Peptides for Diagnostic and Therapeutic Methods for Celiac Sprue

Abstract

Detection of toxic gluten oligopeptides refractory to digestion and antibodies and T cells responsive thereto can be used to diagnose Celiac Sprue. Analogs of such oligopeptides are useful in the inhibition of immune responses.

1 . A purified oligopeptide of from about 14 amino acids in length to about 33 amino acids in length, comprising the epitope sequence (SEQ ID NO:43) PQPEX 1 PYPQ, where X 1  is glutamine or lysine or a conjugate of lysine. 
   
   
       2 . The purified oligopeptide of  claim 1 , wherein said peptide comprises as the amino terminal sequence, SEQ ID NO:32, LQLQPF. 
   
   
       3 . The purified oligopeptide of  claim 1 , wherein the carboxy terminal sequence is SEQ ID NO:44 PELPY, or SEQ ID NO:45 PEKPY. 
   
   
       4 . The purified oligopeptide of  claim 1 , wherein said peptide has the structure:
   LQLQPFPQPE-X 1 -PYPQPE-X 2 -PY   where X 1  and X 2  are independently selected from lysine; leucine; and lysine conjugated to a group that provides for steric hindrance of interactions between the peptide and a cognate receptor.   
   
   
       5 . The purified oligopeptide of  claim 4 , wherein said lysine residue is conjugated to succinic acid; benzyloxycarbonyl group; t-Butoxycarbonyl group; 9-fluorenylmethoxycarbonyl group; phthalimides; or polyethylene glycol. 
   
   
       6 . The purified oligopeptide of  claim 1 , wherein said peptide has the structure:
   LQLQPFPQPE-X 1 -PYPQPE-X 2 -PY   where X 1  and X 2  are independently selected from lysine; leucine; and lysine conjugated a linker, where said linker couples two or more of said oligopeptides.   
   
   
       7 . The purified oligopeptides according to  claim 6 , wherein said linker is polyethylene glycol. 
   
   
       8 . A pharmaceutical composition comprising the oligopeptides of  claim 1 , and a pharmaceutically acceptable excipient. 
   
   
       9 . A method for diagnosing Celiac Sprue in an individual, said method comprising determining whether an oligopeptide of  claim 1  stimulates agglutination of anti-gliadin antibodies, anti-tTGase antibodies, or combinations thereof from said individual, and correlating an ability to stimulate agglutination with a positive diagnosis of Celiac Sprue. 
   
   
       10 . The method of  claim 9 , wherein said tissue is a mucosal tissue selected from the group consisting of oral, nasal, lung, and intestinal mucosal tissue. 
   
   
       11 . The method of  claim 9 , wherein said bodily fluid is selected from the group consisting of blood, sputum, urine, phlegm, lymph, and tears. 
   
   
       12 . The method of  claim 9 , wherein said individual has not consumed gluten for an extended period of time. 
   
   
       13 . The method of  claim 12 , wherein said extended period of time is selected from the group consisting of one day, one week, one month, and one year prior to the performance of the diagnostic method. 
   
   
       14 . The method of  claim 9 , wherein said individual has not had an endoscopy. 
   
   
       15 . The method of  claim 9 , wherein said individual is the subject of a therapy intended to treat Celiac Sprue or is in a clinical trial conducted to evaluate such a therapy. 
   
   
       16 . An HLA-binding peptide inhibitor; wherein said inhibitor is an analog of an immunogenic gluten oligopeptide of at least about 8 residues in length, wherein the immunogenic gluten oligopeptide is altered by the replacement of one or more amino acids; and wherein said analog binds tightly to HLA molecules; is proteolytically stable; and does not activate disease-specific T cells. 
   
   
       17 . The HLA-binding peptide inhibitor of  claim 16 , wherein said analog comprises one or more naturally occurring amino acids, non-naturally occurring amino acids, modified amino acids, or amino acid mimetics. 
   
   
       18 . The HLA-binding peptide inhibitor of  claim 17 , wherein said analog is further derivatized to reduce the affinity of the analog for disease-specific T cell receptors. 
   
   
       19 . The HLA-binding peptide inhibitor of  claim 16 , wherein said immunogenic gluten oligopeptides comprises at least one PXP motif. 
   
   
       20 . The HLA-binding peptide inhibitor of  claim 16 , wherein said immunogenic gluten oligopeptides comprises a sequence selected from the group consisting of: (SEQ ID NO: 36) PQPELPY; PFPQPELPYP, (SEQ ID NO: 47) PQPELPYPQPQLP, (SEQ ID NO: 48) PQQSFPEQQPP, (SEQ ID NO: 49) VQGQGIIQPEQPAQ, (SEQ ID NO: 50) FPEQPQQPYPQQP, (SEQ ID NO: 51) FPQQPEQPYPQQP, FSQPEQEFPQPQ; PFPQPQLPY, PQPQLPYPQ, (SEQ ID NO: 17) PFPQPELPY; (SEQ ID NO: 58) PYPQPELPY and PYPQPQLPY. 
   
   
       21 . The HLA-binding peptide inhibitor of  claim 16 , wherein said inhibitor comprises the sequence PXPQPELPY, where X is Tyr, Trp, Arg, Lys, p-iodo-Phe, 3-iodo-Tyr, p-amino-Phe, 3-amino-Tyr, hydroxylysine, ornithine, Asp or Glu. 
   
   
       22 . The HLA-binding peptide inhibitor of  claim 21 , wherein said inhibitor is further derivatized to reduce the affinity of the analog for disease-specific T cell receptors. 
   
   
       23 . The HLA-binding peptide inhibitor of  claim 16 , wherein said inhibitor is further modified to increase binding potency to an MHC molecule. 
   
   
       24 . The HLA-binding peptide inhibitor of  claim 16 , wherein said inhibitor comprises the sequence PFPQX 1 ELX 2 Y, where X 1  and X 2  are independently selected from 4-hydroxy-Pro, 4-amino-Pro, or 3-hydroxy-Pro, and proline, with the proviso that at least one of X 1  and X 2  is a residue other than proline. 
   
   
       25 . The HLA-binding peptide inhibitor of  claim 24 , wherein said inhibitor is further derivatized to reduce the affinity of the analog for disease-specific T cell receptors. 
   
   
       26 . The HLA-binding peptide inhibitor of  claim 24 , wherein said inhibitor is further modified to increase binding potency to an MHC molecule. 
   
   
       27 . A method of treating Celiac Sprue and/or dermatitis herpetiformis, the method comprising:
 administering to a patient an effective dose of an HLA-binding peptide inhibitor;   wherein said HLA-binding peptide inhibitor attenuates gluten toxicity in said patient.   
   
   
       28 . The method of  claim 27 , wherein said HLA-binding peptide inhibitor is administered with a glutenase. 
   
   
       29 . The method according to  claim 27  wherein said HLA-binding peptide inhibitor is administered orally. 
   
   
       30 . The method according to  claim 27 , wherein said HLA-binding peptide inhibitor is contained in a formulation that comprises an enteric coating. 
   
   
       31 . A formulation for use in treatment of Celiac Sprue and/or dermatitis herpetiformis, comprising:
 an effective dose of an HLA-binding peptide inhibitor and a pharmaceutically acceptable excipient.   
   
   
       32 . The formulation according to  claim 29 , further comprising an enteric coating. 
   
   
       33 . Use of an HLA-binding peptide inhibitor in the treatment of HLA-DQ2 positive individuals who are either pre-disposed to type I diabetes or have developed symptoms of type I diabetes.