Methods, kits and apparatus for expanding a population of cells

Abstract

The present invention relates to in vitro-methods of expanding a population of cells such as lymphocytes, comprising contacting a sample comprising a population of cells with a multimerization reagent. The multimerization reagent has reversibly immobilized thereon (bound thereto) a first agent that provides a primary activation signal to the cells and optionally, a second agent that provides a co-stimulatory signal. The invention also provides multimerization reagents, kits, arrangements and an apparatus for expanding cells.

1 . An in vitro method of stimulating a population of cells, the method comprising incubating a sample comprising a population of cells in the presence of a multimerization reagent that is reversibly bound to a first agent, wherein the first agent (i) comprises at least one binding partner C1 that is reversibly bound to a binding site Z1 of the multimerization reagent and (ii) binds to a receptor molecule on the surface of cells in the population to stimulate a signal in the cells. 
     
     
         2 . The method of  claim 1 , wherein the multimerization reagent is reversibly bound to a second agent, wherein the second agent (i) comprises at least one binding partner C2 that is reversibly bound to a binding site Z2 of the multimerization reagent and (ii) binds to an accessory molecule on the surface of the cells to stimulate an accessory signal in the cells. 
     
     
         3 . The method of  claim 1 , wherein the cells comprise T cells. 
     
     
         4 . The method of  claim 3 , wherein the first agent stimulates a TCR/CD3 complex-associated signal in the T cells. 
     
     
         5 . The method of  claim 3 , wherein the first agent binds to CD3. 
     
     
         6 . The method of  claim 2 , wherein the accessory molecule is CD28 or CD137. 
     
     
         7 . The method of  claim 2 , wherein the first agent binds to CD3, and the second agent binds to CD28. 
     
     
         8 . The method of  claim 2 , wherein:
 the first agent comprises an anti-CD3 antibody, a divalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, or a proteinaceous CD3 binding molecule with antibody-like binding properties; and/or   the second agent comprises an anti-CD28 antibody, a divalent antibody fragment of an anti-CD28 antibody, a monovalent antibody fragment of an anti-CD28-antibody, a proteinaceous CD28 binding molecule with antibody-like binding properties, an anti-CD137 antibody, a divalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment of an anti-CD137 antibody, a proteinaceous CD137 binding molecule with antibody-like binding properties, 4-1BB ligand, or a mixture of any of the foregoing.   
     
     
         9 . The method of  claim 2 , wherein the first agent and/or the second agent comprises a monovalent antibody fragment. 
     
     
         10 . The method of  claim 2 , wherein the first agent comprises an anti-CD3 antibody or antibody fragment, and the second agent comprises an anti-CD28 antibody or antibody fragment. 
     
     
         11 . The method of  claim 2 , wherein the first agent comprises an anti-CD3 Fab, and the second agent comprises an anti-CD28 Fab. 
     
     
         12 . The method of  claim 1 , wherein the multimerization reagent is in soluble form. 
     
     
         13 . The method of  claim 1 , wherein the multimerization reagent is immobilized on a solid support. 
     
     
         14 . The method of  claim 1 , wherein the multimerization reagent comprises streptavidin, avidin, a mutein of streptavidin that reversibly binds to the binding partner C1, a mutein of avidin that reversibly binds to the binding partner C1, a reagent that comprises at least two chelating groups K, wherein the at least two chelating groups are capable of binding to a transition metal ion, thereby rendering moiety A capable of binding to an oligohistidine affinity tag, multimeric glutathione-S-transferase, multimeric calmodulin or a biotinylated carrier protein. 
     
     
         15 . The method of  claim 1 , wherein the multimerization reagent comprises an oligomer or a polymer of streptavidin, an oligomer or a polymer of avidin, an oligomer or a polymer of a mutein of streptavidin that reversibly binds to the binding partner C1, or an oligomer or a polymer of a mutein of avidin that reversibly binds to the binding partner C1. 
     
     
         16 . The method of  claim 2 , wherein the multimerization reagent comprises an oligomer or a polymer of a mutein of streptavidin that reversibly binds to the binding partners C1 and C2. 
     
     
         17 . The method of  claim 16 , wherein the first agent comprises an anti-CD3 antibody or antibody fragment, and the second agent comprises an anti-CD28 antibody or antibody fragment. 
     
     
         18 . The method of  claim 16 , 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 44 to 47 of wild type streptavidin. 
     
     
         19 . The method of  claim 18 , wherein the N-terminal amino acid residue of the streptavidin mutein is in the region of sequence positions 10 to 16 of the wildtype streptavidin amino acid sequence, and the C-terminal amino acid residue of the streptavidin mutein is in the region of sequence positions 133 to 142 of the wildtype streptavidin amino acid sequence. 
     
     
         20 . The method of  claim 18 , wherein the first agent comprises an anti-CD3 antibody or antibody fragment, and the second agent comprises an anti-CD28 antibody or antibody fragment. 
     
     
         21 . The method of  claim 1 , wherein:
 (a) the binding partner C1 comprises biotin, and the multimerization reagent comprises a streptavidin mutein that reversibly binds to biotin or an avidin mutein that reversibly binds to biotin;   (b) the binding partner C1 comprises a biotin analog that reversibly binds to streptavidin or avidin, and the multimerization reagent comprises streptavidin, avidin, a streptavidin mutein that reversibly binds to the biotin analog, or an avidin mutein that reversibly binds to the biotin analog; or   (c) the binding partner C1 comprises a streptavidin or avidin binding peptide, and the multimerization reagent comprises streptavidin, avidin, a streptavidin mutein that reversibly binds to the streptavidin or avidin binding peptide, or an avidin mutein that reversibly binds to the streptavidin or avidin binding peptide.   
     
     
         22 . The method of  claim 1 , wherein the binding partner C1 comprises a streptavidin-binding peptide, and the multimerization reagent comprises a streptavidin mutein that reversibly binds to the streptavidin-binding peptide. 
     
     
         23 . The method of  claim 22 , wherein the streptavidin-binding peptide is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 1) or SAWSHPQFEK(GGGS) 2 GGSAWSHPQFEK (SEQ ID NO: 7). 
     
     
         24 . The method of  claim 2 , wherein the binding partner C1 comprises a first streptavidin-binding peptide, the binding partner C2 comprises a second streptavidin-binding peptide, and the multimerization reagent comprises a streptavidin mutein that reversibly binds to the first and second streptavidin-binding peptides. 
     
     
         25 . The method of  claim 24 , wherein the multimerization reagent comprises an oligomer or a polymer of the mutein of streptavidin. 
     
     
         26 . The method of  claim 24 , wherein the first agent comprises an anti-CD3 antibody or antibody fragment, and the second agent comprises an anti-CD28 antibody or antibody fragment. 
     
     
         27 . The method of  claim 24 , 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 44 to 47 of wild type streptavidin. 
     
     
         28 . The method of  claim 27 , wherein the N-terminal amino acid residue of the streptavidin mutein is in the region of sequence positions 10 to 16 of the wildtype streptavidin amino acid sequence, and the C-terminal amino acid residue of the streptavidin mutein is in the region of sequence positions 133 to 142 of the wildtype streptavidin amino acid sequence. 
     
     
         29 . The method of  claim 27 , wherein the multimerization reagent comprises an oligomer or a polymer of the mutein of streptavidin. 
     
     
         30 . The method of  claim 27 , wherein the first agent comprises an anti-CD3 antibody or antibody fragment, and the second agent comprises an anti-CD28 antibody or antibody fragment. 
     
     
         31 . The method of  claim 2 , wherein:
 the binding partner C1 comprises a streptavidin-binding peptide that is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 1) or SAWSHPQFEK(GGGS) 2 GGSAWSHPQFEK (SEQ ID NO: 7),   the binding partner C2 comprises a streptavidin-binding peptide that is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 1) or SAWSHPQFEK(GGGS) 2 GGSAWSHPQFEK (SEQ ID NO: 7), and   the multimerization reagent comprises a streptavidin mutein comprising the amino acid sequence Val 44 -Thr 45 -Ala 46 -Arg 47  or Ile 44 -Gly 45 -Ala 46 -Arg 47  at sequence positions 44 to 47 of wild type streptavidin.   
     
     
         32 . The method of  claim 1 , further comprising disrupting the binding between the binding partner C1 of the first agent and the binding site Z1 of the multimerization reagent. 
     
     
         33 . The method of  claim 32 , wherein the reversible bond is disrupted by contacting the cells with biotin or a biotin analog, wherein:
 (a) the binding partner C1 comprises biotin, and the multimerization reagent comprises a streptavidin mutein that reversibly binds to biotin or an avidin mutein that reversibly binds to biotin,   (b) the binding partner C1 comprises a biotin analog that reversibly binds to streptavidin or avidin, and the multimerization reagent comprises streptavidin, avidin, a streptavidin mutein that reversibly binds to the biotin analog, or an avidin mutein that reversibly binds to the biotin analog, or   (c) the binding partner C1 comprises a streptavidin or avidin binding peptide, and the multimerization reagent comprises streptavidin, avidin, a streptavidin mutein that reversibly binds to the streptavidin or avidin binding peptide, or an avidin mutein that reversibly binds to the streptavidin or avidin binding peptide.   
     
     
         34 . The method of  claim 16 , further comprising disrupting the binding between the streptavidin mutein and the binding partners C1 and C2, wherein the disrupting is by contacting the cells with biotin. 
     
     
         35 . The method of  claim 24 , further comprising disrupting the binding between the streptavidin mutein and the first and second streptavidin-binding peptides, wherein the disrupting is by contacting the cells with biotin. 
     
     
         36 . The method of  claim 3 , wherein a T cell receptor or a chimeric antigen receptor is introduced into the T cells either after the stimulation or during the stimulation. 
     
     
         37 . A reagent kit comprising:
 (i) a multimerization reagent, wherein the multimerization reagent comprises at least one binding site Z1 for the reversible binding of a first agent; and   (ii) a first agent capable of binding to a receptor molecule on the surface of cells and comprising at least one binding partner C1 able to reversibly bind to the binding site Z1 of the multimerization reagent, wherein binding of the first agent to the receptor molecule on the surface of the cells stimulates a signal to the cells.   
     
     
         38 . An arrangement of a bioreactor and a stationary phase for chromatography,
 wherein the bioreactor is suitable for the expansion of cells,   wherein 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, wherein the gel filtration and/or affinity chromatography matrix comprises an affinity reagent, the affinity reagent comprising (i) a binding site Z1 capable of specifically binding to a binding partner C1 and/or (ii) a binding site Z2 capable of specifically binding to a binding partner C2, the affinity reagent thereby being suitable for immobilizing the first binding partner C1 and/or the second binding partner C2 on the stationary phase, and   wherein the bioreactor and the stationary phase are fluidly connected.   
     
     
         39 . An apparatus for purification and expansion of target cells, the apparatus comprising the arrangement of  claim 38 . 
     
     
         40 . A multimerization reagent, wherein the multimerization reagent is in soluble form and is reversibly bound to a first agent, wherein the first reagent (i) comprises at least one binding partner C1 that is reversibly bound to a binding site Z1 of the multimerization reagent, and (ii) is capable of binding to a receptor molecule on the surface of cells to stimulate a signal in the cells. 
     
     
         41 . A composition comprising:
 (1) a first multimerization reagent, wherein the first multimerization reagent is in soluble form and comprises at least one binding site Z1 for the reversible binding of a first agent, the first agent (i) comprising at least one binding partner C1 that is reversibly bound to at least one binding site Z1 of the multimerization reagent and (ii) being capable of binding to a receptor molecule on the surface of cells to stimulate a signal in the cells; and   (2) a second multimerization reagent, wherein the second multimerization reagent is in soluble form and comprises at least one binding site Z2 for the reversible binding of a second agent, the second agent (i) comprising at least one binding partner C2 that is reversibly bound to at least one binding site Z2 of the multimerization reagent and (ii) being capable of binding to an accessory molecule on the surface of the cells to stimulate an accessory signal in the cells.