Her2 antigenic polypeptide compositions, and methods for their use in treatment and prevention of carcinomas

Abstract

Antigenic polypeptides of the growth factor receptor HER2, for breaking the tolerance of a host against self HER2. The antigenic polypeptides include HER2 polypeptides with single amino acid substitutions of lysine for glutamine, arginine for glutamine, or aspartic acid for asparagine. Gene expression constructs, vaccine compositions, and immunization methods including the substituted HER2 polypeptides. Methods for immunizing mammalian subjects with heterologous unsubstituted HER2 antigenic polypeptides, including polypeptides of feline and bear HER2. A diagnostic method of determining whether a mammalian subject is sufficiently immunocompetent to respond to immunotherapies directed at breaking tolerance to self HER2.

What is claimed is: 
     
         1 . Antigenic polypeptides of HER2, for breaking tolerance to the self HER2 of an animal subject, said antigenic polypeptides including at least one point mutation in the extracellular domain of HER2. 
     
     
         2 . The antigenic peptides according to  claim 1 , wherein said point mutation induces the substitution of glutamine with lysine with (Q-K) in the amino acid sequence QLRSLTEILKGGVLI (SEQ ID NO: 109) of HER2 domain I, rendering said amino acid sequence KLRSLTEILKGGVLI (SEQ ID NO: 110). 
     
     
         3 . Isolated antigenic polypeptides for inducing immune response against HER2 in a subject of a mammalian species, said polypeptides comprising at least the extracellular domain of the HER2 of an animal species, said extracellular domain including an amino acid substitution of glutamine with lysine (Q-K), or with a conservative amino acid of lysine, said substitution being at position 119 of mature feline HER2, or at a homologous position of the mature HER2 of another animal species. 
     
     
         4 . The antigenic polypeptides according to  claim 3 , selected from the group consisting of mature feline HER2 having a Q-K substitution at position 119 (mfeHER2-Q119K); mature bear HER2 having a Q-K substitution at position 119 (mbearHER2-Q119K); mature human HER2 having a Q-K substitution at position 119 (mhumHER2-Q119K); mature mouse HER-2 having a Q-K substitution at position 120 (mmouseHER2-Q120K); mature rat HER2 having a Q-K substitution at position 120 (mratHER2-Q120K); and mature human rat chimeric HER2 having a Q-K substitution at position 119 (mE2Neu-Q119K). 
     
     
         5 . The antigenic polypeptides according to  claim 3  wherein said antigenic polypeptides comprise the signal peptide and extracellular and transmembrane domains of HER2 (precursor HER2) and said amino acid substitution of glutamine with lysine (Q-K) or a conservative amino acid of lysine, is at position 141 of precursor feline HER2, or at a homologous position of the precursor HER2 of another animal species. 
     
     
         6 . The antigenic polypeptides according to  claim 5 , selected from the group consisting of precursor feline HER-2 having a Q-K substitution at position 141 (prefeHER2-Q141K); precursor bear HER-2 having a Q-K substitution at position 141 (prebearHER2-Q141K); precursor human HER-2 having a Q-K substitution at position 141 (prehumHER2-Q141K); precursor mouse HER-2 having a Q-K substitution at position 142 (premouseHER2-Q142K); precursor rat HER-2 having a Q-K substitution at position 145 (preratHER2-Q145K); and precursor human rat chimeric HER2 having a Q-K substitution at position 141 (preE2Neu-Q141K). 
     
     
         7 . The antigenic polypeptides according to  claim 4 , wherein said mfeHER2-Q119K includes SEQ ID NO: 1, said mbearHER2-Q119K includes SEQ ID NO: 2, said mhumHER2-Q119K includes SEQ ID NO: 3, said mmouseHER2-Q120K includes SEQ ID NO: 4, said mratHER2-Q120K includes SEQ ID NO: 5, and said mE2Neu-Q119K includes SEQ ID NO: 6. 
     
     
         8 . The antigenic polypeptides according to  claim 6 , wherein said prefeHER2-Q141K includes SEQ ID NO: 7, said prebearHER2-Q141K includes SEQ ID NO: 8, said prehumHER2-Q141K includes SEQ ID NO: 9, said premouseHER2-Q142K includes SEQ ID NO: 10, said preratHER2-Q145K includes SEQ ID NO: 11, and said preE2Neu-Q141K) includes SEQ ID NO: 12. 
     
     
         9 . A method for inducing immune response to HER2 in a mammalian subject, comprising the steps of:
 administering an effective amount of the vaccine composition including an effective amount of a gene expression construct including a nucleic acid sequence encoding an antigenic polypeptide of the HER2 of an animal species, said antigenic polypeptide comprising at least the extracellular domain of HER2, including an amino acid substitution of glutamine for lysine (Q-K) or for a conservative amino acid of K, said gene construct additionally including at least one promoter operatively linked to said nucleic acid sequence encoding a HER2 polypeptide, for expression of said antigenic peptide in a living cell, wherein said nucleic acid sequence additionally encodes the signal peptide of HER2 (precursor HER2), and said amino acid substitution of glutamine for lysine (Q-K) or for a conservative amino acid of K, is encoded at amino acid 141 of precursor feline HER2, wherein said nucleic acid sequence encoding an antigenic polypeptide is selected from the group consisting of nucleic acid sequences encoding precursor feline HER2, and wherein said amino acid substitution is encoded at amino acid 141 (prefeHER2-Q141K); precursor bear HER2, wherein said substitution is encoded at amino acid 141 (prebearHER2-Q141K); precursor human HER2, wherein said substitution is encoded at amino acid 141 (prehumHER2-Q141K); precursor mouse HER2, wherein said substitution is encoded at amino acid 142 (premouseHER2-Q142K); precursor rat HER2, wherein said substitution is encoded at amino acid 145 (preratHER2-Q145K); and precursor human rat chimeric HER2, wherein said substitution is encoded at amino acid 141 (mE2Neu-Q141K), wherein said nucleotide sequence encoding prefeHER2-Q141K includes SEQ ID NO: 19; said nucleotide sequence encoding prebearHER2-Q141K includes SEQ ID NO: 20; said nucleotide sequence encoding prehumHER2-Q141K includes SEQ ID NO: 21; said nucleotide sequence encoding premouseHER2-Q142K includes SEQ ID NO: 22; said nucleotide sequence encoding preratHER2-Q145K includes SEQ ID NO: 23; and said nucleotide sequence encoding mE2Neu-Q141K includes SEQ ID NO: 24, and an effective amount of an adjuvant; and   inducing an immune response to HER2.   
     
     
         10 . A method for inducing immune response to HER2 in a mammalian subject, including the steps of:
 administering, to a mammalian subject, an effective amount of a gene construct comprising a nucleic acid sequence encoding a heterologous antigenic polypeptide selected from the group consisting of precursor unsubstituted bear HER2 (prebearHER2); mature unsubstituted bear HER2 (mbearHER2); precursor unsubstituted feline HER2 (prefeHER2); and mature unsubstituted feline HER2 (mfeHER2);   the gene construct additionally including at least one promoter for expression of said antigenic peptide in a living cell;   administering an effective amount of an immunological adjuvant;   expressing said gene construct in cells of the mammalian subject; and   inducing an immune response against HER2 in the mammalian subject.   
     
     
         11 . The method according to  claim 10 , wherein the prebearHER2 includes SEQ ID NO: 37; the mbearHER2 includes SEQ ID NO: 38; the prefeHER2 includes SEQ ID NO: 39; and the mfeHER2 includes SEQ ID NO: 40. 
     
     
         12 . The method according to  claim 10 , wherein the nucleic acid sequence encoding prebearHER2 includes SEQ ID NO: 41; the nucleic acid sequence encoding mbearHER2 includes SEQ ID NO: 42; the nucleic acid sequence encoding prefeHER2 includes SEQ ID NO: 43; and the nucleic acid sequence encoding mfeHER2 includes SEQ ID NO: 44. 
     
     
         13 . The method according to  claim 10 , wherein the step of administering an effective amount of an immunological adjuvant is further defined as administering an effective amount of GM-CSF. 
     
     
         14 . The method according to  claim 10 , wherein the mammalian subject is further defined as a subject hosting a population of HER2-expressing pathological cells, additionally including the step of eliminating or reducing the population of HER2-expressing pathological cells. 
     
     
         15 . The method according to  claim 14 , wherein the HER2 expressing pathological cells are further defined as mammary carcinoma cells. 
     
     
         16 . A method for inducing immune response to HER2 in a cat, including the steps of:
 administering, to a cat, an effective amount of a gene expression construct encoding an antigenic polypeptide selected from the group consisting of: precursor unsubstituted bear HER2 (prebearHER2); mature unsubstituted bear HER2 (mbearHER2); precursor unsubstituted feline HER2 (prefeHER2); mature unsubstituted feline HER2 (mfeHER2); precursor unsubstituted human HER2 (prehumHER2); mature unsubstituted human HER2 (mhumHER2); precursor unsubstituted mouse HER2 (premouseHER2); mature unsubstituted mouse HER2 (mmouseHER2); precursor unsubstituted rat HER2 (preratHER2); mature unsubstituted rat HER2 (mratHER2); precursor human rat chimeric HER2 (preE2Neu); and mature human rat chimeric HER2 (mE2Neu); the gene expression construct additionally including at least one promoter for expression of the antigenic peptide in a living cell;   administering an effective amount of an immunological adjuvant;   expressing the gene construct in cells of the cat; and   inducing an immune response against HER2 in the cat.   
     
     
         17 . The method according to  claim 16 , wherein the prebearHER2 includes SEQ ID NO: 37; the mbearHER2 includes SEQ ID NO: 38; the prefeHER2 includes SEQ ID NO: 39; the mfeHER2 includes SEQ ID NO: 40; the prehumHER includes SEQ ID NO: 45; the mhumHER2 includes SEQ ID NO: 46; the premouseHER2 includes SEQ ID NO: 47; the mmouseHER2 includes SEQ ID NO: 48; the preratHER2 includes SEQ ID NO: 49; the mratHER2 includes SEQ ID NO: 50; the preE2Neu includes SEQ ID NO: 51; and the mE2Neu includes SEQ ID NO: 52. 
     
     
         18 . The method according to  claim 16 , wherein the nucleic acid sequence encoding prebearHER2 includes SEQ ID NO: 41; the nucleic acid sequence encoding mbearHER2 includes SEQ ID NO: 42; the nucleic acid sequence encoding prefeHER2 includes SEQ ID NO: 43; the nucleic acid sequence encoding mfeHER2 includes SEQ ID NO: 44; the nucleic acid sequence encoding prehumHER includes SEQ ID NO: 53; the nucleic acid sequence encoding mhumHER2 includes SEQ ID NO: 54; said nucleic acid sequence encoding premouseHER2 includes SEQ ID NO: 55; the nucleic acid sequence encoding mmouseHER2 includes SEQ ID NO: 56; the nucleic acid sequence encoding preratHER2 includes SEQ ID NO: 57; the nucleic acid sequence encoding mratHER2 includes SEQ ID NO: 58; the nucleic acid sequence encoding preE2Neu includes SEQ ID NO: 59; and the nucleic acid sequence encoding mE2Neu includes SEQ ID NO: 60. 
     
     
         19 . The method according to  claim 16 , wherein the cat is further defined as a cat hosting a population of HER2-expressing pathological cells, additionally including the step of eliminating or reducing the population of HER2-expressing pathological cells. 
     
     
         20 . The method according to  claim 19 , wherein the HER2 expressing pathological cells are further defined as mammary carcinoma cells. 
     
     
         21 . Antigenic polypeptides for inducing immune response against HER2 in a mammalian subject, said polypeptides comprising at least the extracellular and transmembrane domains of human HER2, said extracellular domain including an amino acid substitution selected from the following amino acid substitutions:
 glutamine with lysine (Q-K) or a conservative amino acid of lysine, at position 141 of precursor humanHER2 (prehumHER2-Q141K);   glutamine with lysine (Q-K) or a conservative amino acid of lysine, at position 119 of mature human HER2 (mhumHER2-Q119K);   glutamine with lysine (Q-K) or a conservative amino acid of lysine, at position 329 of precursor humanHER2 (prehumHER2-Q329K);   glutamine with lysine (Q-K) or a conservative amino acid of lysine, at position 307 of mature humanHER2 (mhumHER2-Q307K);   glutamine with arginine (Q-R) or a conservative amino acid of arginine, said substitution being at position 429 of precursor human HER2 (prehumHER2-Q429R);   glutamine with arginine (Q-R) or a conservative amino acid of R, at position 407 of mature human HER2 mhumHER2-Q407R;   asparagine with aspartic acid (N-D) or a conservative amino acid of aspartic acid, at position 438 of precursor human HER2 (prehumHER2-N438D); and   asparagine with aspartic acid (N-D), or a conservative amino acid of aspartic acid, at position 416 of mature human HER2 mhumHER2-N416D.   
     
     
         22 . The antigenic polypeptides according to  claim 21 , wherein said prehumHER2-Q141K includes SEQ ID NO: 9; said mhumHER2-Q119K includes SEQ ID NO: 3; said prehumHER2-Q329K includes SEQ ID NO: 28; said mhumHER2-Q307K includes SEQ ID NO: 25; said prehumHER2-Q429R includes SEQ ID NO: 29; said mhumHER2-Q407R includes SEQ ID NO: 26; said prehumHER2-N438D includes SEQ ID NO: 30; and said mhumHER2-N416D includes SEQ ID NO: 27. 
     
     
         23 . A gene expression construct comprising a nucleic acid sequence encoding an antigenic polypeptide of HER2, said nucleic acid sequence encoding a substituted HER2 polypeptide selected from the group including: prehumHER2-Q141K, mhumHER2-Q119K, prehumHER2-Q329K mhumHER2-Q307K, prehumHER2-Q429R, mhumHER2-Q407R, prehumHER2-N438D, and mhumHER2-N416D, said gene construct additionally including at least one promoter operatively linked to said nucleic acid sequence encoding a HER2 polypeptide, for expression of said antigenic polypeptide in a living cell. 
     
     
         24 . The gene expression construct according to  claim 23 , wherein said nucleic acid sequence is selected from the group consisting of: SEQ ID NO: 21, encoding said prehumHER2-Q141K; SEQ ID NO: 34, encoding said prehumHER2-Q329K; SEQ ID NO: 35, encoding prehumHER2-Q429R; SEQ ID NO: 36, encoding prehumHER2-N438D; SEQ ID NO: 15, encoding said mhumHER2-Q119K SEQ ID NO: 31, encoding said mhumHER2-Q307K; SEQ ID NO: 32, encoding mhumHER2-Q407R; and SEQ ID NO: 33, encoding mhumHER2-N416D. 
     
     
         25 . A vaccine composition for inducing immunity to HER2 in a mammalian subject, comprising an effective amount of the gene expression construct according to  claim 23 , and an effective amount of an adjuvant. 
     
     
         26 . The vaccine composition according to  claim 25 , wherein said adjuvant is granulocyte macrophage colony stimulating factor (GM-CSF) 
     
     
         27 . The vaccine composition according to  claim 26 , wherein said GM-CSF is delivered to the mammalian subject as an expression vector comprising a polynucleotide encoding GM-CSF, for expression of said polynucleotide in the mammalian subject. 
     
     
         28 . The vaccine composition according to  claim 26 , wherein said GM-CSF is delivered to the mammalian subject as GM-CSF protein. 
     
     
         29 . Monoclonal antibodies selective for substituted HER2 polypeptides, the substituted HER2 polypeptides being selected from the group consisting of: prefeHER2-Q141K, mfeHER2-Q119K, prebearHER2-Q141K, mbearHER2-Q119K, prehumHER2-Q141K, mhumHER2-Q119K, premouseHER2-Q142K, mmouseHER2-Q120K, preratHER2-Q145K, mratHER2-Q120K, preE2Neu-Q141K, mE2Neu-Q119K, prehumHER2-Q329K, mhumHER2-Q307K, prehumHER2-Q429R, mhumHER2-Q407R; prehumHER2-N438D, and mhumHER2-N416D. 
     
     
         30 . A diagnostic method of determining whether a mammalian subject is sufficiently immunocompetent to respond to immunotherapy directed at self HER2, including the steps of:
 administering to the mammalian subject an effective amount of a vaccine known to induce immune response to self HER2 of the species of the mammalian subject;   determining that an immune response to the HER2 antigen is induced in the mammalian subject by the vaccine; and   recognizing the mammalian subject as being sufficiently immunocompetent to respond to immunotherapy directed at self HER2.   
     
     
         31 . The diagnostic method of  claim 30 , wherein the vaccine is selected from the group consisting of the vaccine according to  claim 14  and the vaccine according to claim  34 . 
     
     
         32 . The diagnostic method according to  claim 30 , wherein the step of determining that an immune response is induced is further defined as the step of determining whether there a T cell response has been induced, a B cell response has been induced, or a mixed T and B cell response has been induced.