Defective ribosomal products in blebs (dribbles) and methods of use to stimulate an immune response

Abstract

Methods are disclosed for producing defective ribosomal products (DRiPs) in blebs (DRibbles) by contacting cells with a proteasome inhibitor, and in some examples also an autophagy inducer, thereby producing treated cells. DRibbles can be used to load antigen presenting cells (APCs), thereby allowing the APCs to present the DRiPs and antigenic fragments thereof. Immunogenic compositions that include treated cells, isolated DRibbles, or DRibble-loaded APCs are also disclosed. Methods are also provided for using treated cells, isolated DRibbles, or DRibble-loaded APCs to stimulate an immune response, for example in a subject. For example, DRibbles obtained from a tumor cell can be used to stimulate an immune response against the same type of tumor cells in the subject. In another example, DRibbles obtained from a pathogen-infected cell or cell engineered to express one or more antigens of a pathogen can be used to stimulate an immune response against the pathogen in the subject.

1 . Isolated defective ribosomal products (DRiPs) in blebs (DRibbles) produced using an ex vivo method, comprising:
 contacting a mammalian cell infected with vector comprising a nucleic acid molecule encoding a pathogenic antigen with a proteasome inhibitor under conditions sufficient for the mammalian cell to produce DRibbles;   contacting the mammalian cell with an amount of NH 4 Cl sufficient to reduce lysosome-mediated protein degradation;   optionally contacting the mammalian cell with an agent that reduces glycosylation, with an autophagy inducing agent, or combinations thereof, under conditions sufficient to enhance production of DRibbles by the tumor cell; and   harvesting DRibbles secreted by the mammalian cell, thereby producing isolated DRibbles, wherein the DRibbles are isolated to at least 50% purity.   
     
     
         2 . The isolated DRibbles of  claim 1 , wherein the mammalian cell is contacted with an autophagy inducing agent, thereby producing intracellular DRibble autophagy bodies; 
       and the method further comprises:
 lysing the mammalian cell; and 
 collecting the intracellular DRibble autophagy bodies. 
 
     
     
         3 . The isolated DRibbles of  claim 1 , wherein the mammalian cell is a human cell. 
     
     
         4 . The isolated DRibbles of  claim 1 , wherein the pathogenic antigen is a viral antigen or a bacterial antigen. 
     
     
         5 . The isolated DRibbles of  claim 4 , wherein the viral antigen is from hepatitis A virus, hepatitis B virus, hepatitis C virus, human papillomavirus, Epstein-Barr virus (EBV), or cytomegalovirus. 
     
     
         6 . The isolated DRibbles of  claim 1 , wherein the vector comprises a plasmid or viral vector. 
     
     
         7 . The isolated DRibbles of  claim 1 , wherein harvesting comprises separating DRibbles from the mammalian cell, and collecting the DRibbles. 
     
     
         8 . The isolated DRibbles of  claim 1 , wherein the DRibbles include less than 5% of whole cells. 
     
     
         9 . The isolated DRibbles of  claim 1 , wherein the mammalian cell is contacted with the autophagy inducing agent under conditions sufficient to substantially increase autophagy in the mammalian cell. 
     
     
         10 . The isolated DRibbles of  claim 1 , wherein harvesting DRibbles comprises:
 centrifuging the mammalian cell under conditions sufficient to pellet the mammalian cell but not the DRibbles;   collecting a supernatant containing DRibbles; and   centrifuging the supernatant under conditions sufficient to pellet the DRibbles; and   
       collecting the pellet containing DRibbles, thereby producing isolated DRibbles. 
     
     
         11 . The isolated DRibbles of  claim 1 , wherein the DRibbles are isolated to at least 75% purity. 
     
     
         12 . The isolated DRibbles of  claim 1 , wherein the conditions sufficient for the mammalian cell to produce DRibbles comprises incubation with an effective amount of the proteasome inhibitor that does not substantially induce apoptosis of the mammalian cell. 
     
     
         13 . The isolated DRibbles of  claim 1 , wherein the proteasome inhibitor comprises a reversible proteasome inhibitor. 
     
     
         14 . A composition comprising
 the isolated DRibbles of  claim 1  and dimethyl sulfoxide (DMSO);   the isolated DRibbles of  claim 1  and an antibody specific for a T-cell costimulatory molecule; or   the isolated DRibbles of  claim 1  and granulocyte-colony stimulating factor (GM-CSF).   
     
     
         15 . The composition of  claim 14 , wherein the antibody specific for a T-cell costimulatory molecule comprises anti-OX-40. 
     
     
         16 . An immunogenic composition comprising the isolated DRibbles of  claim 1 . 
     
     
         17 . The immunogenic composition of  claim 17 , further comprising an immunostimulant, antigen presenting cells (APCs) that have been loaded with the DRibbles, or combinations thereof. 
     
     
         18 . The immunogenic composition of  claim 17 , wherein the immunogenic composition is a vaccine. 
     
     
         19 . The immunogenic composition of  claim 17 , wherein the immunostimulant comprises an adjuvant. 
     
     
         20 . A method of stimulating an immune response against a pathogen in a subject, comprising:
 incubating the isolated DRibbles of  claim 1  with an antigen presenting cell (APC) obtained from peripheral blood mononuclear cells (PBMCs) from the subject under conditions sufficient for the APC to present one or more DRiPs, thereby generating DRibble-loaded APCs; and   administering a therapeutically effective amount of the DRibble-loaded APCs to the subject, thereby stimulating an immune response against the pathogen.   
     
     
         21 . The method of  claim 20 , wherein the APC is a dendritic cell (DC). 
     
     
         22 . A method of stimulating an immune response against a pathogen in a subject, comprising:
 administering a therapeutically effective amount of the isolated DRibbles of  claim 1  to the subject, thereby stimulating an immune response against the pathogen.   
     
     
         23 . The method of  claim 22 , further comprising administering an immunostimulant to the subject. 
     
     
         24 . The method of  claim 23 , wherein the immunostimulant
 is an adjuvant; or   comprises granulocyte macrophage-colony stimulating factor (GM-CSF).   
     
     
         25 . A method of stimulating an immune response against one or more DRiPs in a subject, comprising:
 administering a therapeutically effective amount of the immunogenic composition of  claim 16  to a subject, thereby stimulating an immune response against one or more DRiPs in the subject.   
     
     
         26 . The method of  claim 25 , wherein the subject is infected with the pathogen. 
     
     
         27 . The method of  claim 25 , wherein administering the immunogenic composition to the subject comprises administration of at least three doses of the immunogenic composition over a period of at least 180 days. 
     
     
         28 . The isolated DRibbles of  claim 1 , wherein the wherein DRibbles are cryopreserved. 
     
     
         29 . The composition of  claim 14 , wherein the wherein the composition comprises DRibbles and DMSO, and wherein the DRibbles are cryopreserved.