Modified dendritic cells having enhanced survival and immunogenicity and related compositions and methods

Abstract

Modified antigen presenting cells provided herein have improved lifespan and immunogenicity compared to unmodified antigen presenting cells, and are useful for immunotherapy. The modified antigen presenting cells express an altered protein kinase, referred to herein as “Akt.” The altered Akt associates with the cell membrane with greater frequency than unaltered Akt, and is referred to herein as “membrane-targeted Akt.”

1 - 130 . (canceled) 
     
     
         131 . A method for preparing a modified dendritic cell, which comprises:
 contacting a dendritic cell with a nucleic acid that encodes a membrane-targeted Akt protein comprising a mammalian Akt region lacking a functional pleckstrin homology (PH) domain,   whereby the modified dendritic cell survives longer than dendritic cells that do not include the nucleic acid.   
     
     
         132 . The method of  claim 131 , wherein the modified dendritic cell presents a greater amount of antigen than cells that do not include the nucleic acid. 
     
     
         133 . The method of  claim 131 , wherein the modified dendritic cell is more immunogenic than cells that do not include the nucleic acid. 
     
     
         134 . The method of  claim 131 , which comprises contacting the modified dendritic cell with an antigen. 
     
     
         135 . The method of  claim 134 , wherein the antigen is prostate specific membrane antigen. 
     
     
         136 . The method of  claim 135 , wherein the antigen has a sequence substantially identical to SEQ ID NO: 10. 
     
     
         137 . The method of  claim 131 , wherein the dendritic cell is a human cell. 
     
     
         138 . The method of  claim 131 , wherein the membrane-targeted Akt comprises one or more binding partner components. 
     
     
         139 . The method of  claim 138 , wherein the dendritic cell comprises an acylation region having one or more binding partner components, wherein at least one of the binding partner components binds to a binding partner component of the membrane-targeted Akt. 
     
     
         140 . The method of  claim 131 , wherein the membrane-targeted Akt comprises at least one acylation region. 
     
     
         141 . The method of  claim 140 , wherein the acylation region is a dual acylation region. 
     
     
         142 . The method of  claim 140 , wherein one acylation region is a myristoyl region. 
     
     
         143 . The method of  claim 140 , wherein the acylation region is from a protein kinase. 
     
     
         144 . The method of  claim 143 , wherein the protein kinase is Fyn or Lck. 
     
     
         145 . The method of  claim 143 , wherein the protein kinase is Src. 
     
     
         146 . The method of  claim 140 , wherein the acylation region is linked to the N-terminus of the Akt region. 
     
     
         147 . The method of  claim 140 , wherein the acylation region is linked to the C-terminus of the Akt region. 
     
     
         148 . The method of  claim 140 , wherein the acylation region comprises a cys-ala-ala sequence. 
     
     
         149 . The method of  claim 148 , wherein the cys-ala-ala sequence is from a G-protein. 
     
     
         150 . The method of  claim 131 , wherein the mammalian Akt region is substantially identical to mouse Akt lacking a functional pleckstrin homology (PH) domain. 
     
     
         151 . The method of  claim 131 , wherein the mammalian Akt region is substantially identical to human Akt lacking a functional pleckstrin homology (PH) domain. 
     
     
         152 . The method of  claim 131 , wherein the mammalian Akt region is substantially identical to the amino acid sequence of SEQ ID NO: 6. 
     
     
         153 . The method of  claim 131 , wherein the mammalian Akt region consists of the amino acid sequence of SEQ ID NO: 6. 
     
     
         154 . The method of  claim 140 , wherein the acylation region is substantially identical to the amino acid sequence of SEQ ID NO: 8. 
     
     
         155 . The method of  claim 140 , wherein the acylation region consists of the amino acid sequence of SEQ ID NO: 8. 
     
     
         156 . The method of  claim 140 , wherein the acylation region having one or more binding partner components is encoded by a nucleotide sequence in the dendritic cell. 
     
     
         157 . The method of  claim 131 , wherein the membrane-targeted Akt comprises a membrane protein or membrane protein region linked to the mammalian Akt region. 
     
     
         158 . The method of  claim 138 , wherein the dendritic cell comprises a membrane protein or membrane protein region having one or more binding partner components, wherein at least one of the binding partner components binds to a binding partner component of the membrane-targeted Akt. 
     
     
         159 . The method of  claim 158 , wherein the membrane protein or membrane protein region having one or more binding partner components is encoded by a nucleotide sequence in the dendritic cell. 
     
     
         160 . The method of  claim 131 , wherein the nucleic acid is from a virus. 
     
     
         161 . The method of  claim 160 , wherein the dendritic cell is contacted with a virus that contains the nucleic acid. 
     
     
         162 . The method of  claim 131 , wherein the nucleic acid comprises a consitutively active promoter operably linked to the nucleic acid that encodes the membrane-targeted Akt protein. 
     
     
         163 . A method for loading a modified dendritic cell with an antigen, comprising:
 contacting a modified dendritic cell with an antigen or antigen precursor, wherein the modified dendritic cell expresses a membrane-targeted Akt protein
 lacking a functional pleckstrin homology (PH) domain, 
 whereby the modified dendritic cell is loaded with the antigen. 
   
     
     
         164 . A method for inducing an immune response against an antigen, which comprises
 contacting a dendritic cell that expresses a membrane-targeted Akt protein lacking a functional pleckstrin homology (PH) domain with an antigen or antigen precursor; and
 administering the dendritic cell to a subject; 
 whereby the immune response against the antigen is induced. 
   
     
     
         165 . A method for inducing an immune response against an antigen, which comprises
 contacting a dendritic cell that expresses a membrane-targeted Akt protein lacking a functional pleckstrin homology (PH) domain with an antigen or antigen precursor; and
 administering the dendritic cell to a subject; 
 whereby the immune response against the antigen is induced. 
   
     
     
         166 . A method for detecting an immune response against an antigen, which comprises:
 contacting a dendritic cell that expresses a membrane-targeted Akt protein lacking a functional pleckstrin homology (PH) domain with an antigen;
 administering the dendritic cell to a subject; and 
   detecting the immune response.   
     
     
         167 . A method for reducing cell proliferation in a subject, which comprises:
 contacting a dendritic cell that expresses a membrane-targeted Akt protein lacking a functional pleckstrin homology (PH) domain with an antigen produced in proliferating cells; and
 administering the dendritic cell to a subject; 
   whereby cell proliferation is reduced.   
     
     
         168 . A method for inhibiting tumor growth in a subject, which comprises:
 contacting a dendritic cell that expresses a membrane-targeted Akt protein lacking a functional pleckstrin homology (PH) domain with an antigen produced by cells in a tumor; and
 administering the dendritic cell to a subject having a tumor; 
 whereby tumor growth is inhibited. 
   
     
     
         169 . A kit which comprises a nucleic acid comprising a nucleotide sequence that encodes a membrane-targeted Akt protein lacking a functional pleckstrin homology (PH) domain. 
     
     
         170 . An isolated nucleic acid which comprises a nucleotide sequence that encodes a protein containing:
 a first region comprising a human Akt sequence lacking a functional pleckstrin homology (PH) domain; and   a second region linked to the N-terminus of the Akt sequence comprising two or more acylation sites.   
     
     
         171 . An adenovirus which comprises the nucleotide sequence of  claim 170 . 
     
     
         172 . A method for inducing an immune response, which comprises:
 a. contacting a dendritic cell from a human subject with an antigen;   b. contacting the dendritic cell with a nucleic acid that comprises a nucleotide sequence that encodes a protein containing:   a first region comprising a human Akt sequence lacking a pleckstrin homology (PH) domain; and   a second region linked to the N-terminus of the Akt sequence comprising two or more acylation sites; and   administering the dendritic cell after steps a and b to the subject, whereby an immune response is induced.   
     
     
         173 . A method for inducing an immune response, which comprises:
 a. contacting a dendritic cell from a human subject with an antigen;   b. contacting the dendritic cell with a nucleic acid that comprises a nucleotide sequence that encodes a protein containing:
 a first region comprising a human Akt sequence lacking a functional pleckstrin homology (PH) domain; and 
 a second region linked to the N-terminus of the Akt sequence comprising two or more acylation sites; and 
 proliferating in vitro antigen-specific CTLs against the dendritic cell after steps a and b, whereby an immune response is induced. 
   
     
     
         174 . A composition comprising (i) a first polynucleotide sequence that encodes a first chimeric protein comprising an Akt lacking a functional pleckstrin homology (PH) domain, and further comprising a membrane-association region, and (ii) a second polynucleotide sequence that encodes a second chimeric protein comprising a membrane-association region, a multimeric ligand binding region and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain. 
     
     
         175 . A protein comprising:
 a mammalian Akt region lacking a functional pleckstrin homology (PH) domain; and   a dual acylation region covalently linked to the mammalian Akt region.   
     
     
         176 . A protein comprising:
 a mammalian Akt region lacking a functional pleckstrin homology (PH) domain; and   a membrane protein or membrane protein region covalently linked to the mammalian Akt region.