Mature type-1 polarized dendritic cells with enhanced IL-12 production and methods of serum-free production and use

Abstract

The present invention discloses novel dendritic cell maturation-inducing cytokine cocktails, and methods for inducting type-1 polarized dendritic cells in serum-free conditions which enhance the desirable properties of DC1s generated in serum-supplemented cultures. The invention further discloses methods and systems using IFNγ and other ligands of the IFNγ receptor, in combination with IFNα (or other type I interferons), poly I:C, and other IFNα (and IFNβ) inducers to enhance the IL-12-producing properties of dendritic cells. More specifically, the present invention discloses type-1 polarized dendritic cells that have a unique combination of a fully-mature status and an elevated, instead of “exhausted”, ability to produce IL-12p70. allows for the generation of fully-mature DC1s in serum-free AIM-V medium. The invention discloses systems that use the foregoing products and methods to facilitate the clinical application of DC1-based vaccines and the identification of novel factors involved in the induction of Th1 and CTL responses by DC1.

1 . A population of cells wherein 
 a. said cells are antigen-presenting cells; and    b. said cells are activated or matured by a combination of type I interferons, or analogs or inducers of said type I interferons, and type II interferons or analogs of said type II interferons.    
     
     
         2 . The population of  claim 1  wherein said cells are further stimulated with maturation inducing agents.  
     
     
         3 . The population of  claim 2  wherein said maturation agent is one or any combination from the group of inflammatory cytokines, LPS, LPS derivatives, poly-I:C, heat shock proteins, derivatives of heat shock proteins, endogenous mediators of antigen-presenting cell activation, bacterial components, fungal components, and other inducers of NFκb activation.  
     
     
         4 . The population of claims  1  or  2  wherein said type I interferon is IFNα or IFNβ, or an analogue of IFNα or IFNβ, and said type II interferon is IFNγ or an analogue of IFNγ.  
     
     
         5 . The population of claims  1  or  2  wherein said antigen-presenting cells are dendritic cells.  
     
     
         6 . The population of  claim 5  wherein said dendritic cells are alpha-type-1 polarized dendritic cells having fully mature status wherein fully mature status is defined as a high expression of dendritic cell markers CD83, CD86, and CCR7, a high ability to migrate in response to CCR7 ligands), and an ability to produce IL-12p70, in response to subsequent CD40L-dependent activation, at levels above that produced by immature dendritic cells after CD40L-dependent activation.  
     
     
         7 . A method of producing a population of cells wherein antigen-presenting cells are stimulated by exposure to a combination of type I and type II interferons or their analogs.  
     
     
         8 . The method of  claim 7  wherein said antigen-presenting cells are further stimulated with maturation inducing agents.  
     
     
         9 . The method of  claim 7  wherein said maturation agent is one or any combination from the group of inflammatory cytokines LPS, LPS derivatives, poly-I:C, heat shock proteins, derivatives of heat shock proteins, endogenous mediators of antigen-presenting cell activation, bacterial components, fungal components, and other inducers of NFκb activation.  
     
     
         10 . The method of claims  7  or  8  wherein said type I interferon is IFNα or IFNβ, or an analogue of IFNα or IFNβ, and said type II interferon is IFNγ or an analogue of IFNγ.  
     
     
         11 . The method of claims  7  and  8  wherein said antigen-presenting cells are dendritic cells.  
     
     
         12 . The method of claims  7  and  8  wherein said antigen-presenting cells are αType-1 polarized dendritic cells.  
     
     
         13 . The method of  claim 12  wherein αType-1 polarized dendritic cells are produced in a fetal calf serum (FCS)-free medium.  
     
     
         14 . A method of producing a population of αType-1 polarized dendritic cells comprising the steps of: 
 a. isolating dendritic cells or dendritic cell precursors from a donor;    b. incubating said dendritic cells with IFNα or IFNβ or functional analogues of IFNα or IFNβ, in the presence of IFNγ or IFNγ functional analogues and dendritic cell maturation factors.    
     
     
         15 . The method of  claim 14  further comprising the step of presenting an antigen by said dendritic cells.  
     
     
         16 . A method according to  claim 15  wherein said presenting of an antigen to said surface of said dendritic cells is achieved by one of the group of: (a) contacting said dendritic cells with an antigen or epitope differentially expressed on a cell; (b) pulsing said dendritic cells with antigenic proteins; (c) loading said dendritic cells with antigenic peptides; (d) transforming or transducing said dendritic cells with nucleic acid molecules coding for at least part of said antigen; (e) fusing said dendritic cells with cells carrying specific antigens; (f) pulsing said dendritic cells with apoptotic cells, apoptotic bodies, or cell lysates.  
     
     
         17 . A method for stimulating or expanding T cells using a population of alpha type 1 dendritic cells comprising at least the following steps: 
 a. isolating dendritic cells from a donor;    b. incubating said dendritic cells or dendritic cell precursors with IFNα or IFNβ or functional analogues of IFNα or IFNβ, in the presence of IFNγ or IFNγ functional analogues, and dendritic cell maturation factors;    c. presenting a peptide on the surface of said dendritic cells; and    d. stimulating a population of T cells with said population of dendritic cells.    
     
     
         18 . The method of  claim 17  wherein the T cells are stimulated or expanded in vivo through a vaccination with alpha type 1 dendritic cells.  
     
     
         19 . The method of  claim 17  wherein the T cells are stimulated or expanded ex vivo through adoptive immunotherapy with ex vivo stimulated or expanded T cells.  
     
     
         20 . The method according to  claim 17  wherein said T cell is a CD4 cell or CD8 T cell.  
     
     
         21 . A method for stimulating or expanding NK cells using a population of alpha type 1 dendritic cells comprising at least the following steps: 
 a. isolating dendritic cells from a donor;    b. incubating said dendritic cells or dendritic cell precursors with IFNα or IFNα functional analogues in the presence of IFNγ or IFNγ functional analogues, and dendritic cell maturation factors;    c. stimulating a population of NK cells with said population of dendritic cells.    
     
     
         22 . The method of  claim 21  wherein the NK cells are stimulated or expanded in vivo through a vaccination with alpha type 1 dendritic cells.  
     
     
         23 . The method of  claim 21  wherein the NK cells are stimulated or expanded ex vivo through adoptive immunotherapy with ex vivo stimulated or expanded NK cells.  
     
     
         24 . A system using the population of cells of any of claims  1  through  6 , for therapeutic, preventative or diagnostic purposes, including the treatment of cancer, precancerous states, precancerous lesions, viral infections, bacterial infections, fungal infections, parasitic infections, allergies or autoimmune diseases, including its use as vaccine adjuvant.  
     
     
         25 . A system using the methods described in any of claims  7  through  19 , for therapeutic, preventive, or diagnostic purposes, including the treatment of cancer, precancerous states, precancerous lesions, viral infections, bacterial infections, fungal infections, parasitic infections, allergies or autoimmune diseases.  
     
     
         26 . A system using the methods described in any of claims  7  through  19 , as a tool to identify or develop additional therapeutic, preventive, or diagnostic, tools or strategies, applicable for cancer, precancerous states, precancerous lesions, viral infections, bacterial infections, fungal infections, parasitic infections, allergies or autoimmune diseases.  
     
     
         27 . A system comprising IFNα or IFNβ, or inducers or functional analogues of IFNα or IFNβ, in combination with IFNγ, or IFNγ functional analogues for the preparation of alpha type 1 polarized dendritic cells according to the methods of any of claims  7  through  19 .  
     
     
         28 . The system according to  claim 27  further comprising dendritic cell maturation factors.  
     
     
         29 . The system according to claims  28  comprising IFNα or IFNβ, IFNγ, IL-1β, and TNFα.  
     
     
         30 . The system according to  claim 29  further comprising poly-I:C.  
     
     
         31 . The system according to claims  28  comprising poly-I:C, IFNα, IL-1β, and TNFα.  
     
     
         32 . The system according to any of claims  27  through  31  further comprising a diluent that is a serum supplemented medium.  
     
     
         33 . The system according to any of claims  27  through  31  further comprising a diluent that is serum-free medium.  
     
     
         34 . The system according to any of claims  27  through  31 , for the preparation of alpha type 1 dendritic cells, according to any of the claims  19  trough  21  further comprising a delivery route that is one of the group of: intravenous, intratumoral, peritumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, intracranial, intraventricular, intranodal, peri-nodal, intralymphatic or topical.