Polyepitopic protein fragments of the e6 and e7 proteins of hpv, their production and their use particularly in vaccination

Abstract

Polyepitopic peptides of E6 and E7 proteins of Human Papillomavirus, their production, and methods of treating pathologies in which a polyepitopic peptide of the E6 and E7 protein of Human Papillomavirus is recognized by the cellular immune system.

1 . Polyepitopic fragments of the E6 or E7 protein of HPV, characterized in that they comprise a peptide sequence of about 15 to 30 amino acids, this peptide sequence containing amino acid sequences of at least 3 different epitopes binding stably to HLA molecules of identical or different type, when these epitopes are obtained by enzymatic degradation of said peptide sequence, particularly in the proteasome, such that at least 4 HLA molecules of different types bind to these epitopes, these 4 HLA molecules being selected from among those of types A1, A2, A3, A11, A24, A29, B7, B8, B18, B27, B35, B44, B51 and B62. 
     
     
         2 . Polyepitopic fragments according to  claim 1 , characterized in that the number of amino acids of their peptide sequence is greater than or equal to 17, and less than or equal to 30. 
     
     
         3 . Polyepitopic fragments of the E6 protein of HPV according to  claim 1 , characterized in that they comprise a peptide sequence of about 15 to 30 amino acids, this peptide sequence containing amino acid sequences of at least 5 different epitopes binding stably to HLA molecules of identical or different type, when these epitopes are obtained by enzymatic degradation of said peptide sequence, particularly in the proteasome, such that at least 6 HLA molecules of different types bind to these epitopes, these 6 HLA molecules being selected from those of types A1, A2, A3, A11, A24, A29, B7, B8, B18, B27, B35, B44 and B51. 
     
     
         4 . Polyepitopic fragments of the E6 protein of HPV according to one of  claim 1 , characterized in that they all comprise an epitope binding to the HLA molecule of type B35, an epitope binding to the HLA molecule of type B44, and an epitope binding to the HLA molecule of type B51. 
     
     
         5 . Polyepitopic fragment of the E6 protein of HPV according to  claim 1 , characterized by the following:
 (15)RPRKLPQL(22) (residues 15 to 22 of SEQ ID NO: 2) binding stably to HLA molecules of the B7 or B35 type,   (18)KLPQLCTEL(26) (residues 18 to 26 of SEQ ID NO: 2) binding stably to HLA molecules of the A2 type,   (19)LPQLCTEL(26) (residues 19 to 26 of SEQ ID NO: 2) binding stably to HLA molecules of the B51 type,   (21)QLCTELQTTI(30) (residues 21 to 30 of SEQ ID NO: 2) binding stably to HLA molecules of the A2 type,   (24)TELQTTIHDI(33) (residues 24 to 33 of SEQ ID NO: 2) binding stably to HLA molecules of the A29 or B44 type,   (29)TIHDIILRCV(38) (residues 29 to 38 of SEQ ID NO: 2) binding stably to HLA molecules of the A2 type,   (33)IILECVYCK(41) (residues 33 to 41 of SEQ ID NO: 2) binding stably to HLA molecules of the A11 type,   (35)LECVYCKQQL(44) (residues 35 to 44 of SEQ ID NO: 2) binding stably to HLA molecules of the A29 or B44 type,   (37)CVYCKQQL(44) (residues 37 to 44 of SEQ ID NO: 2) binding stably to HLA molecules of the B8 type.   
     
     
         6 . A polyepitopic fragment comprising SEQ ID NO: 8 or SEQ ID NO: 10. 
     
     
         7 . Polyepitopic fragment of the E6 protein of HPV according to  claim 1 , characterized in that it corresponds to the fragment of 29 amino acids delimited by the amino acids located in positions 80 and 108 of the peptide sequence of the E6 protein of HPV, this latter fragment being characterized by the peptide sequence SEQ ID NO: 8 as follows: 
       
         
           
                 
                 
                 
               
                     
                   (80)ISEYRHYCYSLYGTTLEQQYNKPLCDLLI(108) 
                     
                 
             
                
               
            
           
         
         said fragment containing 6 epitopes binding stably to at least one of the 10 HLA molecules of the following types: A1, A3, A11, A24, A29, B7, B18, B35, B44, or B51, said epitopes being the following: 
         (80)ISEYRHYCY(88) (residues 80 to 88 of SEQ ID NO: 2) binding stably to HLA molecules of the A1 or B18 type, 
         (81)SEYRHYCY(88) (residues 81 to 88 of SEQ ID NO: 2) binding stably to HLA molecules of the A29 or B44 type, 
         (87)CYSLYGTTL(95) (residues 87 to 95 of SEQ ID NO: 2) binding stably to HLA molecules of the A24 type, 
         (94)TLEQQYNK(101) (residues 94 to 101 of SEQ ID NO: 2) binding stably to HLA molecules of the A3 or A11 type, 
         (95)LEQQYNKPL(103) (residues 95 to 103 of SEQ ID NO: 2) binding stably to HLA molecules of the A29 or B44 type, 
         (11)KPLCDLLI(108) (residues 101 to 108 of SEQ ID NO: 2) binding stably to HLA molecules of the B7, B35 or B51 type. 
       
     
     
         8 . Polyepitopic fragment of the E6 protein of HPV according to  claim 1 , characterized in that it corresponds to the fragment of 22 amino acids delimited by the amino acids located in positions 118 and 139 of the peptide sequence of the E6 protein of HPV, this latter fragment being characterized by the peptide sequence SEQ ID NO: 10 as follows: 
       
         
           
                 
                 
                 
               
                     
                   (118)CPEEKQRHLDKKQRFHNIRGRW(139) 
                     
                 
             
                
               
            
           
         
         said fragment containing 6 epitopes binding stably to at least one of the 7 HLA molecules of the following types: A24, B8, B18, B27, B35, B44, or B51, said epitopes being the following: 
         (118)CPEEKQRHL(126) (residues 118 to 126 of SEQ ID NO: 2) binding stably to HLA molecules of the B8, B18, B35, B51 type, 
         (119)PEEKQRHL(126) (residues 119 to 126 of SEQ ID NO: 2) binding stably to HLA molecules of the B44 type, 
         (127)DKKQRFHNI(135) (residues 127 to 135 of SEQ ID NO: 2) binding stably to HLA molecules of the B8 type, 
         (128)KKQRFHNIR(136) (residues 128 to 136 of SEQ ID NO: 2) binding stably to HLA molecules of the B27 type, 
         (130)QRFHNIRGRW(139) (residues 130 to 139 of SEQ ID NO: 2) binding stably to HLA molecules of the B27 type, 
         (131)RFHNIRGRW(139) (residues 131 to 139 of SEQ ID NO: 2) binding stably to HLA molecules of the A24 type. 
       
     
     
         9 . Polyepitopic fragments of the E7 protein of HPV according to  claim 1 , characterized in that they comprise a peptide sequence of about 15 to 30 amino acids, this peptide sequence containing amino acid sequences of at least 3 different epitopes binding stably to HLA molecules of identical or different type, when these epitopes are obtained by enzymatic degradation of said peptide sequence, particularly in the proteasome, such that at least 4 HLA molecules of different types bind to these epitopes, these 4 HLA molecules being selected from those of type A1, A2, A3, A11, A29, E7, B18, B35, B44 and B62. 
     
     
         10 . Polyepitopic fragments of the E7 protein of HPV according to  claim 9 , characterized in that they all comprise an epitope binding to the HLA molecule of type B44. 
     
     
         11 . Polyepitopic fragment of the E7 protein of HPV according to  claim 9 , characterized by the following:
 (3)GDTPTLHEY(11) (residues 3 to 11 of SEQ ID NO: 12) binding stably to HLA molecules of the B44 type,   (5)TPTLHEYML(13) (residues 5 to 13 of SEQ ID NO: 12) binding stably to HLA molecules of the B35 type,   (11)YMLDLQPETT(20) (residues 11 to 20 of SEQ ID NO: 12) binding stably to HLA molecules of the A2 type,   (15) LQPETTDLY(23) (residues 15 to 23 of SEQ ID NO: 12) binding stably to HLA molecules of the B62 type,   (16)QPETTDLYCY(25) (residues 16 to 25 of SEQ ID NO: 12) binding stably to HLA molecules of the A1 or B18 type.   
     
     
         12 . Polyepitopic fragment of the E7 protein of HPV according to  claim 9 , characterized in that it corresponds to the fragment of 17 amino acids delimited by the amino acids located in positions 44 and 60 of the peptide sequence of the E7 protein of HPV, this latter fragment being characterized by the peptide sequence SEQ ID NO: 16 as follows: 
       
         
           
                 
                 
                 
               
                     
                   (44)QAEPDRAHYNIVTFCCK(60) 
                     
                 
             
                
               
            
           
         
         said fragment containing 4 epitopes binding stably to at least one of the 6 HLA molecules of the following types: A1, A3, A11, A29, B7, B18, B35, or B44, said epitopes being the following: 
         (44)QAEPDRAHY(52) (residues 44 to 52 of SEQ ID NO: 12) binding stably to HLA molecules of the A1 or B18 type, 
         (45)AEPDRAHY(52) (residues 45 to 52 of SEQ ID NO: 12) binding stably to HLA molecules of the A29 or B44 type, 
         (46)EPDRAHYNIV(55) (residues 46 to 55 of SEQ ID NO: 12) binding stably to HLA molecules of the B7 or B35 type, 
         (53)NIVTFCCK(60) (residues 53 to 60 of SEQ ID NO: 12) binding stably to HLA molecules of the A3 or A11 type. 
       
     
     
         13 . Polyepitopic fragment of the E7 protein of HPV according to  claim 9 , characterized in that it corresponds to the fragment of 19 amino acids delimited by the amino acids located in positions 79 and 97 of the peptide sequence of the E7 protein of HPV, this latter fragment being characterized by the peptide sequence SEQ ID NO: 18 as follows: 
       
         
           
                 
                 
                 
               
                     
                   (79)LEDLLMGTLGIVCPICSQK(97) 
                     
                 
             
                
               
            
           
         
         said fragment containing 4 epitopes binding stably to at least one of the 5 HLA molecules of the following types: A2, A3, A11, A29 or B44, said epitopes being the following: 
         (79)LEDLLMGTL(87) (residues 79 to 87 of SEQ ID NO: 12) binding stably to HLA molecules of the A29 or B44 type, 
         (82) LLMGTLGIV(90) (residues 82 to 90 of SEQ ID NO: 12) binding stably to HLA molecules of the A2 type, 
         (86)TLGIVCPI(93) (residues 86 to 93 of SEQ ID NO: 12) binding stably to HLA molecules of the A2 type, 
         (89) IVCPICSQK(97) (residues 89 to 97 of SEQ ID NO: 12) binding stably to HLA molecules of the A3 or A11 type. 
       
     
     
         14 . Polyepitopic fragments of the E6 or E7 protein, characterized in that they correspond to the peptide sequences derived from the polyepitopic fragments according to  claim 1 ,
 by substitution, and/or suppression, and/or addition of one or several amino acids, of the above-mentioned fragments, and/or   by modification of at least one —CO—NH— peptide linkage of the peptide chain of the above-mentioned fragments, particularly by introduction of a retro or retro-inverso type linkage, and/or   by substitution of at least one amino acid of the peptide chain of the sequence or of the above-mentioned fragment, with a non-proteinogenic amino acid,   said derived sequences containing peptides or pseudopeptides binding specifically to the same molecule or molecules of MCH as those binding to the peptides contained in the above-mentioned polyepitopic fragments from which they derive.   
     
     
         15 . Nucleotide sequences coding for a polyepitopic fragment or for a peptide sequence derived according to  claim 1 , said nucleotide sequences being derived from SEQ ID NO: 1 coding for the E6 protein, or from SEQ ID NO: 11 coding for the E7 protein. 
     
     
         16 . Nucleotide sequences according to  claim 15 , selected from the following:
 the sequence SEQ ID NO: 5, coding for the polyepitopic fragment SEQ ID NO: 6,   the sequence SEQ ID NO: 7, coding for the polyepitopic fragment SEQ ID NO: 8,   the sequence SEQ ID NO: 9, coding for the polyepitopic fragment SEQ ID NO: 10,   the sequence SEQ ID NO: 15, coding for the polyepitopic fragment SEQ ID NO: 16,   the sequence SEQ ID NO: 17, coding for the polyepitopic fragment SEQ ID NO: 18.   
     
     
         17 . Polyclonal or monoclonal antibodies, directed against a polyepitopic fragment or against a peptide sequence derived according to  claim 1 . 
     
     
         18 . Lipopeptide characterized in that said lipopeptide comprises:
 a peptide portion comprising one or several polyepitopic protein fragments, or a peptide sequence derived from said fragments, as defined in one of  claims 1 ,   and one or several lipophile portions, such as those comprising:
 a C4 to C20 hydrocarbon chain, saturated or unsaturated, linear or branched, 
 or a steroid group, as the case may be bonded to the above-mentioned hydrocarbon chain, 
   said lipophilic portions being if desired associated with a short peptide vector comprising one or several ionized functions at physiological pH, and a function permitting the covalent bonding of said hydrocarbon chain and/or said steroid group.   
     
     
         19 . A pharmaceutical composition, or vaccine, characterized in that it comprises:
 at least one polyepitopic fragment of the E6 or E7 according to  claim 1 ,   and/or at least one peptide sequence derived from said fragment,   and/or at least one suitable vector, containing said polyepitopic fragment of the E6 or E7 protein, and/or at least one of said sequence derived from these fragments,   in association with a physiologically acceptable vehicle,   said polyepitopic protein fragment and/or its derived sequence being, as the case may be, associated with one or several other exogenous epitopes recognized by auxiliary T cells, such as the peptide fragment delimited by the amino acids located in positions 830 and 846 of the peptide sequence of the tetanus toxin, hemagglutinin, or PADRE epitope.   
     
     
         20 . A pharmaceutical composition, or vaccine, characterized in that said composition comprises:
 at least one nucleotide sequence, coding for polyepitopic fragment of the E6 or E7 protein,   and/or at least one nucleotide sequence coding for a peptide sequence derived from said fragment,   and/or at least one suitable vector, in association with a physiologically acceptable vehicle.   
     
     
         21 . A pharmaceutical composition, or vaccine, characterized in that it comprises:
 antibodies according to  claim 17 , directed against a polyepitopic fragment of the E6 or E7 protein, and/or against a peptide sequence derived from these fragments, as defined above.   
     
     
         22 . Epitopes of the E6 protein of HPV selected from the following:
 (19)LPQLCTEL(26) (residues 19 to 26 of SEQ ID NO: 2) binding stably to HLA molecules of the B51 type,   (21)QLCTELQTTI(30) (residues 21 to 30 of SEQ ID NO: 2) binding stably to HLA molecules of the A2 type,   (24)TELQTTIHDI(33) (residues 24 to 33 of SEQ ID NO: 2) binding stably to HLA molecules of the A29 or B44 type,   (33)IILECVYCK(41) (residues 33 to 41 of SEQ ID NO: 2) binding stably to HLA molecules of the A11 type,   (35)LECVYCKQQL(44) (residues 35 to 44 of SEQ ID NO: 2) binding stably to HLA molecules of the A29 or B44 type,   (37)CVYCKQQL(44) (residues 37 to 44 of SEQ ID NO: 2) binding stably to HLA molecules of the B8 type,   (46)RREVYDFAFR(55) (residues 46 to 55 of SEQ ID NO: 2) binding stably to HLA molecules of the B27 type,   (49)VYDFAFRDL(57) (residues 49 to 57 of SEQ ID NO: 2) binding stably to HLA molecules of the A24 type,   (50)YDFAFRDL(57) (residues 50 to 57 of SEQ ID NO: 2) binding stably to HLA molecules of the A29, B44 type,   (52)FAFRDLCIV(60) (residues 52 to 60 of SEQ ID NO: 2) binding stably to HLA molecules of the A2, B35, B51 type,   (54)FRDLCIVYR(62) (residues 54 to 62 of SEQ ID NO: 2) binding stably to HLA molecules of the A3, A11 type,   (59)IVYRDGNPY(67) (residues 59 to 67 of SEQ ID NO: 2) binding stably to HLA molecules of the A3, A11 type,   (81)SEYRHYCY(88) (residues 81 to 88 of SEQ ID NO: 2) binding stably to HLA molecules of the A29, B44 type,   (87)CYSLYGTTL(95) (residues 87 to 95 of SEQ ID NO: 2) binding stably to HLA molecules of the A24 type,   (94)TLEQQYNK(101) (residues 94 to 101 of SEQ ID NO: 2) binding stably to HLA molecules of the A3, A11 type,   (95)LEQQYNKPL(103) (residues 95 to 103 of SEQ ID NO: 2) binding stably to HLA molecules of the A29, B44 type,   (101)KPLCDLLI(108) (residues 101 to 108 of SEQ ID NO: 2) binding stably to HLA molecules of the B7, B35, B51 type,   (118)CPEEKQRHL(126) (residues 118 to 126 of SEQ ID NO: 2) binding stably to HLA molecules of the B8, B18, B35, B51 type,   (119)PEEKQRHL(126) (residues 119 to 126 of SEQ ID NO: 2) binding stably to HLA molecules of the B44 type,   (127)DKKQRFHNI(135) (residues 127 to 135 of SEQ ID NO: 2) binding stably to HLA molecules of the B8 type,   (128)KKQRFHNIR(136) (residues 128 to 136 of SEQ ID NO: 2) binding stably to HLA molecules of the B27 type,   (130)QRFHNIRGRW(139) (residues 130 to 139 of SEQ ID NO: 2) binding stably to HLA molecules of the B27 type,   (131)RFHNIRGRW(139) (residues 131 to 139 of SEQ ID NO: 2) binding stably to HLA molecules of the A24 type.   
     
     
         23 . Epitopes of the E7 protein of HPV selected from the following:
 (3)GDTPTLHEY(11) (residues 3 to 11 of SEQ ID NO: 12) binding stably to HLA molecules of the B44 type,   (5)TPTLHEYML(13) (residues 5 to 13 of SEQ ID NO: 12) binding stably to HLA molecules of the B35 type,   (15)LQPETTDLY(23) (residues 15 to 23 of SEQ ID NO: 12) binding stably to HLA molecules of the B62 type,   (16)QPETTDLYCY(25) (residues 16 to 25 of SEQ ID NO: 12) binding stably to HLA molecules of the A1, B18 type,   (45)AEPDRAHY(52) (residues 45 to 52 of SEQ ID NO: 12) binding stably to HLA molecules of the A29, B44 type,   (46)EPDRAHYNIV(55) (residues 46 to 55 of SEQ ID NO: 12) binding stably to HLA molecules of the B7 or B35 type,   (53)NIVTFCCK(60) (residues 53 to 60 of SEQ ID NO: 12) binding stably to HLA molecules of the A3, A11 type,   (79)LEDLLMGTL(87) (residues 79 to 87 of SEQ ID NO: 12) binding stably to HLA molecules of the A29, B44 type,   (89)IVCPICSQK(97) (residues 89 to 97 of SEQ ID NO: 12) binding stably to HLA molecules of the A3, A11 type.