Method for generating genetically altered antigens

Abstract

Dominant negative alleles of human mismatch repair genes can be used to generate hypermutable cells and organisms. By introducing these genes into cells and transgenic animals, new cell lines and animal varieties with novel and useful properties can be prepared more efficiently than by relying on the natural rate of mutation. These methods are useful for generating genetic diversity within genes encoding for therapeutic antigens to produce altered polypeptides with enhanced antigenic and immunogenic activity. Moreover, these methods are useful for generating effective vaccines.

We claim:  
     
         1 . A method for making a hypermutated antigen, comprising introducing into a mammalian cell that expresses a preselected antigen a polynucleotide comprising a dominant negative allele of a mismatch repair gene.  
     
     
         2 . The method of  claim 1  wherein the polynucleotide is introduced by transfection of a suspension of cells in vitro.  
     
     
         3 . The method of  claim 1  wherein the mismatch repair gene is PMS2.  
     
     
         4 . The method of  claim 1  wherein the mismatch repair gene is human PMS2.  
     
     
         5 . The method of  claim 1  wherein the mismatch repair gene is MLH1.  
     
     
         6 . The method of  claim 1  wherein the mismatch repair gene is PMS1.  
     
     
         7 . The method of  claim 1  wherein the mismatch repair gene is MSH2.  
     
     
         8 . The method of  claim 1  wherein the mismatch repair gene is MSH2.  
     
     
         9 . The method of  claim 4  wherein the allele comprises a truncation mutation.  
     
     
         10 . The method of  claim 4  wherein the allele comprises a truncation mutation at codon 134.  
     
     
         11 . The method of  claim 10  wherein the truncation mutation is a thymidine at nucleotide 424 of wild-type PMS2.  
     
     
         12 . The method of  claim 1  wherein the polynucleotide is introduced into a fertilized egg of an animal.  
     
     
         13 . The method of  claim 12  wherein the fertilized egg is subsequently implanted into a pseudo-pregnant female whereby the fertilized egg develops into a mature transgenic animal.  
     
     
         14 . The method of  claim 12  wherein the mismatch repair gene is PMS2.  
     
     
         15 . The method of  claim 12  wherein the mismatch repair gene is human PMS2.  
     
     
         16 . The method of  claim 12  wherein the mismatch repair gene is human MLH1.  
     
     
         17 . The method of  claim 12  wherein the mismatch repair gene is human PMS1.  
     
     
         18 . The method of  claim 11  wherein the mismatch repair gene is human a human mutL homolog.  
     
     
         19 . The method of  claim 15  wherein the allele comprises a truncation mutation.  
     
     
         20 . The method of  claim 15  wherein the allele comprises a truncation mutation at codon 134.  
     
     
         21 . The method of  claim 19  wherein the truncation mutation is a thymidine at nucleotide 424 of wild-type PMS2.  
     
     
         22 . A homogeneous composition of cultured, hypermutable, mammalian cells which comprise a preselected antigen and a dominant negative allele of a mismatch repair gene.  
     
     
         23 . The isolated hypermutable cell of  claim 22  wherein the mismatch repair gene is PMS2.  
     
     
         24 . The isolated hypermutable cell of  claim 23  wherein the mismatch repair gene is human PMS2.  
     
     
         25 . The isolated hypermutable cell of  claim 22  wherein the mismatch repair gene is MLH1.  
     
     
         26 . The isolated hypermutable cell of  claim 22  wherein the mismatch repair gene is PMS1.  
     
     
         27 . The isolated hypermutable cell of  claim 22  wherein the mismatch repair gene is a human mutL homolog.  
     
     
         28 . The isolated hypermutable cell of  claim 22  wherein the cells express a protein consisting of the first 133 amino acids of hPMS2.  
     
     
         29 . A method for generating a mutation in a gene encoding an antigen of interest comprising 
 growing a mammalian cell comprising said gene encoding an antigen of interest and a dominant negative allele of a mismatch repair gene, and    determining whether said gene encoding an antigen of interest harbors a mutation.    
     
     
         30 . The method of  claim 29  wherein determining whether said gene encoding an antigen of interest harbors a mutation is accomplished by analyzing the nucleotide sequence of said gene.  
     
     
         31 . The method of  claim 30  wherein said nucleotide sequence is an mRNA transcribed from said gene.  
     
     
         32 . The method of  claim 29  wherein determining whether said gene encoding an antigen of interest harbors a mutation is accomplished by analyzing a protein encoded by said gene.  
     
     
         33 . The method of  claim 30  wherein determining whether said gene encoding an antigen of interest harbors a mutation is accomplished by analyzing the phenotype of said gene.  
     
     
         34 . The method of  claim 32  wherein analyzing of said protein comprises analyzing the antigenicity and immunogenicity of said protein.  
     
     
         35 . A method for generating a mutation in a gene encoding an antigen of interest comprising 
 growing a cell comprising said gene and a polynucleotide encoding a dominant negative allele of a mismatch repair gene; and    testing the cell to determine whether the cell harbors a mutation in said gene yielding at least one new biochemical feature of said antigen.    
     
     
         36 . The method of  claim 35  wherein said new biochemical feature is selected from the group consisting of increased antigenicity and increased immunogenicity.  
     
     
         37 . The method of  claim 35  wherein said testing comprises analyzing primary structure of said gene.  
     
     
         38 . The method of  claim 35  wherein said testing comprises analyzing the secondary structure of said gene.  
     
     
         39 . The method of  claim 35  wherein said testing comprises analyzing the antigenicity and immunogenicity of the polypeptide encoded by said gene.  
     
     
         40 . The method of  claim 1  wherein said introduction of said polynucleotideis in the presence of at least one DNA mutagen.  
     
     
         41 . The method of  claim 35  wherein said testing comprises analyzing a nucleotide sequence said gene.  
     
     
         42 . The method of  claim 35  wherein said testing comprises analyzing mRNA transcribed from said gene.  
     
     
         43 . The method of  claim 35  wherein said testing comprises analyzing the antigen protein encoded by the gene of interest.  
     
     
         44 . The method of  claim 35  wherein said testing comprises analyzing the biochemical activity of the protein encoded by said gene.  
     
     
         45 . A hypermutable transgenic mammalian cell made by the method of  claim 35 .  
     
     
         46 . The transgenic mammalian cell of  claim 45  wherein the cell is from primate.  
     
     
         47 . The transgenic mammalian cell of  claim 45  wherein the cell is from rodent.  
     
     
         48 . The transgenic mammalian cell of  claim 45  wherein the cell is from human.  
     
     
         49 . The transgenic mammalian cell of  claim 45  wherein the cell is eucaryotic.  
     
     
         50 . The transgenic mammalian cell of  claim 45  wherein the cell is prokaryotic  
     
     
         51 . A method for making randomly altered forms of a secreted antigen comprising introducing a polynucleotide encoding a tagged antigen into an MMR defective cell.  
     
     
         52 . The method of  claim 51  wherein said tagged antigen is screened for increased antigenicity.  
     
     
         53 . The method of  claim 51  wherein said tagged antigen is screened for increased immunogenicity.  
     
     
         54 . The method of  claim 51  wherein the cells are made MMR defective by introducing at least one dominant negative allele of an MMR gene.  
     
     
         55 . The method of  claim 51  wherein the cells are naturally MMR defective.  
     
     
         56 . A method of producing a mutated antigen in a reversibly unstable cell comprising introducing into a cell containing a preselected antigen of interest, an inducible expression vector comprising a polynucleotide encoding a dominant negative allele of a mismatch repair gene; inducing said cell to express said dominant negative mismatch repair gene; and detecting at least one new biochemical feature of said antigen.  
     
     
         57 . The method of  claim 56  wherein said new biochemical feature is selected from the group consisting of a nucleotide mutation, increased antigenicity and increased immunogenicity.  
     
     
         58 . The method of  claim 56  wherein said preselected antigen of interest is encoded on a polynucleotide previously transfected into said cell.  
     
     
         59 . The method of  claim 56  further comprising ceasing induction of said dominant negative allele of a mismatch repair gene, thereby stabilizing said cell.  
     
     
         60 . The method of  claim 58  further comprising isolating the polynucleotide previously transfected into said cell after detection of said new biochemical feature.  
     
     
         61 . An polynucleotide molecule for expressing an antigen in a hypermutable cell comprising an expression cassette wherein said cassette comprises a 3′ sequence encoding a plurality of histidine residues, a 5′ leader sequence of an expressed gene, and a polylinker to allow cloning of a nucleotide sequence encoding a preselected antigen.  
     
     
         62 . The polynucleotide molecule of  claim 61  wherein said cell is a mammalian cell.  
     
     
         63 . The polynucleotide molecule of  claim 61  wherein said cell is a human cell.  
     
     
         64 . The method of  claim 63  wherein said 5′ leader sequence is a 5′ leader sequence of IL-2.  
     
     
         65 . A method of producing a mutated antigen comprising introducing a polynucleotide encoding a preselected antigen in the expression cassette said polynucleotide molecule of  claim 61 , and introducing said polynucleotide molecule into a cell comprising a dominant negative allele of a mismatch repair gene.  
     
     
         66 . A hypermutated antigen produced by the method of  claim 65 .  
     
     
         67 . A method of eliciting an immune response in an animal comprising administering to said animal an immunogenic amount of at least one hypermutated antigen of  claim 66 .  
     
     
         68 . The method of  claim 67  wherein said antigen is a mutated form of an antigen derived from a pathogenic organism selected from the group consisting of, bacteria, fungi, protozoa, helminths, and viruses.  
     
     
         69 . An immunogenic composition comprising at least one hypermutated antigen of  claim 66  and a pharmaceutically acceptable carrier.