Hybrid or chimeric polynucleotides, proteins, and compositions comprising hepatitis B virus sequences

Abstract

H-2 class I negative, HLA-A2.1 transgenic HHD mice were used for a comparative evaluation of the immunogenicity of HLA-A2.1 restricted human tumor-associated CTL epitopes. A hierarchy was established among these epitopic peptides injected into mice in IFA which correlates globally with their capacity to bind and stabilize HLA-A2.1 molecules. Co-injection of a helper peptide enhanced most CTL responses. In contrast, classical HLA class I transgenic mice which still express their own class I molecules did not, in most cases, develop H.A.-A2.1-restricted CTL responses under the same experimental conditions. Different monoepitopic immunization strategies of acceptable clinical usage were compared in HHD mice. Recombinant Ty-virus-like particles, or DNA encoding epitopes fused to the hepatitis B virus middle envelope protein gave the best results. Using this latter approach and a melanoma-based polyepitope construct, CTL responses against five distinct epitopes could be elicited simultaneously in a single animal. Thus, HHD mice provide a versatile animal model for preclinical evaluation of peptide-based cancer immunotherapy.

1 . An isolated or purified polynucleotide containing at least: 
 a part of the coding sequence of the middle glycoprotein of the hepatitis B virus (HBV), in which is inserted a DNA sequence coding for at least one tumor epitope of a tumor antigen.    
     
     
         2 . The polynucleotide according to  claim 1 , wherein the part of the coding sequence of the middle glycoprotein of HBV is a part of the preS2 sequence of HBV.  
     
     
         3 . The polynucleotide according to  claim 2 , the DNA sequence coding for at least one tumor epitope of a tumor antigen is a DNA sequence coding for the surface antigen of HBV.  
     
     
         4 . The polynucleotide according to  claim 3 , wherein the polynucleotide comprises DNA sequences encoding from 1 to 30, inclusive, epitopes, wherein none, some, or all of the epitopes are the same.  
     
     
         5 . The polynucleotide according to  claim 1 , wherein the polynucleotide comprises DNA sequences encoding from 1 to 30, inclusive, epitopes, wherein none, some, or all of the epitopes are the same.  
     
     
         6 . The polynucleotide according to  claim 5 , wherein one or more of the epitopes are mutated due to a mutation in the DNA encoding the epitopes.  
     
     
         7 . A composition containing the polynucleotide according to  claim 1 , wherein expression of at least part of the polynucleotide in vivo induces an immune response against at least one tumor specific antigen or tissue specific antigen.  
     
     
         8 . A vector comprising 
 an early CMV promoter, preS2 and S nucleotide sequences encoding preS2 and S antigens of HBV;    nucleotide sequences from the genome HBV containing post-transcriptional regulatory elements (PREs) that direct nuclear export of RNA corresponding to nucleotide 1,151 to nucleotide 1,684 of the HBV genome;    signal sequences for polyadenylation of messenger RNAs of HBV located at position nucleotide 1,921 to nucleotide 1,955 of the HBV genome; and    nucleotide sequences of tumor epitopes surrounded up and down by alanine spacers.    
     
     
         9 . The vector according to  claim 8 , wherein the vector, when administered to an individual, induces an in vivo cellular or/humoral immune response.  
     
     
         10 . The vector according to  claim 8 , further comprising nucleotide sequences encoding a B cell epitope fused to at least one tumor sequence, wherein the B cell epitope sequences allow the detection of hybrid proteins.  
     
     
         11 . A process for treating an individual in vivo, said process comprising: 
 constructing a polynucleotide containing at least a part of the coding sequence of the middle glycoprotein of the hepatitis B virus (HBV), in which is inserted a DNA sequence coding for at least one tumor epitope of a tumor antigen;    injecting the polynucleotide into an individual; and    optionally, evaluating the cytotoxic responses in the individual's lymphocyte population.    
     
     
         12 . A composition comprising a hybrid protein, wherein the hybrid protein comprises a preS2-S protein sequence and at least sequence of at least one tumor antigen or epitope.  
     
     
         13 . The composition according to  claim 12 , wherein the composition induces, in vivo, a CTL response against one or more of the antigens or epitopes present on the hybrid protein.  
     
     
         14 . The composition according to  claim 12  wherein the hybrid protein further comprises a tag B cell epitope.  
     
     
         15 . Recombinant particles comprising the composition according to  claim 12  and the small envelope protein of HBV.  
     
     
         16 . A process of treating cells of a host, said process characterized by 
 contacting the recombinant particles according to  claim 15  with the host's cells to create treated cells; and    optionally, injecting the treated cells into the host.