Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
2057828
Field of the Invention
The subject of the present inventiori is novel
cytotoxic T lymphocyte-inducing lipopeptides.
The subject is furthermore the use of such
lipopeptides as vaccines.
Description of the Prior Art
Most vaccines used induce a response through
antibodies. However, it has been shown that cytotoxic
lymphocytes can effectively protect mice against
various pathogenic microorganisms (Skehel et al., Proc.
Natl. Acad. Sci. USA. 1982, 79; 968; Lukacher et al.,
Exp. Med. 1984, 160: 814). This has also been shown for
human cytotoxic T lymphocytes raised against cyto-
megaloviruses (Quinnan et al., N. Engl. J. Med. 1982,
307: 7; Rook et al., Am. J. Med. 1984, 76: 385). How-
ever, little is known about inducing immunity due to
lymphocytes.
Some authors have tried to induce cytotoxic T
lymphocytes (CTL) in vivo using peptides derived from
ovalbumin (Carbone et al. J. Exp. Med. 169: 603,
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2~5~8~8
Ishioka et al. 1989, J. Immunol. 143:1094). These
authors obtained immunizations, but these results are
specific for peptides derived from ovalbumin.
AICHELE et al. ((1990) J. Exp. Med. 171:1815)
succeeded, for their part, in inducing a cytolytic T
response by repeated injections in vivo of a synthetic
peptide in emulsion in incomplete Freund's adjuvant.
These authors do not indicate the importance of the
adjuvant under their immunization conditions. However,
they suggest that an adjuvant is necessary for
obtaining such a response.
The application EP-203,676 relates to-a vaccine
intended to induce a T cell mediated response
comprising some peptide-fatty acid conjugates . The
fatty acid which is used is the palmitic acid
However, this vaccine comprises also Freund's adjuvant.
To the knowledge of the applicant, only DERES
et al. (Nature, Volume 342, 30 November 1989) have des-
cribed the use of a synthetic lipopeptide to induce
cytotoxic T lymphocytes (CTL) in vivo. In this article,
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the NP 147-158 fragment of a nucleoprotein of the
influenza virus is coupled to tripalmitoyl-S-glyceryl-
cysteinylserylserine (P3CSS). It is shown that the
NP 147-158 peptide-P3CSS lipid conjugate induces a CTL
response against target cells infected by the influenza
virus, whereas mice immunized with the NP 147-158
peptide alone or with the Ser Ser-NP 147-158 peptide do
not generate cytotoxic T lymphocytes against this
virus.
It should also be noted that lipopeptides have
already been used to induce immunological reactions to
specific antigens, but the responses generated implied
the synthesis of antibodies and not T lymphocyte
responses. HOPP (Molecular Immunology, 21, 13-16,
1984) has shown that antibodies raised against a
determinant of the hepatitis B virus could be obtained
by immunizing rabbits using a molecule consisting of a
peptide of 15 amino acids corresponding to the
antigenic determinant of the hepatitis B virus and of a
pseudolipide residue, dipalmitoyl lysine.
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2957328
The application EP-93 851 describes some
lipopeptides comprising a peptidic sequence of 6 to 15
aminoacids bound to a lipophylic part . This lipophylic
part can be a fatty acid such as palmitic , stearic ,
behenic , oleic or mycolic acids It is mentioned that
these lipopeptides induce the antibodies synthesis .
The publication of WiesmUller et al( Vaccine,
volume 7, n l, 29-33, 1989 ) describes the use of a
lipopeptide comprising a part of the sequence of the
virus FDMV (VP1) and the lipid P3CSS to induce the
synthesis of antibodies.
The abstract of the publication of Jacob et
al; (Chemical Abstracts, vol.104, n 21, 472, abstract
184.455 x , 1986) relates to the induction of the
antibodies synthesis by a lipopeptide comprising the
tetanus toxine bound to a dipalmitoyl rest.
The abstract of the publication of Watari et
al (Chemical Abstracts, vol.106, p 516 , abstract n
154 381 u , 1987) relates to the use of some peptides
corresponding to the N-terminal region of the
glycoprotein D of the virus HSV coupled to the palmitic
acid . It is clearly indicated that there is induction
of a T cells mediated response but that this response
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is not due to cytotoxic T lymphocytes.
Two other references relate to the synthesis
and the structural study of lipopeptides
The International Application WO 89 10348
relates to some fatty acids derivated lipopeptides ,
such as the aminoeicosanic , aminodecanoic ,
aminotetradecanoic , bromodecanoic and bromododecanoic
acids .
It is mentioned that these compounds can be
used as adjuvants and as carriers for vaccines , but
without providing any mean to use these compounds
The abstract of the publication of Mercy et
al. (Chemical Abstracts, vol.106, N 25, 264, abstract
n 209.643 p, 1987) concerns the structural analysis of
a lipopeptide composed of a fragment of the virus G
protein and of a lysine-palmitoyl rest
This analysis of the state of the art therefore
shows that no technology applicable to various types of
antigenic determinants has been developed which enables
the induction of cytotoxic T lymphocytes to be
obtained, with a high induced response, and which does
not require the administration of an adjuvant.
GENERAL DESCRIPTION OF THE INVENTION
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2057828
The applicant has shown, surprisingly, that a
response of cytotoxic T lymphocytes against an antigen
could be induced in a host organism by immunizing said
organism with a lipopeptide complex containing one of
the determinants of this antigen.
Even more surprisingly, the applicant has shown
that this induction could be obtained for a large
number of antigenic determinants of various pathogenic
agents.
The subject of the present invention is there-
fore a lipopeptide comprising a peptide part having
between 10 and 40 and preferably between 10 and 20
amino acids approximately and comprising at least one
antigenic determinant, said lipopeptide also comprising
one or more chains derived from fatty acids comprising
between 10 and 20 carbon atoms and/or one or more
steroid groups modified and coupled on the -NH2 or -NH2
functional groups of said amino acids.
Said fatty acid derivatives may be in
particular 2-aminohexadecanoic acid (D,L) of the
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following formula (I):
N1~
NooG
-1
N--palmitoyllysine (L) of the following formula (II):
'', ~z I 6
Nu ~
H C' 0
or its derivate having the following formula
~t' ~ 6
NN ~
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N,N'-dipalmitoyllysine (L) of the following formula
(III):
Hooc~~,a
H r,
6
0
15 pimelautide of the following formula (IV):
O
coN
co
cAf~N
Noot,
; . _
2057828
trimexautide of the following formula (V):
0
Nco c~oK
CON~
"00G
or one of their derivatives.
Said steroid groups may be N--[(cholest-5-enyl-3-
oxy)acetyl]lysine (L) of the following formula (VI):
Zcmu
NN
\ 1f \
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(cholest-5-enyl-3-oxy)acetic acid of the following
formula (VII)
Ho
o~
or one of their derivatives.
The peptide part may be a fragment of any pro-
tein derived from a pathogenic agent possessing an
antigenic determinant.
Such proteins may be especially the proteins of
the HIV1 or HIV2 virus, in particular the protein
encoded by the ENV gene. In this case, the fragments
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312-327 or 302-336 may be advantageously used depending
on the HIV1-BRU sequence (Hyers, C.A.B. Rabson, S.F.
Josephs, T.F. Smith and E. Wong-Staal (Eds.), 1989,
Human retrovirus and AIDS, Los Alamos Laboratory 11:59)
of this protein, to form the conjugated lipopeptide
molecule.
The present invention relates moreover to
pharmaceutical compositions containing an efficient
quantity of at least one of the compounds above
described in association with one or few diluents or
adjuvants compatible and pharmaceutically acceptable.
These compositions are in particular intended
for treating the diseases relates with the HIV virus by
induction of cytotoxic T lymphocytes.
The subject of the present invention is
furthermore vaccines against viruses or parasites
containing one of the above-described lipopeptides, and
in particular vaccines against diseases linked to HIV
viruses, said vaccines advantageously containing a
fragment of the protein which is the product of the ENV
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2057828
gene.
The subject of the present invention is
furthermore the use of the above-described lipopeptides
for immunizing the human or animal body against
pathogenic agents by inducing cytotoxic T lymphocytes.
Such pathogenic agents may be viruses which have a sub-
stantial cytotoxic activity, in particular the HIV1 and
HIV2 viruses, and certain parasites.
Said lipopeptides may also be used against
certain cancers in order to induce a CTL response
specific for certain tumor cells.
The lipopeptides which are the subject of the
present invention may be obtained from protein and
pseudolipid constituents by methods known to a person
skilled in the art, in particular, either by coupling
the amino acids comprising the peptide part to the
pseudolipid immobilized on a resin, that is to say by
solid phase synthesis, or by coupling the pseudolipid
to a peptide immobilized in a solid phase.
It should be noted that the lipopeptides
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according to the invention possess the notable advan-
tage of being capable of being adapted for inducing
cytotoxic T lymphocytes against any type of antigenic
determinant of any pathogenic agent.
Additionally, the present invention also
relates to the following synthetic intermediates:
2-tert-butyloxycarbonylaminohexadecanoic acid (D,L) of
the following formula (VIII):
O
NH
=,~
H 00C-,
N--terbutyloxycarbonyl--palmitoyllysine (L) of the
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following formula (IX):
ON ~ ~
NW 'S
N--fluorenylmethyloxycarbonyl--palmitoyllysine (L) of
the following formula (X):
0 0
Oy o
lhN
hJ N ~6
t; o oc, 0
N--tert-butyloxycarbonyl--[(cholest-5-enyl-3-
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2057828
oxy)acetyl]lysine (L) of the following formula (XI):
0 H P~~ C,oo H
0
~
o
0
or N--fluorenylmethyloxycarbonyl--[(cholest-5-enyl-3-
oxy)acetyl]lysine (L) of the following formula (XII):
0 0
G~ ~ N N Coc u
0(
II
0
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or one of their derivatives.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention is illustrated, without
being limited in any manner, by the following examples
of application in which Figures 1 and 3 represent the
reverse-phase HPLC spectra of the lipopeptide V3GP120,
312-327 succinyl during preparative and analytical
electrophoreses respectively.
Figure 2 represents, for its part, its mass
spectrum.
EXAMPLE 1.
Syntheses of pseudolipid residues (or hydro-
phobic molecules).
1. Synthesis of 2-tert-butyloxycarbonyl-
aminohexadecanoic acid.
1.1. Synthesis of 2-aminohexadecanoic acid.
Empirical formula: C16H33NO2
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Molecular mass: 271.44
Procedure:
0.0298 mole of 2-bromohexadecanoic acid (10 g)
and 100 ml of a 28% solution of NH4OH are introduced
into an autoclave previously cleaned with 50% nitric
acid and 10% phosphoric acid. After stirring, the auto-
clave is heated at 60 C for 15 hours. The nascent amino
derivative precipitates in the reaction medium. After
cooling, the autoclave is washed, the water dispersed
and the ethanol removed. The reaction mixture is fil-
tered on sintered glass with a porosity of 4 The
difference in solubility of the various products in
ethanol medium enables the removal of traces of
2-bromohexadecanoic acid by rinsing.
Amount of precipitate obtained: 4.37 g; yield
of 54%.
Remark: The insolubility of the amino deriva-
tive was checked in numerous solvents (water, ethanol,
acetic acid at low temperature, 70% formic acid, ethyl
acetate, toluene, toluene/acetonitrile, ethyl acetate/
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acetonitrile). Among all the solubilization assays
tested, only the action of a specific detergent (tra.-
methylbenzylammonium hydroxide) or boiling acetic acid
dissolved the amino derivative.
The dried precipitate is taken up in 150 ml of
acetic acid which is refluxed until a yellowish clear
solution is obtained. The colored pigments are absorbed
on vegetable charcoal. After filtering on folded filter
paper, the purified amino derivative is obtained by
crystallization from the eluate. The white crystals
obtained are recovered on sintered glass with a
porosity of 4, washed with cold acetic acid before
being dried in a desiccator over P205.
Yield: 46% (the product being in acetate form,
a MW of 330.48 is used).
The purification yield is equal to 85%.
1.2. Synthesis of 2-tert-butyloxycarbonyl-
aminohexadecanoic acid.
- Empirical formula: C21H41NO4
- Molecular mass: 371.557.
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CA 02057828 2001-03-06
- Procedure:
Dissolution of 2-aminohexadecanoic acid.
1.1 equivalents of "Tritorf" (benzyltrimethyl-
ammonium hydroxide) in solution at 40% in methanol
(2.9 ml) as well as 100 ml of DMF are added to 5 mmol
of amino acid in acetate form (1.655 g). The reaction
mixture is left stirred at room temperature until com-
plete dissolution. The DMF is then evaporated using a
vane pump. The residue is dried in a desiccator over
P205.
Protection of the amine functional group.
The dry residue is dissolved in a mixture con-
sisting of 36 ml of water, 8 ml of 1N KHCO3 and 30 ml
of tert-butyl alcohol. 2.5 equivalents of terbutyl-
dicarbonate (MW = 218.25) are added to this solution.
The pH is adjusted to between 8 and 9 using a 1N
solution of Na2CO3 and maintained constant during the
initial hours of the reaction. The kinetics of coupling
are monitored by thin-layer chromatography on silica.
After evaporating the tert-butyl alcohol under
"Trademark
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vacuum, the product is taken up in 100 ml of water. The
aqueous phase is acidified to pH 3 with a iN solution
of HC1. The Boc-amino acid is extracted with ethyl
acetate (2 100 ml). The organic phase is washed with
distilled water, dried over anhydrous Na2SO4, filtered
and then concentrated using a rotating evaporator. 4 ml
of hexane are added to the oily residue. Crystalli-
zation of the Boc-amino acid is enhanced by cooling in
a cold chamber. The white crystals are recovered on
sintered glass with a porosity of 4, washed with hexane
and dried in a desiccator over P205.
Yield: 16 to 18%.
1.3. Purification and characterization.
1.3.1. Purification.
Successive recrystallizations made it possible
to increase the purity (increase in the melting point).
The purification led to a reduction in yield of not
more than 2% for aminohexadecanoic acid and of not more
than 1% for the protected amino acid.
1.3.2. Characterization.
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A. Melting point:
Product Melting point obtained
2-Bromohexadecanoic acid 56 C
2-Aminohexadecanoic acid 144 C
2-tert-Butyloxycarbonyl-
aminohexadecanoic acid 85'C
B. Thin-layer chromatography on silica.
The solutions (10 to 20 1 of a solution at
1 mg/ml) are deposited on thin-layer silica (Merck
silica gel 60 without fluorescence indicator).
2-Bromohexadecanoic acid is dissolved in
ethanol, 2-aminohexadecanoic acid in boiling acetic
acid, and tert-butyloxycarbonyl-2-aminohexadecanoate in
dichloromethane.
Choice of the migration solvent.
The various systems chosen are:
System A: butanol/ethyl acetate/acetic acid/water in
the volume/volume proportions: 1/1/1/1.
System B: ethyl acetate/pyridine/acetic acid/water in
the v/v proportions: 5/5/1/3.
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System C: chloroform/methanol/acetic acid in the v/v
proportions: 10/1/0.1.
Developing.
After migration in the system of suitable
solvents, the thin layers are dried for 15 minutes at
120 C before being developed after spraying with a
developing reagent.
- The ninhydrin reagent which is specific for
primary amino functional groups enables the detection
of the unprotected aminohexadecanoic acid but also of
the Boc-amino acid, the spraying of 20% acetic acid
followed by drying at 120 C enabling the displacement
of the Boc group.
The spraying using a reagent comprising 20 g of
(NH4)2SO4, 3 ml of H2SO4 and 100 ml of H20 enable the
simultaneous developing of the three products. In this
technique, the drying, after spraying, in the thin-
layer chromatography is carried out using an epi-
radiator (porcelain resistance with infrared
radiation).
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Result:
Product Solvents Rf
2-Bromohexadecanoic acid System A 0.5
System B 1
System C 1
2-Aminohexadecanoic acid System A 0.82
System B 0.94
System C 0
2-tert-Butyloxycarbonyl-amino-
hexadecanoic acid System A 1
System B 1
System C 0.67
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C. Mass spectrometry (PDMS BIO-ION 20).
2-tert-Butyloxycarbonyl-aminohexadecanoic acid spec-
trum.
MM (g) (M-H) Fragments:
Theoretical MM 370.557
Experimental MM 370.8 270
The experimental molecular ion and the
theoretical molecular ion have an identical mass. The
molecular ion is fragmented; the Boc group (peak at
270) is released. The 296.6 peak represents the ion
with a mass of 270 containing the CN group (nitro-
cellulose).
2. Synthesis of 3-(2'-carboxymethoxy)-
cholest-5-ene.
2.1. Synthesis of cholesteryl tosylate.
- Empirical formula: C34H53SO3,
- Molecular mass: 540.83,
- Procedure:
After dissolving 25.86 mmol of cholesterol
(10 g) in a minimum of pyridine (5 to 10 ml), an excess
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2057328
of tosyl chloride (10 g, 52.6 mmol) is added. After
stirring for 12 hours, the pyridine is removed by eva-
porating to dryness. The residue is solubilized in
20 ml of acetone at high temperature (the temperature
is maintained below 55 C to avoid the formation of
oil). The mixture is filtered. The cholesterol tosylate
is obtained by crystallization from the eluate. The
white crystals obtained are washed with cold acetone
and dried in a desiccator over P205.
Yield: 82 to 85%.
2.2 Synthesis of 3-(2'-hydroxyethoxy)-
cholest-5-ene.
- Empirical formula: C29H5002.
- Molecular mass: 430.71.
- Procedure:
30 ml of ethylene glycol (480 mmol) are added
to 17.5 mmol of cholesteryl tosylate (10 g) dissolved
in 120 ml of dioxane. The reaction mixture is refluxed
for 4 hours at 120 C. After cooling, it is taken up in
150 ml of distilled water. The alcohol derivative
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formed is extracted with diethyl ether (3 200 ml). The
ethereal phase is successively washed with 5% NaHCO3 (2
200 ml) and distilled water (2 200 ml). After drying
over anhydrous Na2SO4, the ethereal solution is
concentrated until an oil is obtained. After adding 4
ml of hexane, the crystallization is started by rubbing
and cooling in a cold chamber (4 C). The white crystals
are recovered on sintered glass with a porosity of 4
and washed with hexane before being dried in a
desiccator over P205.
Yield: 32 to 34%.
2.3. Synthesis of 3-(2'-carboxymethoxy)-
cholest-5-ene.
- Empirical formula: C29H48031
- Molecular mass: 444.69.
- Procedure:
The oxidizing solution is prepared beforehand:
it comprises 2.67 g of chromic anhydride, 2.3 ml of
concentrated H2SO41 the volume being brought to 10 ml
with distilled water. The oxidizing medium is added
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dropwise to 4.66 mmol (2 g) of 3-(2'-hydroxyethoxy)-
cholest-5-ene dissolved in 50 ml of acetone. The pro-
gress of the reaction is monitored by thin-layer chro-
matography. Once the reaction is completed, the
reaction medium is taken up in 250 ml of distilled
water. The acid derivative is extracted with ethyl
acetate (3 200 ml). The organic phase is washed with
distilled water (2 200 ml), dried over anhydrous
Na2SO4 and concentrated until an oil is obtained. 4 ml
of petroleum ether are added. The crystallization of
the acid derivative is enhanced by cooling in a cold
chamber (4 C). The white crystals are recovered on
sintered glass with a porosity of 4, washed with
petroleum ether and dried in a desiccator over P205.
Yield: 29 to 31%.
2.4. Purification and characterization.
2.4.1. Purification.
Cholesteryl p-toluenesulfonate is purified by
successive recrystallizations in acetone. -(2'-
Hydroxyethoxy)cholest-5-ene and the acid derivative
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20 578 2 8
have both been purified by thick-layer chromatography
on silica.
A. Thick-layer chromatography on silica.
The depositions are carried out on a thick
layer of silica (Merck silica gel 60 PF254 with a
fluorescence indicator), the spots being detected by
ultraviolet radiation.
A solution containing 0.250 mg of product is
deposited on the silica plate, the products have both
been dissolved in dichloromethane.
Product Solvent Rf
3-(2'-Hydroxyethoxy)- Petroleum ether/ 0.48
cholest-5-ene ethyl ether
Volume/volume
proportions: 1/1
3-(2'-Carboxymethoxy)- Petroleum ether/ 0.52
cholest-5-ene ethyl ether/
methanol
v/v proportions:
10/10/3
The two products were extracted from the silica
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with methanol. A loss equivalent to about 30% of the
amount deposited is observed for each of the products.
2.4.2. Characterization.
A. Melting point.
Product Melting point Melting
(literature) point
Cholesteryl para-toluene-
sulfonate 131.5 C-132.5 C 128 C
3-(2'-Hydroxyethoxy) 102 C-104 C 99 C
3-(2'-Carboxymethoxy)-
cholest-5-ene 160 C-161 C 157 C
B. Thin-layer chromatography
The depositions (10 to 20 1) of a 1 mg/ml
solution are carried out on a thin layer of silica with
a fluorescence indicator (Merck Kieselgel 60F254).
The dissolution of the various products is
carried out in dichloromethane.
After migration in the suitable solvent
system, the thin layers are dried in air before being
developed either by ultraviolet radiation or after
spraying with HC104 (20%) and drying in an oven (120 C
for 20 minutes).
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Result:
Various solvent systems.
System A: Ethyl ether/petroleum ether in the volume/
volume proportions: 1/1.
System B: Ethyl ether/petroleum ether in the v/v pro-
portions: 1/2.
System C: Petroleum ether/ethyl ether/methanol in the
v/v proportions: 5/5/1.
System D: Petroleum ether/ethyl ether/methanol in the
v/v proportions: 10/10/3.
System E: Petroleum ether/ethyl ether/methanol in the
v/v proportions: 5/5/2.
System F: Ethyl ether.
Product Solvent system Rf
Cholesterol System A 0.54
System B 0.3
System F 0.95
Cholesteryl para-
toluenesulfonate System A 0.85
System B 0.62
System F 1
3-(2'-Hydroxyethoxy)-
cholest-5-ene System A 0.41
System B 0.24
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System F 0.9
3-(2'-Carboxymethoxy)-
cholest-5-ene System A 0
System B 0
System C 0.1
System D 0.42
System E 0.78
System F Streaking
effect: Rf 0 0.5
C. Mass spectrometry (PDMS)
Analysis of the 3-(2'-carboxymethoxy)cholest-5-ene.
MM (g) (M-H)-
Theoretical MM 443.69
Experimental MM 443.1
The experimental molecular ion and the theoretical
molnular ion have an identical mass.
D. C nuclear magnetic resonance
Analysis of the 3-(2'-carboxymethoxy)cholest-
5 ene spectrum was carried out by comparison with the
13C NMR spectrum of cholesterol.
The dissolution of the cholesterol and the 3-
(2'-carboxymethoxy)cholest-5-ene was performed in
deuterated chloroform.
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Cholesterol spectrum.
Peaks d(ppm) obtained attribution theoretical
d(ppm)
1 140.7606 C5 or C6 alkene func-
tional group:
d(ppm) from
100 to 145
2 121.7064 C5 or C6
3 78.5715 CDC13
4 76.9981 CDC13
5 75.4010 CDC13
1' to 22' 71 to 11 alkane func-
tional groups.
3-(2'-Carboxymethoxy)cholest-5-ene spectrum.
Peaks d(ppm) obtained attribution theoretical
d(PPm)
1 172.2923 C2' acid func-
tional group:
d(ppm) from
160 to 185
2 139.8394 C5 or C6 alkene func-
tional group:
d(ppm) from
100 to 145
3 122.5233 C5 or C6 alkene func-
tional group
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4 80.5745 Cl' ether func-
tional group:
d(ppm) from 45
to 80
5 79.2943 C1' + slight dis-
placement
6 78.5903 CDC13
7 77.0089 CDC13
8 75.4146 CDC13
9 to 31 65 to 11 alkane func-
tional groups.
E. Elemental analysis.
Elemental analysis of 3-(2'-carboxymethoxy)cholest-5-
ene gave the following results:
Theoretical Obtained
% carbon 78.3 76.25
% hydrogen 10.9 10.9
% oxygen 10.8 12.5
EXAMPLE 2: - Synthesis of the lipopeptides.
I Method for couplina 2-aminohexadecanoic acid
The 312-327 region of gp120 of the HIV-1
LAV-aRU virus was chosen for sequences constructed in
lipopeptide form in order to study the cytotoxic T
response. 3 preparations were carried out using this
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205"18200
sequence.
The synthesis was carried out in solid phase
(MERRIFIELD R.B., 1963, J. Am. Chem. Soc. 85, 2149-
2210).
All the lipopeptides were synthesized on a
benzhydrylamine resin (charged at 0.72 millimole/gram).
In all cases, the first amino acid grafted was 2-Boc-
aminohexadecanoic acid (2 equivalents). This made it
possible to obtain constructs where the C-terminal
amino acid was 2-aminohexadecanoic acid in amide form
in order to avoid the presence of a charge near the
hydrophobic aliphatic chain. After acetylation with
acetic anhydride in basic medium, in order to block the
free reactive sites, the cleavage of the N-terminal Boc
and then the coupling of the first amino acid of the
sequence were carried out.
All these stages were carried out manually,
which made it possible to carry out very precise
controls of the coupling of the pseudolipid amino acid
and the first amino acid on the latter.
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The coupling activator is benzotriazolyl-N-
oxytriadimethylaminophosphonium (BOP) in the presence
of hydroxybenzotriazole (HOBt) and diisopropylethyl-
amine (DIEA). By virtue of this very efficient coupling
method, the yield of these two coupling reactions was
always greater than 99.5% despite the substantial
steric hindrance due to 2-Boc-aminohexadecanoic acid.
The rest of the synthesis was carried out in a
conventional manner, automatically, up to the last
amino acid. At this stage, the peptidyl-resin was
divided into 3 batches, treated manually:
- 1 batch was preserved as such;
- 1 batch was grafted using 2-Boc-aminohexadecanoic
acid. The manual coupling (using BOP) of the latter was
followed by cleavage of the N-terminal Boc and
acetylation of all the amine functional groups thus
exposed. This made it possible to avoid the presence of
a charge near the hydrophobic aliphatic chain of the
pseudolipid amino acid,
1 batch was grafted using diBoc, Na,C~-lysine. Manual
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grafting (using BOP) of the latter was followed by
cleavage of the two Boc groups and the manual coupling
of palmitic acid (using BOP). We thus obtained peptides
possessing a dipalmitoyl-lysine in the N-terminal
position.
These couplings, carried out manually, were the
subject of a strict control which revealed yields that
were always higher than 99.5%. These results confirm
the advantage of using BOP as activating agent in pep-
tide synthesis, especially for coupling pseudolipid
amino acids or for coupling to the latter. The synthe-
sized lipopeptides were then cleaved from their
support. The lipopeptides derived from the 312-327
sequence were cleaved by anhydrous hydrofluoric acid
treatment. The cleavage yields are quite low, between
40 and 70%.
There are at least two explanations for these
values: 1) the cleavage of a peptide grafted on a
benzhydrylamine resin is never total under the usual
conditions of cleavage; 2) the presence of 2-aminohexa-
- 36 -
2057828
decanoic acid, directly in contact with the resin,
certainly amplified this phenomenon.
After washing twice (TFA-ether), the identity
of the lipopeptides was assessed by amino acid analysis
after total acid hydrolysis and, for some, by PDMS mass
spectrometry. Their homogeneity was checked by thin
layer chromatography on silica and analytical reverse
phase HPLC.
II. Methods for coupling pimelautide and tri-
mexautide
1) Method for couplina pimelautide (or tri-
mexautide) to the N-terminal end of a peptide by means
of a succinyl link.
This method applies to the fixing of unpro-
tected pimelautide (or trimexautide) on a peptide
constructed in a solid phase, still fixed to the resin,
and to protected side chains.
Both trimexautide and pimelautide (Rhone
Poulenc) have two free carboxylic functional groups and
a free primary amine functional group. The creation of
- 37 -
I 2, b
2 nr'"
a predetermined amide bond between the peptide and the
pimelautide (or trimexautide) can only be achieved by
using the pimelautide (or trimexautide) as amino
partner.
The succinylation of the peptide on the resin
makes it possible to make it the carboxylic partner.
DEPROTECTION
The terbutyloxycarbonyl group, which tem-
porarily protects the N-terminal end of the peptide on
the resin, is cleaved by the action of a 40% trifluoro-
acetic acid solution in dichloromethane for 20 minutes
with stirring.
The resin is washed with:
- twice 20 ml of dichloromethane,
- twice 20 ml of 5% diisopropylethylamine in
dichloromethane,
- twice 20 ml of dimethylformamide (for
3 minutes for each wash).
SUCCINYLATION.
It is achieved by carrying out the coupling
- 38 -
2~57828
three times by bringing the resin of the succinylation
solution into contact with:
- a fivefold excess of succinic anhydride (5%
solution in N-methylpyrrolidone)
- diisopropylethylamine in a stoichiometric
amount relative to the amines of the resin (for
20 minutes with stirring).
ACTIVATION.
The activation of the carboxyl now present on
the resin is carried out as follows:
the resin is subjected to the action of the
activating solution (for 15 minutes at room temperature
and with stirring):
- BOP (benzotriazolyloxytrisdimethylaminophos-
phonium hexafluorophosphate): 3 excesses with respect
to the carboxyls,
- HOBT (hydroxybenzotriazole): 3 excesses with
respect to the carboxyls,
- diisopropylethylamine: 7 excesses with
respect to the carboxyls in solution in N-
- 39 -
2057828
methylpyrrolidone.
WASHING:
The solution is washed with:
- 3 times 30 ml of dimethylformamide,
- 3 times 30 ml of dichloromethane.
COUPLING:
The resin is subjected to the action of the coupling
solution:
- pimelautide (or trimexautide): 3 excesses
with respect to the activated ester of
hydroxybenzotriazole,
- diisopropylethylamine: 3 excesses with
respect to the ester,
- 10% dimethyl sulfoxide
- 90% N-methylpyrrolidone: sufficient amount to
dissolve pimelautide (or trimexautide).
The saturated solution is at about 4% pimelautide or
trimexautide after sonication and passage for 2 minutes
at 50 C.
2) Svnthesis in solid phase of V3GP120, 312-327
- 40 -
2057828
succinyl.
a) N-Protection of the pimelautide (or tri-
mexautide) by the tert-butyloxycarbonyl group.
500 micromoles of pimelautide (or trimexautide)
are dissolved in 10 ml of a 0.1 molar solution of car-
bonate buffer at pH 9.5.
ml of a solution of diterbutyl pyrocarbonate
at 100 mmol/l are added.
A pH of between 9 and 10 is maintained for
10 100 hours by adding disodium carbonate.
The reaction mixture is then diluted with 10 ml
of water and 10 ml of diethyl ether and the washed
aqueous phase is acidified to pH 2.5 with potassium
bisulfate.
An extraction with 100 ml of dichloromethane
followed by evaporation of the solvent using a rotating
evaporator leads to the crystallization of Boc-
pimelautide (or Boc-trimexautide).
The incorporation of Boc-pimelautide (or Boc-
trimexautide) by peptide synthesis in a solid phase
- 41 -
2057328
generates two position isomers.
b) CLEAVAGE AND PURIFICATION
The cleavage of the peptide at the end of the
synthesis is performed using anhydrous hydrofluoric
acid.
The peptide is then purified by gel filtration
and type C4 reverse-phase preparative HPLC.
Figure 1 represents the preparative HPLC spec-
trum at 235 nm obtained for 20 mg of lipopeptide dis-
solved in HCOOH.
The lipopeptide obtained is then analyzed by
total acid hydrolysis, by analytical C4 HPLC chroma-
tography and mass spectrography.
Figure 2 represents the mass spectrum. A dis-
tinct peak is observed at 2422.2 which corresponds to
the mass of the lipopeptide.
Figures 3a and 3b represent the analytical C4
HPLC chromatography of 'the lipopeptide and of the
control without lipopeptide respectively.
The chromatographic conditions are as follows:
- 42 -
2057828
solvent (A) : 0.5 0/00 trifluoroacetate,
gradient: solvent B : 0.75 0/00 acetonitrile
0.5% trifluoroacetate,
gradient from 10% to 80% in 120 min,
measurement at a wavelength of 215 nm.
During the total acid hydrolysis, the diaminopimelic
acid (Dap) present in pimelautide and trimexautide
constitutes a good coupling marker.
Results of the total acid hydrolysis
Amino nanomoles measured theoretical measured/
acids theoretical
Thr 3.2500 0.97 1 0.97/1
Glu 7.1000 2.11 2 1.06/1
Pro 3.1800 0.95 1 0.95/1
Gly 10.3500 3.08 3 1.03/1
Arg 6.6900 1.99 2 1.00/1
Val 3.3900 1.01 1 1.01/1
Ile 9.5800 2.85 3 0.95/1
- 43 -
2057328
Phe 3.3500 1.00 1 1.00/1
Lys 3.5200 1.05 1 1.05/1
Arg 9.9200 2.95 3 0.98/1
Dap 3.500 1.05 1 1.05/1
EXAMPLE 3 -
Immunization against the peptide NP 147-158.
The immunizations of mice are carried out as
follows:
Immunizations:
The mice were injected with the lipopeptide
preparations intraperitoneally (i.p.) or subcutaneously
(S.C.), with or without adjuvant.
At least two injections (at intervals of 8 to
30 days) are necessary in order to obtain CTLs.
Each injection contains 5 10-8 mole of
lipopeptide.
Detection of the CTLs
8 to 21 days after the last injection, the
- 44 -
2057828
spleen of the immunized mice was removed, the spleno-
cytes of these mice were cultured in vitro in an amount
of 5 106 splenocytes/2 ml of conventional culture
medium (DMEM + 10% FeS + pyruvate + nonessential amino
acids + -2-mercaptoethanol) containing 5 M of the
peptide corresponding to that involved in the lipopep-
tide construction.
From the 5th day of the in vitro culture, the
activity of the CTLs may be detected by the
conventional test of 51Cr release (Martinon et al., J.
Immunol., 142;3489-3494, 1989).
The CTL activity is tested against some
syngenic target cells in the presence of the peptide
(NP 147-158 R- ,P3CSS. PepNP or Ll-Pep.NP) or against
some syngenic target cells infected by the influenza A
virus.
The results obtained are summarized in Table I.
The first part of the table relates to the results
already obtained with the whole influenza virus, the
NP 147-158 R protein of the influenza virus and the
- 45 -
2057828
P3CSS-PEPNP lipopeptide, which consists of the
NP 147-158 peptide coupled to tripalmitoyl-S-glyceryl-
cysteinylserylserine (DERES et al. previously
mentioned).
The second part of the table relates, on the
one hand, to the immunization trials carried out with
liposomes containing the NP 147-158 peptide and with
the lipopeptides L1, L2 and W. These lipopeptides are
molecules containing a peptide part (NP 147-158) and a
lipid part, respectively. The lipopeptides L1, L2 and
L3 are therefore of the following formulae
NH?- i YQR I RALV i G-CO-N,-Cf i-CO-NH 2
Lx
C14
- 46 -
2057828
CN-CO-N'-H C;-i-CO-I',,'H- T YQR i RAL V T G-CO= NH--CH-CC-M~2
L ~
~
C14 C14
CO-NH CH-CO-NH-TYCJRTRALVTG-CO-N1 i-CH CO-NH:,
NH-CO
C15 C14
L3
Cy5
- 47 -
2057828
The cytolytic activities after 5 days, 12 days
and more than 21 days show that a very high activity is
obtained for the lipopeptide L1 compared to the other
trials carried out.
EXAMPLE 4:
Immunization by CB1R CB2 and CB3 against the
ENV 312-327 peptide .
This peptide is a protein fragment encoded by
the ENV gene of the HIV virus.
The experimental procedures are identical to
those described in Example 3.
Table II summarizes the results obtained.
In this table, CB1, CB2 and CB3 correspond to
lipopeptides formed from the peptide derived from the
ENV protein and a lipid.
The formulae of CB1, CB2 and CB3 are as follows:
- 48 -
2057828
NH,-IRIQRGPGRAFVTIGK-CQ-NH-CH-CO-NH,
c:<
CB1
CH,-CO-NH-CH-CO-NH-IRIQRGPGRAFVTIGK-CO-NH-CH-CO-NH,
CB c) 4 C14
~
- 49 -
2057828
CO-N}i-CH-CO-NH-iRIQRGPGRAtVTIGK-CO-NH-CH-CO-NH,
~~1v
C:c
CB3
Ci5 Ci5
The results in Table II show a substantial
activity for one of the lipopeptides (CB1).
EXAMPLE 5:
Immunization against the peptide ENV 302-335.
This peptide is the 302 to 335 fragment of the
ENV protein of the HIV virus.
- 50 -
2057828
The experimental procedures are identical to
those described in Example 3.
The results are shown in Table III.
The formulae of the lipopeptides CBS1 CB7 and
CB8 are as follows:
NHa -TRPNNNTRKS I RIQRG?GR.t:FVTI GK I GNNRQAH-CO-NH-Cii-CO-Ii-rf .
CBg
C14
- 51 -
2057828
C='=i{~-HH{H{y.NH-i F?hTT'~aX
D S: A: CRC?G.~~AFV : Ci( : CNYROAH-C~-1n-{ y{,~_ NH 2
CD7
C:4
C: 4
;.N.S : R: CRC?C"'~F~ ; L'R: C~'MRCnH-{~-NH{,yg -_NH2 ..
NH-CO
CBg
C 4
R3 C)S
- 52 -
2057828
It can be observed in Table III that the two
lipopeptides, CB6 and CB7, show cytolytic activities
substantially higher than the control.
EXAMPLE 6 :
Immunization against the peptide ENV 312-327 by
CB1, CB4, CB5, CB17, CB19, CB21 and CB25.
The_experimental procedures are substantially
identical to those described in example 3.
The table IV summarizes the results obtained.
The formula of CB1 is indicated in example 4.
The formulae of CB4, CB5, CB17, CB19, CB21 and
CB25 are as follows
C'E4 IV~ICC3~- -(Ci42)14---CH3
i-t--I[t[QRCPc;kAt,V'r[GK--CC~--NH CONHa
=
NIa2- C~~ - C u- :Z K~ Ciy, r. (> G(~ h1 F\/ l 1(i Y,\ - i-q
53
2057828
cll3
Lc) i CONI~ C~C~C~II
12
H2NOG CONH
R ~ " {H2C)3 ''~
t-i N cooli
(;li 5 - f~ = t'() ;C'IIw)~--(Y1N1i---1RIQ(t(~1'{.;4tA41V11CiK---UII
('I3 19 = lt - CO CHZ-'~'H--C O (CliZ)Z-t_~N1-1--7KlQRCjP(iRAI--V"1IGK-ON
CONN C'UNI-9 COR
li2NOC GONH
0i 2C')3 ~
HZN---.112C --CO ~i CORI
t'.t3 21 MMt,nV do A R- oH
R' ~- NH-Itt1(~HC3r'GRAI'V77(~l~--C)11
li : hZ zdl~---IRIQROYC31tA1'v"i'1QK- Uli
R'OH
ICCX--(CI-i2)14-'CHg
1E---TRFNN'N"TRKSIRiQRCiP(3RAPVTirKiG,NMRQAu--coNH
54
TABLE I
ANTI-NP 147-158R PEPTIDE IMMUNIZATIONS (influenza virus)
In vivo In vitro Target Cytolytic activity
injection stimulation d5 d12 > d21
0 NP.147-158R NP.147-158R' (a) - - ++
Influ. virus (b) (+)
Influ. virus NP.147-158R- NP.147-158R (++) (++) (+++)
_ Influ. virus (++) (++) (+++)
NP.147-158R NP.147-158R NP.147-158R (-) - +
Influ. virus (-) -
Influ. virus Influ. virus (-)
P3CSS-Pep.NP NP.147-158R NP.147-158R (++)
Influ. virus (++)
P3CSS-Pep. NP. (++)
LIPOSOME-Pep.NP. (c)
[1* s.c. syntex] NP.147-158R NP.147-158R - -
[1* i.p.] NP.147-158R NP.147-158R- - -
V.
[2* s.c. syntex] NP.147-158R NP.147-158R - ++
[2* i.p.] NP.147-158R NP.147-158R - ++
LIPOPEPTIDES-Pep.NP.
L1-Pep.NP.[2* i.p.] NP.147-158R NP.147-158R - +++
Influ. virus -
L1-Pep.NP. +++ +++
L2-Pep-NP. [2* i.p.] NP.147-158R NP.147-158R' - -
Influ. virus - -
- -
Ll-Pep.NP.
L3-Pep.NP. 12* i.p.] NP.147-158R NP.147-158R - -
Influ. virus - -
L1-Pep.NP. - -
The results in brackets have already been published.
(a) syngenic target cells in the presence of 3'),An of the penti_de NP. 147-
158 R
(b) svngenic tar-et cells infected by the virus influenza A
(c) linosomes loaded with nentide NP 147-158 R.
00
U~
TABLE II by and CB
ANTI-ENV.312-327 PEPTIDE IMMUNIZATIONS HIV-BRU y 1 2 6
In vivo In vitro Target Cytolytic activity
injection stimulation d5 d12 > d21
0 ENV.312-327 ENV.312-327(a) - ++
Vac-env (b) - - -
CBI ENV.312-327 - -
Vac-env ENV.312-327 ENV.312-327 (++)
Vac-env (++)
ENV.312-327 ENV.312-327 ENV.312-327 - - ++
Vac-env - - -
LIPOPEPTIDES-ENV.312-327
CB1 [1* i.p.] ENV.312-327 ENV.312-327 -
v CB1 [2* i.p.] ENV.312-327 ENV.312-327 +++ +++ +++
Vac-env +++ +++ +++
CB1 [1* s.c.syntex] ENV.312-327 ENV.312-327 -
CB1 [2* s.c.syntex] ENV.312-327 ENV.312-327 ++
Vac-env ++
CB2 [2* i.p.] ENV.312-327 ENV.312-327 - ++
Vac-env - ++
CB3 [2* i.p.] ENV.312-327 ENV.312-327 - -
Vac-env - -
(a) syngenic tar;et cells in the presence of 3(~+*1 of neptide INV 312-327
(b) svngenic target cells infected by a vaccine vi.rus allowing the expression
of the gene
env of NIV
t\O
WT
-12
cU
c~J
00
TABLE III
ANTI-ENV.302-335 PEPTIDE IMMUNIZATIONS (HIV-BRU)
In vivo 'In vitro Target Cytolytic activity
injection stimulation d5 d12 >_ d21
ENV.302-336 ENV.302-336
or ENV.312-327 (a) - -
ENV.312-327
LIPOPEPTIDES-ENV.302-335
ENV.302-336
CB6 [2* i.p.] or ENV.312-327 (a) ++
ENV.312-327 Vac-env (b) +++
ENV.302-336
CB7 [2* i.p.] or ENV.312-327 ++
ENV.312-327 Vac-env +++
ENV.302-336
CB8 [2* i.p.) or ENV.312-327 - -
ENV.312-327 Vac-env -
(a) and ( b) cf TABLE IT
t\3
co
CK)
TAALE IV
Anti- IsJJV,312-327 nentide immunizations by Ci3(, CB4, C13i7, CB1 91 CB21
,,C'925 and CB5
cytotoxic activit;,
n v;vo injec_ .n v t=ro target ~.(~i
stimulation days ~ 21days
Cbl (:t x 5 . c. ) ENV 312-32 1 EtJV 312-32T (a =+-++ ++
vac-Er:V (b) +++= + + +
CB1 +++
CL~4 (2 x i.p.) ENV 312-327 ENV 312-327 +++ +++
Vac-Et=TV + + + +Y 4
C917 :'(2 =x'yi::p:.Y ENV 312-321 ENV 312-32 7 - ++
Vac-EN~~ - 4
CB15 (2 x i.p. ) ENV 312-321 F.NV 312-327. + ++
Vac-ENV - 4
CF321 (2 x ip. ) ENv 312-327 ENV 312-327 ++ +++
Vac~-EPJV ++ +++
C1i25 (2 x i. p. ENV 312-327 E13v 312-327 +++ NT
Vac-ENV ++
C1325 (2 x i.p.FIA) c:NV 312-327 ENV 312-:.',27 ++ N'P
Vac-EZ=I11 +
CE25 (3 x i.p. ) f:NV 312-327 1:NV 312-32'1 +++ 24
Vac-ENV ++
Ca25 (3 x i. n_ EIA) l:w 312-327 ENv 312-327 ++ N'r
Vac-ENV ++
CF325 (2 x s.c_) ENV 312-327 ENV 312-327 +++
sla cc-EN V ++
Ct325 (2 x s. c. FZF ) ENV 312-327 E'NV 312- 327 +++ 1s':'
Vac-r:N"V +++
CB25 (3 x s.c_) ENV 312-327 ENV 312-327 +++
Vac-EISJ F ++
CB25 (3 x 3.C_'r'Irl) ENV 312-327 ENV 312-327 +++ NT
VOC"EN'J +T+
C B5 ( 2 x i. p) ~ ENV 312-327 J ENV 312-327 ++ +, +
Srac ENV ++ +++
" _ _ : '-- = --
_- - v
(a) and ( b ): cf table I.L 00
00
