked oligomer or polymer of streptavidin, avidin, or of an analogue of streptavidin or avidin that reversibly binds to biotin, comprises three or more streptavidin monomers.

146. (a) the binding partners C1 and C2 each independently comprise biotin, and the multimerization reagent comprises a streptavidin analog or an avidin analog that reversibly binds to biotin; or (b) the binding partners C1 and C2, independently of one another, comprise a biotin analogue that reversibly binds to streptavidin or avidin, and the multimerization reagent comprises streptavidin, avidin, or a streptavidin analogue or an avidin analogue that reversibly binds to the biotin analogue; or (c) the binding partners C1 and C2 each independently comprise a streptavidin or avidin-binding peptide, and the multimerization reagent comprises streptavidin, avidin, or a streptavidin analogue or an avidin analogue that reversibly binds to the streptavidin or avidin-binding peptide. The multimerization reagent of any one of claims 123 to 145.

147. The binding partners C1 and C2 each independently comprise the streptavidin-binding peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys, and the multimerization reagent comprises the amino acid sequence Va1 at sequence positions 44-47 of wild-type streptavidin. 44 -Thr 45 -Ala 46 -Arg 47 or a streptavidin analogue comprising the amino acid sequence lle at sequence positions 44-47 of wild-type streptavidin. 44 -Gly 45 -Ala 46 -Arg 47 The multimerization reagent of claim 146, comprising a streptavidin analog comprising:

148. 1. 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) on the first multimerization reagent; a first multimerization reagent, the first agent comprising at least one binding partner C1, the binding partner C1 being capable of reversibly binding to the at least one 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; (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, the second agent, which stimulates an accessory molecule on the surface of the cell, is reversibly immobilized (bound) onto the multimerization reagent; and a second multimerization reagent, wherein the second agent comprises a binding partner C2, the binding partner C2 being 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.

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