Recombitope peptides

Abstract

The present invention provides peptides having T cell stimulating activity termed recombitope peptides. Recombitope peptides of the invention preferably comprise at least two T cell epitopes derived from the same or from different protein antigens, and more preferably comprise at least two regions, each region preferably having human T cell stimulating activity and each region comprising at least one T cell epitope derived from a protein antigen. Recombitope peptides of the invention can be derived from protein allergens, autoantigens, or other protein antigens. The invention also provides methods of diagnosing sensitivity to a protein allergen or other protein antigen in an individual, methods to treat such sensitivity and therapeutic compositions comprising one or more recombitope peptides. The invention further provides methods for designing recombitope peptides of the invention where the protein antigen to which the individual is sensitive has unknown or ill-defined T cell epitopes.

1 . An isolated recombitope peptide comprising at least two regions each having human T cell stimulating activity, said regions each comprising at least one T cell epitope of a protein allergen, said regions derived from the same or from different protein allergens.  
     
     
         2 . An isolated recombitope peptide of  claim 1  comprising at least three regions each having human T cell stimulating activity.  
     
     
         3 . An isolated recombitope peptide of  claim 1  wherein the regions are arranged in a configuration different from a naturally-occurring configuration of the regions in a protein allergen.  
     
     
         4 . An isolated recombitope peptide of  claim 3  wherein the regions are derived from the same protein allergen.  
     
     
         5 . An isolated recombitope peptide of  claim 1  wherein the regions are derived from the same protein allergen and wherein the regions are arranged in a noncontiguous configuration.  
     
     
         6 . An isolated recombitope peptide of  claim 5  wherein the noncontiguous regions are defined by amino acids and wherein the protein allergen from which the regions are derived comprises amino acids arranged in a sequential order from an amino terminus to a carboxy terminus and wherein said noncontiguous regions of the recombitope are arranged in a nonsequential order.  
     
     
         7 . An isolated recombitope peptide of  claim 1  wherein at least two of the regions are derived from different protein allergens from the same genus.  
     
     
         8 . An isolated recombitope peptide of  claim 7 , wherein the genus is selected from the group consisting of: the genus Dermatophagoides; the genus Felis; the genus Ambrosia; the genus Lolium; the genus Cryptomeria; the genus Alternaria; the genus Alder: the genus Betula; the genus Ouercus; the genus Olea; the genus Artemisia; the genus Plantago; the genus Panetaria: the genus Canine; the genus Blattela; the genus Apis; and the genus Periplaneta.  
     
     
         9 . An isolated recombitope peptide of  claim 7  wherein the at least two regions are derived from cross-reactive species.  
     
     
         10 . An isolated recombitope peptide of  claim 9  wherein the recombitope peptide comprises at least one region derived from  Dermatophagoides pteronyssinus  and at least one region derived from  Dermatophagoides farinae.    
     
     
         11 . An isolated recombitope peptide of  claim 1  wherein at least two of the regions are derived from different protein allergens of the same species.  
     
     
         12 . An isolated recombitope peptide of  claim 11  wherein the recombitope peptide is selected from the group consisting of a recombitope peptide comprising: 
 a) one region derived from Der p I and one region derived from Der p II;  
 b) one region derived from Der f I and one region derived from Derf II;  
 c) one region derived from Amb a I and one region derived from Amba II;  
 d) one region derived from Lob p I and one region derived from L IX: and  
 e) one region derived from Cry j I and one region derived from II.  
 
     
     
         13 . An isolated recombitope peptide of  claim 1  wherein at least two of the regions are derived from different protein allergens of the same group.  
     
     
         14 . An isolated recombitope peptide of  claim 13  wherein the recombitope peptide comprises at least one region derived from Derp I and one region derived from Der f I.  
     
     
         15 . An isolated recombitope peptide of  claim 1  wherein at least two of the regions are derived from different protein allergens of the same family.  
     
     
         16 . An isolated recombitope peptide of  claim 15  wherein at least two of the regions are each derived from the group consisting of Amb a I1, Amb a I.2, Amb a I.3, and Amb a I.4.  
     
     
         17 . An isolated recombitope peptide of  claim 1  which has minimal immunoglobulin E stimulating activity.  
     
     
         18 . An isolated recombitope peptide of  claim 5  which has minimal immunoglobulin E stimulating activity.  
     
     
         19 . An isolated recombitope peptide of  claim 1  which binds immunoglobulin E to a substantially lesser extent than protein allergens from which the regions are derived binds said immunoglobulin E.  
     
     
         20 . An isolated recombitope peptide of  claim 5  which binds immunoglobulin E to a substantially lesser extent than the protein allergen from which the regions are derived binds said immunoglobulin E.  
     
     
         21 . An isolated recombitope peptide of  claim 1  which does not bind immunoglobulin E specific for a protein allergen, or if binding of the recombitope peptide to said immunoglobulin E occur such binding does not result in release of mediators from mast cells or basophils in a substantial percentage of individuals sensitive to said protein allergen.  
     
     
         22 . An isolated recombitope peptide of  claim 5  which does not bind immunoglobulin E specific for a protein allergen, or if binding of the recombitope peptide to said immunoglobulin E occurs, such binding does not result in release of mediators from mast cells or basophils in a substantial percentage of individuals sensitive to said protein allergen.  
     
     
         23 . An isolated recombitope peptide of  claim 1  which, upon administration to an individual allergic to said protein allergen or allergens from which the recombitope peptide is derived, modifies the allergic response of the individual to said protein allergen or allergens.  
     
     
         24 . An isolated recombitope peptide of  claim 5  which, upon administration to an individual allergic to said protein allergen or allergens from which the recombitope peptide is derived, modifies the allergic response of the individual to said protein allergen or allergens.  
     
     
         25 . An isolated recombitope peptide of  claim 1  wherein the regions are selected from the group consisting of peptide X, peptide Y, peptide Z, peptide A, and peptide B, each as shown in FIG. 4.  
     
     
         26 . An isolated recombitope peptide of  claim 25  wherein the regions comprise peptide X, peptide Y, and peptide Z each as shown in FIG. 4.  
     
     
         27 . An isolated recombitope peptide of  claim 25  wherein the regions comprise peptide X. peptide Y, peptide Z, peptide A, and peptide B, each as shown in FIG. 4.  
     
     
         28 . An isolated recombitope peptide YZX comprising peptide Y. peptide Z and peptide X, in sequential order, each peptide as shown in FIG. 4.  
     
     
         29 . An isolated recombitope peptide of AYZXB comprising peptide A, peptide Y, peptide Z, peptide Y and peptide B in sequential order, each peptide as shown in FIG. 4.  
     
     
         30 . An isolated recombitope peptide of  claim 1  including a proteolytic site inserted between at least two of said regions.  
     
     
         31 . An isolated recombitope peptide of  claim 1  wherein the regions each comprise at least two T cell epitopes of a protein allergen.  
     
     
         32 . An isolated peptide of a protein allergen of the genus Felis said peptide comprising at least one T cell epitope of said protein allergen, said peptide comprising an amino acid sequence selected from the group consisting of the amino acid sequence as shown in FIG. 4 of peptide A and peptide B.  
     
     
         33 . A nucleic acid sequence coding for a recombitope peptide of  claim 1  or the functional equivalent of said nucleic acid sequence.  
     
     
         34 . A nucleic acid sequence coding for a recombitope peptide of  claim 5  or the functional equivalent of said nucleic acid sequence.  
     
     
         35 . An isolated recombitope peptide comprising at least two regions derived from the same or from different protein antigens, said recombitope peptide having human T cell stimulating activity.  
     
     
         36 . An isolated recombitope peptide of  claim 35  wherein the protein antigens comprise protein allergens.  
     
     
         37 . An isolated recombitope peptide of  claim 35  comprising at least three regions.  
     
     
         38 . An isolated recombitope peptide of  claim 35  wherein the regions are arranged in a configuration different from a naturally-occurring configuration of the regions in a protein antigen.  
     
     
         39 . An isolated recombitope peptide of  claim 38  wherein the regions are derived from the same protein antigen.  
     
     
         40 . An isolated recombitope peptide of  claim 35  wherein the regions are derived from the same protein antigen and wherein the regions are arranged in a noncontiguous configuration.  
     
     
         41 . An isolated recombitope peptide of  claim 40  wherein the noncontiguous regions are defined by amino acids and wherein the protein antigen from which the regions are derived comprises amino acids arranged in a sequential order from an amino terminus to a carboxy terminus and wherein said noncontiguous regions of the recombitope peptide are arranged in a nonsequential order.  
     
     
         42 . A therapeutic composition comprising a recombitope peptide of  claim 1  and a pharmaceutically acceptable carrier or diluent.  
     
     
         43 . A therapeutic composition comprising a recombitope peptide of  claim 5  and a pharmaceutically acceptable carrier or diluent.  
     
     
         44 . A therapeutic composition comprising a recombitope peptide of  claim 7  and a pharmaceutically acceptable carrier or diluent.  
     
     
         45 . A method of treating sensitivity to a protein allergen in an individual., comprising administering to the individual a therapeutically effective amount of the composition of  claim 42 .  
     
     
         46 . A method of treating sensitivity to a protein allergen in an individual, comprising administering sequentially to the individual two different compositions of  claim 42  in a therapeutically effective amount.  
     
     
         47 . A method of treating sensitivity to a protein allergen in an individual, comprising administering to the individual a therapeutically effective amount of the composition of  claim 43 .  
     
     
         48 . A therapeutic composition comprising a recombitope peptide of  claim 35  and a pharmaceutically acceptable carrier or diluent.  
     
     
         49 . A therapeutic composition comprising a recombitope peptide of  claim 36  and a pharmaceutically acceptable carrier or diluent.  
     
     
         50 . A therapeutic composition comprising a recombitope peptide of  claim 40  and a pharmaceutically acceptable carrier or diluent.  
     
     
         51 . A method of treating sensitivity to a protein allergen in an individual, comprising, administering to the individual a therapeutically effective amount of the composition of  claim 48 .  
     
     
         52 . A method of treating sensitivity to a protein allergen in an individual, comprising administering to the individual a therapeutically effective amount of the composition of  claim 49 .  
     
     
         53 . A method for detecting specific Delayed Type Hypersensitivity in an individual to at least one protein allergen, comprising administering to the individual a Delayed Type Hypersensitivity test utilizing a modified from of said at least one protein allergen or a portion thereof, or said at least one protein allergen produced recombinantly, or a recombitope peptide comprising at least two regions derived from said at least one protein allergen, each of which has human T cell stimulating activity and each of which does not bind immunoglobulin E specific for said at least one protein allergen, or if binding of the recombitope to said immunoglobulin E occurs, such binding does not result in release of mediators from mast cells or basophils in a substantial percentage of individuals sensitive to said at least one protein allergen, and determining the extent to which a specific Delayed Type Hypersensitivity reaction occurs in the individual.  
     
     
         54 . A method of  claim 53 , wherein the regions each comprise at least one T cell epitope of said at least one protein allergen.  
     
     
         55 . A method for determining in individuals the presence of immunoglobulin E specific for at least one protein allergen and the ability of T cells of the individuals to respond to T cell epitope(s) of said at least one protein allergen, comprising the steps of: 
 a) administering to the individuals an Immediate Type Hypersensitivity test utilizing said at least one protein allergen or a portion thereof, or a modified form of said at least one protein allergen or a portion thereof, each of which binds immunoglobulin E specific for said at least one protein allergen;    b) determining whether a specific Immediate Type Hypersensitivity reaction occurs;    c) administering to an individual prior to, simultaneously with, or subsequent to administration of the Immediate Type Hypersensitivity test in step (a), a Delayed Type Hypersensitivity test utilizing a modified form of said at least one protein allergen or a portion thereof, or said at least one protein allergen produced recombinantly, or a recombitope peptide comprising at least two regions derived from said at least one protein allergen, each of which has human T cell stimulating activity and each of which does not bind immunoglobulin E specific for said at least one protein allergen, or if binding to said immunoglobulin E occurs, such binding does not result in release of mediators from mast cells or basophils in a substantial percentage of a population of individuals sensitive to said at least one protein allergen; and    d) determining whether a specific Delayed Type Hypersensitivity reaction occurs.    
     
     
         56 . A method of  claim 55  wherein the regions of the recombitope peptide each comprise at least one T cell epitope of said at least one protein allergen.  
     
     
         57 . A method of detecting and treating sensitivity in an individual to at least one protein allergen, comprising the steps of: 
 a) administering to the individuals an Immediate Type Hypersensitivity test utilizing said at least one protein allergen or a portion thereof, or a modified form of said at least one protein allergen or a portion thereof, each of which binds immunoglobulin E specific for said at least one protein allergen;    b) determining whether a specific Immediate Type Hypersensitivity reaction occurs;    c) administering to an individual having a specific Immediate Type Hypersensitivity reaction a Delayed Type Hypersensitivity test utilizing a modified form of said at least one protein allergen or a portion thereof, or said at least one protein allergen produced recombinantly, or a recombitope peptide comprising at least two regions derived from said at least one protein allergen, each of which has human T cell stimulating activity to said at least one protein allergen and each of which does not bind immunoglobulin E specific for said at least one protein allergen, or if binding to said immunoglobulin E occurs, such binding does not result in release of mediators from mast cells or basophils;    d) determining whether a specific Delayed Type Hypersensitivity reaction occurs; and    e) administering to the individual having a specific Immediate Type Hypersensitivity reaction and a specific Delayed Type Hypersensitivity reaction a therapeutically effective amount of a therapeutic composition comprising the modified form of said at least one protein allergen or said portion thereof of step (c), or said recombinantly produced protein allergen of step (c), or said recombitope peptide of step (c) and a pharmaceutically acceptable carrier or diluent.    
     
     
         58 . A method of  claim 57  wherein the regions of the recombitope peptide each comprise at least one T cell epitope of said at least one protein allergen.  
     
     
         59 . A method for designing the recombitope peptide of  claim 35 , comprising the steps of: 
 a) reviewing the known protein structure of an antigen;    b) theoretically dividing the antigen into at least two peptide regions of desired lengths;    c) theoretically arranging said at least two peptide regions to form at least one recombitope peptide in which the peptide regions are rearranged in a noncontiguous order;    d) producing at least one recombitope peptide having the configuration of the at least one recombitope peptide of step (c); and    e) determining whether the at least one recombitope peptide of step (d) has human T cell stimulating activity.    
     
     
         60 . A method of  claim 59  further comprising the step of: 
 f) determining whether the recombitope peptide found to have human T cell stimulating activity in step (e) binds immunoglobulin E specific for the antigen of step (a).  
 
     
     
         61 . A method of  claim 59  wherein in step (b) the antigen is divided into overlapping regions of desired lengths.  
     
     
         62 . The method of  claim 59  wherein there is known at least one T cell epitope of the antigen or at least one region of the antigen having human T cell stimulating activity and wherein in step (b), said region having human T cell stimulating activity or said T cell epitope is utilized as at least one of said peptide regions.  
     
     
         63 . A method of  claim 59  wherein in step (b) the antigen is divided into regions according to an algorithm.  
     
     
         64 . A method of  claim 59  wherein the protein antigen comprises a protein allergen and wherein step (b) further comprises producing said peptide regions and determining whether said peptide regions bind immunoglobulin E specific for the allergen of step (a), and if such peptide regions bind IgE, whether such binding causes the release of mediators from mast cells or basophils, and wherein in step (c) the peptide regions that bind immunoglobulin E specific for the allergen of step (a) and cause the release of mediators from mast cells or basophils are not included in the peptide regions arranged to form the at least one recombitope peptide.  
     
     
         65 . An isolated recombitope peptide comprising at least two T cell epitopes derived from the same or from different protein antigens.  
     
     
         66 . An isolated recombitope peptide of  claim 65  comprising at least three T cell epitopes.  
     
     
         67 . A method of treating sensitivity to a protein antigen in an individual comprising administering sequentially to the individual two different isolated recombitope peptides, each recombitope peptide comprising at least two human T cell epitopes derived from the same or from different protein antigens, in a therapeutically effective amount.  
     
     
         68 . A method of treating sensitivity to a protein allergen in an individual comprising administering sequentially to the individual two different peptides selected from the group consisting of: peptide X; peptide Y; peptide Z; peptide A; and peptide B, each as shown in FIG. 4, in-a therapeutically effective amount.  
     
     
         69 . A composition comprising at least two isolated recombitope peptides, said recombitope peptides each comprising at least two T cell epitopes derived from the same or from different protein antigens.  
     
     
         70 . A composition comprising at least two isolated recombitope peptides, said recombitope peptides each comprising at least two regions each having human T cell stimulating activity, said regions each comprising at least one T cell epitope of a protein antigen, said regions derived from the same or from different protein antigens.  
     
     
         71 . A composition comprising at least two isolated recombitope peptides, said recombitope peptides each comprising at least two regions derived from the same or from different protein antigens, said recombitope peptides each having human T cell stimulating activity.  
     
     
         72 . An isolated recombitope peptide comprising at least two regions each having human T cell stimulating activity, said regions each comprising at least one T cell epitope of a protein antigen.  
     
     
         73 . An isolated recombitope peptide of  claim 72  wherein the protein antigen is an autoantigen.  
     
     
         74 . An isolated recombitope peptide of  claim 73  wherein the autoantigen is selected from the group consisting of: insulin; myelin basic protein; rh factor; acetylcholine receptors; thyroid cell receptors; basement membrane proteins; thyroid proteins; PM-1; glutamic acid decarboxylase (64K); and carboxypeptidase H.  
     
     
         75 . An isolated recombitope peptide of  claim 72  wherein the regions are arranged in a configuration different from a naturally-occurring configuration of the regions in a protein antigen.  
     
     
         76 . An isolated recombitope peptide of  claim 72  wherein the regions are arranged in a noncontiguous configuration.  
     
     
         77 . An isolated recombitope peptide of  claim 76  wherein the noncontiguous regions are defined by amino acids and wherein the protein antigen from which the regions are derived comprises amino acids arranged in a sequential order from an amino terminus to a carboxy terminus and wherein said noncontiguous regions of the recombitope peptide are arranged in a nonsequential order.  
     
     
         78 . An isolated recombitope peptide of  claim 72  which does not bind immunoglobulin specific for said protein antigen in a substantial percentage of individuals sensitive to said protein antigen.  
     
     
         79 . An isolated recombitope peptide of  claim 73  wherein the regions are arranged in a configuration different from a naturally-occurring configuration of the regions in a protein antigen.  
     
     
         80 . An isolated recombitope peptide of  claim 73  wherein the regions are arranged in a noncontiguous configuration.  
     
     
         81 . An isolated recombitope peptide of  claim 80  wherein the noncontiguous regions are defined by amino acids and wherein the protein antigen from which the regions are derived comprises amino acids arranged in a sequential order from an amino terminus to a carboxy terminus and wherein said noncontiguous regions of the recombitope peptide are arranged in a nonsequential order.  
     
     
         82 . An isolated recombitope peptide of  claim 73  which does not bind immunoglobulin specific for said autoantigen in a substantial percentage of individuals sensitive to said protein antigen.  
     
     
         83 . A nucleic acid sequence coding for a recombitope peptide of  claim 72  or the functional equivalent of said nucleic acid sequence.  
     
     
         84 . A therapeutic composition comprising a recombitope peptide of  claim 78  and a pharmaceutically acceptable carrier or diluent.  
     
     
         85 . A therapeutic composition comprising a recombitope peptide of  claim 76  and a pharmaceutically acceptable carrier or diluent.  
     
     
         86 . A therapeutic composition comprising a recombitope peptide of  claim 80  and a pharmaceutically acceptable carrier or diluent.  
     
     
         87 . A therapeutic composition comprising a recombitope peptide of  claim 82  and a pharmaceutically acceptable carrier or diluent.  
     
     
         88 . A method of treating sensitivity to a protein antigen in an individual., comprising administering sequentially to the individual two different compositions of  claim 84  in a therapeutically effective amount.  
     
     
         89 . A method of treating sensitivity to a protein antigen in an individual., comprising administering sequentially to the individual two different compositions of  claim 87  in a therapeutically effective amount.  
     
     
         90 . A composition comprising at least two isolated recombitope peptides, said recombitope peptides each comprising at least two regions each having human T cell stimulating activity, said regions each comprising at least one T cell epitope of a protein antigen.  
     
     
         91 . A method of determining in individuals the presence of immunoglobulin specific for a protein antigen and the ability of T cells of the individuals to respond to T cell epitope(s) of said protein antigen, comprising the steps of: 
 a) combining a first blood sample or at least one portion of a first blood sample obtained from an individual with said protein antigen or a portion thereof, or a modified form of said protein antigen or a portion thereof, each of which binds immunoglobulin specific for said protein antigen, under conditions appropriate for binding of blood components with said protein antigen, modified protein antigen or a portion of either of said antigens;    b) determining whether binding occurs;    c) combining a second blood sample or a second portion of said first blood sample obtained from an individual found to have binding of blood components in step (b), with a recombitope peptide comprising at least two regions derived from said protein antigen, said recombitope peptide having human T cell stimulating activity to said protein antigen and which does not bind immunoglobulin specific for said protein antigen in a substantial percentage of a population of individuals sensitive to the antigen; and    d) determining whether T cell stimulation occurs.    
     
     
         92 . A method of detecting and treating sensitivity in an individual to a protein antigen, comprising the steps of: 
 a) combining a first blood sample or at least one portion of a first blood sample obtained from the individual with said protein antigen or a portion thereof, or a modified form of said protein antigen or a portion thereof, each of which binds immunoglobulin specific for said protein antigen, under conditions appropriate for binding of blood components with said protein antigen, modified protein antigen or a portion of either of said antigens;    b) determining whether binding occurs;    c) combining a second blood sample or a second portion of said first blood sample obtained from the individual found to have binding of blood components in step (b), with a modified form of said protein antigen or a portion thereof, or said protein antigen produced recombinantly, or a recombitope peptide comprising at least two regions derived from said protein antigen, each of which has human T cell stimulating activity to said protein antigen and each of which does not bind immunoglobulin specific for said protein antigen in a substantial percentage of a population of individuals sensitive to the protein antigen;    d) determining whether T cell stimulation occurs; and    e) administering to the individual found to have binding in step (b) and T cell stimulation in step (d) a therapeutically effective amount of a therapeutic composition comprising the modified form of said protein antigen or said portion thereof of step (c), or said recombinantly produced protein antigen of step (c), or said recombitope peptide of step (c) and a pharmaceutically acceptable carrier or diluent.    
     
     
         93 . A method for detecting specific Delayed Type Hypersensitivity in an individual to at least one protein antigen, comprising administering to the individual a Delayed Type Hypersensitivity test utilizing a modified form of said at least one protein antigen or a portion thereof, or said at least one protein antigen, produced recombinantly, or a recombitope peptide comprising at least two regions derived from said at least one protein antigen, each of which has human T cell stimulating activity and each of which does not bind immunoglobulin specific for said at least one protein antigen in a substantial percentage of a population of individuals sensitive to said at least one protein antigen, and determining the extent to which a specific Delayed Type Hypersensitivity reaction occurs in the individual.