U.S. patent application number 11/859320 was filed with the patent office on 2008-10-09 for polyepitopic protein fragments of the e6 and e7 proteins of hpv, their production and their use particularly in vaccination.
This patent application is currently assigned to INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE INSERM. Invention is credited to Isabelle Bourgault Villada, Jeannine Choppin, Francine Connan, Estelle Ferries, Jean-Gerard Guillet.
Application Number | 20080248044 11/859320 |
Document ID | / |
Family ID | 9546327 |
Filed Date | 2008-10-09 |
United States Patent
Application |
20080248044 |
Kind Code |
A1 |
Choppin; Jeannine ; et
al. |
October 9, 2008 |
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.
Inventors: |
Choppin; Jeannine;
(Rosny-Sous-Bois, FR) ; Bourgault Villada; Isabelle;
(Paris, FR) ; Guillet; Jean-Gerard; (Paris,
FR) ; Connan; Francine; (Deuil La Barre, FR) ;
Ferries; Estelle; (Paris, FR) |
Correspondence
Address: |
YOUNG & THOMPSON
209 Madison Street, Suite 500
ALEXANDRIA
VA
22314
US
|
Assignee: |
INSTITUT NATIONAL DE LA SANTE ET DE
LA RECHERCHE MEDICALE INSERM
PARIS CEDEX 13
FR
PEPTIDE IMMUNE LIGANDS
LABEGE CEDEX
FR
|
Family ID: |
9546327 |
Appl. No.: |
11/859320 |
Filed: |
September 21, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10858384 |
Jun 2, 2004 |
7288258 |
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11859320 |
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09980523 |
Apr 29, 2002 |
6783763 |
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PCT/FR00/01513 |
May 31, 2000 |
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10858384 |
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Current U.S.
Class: |
424/139.1 ;
424/186.1; 514/1.1; 514/44R; 530/323; 530/324; 530/325; 530/326;
530/328; 530/387.9; 536/23.72 |
Current CPC
Class: |
A61P 37/02 20180101;
C07K 14/005 20130101; C12N 2710/20022 20130101; A61K 38/00
20130101; C12N 2740/16322 20130101; A61P 31/18 20180101; A61K 39/00
20130101 |
Class at
Publication: |
424/139.1 ;
530/326; 530/325; 530/324; 530/328; 536/23.72; 530/387.9; 530/323;
514/14; 514/13; 514/12; 424/186.1; 514/44 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 7/08 20060101 C07K007/08; C07K 14/00 20060101
C07K014/00; C07H 21/00 20060101 C07H021/00; C07K 16/00 20060101
C07K016/00; A61P 37/02 20060101 A61P037/02; A61K 38/10 20060101
A61K038/10; A61K 38/16 20060101 A61K038/16; A61K 39/12 20060101
A61K039/12; A61K 31/7052 20060101 A61K031/7052 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 3, 1999 |
FR |
99/07012 |
Claims
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: TABLE-US-00012
(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: TABLE-US-00013
(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: TABLE-US-00014 (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: TABLE-US-00015 (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.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of co-pending application
Ser. No. 10/858,384 filed on Jun. 2, 2004, which is a divisional
application U.S. application Ser. No. 09/980,523, filed on Apr. 29,
2002, now U.S. Pat. No. 6,783,763 which was a national phase of PCT
International Application No. PCT/FR00/01513 filed on May 31, 2000
under 35 U.S.C. .sctn. 371, the entire contents of each application
are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention has for its object polyepitopic
protein fragments, such as those of the E6 and E7 proteins of human
papillomavirus, or of the human p53 protein, their process of
production, and their uses, particularly in the field of
therapeutic or preventive vaccination.
SUMMARY OF THE INVENTION
[0003] The invention more particularly has for its object the use
of polyepitopic fragments of a predetermined protein for the
preparation of medications adapted for the prevention or treatment
of pathologies in which said protein is recognized by the cellular
immune system.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] FIG. 1 shows an E6 protein of HPV 16;
[0005] FIG. 2 shows an E7 protein of HPV 16.
DETAILED DESCRIPTION OF THE INVENTION
[0006] Preferably, said polyepitopic fragments are such that their
N-terminal amino acid corresponds to the N-terminal amino acid of
the epitope located upstream of one or several other epitopes of a
polyepitopic region of said protein, and their C-terminal amino
acid corresponds to the C-terminal amino acid of the epitope
located downstream of the above-mentioned epitope or epitopes of
said polyepitopic region.
[0007] Thus, the above-mentioned polyepitopic protein fragments of
the present invention correspond preferably to the polyepitopic
regions of a predetermined protein, namely to the regions
containing several epitopes recognized by the T cells in
association with the different molecules of the major complex of
histocompatibility (MCH), said regions being selected from those
having the characteristic of being degraded in vitro in shorter
peptides by proteasomes, such as the 20S proteasome, when the
protein fragment tested is placed in the presence of said
proteasome, particularly according to the following detailed
method. The protein fragment (about 75 .mu.g when it is a
polypeptide of about 30 amino acids) is incubated at 37.degree. C.
with about 15 .mu.g of 20S proteasome (Calbiochem Ref 539150, La
Jolla, Calif., USA) in 500 .mu.l of the following buffer: 20 mM
Tris-HCl pH8, 0.5 mM EDTA.
[0008] Aliquots of 50 .mu.l are removed after incubation times of
24 and 48 hours, and are analyzed by high pressure liquid
chromatography (HPLC). The digestion products of the proteasomes
are separated by RP-HPLC (Perkin Elmer) by using a C18 column and
an acetonitrile gradient (from 0 to 100% containing 0.1%
trifluoroacetic acid, for 90 minutes, elution rate 0.8 ml/min). The
cleavage products are detected at 214 nm by an absorption detector
(759A, Applied Biosystems).
[0009] Preferably, the polyepitopic regions defined above have the
characteristic of containing hydrophobic amino acids.
[0010] The different epitopes of the polyepitopic region of the
predetermined protein, and delimiting the epitopic protein
fragments, are preferably selected from the peptides; [0011]
binding to a predetermined molecule of MCH, particularly to a
molecule of the predetermined HLA type, and this from
concentrations of about 10.sup.-6 M to about 10.sup.-10 M in
peptide for concentrations of about 10.sup.-7 M of HLA molecules,
particularly under the conditions described hereafter, [0012] and
forming a stable complex with said MCH molecule, namely
particularly a complex in which said peptide remains bound to said
molecule for at least about 3 hours at 37.degree. C.
[0013] By way of illustration, the above-mentioned epitopes of the
invention are selected from among the peptides adapted: [0014] on
the one hand of associating with the molecules of MCH, particularly
by using the following method: [0015] incubation (particularly for
about 2 hours at 25.degree. C., then about 15 hours at 4.degree.
C.) of the peptide in the presence of MCH molecules, from the lysis
of human or animal cells, or purified particularly by affinity
chromatography from human or animal cell lines, [0016] trapping
complexes formed during the preceding step on a solid support
covered with a first antibody, particularly monoclonal, recognizing
specifically the CMH molecules in their configuration dependent on
their connection to said peptide, [0017] addition to the preceding
solid support of a second marked antibody, particularly by coupling
to a radioactive, enzymatic or fluorescent marker, said marked
antibody recognizing specifically either the long chains of MCH in
their configuration dependent on their connection to the peptide,
or the short chain of MCH or the .beta.2-microglobulin binding
specifically to the different long chains of the MCH in their
above-mentioned configuration, [0018] detection, after rinsing of
the solid support, of the possible presence of the second marked
antibody remaining fixed on the solid support, signifying an
association effect between the molecules of MCH and the peptide
studied, [0019] and, on the other hand, forming a complex with said
MCH molecules, whose stability can be evaluated by the use of a
method of following according to time the connection established
between the peptide and the MCH molecules, this method being
preferably carried out according to a protocol identical to the
preceding method, but in which the incubation step of the peptide
in the presence of MCH molecules on the solid support covered with
said first antibody, is preceded by a preliminary step of
eliminating the free peptide adapted that may be present in the
reaction medium, particularly by washing the solid support, said
incubation step being carried out (preferably at a temperature of
37.degree. C.) for variable times of 1 hours, 3 hours, 5 hours, 24
hours and 48 hours.
[0020] As mentioned above, the epitopes of the invention should be
recognized by the T cells in association with the MCH molecules and
associate with these latter, particularly in the framework of the
practice of the recognition test described above. This association
can be weak (detectable at concentrations of peptide analogs of the
order of 10.sup.-4 to 10.sup.-5 M), intermediate (detectable at
concentrations of peptide analogs of the order of 10.sup.-6 to
10.sup.-7 M), or strong (detectable at concentrations of peptide
analogs of the order of 10.sup.-8 to 10.sup.-9 M). The peptides
associated with the MCH molecules in the scope of the present
invention are preferably adapted to bond during at least about 3
hours, to said MCH molecules.
[0021] The invention more particularly has for its object the
epitopes (also designated peptides above and hereafter) as
described above and characterized in that they are selected from
among those adapted: [0022] to induce in vitro cytolysis by
cytotoxic T lymphocytes, of target cells having at their surface
the above-mentioned peptide associated with the MCH molecules, said
cytotoxic T lymphocytes being preferably removed from a patient
having a pathology in which the peptide studied is implied, [0023]
and inducing in vitro the secretion of cytokines (or interleukines)
by the above-mentioned cytotoxic T lymphocytes, particularly IL-2,
IL-4 or .gamma. interferon.
[0024] As the case may be, the above-mentioned epitopes are
selected from those able to induce in vitro the appearance and the
growth of cytotoxic T lymphocytes from animal or human cells,
particularly from peripheral blood mononucleated cells (PBMC), in
the presence of factors necessary for the growth and
differentiation of the cytotoxic T cells.
[0025] The polyepitopic protein fragments of the invention are
moreover characterized in that they are adapted to contain CD4
epitopes recognized by auxiliary T cells in association with the
MCH molecules of class II, this property favoring the induction and
maintenance of the CD8.sup.+ T cells recognizing the epitopes
comprised in said fragments.
[0026] The present invention is illustrated with the help of FIGS.
1 (SEQ ID NO: 2) and 2 (SEQ ID NO: 12), showing respectively
peptide sequences of the E6 (SEQ ID NO: 2) and E7 (SEQ ID NO: 12)
proteins of the strain 16 of the human papillomavirus (HPV 16), as
well as the polyepitopic fragments of the invention, and the
epitopes within these fragments.
[0027] The invention more particularly has for its object the
polyepitopic fragments of the E6 and E7 protein of HPV, and more
particularly those of the E6 protein shown in FIG. 1, or by SEQ ID
NO: 2, or those of the E7 protein, shown in FIG. 2, or by SEQ ID
NO: 11, of HPV 16, characterized in that they comprise a peptide
sequence of about 15 to about 30 amino acids, this peptide sequence
containing the amino acid sequences of at least 3 different
epitopes, and preferably at least 4 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, and preferably at least 5 HLA
molecules of different types, bind to these epitopes, these 4 or 5
HLA molecules being selected from those of type A1, A2, A3, A11,
A24, A29, B7, B8, B18, B27, B35, B44, B51, and B62.
[0028] Preferably, the polyepitopic fragments according to the
invention are such that the number of amino acids of their peptide
sequence is greater than or equal to 17, and less than or equal to
30.
[0029] The invention relates more particularly to the polyepitopic
fragments of the E6 protein of HPV defined above, 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, and preferably at least 6 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, and preferably at least 7
HLA molecules of different types, bind to these epitopes, these 6
or 7 HLA molecules being selected from those of type A1, A2, A3,
A11, A24, A29, B7, B8, B18, B27, B35, B44, and B51.
[0030] Preferably, the polyepitopic fragments of the E6 protein
according to the invention are such that the number of amino acids
of their peptide sequence is greater than or equal to 20
(preferably greater than or equal to 22), and less than or equal to
30.
[0031] Also preferably, the above-mentioned polyepitopic fragments
of the E6 protein of HPV, are 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.
[0032] The invention more particularly has for its object the
polyepitopic fragment of the E6 protein of HPV as defined above,
characterized in that it corresponds to the fragment of 30 amino
acids delimited by the amino acids located in positions 15 and 44
of the peptide sequence of the E6 protein of HPV, and characterized
by the peptide sequence SEQ ID NO: 4 as follows:
[0033] (15)RPRKLPQLCTELQTTIHDIILECVYCKQQL(44)
[0034] said fragment containing 9 epitopes binding stably to at
least one of the 8 HLA molecules of the following types: A2, A11,
A29, B7, B8, B35, B44, or B51, said epitopes being the following:
[0035] (15)RPRKLPQL(22) (residues 15 to 22 of SEQ ID NO: 2) binding
stably to HLA molecules of the B7 or B35 type, [0036]
(18)KLPQLCTEL(26) (residues 18 to 26 of SEQ ID NO: 2) binding
stably to HLA molecules of the A2 type, [0037] (19)LPQLCTEL(26)
(residues 19 to 26 of SEQ ID NO: 2) binding stably to HLA molecules
of the B51 type, [0038] (21)QLCTELQTTI(30) (residues 21 to 30 of
SEQ ID NO: 2) binding stably to HLA molecules of the A2 type,
[0039] (24)TELQTTIHDI(33) (residues 24 to 33 of SEQ ID NO: 2)
binding stably to HLA molecules of the A29 or B44 type, [0040]
(29)TIHDIILRCV(38) (residues 29 to 38 of SEQ ID NO: 2)binding
stably to HLA molecules of the A2 type, [0041] (33)IILECVYCK(41)
(residues 33 to 41 of SEQ ID NO: 2) binding stably to HLA molecules
of the A11 type, [0042] (35)LECVYCKQQL(44) (residues 35 to 44 of
SEQ ID NO: 2) binding stably to HLA molecules of the A29 or B44
type, [0043] (37)CVYCKQQL(44) (residues 37 to 44 of SEQ ID NO: 2)
binding stably to HLA molecules of the B8 type.
[0044] The invention also relates to the polyepitopic fragment of
the E6 protein of HPV as defined above, characterized in that it
corresponds to the fragment of 17 amino acids delimited by the
amino acids located at positions 46 and 62, or to the fragment of
22 amino acids delimited by the amino acids located at positions 46
and 67 of the peptide sequence of the E6 protein of HPV, this
latter fragment being characterized by the peptide sequence SEQ ID
NO: 6 as follows:
TABLE-US-00001 (46)RREVYDFAFRDLCIVYRDGNPY(67)
[0045] said fragment containing 6 epitopes binding stably to at
least one of the 10 HLA molecules of the following types: A2, A3,
A11, A24, A29, B7, B27, B35, B44, or B51, said epitopes being the
following: [0046] (46)RREVYDFAFR(55) (residues 46 to 55 of SEQ ID
NO: 2) binding stably to HLA molecules of the B27 type, [0047]
(49)VYDFAFRDL(57) (residues 49 to 57 of SEQ ID NO: 2) binding
stably to HLA molecules of the A24 type, [0048] (50)YDFAFRDL(57)
(residues 50 to 57 of SEQ ID NO: 2) binding stably to HLA molecules
of the A29 or B44 type, [0049] (52)FAFRDLCIV(60) (residues 52 to 60
of SEQ ID NO: 2) binding stably to HLA molecules of the A2, B35,
B51, or B7 type, [0050] (54)FRDLCIVYR(62) (residues 54 to 62 of SEQ
ID NO: 2) binding stably to HLA molecules of the A3 or A11 type,
[0051] (59)IVYRDGNPY(67) (residues 59 to 67 of SEQ ID NO: 2)
binding stably to HLA molecules of the A3 or A11 type.
[0052] The invention also has for its object the polyepitopic
fragment of the E6 protein of HPV as defined above, characterized
in that it corresponds to the fragment of 29 amino acids delimited
by the amino acids located at 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:
TABLE-US-00002 (8O)ISEYRHYCYSLYGTTLEQQYNKPLCDLLI(108)
[0053] said fragment containing 6 epitopes binding stably to at
least 10 HLA molecules of the following types: A1, A3, A11, A24,
A29, B7, B18, B35, B44, or B51, said epitopes being the following:
[0054] (80)ISEYRHYCY(88) (residues 80 to 88 of SEQ ID NO: 2)
binding stably to HLA molecules of the A1 or B18 type, [0055]
(81)SEYRHYCY(88) (residues 81 to 88 of SEQ ID NO: 2) binding stably
to HLA molecules of the A29 or B44 type, [0056] (87)CYSLYGTTL(95)
(residues 87 to 95 of SEQ ID NO: 2) binding stably to HLA molecules
of the A24 type, [0057] (94)TLEQQYNK101) (residues 94 to 101 of SEQ
ID NO: 2)binding stably to HLA molecules of the A3 or A11 type,
[0058] (95)LEQQYNKPL(103) (residues 95 to 103 of SEQ ID NO: 2)
binding stably to HLA molecules of the A29 or B44 type,
[0059] (101)KPLCDLLI(108) (residues 101 to 108 of SEQ ID NO: 2)
binding stably to HLA molecules of the B7, B35 or B51 type.
[0060] The invention more particularly has for its object the
polyepitopic fragment of the E6 protein of HPV as defined above,
characterized in that it corresponds to the fragment of 22 amino
acids delimited by the amino acids located at 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:
TABLE-US-00003 (118)CPEEKQRHLDKKQRFHNIRGRW(139)
[0061] 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:
[0062] (118)CPEEKQRHL(126) (residues 118 to 126 of SEQ ID NO: 2)
binding stably to HLA molecules of the B8, B18, B35, B51 type,
[0063] (119)PEEKQRHL(126) (residues 119 to 126 of SEQ ID NO: 2)
binding stably to HLA molecules of the B44 type, [0064]
(127)DKKQRFHNI(135) (residues 127 to 135 of SEQ ID NO: 2) binding
stably to HLA molecules of the B8 type, [0065] (128)KKQRFHNIR(136)
(residues 128 to 136 of SEQ ID NO: 2) binding stably to HLA
molecules of the B27 type, [0066] (130)QRFHNIRGRW(139) (residues
130 to 139 of SEQ ID NO: 2) binding stably to HLA molecules of the
B27 type, [0067] (131)RFHNIRGRW(139) (residues 131 to 139 of SEQ ID
NO: 2) binding stably to HLA molecules of the A24 type.
[0068] The invention also relates to the polyepitopic fragments of
the E7 protein of HPV as defined above, characterized in that they
comprise a peptide sequence of about 15 to 30 amino acids, this
peptide sequence containing the amino acid sequences of at least 3
different epitopes, and preferably of at least 4 different epitopes
binding stably to HLA molecules of the 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, and preferably at least 5
HLA molecules of different types bind to these epitopes, these 4 or
5 HLA molecules being selected from those of type A1, A2, A3, A11,
A29, B7, B18, B35, B44, and B62.
[0069] Preferably, the polyepitopic fragments of the E7 protein
according to the invention are such that the number of amino acids
of the peptide sequence is greater than or equal to 17, and less
than or equal to 23.
[0070] Again preferably, the polyepitopic fragments of the E7
protein of the above-mentioned HPV, are characterized in that they
all comprise an epitope binding to the HLA molecule of type
B44.
[0071] The invention more particularly has for its object the
polyepitopic fragment of the E7 protein of HPV as defined above,
characterized in that it corresponds to the fragment of 23 amino
acids delimited by the amino acids located in positions 3 and 25 of
the peptide sequence of the E7 protein of HPV, this latter fragment
being characterized by the peptide sequence SEQ ID NO: 14 as
follows:
TABLE-US-00004 (3)GDTPTLHEYMLDLQPETTDLYCY(25)
[0072] said fragment containing 5 epitopes binding stably to at
least one of the 6 HLA molecules of the following type: A1, A2,
B18, B35, B44 or B62, said epitopes being the following: [0073]
(3)GDTPTLHEY(11) (residues 3 to 11 of SEQ ID NO: 12) binding stably
to HLA molecules of the B44 type, [0074] (5)TPTLHEYML(13) (residues
5 to 13 of SEQ ID NO: 12) binding stably to HLA molecules of the
B35 type, [0075] (11)YMLDLQPETT(20) (residues 11 to 20 of SEQ ID
NO: 12) binding stably to HLA molecules of the A2 type, [0076]
(15)LQPETTDLY(23) (residues 15 to 23 of SEQ ID NO: 12) binding
stably to HLA molecules of the B62 type, [0077] (16)QPETTDLYCY(25)
(residues 16 to 25 of SEQ ID NO: 12) binding stably to HLA
molecules of the A1 or B18 type.
[0078] The invention also relates to the polyepitopic fragment of
the E7 protein of HPV as defined above, 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:
TABLE-US-00005 (44)QAEPDRAHYNIVTFCCK(60)
[0079] 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:
[0080] (44)QAEPDRAHY(52) (residues 44 to 52 of SEQ ID NO: 12)
binding stably to HLA molecules of the A1 or B18 type, [0081]
(45)AEPDRAHY(52) (residues 45 to 52 of SEQ ID NO: 12) binding
stably to HLA molecules of the A29 or B44 type, [0082]
(46)EPDRAHYNIV(55) (residues 46 to 55 of SEQ ID NO: 12) binding
stably to HLA molecules of the B7 or B35 type, [0083]
(53)NIVTFCCK(60) (residues 53 to 60 of SEQ ID NO: 12) binding
stably to HLA molecules of the A3 or A11 type.
[0084] The invention also has for its object the polyepitopic
fragment of the E7 protein of HPV as defined above, 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:
TABLE-US-00006 (79)LEDLLMGTLGIVCPICSQK(97)
[0085] 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: [0086]
(79)LEDLLMGTL(87) (residues 79 to 87 of SEQ ID NO: 12) binding
stably to HLA molecules of the A29 or B44 type, [0087]
(82)LLMGTLGIV(90) (residues 82 to 90 of SEQ ID NO: 12) binding
stably to HLA molecules of the A2 type, [0088] (86)TLGIVCPI(93)
(residues 86 to 93 of SEQ ID NO: 12) binding stably to HLA
molecules of the A2 type, [0089] (89)IVCPICSQK(97) (residues 89 to
97 of SEQ ID NO: 12) binding stably to HLA molecules of the A3 or
A11 type.
[0090] The invention also has for its object the polyepitopic
fragments of the p53 human protein characterized in that they
comprise a peptide sequence of about 20 to about 35 amino acids,
this latter containing amino acid sequences of at least three
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 3 HLA molecules of different types
will be recognized by said epitopes and will bind to these latter,
these 3 HLA molecules being selected from those of type A1, A2, A3,
A24, B7, B8, B27, B35, B44 and B62.
[0091] The invention also relates to the polyepitopic fragments of
the p53 human protein mentioned above, characterized in that they
comprise a peptide sequence of about 20 to about 35 amino acids,
this latter containing the amino acid sequences of at least 5
different epitopes, and preferably of at least 6 different epitopes
binding to HLA molecules of identical or different type, such that
at least 3 HLA molecules of different types, and preferably at
least 4 HLA molecules of different types will be recognized by said
epitopes and will bind to these latter, these 3 or 4 HLA molecules
being selected from those of type A2, A24, B27, B35, B44 and
B62.
[0092] The invention more particularly has for its object the
polyepitopic fragment of the p53 human protein as defined above,
characterized in that it corresponds to the fragment of 32 amino
acids delimited by the amino acids located in positions 106 and 137
of the peptide sequence of the p53 protein, or to the fragment of
36 amino acids delimited by the amino acids in positions 102 and
137 of said peptide sequence, this latter fragment being
characterized by the following peptide sequence:
TABLE-US-00007 (SEQ ID NO: 19)
(102)TYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQL(137)
[0093] said fragment containing 6 epitopes binding stably to at
least one of the 4 HLA molecules of the following types: A2, A24,
B35 or B62, said epitopes being the following: [0094]
(102)TYQGSYGFRL(111) (residues 1 to 10 of SEQ ID NO: 19) binding
stably to HLA molecules of the A24 type, [0095]
(105)GSYGFRLGFL(114) (residues 4 to 13 of SEQ ID NO: 19) binding
stably to HLA molecules of the B35 type, [0096] (106)SYGFRLGFL(114)
(residues 5 to 13 of SEQ ID NO: 19) binding stably to HLA molecules
of the A24 type, [0097] (118)TAKSVTCTY(126) (residues 17 to 25 of
SEQ ID NO: 19) binding stably to HLA molecules of the B62 type,
[0098] (125)TYSPALNKMF(134) (residues 24 to 33 of SEQ ID NO: 19)
binding stably to HLA molecules of the A24 type, [0099]
(129)ALNKMFCQL(137) (residues 28 to 36 of SEQ ID NO: 19) binding
stably to HLA molecules of the B35 type.
[0100] The invention also has for its object the polyepitopic
fragment of the p53 human protein as defined above, characterized
in that it corresponds to the fragment of 21 amino acids delimited
by the amino acids located in the positions 149 and 169 of the
peptide sequence of the p53 protein, and characterized by the
following peptide sequence:
TABLE-US-00008 (149)STPPPGTRVRAMAIYKQSQHM(169) (SEQ ID NO: 20)
[0101] said fragment containing 6 epitopes binding stably to at
least one of the 6 HLA molecules of the following types: A2, A3,
A24, B27, B35 or B62, said epitopes being the following: [0102]
(149)STPPPGTRV(157) (residues 1 to 9 of SEQ ID NO: 20)binding
stably to HLA molecules of the A2 type, [0103] (152)PPGTRVRAM(160)
(residues 4 to 12 of SEQ ID NO: 20) binding stably to HLA molecules
of the B35 type, [0104] (155)TRVRAMAIYK(164) (residues 7 to 16 of
SEQ ID NO: 20) binding stably to HLA molecules of the B27 type,
[0105] (156)RVRAMAIY(163) (residues 8 to 15 of SEQ ID NO: 20)
binding stably to HLA molecules of the B62 type, [0106]
(156)RVRAMAIYK(164) (residues 8 to 16 of SEQ ID NO: 20) binding
stably to HLA molecules of the A3 type, [0107] (162)IYKQSQHM(169)
(residues 14 to 21 of SEQ ID NO: 20) binding stably to HLA
molecules of the A24 type.
[0108] The invention also relates to the polyepitopic fragment of
the p53 human protein as defined above, characterized in that it
corresponds to the fragment of 26 amino acids delimited by the
amino acids located in positions 187 and 212 of the peptide
sequence of the p53 protein, or to the fragment of 34 amino acids
delimited by the amino acids located in positions 187 and 220 of
said peptide sequence, this latter fragment being characterized by
the following peptide sequence:
TABLE-US-00009 (SEQ ID NO: 21)
(187)GLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPY(220)
[0109] said fragment containing 11 epitopes binding stably to at
least one of the 7 HLA molecules of the following types: A1, A2,
A24, B7, B8, B27 or B44, said epitopes being the following: [0110]
(187)GLAPPQHLIRV(197) (residues 1 to 11 of SEQ ID NO: 21) binding
stably to HLA molecules of the A2 type, [0111] (189)APPQHLIRV(197)
(residues 3 to 11 of SEQ ID NO: 21) binding stably to HLA molecules
of the B7 type, [0112] (195)IRVEGNLRVEY(205) (residues 9 to 19 of
SEQ ID NO: 21) binding stably to HLA molecules of the B27 type,
[0113] (196)RVEGNLRVEY(205) (residues 10 to 19 of SEQ ID NO: 21)
binding stably to HLA molecules of the A2 type, [0114]
(197)VEGNLRVEY(205) (residues 11 to 19 of SEQ ID NO: 21) binding
stably to HLA molecules of the B44 type, [0115] (201)LRVEYLDDR(209)
(residues 15 to 23 of SEQ ID NO: 21) binding stably to HLA
molecules of the B27 type, [0116] (203)VEYLDDRNTF(212) (residues 17
to 26 of SEQ ID NO: 21) binding stably to HLA molecules of the B44
type, [0117] (204)EYLDDRNTF(212) (residues 18 to 26 of SEQ ID NO:
21) binding stably to HLA molecules of the A24 type, [0118]
(210)NTFRHSVVV(218) (residues 24 to 32 of SEQ ID NO: 21) binding
stably to HLA molecules of the B8 type, [0119] (21 1)TFRHSVVV(218)
(residues 25 to 32 of SEQ ID NO: 21) binding stably to HLA
molecules of the A24 type, [0120] (212)FRHSVVVPY(220) (residues 26
to 34 of SEQ ID NO: 21) binding stably to HLA molecules of the B27
type.
[0121] The invention also has for its object the polyepitopic
fragment of the p53 human protein as defined above, characterized
in that it corresponds to the fragment of 18 amino acids delimited
by the amino acids located in positions 226 and 243 of the peptide
sequence of the p53 protein, and characterized by the following
peptide sequence:
TABLE-US-00010 (226)GSDCTTIHYNYMCNSSCM(243) (SEQ ID NO: 22)
[0122] said fragment containing 3 epitopes binding stably to at
least one of the 3 HLA molecules of the following types: A1, A24 or
B44, said epitopes being the following: [0123] (226)GSDCTTIHY(234)
(residues 1 to 9 of SEQ ID NO: 22) binding stably to HLA molecules
of the A1 type, [0124] (227)SDCTTIHYNY(236) (residues 2 to 11 of
SEQ ID NO: 22) binding stably to HLA molecules of the B44 type,
[0125] (235)NYMCNSSCM(243) (residues 10 to 18 of SEQ ID NO: 22)
binding stably to HLA molecules of the A24 type.
[0126] The invention also relates to the polyepitopic fragment of
the p53 human protein as defined above, characterized in that it
corresponds to the fragment of 25 amino acids delimited by the
amino acids located in positions 249 and 273 of the peptide
sequence of the p53 protein, or to the fragment of 26 amino acids
delimited by the amino acids located in positions 248 and 273 of
said peptide sequence, or to the fragment of 33 amino acids
delimited by the amino acids located in positions 248 and 280 of
said peptide sequence, this latter fragment being characterized by
the following peptide sequence:
TABLE-US-00011 (SEQ ID NO: 23)
(248)RRPILTIITLEDSSGNLLGRNSFEVRVCACPGR(280)
[0127] said fragment containing 8 epitopes binding stably to at
least one of the 6 HLA molecules of the following types: A2, B7,
B27, B35, B44 or B62, said epitopes being the following: [0128]
(248)RRPILTIITL(257) (residues 1 to 10 of SEQ ID NO: 23) binding
stably to HLA molecules of the B27 type, [0129] (249)RPILTIITL(257)
(residues 2 to 10 of SEQ ID NO: 23) binding stably to HLA molecules
of the B35 and B7 type, [0130] (255)ITLEDSSGN(263) (residues 8 to
16 of SEQ ID NO: 23) binding stably to HLA molecules of the A2
type, [0131] (257)LEDSSGNLL(265) (residues 10 to 18 of SEQ ID NO:
23) binding stably to HLA molecules of the B44 type, [0132]
(263)NLLGRNSF(270) (residues 16 to 23 of SEQ ID NO: 23) binding
stably to HLA molecules of the B62 type, [0133] (264)LLGRNSFEV(272)
(residues 17 to 25 of SEQ ID NO: 23) binding stably to HLA
molecules of the A2 type, [0134] (266)GRNSFEVR(273) (residues 19 to
26 of SEQ ID NO: 23) binding stably to HLA molecules of the B27
type, [0135] (272)VRVCACPGR(280) (residues 25 to 33 of SEQ ID NO:
23) binding stably to HLA molecules of the B27 type.
[0136] The invention also relates to peptide sequences derived from
the polyepitopic fragments mentioned above, of the E6 or E7
proteins, or of the p53 protein, particularly: [0137] by
substitution and/or suppression and/or addition of one or several
amino acids, of the above-mentioned fragments, and/or [0138] by
modification of at least one peptide linkage --CO--NH-- of the
peptide chain of the above-mentioned fragments, particularly by
introduction of a retro or retro-inverso type linkage, and/or
[0139] 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,
[0140] 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 polyepitopic
fragments mentioned above from which they derive.
[0141] By derived sequence by introduction of a retro-inverso
linkage, should be understood any peptide analog of an
above-mentioned fragment, said analog being constituted by a
peptide chain in which at least one of the residues on the one hand
is bound to at least one adjacent residue by an --NH--CO-- linkage,
and on the other hand, is of a chirality opposite that of the same
amino acyl residue in the peptide chain of the parent peptide
(namely of the above-mentioned fragment from which it derives).
[0142] By a sequence derived by introduction of a retro linkage,
should be understood any peptide analog of an above-mentioned
fragment, said analog being constituted by a peptide chain in which
at least one of the residues is bound to at least one adjacent
residue by an --NH--CO-- linkage, the chirality of the whole of the
amino acyl residues involved in at least one --NH--CO-- linkage
being conserved relative to the corresponding residues of the
peptide chain of the parent peptide.
[0143] It follows that the --CO--NH-- and --NH--CO-- linkages must
be taken into account in the preceding, in the direction of the
parent peptide chain going from the amino terminal (N-terminal) end
toward the carboxy terminal (C-terminal) end.
[0144] By "proteinogenic amino acid", is meant, in the preceding,
any amino acid entering into the constitution of a natural protein
or peptide.
[0145] By "non-proteinogenic amino acid" is meant, in contrast to
the preceding definition, any amino acid that does not enter into
the constitution of a natural protein or peptide.
[0146] There is meant more particularly by "non-proteinogenic amino
acid", any amino acid whose carbon carrying a R side chain, namely
the --CHR-- group, located between --CO-- and --NH-- in the natural
peptide chain, is replaced by a structure that does not enter into
the constitution of a natural protein or peptide.
[0147] The invention more particularly has for its object the
derived sequences as described above, characterized in that at
least one of the peptide linkages --CO--NH-- of the peptide chain
of the parent peptide is replaced by a linkage different from the
--CO--NH-- linkage, said different linkage being particularly
selected from the following.
[0148] --CH.sub.2--NH-- (amino methylene);
[0149] --CH.sub.2--CH.sub.2-- (carba);
[0150] --CO--CH.sub.2-- (cetomethylene);
[0151] --CH.sub.2--O-- (methylene-oxy);
[0152] --CHOH--CH.sub.2-- (hydroxyethylene);
[0153] --CHOH--CHOH-- (di-hydroxyethylene);
[0154] --CH.dbd.CH-- (E or Z olefin);
[0155] --CHCN--NH-- (amino cyanomethylene);
[0156] --S--CH.sub.2-- (thiomethylene);
[0157] --CH.sub.2--S-- (thiomethylene);
[0158] --CS--NH-- (thioamide);
[0159] --PO.sub.2--NH-- (phosphonamide);
[0160] --CHOH-- (hydroxymethylene);
[0161] --NH--CO--NH-- (urea);
##STR00001##
[0162] (oxiran);
##STR00002##
[0163] (tetrazole);
[0164] --CH.sub.2--CO--NH-- (.beta.-homologation);
[0165] --CHOH--CH.sub.2--NH-- (amino hydroxyethylene);
[0166] --CO--NH--NH-- (hydrazino).
[0167] The invention also has for its object nucleotide sequences
coding for a polyepitopic fragment of the E6 or E7 protein, or for
a derived peptide sequence, as defined above, said nucleotide
sequences being derived from the sequence SEQ ID NO: 1 coding for
the E6 protein, or from the sequence SEQ ID NO: 11 coding for the
E7 protein.
[0168] In this connection, the invention more particularly has for
its object the nucleotide sequences defined above, selected from
the following: [0169] the sequence SEQ ID NO: 3, coding for the
polyepitopic fragment SEQ ID NO: 4 mentioned above, of the E6
protein, [0170] the sequence SEQ ID NO: 5, coding for the
polyepitopic fragment SEQ ID NO: 6 mentioned above, of the E6
protein, [0171] the sequence SEQ ID NO: 7, coding for the
polyepitopic fragment SEQ ID NO: 8 mentioned above, of the E6
protein, [0172] the sequence SEQ ID NO: 9, coding for the
polyepitopic fragment SEQ ID NO: 10 mentioned above, of the E6
protein, [0173] the sequence SEQ ID NO: 13, coding for the
polyepitopic fragment SEQ ID NO: 14 mentioned above, of the E7
protein, [0174] the sequence SEQ ID NO: 15, coding for the
polyepitopic fragment SEQ ID NO: 16 mentioned above, of the E7
protein, [0175] the sequence SEQ ID NO: 17, coding for the
polyepitopic fragment SEQ ID NO: 18 mentioned above, of the E7
protein.
[0176] The invention also has for its object the nucleotide
sequences coding for a polyepitopic fragment of the p53 protein, or
for a derived peptide sequence, as defined above.
[0177] The invention also has for its object any vector,
particularly a plasmid, cosmid or phage, containing at least one
above-mentioned nucleotide sequence under the control of elements
necessary for the transcription of said sequence, particularly
under the control of a transcription promoter and terminator.
[0178] The invention also relates to host cells, particularly
bacterial, virus, yeasts, eucaryotic cells, transformed with the
help of a vector mentioned above according to the invention, so as
to integrate stably into their genome or to maintain in a stable
manner in their cytoplasm, at least one nucleotide sequence
according to the invention.
[0179] The invention also relates to any vector comprising one or
several polyepitopic fragments and/or one or several derived
peptide sequences as defined above, or any vector comprising one or
several above-mentioned nucleotide sequences, said vectors being
selected from those adapted to ensure protection of said fragments
or nucleotide sequences in the organism and/or their penetration
into the cells of the organism.
[0180] In the case of the use of polyepitopic fragments and/or the
above-mentioned derived peptide sequences, such vectors are
selected from fatty acids (in the framework of the preparation of
lipopeptides), liposomes, etc.
[0181] In this connection, the invention more particularly has for
its object any lipopeptide characterized in that it comprises:
[0182] a peptide portion comprising one or several polyepitopic
protein fragments selected from those defined above, or any peptide
sequence derived from said fragments as defined above, [0183] and
one or several lipophilic portions, preferably selected from those
comprising: [0184] a C4 to C20 hydrocarbon chain, saturated or
unsaturated, linear or branched, [0185] or a steroid group, as the
case may be connected to the above-mentioned hydrocarbon chain,
[0186] said lipophilic portions being if desired associated with a
short peptide vector (thereby to form lipopeptide vector
structures) 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.
[0187] By lipophilic portion, in what precedes and what follows, is
intended any lipophilic molecule, insoluble in water, permitting,
when it is linked to the peptide portion defined above, an
intracellular passive passage of the obtained lipopeptide, thanks
to the hydrophobic properties of said molecule. Preferably the
lipopeptide resulting from the linking of the lipophile portion to
the peptide portion, is soluble in water.
[0188] Preferably, the hydrocarbon chain of the lipophilic
portions, is selected from the following: [0189] palmitic acid,
[0190] oleic acid, [0191] linoleic acid, [0192] linolenic acid.
[0193] Also preferably, the steroid group of the lipophilic portion
or portions is selected from cholesterol derivatives such as
cholest-5-enyl-3-oxy acetic acid, or cholest-5-enyl-3-oxycarbonic
acid.
[0194] The invention more particularly has for its object any
lipopeptide as described above, characterized in that the
lipophilic portion or portions are bonded covalently to one or
several amino acids of the peptide portion.
[0195] Preferably, the lypophilic portion or portions are bonded
covalently to the .alpha.NH.sub.2 or .epsilon.NH.sub.2 function of
a lysine located in the N terminal or C terminal position of the
peptide portion, or to the thiol function of a cystein, or to any
amino, alcohol or thiol function if desired added to this peptide
with a single spacer.
[0196] In this connection, the invention more particularly has for
its object any lipopeptide as defined above, in which the
lipophilic portion or portions are represented by a group
[0197] N.sup..alpha.-acetyl-Lysine N.sup..epsilon.(palmitoyl) (also
designated by the abbreviation Ac-K(Pam)).
[0198] The present invention also has for its object micelles or
microaggregates of one or several different lipopeptides defined
above.
[0199] Preferably, said micelles or microaggregates have a size
less than about 1 .mu.m.
[0200] Preferably, the micelles or microaggregates according to the
invention are as obtained by dispersion of said lipopeptides in a
concentrated acetic acid solution of about 80%, or any other
solvent capable of ensuring molecular dispersion of the
lipopeptides in solution.
[0201] In the case of the use of nucleotide sequences defined above
according to the invention, the above-mentioned vectors are
selected from the viruses, particularly the retroviruses, the
adenoviruses and the associated viruses (AAV Adeno Associated
Virus).
[0202] The invention also has for its object antibodies directed
against the polyepitopic protein fragments or the epitopes or their
derived peptide sequences (or analogs) as defined above, said
antibodies being those obtained by immunization of an animal with
at least one of the above-mentioned complexes, said antibodies
being adapted to form a complex with these polyepitopic fragments
or these epitopes or their analogs.
[0203] The antibodies according to the invention are polyclonal or
monoclonal antibodies.
[0204] The polyclonal antibodies mentioned above are obtained by
immunization of an animal with at least one polyepitopic protein
fragment or an epitope or an analog according to the invention,
followed by the recovery of the desired antibodies in purified
form, by removal of the serum of said animal, and separation of
said antibodies from the other constituents of the serum,
particularly by affinity chromatography on a column on which is
fixed a specific antigen recognized by the antibody, particularly a
polyepitopic protein fragment or an epitope or an analog according
to the invention.
[0205] The monoclonal antibodies according to the invention can be
obtained by the hybridome technique whose general principle is set
forth below.
[0206] In a first instance, an animal is immunized, generally a
mouse (or culture cells in an in vitro immunization framework) with
a polyepitopic protein fragment or an epitope or an analog
according to the invention, against which the B lymphocytes of the
animal are then capable of producing antibodies. These
antibody-producing lymphocytes are then fused with "immortal"
myelomatous cells (particularly of mice) to give rise to
hybridomes. From the heterogeneous mixture of cells thus obtained,
there is then carried out a selection of the cells capable of
producing a particular antibody and of multiplying indefinitely.
This hybridome is multiplied in the form of clones, each leading to
the production of a monoclonal antibody whose recognition
properties relative to the polyepitopic protein fragment or epitope
or the like of the invention, can be tested for example with ELISA,
by immunotransfer in one or two dimensions, by immunofluorescence,
or with the help of a biodetector. The monoclonal antibodies thus
selected are then purified particularly according to the affinity
chromatography technique described above.
[0207] The invention also relates to the use of one or several
above-mentioned antibodies for practicing a diagnostic method in
vitro of the above-mentioned pathologies.
[0208] In this connection, the invention also has for its object
sets or kits comprising said antibodies, for practicing a
diagnostic method as defined above.
[0209] The invention also relates to pharmaceutical compositions,
or vaccines, characterized in that they comprise: [0210] a) [0211]
at least one polyepitopic fragment of the E6 or E7 protein as
defined above, [0212] and/or at least one peptide sequence derived
from this fragment, as defined above, [0213] and/or at least one
suitable vector, particularly lipopeptides and/or micelles defined
above, containing at least one polyepitopic fragment mentioned
above of the E6 or E7 protein, and/or at least one sequence
mentioned above derived from these fragments,
[0214] in association with a physiologically acceptable
vehicle,
[0215] 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 (also
called CD4 or T helper epitopes), said epitopes being selected
particularly from the following: [0216] the peptide fragment
delimited by amino acids located in positions 830 and 846 of the
peptide sequence of the tetanus toxin, said fragment responding to
the following formula: QYIKANSKFIGITELKK(SEQ ID NO: 24), [0217]
hemagglutinin (Prevost-Blondel et al., 1995, J. Virol, 62, No. 12,
pp 8046-8055), [0218] PADRE epitope (Alexander et al., 1994,
Immunity, 1, 751). [0219] or b) [0220] at least one nucleotide
sequence as defined above, coding for an above-mentioned [0221]
polyepitopic fragment of the E6 or E7 protein, [0222] and/or at
least one nucleotide sequence coding for a peptide sequence derived
from this fragment, as defined above,
[0223] the above-mentioned nucleotide sequences being adapted to be
used alone, as minigenes, [0224] and/or at least one
above-mentioned suitable vector, selected particularly from the
viruses such as defined above, containing at least one
above-mentioned nucleotide sequence,
[0225] in association with a physiologically acceptable vehicle,
[0226] or c) [0227] antibodies defined above, directed against a
polyepitopic fragment of the E6 or E7 protein, and/or against a
peptide sequence derived from these fragments, as defined above, in
association with a physiologically acceptable vehicle.
[0228] Preferably, the pharmaceutical compositions or vaccines
mentioned above are present in a form administrable subcutaneously,
particularly in several injections (preferably 3 injections) of
about 500 .mu.g of the polyepitopic fragment in the lipopeptide
form, at about one month intervals.
[0229] The invention has more particularly for its object the use
of polyepitopic fragments of the E6 or E7 protein defined above, or
of the above-mentioned derived peptide sequences, or the
above-defined nucleotide sequences, or the above-mentioned
antibodies, or the lipopeptides defined above, for the preparation
of a medication or vaccine for the prevention or treatment of
pathologies connected with the infection of individuals by human
papillomavirus, such as cervical intraepithelial neoplasias (CIN),
the invasive cancer of the neck of the uterus, vulvar
intraepithelial neoplasias (VIN).
[0230] The invention also relates to pharmaceutical compositions or
vaccines characterized in that they comprise: [0231] a) [0232] at
least one polyepitopic fragment of the p53 protein as defined
above, [0233] and/or at least one peptide sequence derived from
this fragment, as defined above, [0234] and/or at least one
suitable vector, particularly the lipopeptides and/or micelles
defined above, containing at least one above-mentioned polyepitopic
fragment of the p53 protein, and/or at least one above-mentioned
sequence derived from these fragments,
[0235] in association with a physiologically acceptable
vehicle,
[0236] 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 the auxiliary T cells (also
called CD4 or T helper epitopes), said epitopes being selected
particularly from those defined above, [0237] or b) [0238] at least
one nucleotide sequence as defined above, coding for an
above-mentioned polyepitopic fragment of the p53 protein, [0239]
and/or at least one nucleotide sequence coding for a peptide
sequence derived from this fragment, as defined above,
[0240] the above-mentioned nucleotide sequences being adapted to be
used alone, as minigenes, [0241] and/or at least one
above-mentioned suitable vector, selected particularly from the
viruses as defined above, containing at least one above-mentioned
nucleotide sequence, in association with a physiologically
acceptable vehicle, [0242] or c) [0243] antibodies defined above,
directed against a polyepitopic fragment of the p53 protein, and/or
against a peptide sequence derived from these fragments, as defined
above, in association with a physiologically acceptable
vehicle.
[0244] The invention also has for its object the use: [0245] of at
least one polyepitopic fragment of the p53 protein, selected from
those defined above, [0246] and/or at least one peptide sequence
derived from this fragment, as defined above, [0247] or at least
one nucleotide sequence, as defined above, coding for a
polyepitopic fragment of the p53 protein, and/or for a peptide
sequence derived from this fragment, as defined above,
[0248] for the preparation of a medication or vaccine adapted for
the prevention or treatment of cancers, particularly of the breast,
of the colon, of the lung or of the bladder.
[0249] The invention also relates to peptides or epitopes of the E6
protein of HPV selected from the following: [0250] (19)LPQLCTEL(26)
(residues 19 to 26 of SEQ ID NO: 2) binding stably to HLA molecules
of the B51 type, [0251] (21)QLCTELQTTI(30) (residues 21 to 30 of
SEQ ID NO: 2) binding stably to HLA molecules of the A2 type,
[0252] (24)TELQTTIHDI(33) (residues 24 to 33 of SEQ ID NO: 2)
binding stably to HLA molecules of the A29 or B44 type, [0253]
(33)IILECVYCK(41) (residues 33 to 41 of SEQ ID NO: 2) binding
stably to HLA molecules of the A11 type, [0254] (35)LECVYCKQQL(44)
(residues 35 to 44 of SEQ ID NO: 2) binding stably to HLA molecules
of the A29 or B44 type, [0255] (37)CVYCKQQL(44) (residues 37 to 44
of SEQ ID NO: 2) binding stably to HLA molecules of the B8 type,
[0256] (46)RREVYDFAFR(55) (residues 46 to 55 of SEQ ID NO: 2)
binding stably to HLA molecules of the B27 type, [0257]
(49)VYDFAFRDL(57) (residues 49 to 57 of SEQ ID NO: 2) binding
stably to HLA molecules of the A24 type, [0258] (50)YDFAFRDL(57)
(residues 50 to 57 of SEQ ID NO: 2) binding stably to HLA molecules
of the A29, B44 type, [0259] (52)FAFRDLCIV(60) (residues 52 to 60
of SEQ ID NO: 2) binding stably to HLA molecules of the A2, B35,
B51 type, [0260] (54)FRDLCIVYR(62) (residues 54 to 62 of SEQ ID NO:
2) binding stably to HLA molecules of the A3, A11 type, [0261]
(59)IVYRDGNPY(67) (residues 59 to 67 of SEQ ID NO: 2) binding
stably to HLA molecules of the A3, A 11 type, [0262]
(81)SEYRHYCY(88) (residues 81 to 88 of SEQ ID NO: 2) binding stably
to HLA molecules of the A29, B44 type, [0263] (87)CYSLYGTTL(95)
(residues 87 to 95 of SEQ ID NO: 2) binding stably to HLA molecules
of the A24 type, [0264] (94)TLEQQYNK(101) (residues 94 to 101 of
SEQ ID NO: 2) binding stably to HLA molecules of the A3, A11 type,
[0265] (95)LEQQYNKPL(103) (residues 95 to 103 of SEQ ID NO: 2)
binding stably to HLA molecules of the A29, B44 type, [0266]
(101)KPLCDLLI(108) (residues 101 to 108 of SEQ ID NO: 2) binding
stably to HLA molecules of the B7, B35, B51 type, [0267]
(118)CPEEKQRHL(126) (residues 118 to 126 of SEQ ID NO: 2) binding
stably to HLA molecules of the B8, B18, B35, B51 type, [0268]
(119)PEEKQRHL(126) (residues 119 to 126 of SEQ ID NO: 2) binding
stably to HLA molecules of the B44 type, [0269] (127)DKKQRFHNI(135)
(residues 127 to 135 of SEQ ID NO: 2) binding stably to HLA
molecules of the B8 type, [0270] (128)KKQRFHNIR(136) (residues 128
to 136 of SEQ ID NO: 2) binding stably to HLA molecules of the B27
type, [0271] (130)QRFHNIRGRW(139) (residues 130 to 139 of SEQ ID
NO: 2) binding stably to HLA molecules of the B27 type, [0272]
(131)RFHNIRGRW(139) (residues 131 to 139 of SEQ ID NO: 2) binding
stably to HLA molecules of the A24 type.
[0273] The invention also relates to peptides or epitopes of the E7
protein of HPV selected from the following: [0274] (3)GDTPTLHEY(11)
(residues 3 to 11 of SEQ ID NO: 12) binding stably to HLA molecules
of the B44 type, [0275] (5)TPTLHEYML(13) (residues 5 to 13 of SEQ
ID NO: 12) binding stably to HLA molecules of the B35 type, [0276]
(15)LQPETTDLY(23) (residues 15 to 23 of SEQ ID NO: 12) binding
stably to HLA molecules of the B62 type, [0277] (16)QPETTDLYCY(25)
(residues 16 to 25 of SEQ ID NO: 12) binding stably to HLA
molecules of the A1, B18 type, [0278] (45)AEPDRAHY(52) (residues 45
to 52 of SEQ ID NO: 12) binding stably to HLA molecules of the A29,
B44 type, [0279] (46)EPDRAHYNIV(55) (residues 46 to 55 of SEQ ID
NO: 12) binding stably to HLA molecules of the B7 or B35 type,
[0280] (53)NIVTFCCK(60) (residues 53 to 60 of SEQ ID NO: 12)
binding stably to HLA molecules of the A3, A11 type, [0281]
(79)LEDLLMGTL(87) (residues 79 to 87 of SEQ ID NO: 12) binding
stably to HLA molecules of the A29, B44 type, [0282]
(89)IVCPICSQK(97) (residues 89 to 97 of SEQ ID NO: 12) binding
stably to HLA molecules of the A3, A11 type.
[0283] The invention also relates to peptide sequences derived from
the above-mentioned peptides, said derived sequences, or the like,
being as defined above in the framework of sequences derived from
the polyepitopic protein fragments mentioned above.
[0284] The invention also has for its object the nucleotide
sequences coding for peptides of the E6 or E7 proteins mentioned
above, namely, [0285] the sequence delimited by the nucleotides
located in positions 43 and 66 of the sequence SEQ ID NO: 1, coding
for (15)RPRKLPQL(22) (residues 15 to 22 of SEQ ID NO: 2), [0286]
the sequence delimited by the nucleotides located in positions 52
and 78 of the sequence SEQ ID NO: 1, coding for (18)KLPQLCTEL(26)
(residues 18 to 26 of SEQ ID NO: 2), [0287] the sequence delimited
by the nucleotides located in positions 55 and 78 of the sequence
SEQ ID NO: 1, coding for (19)LPQLCTEL(26) (residues 19 to 26 of SEQ
ID NO: 2), [0288] the sequence delimited by the nucleotides located
in positions 61 and 90 of the sequence SEQ ID NO: 1, coding for
(21)QLCTELQTTI(30) (residues 21 to 30 of SEQ ID NO: 2), [0289] the
sequence delimited by the nucleotides located in positions 70 and
99 of the sequence SEQ ID NO: 1, coding for (24)TELQTTIHD(33)
(residues 24 to 33 of SEQ ID NO: 2), [0290] the sequence delimited
by the nucleotides located in positions 97 and 123 of the sequence
SEQ ID NO: 1, coding for (33)IILECVYCK(41) (residues 33 to 41 of
SEQ ID NO: 2), [0291] the sequence delimited by the nucleotides
located in positions 103 and 132 of the sequence SEQ ID NO: 1,
coding for (35)LECVYCKQQL(44) (residues 35 to 44 of SEQ ID NO: 2),
[0292] the sequence delimited by the nucleotides located in
positions 109 and 132 of the sequence SEQ ID NO: 1, coding for
(37)CVYCKQQL(44) (residues 37 to 44 of SEQ ID NO: 2), [0293] the
sequence delimited by the nucleotides located in positions 136 and
165 of the sequence SEQ ID NO: 1, coding for (46)RREVYDFAFR(55)
(residues 46 to 55 of SEQ ID NO: 2), [0294] the sequence delimited
by the nucleotides located in positions 145 and 171 of the sequence
SEQ ID NO: 1, coding for (49)VFDFAFRDL(57) (residues 49 to 57 of
SEQ ID NO: 2), [0295] the sequence delimited by the nucleotides
located in positions 148 and 171 of the sequence SEQ ID NO: 1,
coding for (50)YDFAFRDL(57) (residues 50 to 57 of SEQ ID NO: 2),
[0296] the sequence delimited by the nucleotides located in
positions 154 and 180 of the sequence SEQ ID NO: 1, coding for
(52)FAFRDLCIV(60) (residues 52 to 60 of SEQ ID NO: 2), [0297] the
sequence delimited by the nucleotides located in positions 160 and
186 of the sequence SEQ ID NO: 1, coding for (54)FRDLCIVYR(62)
(residues 54 to 62 of SEQ ID NO: 2), [0298] the sequence delimited
by the nucleotides located in positions 175 and 201 of the sequence
SEQ ID NO: 1, coding for (59)IVYRDGNPY(67) (residues 59 to 67 of
SEQ ID NO: 2), [0299] the sequence delimited by the nucleotides
located in positions 241 and 264 of the sequence SEQ ID NO: 1,
coding for (81)SEYRHYCY(88) (residues 81 to 88 of SEQ ID NO: 2),
[0300] the sequence delimited by the nucleotides located in
positions 259 and 285 of the sequence SEQ ID NO: 1, coding for
(87)CYRLYGTTL(95) (residues 87 to 95 of SEQ ID NO: 2), [0301] the
sequence delimited by the nucleotides located in positions 280 and
303 of the sequence SEQ ID NO: 1, coding for (94)TLEQQYNK( 101)
(residues 94 to 101 of SEQ ID NO: 2), [0302] the sequence delimited
by the nucleotides located in positions 283 and 309 of the sequence
SEQ ID NO: 1, coding for (95)LEQQYNKPL(103) (residues 95 to 103 of
SEQ ID NO: 2), [0303] the sequence delimited by the nucleotides
located in positions 301 and 324 of the sequence SEQ ID NO: 1,
coding for (101)KPLCDLLI(108) (residues 101 to 108 of SEQ ID NO:
2), [0304] the sequence delimited by the nucleotides located in
positions 352 and 378 of the sequence SEQ ID NO: 1, coding for
(118)CPEEKQRHL(126) (residues 118 to 126 of SEQ ID NO: 2), [0305]
the sequence delimited by the nucleotides located in positions 355
and 378 of the sequence SEQ ID NO: 1, coding for ( 19)PEEKQRHL(126)
(residues 119 to 126 of SEQ ID NO: 2), [0306] the sequence
delimited by the nucleotides located in positions 379 and 405 of
the sequence SEQ ID NO: 1, coding for (127)DKKQRFHNI(135) (residues
127 to 135 of SEQ ID NO: 2), [0307] the sequence delimited by the
nucleotides located in positions 382 and 408 of the sequence SEQ ID
NO: 1, coding for (128)KKQRFHNIR(136) (residues 128 to 136 of SEQ
ID NO: 2), [0308] the sequence delimited by the nucleotides located
in positions 388 and 417 of the sequence SEQ ID NO: 1, coding for
(130)QRFHNIRGRW(139) (residues 130 to 139 of SEQ ID NO: 2), [0309]
the sequence delimited by the nucleotides located in positions 391
and 417 of the sequence SEQ ID NO: 1, coding for
(131)RFHNIRGRW(139) (residues 131 to 139 of SEQ ID NO: 2), [0310]
the sequence delimited by the nucleotides located in positions 7
and 33 of the sequence SEQ ID NO: 11, coding for (3)GDTPTLHEY(11)
(residues 3 to 11 of SEQ ID NO: 12), [0311] the sequence delimited
by the nucleotides located in positions 13 and 39 of the sequence
SEQ ID NO: 11, coding for (5)TPTLHEYML(13) (residues 5 to 13 of SEQ
ID NO: 12), [0312] the sequence delimited by the nucleotides
located in positions 43 and 69 of the sequence SEQ ID NO: 11,
coding for (15)LQPETTDLY(23) (residues 15 to 23 of SEQ ID NO: 12),
[0313] the sequence delimited by the nucleotides located in
positions 46 and 75 of the sequence SEQ ID NO: 11, coding for
(16)QPETTDLYCY(25) (residues 16 to 25 of SEQ ID NO: 12), [0314] the
sequence delimited by the nucleotides located in positions 133 and
153 of the sequence SEQ ID NO: 11, coding for (45)AEPDRAHY(52)
(residues 45 to 52 of SEQ ID NO: 12), [0315] the sequence delimited
by the nucleotides located in positions 136 and 165 of the sequence
SEQ ID NO: 11, coding for (46)EPDRAHYNIV(55) (residues 46 to 55 of
SEQ ID NO: 12), [0316] the sequence delimited by the nucleotides
located in positions 157 and 180 of the sequence SEQ ID NO: 11,
coding for (53)NIVTFCCK(60) (residues 53 to 60 of SEQ ID NO: 12),
[0317] the sequence delimited by the nucleotides located in
positions 235 and 261 of the sequence SEQ ID NO: 11, coding for
(79)LEDLLMGTL(87) (residues 79 to 87 of SEQ ID NO: 12), [0318] the
sequence delimited by the nucleotides located in positions 265 and
291 of the sequence SEQ ID NO: 11, coding for (89)IVCPICSQK(97)
(residues 89 to 97 of SEQ ID NO: 12).
[0319] The invention also has for its object epitopes of the p53
protein selected from the following: [0320] (102)TYQGSYGFRL(111)
(residues 1 to 10 of SEQ ID NO: 19) binding stably to HLA molecules
of the A24 type, [0321] (105)GSYGFRLGFL(114) (residues 4 to 13 of
SEQ ID NO: 19) binding stably to HLA molecules of the B35 type,
[0322] (106)SYGFRLGFL(114) (residues 5 to 13 of SEQ ID NO: 19)
binding stably to HLA molecules of the A24 type, [0323]
(118)TAKSVTCTY(126) (residues 17 to 25 of SEQ ID NO: 19) binding
stably to HLA molecules of the B62 type, [0324]
(125)TYSPALNKMF(134) (residues 24 to 33 of SEQ ID NO: 19) binding
stably to HLA molecules of the A24 type, [0325] (152)PPGTRVRAM(160)
(residues 4 to 12 of SEQ ID NO: 20) binding stably to HLA molecules
of the B35 type, [0326] (155)TRVRAMAIYK(164) (residues 7 to 16 of
SEQ ID NO: 20) binding stably to HLA molecules of the B27 type,
[0327] (156)RVRAMAIY(163) (residues 8 to 15 of SEQ ID NO: 20)
binding stably to HLA molecules of the B62 type, [0328]
(162)IYKQSQHM(169) (residues 14 to 21 of SEQ ID NO: 20) binding
stably to HLA molecules of the A24 type, [0329]
(195)IRVEGNLRVEY(205) (residues 9 to 19 of SEQ ID NO: 21) binding
stably to HLA molecules of the B27 type, [0330] (197)VEGNLRVEY(205)
(residues 11 to 19 of SEQ ID NO: 21) binding stably to HLA
molecules of the B44 type, [0331] (201)LRVEYLDDR(209) (residues 15
to 23 of SEQ ID NO: 21) binding stably to HLA molecules of the B27
type, [0332] (203)VEYLDDRNTF(212) (residues 17 to 26 of SEQ ID NO:
21) binding stably to HLA molecules of the B44 type, [0333]
(204)EYLDDRNTF(212) (residues 18 to 26 of SEQ ID NO: 21) binding
stably to HLA molecules of the A24 type, [0334] (211)TFRHSVVV(218)
(residues 25 to 32 of SEQ ID NO: 21) binding stably to HLA
molecules of the A24 type, [0335] (212)FRHSVVVPY(220) (residues 26
to 34 of SEQ ID NO: 21) binding stably to HLA molecules of the B27
type, [0336] (227)SDCTTIHYNY(236) (residues 2 to 11 of SEQ ID NO:
22) binding stably to HLA molecules of the B44 type, [0337]
(235)NYMCNSSCM(243) (residues 10 to 18 of SEQ ID NO: 22) binding
stably to HLA molecules of the A24 type, [0338] (249)RPILTIITL(257)
(residues 2 to 10 of SEQ ID NO: 23) binding stably to HLA molecules
of the B35 type, [0339] (257)LEDSSGNLL(265) (residues 10 to 18 of
SEQ ID NO: 23) binding stably to HLA molecules of the B44 type,
[0340] (263)NLLGRNSF(270) (residues 16 to 23 of SEQ ID NO: 23)
binding stably to HLA molecules of the B62 type, [0341]
(266)GRNSFEVR(273) (residues 19 to 26 of SEQ ID NO: 23) binding
stably to HLA molecules of the B27 type, [0342] (272)VRVCACPGR(280)
(residues 25 to 33 of SEQ ID NO: 23) binding stably to HLA
molecules of the B27 type.
[0343] The invention also has for its object any process for the
preparation of polyepitopic fragments, of single epitopes
(above-mentioned peptides), or of derived sequences, by
conventional peptide synthesis in liquid or solid phase.
[0344] As a modification, the polyepitopic fragments, single
epitopes or derived peptide sequences, as defined above according
to the invention, can be obtained in the form of recombinant
polypeptides by transformation of suitable host cells as defined
above with the help of vectors containing a recombinant nucleotide
sequence as defined above according to the invention, and the
recovery, as the case may be after purification, of the recombinant
polypeptide coded by said nucleotide sequence and produced by the
host cells mentioned above.
Sequence CWU 1
1
241477DNAHuman PapillomavirusCDS(1)..(477) 1atg cac caa aag aga act
gca atg ttt cag gac cca cag gag cga ccc 48Met His Gln Lys Arg Thr
Ala Met Phe Gln Asp Pro Gln Glu Arg Pro1 5 10 15aga aag tta cca cag
tta tgc aca gag ctg caa aca act ata cat gat 96Arg Lys Leu Pro Gln
Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp 20 25 30ata ata tta gaa
tgt gtg tac tgc aag caa cag tta ctg cga cgt gag 144Ile Ile Leu Glu
Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu 35 40 45gta tat gac
ttt gct ttt cgg gat tta tgc ata gta tat aga gat ggg 192Val Tyr Asp
Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly 50 55 60aat cca
tat gct gta tgt gat aaa tgt tta aag ttt tat tct aaa att 240Asn Pro
Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile65 70 75
80agt gag tat aga cat tat tgt tat agt ttg tat gga aca aca tta gaa
288Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu
85 90 95cag caa tac aac aaa ccg ttg tgt gat ttg tta att agg tgt att
aac 336Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile
Asn 100 105 110tgt caa aag cca ctg tgt cct gaa gaa aag caa aga cat
ctg gac aaa 384Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg His
Leu Asp Lys 115 120 125aag caa aga ttc cat aat ata agg ggt cgg tgg
acc ggt cga tgt atg 432Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp
Thr Gly Arg Cys Met 130 135 140tct tgt tgc aga tca tca aga aca cgt
aga gaa acc cag ctg tga 477Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg
Glu Thr Gln Leu145 150 1552158PRTHuman Papillomavirus 2Met His Gln
Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro1 5 10 15Arg Lys
Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp 20 25 30Ile
Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu 35 40
45Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly
50 55 60Asn Pro Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys
Ile65 70 75 80Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr
Thr Leu Glu 85 90 95Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile
Arg Cys Ile Asn 100 105 110Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys
Gln Arg His Leu Asp Lys 115 120 125Lys Gln Arg Phe His Asn Ile Arg
Gly Arg Trp Thr Gly Arg Cys Met 130 135 140Ser Cys Cys Arg Ser Ser
Arg Thr Arg Arg Glu Thr Gln Leu145 150 155390DNAArtificial
SequenceDescription of the Artificial Sequence fragment of the
sequence coding for E6 of HPV and corresponding peptide sequence
3cga ccc aga aag tta cca cag tta tgc aca gag ctg caa aca act ata
48Arg Pro Arg Lys Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile1
5 10 15cat gat ata ata tta gaa tgt gtg tac tgc aag caa cag tta
90His Asp Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu 20 25
30430PRTArtificial SequenceDescription of the Artificial Sequence
Peptide fragment for E6 of HPV 4Arg Pro Arg Lys Leu Pro Gln Leu Cys
Thr Glu Leu Gln Thr Thr Ile1 5 10 15His Asp Ile Ile Leu Glu Cys Val
Tyr Cys Lys Gln Gln Leu 20 25 30566DNAArtificial
SequenceDescription of the Artificial Sequence fragment of the
sequence coding for E6 of HPV and corresponding peptide sequence
5cga cgt gag gta tat gac ttt gct ttt cgg gat tta tgc ata gta tat
48Arg Arg Glu Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr1
5 10 15aga gat ggg aat cca tat 66Arg Asp Gly Asn Pro Tyr
20622PRTArtificial SequenceDescription of the Artificial Sequence
Peptide fragment for E6 of HPV 6Arg Arg Glu Val Tyr Asp Phe Ala Phe
Arg Asp Leu Cys Ile Val Tyr1 5 10 15Arg Asp Gly Asn Pro Tyr
20787DNAArtificial SequenceDescription of the Artificial Sequence
fragment of the sequence coding for E6 of HPV and corresponding
peptide sequence 7att agt gag tat aga cat tat tgt tat agt ttg tat
gga aca aca tta 48Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr
Gly Thr Thr Leu1 5 10 15gaa cag caa tac aac aaa ccg ttg tgt gat ttg
tta att 87Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile 20
25829PRTArtificial SequenceDescription of the Artificial Sequence
Peptide fragment for E6 of HPV 8Ile Ser Glu Tyr Arg His Tyr Cys Tyr
Ser Leu Tyr Gly Thr Thr Leu1 5 10 15Glu Gln Gln Tyr Asn Lys Pro Leu
Cys Asp Leu Leu Ile 20 25966DNAArtificial SequenceDescription of
the Artificial Sequence fragment of the sequence coding for E6 of
HPV and corresponding peptide sequence 9tgt cct gaa gaa aag caa aga
cat ctg gac aaa aag caa aga ttc cat 48Cys Pro Glu Glu Lys Gln Arg
His Leu Asp Lys Lys Gln Arg Phe His1 5 10 15aat ata agg ggt cgg tgg
66Asn Ile Arg Gly Arg Trp 201022PRTArtificial SequenceDescription
of the Artificial Sequence Peptide fragment for E6 of HPV 10Cys Pro
Glu Glu Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe His1 5 10 15Asn
Ile Arg Gly Arg Trp 2011297DNAHuman PapillomavirusCDS(1)..(297)
11atg cat gga gat aca cct aca ttg cat gaa tat atg tta gat ttg caa
48Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln1
5 10 15cca gag aca act gat ctc tac tgt tat gag caa tta aat gac agc
tca 96Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser
Ser 20 25 30gag gag gag gat gaa ata gat ggt cca gct gga caa gca gaa
ccg gac 144Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu
Pro Asp 35 40 45aga gcc cat tac aat att gta acc ttt tgt tgc aag tgt
gac tct acg 192Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys
Asp Ser Thr 50 55 60ctt cgg ttg tgc gta caa agc aca cac gta gac att
cgt act ttg gaa 240Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile
Arg Thr Leu Glu65 70 75 80gac ctg tta atg ggc aca cta gga att gtg
tgc ccc atc tgt tct cag 288Asp Leu Leu Met Gly Thr Leu Gly Ile Val
Cys Pro Ile Cys Ser Gln 85 90 95aaa cca taa 297Lys Pro1298PRTHuman
Papillomavirus 12Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met
Leu Asp Leu Gln1 5 10 15Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln
Leu Asn Asp Ser Ser 20 25 30Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala
Gly Gln Ala Glu Pro Asp 35 40 45Arg Ala His Tyr Asn Ile Val Thr Phe
Cys Cys Lys Cys Asp Ser Thr 50 55 60Leu Arg Leu Cys Val Gln Ser Thr
His Val Asp Ile Arg Thr Leu Glu65 70 75 80Asp Leu Leu Met Gly Thr
Leu Gly Ile Val Cys Pro Ile Cys Ser Gln 85 90 95Lys
Pro1369DNAArtificial SequenceDescription of the Artificial Sequence
fragment of the sequence coding for E7 of HPV and corresponding
peptide sequence 13gga gat aca cct aca ttg cat gaa tat atg tta gat
ttg caa cca gag 48Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp
Leu Gln Pro Glu1 5 10 15aca act gat ctc tac tgt tat 69Thr Thr Asp
Leu Tyr Cys Tyr 201423PRTArtificial SequenceDescription of the
Artificial Sequence Peptide fragment for E7 of HPV 14Gly Asp Thr
Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln Pro Glu1 5 10 15Thr Thr
Asp Leu Tyr Cys Tyr 201551DNAArtificial SequenceDescription of the
Artificial Sequence fragment of the sequence coding for E7 of HPV
and corresponding peptide sequence 15caa gca gaa ccg gac aga gcc
cat tac aat att gta acc ttt tgt tgc 48Gln Ala Glu Pro Asp Arg Ala
His Tyr Asn Ile Val Thr Phe Cys Cys1 5 10 15aag
51Lys1617PRTArtificial SequenceDescription of the Artificial
Sequence Peptide fragment for E7 of HPV 16Gln Ala Glu Pro Asp Arg
Ala His Tyr Asn Ile Val Thr Phe Cys Cys1 5 10
15Lys1757DNAArtificial SequenceDescription of the Artificial
Sequence fragment of the sequence coding for E7 of HPV and
corresponding peptide sequence 17ttg gaa gac ctg tta atg ggc aca
cta gga att gtg tgc ccc atc tgt 48Leu Glu Asp Leu Leu Met Gly Thr
Leu Gly Ile Val Cys Pro Ile Cys1 5 10 15tct cag aaa 57Ser Gln
Lys1819PRTArtificial SequenceDescription of the Artificial Sequence
Peptide fragment for E7 of HPV 18Leu Glu Asp Leu Leu Met Gly Thr
Leu Gly Ile Val Cys Pro Ile Cys1 5 10 15Ser Gln Lys1936PRTHomo
sapiens 19Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly Phe Leu His
Ser Gly1 5 10 15Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu
Asn Lys Met 20 25 30Phe Cys Gln Leu 352021PRTHomo sapiens 20Ser Thr
Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys1 5 10 15Gln
Ser Gln His Met 202134PRTHomo sapiens 21Gly Leu Ala Pro Pro Gln His
Leu Ile Arg Val Glu Gly Asn Leu Arg1 5 10 15Val Glu Tyr Leu Asp Asp
Arg Asn Thr Phe Arg His Ser Val Val Val 20 25 30Pro Tyr2218PRTHomo
sapiens 22Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn
Ser Ser1 5 10 15Cys Met2333PRTHomo sapiens 23Arg Arg Pro Ile Leu
Thr Ile Ile Thr Leu Glu Asp Ser Ser Gly Asn1 5 10 15Leu Leu Gly Arg
Asn Ser Phe Glu Val Arg Val Cys Ala Cys Pro Gly 20 25
30Arg2417PRTClostridium tetani 24Gln Tyr Ile Lys Ala Asn Ser Lys
Phe Ile Gly Ile Thr Glu Leu Lys1 5 10 15Lys
* * * * *