Methods and compositions for stimulating T-lymphocytes

Abstract

Disclosed are methods, compositions, antibodies, and therapeutic kits for use in stimulating cytotoxic T-lymphocytes and generating immune responses against epitopes of protooncogenes. Novel peptides are described which have been shown to stimulate cytotoxic T-lymphocytes, and act as antigens in generation of oncogenic epitope-recognizing antibodies. Methods are disclosed for use in treating various proliferative disorders, and diagnosing HER-2/neu-containing cells; also disclosed are therapeutic kits useful in the treatment of cancer and production of potential anti-cancer vaccines.

What is claimed is:  
     
         1 . A peptide of between 8 and about 20 amino acid residues in length, and including within its sequence the amino acid sequence of: 
 AA 1 —AA 2 —AA 3 —AA 4 —AA 5 —AA 6 —AA 7 —AA 8 ; wherein    AA 1  is Leu or Ile;    AA 2  is Ala, Arg, Gln, Glu, Gly, Leu, Met, Phe, Pro, Ser, Thr, Tyr, or Val;    AA 3  is Ala, Gln, Glu, Gly, His, Lys, Met, Pro, Ser, Tyr, or Val;    AA 4  is Ala, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Thr, Trp, Tyr, or Val;    AA 5  is Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, or Val;    AA 6  is Ala, Asn, Asp, Cys, Gln, Glu, Gly, Leu, Lys, Ser, or Thr;    AA 7  is Ala, Arg, Gln, Gly, His, Ile, Leu, Lys, Phe, Ser, Tyr, or Val; and    AA 8  is Val, Leu, Met, Gly, or Glu.    
     
     
         2 . The peptide of  claim 1 , further defined as a peptide of between 8 and about 15 amino acid residues in length.  
     
     
         3 . The peptide of  claim 2 , further defined as a peptide of between 8 and 10 amino acid residues in length.  
     
     
         4 . The peptide of  claim 3 , further defined as having the amino acid sequence of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; or SEQ ID NO: 29.  
     
     
         5 . The peptide of  claim 4 , further defined as having the amino acid sequence of SEQ ID NO: 1.  
     
     
         6 . The peptide of  claim 4 , further defined as having the amino acid sequence of SEQ ID NO: 2.  
     
     
         7 . The peptide of  claim 4 , further defined as having the amino acid sequence of SEQ ID NO: 3.  
     
     
         8 . The peptide of  claim 4 , further defined as having the amino acid sequence of SEQ ID NO: 4.  
     
     
         9 . The peptide of  claim 4 , further defined as having the amino acid sequence of SEQ ID NO: 5.  
     
     
         10 . The peptide of  claim 4 , further defined as having the amino acid sequence of SEQ ID NO: 6.  
     
     
         11 . The peptide of  claim 4 , further defined as having the amino acid sequence of SEQ ID NO: 7.  
     
     
         12 . The peptide of  claim 4 , further defined as having the amino acid sequence of SEQ ID NO: 8.  
     
     
         13 . The peptide of  claim 4 , further defined as having the amino acid sequence of SEQ ID NO: 9.  
     
     
         14 . The peptide of  claim 4 , further defined as having the amino acid sequence of SEQ ID NO: 18.  
     
     
         15 . The peptide of  claim 4 , further defined as having the amino acid sequence of SEQ ID NO: 19.  
     
     
         16 . The peptide of  claim 1 , wherein 
 AA 2  is Val or Gln;    AA 3  is Ser, Gln, Glu, Lys, or Pro;    AA 4  is Glu, Gly, Ile, Leu, or Ser;    AA 6  is Asn, Gln, or Ser; and    AA 7  is Arg, Leu, Gln, Tyr, Val, or Lys.    
     
     
         17 . The peptide of  claim 16 , wherein 
 AA 2  i s Val;    AA 3  is Ser;    AA 4  is Glu;    AA 6  is Ser; and    AA 7  is Arg or Lys.    
     
     
         18 . A peptide of between 8 and about 20 amino acid residues in length, said peptide stimulating cytotoxic T-lymphocytes and comprising the amino acid sequence: 
 AA 1 —AA 2 —AA 3 —AA 4 —AA 5 —AA 6 —AA 7 —AA 8 ; wherein 
 AA 1  is Leu or Ile;  
 AA 2  is Ala, Arg, Gln, Glu, Gly, Leu, Met, Phe, Pro, Ser, Thr, Tyr, or Val;  
 AA 3  is Ala, Gln, Glu, Gly, His, Lys, Met, Pro, Ser, Tyr, or Val;  
 AA 4  is Ala, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Thr, Trp, Tyr, or Val;  
 AA 5  is Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, or Val;  
 AA 6  is Ala, Asn, Asp, Cys, Gln, Glu, Gly, Leu, Lys, Ser, or Thr;  
 AA 7  is Ala, Arg, Gln, Gly, His, Ile, Leu, Lys, Phe, Ser, Tyr, or Val; and  
 AA 8  is Val, Leu, Met, Gly, or Glu.  
   
     
     
         19 . The peptide of  claim 18 , further defined as being from 8 amino acid residues in length to about 15 amino acid residues in length.  
     
     
         20 . The peptide of  claim 19 , further defined as being from 8 amino acid residues in length to about 10 amino acid residues in length.  
     
     
         21 . The peptide of  claim 20 , further defined as being 8 amino acid residues in length.  
     
     
         22 . The peptide of  claim 21 , further defined as being 9 amino acid residues in length.  
     
     
         23 . The peptide of  claim 21 , further defined as being 10 amino acid residues in length.  
     
     
         24 . The peptide of  claim 18 , further defined as having the amino acid sequence of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; or SEQ ID NO: 29.  
     
     
         25 . The peptide of  claim 24 , further defined as having the amino acid sequence of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 18, or SEQ ID NO: 19.  
     
     
         26 . A peptide of between 8 and about 20 amino acid residues in length, said peptide binding HLA and stimulating cytotoxic T-lymphocytes, and including within its sequence an amino acid sequence represented by: 
 AA 1 —AA 2 —AA 3 —AA 4 —AA 5 —AA 6 —AA 7 —AA 8 ; wherein 
 AA 1  is Leu or Ile;  
 AA 2  is Ala, Arg, Gln, Glu, Gly, Leu, Met, Phe, Pro, Ser, Thr, Tyr, or Val;  
 AA 3  is Ala, Gln, Glu, Gly, His, Lys, Met, Pro, Ser, Tyr, or Val;  
 AA 4  is Ala, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Thr, Trp, Tyr, or Val;  
 AA 5  is Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, or Val;  
 AA 6  is Ala, Asn, Asp, Cys, Gln, Glu, Gly, Leu, Lys, Ser, or Thr;  
 AA 7  is Ala, Arg, Gln, Gly, His, Ile, Leu, Lys, Phe, Ser, Tyr, or Val; and  
 AA 8  is Val, Leu, Met, Gly, or Glu.  
   
     
     
         27 . The peptide of  claim 26 , wherein 
 AA 2  is Val or Gln;    AA 3  is Ser, Gln, Glu, Lys, or Pro;    AA 4  is Glu, Gly, Ile, Leu, or Ser;    AA 6  is Asn, Gln, or Ser; and    AA 7  is Arg, Leu, Gln, Tyr, Val, or Lys.    
     
     
         28 . The peptide of  claim 27 , wherein 
 AA 2  is Val;    AA 3  is Ser;    AA 4  is Glu;    AA 6  is Ser; and    AA 7  is Arg or Lys.    
     
     
         29 . The peptide of  claim 26 , further defined as having the amino acid sequence of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; or SEQ ID NO: 29.  
     
     
         30 . A method for stimulating cytotoxic T-lymphocytes, comprising contacting said cytotoxic T-lymphocytes with an amount of a peptide in accordance with  claim 1  effective to stimulate said cytotoxic T-lymphocytes.  
     
     
         31 . The method of  claim 30 , wherein said cytotoxic T-lymphocytes are located within an animal and said peptide or composition is administered to said animal.  
     
     
         32 . The method of  claim 30 , wherein said cytotoxic T-lymphocytes are obtained from an animal, contacted with said peptide, and re-administered to said animal.  
     
     
         33 . The method of  claim 30 , wherein said peptide is formulated for administration parenterally, topically, or as an inhalant, aerosol or spray.  
     
     
         34 . The method of  claim 31 , wherein said animal is a human subject.  
     
     
         35 . A pharmaceutical composition including the composition of  claim 1  in a pharmaceutically acceptable excipient.  
     
     
         36 . The pharmaceutical composition of  claim 35 , wherein said composition comprises the peptide of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; or SEQ ID NO: 29.  
     
     
         37 . A method of treating a proliferative cell disorder in an animal, comprising administering to said animal a therapeutically-effective amount of a pharmaceutical composition in accordance with  claim 35 .  
     
     
         38 . The method of  claim 37 , wherein said proliferative cell disorder is cancer.  
     
     
         39 . The method of  claim 38 , wherein said cancer is breast or ovarian cancer.  
     
     
         40 . A method for detecting cytotoxic T-lymphocytes in a sample, comprising obtaining a sample suspected of containing cytotoxic T-lymphocytes, contacting said sample with a peptide in accordance with  claim 1 , under conditions effective to allow the formation of cell-peptide complexes, and detecting the cell-peptide complexes so formed.  
     
     
         41 . The method of  claim 40 , wherein said sample is a biological sample from an animal suspected of having a HER-2/neu-related cancer.  
     
     
         42 . The method of  claim 40 , wherein said peptide is linked to a detectable label and the cell-peptide complexes are detected by detecting the presence of the label.  
     
     
         43 . The method of  claim 40 , wherein said cell-peptide complexes are detected by means of an antibody linked to a detectable label, the antibody having binding affinity for the peptide.  
     
     
         44 . A method of generating an immune response, comprising administering to an animal a pharmaceutical composition comprising an immunologically effective amount of a composition comprising the peptide of  claim 1 .  
     
     
         45 . The method of  claim 44 , wherein said composition comprises an immunologically effective amount of a composition comprising the peptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; or SEQ ID NO: 29.  
     
     
         46 . A purified antibody that binds to the peptide of  claim 1 .  
     
     
         47 . The antibody of  claim 46 , wherein the antibody is a monoclonal antibody.  
     
     
         48 . The antibody of  claim 46 , wherein the antibody is linked to a detectable label.  
     
     
         49 . The antibody of  claim 48 , wherein the antibody is linked to a radioactive label, a fluorogenic label, a nuclear magnetic spin resonance label, biotin or an enzyme that generates a colored product upon contact with a chromogenic substrate.  
     
     
         50 . The antibody of  claim 49 , wherein the antibody is linked to an alkaline phosphatase, hydrogen peroxidase or glucose oxidase enzyme.  
     
     
         51 . A method for detecting a neu-containing cancer cell, a neu protein, or neu peptide; the method comprising: 
 (a) generating an antibody that binds to the peptide of  claim 1 .    (b) obtaining a sample suspected of containing a neu-containing cancer cell, a neu protein, or neu peptide;    (c) contacting said sample with said antibody, under conditions effective to allow the formation of immune complexes; and    (d) detecting the immune complexes so formed.    
     
     
         52 . The method of  claim 51 , wherein said antibody is a monoclonal antibody.  
     
     
         53 . An immunodetection kit comprising, in suitable container means, the peptide of  claim 1 , or a first antibody that binds to the peptide of  claim 1 , and an immunodetection reagent.  
     
     
         54 . The immunodetection kit of  claim 53 , wherein the immunodetection reagent is a detectable label that is linked to said peptide or said first antibody.  
     
     
         55 . The immunodetection kit of  claim 54 , wherein the immunodetection reagent is a detectable label that is linked to a second antibody that has binding affinity for said peptide or said first antibody.  
     
     
         56 . The immunodetection kit of  claim 54 , wherein the immunodetection reagent is a detectable label that is linked to a second antibody that has binding affinity for a human antibody.  
     
     
         57 . A DNA segment encoding the peptide of  claim 1 .  
     
     
         58 . The DNA segment of  claim 57 , further defined as encoding the peptide of  claim 3 .  
     
     
         59 . The DNA segment of  claim 57 , further defined as encoding the peptide of  claim 4 .  
     
     
         60 . The DNA segment of  claim 57 , further defined as comprising the DNA sequence of SEQ ID NO: 51; SEQ ID NO: 52; SEQ ID NO: 53; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; SEQ ID NO: 63; or SEQ ID NO: 64.  
     
     
         61 . A recombinant vector comprising the DNA segment of  claim 57 .  
     
     
         62 . The recombinant vector of  claim 61 , further defined as comprising a DNA segment encoding a peptide which stimulates a cytotoxic T-lymphocyte.