Methods for culturing cells and kits and apparatus for same

Abstract

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

1 . A method for culturing target cells, comprising incubating a composition comprising target cells in the presence of a receptor-binding agent, wherein the receptor-binding agent is (i) reversibly bound to a reagent comprising a plurality of binding sites capable of reversibly binding to the receptor-binding agent, and (ii) specifically binds to a molecule on the surface of the target cells other than CD28, CD3, or CD40 in a manner that induces a signal in the target cells, thereby generating cultured target cells. 
     
     
         2 . The method of  claim 1 , wherein the signal is a costimulatory signal, and the molecule is a costimulatory molecule selected from among CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, and HVEM. 
     
     
         3 . The method of  claim 1 , wherein the signal is a costimulatory signal, and the molecule is a costimulatory molecule selected from among CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), and CD154 (CD40L). 
     
     
         4 . The method of  claim 1 , wherein:
 the molecule is a cytokine receptor selected from among IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, Type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1, and TNFR2; and   the receptor-binding agent comprises IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-1β, IL-12, IL-15, IL-17, or TNF.   
     
     
         5 . The method of  claim 1 , wherein:
 the molecule is a cytokine receptor selected from among IL-7R, IL-21R, and CD132 (IL receptor common gamma chain); and   the receptor-binding agent comprises IL-7, IL-21, IL-2, IL-4, IL-9, or IL-15.   
     
     
         6 . The method of  claim 1 , wherein:
 the molecule is a chemokine receptor selected from among CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4; and   the receptor-binding agent comprises CXCL9, CXCL10, CCL19, CCL21, or CCL25.   
     
     
         7 . The method of  claim 1 , wherein the molecule is an adhesion molecule selected from among CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin), CD29/CD49d (VLA-4), and CD106 (VCAM-1). 
     
     
         8 . The method of  claim 1 , wherein the receptor-binding agent comprises a nuclear factor that is retinoic acid receptor-related orphan receptor gamma (RORgamma) or RORalpha. 
     
     
         9 . The method of  claim 1 , wherein the receptor-binding agent is a first receptor-binding agent, the molecule is a first molecule, and the incubation is further carried out in the presence of a second receptor-binding agent that specifically binds to a second molecule on the surface of one or more of the target cells, wherein the second receptor-binding agent is reversibly bound to the reagent, the reagent comprising a plurality of binding sites capable of reversibly binding to the second receptor-binding agent. 
     
     
         10 . The method of  claim 9 , wherein the signal is a first signal, and the second receptor-binding agent specifically binds to the second molecule in a manner that induces a second signal that is a TCR/CD3 complex-associated signal in one or more of the target cells. 
     
     
         11 . The method of  claim 9 , wherein the second molecule is CD3. 
     
     
         12 . The method of  claim 1 , wherein the target cells comprise T cells. 
     
     
         13 . The method of  claim 1 , wherein the target cells comprise primary T cells from a human subject. 
     
     
         14 . The method of  claim 1 , wherein the reagent is in soluble form. 
     
     
         15 . The method of  claim 1 , wherein the reagent is bound to a solid support during at least a portion of the incubation, whereby a plurality of the target cells are reversibly immobilized on the solid support during at least a portion of the incubation. 
     
     
         16 . The method of  claim 15 , wherein the solid support comprises a stationary phase. 
     
     
         17 . The method of  claim 1 , wherein the receptor-binding agent comprises an antibody, an antibody fragment, a proteinaceous binding molecule with antibody-like binding properties, a molecule containing Ig domains, a cytokine, a chemokine, an aptamer, or an MHC molecule. 
     
     
         18 . The method of  claim 1 , wherein the receptor-binding agent comprises an antibody or an antibody fragment. 
     
     
         19 . The method of  claim 1 , wherein the receptor-binding agent comprises a Fab fragment. 
     
     
         20 . The method of  claim 1 , wherein the reagent comprises streptavidin; avidin; a mutein of streptavidin that reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide; an analog of avidin that reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide; a reagent that comprises at least two chelating groups, wherein the at least two chelating groups are capable of binding to a transition metal ion; an agent capable of binding to an oligohistidine affinity tag; an agent capable of binding to a glutathione-S-transferase;
 calmodulin or an analog thereof; an agent capable of binding to calmodulin binding peptide (CBP); an agent capable of binding to a FLAG-peptide; an agent capable of binding to an HA-tag; an agent capable of binding to maltose binding protein (MBP); an agent capable of binding to an HSV epitope; an agent capable of binding to a myc epitope; or an agent capable of binding to a biotinylated carrier protein.   
     
     
         21 . The method of  claim 1 , wherein the reagent comprises a streptavidin mutein that reversibly binds to a streptavidin-binding peptide. 
     
     
         22 . The method of  claim 21 , wherein the streptavidin mutein comprises the amino acid sequence Val 44 -Thr 45 -Ala 46 -Arg 47  or Ile 44 -Gly 45 -Ala 46 -Arg 47  at sequence positions corresponding to positions 44 to 47 of the sequence of amino acids set forth in SEQ ID NO:1. 
     
     
         23 . The method of  claim 21 , wherein the streptavidin mutein comprises the sequence of amino acids set forth in any of SEQ ID NO: 3-6, 27, and 28. 
     
     
         24 . The method of  claim 21 , wherein the receptor-binding agent comprises a binding partner that is reversibly bound to one or more binding sites of the plurality of binding sites, wherein the binding partner comprises a streptavidin-binding peptide comprising the sequence of amino acids set forth in any of SEQ ID NO: 7, 8, and 15-19. 
     
     
         25 . The method of  claim 1 , further comprising disrupting the reversible binding between the receptor-binding agent and the reagent. 
     
     
         26 . The method of  claim 25 , wherein the disruption is carried out within 14 days after initiation of the incubation. 
     
     
         27 . The method of  claim 25 , wherein the disruption is carried out within 8 days after initiation of the incubation. 
     
     
         28 . The method of  claim 25 , wherein the disruption comprises introducing to the cells a composition comprising a substance that is a competition agent capable of reversing the bond between the receptor-binding agent and the reagent. 
     
     
         29 . The method of  claim 28 , wherein the reagent comprises a streptavidin mutein that reversibly binds to a streptavidin-binding peptide, and the substance comprises biotin. 
     
     
         30 . The method of  claim 1 , wherein the induction of the signal effects an increase in expansion or activation of the cultured target cells compared to incubation of target cells in the absence of the induction of the signal. 
     
     
         31 . The method of  claim 1 , wherein the incubation is carried out at or at about 37° C.±2° C.