in vitro method of stimulating a cell population

专利摘要:
METHODS, KITS AND APPLIANCE TO ENLARGE A CELL POPULATION. The present invention relates to an in vitro method 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 (attached to it) a first agent that provides a primary activation signal to the cells and optionally, a second agent that provides a costimulatory signal. The invention likewise provides multimerization reagents, kits, arrangements and an apparatus for expanding cells.
公开号:BR112016024072B1
申请号:R112016024072-3
申请日:2015-04-16
公开日:2021-02-09
发明作者:Lothar Germeroth；Christian Stemberger
申请人:Juno Therapeutics Gmbh；
IPC主号:

专利说明:

CROSS REFERENCE FOR RELATED ORDERS
[0001] The present invention claims priority benefit for US Provisional US Patent Application 61 / 980,506 "Methods, Kits And Apparatus For Expanding A Population Of Cell" filed with the US Patent and Trademark Office on April 16, 2014 , the contents of which are incorporated herein by reference in their entirety for all purposes. FIELD OF THE INVENTION
[0002] The present invention relates to the expansion (proliferation) of a population of cells such as a population of lymphocytes. The invention in general provides new methods and reagents for the expansion (proliferation) of cell populations that require binding of a receptor binding molecule (such as a first agent as described here) to a receptor molecule on the surface of cells, thereby providing a primary activation signal to cells. The invention employs a multimerization reagent that has immobilized on it (binding to it) a first agent that provides a primary activation signal to the cells. This primary activation signal may therefore be sufficient to activate cells to expand / proliferate. This first agent can be reversibly or irreversibly linked to the multimerization reagent. The multimerization reagent may have immobilized on it (binding to it) in the same way a second agent that stimulates an additional molecule on the surface of cells. The second agent, by binding to the additional molecule on the surface at the cell surface, can thus stimulate the activated cells to expand. In the same way, this second agent can be reversibly or irreversibly linked to the multimerization reagent. The multimerization agent can be immobilized on a solid or soluble support. In one aspect, the method described here is a serial expansion of a population of cells in which a complete population of lymphocytes is stimulated / expanded, the reagents necessary for the expansion are then removed by chromatography at an appropriate stationary phase and the cells of expanded / stimulated are optionally transfected with, for example, a T cell receptor or a chimeric antigen receptor (CAR) and subjected to a second stimulus expansion with a different stimulator molecule that binds to the introduced T cell receptor or antigen receptor chimeric. The invention likewise relates to an apparatus for expanding the selected cell population. BACKGROUND OF THE INVENTION
[0003] The development of techniques for propagating T cell populations in vitro has been crucial to many of the advances in the understanding of T cell antigen recognition and T cell activation. The development of culture methods for the generation of T cell clones specific human antigens was useful in defining antigens expressed by pathogens and tumors that are recognized by T cells to establish immunotherapy methods to treat a variety of human diseases. Antigen-specific T cells can be expanded in vitro for use in adoptive cell immunotherapy or cancer therapy in which infusions of such T cells have been shown to have anti-tumor reactivity in a tumor-supporting host. In addition, adoptive immunotherapy was similarly used to treat viral infections in immunocompromised individuals.
[0004] A method of expanding human T cells in vitro in the absence of exogenous growth factor and additional cells that have been established in recent years is described in US patent 6,352,694 B1 and European patent EP 0 700 430 B1. Described in these patents is an in vitro method for inducing a population of T cells to proliferate. The method comprises contacting a population of T cells with a solid phase surface, having immobilized directly on it: (a) a first agent that provides a primary activation signal to T cells, thereby activating T cells; and (b) a second agent that stimulates an additional molecule on the surface of T cells, thereby stimulating activated T cells. The binding of the first agent and the second agent to T cells induces T cells to proliferate / expand. The first preferred agent described in US patent 6,352,694 B1 and European patent EP 0 700 430 B1 is a monoclonal anti-CD3 antibody that binds to the TCR / CD3 complex (TCR = T Cell Receptor) and thereby stimulates the signal associated with the TCR / CD3 complex in T cells. The second preferred agent according to these two patents is an anti-CD28 monoclonal antibody that binds to the additional CD28 molecule that is present in T cells. CD28 provides the necessary co-stimulus that is required for expansion / proliferation of activated T cells. Meanwhile, Dynabeads® CD3 / CD28 (Invitrogen) are commercially available for T cell expansion. Dynabeads® CD3 / CD28 CTS ™ are uniform, 4.5 μ superparamagnetic, sterile, non-pyrogenic polystyrene beads coated with a mixture of monoclonal antibodies purified by affinity against CD3 and CD28 cell surface molecules in human T cells.
[0005] However, such magnetic spheres are, for example, difficult to integrate into a method for expanding cells under conditions required for clinical trials or therapeutic purposes since you have to make sure that these magnetic spheres are completely removed before administration to the cells. T expanded to a patient. Accordingly, 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 especially therapeutic purposes. Ideally, this new method should likewise be compatible with integration into an automated process that can be used for rapid and easy expansion of the desired cell population for therapeutic applications.
[0006] This objective is solved by the subject of the independent claims, among others the methods, kits, arrangements and apparatus as recited in the independent claims. SUMMARY OF THE INVENTION
[0007] The present invention provides methods, kits, arrangements, and apparatus for in vitro expansion of a desired cell population, having a receptor molecule on its surface that can provide a primary activation signal for the population under binding of a suitable agent. cells and thereby activate the cell population for expansion (proliferation). Thus, the methods of the invention are likewise used to induce a population of cells to proliferate.
[0008] According to a first aspect, the invention provides an in vitro method of expanding a cell population, comprising contacting a sample comprising the cell population with a multimerization reagent,
[0009] in which the multimerization reagent has reversibly immobilized on it (attached to it) a first agent that provides a primary activation signal to the cells;
[00010] wherein the multimerization reagent comprises at least one Z1 binding site for the reversible binding of the first agent,
[00011] wherein the first agent comprises at least one C1 binding partner, where the C1 binding partner is capable of reversibly binding to the Z1 binding site of the multimerization reagent, where the first agent is binding to the reagent multimerization by the reversible bond formed between the bonding partner C1 and the bonding site Z1, and
[00012] in which the first agent binds to a receptor molecule on the surface of cells, thereby providing a primary activation signal to the cells and thereby activating the cells.
[00013] In accordance with a second aspect the invention provides an in vitro method of expanding a cell population, comprising contacting a sample comprising the cell population with a multimerization reagent,
[00014] in which the multimerization reagent is in a soluble form and immobilized (attached to it) a first agent that provides a primary activation signal to the cells;
[00015] wherein the multimerization reagent comprises at least one Z1 binding site for the binding of the first agent,
[00016] in which the first agent comprises at least one C1 binding partner, in which the C1 binding partner is capable of binding to the Z1 binding site of the multimerization reagent, in which the first agent is linked to the multimerization reagent by link formed between the link partner C1 and the link site Z1, and
[00017] in which the first agent binds to a receptor molecule on the surface of cells, thereby providing a primary activation signal to the cells and thereby activating the cells.
[00018] According to a third aspect the invention provides a reagent kit to expand a cell population, the kit comprising,
[00019] (i) a multimerization reagent,
[00020] wherein the multimerization reagent comprises at least one Z-binding site for the reversible binding of a first agent,
[00021] (ii) a first agent that binds to a receptor molecule on the surface of cells, thereby providing a primary activation signal to cells and thereby activating cells,
[00022] in which the first agent comprises at least one C1 binding partner, in which the C1 binding partner is capable of reversibly binding to a Z1 binding site of the multimerization reagent, in which the first agent is binding to the multimerization reagent by the reversible bond formed between the C1 binding partner and the Z1 binding site, and
[00023] (iii) a second agent that stimulates an additional molecule on the surface of cells,
[00024] in which the second agent comprises a C2 binding partner, in which the C2 binding partner is capable of reversibly binding to a Z2 binding site of the multimerization reagent, in which the second agent is binding to the multimerization by the link formed between the link partner C2 and the link site Z2,
[00025] in which the second agent binds to the additional molecule on the surface on the cell surface, thereby stimulating the activated cells.
[00026] According to a fourth aspect the invention provides a reagent kit to expand a cell population, the kit comprising,
[00027] (i) a multimerization reagent,
[00028] wherein the multimerization reagent is in soluble form and comprises at least one Z-binding site for the reversible binding of a first agent,
[00029] (ii) a first agent that binds to a receptor molecule on the surface of cells, thereby providing a primary activation signal to cells and thereby activating cells,
[00030] wherein the first agent comprises at least one C1 binding partner, where the C1 binding partner is capable of binding to a Z1 binding site of the multimerization reagent, where the first agent is attached to the multimerization reagent by the reversible link formed between the link partner C1 and the link site Z1.
[00031] In accordance with a fifth aspect the invention provides an in vitro method of serially expanding a lymphocyte population, wherein the lymphocyte population comprises T cells, the method comprising,
[00032] contacting a sample comprising the T cell, which comprises a lymphocyte population with a multimetering reagent,
[00033] in which the multimerization reagent is in a soluble form and has reversibly immobilized on it (i) a first agent that provides a primary activation signal to T cells and (ii) a second agent that stimulates an additional molecule on the surface of T cells,
[00034] wherein the multimerization reagent comprises at least one Z1 binding site for the reversible binding of the first agent,
[00035] in which the first agent comprises at least one C1 binding partner, in which the C1 binding partner is capable of reversibly binding to the Z1 binding site of the multimerization reagent, in which the first agent is binding to the reagent multimerization by the reversible bond formed between the bonding partner C1 and the bonding site Z1,
[00036] wherein the multimerization reagent comprises at least one Z2 binding site for the reversible binding of the second agent,
[00037] in which the second agent comprises at least one C2 binding partner, in which the C2 binding partner is capable of reversibly binding to the Z2 binding site of the multimerization reagent, in which the first agent is binding to the reagent multimerization by the reversible bond formed between the bonding partner C2 and the bonding site Z2,
[00038] in which the first agent binds to a receptor molecule on the surface of T cells, thereby providing a primary activation signal to cells and thereby activating T cells,
[00039] in which the second agent binds to the additional molecule on the surface of T cells, thereby stimulating activated cells, the first agent and the second agent thereby inducing T cells to expand.
[00040] According to a sixth aspect the invention provides an arrangement of a bioreactor and a stationary phase for chromatography,
[00041] in which the bioreactor is suitable for cell expansion,
[00042] in which the stationary phase is suitable for cell separation and removal of reagents, the stationary phase being a gel filtration matrix and / or affinity chromatography matrix, whereas in which the gel filtration matrix and / or affinity chromatography comprises an affinity reagent, wherein the affinity reagent comprises a Z1 binding site specifically binding to a C1 binding partner comprised in a first agent and / or the affinity reagent comprises a specifically Z2 binding site binding to a C2 binding partner comprised of a second agent, thereby being suitable for immobilization in the stationary phase the first agent and / or the second agent, the first C1 binding partner and / or the second free C2 binding partner ,
[00043] in which the bioreactor and the stationary phase are fluidly connected.
[00044] According to a seventh aspect the invention provides an apparatus for purifying and expanding a population of cells, the apparatus comprising at least one arrangement of a bioreactor and a stationary phase for chromatography according to the sixth aspect.
[00045] According to an eighth aspect, the invention provides a multimerization reagent capable of expanding a population of cells,
[00046] wherein the multimerization reagent is in soluble form and comprises at least one Z1 binding site for the reversible binding of a first agent that provides a primary activation signal to cells,
[00047] in which the multimerization reagent has reversibly immobilized on it (attached to it) said first agent which provides a primary activation signal to the cells;
[00048] wherein the first agent comprises at least one C1 binding partner, where the C1 binding partner is capable of reversibly binding to at least one Z1 binding site of the multimerization reagent,
[00049] wherein the first agent is linked to the multimerization reagent by the reversible link formed between the link partner C1 and the link site Z1,
[00050] According to a ninth aspect, the invention provides a composition capable of expanding a population of cells, the composition comprising,
[00051] (i) a first multimerization reagent,
[00052] wherein the first multimerization reagent is in soluble form and comprises at least one Z1 binding site for the reversible binding of a first agent that provides a primary activation signal to cells,
[00053] wherein the first multimerization reagent has reversibly immobilized (attached thereto) said first agent which provides a primary activation signal to the cells;
[00054] in which the first agent comprises at least one C1 binding partner, in which the C1 binding partner is capable of reversibly binding to at least one Z1 binding site of the multimerization reagent, in which the first agent is linked to the multimerization reagent by the reversible link formed between the link partner C1 and the link site Z1, and
[00055] (ii) a second multimerization reagent,
[00056] wherein the second multimerization reagent is in soluble form and comprises at least one Z2 binding site for the reversible binding of a second agent that stimulates an additional molecule on the cell surface,
[00057] in which the multimerization reagent has reversibly immobilized (attached to it) said second agent that stimulates an additional molecule on the surface of cells,
[00058] wherein the second agent comprises a C2 binding partner, where the C2 binding partner is capable of binding to at least one Z2 binding site of the multimerization reagent, where the second agent is attached to the multimerization reagent by the link formed between the link partner C2 and the link site Z2. DESCRIPTION OF THE DRAWINGS
[00059] The invention will be better understood with reference to the detailed description when considered together with the non-limiting examples and accompanying drawings. The figures illustrate modalities of the methods of the invention. Without wishing to be bound by theory, the figures include conclusions regarding the underlying expansion mechanism. The conclusions are determined for illustrative purposes only for the purpose of allowing a visualization of the expansion method that is achievable at a molecular level.
[00060] Figure 1 describes an embodiment of an in vitro expansion method of expanding a population of cells that have a cell surface receptor bound by which by a first agent can provide an activation signal for the cells to expand.
[00061] As shown in Fig. 1a, a sample comprising the population of cells (2) that carries a surface receptor molecule (30) is contacted with a multimerization reagent (4). The cell population (2) is mixed with other cell populations (22) that lack the surface receptor molecule (30). The multimerization reagent (4) has reversibly immobilized on it (attached to it) a first agent (6) that provides a primary activation signal to the cells. The multimerization reagent (4) comprises at least one Z1 binding site (42) for the reversible binding of the first agent (6) and the first agent (6) comprises at least one C1 binding partner (6a), wherein the binding partner C1 (6a) is capable of reversibly binding to the Z1 binding site (44) of the multimerization reagent. Thus, for immobilization, the first agent (6) is connected to the multimerization reagent (4) by the reversible bond formed between the bonding partner C1 (6a) and the bonding site Z1 (42). In the example shown in Fig. 1, the multimerization reagent (4) has a second binding site Z2 (44) that is not used in this example. The multimerization reagent (4) is immobilized on a solid support (10) such as a magnetic sphere, a polymeric sphere on a surface of a cell culture plate or reactor. The cell population (2) can, for example, be a lymphocyte cell population such as a population of B cells that can be activated by the CD40 receptor (see, for example, 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 molecule (30) is CD40 and the first reagent (6) can be any CD40 binding molecule that provides the desired activation signal, for example, the monoclonal antibody CP-870,893 or an antibody binding fragment thereof such as a monovalent Fab fragment. The C1 binding partner of the first agent ( 6) can, for example, be any affinity peptide that is fused or conjugated, for example, the C-terminus of one or two polypeptide chains (heavy or light chain) of the antibody molecule. ) can, for example, be a streptavidin-binding peptide such as the Trp-Ser-His-Pro-Gln-Phe-Glu-Lys peptide (SEQ ID NO: 01), also known as "Strep-tag®") which is described in the patent US 5,506,121, for example, or streptavidin binding peptides having a sequential arrangement of two or more individual binding modules as described in International Patent Publication WO 02/077018 or US Patent 7,981,632. When using a streptavidin binding peptide as a C1 binding partner, the multimerization reagent (4) can be any streptavidin mutein to which the streptavidin peptide (= first C1 binding partner (6a)) reversibly binds by its ( biotin) Z1 binding sites (42) schematically shown in Fig. 1. Such a multimerization reagent may be a streptavidin mutein (analogue) comprising the amino acid sequence Va144-Thr45- Ala46-Arg47 (SEQ ID NO: 02) at sequence positions 44 to 47 of wild-type streptavidin or a streptavidin mutein (analogue) comprising amino acid sequence lle44-Gly45-Ala46-Arg47 (SEQ ID NO: 03) at sequence positions 44 to 47 of streptavidin type both of which are described in US patent 6,103,493 for example, and are commercially available under the trademark Strep-Tactin®. In the Example of Fig. 1, the multimerization reagent (4) could also include multimeric calmodulin or glutathione-S-transferase, both of which form reversible bonds with calmodulin or glutathione binding peptides. In this way, the Z2 binding site (44) can be formed by calmodulin or glutathione-S-transferase. Such a protein conjugate of, for example, calmodulin with a streptavidin mutein can be done by standard protein chemistry, for example, using bifunctional ligands.
[00062] As shown in Fig. 1b, after contacting the cell population (2) with the multimerization reagent (4) and normally incubating the cell population with the multimerization reagent (4), the cell population (2 ) complexes / is linked to the multimerization agent by the first agent (6). The first agent specifically binds to the cell surface receptor molecule such as CD40 in this Example and provides the activation signal for cell expansion, for example B cells. The other cell populations (22) contained in the missing initial sample the specific cell surface molecule (30) does not bind to the multimerization reagent. In this regard, it is noted that the cell population (2) normally has multiple copies of the cell surface molecule (30) on its surface and binding of these multiple copies is typically required for activation. In this way, the multimerization agent (4) typically provides more than one Z1 binding site so that first multiple agents (6) can be reversibly linked to obtain "multimerization" of the first agent, intending to present the first agent at a density enough to the cell population (2) (not shown in the diagram in Fig.1). In this regard, it is noted that the multimerization agent when used herein can such as have multiple Z1 binding sites, for example, a streptavidin mutein (being a homo-tetramer) in its native state has four such Z1 binding sites. It is, however, just as possible that the multimerization reagent is based on a compound that has only one Z1 binding site for the reversible binding of a C1 binding partner. Such an example is multimeric calmodulin. Calmodulin as such has only one binding site for calmodulin-binding peptides. However, calmodulin can be biotinylated and then can react with streptavidin oligomers (see similarly below), providing a multimerization reagent in which multiple calmodulin molecules are presented in high density in a "framework", thereby providing multimeric calmodulin .
[00063] As shown in Fig.1c, after incubation (which is normally carried out for a suitable time to obtain expansion of the desired cell population) the connection between the C1 binding partner (6a) of the first agent (6) and the Z1 binding site of the multimerization reagent (4) is disrupted by breaking the respective reversible bond. The disruption can be achieved by adding a competitor to the incubation / reaction mixture containing the population of cells (2) being linked to the multimerization reagent. For competitive disruption (which can be understood to be a competitive elution) of the reversible link between the first agent's C1 binding partner (6a) and the multimerization reagent Z1 binding site (22), the incubation mix / population of cells can be contacted with a first free C1 binding partner (20) or an analog of said first C binding partner which is capable of breaking the connection between the first C1 binding partner (6a) and the Z1 binding site (22 ). In the example of the C1 binding partner being a streptavidin-binding peptide that binds to the streptavidin biotin-binding site, the first free C1 partner (20) may be the corresponding free streptavidin-binding peptide or an analog that binds competitively. Such an analogue can, for example, be biotin or a biotin derivative such as destiobiotin.
[00064] As shown in Fig. 1d, addition of the first free partner (20) or its analog results in displacement of the C1 binding partner (6a) from the multimerization reagent (4) and thus, since the connection C1 is comprised in the first agent (6), displacement of the first agent (6) from the multimerization reagent (4). This displacement of the first agent (6) successively results in a dissociation of the first agent (6) from the cell surface receptor (30), in particular if the binding affinity of the bond between the first agent and the cell surface receptor ( 30) has a constant dissociation (Kd) in the range of 10-2 M to 10-13 M and is likewise reversible. Due to this dissociation, the stimulus of the cell population (2) is likewise terminated. In this way, the present invention provides the advantage that the time period of the stimulus or expansion of the cell population can be precisely controlled and in the same way the functional state of the cell population can be closely controlled. In this context, it is noted that the binding affinity of antibody molecules to their antigen, including for example, a cell surface receptor molecule such as CD40 in this Example, is normally in the Kd affinity range of 10-7 M to 1013 M. Thus, conventional monoclonal antibodies can be used as the first agent (and in the same clear way as the second agent as explained below) in the present invention. To avoid any unwanted greediness that leads to stronger binding, monoclonal antibodies can likewise be used in the form of their monovalent antibody fragments such as Fab fragments or single chain Fv fragments.
[00065] Furthermore, due to the dissociation of the first agent from the cell surface molecule (30), the present invention has the added advantage that the stimulated cell population is free of stimulating agents at the end of the stimulus period and that all other reagents used in the method, i.e. the first agent (6) as well as the first free partner (20) of the C1 binding partner or the analogue thereof can easily be removed from the stimulated cell population (2) by means of a " removal cartridge "described in International patent application WO 2013/124474 while the multimerization reagent (4) being immobilized on a solid support such as a bioreactor surface or a magnetic sphere is being held. Thus, reverting to the removal of the free agent (6) and the first free partner (20), according to the description of the "removal cartridge" in WO 2013/124474 (see with reference to Fig. 4 of it, for example ), the elution sample obtained in Fig. 1d here can be loaded onto the second chromatography column of WO 2013/124474. This chromatography column has a suitable stationary phase which is an affinity chromatography matrix and, at the same time, can act as a gel permeation matrix. This affinity chromatography matrix has an affinity reagent immobilized on it. The affinity reagent may, in the case of the current Example, for example, be streptavidin, a streptavidin mutein, avidin, an avidin mutein or a mixture thereof. The first agent (6), the first free partner (20) of the C1 binding partner (which is likewise called "competition reagent" here) binds to the affinity reagent, thereby being immobilized on the chromatography matrix. As a result, the elution sample containing the isolated and expanded cell population (2) is being emptied of the first agent (6) and the competing reagent (20). The expanded cell population (2), being free of any reagent, is now in a condition for another use, for example, for diagnostic applications (for example, another FACSTM separator) or for any cell-based therapeutic application.
[00066] Fig.2 shows another embodiment of an expansion method of the invention. As shown in Fig. 2a a sample comprises a cell population (2) that carries two specific cell surface molecules (30) and (32). The cell surface molecule (30) is involved in a primary activation signal for the cell population, while the cell surface molecule (32) is an additional molecule on the cell surface that is involved in providing a stimulus to cells . The cell population can, for example, be a T cell population in which the cell surface molecule (30) is a TCR / CD3 complex and the cell surface molecule (32) is the additional CD28 molecule. Binding of both TCR / CD3 complexes as the primary activation signal and CD28 as a co-stimulant are necessary for T cell expansion / proliferation. The T cell population (2) is mixed with other missing cell populations (22) the surface receptor molecules (30) and (32). In the same way, in this modality, the cell population (2) is contacted with a multimerization reagent (4). The multimerization reagent (4) has reversibly immobilized on it (attached to it) a first agent (6) that provides a primary activation signal to the cells. In addition, the multimerization agent has reversibly immobilized (attached to it) a second agent (8) that stimulates CD28 as an additional molecule on the cell surface.
[00067] The multimerization reagent (4) comprises at least one Z1 binding site (42) for the reversible binding of the first agent (6) and the first agent (6) comprises at least one C1 binding partner (6a), wherein the binding partner C1 (6a) is capable of reversibly binding to the Z1 binding site (44) of the multimerization reagent. Thus, for immobilization, the first agent (6) is connected to the multimerization reagent (4) by the reversible bond formed between the bonding partner C1 (6a) and the bonding site Z1 (42). In addition, in the Example illustrated in Fig. 2, the second agent (8) comprises a C2 binding partner (8a), wherein the C2 binding partner is capable of reversibly binding to a Z2 binding site (44) of the multimerization reagent (4). The second agent (8) is linked to the multimerization reagent (4) by the reversible link formed between the link partner C2 (8a) and the link site Z2 (44). In this Example, the first agent (6) could be an anti-CD3 monoclonal antibody or a binding antigen fragment thereof such as a Fab fragment. The second agent (8) could be a monoclonal anti-CD28 antibody or an antigen fragment binding pathway such as Fab fragment. The first binding partner (6a) could be a streptavidin binding peptide (6a) that is fused or conjugated to the anti-CD3 antibody or the anti-CD3 antibody fragment. The second binding partner (8a) could be calmodulin binding peptide which is likewise conjugated or fused to the CD28 antibody or antibody fragment for CD28 binding. In this context, it is noted that monoclonal antibodies against, for example, CD3 or CD28 are well known (see, for example, US Patent 6,352,694 B or European Patent EP 0 700 430 B1 discussed above) and are commercially available from numerous suppliers 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. In this way, such monoclonal antibodies can be used as a first and second agent and can, for example, be chemically coupled (conjugated) with a C1 or C2 binding partner. Alternatively, it is likewise possible to clone the genes of the variable domains from the hydridoma cell lineage or to use an antibody of which the amino acid sequence is known and produces a respective antibody fragment such as a Fab fragment or a Fv-like recombinant. Using such an approach as described here in the Example section for both, the OKT3 hybridoma cell line (ATCC® CRL-8001 ™, described in US patent 4,361,549) that produces a monoclonal anti-CD3 antibody) and the antibody anti-CD28 28.3 described by Vanhove et al., BLOOD, July 15, 2003, Vol. 102, no. 2, pages 564-570 and GenBank accession number AF451974.1, the C1 and C2 binding partners are conveniently provided by the respective expression vector used for recombinant production so that the antibody fragment carries the C1 or C2 binding partner as a fusion peptide such as the C-terminal of the light or heavy chain (In this context, the amino acid sequence of the variable domain of the heavy chain and the variable domain of the OKT3 antibody chain that is described in Arakawa and another J. Biochem. 120, 657-662 (1996) is shown for purposes of illustration as SEQ ID NOs: 17 and 18 and in the accompanying Sequence Listings, while the amino acid sequence of the anti-CD28 antibody variable domain 28.3 described by Vanhove et al., Supra , is shown as SEQ ID NOS 19 (VH) and 20 (VL) in the accompanying Sequence Listings). In the same way, this methodology of cloning the variable domains of an antibody molecule and recombinantly producing a fragment of the respective antibody is well known to the person skilled in the art, see for example, Skerra, A., (1994) a general vector, pASK84, for cloning, bacterial production, and single-step purification of Fab antibody fragments. Gene 141, 79-84, or Skerra, A., (1993) bacterial expression of immunoglobulin fragments. Curr Opin Immunol. 5, 256-562). Finally, it is likewise possible to generate antibody molecules from artificial binding molecules with antibody-like properties against a particular target such as CD3 or CD28 as in the Example of Fig. 2 by well-known evolutionary methods such as phage display (revised, for example, in Kay, BK and others (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 in Amstutz, P. et al. (2001) Curr. Opin .Biotechnol. 12, 400-405) or mRNA display as reported in Wilson, DS and another (2001) Proc. Natl. Acad. Sci. USA 98, 3750-3755.
[00068] In the case of 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) being a streptavidin binding peptide, the Z1 binding site (42) of the multimerization reagent (4) is provided by a suitable streptavidin mutein to which the streptavidin peptide (6a) reversibly binds up. Since the C2 binding is a calmodulin binding peptide, the Z2 binding site (44) of the multimerization reagent (4) is provided by multimeric calmodulin. The multimerization reagent (4) can be a single molecule, for example a conjugate of multimeric calmodulin with streptavidin (this alternative would normally be used in the case of a soluble multimerization) or it can likewise consist of two independent molecules. The latter latter option is preferred when the multimerization reagent (44) is immobilized on a solid support as shown in Fig.2. In this case, a mixture of a streptavidin and calmodulin mutein can be coated (immobilized) on the solid support, for example, in a 1: 1 molar ratio with respect to the Z1 and Z2 binding sites. In this context, it is noted that, due to the immobilization of calmodulin on the surface of the solid support, there is no need to prepare multimeric calmodulin as explained above, but immobilization of calmodulin on the surface is sufficient to present calmodulin (which, as mentioned above, has only a single binding site to calmodulin-binding peptides), at a sufficiently high density to guarantee binding of the cell population (2). For example, in this case, a bivalent antibody fragment that has two binding sites against CD28 or an intact antibody that per se has two identical binding sites, could be used as a second reagent (8).
[00069] As shown in Fig. 2b, after contacting the T cell population (2) with the multimerization reagent (4) and normally incubating the cell population with the multimerization reagent (4), the T cell population (2) complex forms / are linked to the multimerization agent by 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 additional molecule CD28, thereby inducing T cells to proliferate / expand.
[00070] As shown in Fig. 2c, after incubation (which is normally carried out for an appropriate period of time to achieve expansion of the desired cell population) the connection between the C1 binding partner (6a) of the first agent (6) and the Z1 binding site of the multimerization reagent (4) is disrupted by breaking the respective reversible bond. Also, the bond between the bonding partner C2 (8a) of the second agent (8) and the bonding site Z2 of the multimerization reagent (4) is broken by breaking the respective reversible bond. The reversible link between the C1 binding partner (6a) of the first agent (6) and the Z1 binding site can be disrupted by biotin (which acts as an analogue (20) of the first free partner) while the reversible connection between the partner C2 binding agent (8a) of the first agent (8) and the Z2 binding site can be disrupted by the addition of a metal chelator (calcium chelator) such as EDTA or EGTA (which acts as an analogue (20) of the second free partner ) since the binding to calmodulin to calmodulin binding peptides is calcium ion (Ca2 +) dependent). This means of course that the contact of the cell population (2) is carried out in a buffer containing Ca2 +.
[00071] As shown in Fig. 2d, addition of the analogue (20) of the first free partner and the second free partner, respectively results in displacement of the binding partners C1 (6a) and C2 (8a) of the multimerization reagent (4) and thereby displacing 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) successively results in a dissociation of the first agent (6) and the second agent (8) from the TCR / CD3 complex and the additional molecule CD28, thereby ending the stimulus / expansion of the cell population (2). Thus, as stated above, the present invention provides the advantage that the time period of stimulus or expansion of a T cell population can be exactly controlled and, therefore, likewise the functional state of the T cell population can be closely related. controlled. After elution of the cells as shown in Fig. 1d, the first agent (6), the second reagent (8) as well as the analogue (20) of the first free partner of the C1 binding partner and the second free partner of the binding partner C2 can be easily removed from the stimulated cell population (2) by means of a "removal cartridge" described in International patent application WO 2013/124474. In addition, and most importantly, if the initial sample is a population of lymphocytes, for example, in the form of PMBCs obtained from a Ficoll gradient, the T cell population (2) is now available for serial expansion as defined here. Since the expanded cell population (for example by an initial stimulus by means of CD3 / CD28) can be transfected during expansion, for example, with a T cell receptor (TCR) or a chimeric antigen receptor (CAR, in the same way) known as artificial T cell receptor), the genetically modified cells can then be released from the initial stimulus and subsequently can be stimulated with a second type of stimulus for example by the newly introduced receptor. These second stimuli may comprise an antigenic stimulus in the form of a peptide / MHC molecule, the cognate linker (crosslinking) of the genetically introduced receptor (for example a natural linker from a CAR) or any linker (such as an antibody) that directly binds if within the structure of the new receiver (for example recognizing constant regions within the receiver). In this way, the T cell population obtained from this serial expansion can be used for adoptive cell transfer.
[00072] Fig. 3 shows another embodiment of an expansion method of the invention. Likewise the sample used in this Example comprises a population of T cells (2) that carry two specific cell surface molecules (30) and (32), with the cell surface molecule (30) being a TCR / CD3 and the cell surface molecule (32) being the additional molecule CD28. In Fig. 3a the population of T cells (2) is shown after being placed in contact with a multimerization reagent (4). Likewise in this Example, the multimerization reagent (4) has reversibly immobilized on it (attached to it) as the first agent (6) an anti-CD3 antibody or a binding antigen fragment thereof that provides a primary activation signal to T cells and as a second agent (8) an anti-CD28 antibody or an antigen-binding fragment thereof that stimulates CD28 as an additional molecule.
[00073] The multimerization reagent (4) shown in the Example of Fig. 3 comprises only one type Z1 binding site (42) for the reversible binding of both the first agent (6) and the second agent (8). Likewise, the first agent (6) and the second agent (8) comprise at least one C1 bonding partner (6a, 8a), in which both the C1 bonding partner (6a) and the bonding partner (8a) are capable of reversibly bind to the Z1 binding site (44) of the multimerization reagent. Thus, for immobilization, the first agent (6) and the second agent (8), respectively, are connected to the multimerization reagent (4) by the reversible bond formed between the binding partner C1 (6a) and the binding partner C2 and the Z1 binding site (42). The connection partners C1 and C2 can be different or identical. For example, the C1 binding partner may be a streptavidin-binding peptide from the Trp-Ser-His-Pro-Gln-Phe-Glu-Lys sequences ((SEQ ID NO: 01), the "Strep-tag®") while the C2 binding partner may be the 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), similarly known as "tag2"), described by Junttila et al., Proteomics 5 (2005), 1199-1203 or US patent 7,981,632). All of these streptavidin binding peptides bind to the same binding site, that is, the streptavidin biotin binding site. If one or more of such streptavidin binding peptides are used as C1 and C2 binding partners, 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 can, for example, be an oligomer or a streptavidin or avidin polymer or any streptavidin or avidin mutein (analog). The oligomer may comprise three or more stromtavidin, avidin or a mutein monomers thereof. The oligomer or polymer can be cross-linked by a polysaccharide. Such streptavidin or avidin oligomers or polymers or streptavidin or avidin muteins can in a first step be prepared by introducing carboxyl residues into a polysaccharide, for example 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 colon cancer anti-human monoclonal antibody and mitomycin C - dextran conjugate. Bioconjugate Chemistry 3,132-137. " In a second step, streptavidin or avidin or muteins are coupled by primary amino groups of the internal lysine residue and / or the free N-terminal to the carboxyl groups in the dextran backbone using conventional carbodiimide chemistry. Alternatively, crosslinked oligomers or polymers of streptavidin or avidin or any streptavidin or avidin mutein can likewise be obtained by crosslinking by means of bifunctional ligands such as glutardialdehyde or by other methods described in the literature.
[00074] Using as connection partners C1 and C2, portions of connection to the same connection site (42) of the multimerization agent have the advantage that, as shown in Fig. 3b, the same free partner (of the first connection partner C1 and likewise the second binding partner C2) or analog thereof can be used to terminate the expansion of the T cell population (2) and release this T cell population (2) from the multimerization agent. In the Example of Fig. 3, an analogue of the first and second partner C1 and C2 such as biotin or a biotin derivative (iminobiotin or destiobiotin) can be used conveniently for the termination of expansion and the elution of the T cell population (2) .
[00075] As shown in Fig. 3c, after elution of the cells as shown in Fig. 1d, the first agent (6), the second reagent (8) as well as biotin as the analogue (20) of the first free partner of the partner binding agent C1 and the second free partner of binding partner C2 can be easily removed from the stimulated cell population (2) by a "removal cartridge" described in International patent application WO 2013/124474. In addition, the method of using a soluble multimerization reagent (4) has the additional advantage of being able to avoid any solid support such as magnetic beads. This means that there is no risk of contamination of the T cells activated by such magnetic spheres. This likewise means that a process that is compliant with GMP standards can be much easier established compared to the known method such as the use of Dynabeads® in which additional measures have to be employed to ensure that the final expanded T cell population be free of magnetic spheres. In addition, the use of a soluble multimerization agent makes it much easier to remove it from the activated cell population (T cells, B cells or likewise natural killer cells) since the cells can be pelleted simply by centrifugation and the supernatant including the soluble multimerization agent can be discarded. Alternatively, the soluble multimerization agent can be removed from the expanded cell population in a gel permeation matrix of the International WO 2013/124474 removal cartridge. Since no solid phases (e.g. magnetic spheres) are present, the present invention likewise provides for 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).
[00076] Fig. 4 shows the results of an experiment in which CD3 + T response cells proliferated after being stimulated in vitro with Fab αCD3 and aCD28 fragments that were reversibly immobilized on beads coated with streptavidin mutein Strep-tactin®. Fig. 4A in a histogram showing size distribution (forward dispersion) of stimulated cells, Fig. 4B describes histograms representing the degree of proliferation according to the number of cells per cell division which is indicated at the top of Fig. 4B ( 0 represents undivided cells, 5 represents cells that have gone through at least 5 divisions), and Fig. 4C shows a picture of the culture plate after 4 days of stimulation.
[00077] Fig. 5 shows the results of differential intracellular calcium mobilization in Jurkat cells that are labeled with the αCD3 OKT3 antibody or with Fab OKT3 fragments being multimerized with Strep-tactin® (in the same way referred to as Fab multimers here). Fig. 5A: Jurkat cells were loaded with the calcium sensitive dye Indo-1-AM and calcium release was activated by injection of either aCD3 mAb (black squares) or Fab αCD3 OKT3 multimers (derived from the OKT3 parental cell line) with or without disruption of anterior D-biotin (dark gray triangles and light gray circles respectively) compared to PBS injection (inverted white triangles). Ionomycin application served as a positive control. Changes resolved by time in intracellular Ca2 + concentration were monitored by flow cytometry based on the change in relation to FL6 / FL7. Fig. 5B: Jurkat cells labeled by Indo-1-AM- were activated by different aCD3 stimuli as described in Fig 4a; OKT3: superior graph and multimer of Fab aCD3: median graph) followed by disruption mediated by subsequent D-biotin (t = 140s) of Fab multimer signaling aCD3. Injection of PBS (lower graph) and ionomycin served as a negative or positive control. Data are representative of three different experiences.
[00078] Fig. 6 shows the result of reversible labeling of cells by OKT3 anti-CD3 Fab multimers. Recently isolated PBMCs were labeled with either a monoclonal antibody (dot plot on the left, parental clone for Fab multimers) or Fab multimers labeled by cognate PE and analyzed before (second column on the left) or after treatment with D-biotin (median column). Remaining Fab monomers were then detected after the subsequent washing steps using fresh PE labeled Strep-Tactin® (second column on the right). Secondary Fab multimer labeling of reversibly labeled cells served as a control (right column). Only living cells (PInegative) are shown. Numbers on dot charts indicate the percentage of cells within gates.
[00079] Fig. 7 shows the isolation of cells by reversible ligation of anti-CD28 Fab fragments multimerized with phycoerythrin-labeled Strep-Tactin® as a fluorescent label. CD28 + cells were selected / isolated by selection of newly isolated Fab PMBC multimer magnetic cell as described in International patent application WO2013 / 011011. Before selection cells were labeled by the control with cognitive fluorescent aCD28 multimers (dot plot on the left) or with an antibody directed against the immunoglobulin kappa light chain (second plot on the left, α-Ig kappa mAb) . After selection, cells were treated with D-biotin and subsequently washed to remove magnetic beads and Fab monomers. Released CD28 + cells were subsequently (re-) labeled or with CD28 Fab multimers (according to the right dot plot) or with α-Ig kappa mAb (dot plot to the right) to detect potentially remaining Fab monomers. Only live CD3 + cells (PInegative) are shown. Numbers on dot charts indicate the percentage of cells within gates.
[00080] Fig. 8 shows the results of an experiment in which CD3 + T response cells were proliferated after being stimulated in vitro with reversible αCD3 / αCD28 Fab fragments that were reversibly immobilized on soluble oligomeric Strep-tactin® acting on a multimerization reagent soluble. For the experiments the results of which are shown in Fig. 8, 300,000 CD3 + response T cells (Tresp) were labeled with 2μM succinimidyl carboxyfluorescein ester (CFSE) and stimulated with varying amounts of a preparation of Streptactin oligomers soluble in that a combination of Fab αCD3 and Fab αCD28 fragments both carrying a Strep tag as a streptavidin binding peptide in the heavy chain were immobilized. ("1x" corresponds to 3μg of multimerized Strep-tactin functionalized with 0.5 μg of αCD3 - and 0.5 μg of aCD28 Fab; numbers indicate the number of times "1x"). Tresp cells left unstimulated or stimulated with blank Strep-tactin multimers (no Fab) served as a negative control. Tresp cells were seeded in duplicates in 48 well plates together with 300,000 CD3 negative autologous feeder cells (irradiated with 30Gy) in 1 ml of cell culture medium supplemented with 20 U / ml of interleukin 2 (IL-2). Cells were incubated at 37 ° C without changing media and proliferation was analyzed according to CFSE dilution after 5 days by FACS analysis (Fig. 8B). Fig. 8A shows cell size distribution after 5 days in culture. Histograms show live CD3 + cells, while Fig. 8C shows cells after cultivation that were released by stimulus reagents after being treated with 1 mM D-biotin and washed. The dissociation and removal of monomeric Fab fragments was analyzed by re-labeling with phycoerythrin-labeled Strep-Tactin® (ST-PE) as a fluorescent label and a representative histogram is shown. Fig. 8D shows the absolute number of living cells (trypan blue negative) after 5 days were counted using a Neubauer counting chamber and plotted against the respective stimulus condition. Median cell numbers are shown in Fig. 8D; error bars indicate standard deviation (SD). Fig. 8E shows a picture of the culture plate after 5 days of stimulation.
[00081] Fig. 9 describes an illustration of the serial expansion method of the present invention (Fig. 9a) while Fig. 9b briefly describes some characteristics and advantages of serial expansion.
[00082] Fig. 10 shows an arrangement of the invention that can be used in conjunction with the expansion methods of the invention. This arrangement (100) includes a bioreactor (50), a first "removal cartridge" (70) and a second "removal cartridge" (90). The bioreactor (50) is fluidly connected to the first removable cartridge (70), and the first removal cartridge is fluidly connected to the second removal cartridge (90). This arrangement (100) can form part of a device for automated cell expansion and purification as described here.
[00083] In the bioreactor (50) an expansion method as described here is carried out, for example an expansion method illustrated in Fig. 3 that makes use of a soluble multimerization reagent. In this case, after the end of the activation / expansion of the cell population (2) by adding a competitor (20) (free partner of the C1 binding partner or an analog of the same) the reaction mixtures that are released from the bioreactor contain the expanded cell population (2), the first agent (6), the second agent (8) as well as the soluble multimerization reagent (4). In this example, the first agent (6) is a CD3-binding antibody fragment that includes a streptavidin-binding peptide as a C1-binding partner, the second agent (8) is a CD28-binding antibody fragment that includes a streptavidin binding peptide as C1 binding partner and competitor (20) (free analogue of C1 binding partner) is biotin. This reaction mixture is applied to the first removal cartridge (70). This first removal cartridge (70) is a removal cartridge as described in International patent application WO 2013/124474 which includes a chromatography column with a suitable stationary phase. The stationary phase can also serve an affinity chromatography matrix and, at the same time, can act as a gel permeation matrix. This affinity chromatography matrix has an affinity reagent immobilized on it. The affinity reagent may, in the case of the current Example, for example, be streptavidin, a streptavidin mutein, avidin, an avidin mutein or a mixture thereof. In this way, the first agent (6) and the second agent (8) bind to the affinity reagent by its streptavidin binding peptide. In the same way biotin as the competitor (20) binds to the affinity reagent. Thus, these three reagents are all being immobilized in the chromatography matrix of the first removal cartridge while the cell population expands (2) and the soluble multimerization reagent (4) passes through the stationary phase. This "flow through" is then applied to the second removal cartridge (90). Likewise, this second removal cartridge (90) comprises a stationary phase. This stationary phase comprises a second affinity reagent on it that can bind to the Z1 binding site (42) of the multimerization reagent (4). This affinity reagent can be, for example, biotin which is covalently bound to the stationary phase. Such a stationary phase can, for example, be d-biotin SepharoseTM obtainable from Affiland S.A. (Ans-Liege, Belgium). In this way, the soluble multimerization reagent (4) will be bound (retained) in the stationary phase of the second removal cartridge (90) while the expanded population of cells (2) passes through the stationary phase and is being freed of any reagents. The cell population (2) is now in a condition for any additional use, for example, for diagnostic applications (for example, FACSTM classification) or for any cell based on therapeutic application. It is noted here that it is clear in the same way possible to change the order of the first "removal cartridge" (70) and the second "removal cartridge" (90) in an arrangement (100), such that bioreactor (50) is (directly ) fluidly connected to the second removable cartridge (90), and the first removal cartridge (70) is then arranged and fluidly connected to the second removal cartridge (90). In this arrangement the multimerization reagent (4) will be removed first from the cell population (2) and subsequently the first agent (6), the second (8) and for example the competitor (20) is removed. Such an arrangement is likewise encompassed in the present invention and can likewise form part of a device for automated cell expansion and purification as described herein.
[00084] Fig. 11 shows another modality of an arrangement of the invention that can be used together with the expansion methods of the invention. This arrangement (110) includes a bioreactor (50), a first "removal cartridge" (70) and a second "removal cartridge" (90). The bioreactor (50) is fluidly connected to the first removable cartridge (70), and the first removal cartridge is fluidly connected to the second removal cartridge (90). In addition, the second removal cartridge (110) is fluidly connected to the bioreactor (50). This arrangement (110) can likewise be part of a device for automated cell expansion and purification as described here. For example, when used in conjunction with an expansion method employing a soluble multimerization reagent (4), a purified expanded cell population (2) is obtained as an eluate from the second removal cartridge (90). Since the removal cartridge (90) is fluidly connected to the bioreactor (50), the cell population (2) can be transferred again into the bioreactor (50), for example, for serial clonal expansion as described here, transfecting the cell population, for example, with the gene for a T cell receptor and subsequent other (second) expansion using an expansion method of the invention.
[00085] Fig. 12 shows another embodiment of an arrangement of the invention that can be used in conjunction with the expansion methods of the invention. This arrangement (120) includes a bioreactor (50), a first "removal cartridge" (70) and a second "removal cartridge" (90). The bioreactor (50) is fluidly connected to the first removable cartridge (70), and the first removal cartridge is fluidly connected to the second removal cartridge (90). Similar to the modality shown in Fig. 11, the second removal cartridge (110) is fluidly connected to the bioreactor (50). However, a "selection cartridge" (92) as described in International patent application WO 2013/124474 is arranged between the second removal cartridge (90) and the bioreactor (50). In this way, a subpopulation of cells (2a) that is comprised in the cell population (2) can be selected / enriched by this "selection cartridge" (92) as described in WO 2013/124474. This subpopulation of cells (2a) can be transferred in the bioreactor (50), for example, to undergo serial expansion as described here. Alternatively (not shown), this subpopulation of cells (2a) can be used for cell-based therapy. It is again noted here that the use of a soluble multimerization reagent as described here allows for the design of automated cell purification and expansion devices that are functionally closed and thus not prone to contamination. In addition, since soluble multimerization reagent avoids the need for solid phase materials such as magnetic beads, such cell purification devices may be referred to as continuous flow devices.
[00086] Fig. 13 shows the proliferation kinetics of purified CD4 + and CD8 + T response cells (Tresp) that were stimulated in vitro with αCD3 / αCD28 Fab fragments or with aCD3 / aCD28 / aCD8 which were reversibly immobilized in two types of an oligomeric Strep-tactin® mutein acting as a soluble multimerization reagent. The first type of oligomeric Strep-tactin® was the fraction of the oligomeric streptavidin mutein (n = 3) obtained in Example 5 (in the same way referred to here as "conventional Streptactin® skeleton", illustrated by the triangle symbol with the tip below in Fig. 13), the second type of this oligomeric streptavidin mutein used as a soluble multimerization reagent was an oligomer that was obtained by reacting soluble oligomeric streptavidin mutein with biotinylated human serum albumin (HSA) This soluble multimerization reagent based in HSA in the same way referred to here as "large Streptactin® skeleton). In the experiments in Fig. 13 the expansion was carried out without changing media. The results for the CD4 + T-response cells are shown in Fig.13A, the results for CD8 + T response cells are shown in Fig. 13B In this context, it is noted that the experimentally used soluble multimerization reagents that have been reversibly functionalized Binding to the first agents, and optionally second and third agents are referred to in the Figures as "Streptamer® multimers"
[00087] Fig. 14 shows the proliferation expansion kinetics of purified CD4 + and CD8 + T response cells (Tresp) that were stimulated in vitro with αCD3 / αCD28 Fab fragments that were reversibly immobilized fragments that were reversibly immobilized with two Strep types -tactin® soluble oligomer acting as soluble multimerization reagent. The first type of oligomeric Streptactin® was the fraction of the oligomeric streptavidin mutein (n> 3) obtained in Example 5 (likewise referred to here as "conventional Streptactin® skeleton", illustrated by the triangle symbol with the tip at the top in Fig. 14), the second type of this oligomeric streptavidin mutein used as a soluble multimerization reagent was the HSA-based soluble multimerization agent, the "large Streptactin® backbone" mentioned above). In the experiments in Fig. 14 the expansion was carried out with a change of means. The results for the CD4 + T-response cells are shown in Fig.14, the results for the CD8 + T-response cells are shown in Fig. 14B.
[00088] Fig. 15 shows the combined data of the results obtained in Figs 13 and 14 for the proliferation expansion kinetics of purified CD4 + and CD8 + T response cells, with Fig. 15A describing the results for CD4 + and Fig T cells. 15B describing the results for CD8 + T cells. Linear lines are used for culture with media change on day 3, while dotted lines describe the values obtained for the degree of expansion without media change on day 3. The data shown in Fig. 15 are normalized to the input cell number . Only data for Tresp stimulated with oligomeric streptavidin mutein (n> 3), Tresp stimulated with commercially available Dynabeads (positive control) and unstimulated T cells (negative control) are shown but no data in the multimerization reagent with the "large Streptactin® skeleton".
[00089] Fig. 16 shows formation of early T cell clustering after activation of purified CD4 + and CD8 + T response cells stimulated in vitro with αCD3 / αCD28 Fab fragments that were reversibly immobilized on soluble oligomeric streptavidin mutein (n> 3 ) described in Example 5. Fig. 16A describes the results for CD4 + T cells and Fig. 16B describes the results for CD8 + T cells. Data for Tresp stimulated with the soluble multimerization reagent (oligomeric streptavidin mutein), Tresp stimulated with commercially available Dynabeads (positive control) and unstimulated T cells (negative control) are shown.
[00090] Fig. 17 shows the expansion kinetics and central memory phenotype of CD3 + (Tcm) T cells (CD3 + CD62L + CD45RA-Tcm) polyclonically stimulated in vitro with α α CD3 / α CD28 Fab fragments that were reversibly immobilized on the mutein soluble oligomeric streptavidin (with n> 3) described in Example 5. The graphs shown in Fig. 17 represent the degree of proliferation according to the number of cells harvested per time point, with Fig. 17A showing the proliferation is only supplemented medium IL-2 and in Fig. 17B showing proliferation in medium supplemented by IL-2 and IL-15. Fig. 17C shows a flow cytometry analysis of CD62L and CD127 surface expression after 14 days of culture in these variable cytokine environments.
[00091] Fig. 18 shows the selective antigen-specific expansion kinetics (Ag-specific) outside a volume population of purified CD3 + CD62L + CD45RA-Tcm response cells that were stimulated in vitro also with a peptide complex : MHC molecule (which acts as the first agent that provides a primary activation signal to cells) and Fab fragment aCD28 (which acts as the second agent that binds to an additional molecule on the cell surface) and unstimulated T cells (negative control) are shown. Likewise, the specific peptide-antigen complex with the MHC molecule and the Fab fragment aCD28 were reversibly immobilized on the same soluble oligomeric streptavidin mutein (with n> 3) described in Example 5. The peptide used for antigen-specific expansion in Fig. 18A was the CRVLCCYVL peptide (SEQ ID NO: 06), amino acids 309-317 of immediate-early protein 1 restricted by the HLA-C702 MHC molecule (described in Ameres et al., PLOS Pathogens, May 2013, vol. 9 , issue 5, e1003383) representing an HLA-C7 / IE-1 epitope that is specific for cytomegalovirus (CMV). The MHC I molecule that exhibits the peptide transport at its C-terminal streptavidin binding peptide heavy chain (SAWSHPQFEK (GGGS) 2GGSAWSHPQFEK (SEQ ID NO: 07), which is commercially available as a "Twin-Strep-tag ® "from IBA GmbH, Gottingen, Germany). Fig. 18A shows exemplary flow cytometry analysis for the fraction of Ag-specific cells that have been proliferated using the peptide: MHC-I complex specific for this HLA-C7 / IE-1 epitope as the first agent that provides an activation signal primer to cells reversibly immobilized in soluble oligomeric streptavidin mutein. The graphs in Fig.18B to Fig.18E illustrate the expansion kinetics of other specificities for antigens according to the number of peptide cells: specific MHCI-positive multimers harvested by time point in analogy to Fig. 18A using different complexes of one antigen-specific peptide with the MHC I molecule as the first agent that provides a primary activation signal to cells reversibly immobilized in soluble oligomeric streptavidin mutein. In more detail, Fig. 18B shows the expansion of Ag-specific cells that were expanded using the peptide complex: MHC-I specific for the CMV pp65 epitope (amino acids 341-350 (QYDPVAALF, (SEQ ID NO: 08) ) restricted by HLA-A2402), Fig. 18C shows the expansion of Ag-specific cells that were expanded using another peptide complex: MHC-I specific for the CMV pp65 epitope (amino acids 265-274 RPHERNGFTV, (SEQ ID NO : 09)) restricted by HLA-B702), Fig. 18D shows the expansion of Ag-specific cells that have been proliferated using the adenovirus hexon 5 epitope specific MHC-I complex (amino acids 114-124 (CPYSGTAYNSL, (SEQ ID NO: 10)) restricted by HLA-B702), Fig. 18E shows the expansion of Ag-specific cells that have been proliferated using the HLA-B7 / IE-1309 epitope-specific MHC-I complex -317 CMV (exemplary FACS data see Fig. 18A above). All peptide: MHC molecules supporting the Twin Strep®-Tag are commercially available from IbaGmbH. In this context, the amino acid sequences of the HLA-A * 2402, HLA-B * 0702 and HLA-C * 0702 molecules that carry the "Twin-Strep-tag®" as its C-terminal are shown as SEQ ID NO: 21, 22 and 23 in the accompanying Sequence listings, while the β2 microglobulin amino acid sequence (which forms together with the α chain, meaning the HLA-encoded molecules, the respective MHC I molecule) is shown as SEQ ID NO: 24 in the accompanying Sequence Listing. In addition, Fig.18F shows exemplary flow cytometry analysis of CD62L and CD127 surface expression after 14 days of culture for HLA-B7 / Hexon5114-124 stimulated / expanded cells of Fig. 18D.
[00092] Fig. 19 shows the selective kinetics of Ag-specific expansion out of purified CD3 + CD62L + CD45RA-response Tcm cells that were stimulated in vitro with a) antigen specific peptide MHC I complexes and b) Fab αCD28 fragments which were reversibly immobilized as first and second agents on soluble oligomeric streptavidin muteins. For this purpose 500,000 CD3 + CD62L + CD45RA- response Tcm (Tresp) cells were specifically stimulated by the Ag using 3 μL of a Streptactin multimerization reagent preparation functionalized with 0.5 μg of peptide complexes: MHC class I equipped with a streptavidin-binding peptide (the specific peptide represents amino acids 114-124 (CPYSGTAYNSL, SEQ ID NO: 10) of the HLA-B0702-restricted adenovirus Hexon 5 protein, see above) and 0.5 μg of α α28 Fab . As an alternative, 4.5 μL of a Streptactin multimerization reagent preparation loaded with 0.5 μg of this peptide complex: MHC class I, 0.5 μg of Fab αCD8 and 0.5 μg of Fab αCD28. For comparison, polyclonal stimulation was performed, using 3 μL of a preparation of Streptactin multimerization reagent (1mg / mL) loaded with a combination of 0.5 μg of Fab αCD3 and 0.5 μg of Fab αCD28. Again as the alternative stimulus condition described above, 4.5 μL of a preparation of Streptactin multimers loaded with 0.5 μg of Fab αCD3, 0.5 μg of Fab αCD8 and 0.5 μg of Fab αCD28 was used. Untreated (unstimulated) Tresp cells served as a negative control and Tresp cells stimulated with polyclonal Dynabeads as a positive control. Tresp cells were seeded in 48-well plates in 1 ml of cell culture medium supplemented with 30 U / ml of IL-2 and 5 ng / ml of IL-15. Cells were incubated at 37 ° C with change of media at every 3 days and cell count was analyzed after 7 and 14 days. The photographs shown in Fig. 19 represent the degree of cluster formation on day 5 for Ag-specific stimulus as exemplified for the HLA-B7 / Hexon 5 adenovirus epitope.
[00093] Fig. 20 shows the yield and expansion phenotype of purified CD8 + T response cells stimulated in vitro with Fab αCD3 / αCD28 fragments that were reversibly immobilized in two soluble oligomeric Strep-tactin® types acting on a soluble multimerization reagent. The first type of oligomeric Strep-tactin® was the fraction of the oligomeric streptavidin mutein (numeric obtained in Example 5 (conventional structure), the second type of this oligomeric streptavidin mutein used as the soluble multimerization reagent was the soluble oligomer described above and referred to here as a "large" Streptactin® structure. In these experiments, the fraction of the conventional oligomeric streptavidin mutein (n> 3) was similarly used as a multimerization reagent that was functionalized with a single Fab fragment (third bar in Fig. 20A and Fig. 20B) or with a combination of Fab fragments aCD3 and aCD28 In addition to the stimulus combined with Fab fragments αCD3 / αCD28, likewise an additional Fab aCD8 fragment (commercially available from IBA GmbH, Gottingen, Germany) was immobilized to test whether it is possible to preferentially stimulate a specific T cell subpopulation. Fig. 20A shows a bar graph representing the grain proliferation u according to the number of cells harvested on day 6 compared to negative controls (unstimulated purified CD8 + T response cells) and normalized to positive control (purified CD8 + T response stimulated with commercially available Dynabeads (beads on which antibodies monoclonal αCD3 and aCD28 are irreversible immobilized). Fig. 20B shows flow cytometry analysis of CD8 surface expression and the CD45RO T cell surface molecule (which is indicative of T cell proliferation and activation) after cell culture. The various stimulating conditions were compared using one-way ANOVA analysis and no significant differences (n.s.) were detected.
[00094] Fig. 21 shows the yield and phenotype for the expansion of purified CD8 + response T cells stimulated in vitro with Fab αCD3 / αCD28 fragments that were reversibly immobilized on soluble oligomeric Strep-tactin® acting as a soluble multimerization reagent that were functionalized with a single Fab fragment or with a combination of Fab fragments (as already described above). In these experiments, CD8 + response T cells were stimulated with the soluble multimerization reagent (Strepactin® soluble oligomeric (1mg / mL) from Example 5) which was functionalized with varying amounts of αCD3 and aCD28 Fab fragments, optionally together with the αCD8 Fab fragment described above. The term “1x” corresponds to 1.5 μg of functionalized multimerized Streptactin with 0.5 μg of aCD3 Fab fragment only and 1.5 μg of functionalized multimerized Streptactin with 0.5 μg of aCD28 Fab only), or 3 μL of a preparation Oligomeric streptactin loaded with 0.5 μg of Fab aCD3 fragment and 0.5 μg of Fab aCD28, or 4.5 μL of a preparation of Streptactin multimers loaded with 0.5 μg of strep-labeled aCD3, 0.5 μg of aCD8 strep-r labeled and 0.5 μg Fab αCD28 strep- labeled. In this way, the term “2x” corresponds to 3.0 μg of multimerized functionalized Streptactin with 1μg of aCD3 Fab fragment alone and 3.0 μg of functionalized multimerized Streptactin with 1 μg of α α2828 Fab alone, meaning that twice the amount of fragment Immobilized αCD3 Fab was used. Untreated Tresp cells served as a negative control and T response of purified CD8 + stimulated with commercially available Dynabeads (spheres in which αCD3 and aCD28 monoclonal antibodies are irreversible immobilized) as a positive control. Fig. 21A shows a graph in which the bars represent the degree of proliferation according to the number of cells harvested on day 5 compared to the negative controls and normalized to the positive control. Fig. 21B show FACS analysis of CD8 and CD45RO surface expression after cell culture.
[00095] Fig. 22 shows the activation of intracellular signaling cascades from transfected Jurkat cells that have been modified to express an αCD19 chimeric antigen receptor (CAR), and that have been stimulated using the oligomeric Strep-tactin® of Example 5 as a reagent of soluble multimerization. The specificity of a CAR is typically derived from a 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 to cell-specific signaling T (described in Hudecek et al., Clin Cancer Res. June 15, 2013; 19 (12): 3153-3164. In the experiments, the extracellular domain (ECD) of CD19, which contains the natural CD ligand of aCD19 as well as the polyclonal dgG F (ab) 2 fragment that recognizes donkey anti-human IgG4 (F (ab) 2 spacer is commercially available from Jackson Immuno Research) within aCD19-CAR were likewise used in this experiment as the first agent which provides a primary activation signal to the Jurkat cells. The reversibly immobilization for the soluble oligomeric streptavidin mutein was provided by the streptavidin peptide SAWSHPQFEK (GGGS) 2GGS- AWSHPQFEK (SEQ ID NO: 07) which was fused to the ECD C-terminal of ECD CD19 or by the biotinylated (Fab) 2 fragment of dgG (since streptavidin mutein "m2" binds biotin with reduced affinity, this bond is reversible and can for example be displaced by the addition of an excess of free biotin). In the control experiment of Fig.22A 300,000 Jurkat CD3 + response cells (Jresp) were stimulated with varying amounts of a mixture of preparations Oligomeric streptactin (1mg / mL) that was functionalized with Fab aCD3 and Fab aCD28 (“1x” corresponds to 3 μg of functionalized multimerized Streptactin with 0.5 μg of aCD3- and 0.5 μg of Fab aCD28-polyclonal Streptamer multimer). In the experiment in Fig. 22B 3 μL of an oligomeric Streptactin preparation was functionalized with 0.5 μg (x1) or 1 μg (x2) of the CD19 extracellular domain (ECD) or with 3μL of an oligomeric Streptactin preparation loaded with 0 , 5μg (x1) or 1μg (x2) αIgG that recognizes the IgG4 spacer (which are both Streptamer® CAR-specific multimers). Jresp stimulated with Dynabeads (spheres in which αCD3 and aCD28 monoclonal antibodies are irreversible immobilized) or PMA and ionomycin served as positive controls. Jresp cells were seeded in 1.5 ml of Eppendorf tubes in 200 μL of cell culture medium supplemented with 30 U / ml of IL-2. Cells were incubated at 37 ° C and placed on ice and lysed after 0 min to 20 min of stimulation.
[00096] Fig. 23 shows the expansion of purified CD3 + T-response cells stimulated in vitro with αCD3 / αCD28 Fab fragments that were reversibly immobilized on the soluble oligomeric Strep-tactin® of Example 5 which served for a soluble multimerization reagent. In one experiment, in addition to αCD3 / αCD28 Fab fragments, a commercially available aCD8 Fab fragment from IBA GmbH, Gottingen, Germany (catalog number 6-8000-203) was immobilized on the streptavidin mutein soluble oligomer to test whether it is possible to preferentially stimulate the CD8 + T cell subpopulation within the CD3 + culture by volume with a multimerization reagent of the invention having a Fab aCD8 fragment reversibly immobilized thereto in the same way. In more detail, 500,000 purified CD3 + response T cells (Tresp) were stimulated with 3 μL of an oligomeric Streptavidin preparation (1mg / mL) loaded with a combination of 0.5 μg of aCD3 and 0.5 μg of Fab aCD28. As an alternative approach, 4.5 μL of the Streptactin oligomer was loaded with 0.5 μg aCD3, 0.5 μg Fab aCD8 and 0.5 μg Fab aCD28 described above. Unstimulated Tresp cells served as a negative control and Tresp stimulated with Dynabeads (spheres in which αCD3 and aCD28 monoclonal antibodies are irreversible immobilized) served as positive control.
[00097] Fig. 24 describes exemplary strategies for the generation of oligomeric streptavidin muteins that can be used as the soluble multimerization reagent of the invention. Fig. 24A shows that in a first step, the streptavidin mutein "m2" (SAm2) comprising the amino acid sequence lle44-Gly45-Ala46-Arg47 (SEQ ID NO: 03) in the sequence positions 44 to 47 of streptavidin type Wild is used for generation of oligomeric streptavidin muteins having a "conventional structure." In a second step, soluble oligomeric streptavidin muteins having a "large structure" can be generated by coupling streptavidin mutein with biotinylated carrier protein such as human serum albumin (HSA) or by coupling streptavidin muteins with synthetic vehicles such as PEG. Fig. 24B: Biotinylation of human serum albumin (HSA). DETAILED DESCRIPTION OF THE INVENTION
[00098] The present invention provides methods, kits and an apparatus for expanding a population of cells or for inducing a population of T cells to proliferate.
[00099] The term "cell population" when used herein encompasses all cells that can be expanded by binding to a cell surface receptor a first agent that provides a primary activation signal to the cells. It is just as possible that for expansion of the cell population, binding of the second agent to a second cell surface receptor (additional molecule) might be necessary to produce a co-stimulatory signal required for cell expansion. In some embodiments, the cell population may be a lymphocyte population including, but not limited to, a population of B cells, a population of T cells, or a population of natural exterminating cells. Illustrative examples of cell populations are B cells carrying CD40 or CD137 (likewise the cell population can be proliferated under the binding of only a first agent that provides an activation signal, for example 4-1BB ligand; or an antibody molecule for αCD40 or an antibody molecule for αCD137 (see for example Zhang et al., 2010, J Immunol, 184: 787-795)). Other illustrative examples for agents (first or second) that can be used for B cell expansion are agents that bind to IgG, CD19, CD28 or CD14, for example antibody molecules for αCD19, aIgG, αCD28, or aCD14. It is also sensed that first or second agents for B cell expansion may comprise ligands for toll like receptors or interleukins, such as IL-21 (see for example Dienz O, 2009. J. Exp. Med. 206: 69) . It is noted that B cell lipopolysaccharide dependent activation is likewise covered in the present invention, as a lipopolysaccharide can likewise be used as the first agent and can be equipped with a C1 binding partner when used here. Other illustrative examples of suitable cell populations include a T cell population that expands after being activated by binding a first agent to TCR / CD3 and binding a second agent to an additional molecule in the T cell such as CD28. In this case, the first agent stimulates a signal associated with the TCR / CD3 complex in T cells and the second agent provides a secondary stimulus by binding CD28 as an additional molecule. Agents that can be used for T cell expansion can likewise include interleukins, such as IL-2, IL-7, IL-15, or IL-21 (see Cornish and others for example. 2006, Blood. 108 ( 2): 600-8, Bazdar and Sieg, 2007, Journal of Virology, 2007, 81 (22): 12670-12674, Battalia and others, 2013, Immunology, 139 (1): 109-120). Other illustrative examples for agents that can be used for T cell expansion are agents that bind to CD8, CD45 or CD90, such as antibodies to αCD8, aCD45 or aCD90. Illustrative examples of T cell population including antigen-specific T cells, inducing T cells, cytotoxic T cells, memory T cell (an illustrative example of memory T cells are CD62L + CD8 + central memory T cells) or regulatory T cells (an illustrative example of Treg are CD4 + CD25 + CD45RA + Treg cells). The term "T cell (population)" when used in the same way herein includes T cells comprising a chimeric antigen receptor (CAR) which is likewise known as artificial T cell receptors or chimeric T cell receptors. In this way, a T cell population comprising a chimeric antigen receptor can likewise be expanded using the methods, reagents and devices of the present invention. See in this respect in the same way Example 15 in which Jurkat cells expressing for a chimeric CD19 specific antigen receptor (CAR) were stimulated using a soluble multimerization reagent of the present invention. Another illustrative example of a suitable cell population includes natural killer cells (NK cells), which can for example be expanded with agents that bind to CD16 or CD56 such as, for example, antibodies to aCD16 or aCD56. An illustrative example for such an antibody for aCD16 is the 3G8 antibody with a VH sequence mentioned in SEQ ID NO: 25 and a VL sequence mentioned in SEQ ID NO: 26 (see Hoshino et al., Blood, for example. Dec 1991 15; 78 (12): 3232-40.). Another agent that can be used to expand NK cells can be IL-15 (see for example Vitale et al. 2002. The Anatomical Record. 266: 87-92). Yet another illustrative example of a suitable cell population includes monocytes, which can for example be amplified using an agent that binds to CD14 such as an antibody molecule to aCD14. 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 monkeys, macaque, or a chimpanzee.
[000100] Thus, according to the above, this invention pertains to methods for selectively inducing ex vivo expansion of a cell population such as B cells, T cells or natural killer cells in the absence of exogenous growth factors, such as lymphokines , and additional cells. In addition, the proliferation of these cells such as B cells or T cells can be induced without the need for antigen, thereby providing an expanded cell population such as a T cell population that is polyclonal with respect to antigen reactivity. The methods described here can provide prolonged proliferation of a selected population of T cells such as CD4 + or CD8 + T cells over an extended period of time to produce a multiple-fold increase in the number of these cells relative to the original T cell population. In general, in the case of a (clonal) expansion of a lymphocyte population as described here, the entire progeny can share the same antigen specificity as the cell population that has been selected for expansion.
[000101] In the same way as above, provided by this invention are methods for expanding a population of antigen-specific T cells. To produce a population of antigen-specific T cells, T cells are connected with an antigen in a form suitable for activating a primary activation signal in the T cell, that is, the antigen is presented to the T cell such that a signal is activated in the T cell through the TCR / CD3 complex. For example, the antigen can be presented to the T cell by an antigen presenting a cell together with an MHC molecule. A cell showing antigen, such as a B cell, macrophage, monocyte, dendritic cell, Langerhans cell, or another cell that can present antigen to a T cell, can be incubated with the T cell in the presence of the antigen (for example, a soluble antigen) such that cells presenting antigen present the antigen to the T cell. Alternatively, a cell expressing an antigen of interest can be incubated with the T cell. For example, a tumor cell expressing tumor-associated antigens can be incubated with a T cell together to induce a tumor-specific response. Similarly, a cell infected with a pathogen, for example, a virus that has antigens of the pathogen can be incubated with a T cell. Following specific antigen activation of a population of T cells, the cells can be enlarged according to the methods of invention. For example, after antigen specificity has been established, T cells can be expanded by culture with an anti-CD3 antibody (used as a first agent) and an anti-CD28 antibody (used as a second agent) according to the methods described here. In another embodiment, the first agent may be a peptide complex: MHC I, which binds to an antigen-specific T cell population. In such an embodiment, any antigen-specific peptide that is known and that can be complexed with the respective MHC I molecule can be used. See in this respect Examples 11 and 12 in which selective Antigen-specific expansion of Tcm response cells out of CD3 + central memory T cells in volume was exemplified for four different antigen-specific cells. Alternatively, it is likewise possible to use the natural ligand of a receptor that activates cell expansion as the first agent. See in this respect Example 15 where the extracellular domain of CD19 caused the activation of intracellular signaling cascades of transduced Jurkat cells that were modified to express chimeric CD19 binding antigen (CAR) receptor.
[000102] The sample of the cell population can be from any suitable source, typically the entire sample of a body tissue or a body fluid such as blood. In the latter case, the sample may for example be a population of peripheral blood mononucleated cells (PBMC) that can be obtained by standard isolation methods such a blood cell ficol gradient. The cell population to be expanded may, however, likewise be in purified form and could have been isolated using reversible cell marking / isolation technology as described in US patent 7,776,562, US patent 8,298,782, application for International patent WO02 / 054065 or International patent application WO2013 / 011011. Alternatively, the cell population can likewise be obtained by cell classification by means of negative magnetic immunoadhesiveness as described in US patent 6,352,694 B1 or European patent EP 0 700 430 B1. If an isolation method described here is used in basic research, the sample could be cells from in vitro cell culture experiments. The sample would typically have been prepared in the form of a fluid, such as a solution or dispersion.
[000103] According to the above, in one embodiment the invention provides an in vitro method of expanding a population of cells, comprising contacting a sample comprising a population of cells with a multimerization reagent. The multimerization reagent has reversibly immobilized (attached thereto) a first agent that provides a primary activation signal to the cells, wherein the multimerization reagent comprising at least one Z1 binding site for the reversible binding of the first agent. The first agent comprises at least one C1 binding partner, in which the C1 binding partner is capable of reversibly binding to the Z1 binding site of the multimerization reagent, in which the first agent is linked to the multimerization reagent by reversible binding formed between the C1 binding partner and the Z1 binding site. The first agent binds to a receptor molecule on the surface of cells, thereby providing a primary activation signal to the cells and thereby activating the cells.
[000104] In another embodiment, the invention provides a method, in which the multimerization agent has reversibly immobilized on it (attached to it) a second agent that stimulates an additional molecule on the cell surface. The second agent comprises a C2 binding partner, in which the C2 binding partner is capable of being reversibly linked to a Z2 binding site of the multimerization reagent, in which the second agent is linked to the multimerization reagent by the reversible bond formed between the binding partner C2 and the binding site Z2. The second agent binds to the additional molecule on the surface on the cell surface, thereby stimulating the activated cells. In this embodiment, the first agent can stimulate a signal associated with the TCR / CD3 complex in T cells and can be a binding agent that specifically binds to CD3. In this embodiment the additional molecule in the T cell can be CD28 and the second agent that binds to the additional molecule is a binding reagent that specifically binds to CD28. In this case, the first agent that specifically binds to CD3 can be selected from the group consisting of an anti-CD3 antibody, a divalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody. -CD3, and CD3-binding protein molecule with antibody-like binding properties. Likewise, the second agent that specifically binds to CD28 can be selected from the group consisting of an anti-CD28 antibody, a divalent antibody fragment of an anti-CD28 antibody, a monovalent antibody fragment of an anti-CD28 antibody. -CD28, and a protein binding molecule of CD28 with antibody-like binding properties. The divalent antibody fragment can be a (Fab) 2 'fragment, or a divalent single chain Fv fragment while the monovalent antibody fragment can be selected from the group consisting of a Fab fragment, an Fv fragment, and a single chain Fv fragment (scFv). A protein molecule binding to CD3 or CD28 with antibody-like binding properties can be an aptamer, a mutein based on a polypeptide of the lipocalin family, a glucorpo, a protein based on the ankyrin framework, a protein based on crystalline framework, an adnectin, and an avimer.
[000105] In general the first and second agents that are used in the present invention can, for example, be an antibody, a fragment thereof and a protein binding molecule with antibody-like functions. Examples of (recombinant) antibody fragments are Fab fragments, Fv fragments, single chain Fv fragments (scFv), a divalent antibody fragment such as a (Fab) 2 'fragment, diabody, triabody (Iliades, P. , and another, FEBS Lett (1997) 409, 437-441), decibodies (Stone, E., and others, Journal of Immunological Methods (2007) 318, 88-94) and other domain antibodies (Holt, LJ, and another, Trends Biotechnol. (2003), 21, 11, 484490). In some embodiments, one or more binding sites of the first or second agents may be a bivalent artificially binding protein molecule such as a dimeric lipocalin mutein which is similarly known as "duocalin." In some embodiments, the receptor binding reagent may have a single second binding site, that is, it may be monovalent. Examples of first or second monovalent agents include, but are not limited to, a monovalent antibody fragment, a protein binding molecule with antibody-like binding properties, or an MHC molecule. Examples of monovalent antibody fragments include, but are not limited to, a Fab fragment, an Fv fragment, and a single chain Fv fragment (scFv), including a divalent single chain Fv fragment.
[000106] As mentioned above, an example of a protein binding molecule with antibody-like functions is a mutein based on a polypeptide 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 the bilin binding protein, human neutrophil gelatinase-associated lipocalin, human Apolipoprotein D, or human tear lipocalin have natural ligand binding sites that can be modified so that they bind a particular target. Other examples of a protein binding molecule with antibody-like binding properties that can be used as a receptor binding reagent that specifically binds to the receptor molecule include, but are not limited to, the so-called glucibodies (see for example international patent application WO 96/23879), proteins based on the ankyrin framework (Mosavi, LK, and other, Protein Science (2004) 13, 6, 14351448) or crystalline framework (for example international patent application WO 01/04144 ) the proteins described in Skerra, J. Mol. Recognit. (2000) 13, 167-187, AdNectins, tetranoctins and avimers. Avimers, including multivalent avimer proteins evolved by mixing exon from a family of human receptor domains, which contain so-called A-domains that occur as strands of multiple domains at various cell surface receptors (Silverman, J., and others, Nature Biotechnology (2005) 23, 1556-1561). Adnectins, derived from a human fibronectin domain, contain three loops that can be created for immunoglobulin-like binding to targets (Gill, D.S. & Damle, N.K., Current Opinion in Biotechnology (2006) 17, 653-658). Tetranoctins, derived from the respective human homotrimeric protein, also contain loop regions in a type C lectin domain that can be created for desired binding (ibid.). Peptides that can act as protein ligands are oligo (N-alkyl) glycines that differ from peptides in which the side chain is connected to amide nitrogen instead of the α carbon atom. Peptides are typically resistant to proteases and other modifying enzymes and may have a much higher cell permeability than peptides (see for example Kwon, Y.-U., and Kodadek, T., J. Am. Chem. Soc. (2007) 129, 1508-1509). Still other examples of suitable protein binding molecules are an EGF-like domain, a Kringle domain, a type I fibronectin domain, a type II fibronectin domain, a type III fibronectin domain, a PAN domain, a G1a domain, a SRCR domain, a pancreatic Kunitz / Bovine trypsin inhibitor domain, tendamistat, a Kazal type serine protease inhibitor domain, a Trefoil (type P) domain, a von Willebrand factor type C domain, an Anaphylatoxin-like domain, a CUB domain, a type I thyroglobulin repeat, an LDL receptor class A domain, a Sushi domain, a Binding domain, a Thrombospondin type I domain, an immunoglobulin domain, or an immunoglobulin-like domain (for example, antibodies to camel heavy chain domain or antibodies), a type C lectin domain, a MAM domain, a von Willebrand factor type A domain, a Somatomedin B domain, a central four disulfide domain po WAP, a F5 / 8 type C domain, a Hemopexin domain, an SH2 domain, an SH3 domain, a Laminin-like EGF-like domain, a C2 domain, "Kapacorpos" (cf. Ill. And another, Protein Eng (1997) 10, 949-57, a so-called "minibody" (Martin and another, EMBO J (1994) 13, 5303-5309), a diabody (cf. Holliger and another, PNAS USA ( 1993) 90, 6444-6448), one called "Janusis" (cf. Traunecker et al, EMBO J (1991) 10, 3655-3659, or Traunecker et al, Int J Cancer (1992) Suppl 7, 51-52) , a nanobody, a microbody, an afiline, an afibody, a knotin, ubiquitin, a zinc-binding protein, an autofluorescent protein or a leucine-rich repeated protein. An example of a nucleic acid molecule with antibody-like functions is an aptamer. An aptamer bends into a defined three-dimensional motif and shows high affinity for a given target structure.
[000107] Now returning the multimerization reagent, the binding sites Z1 and Z2 of the multimerization agent can be identical (see the example of Fig. 3 in the same way). In this case, a single multimerization agent can be used.
[000108] In the embodiment that a first reversible binding agent and, optionally, a second agent is used, the multimerization reagent can be immobilized on a solid surface. Any solid surface (support) can be used to immobilize the multimerization reagent. Illustrative examples of solid surfaces on which the multimerization reagent can be immobilized include a magnetic sphere, a polymeric sphere, a cell culture plate, a microtiter plate, a membrane, or a hollow fiber. Hollow fibers are, for example, used as a bioreactor in the Quantum® Cell Expansion System, available from TerumoBCT Inc. (Lakewood, CO, USA). The multimerization reagent is normally covalently attached to the solid support, however, non-covalent interactions can likewise be used for immobilization, for example on plastic substrates, if desired. As explained in more detail below, the multimerization reagent can, for example, be a streptavidin or avidin mutein that reversibly binds a binding peptide to streptavidin. Such streptavidin muteins can be covalently attached to any surface, for example, resin (spheres) used for chromatography purification and is commercially available in such form from IBA GmbH, Gottingen, for example, as Strep-Tactin® Sepharose , Strep-Tactin® Superflow®, Strep-Tactin® Superflow® high capacity or Strep-Tactin® MacroPrep®. Other illustrative example multimerization reagents that are easily commercially available are immobilized by metal affinity chromatography resins (IMAC) such as TALON® resins (Westburg, Leusden, The Netherlands) that can be used for reversible protein immobilization marked by oligohistidine (His tag) in general, meaning here, for the reversible binding of a first or a second agent that carry as an first C1 binding partner or second C2 binding partner an oligohistidine tag such as a penta- or hexa-histidine. Other examples of multimerization reagents are calmodulin sepharose available from GE Life Sciences which can be used together with a first or second agent comprising a calmodulin binding peptide as a C1 or C2 binding partner or sepharose, which is glutathione. coupled. In this case, the C1 or C2 binding partner is glutathione-S-transferase.
[000109] In other embodiments of the method of the invention the multimerization reagent can be in a soluble form. In principle, the same multimerization agents can be used as in the case of a multimerization reagent that is immobilized on a solid support. The multimerization reagent is a soluble form, for example, it may be a streptavidin mutein oligomer, a calmodulin oligomer, a compound (oligomer) that provides at least two chelating groups K, in which at least two chelating groups are capable of binding to a transition metal ion, thereby rendering portion A capable of binding to an oligohistidine affinity tag, multimeric glutathione-S-transferase, or a biotinylated carrier protein.
[000110] As explained above, the first and second agent has, in addition to the binding site that is capable of binding the respective cell surface receptor molecule, a C1 or C2 binding partner (which will be referred to as "C binding partner "in the following for ease of reference). This binding partner C is capable of binding to a Z binding site of the multimerization reagent (Z means any Z1 binding site or Z2 binding site of the multimerizing reagent) C. The non-covalent bond that is formed between the binding partner C that is included in the first or second agent and the binding site (s) Z of the multimerization reagent can be of any desired strength and affinity, as long as it is breakable or reversible under conditions under which the method of the invention is carried out. The dissociation constant (KD) of the bond between bonding partner C that is included in the receptor binding reagent and the Z binding site of the multimerization reagent can have a value in the range of about 10-2 M to about 10-13 M. Thus, this reversible connection can, for example, have a KD of about 102 M to about 10-13 M, or from about 10-3 M to about 10-12 M or about from 10-4 M to about 10-11M, or from about 10-5 M to about 10-10M. The KD of this bond as well as the rate of KD, koff and kon of the bond formed between the B binding site of the receptor binding reagent and the receptor molecule can be determined by any suitable means, for example, by fluorescence titration, dialysis of equilibrium or resonance of surface plasmon. The receptor molecule binding reagent can include at least one, including two, three or more, second C binding partners, and the affinity reagent can include at least two, such as three, four, five, six, seven, eight or more binding sites for the binding partner that is included in the receptor molecule binding reagent. As described in US patent 7,776,562, US patent 8,298,782 or International patent application WO 2002/054065 any combination of a C-binding partner and an affinity agent with one or more corresponding Z-binding sites can be chosen, provided that the binding partner C and the binding site Z of the affinity agent are capable of reversibly binding or multimerizing in a (multivalent) complex that typically accompanies with an avid effect.
[000111] The binding partner included in the first or second agent can be an oligopeptide, a polypeptide, a protein, a nucleic acid, a lipid, a saccharide, an oligosaccharide, or a polysaccharide. Such a binding partner has a higher affinity for the multimerization reagent binding site than for another subject. Examples of a respective binding partner include, but are not limited to, an immunoglobulin molecule, a fragment thereof and a protein binding molecule with antibody-like functions.
[000112] In some embodiments the binding partner C that is included in the first or second agent includes biotin and the affinity reagent includes a streptavidin analogue or an avidin analogue that reversibly binds biotin.
[000113] In some embodiments the C-binding partner that is included in the first or second agent includes a biotin analog that reversibly binds to streptavidin or avidin, and the affinity reagent includes streptavidin, avidin, a strepta-vidin analogue or an avidin analogue that reversibly binds to the respective biotin analogue.
[000114] In some other embodiments the C binding partner that is included in the first or second agent includes a streptavidin or avidin binding peptide and the affinity reagent includes streptavidin, avidin, a streptavidin analog or an avidin analog that reversibly binds to the respective streptavidin or avidin binding peptide.
[000115] In some embodiments the binding partner that is included in the first or second agent may include a streptavidin binding peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 01) and the affinity reagent may include a streptavidin mutein (analogue) comprising the amino acid sequence Va144-Thr45-Ala46-Arg47 (SEQ ID NO: 02) at sequence positions 44 to 47 of wild-type streptavidin or streptavidin mutein (analogue ) which comprises the amino acid sequence lle44-Gly45-Ala46-Arg47 (SEQ ID NO: 03) at sequence positions 44 to 47 of wild-type streptavidin both of which are described in US patent 6,103,493 for example, and are commercially available under the trademark Strep-Tactin®. For example, streptavidin-binding peptides could be unique peptides such as the "Strep-tag®" described in US patent 5,506,121, for example, or streptavidin-binding peptides having a sequential arrangement of two or more binding modules as described in International Patent Publication WO 02/077018 or US patent 7,981,632.
[000116] In some embodiment, the C liaison partner of the first or second agent includes a portion known to the technician versed as an affinity tag. In such an embodiment the affinity reagent includes a corresponding binding partner, for example, an antibody or an antibody fragment, known to bind to the affinity tag. As some illustrative examples of known affinity tags, the binding partner that is included in the first or second agent can include an oligohistidine, an immunoglobulin domain, maltose binding protein, glutathione-S-transferase (GST), binding protein chitin (CBP) or thioredoxin, calmodulin-binding peptide (CBP), FLAG'-peptide, HA tag (sequence: Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala, (SEQ ID NO: 11)), VSV-G tag (sequence: Tyr-Thr-Asp-Ile-Glu-Met-Asn-Arg-Leu-Gly-Lys, (SEQ ID NO: 12)), HSV tag (sequence: Gln-Pro -Glu-Leu-Ala-Pro-Glu- Asp-Pro-Glu-Asp, (SEQ ID NO: 13)), 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 the sequence Gln-Pro-Glu-Leu- Ala-Pro-Glu-Asp-Pro-Glu-Asp (SEQ ID NO: 13) herpes simplex virus glycoprotein D, "myc" epitope of the transcription factor c-myc of the sequence Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu (SEQ ID NO: 15), the V5 tag (sequence ia: 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 the multimerization reagent, in this case an antibody or antibody fragment, and the antigen can be disrupted competitively by adding the free antigen, that is, the free peptide (tag). epitope) or the free protein (such as MBP or CBP). The affinity tag could likewise be an oligonucleotide tag. Such an oligonucleotide tag can, for example, be used to hybridize to an oligonucleotide with a complementary sequence, linked to or included in the affinity reagent.
[000117] In some embodiments, the link between the C binding partner that is included in the first or second agent and one or more binding sites of the multimerization reagent occurs in the presence of a divalent, trivalent or tetravalent cation. In this regard, in some embodiments, the multimerization reagent includes a divalent, trivalent or tetravalent cation, typically sustained, for example complexed, by means of a suitable chelator. The binding partner that is included in the receptor binding reagent may in such an embodiment include a portion that includes, for example complexes, a divalent, trivalent or tetravalent cation. Examples of a respective metal chelator include, but are not limited to, ethylenediamine, ethylene diaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), diethylenetriaminepenetaacetic acid (DTPA), N, N-bis (carboxymethyl) glycine (similarly called nitrilotriacetic acid, NTA), or 1,2-bis (o-aminophenoxy) ethane-N, N, N ', N'-tetraacetic acid (BAPTA). As an example, EDTA forms a complex with more monovalent, divalent, trivalent and tetravalent metal ions, such as for example calcium (Ca2 +), manganese (Mn2 +), copper (Cu2 +), iron (Fe2 +), cobalt (Co3 +) and zirconium (Zr4 +), while BAPTA is specific for Ca2 +. As an illustrative example, a standard method used in the art is the formation of a complex between an oligohistidine tag and copper (Cu2 +), nickel (Ni2 +), cobalt (Co2 +), or zinc (Zn2 +) ions that are presented through of chelating nitrilotriacetic acid (NTA).
[000118] In some embodiments the C-binding partner that is included in the first or second agents includes a calmodulin-binding peptide and the affinity reagent includes multimeric calmodulin as described in US patent 5,985,658 or as described here with reference to Figure 2, for example. In some embodiments, the C binding partner that is included in the first or second agent includes a FLAG peptide and the affinity reagent includes an antibody that binds to the FLAG peptide, for example the FLAG peptide, which binds to the monoclonal antibody 4E11 as described in US patent 4,851,341. In one embodiment, the C-binding partner that is included in the first or second agent includes an oligohistine tag and the affinity reagent includes an antibody or a transition metal ion that binds to the oligohistine tag. The disruption of all of these binding complexes can be accomplished by metal ion chelation, for example calcium chelation, for example by adding EDTA or EGTA (supra). Calmodulin, antibodies such as 4E11 or chelated metal ions or free chelators can be multimerized by conventional methods, for example by biotinylation and complexation with streptavidin or avidin or multimers thereof or by the introduction of carboxyl residues in a polysaccharide, for example dextran, essentially as described in Noguchi, A. and another Bioconjugate Chemistry (1992) 3, 132-137, in a first step and linking calmodulin or free chelated or chelated antibodies or metal ions by means of primary amino groups to the carboxyl groups in the polysaccharide, eg dextran, skeleton using conventional carbodiimide chemistry in a second step. In such embodiments, the bond between the bonding partner C that is included in the first or second agent and the one or more Z bond sites of the multimerization reagent can be disrupted by metal ion chelation. For example, metal chelation can be performed by adding EGTA or EDTA.
[000119] In some embodiments, in particular, if the multimerization reagent is in soluble form and is based on streptavidine or avidin, it is an oligomer or a polymer of streptavidin or avidin or any streptavidin or analog mutein (analog) avidin. The Z binding site is the natural biotin binding of avidin or streptavidin. The respective oligomer or polymer can be cross-linked by a polysaccharide. In one embodiment, streptavidin or avidin or mutein polymers (analogues) of streptavidin or avidin are prepared by introducing carboxyl residues into a polysaccharide, for example, dextran, essentially as described in Noguchi, A, and another , Bioconjugate Chemistry (1992) 3,132-137 in a first stage. Then streptavidin or avidin or analogues thereof can be linked via primary amino groups of internal lysine residue and / or the free N-terminus to the carboxyl groups on the dextran backbone using conventional carbodiimide chemistry in a second step. In addition, strigtavidin or avidin crosslinked oligomers or polymers or any streptavidin or avidin mutein (analog) can likewise be obtained by crosslinking individual streptavidin or avidin molecules (the tetrameric homodimer of streptavidin or avidin is referred to here as an "individual molecule" or smaller building block of a respective oligomer or polymer) by means of bifunctional molecules, serving as a binder, such as glutardialdehyde or by other methods described in the art. For example, streptavidin mutein oligomers can be generated by first introducing thio groups into streptavidin mutein (this can, for example, be done by reacting streptavidine mutein 2-iminothiolan (Trauts' reagent) ) and activating, in a separate reaction, amino groups available in streptavidin mutein.This activation of amino groups can be obtained by reaction of streptavidin mutein with commercially available heterobifunctional crosslinkers such as 4- (N-maleimidomethyl) cyclohexane-1- sulfosuccinimidyl carboxylate (sulfo SMCC) or Succinimidyl-6 - [(β-maleimidopropionamido) hexanoate (SMPH). In a second step, the two reaction products thus obtained are mixed together, leading to the reaction of the thiol groups contained in the a batch of modified streptavidin mutein with the activated amino acids (for maleimide functions) from the other batch of modified streptavidin mutein. For this reaction, multimers / oligomers those of streptavidin mutein are formed. These oligomers can have any suitable "individual molecule" number or streptavidin building block "higher than 3 and the degree of oligomerization can be varied according to the reaction condition (see Fig. 24). two groups of the modified streptavidin mutein, the soluble multimerization oligomer reagent is typically isolated by size exclusion chromatography and any desired fraction can be used as a multimerization reagent. Typically, the oligomers do not (and need not have) a single weight molecular however they normally observe a statistical weight distribution such as Gaussian distribution.Any oligomer with more than three streptavidin homotetramers (building blocks; (n = 3)) can be used as a soluble multimerization reagent. , for example 3 to 25 staptavidin mutein homotetramers. With a molecular weight of about 50 kDa for star muteins ptavidin such as the mutein "m1" or "m2" described in more detail below, 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 12 to 100 Z1 (and Z2) binding sites as described here.
[000120] According to the above description, in addition to such oligomeric multimerization reagents that only contain cross-linked streptavidin homotetramers, it is possible to react tetrameric streptavidine muteins in a vehicle to obtain multimerization reagents that are used in the present invention. In addition to the reaction described above with a polysaccharide, it is likewise possible to use physiologically or pharmaceutically acceptable proteins such as serum albumin (for example human serum albumin (HSA) or bovine serum albumin (BSA) as a carrier protein. In this case, streptavidin mutein (as an individual homo-tetramer or similarly in the form of oligomers with n = 3) can be coupled to the carrier protein through non-covalent interaction. For this purpose, biotinylated BSA (which is commercially available at from various suppliers such as ThermoFisher Scientific, Sigma Aldrich or Vectorlabs, to name just a few) can be reacted with streptavidin mutein, in doing so some of the streptavidin oligomers will not covalently link via one or more binding sites to biotin (Z1, Z2) to the biotinylated carrier protein, leaving most of the binding sites (Z1, Z2) of the oligomer available to bind ta agents l as the first agent and optionally the second agent and any other agent as described herein. Thus, for such an approach a soluble multimerization reagent with a multiple Z1 binding sites can be conveniently prepared (see Fig. 24). Alternatively, a streptavidin mutein (either as an individual homo-tetramer or similarly in the form of oligomers with n = 3) can be covalently coupled to a synthetic vehicle such as a polyethylene glycol (PEG) molecule. Any suitable PEG molecule can be used for this purpose, as long as the PEG molecule and its multimerization reagent is soluble. Typically, PEG molecules up to a molecular weight of 1000 Da are all soluble in water or culture medium that can be used in the present invention. Such PEG-based multimerization reagent can be easily prepared using commercially available activated PEG molecules (for example, 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) with streptavidin mutein amino groups.
[000121] Under streptavidin or wild type streptavidin (wt-streptavidin), the amino acid sequence described by Argarana and another, Nucleic Acids Res. 14 (1986) 1871-1882 is referred to. Streptavidin muteins are polypeptides that are distinct from the wild-type streptavidin sequence by one or more amino acid substitutions, deletions or additions and that retain the wt-streptavidin binding properties. Streptavidin-like polypeptides and streptavidin muteins are polypeptides that are essentially immunologically equivalent to wild-type streptavidin and are in particular capable of binding biotin, biotin-derived or biotin analogues with the same or different affinity as wt-streptavidin. Streptavidin-like polypeptides or streptavidin muteins may contain amino acids that are not part of wild-type streptavidin or they may include only part of wild-type streptavidin. Streptavidin-like polypeptides are likewise polypeptides that are not identical to wild-type streptavidin, since the host lacks the enzymes that are required to transform the host polypeptide produced into the wild-type streptavidin structure. The term "streptavidin" likewise includes streptavidin tetramers and streptavidin dimers, in particular streptavidin homotetramers, streptavidin homodimers, streptavidin heterotetramers and strepavidin heterodimers. Each subunit regularly has a binding site for biotin or biotin analogues or for streptavidin binding peptides. Examples of streptavidins or streptavidin muteins are mentioned, for example, in WO 86/02077, DE 19641876 A1, US 6,022,951, WO 98/40396 or WO 96/24606.
[000122] In a preferred embodiment, streptavidin muteins that are used as a multimerization reagent are those streptavidin muteins that are described in US patent 6,103,493 and likewise in DE 196 41 876.3. These streptavidin muteins have at least one mutation within the region of amino acid positions 44 to 53, based on the wild-type streptavidin amino acid sequence. Preference is given to muteins of a minimal streptavidin, which begins N-terminally in the amino acid region 10 to 16 of the wild-type streptavidin and C-terminal end in the amino acid region 133 to 142 of the wild-type streptavidin. Examples of such preferred streptavine 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. A streptavidin mutein can be a mutein comprising the amino acid sequence Va144-Thr45-Ala46-Arg47 (SEQ ID NO: 02) at sequence positions 44 to 47 or streptavidin mutein (analog) comprising the amino acid sequence lle44-Gly45 -Ala46-Arg47 (SEQ ID NO: 03) at sequence positions 44 to 47 of wild-type streptavidin. Such muteins are described in US patent 6,103,493, for example, and are commercially available from IBA GmbH in the form of mutein "m1" and mutein "m2" under the trademark Strep-Tactin®.
[000123] A method according to the present invention can in some embodiments be used to empty a sample of reagents that was previously used in cell expansion. The first or second agent and the respective free partner (the competition agent) can, for example, be present included in the eluate of an expansion method as described above. Using a method according to the invention such reagents can be at least essentially, including completely removed from a sample, for example from a cell population. As an illustrative example, the first or second agent as defined above can be emptied from a sample at levels below those of detection, for example FACS or Western Blot. A competition reagent (first or second free binding partner or analog of the same) may have been used to terminate and control the expansion and release of the cell population forms the multimerization agent. This competition reagent may have a binding site that is capable of specifically binding to the affinity reagent Z binding site in a WO 2013/124474 "removal cartridge". In such an embodiment the respective method of the invention can likewise serve to empty the first and second agent and the competitor reagent, including removal thereof.
[000124] A method according to the present invention can be carried out at any temperature where the viability of the cell population is at least essentially uncompromised. When reference is made here to conditions that are at least essentially not harmful, not harmful or at least essentially not compromising viability, conditions are referred to, under which the percentage of the cell population that will be expanded with total 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, a method according to the invention is carried out at a temperature of about 20 ° C or higher. Depending on the cell population to be expanded, a suitable temperature range can be for example from about 20 ° C to about 45 ° C, including from about 25 ° C to about 40 ° C, or from about 32 ° C to 37 ° C. In some embodiments, a method according to the invention is 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. The person skilled in the art can determine an appropriate temperature empirically, taking into account the nature of the cells and the conditions of expansion. Typically human cells are expanded at a temperature such as 37 ° C.
[000125] In another embodiment, the invention provides an in vitro method of expanding a cell population, comprising contacting a sample comprising a cell population with a multimerization reagent, wherein the multimerization reagent is in a soluble and immobilized form here (linked to that) a first agent that provides a primary activation signal to the cells. The multimerization reagent comprises at least one Z1 binding site for binding the first agent, wherein the first agent comprises at least one C1 binding partner, where the C1 binding partner is capable of binding to the Z1 binding site of the multimerization reagent. The first agent is linked to the multimerization reagent by the link formed between the C1 binding partner and the Z1 binding site, and the first agent binds to a receptor molecule on the cell surface, thereby providing a primary activation signal to the cells. cells and thereby activating the cells. It is expressly noted here that when a soluble multimerization agent is used, the connection between the C1 binding part and the Z1 binding site need not be reversible.
[000126] In one embodiment of this second multimerizing agent, it has immobilized on it (attached to it) a second agent that stimulates an additional molecule on the surface of cells, in which the second agent comprises a C2 binding partner, in which the binding partner C2 is able to be linked to a Z2 binding site of the multimerization reagent. The second agent is linked to the multimerization reagent by the bond formed between the C2 binding partner and the Z2 binding site, where the second agent binds to the additional molecule on the surface of the cell surface, thereby stimulating the activated cells.
[000127] In an embodiment of this second method, the bond formed between the C1 bonding partner and the Z1 bonding site may be irreversible and / or likewise the bond formed between the C2 bonding partner and the Z2 bonding site may be irreversible. be irreversible.
[000128] In a different embodiment of this second method, the bond formed between the C1 binding partner and the Z1 binding site can be reversible. In the same way, the bond formed between the bonding partner C2 and the bonding site Z2 can be reversible. In this case, the dissociation constant (Kd) for the reversible link between said Z1 link site and said C1 link partner and / or for the reversible link between said Z2 link site and said C2 link partner can be in the range of 10-2 M to 10-13 M.
[000129] In this second method which is based on a soluble multimerization reagent, the first and second reagents as well as the multimerization reagent and all other reagents and cell populations can otherwise be used in the same manner as described above for the method that makes use of reversible between the first or second agents and the multimerization reagent.
[000130] The invention also provides a reagent kit to expand a cell population, the kit comprising,
[000131] (i) a multimerization reagent, wherein the multimerization reagent comprises at least one Z binding site for the reversible binding of a first agent,
[000132] (ii) a first agent that binds to a receptor molecule on the surface of cells, thereby providing a primary activation signal to the cells and thereby activating the cells, wherein the first agent comprises at least one binding partner C1, in which the C1 binding partner is capable of reversibly binding to a Z1 binding site of the multimerization reagent, in which the first agent is linked to the multimerization reagent by the reversible bond formed between the C1 binding partner and the connection site Z1, and
[000133] (iii) a second agent that stimulates an additional molecule on the cell surface, wherein the second agent comprises a C2 binding partner, wherein the C2 binding partner is capable of reversibly binding to a binding site Z2 of the multimerization reagent, in which the second agent is linked to the multimerization reagent by the bond formed between the C2 binding partner and the Z2 binding site, in which the second agent binds to the additional molecule on the surface of the cell surface , thereby stimulating the activated cells.
[000134] The invention also provides a reagent kit to expand a cell population, the kit comprising,
[000135] (i) a multimerization reagent, wherein the multimerization reagent is in soluble form and comprises at least one Z-binding site for the reversible binding of a first agent,
[000136] (ii) a first agent that binds to a receptor molecule on the surface of cells, thereby providing a primary activation signal to the cells and thereby activating the cells, wherein the first agent comprises at least one binding partner C1, in which the C1 binding partner is capable of binding to a Z1 binding site of the multimerization reagent, in which the first agent is linked to the multimerization reagent by the reversible bond formed between the C1 binding partner and the binding site Z1.
[000137] This second reagent kit may also comprise (iii) a second agent that stimulates an additional molecule on the cell surface, wherein the second agent comprises a C2 binding partner, wherein the C2 binding partner is capable of binding to a Z2 binding site of the multimerization reagent, wherein the second agent is linked to the multimerization reagent by the bond formed between the C2 binding partner and the Z2 binding site.
[000138] A kit as described here is in particular used when the cell population is a lymphocyte population.
[000139] According to the above description, the invention likewise provides new multimerization reagents and new compositions comprising multimerization reagents that care to expand a cell population. Such a multimerization reagent that is capable of expanding a cell population is a multimerization reagent that is in soluble form and comprises at least one Z1 binding site for the reversible binding of a first agent that provides a primary activation signal to the cells , wherein the multimerization reagent has reversibly immobilized (attached thereto) said first agent which provides a primary activation signal to the cells; wherein the first agent comprises at least one C1 binding partner, where the C1 binding partner is able to reversibly bind to at least one Z1 binding site of the multimerization reagent, where the first agent is bound to the reagent of multimerization by the reversible link formed between the link partner C1 and the link site Z1. It should be noted here that such a multimerization agent may have immobilized on it any of the first agent that is described here.
[000140] A multimerization reagent of the invention can also comprise at least one Z2 binding site for the reversible binding of a second agent that stimulates an additional molecule on the cell surface, where the multimerization reagent has reversibly immobilized on it (bound thereto) the second agent that stimulates an additional molecule on the cell surface, wherein the second agent comprises a C2 binding partner, wherein the C2 binding partner is capable of binding to at least one Z2 binding site of the reagent of multimerization. In this embodiment, the second agent is linked to the multimerization reagent by the link formed between the link partner C2 and the link site Z2.
[000141] Likewise according to the description determined above, such a multimerization reagent is capable of expanding a lymphocyte population or a subpopulation contained in the 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 exterminating T cell population. Thus, in such modalities of the multimerization reagent, the first agent can stimulate a signal associated with the TCR / CD3 complex in T cells. The first agent present in the multimerization reagent can thus be a binding reagent that specifically binds to CD3, while the second agent that binds to the additional molecule may be a binding agent that specifically binds to CD28 or CD137.
[000142] In multimerization reagent embodiments the first agent that specifically binds to CD3 may be an anti-CD3 antibody, a divalent antibody fragment from an anti-CD3 antibody, a monovalent antibody fragment from an anti-CD3 antibody CD3, and / or CD3-binding protein molecule with antibody-like binding properties. In these embodiments, the second agent that specifically binds to CD28 or CD137 can be an anti-CD28 antibody, a divalent antibody fragment of an anti-CD28 antibody, a monovalent antibody fragment of an anti-CD28 antibody, a molecule CD28 binding protein with antibody-like binding properties, an anti-CD137 antibody, a divalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment of an anti-CD137 antibody, a CD137 binding protein molecule with antibody-like binding properties, 4-1BB linker, and any mixture thereof. Thus, a multimerization reagent of the invention may generally have immobilized on it a type of first agent and a mixture of second agents, for example, an anti-CD3 antibody as the first agent and for example, an anti-CD28 antibody and linker 4 -1BB as (joining) second agents.
[000143] If the multimerization reagent will be used for the expansion of B cells, the first agent immobilized in the multimerization reagent can be a binding reagent that specifically binds to CD40 or CD137. In such embodiments the first binding reagent that specifically binds to CD40 or CD137 can be selected from an anti-CD40 antibody according to the description determined here, a divalent antibody fragment from an anti-CD40 antibody, a monovalent antibody fragment of an anti-CD40 antibody, and a CD40 binding protein molecule with antibody-like binding properties or an anti-CD137 antibody, a divalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment an anti-CD137 antibody, a CD137 binding protein molecule with antibody-like binding properties, and CD40 linker (CD154).
[000144] In the same way according to the general description of the present invention, in the multimerization reagent as described here the binding sites Z1 and Z2 of the multimerization reagent can be identical. As described above, such a multimerization reagent may comprise a streptavidin oligomer or polymer, an avidin oligomer or polymer, an oligomer or a streptavidin analog polymer that reversibly binds biotin, an oligomer or a polymer of an analogous avidin that reversibly binds biotin, a reagent comprising at least two chelating groups K, wherein the at least two chelating groups are capable of binding to a transition metal ion, thereby rendering the reagent capable of binding to an oligohistidine affinity tag, multimeric glutathione-S-transferase, multimeric calmodulin and a biotinylated carrier protein.
[000145] A new composition provided here that is capable of expanding a cell population can comprise
[000146] (i) a first multimerization reagent, wherein the first multimerization reagent is in soluble form and comprises at least one Z1 binding site for the reversible binding of a first agent that provides a primary activation signal to cells, wherein the first multimerization reagent has reversibly immobilized (attached thereto) said first agent which provides a primary activation signal to the cells, wherein the first agent comprises at least one C1 binding partner, in which the bond C1 is capable of reversibly bonding to at least one Z1 binding site of the multimerization reagent, where the first agent is bound to the multimerization reagent by the reversible bond formed between the C1 binding partner and the Z1 binding site, and
[000147] (iI) a second multimerization reagent, wherein the second multimerization reagent is in soluble form and comprises at least one Z2 binding site for the reversible binding of a second agent that stimulates an additional molecule on the cell surface, wherein the multimerization reagent has reversibly immobilized (attached thereto) said second agent which stimulates an additional molecule on the surface of cells, where the second agent comprises a C2 binding partner, wherein the C2 binding partner is capable of binding to at least one Z2 binding site of the multimerization reagent, wherein the second agent is linked to the multimerizing reagent by the bond formed between the C2 binding partner and the Z2 binding site.
[000148] Such a new composition is, for example, the reaction mixture used in Example 13, in which two separate multimerization reagents were functionalized with an αCD3 Fab fragment only or an αCD28 Fab fragment only. It is noted in this context that such a composition was shown in Example 13 to have the same expansion efficiency as a single multimerization reagent in which the first agent and the second agent are immobilized together. Thus, the combined use of two or more multimerization reagents that is individually functionalized with only one type of reagent (for example, a first or a second agent) is functionally equivalent to use for the expansion a common multimerization reagent that immobilized the first agent and a second agent on him as well. In this context, it is likewise noted that a multimerization reagent of the present invention is functionalized with as many agents (for example, one, two, three, four or even more agents) that are intended to be used for the expansion of a population of selected cell. A third or fourth agent can, for example, provide a stimulus for the expansion of a desired subpopulation of cells. For example, see in this context Example 13 in which soluble multimerization reagents were reversibly functionalized with three reagents, that is, a αCD3 Fab fragment as a first reagent, a aCD28 Fab fragment as a second reagent and a aCD8 Fab fragment as a third reagent to enrich the subpopulation of CD8 + T cells in a sample of a population of CD3 + T cells (lymphocyte). Using such combinations of agents that can all be reversibly immobilized in the same multimerization reagent, the present invention allows the possibility to preferentially expand or selectively enrich any desired cell (sub) population of a sample that, for example , comprises a variety of different subpopulations. In this context, it is noted that it is however equally possible to use for this purpose to use three different multimerization reagents, for example, a first multimerization reagent that is functionalized with only a Fab fragment aCD3, a second multimerization reagent that it is functionalized with an aCD28 Fab fragment and a third multimerization reagent which is functionalized with an aCD8 Fab fragment. Likewise, it is possible to use only two different multimerization reagents, a first multimerization reagent that is functionalized with only one aCD3 Fab fragment and a second multimerization reagent that is also functionalized with a Fab aCD28 fragment and a Fab aCD8 fragment. In this way, the present invention allows to design any type of desirable expansion reagent in a modular way.
[000149] The invention likewise provides an in vitro method of serially expanding a lymphocyte population, in which the lymphocyte population comprises T cells. This method comprises
[000150] contacting a sample comprising the T cell, which comprises a lymphocyte population with a multimetering reagent,
[000151] in which the multimerization reagent is in a soluble form and has reversibly immobilized on it (i) a first agent that provides a primary activation signal to T cells and (ii) a second agent that stimulates an additional molecule on the surface of T cells,
[000152] wherein the multimerization reagent comprises at least one Z1 binding site for the reversible binding of the first agent,
[000153] in which the first agent comprises at least one C1 binding partner, in which the C1 binding partner is capable of reversibly binding to the Z1 binding site of the multimerization reagent, in which the first agent is binding to the reagent multimerization by the reversible link formed between the link partner C1 and the link site Z1,
[000154] wherein the multimerization reagent comprises at least one Z2 binding site for the reversible binding of the second agent,
[000155] wherein the second agent comprises at least one C2 binding partner, where the C2 binding partner is capable of reversibly binding to the Z2 binding site of the multimerization reagent, where the first agent is binding to the reagent multimerization by the reversible bond formed between the bonding partner C2 and the bonding site Z2,
[000156] in which the first agent binds to a receptor molecule on the surface of T cells, thereby providing a primary activation signal to cells and thereby activating T cells,
[000157] wherein the second agent binds to the additional molecule on the surface of T cells, thereby stimulating activated cells, the first agent and the second agent thereby inducing T cells to expand.
[000158] In this method contacting the sample containing the population of lymphocytes that successively contain the population of T cells with the soluble multimerization reagent that immobilized on it the first and second agents results in specific binding of T cells to the multimerization reagent.
[000159] The contact of the sample comprising the T cell, which comprises lymphocyte population with the multimerization reagent can be carried out in a bioreactor such as a hollow fiber bioreactor (for example hollow fiber bioreactor of the cell expansion system Quantum®) or a plastic bag bioreactor (eg Cellbag® used in GE Healthcare Xuri W25 Cell Expansion System).
[000160] This method also comprises contacting the lymphocyte population (reaction mixture containing T cells linked to the multimerization reagent by the first agent and the second agent) with (i) a first free C1 binding partner or an analogue of the same capable of break the connection between the first C1 binding partner and the Z1 binding site and (ii) a second free C2 binding partner or an analog thereof, capable of breaking the connection between the second C2 binding partner and the binding site Z2. Thus making the reversible link between said first agent C1 binding partner and said Z1 binding sites as well as the reversible link between said second agent C2 binding partner and said Z2 binding site of said multimerization reagent site it is disrupted, thereby releasing in an eluate the T cells bound to the multimerization reagent by the first agent and the second agent and stopping the expansion of the T cells.
[000161] In this method the eluate (the reaction mixture in which the expansion reaction was terminated by adding the first free partner (s) or analog (s) of the same that contains the expanded T cell population can be exposed to chromatography on a suitable (first) stationary phase The (first) stationary phase can be a gel filtration matrix and / or an affinity chromatography matrix as described in International patent application WO 2013/124474. This gel filtration matrix and / or affinity chromatography comprises an affinity reagent, wherein the affinity reagent comprises a Z1 and / or Z2 binding site that specifically binds to the C1 and / or C2 binding partner comprised in the first agent or the second agent When making the first agent, the second agent, the first C1 bonding partner and / or the second free C2 bonding partner are immobilized in the stationary phase.In this method, the first stationary phase is fluidly connected to the biorea actor.
[000162] In one of the modalities of this serial expansion, the connection sites Z1 and Z2 of the multimerization agent are identical. In addition, a single multimerization agent can be used. When a soluble multimerization agent is used, the T cell population (or the expanded cell population in general) is separated from the soluble multimerization reagent. The separation / removal could be carried out using a second stationary phase. For this purpose, a mixture comprising T cells and the soluble multimetering reagent is exposed, before or after being applied on the first stationary phase described above, for chromatography on a suitable second stationary phase. This secondary stationary phase can be a gel filtration matrix and / or affinity chromatography matrix, wherein the gel filtration matrix and / or affinity chromatography comprises an affinity reagent. The affinity reagent comprised in the chromatography resin includes a D binding partner that (specifically) binds to the Z1 binding site and / or the Z2 binding site, if present, of the multimerization reagent, thereby immobilizing the multimerization reagent. stationary phase. If a streptavidin-based multimerization agent is used and both first and second agents have a streptavidin-binding peptide as the C1 or C2-binding partner, the D-binding partner that is comprised in the affinity reagent of this second stationary phase can be biotin. The soluble oligomer of streptavidin or a streptavidine mutein which is used as a multimerization reagent then binds to biotin which is normally covalently coupled to a chromatography matrix such as biotin-sepharoseTM which is commercially available.
[000163] In this serial expansion method the first agent can stimulate a TCR / CD3 complex signal on T cells and the first agent can thus be a binding reagent that specifically binds to CD3. In addition, the additional molecule in the T cell can be CD28. In this case the second agent that binds to the additional molecule is a binding reagent that specifically binds to CD28.
[000164] In this method of serial expansion, T cells can be transfected during or after expansion for example with a T cell receptor (TCR) or a chimeric antigen receptor (CAR, also known as an artificial T cell receptor) ). This transfection for the introduction of the desired receptor gene can be performed with any suitable retroviral vector, for example. The genetically modified cell population can then be released from the initial stimulus (the CD3 / CD28 stimulus, for example) and subsequently be stimulated with a second type of stimulus for example by the newly introduced receptor). This second type of stimulus can comprise an antigenic stimulus in the form of a peptide / MHC molecule, the cognate linker (crosslinking) of the genetically introduced receptor (for example a natural linker from a CAR) or any linker (such as an antibody) that directly it binds within the structure of the new receptor (for example by recognizing constant regions within the receptor). In this regard, Cheadle et al., "Chimeric antigen receptors for T-cell based therapy" MethodsMol Biol. 2012; 907: 645-66 or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine Vol. 65: 333347 (2014).
[000165] In this method, the lymphocyte population comprising T cells can be a population of peripheral blood mononucleated cells (PBMC) or a population of enriched or purified T cells. The lymphocyte population can, for example, be derived from whole blood, or from an unf mobilized apheresis product or frozen tissue preparation.
[000166] In this serial expansion method which is based on a soluble multimerization reagent, the first and second reagent as well as the multimerization reagent and all other reagents and cell populations can be used in another way in the same manner as described above for the method that makes use of reversible between the first or second agent and the multimerization reagent.
[000167] The invention is also directed to an arrangement of a bioreactor and a first stationary phase for chromatography. The bioreactor is suitable for cell expansion, and the stationary phase is suitable for cell separation and reagent removal. The first stationary phase is a gel filtration matrix and / or affinity chromatography matrix, wherein the gel filtration matrix and / or affinity chromatography comprises an affinity reagent, wherein the affinity reagent comprises a binding Z1 specifically binding to a C1 binding partner comprised in a first agent and / or the affinity reagent comprises a Z2 binding site specifically binding to a C2 binding partner comprised in a second agent. The first stationary phase is therefore suitable for immobilizing the first agent and / or the second agent, the first C1 binding partner and / or the second free C2 binding partner on it. In addition, the bioreactor and the stationary phase are fluidly connected. This arrangement can be used for serial expansion as explained above and can be integrated into known cell expansion systems (such as the Quantum® cell expansion system) or the Xuri Cell W25 Expansion System.
[000168] In this arrangement, the first stationary phase is comprised of a chromatography column or is a planar stationary phase. The arrangement may also comprise a second stationary phase that is fluidly connected to the first stationary phase. The secondary stationary phase can be a gel filtration matrix and / or affinity chromatography matrix, wherein the gel filtration matrix and / or affinity chromatography comprises an affinity reagent. This affinity reagent can comprise a binding partner D that (specifically) binds to the Z1 binding site of the multimerization reagent, thereby being suitable for immobilizing the multimerization reagent in the stationary phase.
[000169] The invention is also directed to an apparatus for purifying and expanding a population of cells, the apparatus comprising at least an arrangement of a bioreactor and a first stationary phase or a second stationary phase for chromatography as defined above.
[000170] The apparatus can also comprise a plurality of arrangements of a bioreactor and a stationary phase that are fluidly connected in series.
[000171] The apparatus can comprise a sample inlet being fluidly connected to the bioreactor of the arrangement of a bioreactor and the stationary phase for chromatography. The apparatus can likewise comprise a sample outlet for purified and amplified target cells, the sample outlet that is fluidly connected to the stationary phase of the last of at least one arrangement of a bioreactor and the stationary phase for chromatography.
[000172] Finally, the device can be designated as a functionally closed system.
[000173] As one of ordinary skill in the art will readily appreciate from the description of the present invention, other subject compositions, means, uses, methods, or steps, presently existing or after being developed that perform substantially the same function or achieve substantially the same result as the corresponding exemplary embodiments described here can also be used in accordance with the present invention. Experimental Examples Example 1: Stimulation / expansion of CD3 + response T cells with αCD3 / αCD28 Fab fragments that were reversibly immobilized on streptavidin mutein coated spheres.
[000174] 300,000 CD3 + CD62 response T cells (Tresp, isolated by serial magnetic enrichment of a non-mobilized donor apheresis product) were marked with 3 μM CFSE and stimulated with 5 μL of a 15 μL preparation of spheres Streptactin® (10 mg of magnetic particles / mL, loaded with 35 μg of Streptactin® / mg beads) loaded with 0.5 μg of the αCD3 Fab fragment only, 0.5 μg of the αCD28 Fab fragment only or a mixture of 0 , 5μg of α α3 Fab fragment and 0.5 μg of α α CD28 Fab.
[000175] The αCD3 Fab fragment used was derived from the CD3-binding monoclonal antibody produced by the hybridoma cell line OKT3. The hybridoma cell line OKT3 and the antibody to OKT3 are described in US patent 4,361,549, the cell line was deposited under accession number ATCC® CRL-8001 ™). The CD28 Fab used was derived from the CD28.3 monoclonal anti-human CD28 antibody (Vanhove et al., BLOOD, July 15, 2003, Vol. 102, no. 2, pages 564-570). The nucleotide sequence of the variable domains of this CD28.3 antibody was deposited on GenBank in the form of an anti-human CD28 antibody of single synthetic chain Fv construction scFv28.3 under GenBank accession number AF451974.1).
[000176] Both Fab fragments were recombinantly produced in E. coli as described in International patent applications WO2013 / 011011 and WO 2013/124474 carrying as a constant domain (CH1 and Ckappa) a sequence of IgG1 consensus. The heavy chain of both Fab fragments was carboxy-terminally fused with a sequential arrangement of two streptavidin binding modules (SAWSHPQFEK (GGGS) 2GGS- AWSHPQFEK, (SEQ ID NO: 07)), which is commercially available as "Twin- Strep-tag® from IBA GmbH, Gottingen, Germany) The Fab αCD3 fragment was used as the first agent with the streptavidin binding peptide that serves as the C1 binding partner and the Fab fragment aCD28 was used as the second agent with the peptide from binding to streptavidin which serves as a C2 binding partner. Streptavidin (tetrameric) mutein "Strep-tactin® serves as a multimerization reagent in which both Fab fragments have been reversibly immobilized.
[000177] In the expansion experiment, Tresp cells stimulated with blank spheres (no Fab) served as a negative control. Tresp cells were seeded in triplets in 48-well plates along with 300,000 autologous CD3 feeder cells (irradiated with 30Gy) in 3 mL of complete cell culture medium (RPMI (Gibco) supplemented with 10% (v / v) serum) fetal bovine, L-glutamine, beta-mercapto ethanol, HEPES, penicillin, streptomycin and gentamicin) supplemented with 10 U / mL of interleukin 2 (IL-2). The cells were incubated at 37 ° C without changing media and analyzed after 4 days by FACS analysis. FACS staining and analysis was performed after 10 min of incubation with 100 μM of D-biotin. A representative graph for each condition is shown in Fig. 4. Graphs show live CD3 + cells that have been marked with propidium iodide (PI) for life / death discrimination. Fig. 4a is a histogram showing size distribution (forward scattering) of stimulated cells. Fig. 4a shows that a specific cell population of Tresp cells was stimulated and enlarged (increase in size / number compared to the unstimulated control "spheres only") when incubated in the presence of spheres in which a mixture of 0.5 μg of fragment Fab aCD3 and 0.5 μg of Fab aCD28 was immobilized after being stimulated in vitro with Fab fragments αCD3 / αCD28 that were reversibly immobilized in spheres coated with streptavidin mutein Strep-tactin®. Fig. 4B describes histograms of the CFSE proliferation staining dilution representing the degree of proliferation according to the number of cells per cell division (indicated at the top of Fig. 4B, 0 represents undivided cells; 5 represents cells that passed through the minus 5 divisions). It can be seen from Fig. 4B that the population of T cells stimulated with the spheres in which a mixture of 0.5 μg Fab aCD3 fragment and 0.5 μg Fab aCD28 was immobilized went through mainly three cell divisions and represented a more uniform proliferation pattern than with a single stimulus only (small number of cells within the undivided peak "0"). The absolute increased amount of proliferation (more cells proliferated evenly after 4d stimulation with aCD3 and functionalized spheres of aCD28) is likewise represented by a more intense consumption of the medium as described by a color change indicator to yellow in Fig. 4C. Example 2: Analysis of differential intracellular calcium mobilization in Jurkat cells
[000178] Low real-time cytometric analysis of differential intracellular calcium mobilization induced in Jurkat cells that are labeled with the OKT3 αCD3 antibody clone or with OKT3 Fab fragments being multimerized with Strep-tactin® was examined here.
[000179] For this purpose, Jurkat cells were loaded with the calcium sensitive dye Indo-1-AM and calcium release was activated by injection of the monoclonal antibody of αCD3 OKT3 (produced by the hybridoma cell line OKT3, see above, black squares ) or Fab fragment aCD3 (derived from the parental cell line OKT3) that was multimerized by reversible binding of its streptavidin-binding peptide to fluorescently soluble Strep-Tactin conjugated to phycoerythrin. In the case of intact multimeric OKT3 Fab-Strep-Tactin complexes, calcium release was activated over an identical period of time as with the parental antibody clone (dark gray triangles). Cell activation could be completely avoided by injecting treated D-biotin, pre-dissociated Fab-Strep-Tactin complexes (light gray circles) identical to injecting the PBS negative control (inverted white triangles). Ionomycin application served as a positive control for calcium influx. Changes resolved with time in intracellular Ca2 + concentration were monitored by flow cytometry based on the change in relation to FL6 / FL7. It can be seen from Fig. 5A that both the OKT3 parental antibody as well as the multimerized monovalent Fab OKT3 fragment effected calcium, meaning that the multimerized monovalent OK OK3 fragment is essentially as functional as the parental antibody. Notably, the multimeric Fab OKT3 fragment was unable to activate calcium release if biotin was added to Strep-tactin where the Fab OKT3 fragment was immobilized prior to the addition of the Fab Streptactin-OKT3 fragment. In this case, the biotin disrupted the reversible bond formed between Strep-tactin as the multimerization agent and the Fab OKT3 fragment. The monovalent Fab fragment was therefore displaced from the multimerization agent and after dissociation was unable to activate calcium release by binding to CD3 from Jurkat cells.
[000180] In the experiments shown in Fig. 5B Jurkat cells labeled by indo-1-AM were activated by OKT3-derived Fab-Strep-Tactin αCD3 complexes as described in Fig. 5a. Injection of intact complexes (upper graph) or pre-dissociated complexes (lower graph) respectively served as positive or negative controls. In addition, stimulation of cells with intact Fab-Estrep Tactin complexes followed by subsequent injection of D-biotin (close to peak activation at t = 140s) resulted in abrupt disruption of aCD3 Fab multimer signaling (median plot). Ionomycin injection in the pre-dissociated Fab complex group served as a positive control. Data are representative of three different experiences. Importantly, Fig. 5B shows that the addition of D-biotin to the sample quickly displaces the Fab fragment of the Strep-tactin multimerization agent, thereby effectively ending the release of calcium while still under continuous calcium stimulation and demonstrating that the OKT3 Fab fragment dissociated is no longer biologically active. Likewise, the multimeric Fab OKT3 fragment was unable to activate calcium release when biotin was added to the Fab strep-tactin-OKT3 fragment multimer prior to the addition of the Fab Streptactin-OKT3 sample to Jurkat cells. Example 3: Reversible cell staining by Fab CD3 multimers
[000181] This Example examines the reversible labeling of cells by Fab CD3 multimers. Recently isolated PBMCs were labeled with either the OKT3 αCD3 monoclonal antibody clone (dot plot on the left, parental clone for Fab multimers) or PE OKT3 Fab multimer cognate phycoerythrin and analyzed before (second column on the left) or after treatment with D-biotin (median column). Remaining Fab monomers were then detected after the subsequent wash steps using fresh PE labeled Strep-Tactin® (second column on the right). Secondary Fab multimer labeling of reversibly labeled cells served as a control (right column). Only live CD3 cells that are negative in staining with propidium iodide (PI) for life / death discrimination are shown in Fig. 6. Numbers in dot plots indicate the percentage of cells inside gates. This experiment shows that the labeling of CD3 + PBMCs with an anti-CD3 multimerized Fab fragment with Streptactin as a multerization reagent is completely reversible by adding D-biotin and that the monovalent Fab fragment just does not bind to the CD3 molecule present in PBMCs . Example 4: Reversible cell isolation by Fab CD28 multimers
[000182] This Example shows cell isolation by reversible ligation of multimerized anti-CD28 Fab fragments with Strep-Tactin® magnetic particles (magnetic particles are available from IBA GmbH Gottingen, Germany). Fab fragments derived from the CD28.3 antibody described in Example 1 were used for this purpose. CD28 + cells were selected / isolated by magnetic cell selection of freshly isolated Fab PMBCs multimers essentially as described in International Patent Application WO2013 / 011011. Before selection cells were labeled by the control with either the cognate fluorescent aCD28 multimers (dot plots on the left) or with an antibody directed against the immunoglobulin kappa light chain (according to dot plots on the left, α-Ig kappa mAb ) as a control tag. After selection, CD28 + cells were treated with D-biotin and subsequently washed to remove magnetic beads and Fab monomers. Released CD28 + cells were subsequently (re-) labeled with Fab CD28 multimers (according to left dot plot) or with a-Igkappa mAb (dot plot on the right) to detect potentially remaining Fab monomers. Only live CD3 + cells (PInegative) are shown. Numbers on dot charts indicate the percentage of cells within gates. Fig. 7 shows that CD28 + cells can be isolated from PMBC using such a multimerized anti-CD28 Fab fragment and that all isolation reagents including anti-CD28 Fab monomers can be removed after selection. Example 5: Stimulation / Expansion of CD3 + response T cells with αCD3 / αCD28 Fab fragments that were reversibly immobilized in soluble strep-tactin
[000183] In this example CD3 + response T cells (isolated by magnetic selection of a PBMC sample recently obtained from a Ficoll gradient) were expanded after in vitro stimulation with Fab αCD3 / αCD28 fragments that were reversibly immobilized on Strep-tactin ® soluble oligomeric acting as a soluble multimerization reagent. The oligomeric Strep-tactin® was obtained by polymerizing Strep-tactin® with SMCC sulfo (sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, product # 22122 Thermo Scientific) and iminothiolane (product # 26101 Thermo Scientific) from according to the manufacturer's protocol (Thermo Scientific). Oligomeric streptavidin was separated from monomeric (unreacted) and dimeric streptavidin mutein by size exclusion chromatography and the fraction thereby obtained from oligomeric streptavidin mutein (n> 3) was used as a soluble multimerization reagent.
[000184] For in vitro expansion, 300,000 CD3 + response T cells (Tresp) were labeled with 2 μM succinimidyl carboxyfluorescein ester (CFSE) and stimulated with varying amounts of a strep-tactin® oligomer preparation soluble in that a combination of the Fab OKT3 αCD3 fragment described above and the Fab aCD28 fragment of antibody 28.3 (both carrying the Twin-Strep-tag® described above as streptavidin binding peptide in the heavy chain) were immobilized. ("1x" corresponds to 3 μg of functionalized multimerized Streptactin with 0.5 μg of αCD3- monomeric Fab fragment and 0.5 μg of aCD28, the numbers "0.5x", "2x" and "5x" indicate the amount respective n-times of "1x"). Tresp cells left unstimulated or stimulated with blank strep-tactin multimers (no Fab) served as a negative control. Tresp cells were seeded in duplicates in 48-well plates together with 300,000 CD3-negative autologous feeder cells (irradiated with 30 Gy) in 1 ml of cell culture medium supplemented with 20U / ml IL-2. Cells were incubated at 37 ° C without media change and proliferation was analyzed according to CFSE dilution after 5 days by FACS analysis. Fig. 8A shows the increased size distribution of proliferating cells after 5 days in culture compared to negative controls. Fig. 8B shows that Tresp CD3 + cells were correctly stimulated and proliferated vigorously when incubated with soluble oligomeric Strep-tactin® (when compared to magnetic particles of solid Streptactin in Fig.4) in which a mixture of αCD3 Fab and αCD28 Fab fragments was immobilized . The results in Fig. 8a and 8b indicate that under these in vitro conditions most of the CD3 + response T cells divided (2 to 5 cell divisions) after compromising the CD28 and TCR / CD3 surface with Fab αCD3 and aCD28 that were reversibly immobilized on soluble Strep-tactin® oligomers. After in vitro expansion the soluble Fab-Strep-Tactin stimulating reagents were dissociated and removed after D-biotin treatment. Dissociation and removal of monomeric Fab fragments were flow-cytometrically analyzed by remaining cells with phycoerythrin-labeled Strep-Tactin® (ST-PE). A representative histogram (dark gray histogram) is shown compared to the appropriate ST-PE only negative control (light gray histogram). It can be seen from Fig. 8C that both Fab fragments had completely dissociated and were completely removed from the expanded cells. Fig. 8D shows the absolute number of living cells (trypan blue negative) after 5 days. The number was counted using a Neubauer counting chamber and plotted against the respective stimulus condition. Median cell numbers in Fig. 8D; error bars indicate standard deviation (SD). Fig. 8D shows that all mixtures of αCD3 Fab fragment and aCD28 Fab fragments that were immobilized in a soluble strepactin multimerization reagent were equally effective in expanding the CD3 + cells and resulted in an approximately 4-fold increase in numbers of absolute cells. Example 6: Proliferation kinetics of purified CD4 + and CD8 + response cells stimulated in vitrocell with Fab multimers - αCD3 / αCD28 streptamers without exchange of media
[000185] In this example the proliferation kinetics of purified CD4 + and CD8 + T-cell (Tresp) response cells that were stimulated in vitro with Fab αCD3 / αCD28 fragments that were reversibly immobilized with soluble oligomeric streptavidin muteins were examined. For this purpose, soluble oligomeric Strep-tactin® mutein of two different sizes served as soluble multimerization reagent. The first oligomeric Streptactin® type was the fraction of the oligomeric streptavidin mutein (n> 3) obtained in Example 5 (similarly referred to here as "conventional Streptactin® structure", illustrated by the triangle symbol with the tip At the top of Fig. 13). The second type of this oligomeric streptavidin mutein used as a soluble multimerization reagent was an oligomeric streptavidin mutein (n> 3) which was reacted with biotinylated human serum albumin (similarly referred to here as "large Streptactin® skeleton).
[000186] In this example 500,000 purified CD4 + CD8 + response T cells (Tresp) were stimulated separately with this two different Streptamer multimers as explained above, i.e. with the Streptactin backbone of Example 5 (using a solution with a concentration of 1mg streptavidin mutein / mL)) or with large Streptactin backbones (0.1 mg / mL). 3 μL of both different main chains were loaded with a combination of 0.5 μg of aCD3 and 0.5 μg of Fab aCD28 used in the previous Examples that carried a streptavidin binding peptide SAWSHPQFEK (GGGS) 2GGSA- WSHPQFEK (SEQ ID NO: 07) at the C-terminal of the Fab fragment heavy chain. In addition, 4.5 μL of the conventional Streptactin structure was loaded with 0.5 μg of Fab aCD3 fragment, 0.5 μg of Fab αCD8 fragment (IBA GmbH Gottingen, which likewise carries the streptavidin binding peptide SAWSHPQFEK (GGGS) 2GGSAWSHPQFEK (SEQ ID NO: 07) and 0.5 μg of aCD28 Fab fragment to the C-terminal of the Fab fragment. as negative control and Tresp cells stimulated with commercially available Dynabeads (spheres in which αCD3 and aCD28 monoclonal antibodies are irreversibly immobilized) as positive control. Tresp cells were seeded in duplicates in 48 well plates in 1 ml of cu medium cell mass (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. Cells were incubated at 37 ° C without changing media and cell count was analyzed after 1, 3 and 6 days. In the experiments in Fig. 13 the expansion was carried out without changing the means. The results for the CD4 + T-response cells are shown in Fig.13A, the results for the CD8 + T-response cells are shown in Fig. 13B, with the graphs representing degree of proliferation according to the number of cells harvested by time point for Tresp CD4 + (Fig. 13A) and for Tresp CD8 + in Fig.13B.
[000187] As can be seen from Fig. 13A the "smallest" soluble multimerization reagent in which Fab aCD3 and aCD28 fragments were reversibly immobilized provided the same amount of CD4 + T cell expansion as Dynabeads (which are the reagent so far) standard for T cell expansion), while the "largest" oligomeric soluble streptactin provided for even better expansion compared to Dynabead. This improvement could be caused by the soluble "larger oligomeric multimerization reagent" being able to bind to more T cells at the same time than the "smaller" soluble oligomer, thereby being able to stimulate more CD4 + T cells than the "smaller" oligomer.
[000188] As evident from Fig. 13B, using the soluble multimerization reagents of the CD8 + T cells of the present invention could be expanded within the first 3 days at least as effectively as with Dynabeads. Notably, for this period of time, the expansion experiment that used a soluble multimerization reagent that in addition to Fab fragments αCD3 and aCD28 (as first and second agents) carried reversibly immobilized on it Fab fragment aCD8, showed the best degree of expansion under these growing conditions. This indicates that it is possible to use a stimulus that is specific to a particular sub-population of cells (here the Fab fragment aCD8) to increase or modulate the selectivity of the expansion, thereby being able to obtain larger amounts of a desired cell (sub) -population.
[000189] Thus, summarizing the above, Example 6 shows that the functionality of the soluble multimerization reagent used in the present invention in terms of activating T cell expansion is comparable to the current standard methodology of using Dynabeads for this purpose. However, since the stimulus can be controlled (and terminated, if desired) by adding a competitor such as biotin in the case of a streptavidin-based reversible interaction between the first and second agent and the multimerization reagent, the present invention provides a significant advantage over the Dynabeads technology as the expansion conditions can be improved (for example, it would be possible to stop the stimulus in the Fig.13B experiment after 3 days). In addition, since the soluble multimerization reagent can be easily removed from the reaction (for example, immobilizing the reagent on a biotinylated column after the expansion reaction), the expansion method of the invention can be carried out and automated in closed systems that , for example, are needed for the production of GMP cells for therapeutic purposes, without having to deal with the removal of spheres, such as Dynabeads. Example 7: Kinetics of proliferation of purified CD4 + and CD8 + response cells stimulated in vitrocomers with reversible Fab- αCD3 / αCD28 streptamer with exchange of media
[000190] Likewise in this example the proliferation expansion kinetics of purified CD4 + and CD8 + response T cells (Tresp) that were stimulated in vitro with Fab aCD3 / aCD28 fragments that were reversibly immobilized on soluble oligomeric streptavidin muteins. For this purpose, soluble oligomeric Strep-tactin® mutein of two different sizes served as soluble multimerization reagent. The first type of oligomeric Streptactin® was the fraction of the oligomeric streptavidin mutein (n> 3) obtained in Example 5 (similarly referred to here as "conventional Streptactin® structure", illustrated by the triangle symbol with the tip downwards in Fig. 13). The second type of this 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 likewise referred to here as "large Streptactin® structure.
[000191] In this example, 400,000 purified CD4 + CD8 + response T cells (Tresp) were stimulated separately with this two different Streptamer multimers as explained above, that is, with the Streptactin backbone of Example 5 (1.0 mg / mL ) or with large Streptactin backbones (0.1 mg / ml). 3 μL of both different main chains were loaded with a combination of 0.5 μg αCD3 and 0.5 μg of α α28 Fab fragments described above. In addition, 4.5 μL of the Streptactin structure of Example 5 was loaded with 0.5 μg αCD3, 0.5 μg Fab αCD8 and 0.5 μg Fab fragment αCD28 as described above. Untreated (unstimulated) Tresp cells served as a negative control and Dynabeads-stimulated Tresp cells (in which αCD3 and aCD28 monoclonal antibodies are irreversibly immobilized) as a positive control. Tresp cells were seeded in duplicates in 48-well plates in 1 ml of cell culture medium supplemented with 30 U / ml of IL-2. Cells were incubated at 37 ° C with change of media on day 3 and cell count was analyzed after 1, 3 and 6 days. The results for CD4 + response T cells are shown in Fig.14A, the results for CD8 + response T cells are shown in Fig. 14B, with the graphs representing degree of proliferation according to the number of cells harvested per point of time for Tresp CD4 + (Fig. 14A) and for Tresp CD8 + in Fig.14B.
[000192] As can be seen from Fig. 14A the soluble multimerization reagents of the present invention in which Fab αCD3 and aCD28 fragments were reversibly immobilized provided better expansion of CD4 + T cells than Dynabeads.
[000193] As evident from Fig. 14B, using the soluble multimerization reagents of the present invention CD8 + T cells could be expanded within the first 6 days at least as effectively as with Dynabeads. Notably, in this period of time, the expansion experiment that used the soluble multimerization reagent larger than transported Fab fragments αCD3 and aCD28 (as first and second agents) showed the best degree of expansion under these culture conditions. This could be caused by the soluble "larger oligomeric multimerization reagent" being able to bind to more T cells at the same time than the "smaller" soluble oligomer, thereby being able to stimulate more CD4 + T cells than the " smaller "oligomer. Example 8: Purified CD4 + CD8 + T cell culture expansion kinetics with or without media change
[000194] In this Example the combined data from Examples 6 and 7 were normalized in input cell number for the "smallest" soluble multimerization reagent and positive and negative control. No normalization data was obtained on the "largest" multimerization reagent. As explained in Examples 6 and 7, 400,000 to 500,000 CD4 + or CD8 + response T cells (Tresp) were stimulated with 3 μL of a preparation of Streptactin multimers (1mg / mL; where 0.5 μg of aCD3 and 0 Fab fragment , 5 μg of aCD28 Fab fragment were immobilized. Untreated (unstimulated) Tresp cells served as a negative control and Tresp cells stimulated with Dynabeads as a positive control. Tresp cells were seeded in duplicates in 48 well plates in 1 ml of culture medium. of cell supplemented with 30 U / ml of IL-2. Tresp cells were seeded in duplicates in 48 well plates in 1 ml of cell culture medium supplemented with 30 U / ml of IL-2. Cells were incubated at 37 ° C with change of media (straight lines in Fig. 15) or without change of media (dotted lines in Fig. 15) on day 3 and cell count was analyzed after 1, 3 and 6 days. normalized from Fig. 15A, the "smallest" soluble multimerization reagent It is possible that Fab αCD3 and aCD28 fragments were reversibly immobilized yielding a 2.5 fold expansion of CD4 + T cells, while the expansion using Dynabeads yielded a 1.8 fold expansion rate. Thus, the use of a soluble multimerization reagent of the invention still provides an improvement in the expansion of CD4 + T cells over Dynabeads. Similarly, Fig. 15B, confirms that using the soluble multimerization reagents of the present invention CD8 + T cells could be expanded within the first 3 days at least as effectively as with Dynabeads. Example 9: Early cluster formation after activation of purified CD4 + and CD8 + response T cells stimulated in vitro with reversible αCD3 / αCD28 Fab-Streptamer multimers
[000195] In this Example, 400,000 T cells of CD4 + or CD8 + response (Tresp) were stimulated with 3 μL of an oligomeric preparation Streptactin multimerization reagent (1 mg / mL) loaded with a combination of 0.5 μg of αCD3- and 0.5 μg of Fab αCD28. Untreated (unstimulated) Tresp cells served as a negative control and Tresp cells stimulated with Dynabeads as a positive control. Tresp cells were seeded in duplicates in 48-well plates in 1 ml cell culture medium supplemented with 30 U / ml IL-2. Cells were incubated at 37 ° C and microscopically analyzed after 1 and 2 days. Stimulus of Tresp CD4 + (Fig. 16A) and Tresp CD8 + (Fig. 16B) is shown for Dynabeads (median row) and Streptamer multimers (lower row) respectively. The photographs represent degree of cluster formation: For exemplary clusters of better visibility they are indicated by circles for the stimulation with soluble streptavidin mutein oligomers in Fig. 16A and Fig. 16B. Clusters within the Dynabead stimulus are easily visible by accumulation of dark stimulating particles. Of course, also for early CD4 + and CD8 + T cells formed using the expansion method of the invention that employs a soluble oligomeric multimerization reagent. Example 10: Kinetics of expansion & phenotype of CD3 + central memory T cells activated / expanded (Tcm)
[000196] In this Example, 500,000 CD3 + CD62L + CD45RA-Tcm response cells (Tresp) were stimulated with 3 μL of a preparation of the soluble oligomeric Streptactin of Example 5 (1 mg / mL) that was loaded with a combination of 0.5 μg of aCD3 and 0.5 μg of Fab aCD28. In addition, 4.5 μL of a preparation of Streptactin multimers loaded with 0.5 μg aCD3, 0.5 μg Fab aCD8 and 0.5 μg Fab aCD28 was used as an additional stimulus condition. Untreated (unstimulated) Tresp cells served as a negative control and Tryn cells stimulated with Dynabeads (where αCD3 and aCD28 monoclonal antibodies are irreversible immobilized) as a positive control. Tresp cells were seeded in 48-well plates in 1 ml of cell culture medium supplemented with 30 U / ml IL-2 alone or 30 U / ml IL-2 and 5 ng / ml IL-15. Cells were incubated at 37 ° C with change of media every 3 days and cell count was analyzed after 7 and 14 days. Graphs represent degree of proliferation according to the number of cells harvested per time point, in Fig. 17A only medium supplemented with IL-2 and in Fig.17B medium supplemented with IL-2 and IL-15. As can be seen from Fig. 17A and Fig. 17B, the soluble multimerization reagent that has been reversibly linked to that Fab CD3 fragment and Fab aCD28 fragment yields better cell expansion than Dynabeads. As also shown by the CD62L and CD127 surface expression flow cytometry analysis after 14 days of culture in variable cytokine environments of Fig. 17C, the experimental approaches using soluble multimerization reagents of the present invention retain, under both conditions chosen here, a higher CD127 content expressing lasting memory T cells than expansion with Dynabeads. This illustrates another advantage of the methods of the present invention. Example 11: Selective antigen-specific expansion of Tcm response cells outside CD3 + central memory T cells by volume (kinetics & phenotype)
[000197] In this Example, the kinetics and phenotype of selective Antigen-specific (Ag-specific) expansion were from purified Tcm CD3 + CD62L + CD45RA response cells were examined.
[000198] In more detail, Tcm CD3 + CD62L + CD45RA response cells were stimulated in vitro with both a peptide molecule complex: MHC (which acts as the first agent that provides a primary activation signal to cells) and an αCD28 Fab fragment (which acts as a second reagent that stimulates an additional molecule on the surface of cells). Likewise, the specific peptide antigen complex with the MHC molecule and the αCD28 Fab fragment were reversibly immobilized in the soluble oligomeric streptavidin mutein (with n> 3) described in Example 5. The peptide that was used for the specific antigen expansion was the CRVLCCYVL peptide (SEQ ID NO: 06), amino acids 309-317 of the immediate-early protein 1 (described in Ameres et al., PLOS Pathogens, May 2013, vol. 9, subject 5, e1003383) representing an HLA-C7 epitope / IE-1 that is specific for cytomegalovirus (CMV). The MHC I molecule that exhibits the C-terminal transport of the α-streptavidin binding peptide (heavy chain) (SAWSHPQFEK (GGGS) 2GGSAWSHPQFEK, (SEQ ID NO: 07) which is commercially available as "Twin- Strep-tag® "from IBA GmbH, Gottingen, Germany).
[000199] For this purpose, 500,000 Tcm cells of CD3 + CD62L + CD45RA- response (Tresp) were stimulated Ag-specifically using 3 μL of a soluble oligomeric streptactin functionalization reagent preparation with 0.5 μg of the peptide complexes : MHC class I equipped with streptavidin binding peptide and 0.5 μg of the αCD28 Fab described above. As an alternative, 4.5 μL of a preparation of the Streptactin multimerization reagent was loaded with 0.5 μg of these peptide complexes: MHC class I, 0.5 μg of Fab αCD8 and 0.5 μg of Fab αCD28. For comparison, polyclonal stimulation was performed, using 3 μL of a preparation of Streptactin multimerization reagent (1 mg / mL) loaded with a combination of 0.5 μg Fab αCD3 and 0.5 μg Fab αCD28. Again as the alternative stimulus condition described above, 4.5 μL of a reversibly charged Streptactin multimerization reagent preparation with 0.5 μg Fab αCD3, 0.5 μg Fab αCD8 and 0.5 μg Fab αCD28 was used. Untreated (unstimulated) Tresp cells served as negative control and polyclonal stimulated Tresp cells with Dynabeads (spheres in which αCD3 and aCD28 monoclonal antibodies are irreversible immobilized) as a positive control. Tresp cells were seeded in 48-well plates in 1 ml of cell culture medium supplemented with 30 U / ml IL-2 and 5 ng / ml IL-15. Cells were incubated at 37 ° C with change of media every 3 days and cell count was analyzed after 7 and 14 days. Exemplary flow cytometry analysis for the fraction of Ag-specific cells that was stimulated / expanded by the soluble streptactin oligomer in which the peptide complex: MHC-I for an HLA-C7 / IE-1 epitope (for CMV) was immobilized (Fig. 18A) shows that these antigen-specific T cells have been specifically expanded. The graphs of Fig. 18B to Fig. 18E (representing the degree of expansion of different Ag-specificities according to the number of peptide: MHCI multimer- positive cells harvested by time point in analogy to the expansion experiment shown in Fig .18A) shows that the multi-polymerization reagent using the respective Ag-specific peptide complex and MHC I molecule supplied for the highest number of expanded cells (ranging from a twenty-fold increase in the number of cells for Ag cells - specific that recognize the CMV pp65 epitope (amino acids 341-350 (QYDPVAALF, (SEQ ID NO: 08)) restricted by HLA-A2402) (see Fig. 18B) for a 98-fold increase in the number of Ag- that recognize the CMV HLA-B7 / IE-1309-317 (CRVLCCYVL (SEQ ID NO: 06)) epitope (see Fig. 18E), thereby showing that the expansion method of the present invention is completely applicable to expansion of Ag-specific cells. Finally, flow cytometry analysis exemplary surface expression of CD62L and CD127 after 14 days of culture for HLA-B7 / Hexon5 epitope (for adenovirus) shown in Fig. 18F also confirms that experimental approaches using the soluble multimerization reagents of the present invention retain a higher content high level of CD127 expressing T cells of lasting memory in polyclonal and Ag-specific stimulatory conditions. Example 12: Selective Ag-specific expansion kinetics & volume central T-cell phenotype
[000200] This Example examines the selective Ag-specific expansion kinetics outside the purified Tcm CD3 + CD62L + CD45RA response cells that were stimulated in vitro with a) antigen-specific MHC I peptide complexes and b) Fab αCD28 fragments that were reversibly immobilized as first and second agents on soluble oligomeric streptavidin muteins.
[000201] For this purpose, 500,000 Tcm cells of CD3 + CD62L + CD45RA response (Tresp) were specifically stimulated by Ag using 3 μL of a functionalized Streptactin multimerization reagent preparation with 0.5 μg of peptide complexes: MHC class I equipped with a streptavidin-binding peptide (the specific peptide represents amino acids 114-124 (CPYSGTAYNSL, SEQ ID NO: 10) of the Hexen 5 protein from adenovirus) restricted by HLA-B07) and 0.5 μg of Fab αCD28. As an alternative, 4.5 μL of a Streptactin multimerization reagent preparation loaded with 0.5 μg of peptide complex: MHC class I, 0.5 μg of Fab αCD8 and 0.5 μg of Fab αCD28. For comparison, polyclonal stimulation was performed, using 3 μL of a Streptactin multimerization reagent preparation (1 mg / mL) loaded with a combination of 0.5 μg of αCD3 Fab and 0.5 μg of αCD28. Again as the alternative stimulus condition described above, 4.5 μL of a Streptactin multimer preparation loaded with 0.5 μg of Fab αCD3, 0.5 μg of Fab αCD8 and 0.5μg of Fab αCD28 were used. Untreated (unstimulated) Tresp cells served as a negative control and polyclonal stimulated Tresp cells with Dynabeads as a positive control. Tresp cells were seeded in 48-well plates in 1 ml of cell culture medium supplemented with 30 U / ml IL-2 and 5 ng / ml IL-15. The cells were incubated at 37 ° C with medium change every 3 days and the cell count was analyzed after 7 and 14 days. The tables shown in Fig. 19 represent the degree of cluster formation on day 5, the specific excitation of exemplary Ag is illustrated for the adenovirus HLA-B7 / Hexon 5 epitope. As can be seen from Fig. 19, such adenovirus antigen-specific cells can be specifically amplified from the original Tcm population of CD3 + CD62L + CD45RA response. Example 13: Yield and phenotype of expanded CD8 + T cells - size variation of the soluble multimerization reagent and addition of the addition of αCD8-Fab for excitation
[000202] In this Example, the expansion of purified CD8 + T response cells stimulated in vitro with Fab αCD3 / aCD28 fragments that were reversibly immobilized soluble oligomeric streptavidin muteins was examined. In addition, the effect of adding αCD8-Fab to the multimerization reagent to increase the specificity of the expansion for CD8 + T cells was examined.
[000203] For this purpose, 300,000 purified CD8 + response cells (Tresp) were stimulated separately with two different Streptactin-based multimerization reagents, that is, the small oligomeric Streptactin multimerization reagent of Example 5 (1 mg / mL) or the larger Streptactin oligomers described above (0.1 mg / ml). 3 μL of both different multimerization reagents (skeletons) were loaded with a combination of the 0.5 μg αCD3 Fab fragments and 0.5 μg αCD28 described above. In addition, 4.5 μL of the minor Streptactin multimerization reagent (backbone) was loaded with 0.5 μg αCD3, 0.5 μg Fab αCD8 and 0.5 μg Fab fragments αCD28 described above. In addition, 3 μL of the "minor" Streptactin multimerization reagent (backbone) only functionalized with 0.5 μg of α α CD3 Fab fragment alone or 0.5 μg of α α CD28 Fab fragment alone 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 48 well plates in 1 ml of cell culture medium supplemented with 30 U / ml of IL-2. The cells were incubated at 37 ° C with media change after 3 days and analyzed after 6 days. Fig. 20A describes the degree of proliferation according to the number of cells harvested on day 6 compared to the negative controls and normalized to the positive control. Fig. 20A shows that expansion of CD8 + T cells using the soluble multimerization reagents of the invention results in higher yields of CD8 + T cells than expansion using dynabeads. FACS analysis of CD8 surface expression (Fig.20B) and CD45RO surface expression (Fig. 20C) after cell culture shows that the same phenotype of CD8 + T cells was amplified by the multimerization reagents of the invention or Dynabeads (the various stimulating conditions were compared using one-way ANOVA analysis and no significant differences (ns) were detected). The improved yield of CD8 + cells using the inventive expansion methods compared to Dynabeads could be due to the fact that the soluble multimerization reagent can access its target receptors on the cell surface better than the antibodies that are immobilized on Dynabeads. This improved yield could be very advantageous when expanding the rare cell population of an initial sample.
[000204] In addition, comparing the expansion yield obtained with the multimerization agent in which also 0.5 μg of αCD3 and 0.5 μg of Fab αCD28 fragments were immobilized together (second column from the left in Fig. 20B) for the yield using two multimerization reagents that were functionalized alone with the αCD3 Fab fragment alone or the αCD28 Fab fragment alone (third column from the left in Fig. 20B), it can be seen that both experiments had the same expansion efficiency. In this way, these experiments showed that the use of a multimerization reagent in which both the first agent and the second agent are together immobilized, is functionally equivalent to the use for the expansion of two separate multimerization reagents that are loaded only with the first agent and the second agent, respectively. Example 14: Yield & phenotype of CD8 + T cells - titration of separate soluble multimerization reagents with different ratios of Fab fragment αCD3 and αCD28 immobilized in this
[000205] In this Example, the yield and phenotype of expanded CD8 + T-response cells (Tresp) that were stimulated in vitro with Fab αCD3 / aCD28 fragments that were reversibly immobilized in different amounts on soluble oligomeric streptavidin muteins were examined.
[000206] For this purpose, 300,000 CD8 + response T cells (Tresp) were stimulated with varying amounts of a mixture of preparations of the "small" oligomeric Streptactin (1 mg / mL) functionalized with Fab αCD3 alone and Fab αCD28 alone ("1x" corresponds to 1.5 μg of functionalized Streptactin multimerization reagent with 0.5 μg of αCD3 alone and 1.5 μg of functionalized multimerized Streptactin with 0.5 μg of α α28 Fab fragment only), or 3 μL of a preparation of the Streptactin multimerization reagent loaded with 0.5 μg of Fab αCD3 and 0.5 μg of αCD28, or 4.5 μL of a preparation of the Streptactin multimerization reagent loaded with 0.5 μg of αCD3, 0 , 5 μg of αCD8 labeled with streptactin and 0.5 μg of Fab αCD28. Untreated Tresp cells served as a negative control and Tresp stimulated with Dynabeads as a positive control. Tresp cells were seeded in 48-well plates in 1 ml of cell culture medium supplemented with 30 U / ml of IL-2. The cells were incubated at 37 ° C without changing media and analyzed after 5 days. Fig. 21A describes the degree of proliferation according to the number of cells harvested on day 5 compared to the negative controls and normalized for the positive control. Fig. 21A shows that expansion of CD8 + T cells using the various soluble multimerization reagents of the invention results in higher yields of CD8 + T cells than expansion using dynabeads (especially the cumulative total reagent quantity of condition 5x resulted in an expansion ideal of cells especially on the time / increase in total cells starting cell division). FACS analysis of CD8 surface expression (Fig.21B) and CD45RO surface expression (Fig. 21C) after cell culture shows that the same phenotype of CD8 + T cells has been amplified by the various multimerization reagents of the invention or commercially available Dynabeads. Example 15: Activation of intracellular signaling cascades after excitation of Jurkat cell streptomer multimers transduced by αCD19-CAR
[000207] In this Example, the activation of intracellular signaling cascades from transduced Jurkat cells that have been modified to express a tumor-specific chimeric antigen (CAR) receptor, that is, here CD19 and that have been stimulated using the oligomeric Strepactin® Example 5 as a soluble multimerization reagent was examined.
[000208] For this purpose, 300,000 response Jurkat cells (Jresp) were stimulated with (A) varying amounts of a mixture of Streptactin multimerization reagent preparations (1 mg / mL) functionalized with Fab αCD3 and Fab fragments αCD28 described here ("x1" corresponds to 3 μg of functionalized Streptactin reagent with 0.5 μg of αCD3 Fab and 0.5 μg of αCD28 - this provides a "polyclonal Streptactin based multimerization reagent"), or (B) 3 μL of a Streptactin multimerization reagent preparation functionalized with 0.5 μg (x1) or 1 μg (x2) of the extracellular domain (ECD) of CD19 (the natural ligand for αCD19-CAR - this provides a "CAR-specific Streptactin based multimerization reagent"), or 3 μL of a Streptactin multimerization reagent preparation loaded with 0.5 μg (x1) or 1 μg (x2) of αIgG that recognizes the IgG4 spacer inside αCD19-CAR - this likewise provides a "mu reagent" ltimerization based on CAR-specific Streptavidin mutein "). CD19 ECD equipped with a hexahistidine tag was obtained from Sino Biological / Life technologies (SEQ ID NO: 27) and was functionalized for binding to streptavidin-based multimerization reagent by mixing the CD19 ECD with the His- STREPPER (IBA GmbH, Germany, Order number 2-0920-005) at a molecular ratio of 1: 1 and incubating for 15 min at room temperature. The adapter molecule His-STREPPER contains a chelation portion that binds to the hexahistidine tag and a streptavidin-binding peptide, thereby temporarily supplying the target molecule, here the CD19 ECD with a streptavidin-binding peptide reversibly bind to a streptavidin mutein-based multimerization reagent. Jresp stimulated with Dynabeads (spheres that have irreversibly immobilized in it monoclonal antibodies of αCD3 and αCD28) or PMA and Ionomicina that served as positive controls. Jresp cells were seeded in 1.5 ml Eppendorf tubes in 200 μL of cell culture medium supplemented with 30 U / ml IL-2. The cells were incubated at 37 ° C and placed on ice and lysed after 0 min to 20 min excitation. Detection of phosphorylated ERK indicates active MAPK signaling, maintenance β-Actin tagging indicates the loading of equal amounts of total protein by condition and time point. As can be seen from the comparison of Fig. 22A showing the activation of Jurkat cells by the "polyclonal Streptactin multimerization reagent" and Fig. 22B showing the activation of Jurkat cells by the two "CAR specific Streptactin-based multimerization reagents. ", Jurkat cells can be activated / expanded by binding the CD19 extracellular domain to the CD19 specific chimeric antigen receptor. Since then, the T cell genetic downstream process has been carried out almost exclusively in pre-selected cell populations, a generic activation by cross-linking CARs introduced by the IgG4 spacer domain (this is conserved within several CARs with different specificities) expands the applicability for reversible cell stimulation / expansion in these in vitro cell process situations.
[000209] Thus, this experiment shows that in principle any cell population that is activated by binding an agent (ligand) that provides a primary activation signal to the cell population can be amplified using a first agent reversibly immobilized in a reagent multimerization as described here. Example 16: Yield and subset composition of expanded CD3 + T cells with addition of αCD8-Fab for excitation
[000210] The experiment shows the expansion of purified CD3 + T-response cells stimulated in vitro with αCD3 / αCD28 Fab fragments that were reversibly immobilized on the soluble oligomeric Strep-tactin® of Example 5 which served as a soluble multimerization reagent. In one experiment, in addition to αCD3 / aCD28 Fab fragments, a commercially available αCD8 Fab fragment from IBA GmbH, Gottingen, Germany (catalog number 6-8000-203) was immobilized on the streptavidin mutein soluble oligomer to test whether it is it is possible to preferentially stimulate a specific T cell subpopulation in vitro with the reversible Fab-Streptamer multimers αCD3 / aCD28. In more detail, 500,000 purified CD3 + response T cells (Tresp) were stimulated with 3 μL of an oligomeric Streptavidin preparation (1 mg / mL) loaded with a combination of 0.5 μg of the αCD3 Fab and 0.5 μg of αCD28 . As an alternative method, 4.5 μL of the Streptactin oligomer was loaded with 0.5 μg of αCD3, 0.5 μg of Streptactin-labeled αCD8 Fab and 0.5 μg of Streptactin-labeled αCD28 Fab. Unstimulated Tresp cells served as a negative control and Tresp stimulated with Dynabeads (spheres in which the monoclonal antibodies of αCD3 and αCD28 are irreversibly immobilized) served as a positive control. As can be seen from Fig. 23A, the multimerization reagent that is reversibly loaded with the αCD3 Fab fragment, the αCD28 Fab fragment and also the αCD8 Fab fragment provided the highest number of expanded CD3 + T cells. With 1 x 106 of the number of cells expanded, the yield was about 30% higher than for the expansion of these T cells using commercially available Dynabeads. In addition and more importantly, as shown in Fig. 23B with this multimerization reagent that carries the Fab αCD3 fragment, the Fab αCD28 fragment and the Fab αCD8 fragment, the number of CD8 + T cells was the highest, as compared to the expansion with Dynabeads or a soluble multimerization reagent of the invention which alone carries the αCD3 Fab fragment and the αCD28 Fab fragment as first and second agents as described herein. Thus, in the same way this experiment shows the advantage of the present invention that in addition to a first agent that provides a primary activation signal to the desired cell population and optionally a second agent that provides a co-stimulating signal, an additional agent that is specific for activation of the desired cell population can be immobilized on the multimerization reagent. Thus, in doing so, the present invention provides the possibility to expand preferentially or selectively enrich any desired (sub) population of a sample cell, which, for example, comprises a variety of different subpopulations. Example 17: Parallel Antigen-specific expansion of Tcm response cells outside a single cluster
[000211] In this Example, the kinetics of parallel Antigen-specific (Ag-specific) expansion out of a single cluster of T-response cells stimulated in vitro with multiple reversible peptides: MHC / αCH28 Fab-Streptamer multimers are examined.
[000212] 500,000 Tcm cells of CD3 + CD62L + CD45RA response (Tresp) are stimulated simultaneously for multiple Ag specificities using for each specificity, 3 μL of Streptactin multimers functionalized with 0.5 μg of the respective MHC class I peptide complexes that carry a streptavidin-binding peptide and 0.5 μg of α α28 Fab which likewise carries a streptavidin-binding peptide. As an alternative method, 4.5 μL of functionalized Streptactin-based multimerization reagent with 0.5 μg of peptide complexes: MHC class I carrying a streptavidine-binding peptide, 0.5 μg of αα8 Fab and 0.5 μg of Fab αCD28 as described here are used for each specificity. For comparison, polyclonal excitation is performed using 3 μL of a Streptactin-based multimerization reagent preparation (1 mg / mL) reversibly loaded with a combination of 0.5 μg Fab αCD3 and 0.5 μg Fab αCD28. Again as the alternative stimulus condition described above, 4.5 μL of a Streptactin-based multimerization reagent preparation reversibly loaded with 0.5 μg Fab αCD3, 0.5 μg Fab αCD8 and 0.5 μg Fab αCD28 (each carrying a streptavidin binding peptide can be used. Untreated (unstimulated) Tresp cells serve as a negative control and polyclonal stimulated Tresp cells with Dynabeads (αCD3 and αCD28 mAb coated beads) 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. The cells are incubated at 37 ° C with exchange of media every 3 days and the cell count is analyzed after 7 and 14 days. Example 18: Preferential proliferation of CD8 + T cells among CD3 + response T cells stimulated in vitro with streptavidin based multimerization reagents reversibly functionalized with αCD3 / aCD8 / αCD28 Fab fragments
[000213] 300,000 CD3 + response T cells (Tresp) are stimulated with 3 μL of a Streptactin multimerization preparation (1 mg / mL) or a multimerization reagent preparation using the large Streptactin structure (0.1 mg / mL) loaded with a combination of 0.5 μg of αCD3 and 0.5 μg of Fab αCD28, or 4.5 μL of a Streptactin-based multimerization reagent preparation loaded with 0.5 μg of αCD3, 0, 5 μg of Fab αCD8 and 0.5 μg of Fab αCD28, or 3 μL of a mixture of Streptactin-based multimerization reagent preparations with 0.5 μg of ααCD3 Fab alone and 0.5 μg of ααCD28 Fab alone (each Fab fragment again carries a streptavidin-binding peptide). Untreated Tresp cells serve as a negative control and Tresp stimulated with Dynabeads (spheres coated with αCD3 and αCD28 mAb) as positive control. Tresp cells are seeded in duplicate in 48-well plates in 1 ml of cell culture medium supplemented with 30 U / ml of IL-2. The cells are incubated at 37 ° C with media change after 3 days and analyzed after 6 days. Example 19: Preferential proliferation of CD8 + T cells among CD3 + T-cells stimulated in vitro with multimerization reagents based on streptavidin reversibly functionalized with αCD3 and αCD28 Fab fragments
[000214] 300,000 CD3 + response T cells (Tresp) are stimulated with varying amounts of a mixture of Streptactin-based multimerization reagent preparations (1 mg / mL) functionalized with αCD3 Fab fragment alone and Fab αCD28 fragment alone (1 , 5 μg of functionalized Streptactin-based multimerization reagent with 0.5 μg of αCD3 Fab fragment alone and 1.5 μg of functionalized Streptactin-based multimerization reagent with 0.5 μg of αCD28 Fab fragment only), or quantities assorted mixtures of multimerization reagent preparations based on functionalized Streptactin with αCD3 Fab fragment and αCD28 Fab fragment with or without the αCD8 Fab fragment (each Fab fragment again carries a streptavidin-binding peptide) (3 μg of multimerization reagent based on Streptactin functionalized with 0.5 μg of αCD3 Fab fragment and 0.5 μg of αCD28 - without αCD8 Fab fragment, or 4.5 μL of a multimer reagent preparation ization of Streptactin loaded with 0.5 μg αCD3 Fab fragment, 0.5 μg αCD8 Fab fragment and 0.5 μg αCD28 Fab fragment, where the Fab fragment again carries a streptavidin binding peptide). Untreated Tresp cells serve as a negative control and Tresp stimulated with Dynabeads (spheres coated with αCD3 and αCD28 mAb) 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. The cells are incubated at 37 ° C with media change after 3 days and analyzed after 6 days.
[000215] The listing or discussion of a document previously published in this specification should not necessarily be used as an acknowledgment that the document is part of the state of the art or is of common general knowledge.
[000216] The invention illustratively described here can be practiced appropriately in the absence of any element or elements, limitations or limitations, not specifically described here. Thus, for example, the terms "comprising", "including", containing ", etc. will be read expansively and without limitation. Additionally, the terms and expressions used here were used as terms of description and not of limitation, and there is no no intention in the use of such terms and expressions to exclude any equivalents of the aspects shown and described or portions thereof, however it is recognized that several modifications are possible within the scope of the claimed invention. been specifically described by exemplary modalities and optional aspects, the modification and variation of the inventions included in this described herein can be appealed by those skilled in the art, and that such modifications and variations are considered within the scope of this invention.
[000217] The invention has been described widely and generically here. Each of the limited species and subgeneric groupings included in the generic description is also part of the invention. This includes the generic description of the invention with a negative condition or limitation that removes any subject of its kind, regardless of whether or not the cut material is specifically listed here.
[000218] Other modalities are within the following claims. In addition, where the features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is likewise described in terms of any individual member or subgroup of members of the Markush group.

权利要求:
Claims (23)
[0001]
1. In vitro method of stimulating a cell population, characterized by the fact that it comprises contacting a sample comprising a cell population in the presence of a multimerization reagent, which is reversibly linked to a first agent and a second agent, where the multimerization reagent is in a soluble form, where the multimerization reagent comprises an oligomer or a streptavidin polymer, avidin, a streptavidin analog that reversibly binds to biotin or an avidin analog that reversibly binds to biotin , wherein the first agent: (i) comprises at least one C1 binding partner capable of reversibly binding to a Z1 binding site of the multimerization reagent to form a reversible bond between the C1 binding partner and the Z1 binding site , in which the C1 binding partner is a streptavidin or avidin binding peptide, and (ii) is able to bind to a receptor molecule on the surface of the population cell to stimulate a signal to the cell, and in which the second agent: (i) comprises at least one C2 binding partner capable of reversibly binding to a Z2 binding site of the multimerization reagent to form a reversible link between the binding partner C2 and the Z2 binding site, and (ii) is able to bind to an accessory molecule on the cell surface to stimulate an accessory signal on the cell surface, thereby stimulating cells in the population, in which the partner C2 binding is a streptavidin or avidin binding peptide.
[0002]
2. Method according to claim 1, characterized by the fact that the cell population is a lymphocyte population.
[0003]
Method according to claim 2, characterized by the fact that the lymphocyte population is a B cell population, a T cell population, a natural killer cell population, or a mixture thereof.
[0004]
Method according to any one of claims 1 to 3, characterized in that: the cell population comprises a population of T cells and the first agent comprises an MHC I complex: peptide or stimulates a signal associated with the complex of TCR / CD3 in T cells.
[0005]
Method according to any one of claims 1 to 4, characterized in that the first agent comprises a binding reagent that binds to CD3.
[0006]
6. Method according to any one of claims 1 to 5, characterized in that the cell population comprises a population of T cells and the accessory molecule is CD28 or CD137.
[0007]
Method according to any one of claims 1 to 6, characterized in that the second agent comprises a binding reagent that binds to CD28 or CD137 or is a mixture of two different binding reagents that both specifically bind to CD28 or CD137.
[0008]
Method according to any one of claims 1 to 7, characterized in that: said first agent specifically binds to CD3 and is selected from the group consisting of an anti-CD3 antibody, a divalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody and a CD3-binding protein molecule with similar antibody-binding properties, and / or the second agent specifically binds to CD28 or CD137 and is selected of the group consisting of an anti-CD28 antibody, a divalent antibody fragment of an anti-CD28 antibody, a monovalent antibody fragment of an anti-CD28 antibody, a CD28-binding protein molecule with similar antibody-binding properties, an anti-CD137 antibody, a divalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment of an anti-CD137 antibody, a protein molecule binding to CD137 with properties similar antibody binding, 4-1BB ligand and any mixture thereof.
[0009]
9. Method according to claim 8, characterized in that the divalent antibody fragment is an F (ab ') 2 fragment or a divalent single chain Fv fragment, or the monovalent antibody fragment is selected from the group that consists of a Fab fragment, an Fv fragment and a single chain Fv fragment (scFv).
[0010]
Method according to any one of claims 1 to 9, characterized in that the first agent comprises an anti-CD3 Fab and the second agent comprises an anti-CD28 Fab.
[0011]
Method according to any one of claims 1 to 3, characterized in that the cell population comprises a population of B cells and the receptor molecule is CD40 or CD 137 and / or the first agent comprises a binding reagent that binds CD40 or CD137.
[0012]
Method according to any one of claims 1 to 11, characterized in that the streptavidin oligomer or polymer, avidin, the streptavidin analogue or the avidin analogue is cross-linked by a polysaccharide or via a bifunctional linker.
[0013]
13. Method according to any one of claims 1 to 12, characterized in that the streptavidin, avidin oligomer or polymer, the streptavidin analogue or avidin analogue comprises three or more streptavidin, avidin crosslinked tetramers, the analog streptavidin or avidin analog.
[0014]
Method according to any one of claims 1 to 13, characterized in that said C1 binding partner and / or said C2 binding partner comprises the streptavidin binding peptide Trp-Ser-His-Pro- GIn-Phe-Glu-Lys and said multimerization reagent comprise a streptavidin analog comprising the amino acid sequence Va144-Thr45-Ala46-Arg47 at sequence position 44 to 47 of the wild-type streptavidin or a streptavidin analog comprising the amino acid sequence lle44-Gly45-Ala46-Arg47 at sequence position 44 to 47 of wild-type streptavidin.
[0015]
Method according to any one of claims 1 to 13, characterized in that said C1 binding partner and / or said C2 binding partner comprises the streptavidin binding peptide SAWSHPQFEKíGGGSteGGSAW-SHPQFEK (SEQ ID NO: 7).
[0016]
16. Method according to any one of claims 1 to 13 and 15, characterized in that the multimerization reagent comprises a streptavidin analogue comprising the amino acid sequence Val44-Thr45-Ala46-Arg47 in sequence positions 44 to 47 of wild-type streptavidin or a streptavidin analog comprising the amino acid sequence Ile44-Gly45-Ala46-Arg47 at sequence positions 44 to 47 of wild-type streptavidin.
[0017]
17. Method according to claim 15 or claim 16, characterized in that the N-terminal amino acid residue of the streptavidin analog is in the region of amino acids 10 to 16 of the amino acid sequence of wild-type streptavidin and the residue of C-terminal amino acid of the streptavidin analog is in the region of amino acids 133 to 142 of the streptavidin amino acid sequence of the wild type.
[0018]
18. Method according to any one of claims 1 to 17, characterized in that the binding partners C1 and C2 are identical or different.
[0019]
19. Method according to any one of claims 1 to 18, characterized in that the dissociation constant (Kd) for the reversible bond between said C1 binding partner and said Z1 and / or Kd binding site for reversible bonding between said bonding partner C2 and said bonding site Z2 is in the range of 10-2 M to 10-13 M.
[0020]
20. Method according to any one of claims 1 to 19, characterized in that it further comprises breaking the connection between said first agent C1 binding partner and / or said second agent C2 binding partner and said binding site Z1 and / or Z2 of said multimerization reagent, optionally, wherein said disruption causes the end of stimulation of the cells.
[0021]
21. Method according to any one of claims 1 to 20, characterized in that the reversible bond is disrupted by contact of the cells with biotin or a biotin analog.
[0022]
22. Method according to any one of claims 1 to 20, characterized in that: said reversible link between said C1 binding partner and said Z1 binding site of said multimerization reagent is disrupted by contact of said cell population with a free C1 binding partner, and / or said reversible link between said C2 binding partner and said Z2 binding site of said multimerization reagent is disrupted by contact of said cell population with a free C2 connection.
[0023]
23. Method according to any one of claims 1 to 22, characterized in that the cell population comprises T cells and a T cell receptor or a chimeric antigen receptor is introduced into the T cells after stimulation or during stimulation.

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WO2022029660A1|2020-08-05|2022-02-10|Juno Therapeutics, Inc.|Anti-idiotypic antibodies to ror1-targeted binding domains and related compositions and methods|

法律状态:
2019-06-04| B25G| Requested change of headquarter approved|Owner name: JUNO THERAPEUTICS GMBH (DE) |

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2020-02-04| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-07-21| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-11-24| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-02-09| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 16/04/2015, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201461980506P| true| 2014-04-16|2014-04-16|

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US61/980,506|2014-04-16|

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PCT/EP2015/058339|WO2015158868A2|2014-04-16|2015-04-16|Methods, kits and apparatus for expanding a population of cells|

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