U.S. patent application number 14/174286 was filed with the patent office on 2014-07-24 for compositions comprising a b subunit of shiga toxin and a means stimulating nkt cells.
This patent application is currently assigned to UNIVERSITE RENE DESCARTES PARIS 5. The applicant listed for this patent is ASSISTANCE PUBLIQUE HOPITAUX DE PARIS, CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE-CNRS, INSTITUT CURIE, UNIVERSITE RENE DESCARTES PARIS 5. Invention is credited to Eric TARTOUR.
Application Number | 20140205630 14/174286 |
Document ID | / |
Family ID | 39203258 |
Filed Date | 2014-07-24 |
United States Patent
Application |
20140205630 |
Kind Code |
A1 |
TARTOUR; Eric |
July 24, 2014 |
COMPOSITIONS COMPRISING A B SUBUNIT OF SHIGA TOXIN AND A MEANS
STIMULATING NKT CELLS
Abstract
The present invention relates to a composition comprising a) a B
subunit of Shiga toxin or a functional equivalent thereof which is
able to bind the Gb3 receptor, complexed with an antigen and b) at
least one ligand of CDl capable of stimulating NK T cells; and to a
pharmaceutical composition and a medicament comprising said
composition.
Inventors: |
TARTOUR; Eric; (Paris,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITE RENE DESCARTES PARIS 5
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE-CNRS
ASSISTANCE PUBLIQUE HOPITAUX DE PARIS
INSTITUT CURIE |
Paris
Paris
Paris
Paris |
|
FR
FR
FR
FR |
|
|
Assignee: |
UNIVERSITE RENE DESCARTES PARIS
5
Paris
FR
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE-CNRS
Paris
FR
ASSISTANCE PUBLIQUE HOPITAUX DE PARIS
Paris
FR
INSTITUT CURIE
Paris
FR
|
Family ID: |
39203258 |
Appl. No.: |
14/174286 |
Filed: |
February 6, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12521404 |
Mar 11, 2010 |
8685408 |
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PCT/EP2007/064556 |
Dec 26, 2007 |
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14174286 |
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60877354 |
Dec 28, 2006 |
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Current U.S.
Class: |
424/197.11 |
Current CPC
Class: |
A61K 39/12 20130101;
A61K 2039/6037 20130101; A61P 31/04 20180101; A61K 2039/55555
20130101; A61P 37/04 20180101; A61P 31/12 20180101; A61P 35/00
20180101; C07K 14/25 20130101; A61K 2039/55511 20130101; C12N
2710/20034 20130101; A61K 2039/55544 20130101; A61K 39/39 20130101;
A61P 31/00 20180101; C07K 2319/00 20130101; A61K 39/385 20130101;
C07K 14/005 20130101; C12N 2710/20022 20130101; C12N 2710/24143
20130101; A61K 2039/5256 20130101; C12N 7/00 20130101 |
Class at
Publication: |
424/197.11 |
International
Class: |
A61K 39/39 20060101
A61K039/39 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 28, 2006 |
EP |
06292066.5 |
Claims
1. A method of breaking tolerance against a self antigens said
method comprising administering to a mammal in need of such
treatment a composition comprising a) a B subunit of Shiga toxin or
a functional equivalent thereof which binds to the Gb3 receptor,
complexed with an antigen and b) at least one ligand of CD1 that
stimulates NK T cells, wherein said ligand is a glycolipid, a
phospholipid, a glycosphingolipid, a derivative or an analogue
thereof, wherein said composition breaks tolerance against self
antigens and stimulates dendritic cells and wherein there is
synergy between said B subunit of Shiga toxin or a functional
equivalent thereof which binds to the Gb3 receptor, complexed with
an antigen and said at least one ligand of CD1 that stimulates NK T
cells.
2. The method according to claim 1, wherein the functional
equivalent of the B subunit of Shiga toxin is administered which
binds to the Gb3 receptor has at least 60% amino acid sequence
identity to the B subunit of Shiga toxin.
3. The method according to claim 1, wherein said ligand of CD1 is a
ligand of CD1d is administered.
4. The method according to claim 1, wherein said ligand that is
administered is chosen from disialogamgloside (GD3),
phosphotidylethanolamine (PE) or phosphatidylinositol (P1).
5. The method according to claim 1, wherein said ligand that is
administered is a glycosylceramide or an analog or a derivative
thereof.
6. The method according to claim 5, wherein said glycosylceramide
that is administered is selected from the group consisting of
.alpha.-GalCer, .alpha.-GlcCer, Gal.alpha.1-6Gal.alpha.1-1'Cer,
Gal.alpha.1-6Glc.alpha.1-1'Cer, Gal.alpha.1-2Gal.alpha.1-1'Cer,
Gal.alpha.1-3Gal.alpha.1-1'Cer and a derivative thereof.
7. The method according to claim 6, wherein the glycosylceramide
.alpha.-GalCer that is administered is
(2S,3S,4S)-1-O-(.alpha.-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-
,-octadecanetriol.
8. The method according to claim 6, wherein the glycosylceramide
that is administered is .alpha.-GalCer is
(2S,3S,4S)-1-O-(.alpha.-D-galactopyranoxy)-2-(N-hexacosanoylamino)-3,4,-o-
ctadecanetriol (KRN7000).
9. The method according to claim 5, wherein said ligand that is
administered is selected from the group consisting of
3-O-sulfo-.alpha.-GalCer, .alpha.-GalCer, and OCH compound and
.alpha.-C-GalCer.
10. The method according to claim 5, wherein said glycosylceramide
that is administered is .alpha.-C-GalCer.
11. The method according to claim 6, wherein said glycosylceramide
that is administered is PBA-57.
12. The method according to claim 1, wherein said ligand that is
administered is a microbe derived glycolipid.
13. The method according to claim 1, wherein said ligand that is
administered is a Sphingomonas species-derived glycosphingolipid
selected from the group consisting of GSL-1 and GSL'1 or a Borrelia
species derived glycolipid selected from the group consisting of
BbGL-I and BgGL-II or a Mycobacteria species derived
phosphoglycolipid PIM.
14. The method according to claim 1, wherein said composition is
administered mucosally, transdermally, subcutaneously or
intramuscularly.
15. The method according to claim 1, wherein said at least one
ligand that stimulates NK T cells is administered before,
simultaneously or after the B subunit of Shiga toxin or functional
equivalent thereof which is able to bind Gb3 receptor cells which
is complexed to an antigen.
16. The method according to claim 1, wherein said composition is
administered in a therapeutically effective amount for stimulating
an immune response against an antigen in said mammal.
17. The method according to claim 16, wherein said antigen is
administered at a dose of 0.1 to 1,000 .mu.g.
18. The method according to claim 1, wherein said mammal is a
human.
19. The method according to claim 1, wherein said antigen is a
tumor antigen, a viral antigen or a bacterial antigen.
20. The method according to claim 1, further comprising a
pharmaceutically acceptable carrier.
21. The method according to claim 1, wherein said composition is
administered as a vaccine.
22. The method according to claim 21, wherein said vaccine is
initially administered followed by one or more booster
administrations.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of
antigen-related disease and especially to the field of therapeutic
vaccines. In particular, the present invention aims to elicit an
immune response in an individual in need thereof, using a B subunit
of Shiga toxin or a functional equivalent thereof complexed to an
antigen and a means stimulating NKT cells.
BACKGROUND OF THE INVENTION
[0002] Vaccine delivery system and adjuvants approved for human use
(aluminium salts, MF59, virosomes . . . ) primarily stimulate
humoral immune responses. However, preclinical studies strongly
suggest that successful vaccines against pathogens (HIV,
mycobacterium tuberculosis, malaria . . . ) but also cancer
vaccines will likely require both humoral and cellular responses.
Live attenuated pathogens or whole inactivated organisms have been
shown to activate both arms of the immune system in human but these
vaccines are difficult to produce, potentially unsafe or poorly
immunogenic. Development of subunit vaccines to elicit a robust
specific CD8+ T cell response represents therefore an ongoing
competitive challenge.
[0003] Dendritic cells (DCs) have been shown to be the most potent
antigen-presenting cells for the induction of primary T cell
responses, but they can also induce the differentiation of B cells
into antibody forming cells and mobilize other effective immune
cells such as NK and NKT cells. In human, clinical studies using
healthy recipients proved the immunogenicity and safety of DCs, and
demonstrated that a single injection of a small number of
antigen-pulsed DCs is sufficient to rapidly expand T-cell immunity
for both naive and recall antigens. However, generation and ex vivo
manipulation of DCs are laborious. Direct antigen targeting to DCs
in vivo will therefore offer several advantages.
[0004] Therefore, a composition that elicits a robust specific CD8+
T cell response in vivo is of great concern in the field of
therapeutic vaccines. The present invention aims to respond to said
objective by providing a composition that delivers antigen to DCs,
leads to an optimal presentation of peptides derived from the
antigen by HLA-class 1 molecules, and provides a maturation
stimulus for DCs.
[0005] WO02/060937 previously disclosed a carrier for targeting a
molecule to Gb3 receptor expressing cells, said carrier having the
following formula STxB-Z(n)-Cys, wherein STxB is the Shiga Toxin
subunit B, Z is an amino acid linker with no sulfydryl group, n
being 0, 1 or a polypeptide, and Cys is the amino acid Cysteine.
WO02/060937 showed that a STxB based vaccine induced humoral
response and a robust and long-lasting CD8+ T cell response. All
these results may be explained by the ability of STxB to increase
costimulatory and MHC class II molecules on DCs and to induce TNF
on some cells which could indirectly favour the maturation of DCs.
However, when myeloid DCs derived from bone marrow were incubated
with STxB, no maturation of these cells has been observed. Thus,
the STxB based vaccine disclosed in WO02/060937 does not lead to
the maturation of DCs in vivo.
[0006] In one aspect, the present invention aims therefore to
provide a composition that leads to the maturation of DCs.
[0007] Combinations of vectors complexed to an antigen with
adjuvants have already been described, as adjuvants are known to
favour DCs maturation.
[0008] For example, EP 1078007 described the use of a toxin-antigen
conjugate, wherein the toxin is the Shiga toxin B subunit, in
combination with KLH for stimulating an immune response.
[0009] In addition, the patent application WO2005/112991 disclosed
the use of the B subunit of Shiga Toxin complexed with an antigen
and an adjuvant for stimulating an immune response. The adjuvant
may be selected from the group consisting of metal salts, oil in
water emulsions, Toll like receptors agonists, saponins, lipid A,
alkyl glucosaminide phosphate, immunostimulatory oligonucleotide or
combinations thereof.
[0010] However, the Applicant observed and confirmed that all
combinations of vectors and adjuvant are not equivalent for the
induction of CD8+ T cells response. After screening many
conventional adjuvants, the inventors found a dramatic synergy
between ligands of CD1 capable of stimulating NKT cells and the
STxB based vaccine, leading to potent CD8+ T cells response with
the use of very low doses of antigen. Vaccines combining STxB and
one of said ligands were found to be efficient to break tolerance
against self antigen and to elicit anti-viral immunity. Contrary to
the teaching of the prior art, this synergy was not observed with
all adjuvants. In particular, the Applicant did not observed such a
synergy with adjuvants such as IFA (Haicheur et al. JI 2000,
165:3301-3308) and observed a very weak synergy with adjuvants such
as IFN.alpha., Poly(I:C), or the Toll like receptor agonist
CpG.
SUMMARY OF THE INVENTION
[0011] Therefore, an object of the present invention is to provide
a composition comprising
a) a B subunit of Shiga toxin or a functional equivalent thereof
which is able to bind the Gb3 receptor, complexed with an antigen
and b) at least one ligand of CD1 capable of stimulating NK T
cells.
[0012] In one embodiment of the invention, the immunological
functional equivalent of the B subunit of Shiga toxin has at least
50% amino acid sequence identity to the B subunit of Shiga
toxin.
[0013] In another embodiment of the invention, said ligand of CD1
is a ligand of CD1d.
[0014] In another embodiment of the invention, said ligand of CD1
is a glycolipid or phospholipid, a glycosphingolipid, a derivative
or an analog thereof. In a preferred embodiment, said ligand is
chosen from iGb3, GD3, PE and PI.
[0015] In another preferred embodiment, said ligand of CD1 is a
glycosylceramide or an analog or a derivative thereof. Preferably,
said glycosylceramide is selected from the group consisting of
.alpha.-GalCer, .alpha.-GlcCer, Gal.alpha.1-6Gal.alpha.1-1'Cer,
Gal.alpha.1-6Glc.alpha.1-1'Cer, Gal.alpha.1-2Gal.alpha.1-1'Cer,
Gal.beta.1-3Gal.alpha.1-1'Cer or a derivative thereof, preferably a
C-glycoside derivative thereof, more preferably a C-glycoside
derivative of .alpha.-GalCer.
[0016] In one embodiment of the invention, .alpha.GalCer is
(2S,3S,4R)-1-O-(.alpha.-D-galactopyranosyl)-2-(N-hexa-cosanoylamino)-1,3,-
4-octadecanetriol. In another embodiment, .alpha.GalCer is
(2S,3S,4S)-1-O-(.alpha.-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-oc-
tadecanediol (KRN7000).
[0017] In a preferred embodiment of the invention, said ligand of
CD1 is selected from the group consisting of
3-O-sulfo-.alpha.-GalCer, .beta.-GalCer, an OCH compound,
.alpha.-C-GalCer.
[0018] In another embodiment, said ligand of CD1 is a microbe
derived glycolipid. Preferably, said ligand is [0019] a
Sphingomonas species-derived glycosphingolipid selected in the
group consisting of GSL-1 and GSL'1 or [0020] a Borrelia species
derived glycolipid selected from the group consisting of BbGL-I and
BbGL-II or [0021] a Mycobacteria species derived phosphoglycolipid
PIM.
[0022] In one embodiment of the invention, the B subunit of Shiga
toxin or the functional equivalent thereof is present in a
universal polypeptidic carrier having the formula STxB-Z(n)-Cys,
wherein [0023] STxB is the Shiga Toxin B subunit or a functional
equivalent thereof, [0024] Z is an amino-acid devoided of sulfydryl
group, n being 0, 1 or a polypeptide, [0025] Cys is the amino-acid
Cysteine.
[0026] In a preferred embodiment, n is 0.
[0027] In a preferred embodiment, the antigen is covalently linked
to the --S residue of the universal carrier by a --S--S, or
--S--CO, or S--CH.sub.2, or --S--NH linkage. In another preferred
embodiment, the universal carrier is covalently linked to an
oligopeptide or a polypeptide by a --S--S, or --S--CO, or
S--CH.sub.2, or --S--NH linkage, and the antigen to be targeted is
operably linked to the said oligopeptide or polypeptide. In a more
preferred embodiment, the universal carrier is covalently linked to
a poly-lysine oligopeptide and the antigen to be targeted is
operably linked to the said poly-lysine moiety.
[0028] In one embodiment of the invention, the antigen is a tumor
antigen, a viral or a bacterial antigen.
[0029] In one embodiment of the invention, the composition as
described above further comprises a pharmaceutically acceptable
carrier.
[0030] The present invention also aims to provide a medicament
comprising a composition as described above.
[0031] Another object of the present invention is a vaccine
comprising said composition.
[0032] It is also an object of the present invention to provide a
pharmaceutical kit comprising:
[0033] a) a first container comprising a B subunit of Shiga toxin
or a functional equivalent thereof which is able to bind the Gb3
receptor, complexed with an antigen and
[0034] b) at least a second container comprising at least one
ligand of CD1 capable of stimulating NK T cells.
[0035] The present invention relates to the use of a composition as
described above for the manufacture of a pharmaceutical composition
or a medicament, including a vaccine, for treating an
antigen-related condition in an individual, said antigen-related
state being a tumor or an infection.
[0036] In one embodiment, the present invention relates to the use
of said composition for treating an antigen-related condition in an
individual in need thereof, wherein said composition is to be
administrated to said individual in an therapeutically effective
amount for stimulating an immune response against the antigen in
said individual, thereby treating said antigen-related condition in
said individual.
[0037] In another embodiment, the present invention relates to the
use of said composition for treating an antigen-related condition
in an individual in need thereof, wherein said composition is to be
administered in combination with thalidomide or an analog thereof,
preferably lenalidomide.
[0038] The present invention also relates to the use of the kit as
described above, wherein at least one ligand of CD1 capable of
stimulating NK T cells is to be administrated before,
simultaneously or after the B subunit of Shiga toxin or a
functional equivalent thereof which is able to bind the Gb3
receptor, complexed with an antigen.
[0039] In one embodiment, the use of the composition as described
above leads to the stimulation of the immune response, which
comprises stimulating dendritic cells.
[0040] In another embodiment, the use of the composition as
described above leads to the stimulation of the immune response,
which comprises eliciting an antigen specific CD8 response.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1: Role of adjuvants on the levels of
anti-OVA.sub.257-264 CD8.sup.+T cells induced by the STxB-OVA
conjugate. Mice were subcutaneously immunized twice (d0 and d21)
with STxB-OVA alone (50 .mu.g=0.5 nmol) diluted in PBS or combined
with various adjuvants (IFA vol/vol, IFN.alpha. 100000 IU d0, d1
and d2, Poly(I:C) (250 d0 and d21), CpG (50 .mu.g, d0 and d21).
.alpha.-GalCer (2 .mu.g) was only admixed with STxB-OVA on d0. The
second immunization was performed with STxB-OVA alone. CD8.sup.+T
cells from spleen were isolated 7 days after the last immunization
and directly stained ex vivo with PE-labeled OVA.sub.257-264/kb
tetramer and allophycocyanin-labeled anti-CD8 mAb. Values shown
correspond to mean.+-.SD obtained with specific tetramer after
subtracting values from irrelevant tetramer recognizing a
VSV-derived peptide in the context of K.sup.b. These results are
representative of three experiments with 4 mice per group.
[0042] FIG. 2: .alpha.-GalCer increases the efficiency of STxB to
induce long-lasting and functional specific anti-OVA.sub.257-264
CD8.sup.+T cells even at very low doses of antigen. Mice were
subcutaneously immunized twice (d0 and d21) with various doses of
STxB-OVA alone (A Left) or combined with .alpha.-GalCer (A right,
C, D) or with free ovalbumin admixed with .alpha.-GalCer (B). As
mentioned in Material and Methods, .alpha.-GalCer was only
administered during the first immunization. A, B, C: CD8.sup.+T
cells from spleens were isolated 7 days after the last immunization
and directly stained ex vivo with PE-labeled
OVA.sub.257-264/k.sup.b tetramer and allophycocyanin-labeled
anti-CD8 mAb. Results shown are gated on CD8.sup.+T cells. An
irrelevant tetramer recognizing a VSV-derived peptide in the
context of K.sup.b and an isotype control mAb were included as
controls. These results are representative of three experiments
with 4 mice per group. D: Mice were immunized with STxB-OVA (1
.mu.g) mixed with .alpha.-GalCer (2 .mu.g). Left: IFN.gamma.
producing OVA.sub.257-264/K.sup.b specific CD8.sup.+T cells were
detected ex vivo by an IFN.gamma. ELISPOT assay using EL4 pulsed
(.box-solid.) or not (.quadrature.) with the OVA.sub.257-264
peptide (SL8). SFC=spot-forming cells were read using an Elispot
automated reader system. Right: CTL activity was measured in a
standard 4-h .sup.51Cr release assay on the target EL4 cells pulsed
with (.box-solid.) or without (.quadrature.) the OVA.sub.257-264
peptide at an effector-target E/T ratio of 100/1. Three mice per
group were immunized and these experiments were reproduced two
times.
[0043] FIG. 3: .alpha.-GalCer also increases the efficiency of
STxB-E7 to elicit anti-E7 CTL. Mice were immunized on day 0 and day
21 with STxB-E7.sub.43-57 (1 .mu.g) alone or mixed with
.alpha.-GalCer or with the free polypeptide E7.sub.43-57 mixed with
.alpha.-GalCer. Seven days after the last immunization, CD8.sup.+ T
cells from spleen were isolated and directly stained with
PE-labeled E7.sub.49-57/D.sup.b tetramer. (Cells were previously
gated on CD8.sup.+ T cells). An irrelevant tetramer recognizing a
LCMV derived peptide in the context of D.sup.b was included as
controls. These results are representative of two experiments with
3 mice per group.
[0044] FIG. 4: CD4.sup.+T cell and humoral responses after STxB-OVA
based vaccine immunization. Left: Mice (n=3 per group) were
immunized twice (d0 and d21) with STxB-OVA (0.01 nmol) alone or
combined with .alpha.-GalCer (2 .mu.g). As control, mice were
vaccinated with ovalbumin (0.01 nmol) mixed with IFA. Seven days
after the last immunization, CD4.sup.+T cells were purified from
spleen and labeled with CFSE. They were then incubated with T cell
depleted splenocytes as APC pulsed with free ovalbumin protein and
cocultured for 5 days in AIM V serum free medium. Proliferation in
the absence of APC sensitization was subtracted as background from
values obtained after ovalbumin pulsing. These experiments were
reproduced two times. Right: One week after the last immunization,
serum was collected and anti-OVA IgG2a and IgG2b were measured by
ELISA.
[0045] FIG. 5: Combination of STxB-OVA and .alpha.-GalCer enhances
the cross-presentation of ovalbumin by dendritic cells. Mice (n=3
per group) were immunized with STxB-OVA alone (1 .mu.g=0.01 nmol)
or combined with Poly (I:C) or with .alpha.-GalCer (2 .mu.g). Seven
days after vaccination, CD11c.sup.+ enriched dendritic cells were
cocultured with CFSE labeled OT-1 cells for 72 hours. These
experiments were reproduced three times with similar results. Dot
plots were gated on CFSE labeled CD8.sup.+ OT-1 cells.
[0046] FIG. 6: STxB-OVA combined with .alpha.-GalCer primes
anti-OVA.sub.257-264 CD8.sup.+T cells in OVA-TG mice.
[0047] OVA-TG mice were immunized with STxB-OVA (0.1 nmol) combined
or not with .alpha.-GalCer (2 .mu.g). Fourteen days later,
CD8.sup.+ T cells from spleen were isolated and directly stained
with PE-labeled OVA.sub.257-264/K.sup.b tetramer and APC-labeled
anti-CD8 mAb. Each square represents values from individual mice
and corresponds to results obtained with specific
OVA.sub.257-264/K.sup.b tetramer after subtracting the values
obtained with an irrelevant tetramer recognizing a VSV derived
peptide in the context of K.sup.b. Two series of experiments were
performed with similar results.
[0048] FIG. 7: STxB-OVA combined with .alpha.-GalCer induce
anti-viral protective immunity against a challenge with rVV-OVA.
Mice (5 mice per group) were injected twice (d0 and d21)
intraperitoneally (200 .mu.l) either with B-OVA alone (0.05 nmol=5
.mu.g), or B-OVA (0.05 nmol) combined with .alpha.-GalCer, or OVA
(0.05 nmol) mixed with .alpha.-GalCer and control mice were
injected with PBS. Similarly to the previous experiments,
.alpha.-GalCer was not added during the second immunization. Eight
days after the last injection mice were challenged
intraperitoneally with 2.5.times.10.sup.6 PFU recombinant vaccina
virus (rVV-OVA, Westerns Reserve strains) expressing ovalbumin
cDNA. After 4 days, ovaries were assayed for rVV titers by plaque
assay on BHK 21 cells. Results represent mean of pfu from 6 mice
per group. P values were calculated by a Student't test.
[0049] FIG. 8: No adjuvant effect of .alpha.-GalCer in mice
deficient in NKT cells. Ja18.sup.-/- mice were immunized with
STxB-OVA (1 .mu.g) or STxB-OVA+.alpha.-GalCer (2 .mu.g). 7 days
after immunization, the spleens of immunized and non-immunized mice
were harvested and stained with anti-CD8 antibody and
OVA.sub.257-264/K.sup.b tetramer.
[0050] FIG. 9: Vaccination with .alpha.GalCer combined with
STxB-Ova induces protection against established tumors. C57BL6 mice
were grafted with EG7 tumor and three days after were vaccinated
with PBS, STxB-OVA, .alpha.-GalCer, or STxB-OVA+.alpha.-GalCer.
DETAILED DESCRIPTION OF THE INVENTION
I. Definition
[0051] By "individual", it is meant mammal, in particular a human
being.
[0052] By "effective amount", it is meant an amount sufficient to
effect a beneficial or desired clinical result (e.g. improvement in
clinical condition).
[0053] As used herein, "treatment" or "treating" generally refers
to a clinical intervention in an attempt to alter the natural
course of the individual or cell being treated, and may be
performed either for prophylaxis or during the course of clinical
pathology. Desirable effects include, but are not limited to,
preventing occurrence or recurrence of disease, alleviating
symptoms, suppressing, diminishing or inhibiting any direct or
indirect pathological consequences of the disease, preventing
metastasis, lowering the rate of disease progression, ameliorating
or palliating the disease state, and causing remission or improved
prognosis.
[0054] The term "NKT cells" or "natural killer cell" is known in
the art, and as used herein, refers to a T cell population that
causes, stimulates or contributes to cytokine production, and/or in
another embodiment, is cytotoxic. NKT cells are characterised by
expression of both a T cell antigen receptor (TCR) and NK cell
marker (i.e. NK 1.1, a C-lectin-type NK receptor, DX5, Ly49
receptors in mice and NKR-P1A in human).
[0055] The term "Th1 cytokine" is known in the art and as used
herein, refers to cytokine elicited by T helper cells as part of
the adaptative immune response. Typically, Th1 cytokines are
interleukin-2 or interferon-.gamma. for example.
[0056] The term "Th2 cytokine" is known in the art, and as used
herein, refers to cytokine elicited by T helper cells as part of
the adaptative immune response. Typically, Th2 cytokine are
interleukine-4 or interleukine-10, for example.
[0057] The term "dendritic cell" (DC) is known in the art, and as
used herein, refers to antigen-presenting cells, which are capable
of presenting antigen to T cells.
[0058] The term "mature dendritic cells" is known in the art, and
as used herein, refers to a population of dendritic cells with
diminished CD115, CD14, CD68 or CD32 expression, or a population of
dendritic cells with enhanced CD86 expression, or a combination
thereof.
[0059] The term "stimulating an immune response" as used herein
refers to the initiation of an immune response against an antigen
of interest in an individual in which an immune response against
said antigen has not already been initiated or refers to any
improvement in an immune response that has already been mounted by
an individual. It is to be understood that reference to the
stimulation of the immune response may involve both the humoral and
cell-mediated arms of the immune system. In one embodiment;
stimulation of the immune response resulting in the stimulation of
the humoral immune response may be reflected by an increase in
antibody production and a TH2 cytokine profile (IL-4, 11-5, IL-6 .
. . ) which can be determined by any means known in the art, such
as for example by ELISA. In another embodiment, stimulation of the
immune response resulting in the stimulation of the cell-mediated
response may be reflected by an increase in IFN-.gamma. or IL-12,
or both, which may be similarly determined. In another embodiment,
stimulating the immune response is associated with a change in
cytokine expression. Such change may be readily measured by any
means well-known in the art, such as ELISA, Western-Blot analysis,
PCR analysis, and others.
[0060] The term "antigen" is known in the art, and as used herein,
refers to any agent (protein, peptide, polysaccharide,
glycoprotein, glycolipid, nucleic acid, or combination thereof),
which elicits an immune response when introduced into a host and
those which are able to elicit an immune response when complexed
with the B subunit of Shiga toxin according to the invention. The
term "antigen epitope" includes fragments of proteins capable of
determining antigenicity. For example, antigens include proteins
and other molecules which are specifically associated with surfaces
of particular types of cancer cells, i.e. tumour cells.
Alternatively, antigens may be associated with the surfaces or
secretion products of micro-organisms or pathogens. The term
"pathogen" is meant to include organisms that cause disorders, such
disorders produced by one or more particular species of bacteria,
viruses, fungi and protozoans which are disease-producing
organisms.
[0061] The term "antigen-related state" or "antigen-related
condition" as used herein refers to micro-organism or pathogenic
infections, allergen associated states or refers to the presence of
a tumour.
[0062] The term "adjuvant" as used herein refers to a compound or a
mixture that may be non-immunogenic when administered in the host
alone, but that augments the host's immune response to an antigen
when administered conjointly with that antigen.
[0063] The term "vaccine" as used herein refers to a composition
that can be used to elicit protective immunity in a recipient.
According to the present invention, a vaccine is a medicament.
II. The Present Invention
[0064] The present invention relates to a composition
comprising
[0065] a) a B subunit of Shiga toxin or a functional equivalent
thereof which is able to bind the Gb3 receptor, complexed with an
antigen and
[0066] b) at least one ligand of CD 1 capable of stimulating NK T
cells.
[0067] The B subunit of Shiga toxin is secreted by Shigella
dysenteriae. This homopentamer is responsible for toxin binding to
and internalisation into target cells by interacting with the
glycolipid Gb3 found in the plasma membrane of these cells. The B
subunit of Shiga toxin has the sequence described in NA Strockbine
et al. J Bacteriol 1988, 170, 1116-22.
[0068] A functional equivalent of the B subunit of Shiga toxin
means a polypeptidic sequence having the capacity to bind
specifically to the Gb3 receptor and/or to trigger an
internalisation of an antigen and its presentation in an MHC class
I restricted pathway, or both MHC class I and class II on the same
antigen presenting cell.
[0069] Additionally, functional equivalents of the B subunit of
Shiga toxin include homologous toxins, which are able to bind the
Gb3 receptor, from other bacteria. For example, the B subunits of
verotoxin-1 or verotoxin-2 from E Coli are also known to bind the
Gb3 receptor. In the context of the present invention, the term
"toxin" is intended to mean toxins that have been detoxified such
that they are no longer toxic to humans, or a toxin subunit or
fragment thereof that are substantially devoid of toxic activity in
humans.
[0070] In one embodiment of the present invention, the functional
equivalent of the B subunit of Shiga toxin has at least 50%, and
preferably 60, 70, 80, 90 or 95%, amino acid identity to the B
subunit of Shiga toxin.
[0071] The capacity of polypeptidic sequence to bind specifically
to the Gb3 receptor may be evaluated by the following assay which
is based on the method described by Tarrago-Trani (Protein
extraction and purification 39, pp 170-176, 2004) and involves an
affinity chromatography on a commercially available
galabiose-linked agarose gel (Calbiochem). Galabiose (Gala1-4Gal)
is the terminal carbohydrate portion of the oligosaccharide moiety
of Gb3 and is thought to represent the minimal structure recognized
by the B subunit of Shiga toxin. The protein of interest in PBS
buffer (500 .mu.l) is mixed with 100 .mu.l of immobilized galabiose
resin previously equilibrated with the same buffer, and incubated
for 30 min to 1 hour at 4.degree. C. on a rotating wheel. After a
first centrifugation at 5000 rpm for 1 min, the pellet is washed
twice with PBS. The bound material is then eluated twice by
re-suspending the final pellet in 2.times.500 .mu.l of 100 mM
glycine pH 2.5. Samples corresponding to the flow-through, the
pooled washes and the pooled eluates are then analysed by SDS Page,
Coomassie staining and Western blotting.
[0072] CD1 molecules are a family of highly conserved antigen
presenting proteins that are similar to in function to classical
MHC molecules. CD1 proteins bind and display a variety of lipids
and glycolipids to T lymphocytes. The five known isoforms are
classified into two groups, group 1 (CD1a, CD1b, CD1c and CD1e in
humans) and group II (CD1d in humans and mice).
[0073] In one embodiment, the ligand of CD1 present in the
composition of the invention is a ligand of CD1d.
[0074] Certain ligands of CD1 molecule, when bound, stimulate NKT
cells: for example they stimulate rapid Th1 and Th2 cytokine
production by NKT cells.
[0075] In one embodiment of the present invention, said ligand
induces Th1 and Th2 cytokine production, such as IL-4 and
IFN-.gamma.. Such cytokine production may be readily measured by
any means well-known in the art, such as ELISA or by flow cytometry
for example. In another embodiment, said ligand induces an increase
in the expression of CD25, CD69 or Fas Ligand molecules, or an
increase in the production of perforin. Such increase may be
measured by any means well-known in the art, such as flow-cytometry
for example.
[0076] In one embodiment of the present invention, said ligand of
CD1 capable of stimulating NKT cells is a glycolipid, a
phospholipid, a glycosphingolipid, a derivative or an analog
thereof.
[0077] Glycosphingolipids are complex glycolipids which contain
ceramide as an extra-lipid component. For example, Kirin
Pharmaceutical in Japan identified several glycosphingolipids
compounds, named agelasphins, from an extract of the Okinawan
marine sponge, Agela mauritianus (Natori et al. Tetrahedron 1994,
50:2771-2784).
[0078] Other examples of glycolipid are: the lysosomal
glycosphingolipid, isoglobotrihexosylceramide iGB3 (Zhou et al.
Science 2004, 306:1786-1789); the disialoganglioside GD3 (Wu et
al., J. Exp. Med. 2003, 198:173-181); the phosphatidylinositol PI
(Gumperz et al. Immunity 2000, 12:211-221) and the
phosphatidylethanolamine PE (Rauch et al. J. Bioch. Chem 2003,
278:47508-47515).
[0079] In one embodiment of the invention, said ligand is chosen
from GD3, PE and PI.
[0080] In one embodiment of the present invention, said ligand is
the sphingolipid as described in the U.S. Pat. No. 5,780,441, said
sphingolipid having the following formula:
##STR00001##
[0081] Wherein R1 represents H or
##STR00002##
[0082] R2 represents H,
##STR00003##
[0083] R3 and R6 represent H or OH, respectively;
[0084] R4 represents H, OH or
##STR00004##
[0085] R5 represents H or
##STR00005##
[0086] X denotes an integer from 19 to 23; and R7 represents any
one of the following groups (a)-(g):
[0087] (a) --(CH.sub.2).sub.11--CH.sub.3,
[0088] (b) --(CH.sub.2).sub.12--CH.sub.3,
[0089] (c) --(CH.sub.2).sub.13--CH.sub.3,
[0090] (d) --(CH.sub.2).sub.9--CH(CH.sub.3).sub.2,
[0091] (e) --(CH.sub.2).sub.10--CH(CH.sub.3).sub.2,
[0092] (f) --(CH.sub.2).sub.11--CH(CH.sub.3).sub.2,
[0093] (g) --(CH.sub.2).sub.11--CH(CH.sub.3)--C.sub.2H.sub.5,
[0094] wherein at least one of R1, R2, R4 and R5 is a glycosyl
moiety.
[0095] In another embodiment of the present invention, said ligand
is an .alpha.-galactosylceramide as described in the U.S. Pat. No.
5,936,076, said .alpha.-galactosylceramide having the following
formula:
##STR00006##
[0096] wherein R represents
##STR00007##
[0097] where R2 represents H or OH and X denotes an integer of 0-26
or R represents (CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7CH.sub.3
and
[0098] R1 represents any one of the substituents defined by the
following (a)-(e):
[0099] (a) --CH.sub.2(CH.sub.2).sub.yCH.sub.3,
[0100] (b) --CH(OH)(CH.sub.2).sub.2CH.sub.3,
[0101] (c) --CH(OH)(CH.sub.2).sub.yCH(CH.sub.3).sub.2,
[0102] (d) --CH.dbd.CH(CH.sub.2).sub.yCH.sub.3, and
[0103] (e)
--CH(OH)(CH.sub.2).sub.yCH(CH.sub.3)CH.sub.2CH.sub.3,
[0104] wherein Y denotes an integer of 5-17.
[0105] In another embodiment of the present invention, said ligand
is an .alpha.-galactosylceramide as described in the U.S. Pat. No.
6,555,372, said .alpha.-galactosylceramide having the following
formula:
##STR00008##
[0106] wherein R1 represents H or OH, X represents an integer
between 7 and 27, R2 represents a substituent selected from the
group consisting of the following (a) to (e) (wherein Y represents
an integer between 5 and 17):
[0107] (a) --CH.sub.2(CH.sub.2).sub.YCH.sub.3
[0108] (b) --CH(OH)(CH.sub.2).sub.YCH.sub.3
[0109] (c) --CH(OH)(CH.sub.2).sub.YCH(CH.sub.2).sub.2
[0110] (d) --CH.dbd.CH(CH.sub.2).sub.Y(CH.sub.3)
[0111] (e)
--CH(OH)(CH.sub.2).sub.YCH(CH.sub.3)CH.sub.2CH.sub.3,
[0112] and R3 to R9 represent substituents as defined in any one of
the following i) and ii):
[0113] i) when R3, R6 and R8 represent H, R4 represents H, OH,
NH.sub.2, NHCOCH.sub.3, or a substituent selected from the group
consisting of the following groups (A) to (D):
##STR00009##
[0114] R5 represents OH or a substituent selected from the group
consisting of the following groups (E) and (F):
##STR00010##
[0115] R7 represents OH or a substituent selected from the group
consisting of the following groups (A) to (D):
##STR00011##
[0116] R9 represents H, CH.sub.3, CH.sub.2OH or a substituent
selected from the group consisting of the following groups (A') to
(D'):
##STR00012##
[0117] ii) when R3, R6 and R7 represent H, R4 represents H, OH,
NH.sub.2, NHCOCH.sub.3, or substituent selected from the group
consisting of the following groups (A) to (D):
##STR00013##
[0118] R5 represents OH or a substituent selected from the group
consisting of groups (E) and (F):
##STR00014##
[0119] R8 represents OH or a substituent selected from the group
consisting of the following groups (A) to (D):
##STR00015##
[0120] R9 represents H, CH3, CH2OH or a substituent selected from
the group consisting of the following groups (A') to (D'):
##STR00016##
[0121] or a salt or solvate thereof.
[0122] In a preferred embodiment of the present invention; said
ligand is the compound KRN7000, having the following formula:
(2S,3S,4S)-1-(.alpha.-D-galactopyranosyloxy)-2-hexacosanoymamino-3,4-octa-
decanediol.
[0123] In another embodiment of the present invention, said ligand
is a glycolipid derivative as described in the patent application
US2002/0032158, said glycolipid derivative having the following
formula:
##STR00017##
[0124] wherein W represents carbone chain from 9 to 17 which
containing double bond or hydroxy group occasionally; X represents
carbone chain from 11 to 25 which containing double bond or hydroxy
group occasionally; Y represents
--(CH.sub.2).sub.a--CH.dbd.CH--(CH.sub.2).sub.a--,
--(CH.sub.2).sub.a-- (a, a' denotes an integer of 0-5 and a+a' is 5
and under.), --S(O).sub.0-2CH.sub.2--, --NHCH.sub.2; Z represents
--CO--, --SO.sub.2; R represents --CH.sub.2OH, --CO.sub.2H,
--CH.sub.2OCH.sub.2CO.sub.2H, --CH.sub.2OSO.sub.3H; R.sub.0
represents --OH, --NH.sub.2, --NHAc.
[0125] In another embodiment of the present invention, said ligand
is a glycosylceramide as described in the patent application
US2003/0157135, said glycosylceramide having the following
formula:
##STR00018##
[0126] wherein R1, R2 and R5 represent H or a specific
monosaccharide; R3 and R6 represent H or OH, respectively; R4
represents H, OH or a specific monosaccharide; X denotes an integer
from 1 to 23; R7 represents any one of the following groups
(a)-(g):
[0127] (a) --(CH.sub.2).sub.11--CH.sub.3,
[0128] (b) --(CH.sub.2).sub.12--CH.sub.3,
[0129] (c) --(CH.sub.2).sub.13--CH.sub.3,
[0130] (d) --(CH.sub.2).sub.9--CH(CH.sub.3).sub.2,
[0131] (e) --(CH.sub.2).sub.10--CH(CH.sub.3).sub.2,
[0132] (f) --(CH.sub.2).sub.11--CH(CH.sub.3).sub.2,
[0133] (g) --(CH.sub.2).sub.11--CH(CH.sub.3)--C.sub.2H.sub.5.
[0134] In a preferred embodiment, said ligand is a glycosylceramide
or an analog or a derivative thereof.
[0135] In a more preferred embodiment, said ligand is selected from
the group consisting of .alpha.-galactosylceramide
(.alpha.-GalCer), .alpha.-glucosylceramide (.alpha.-GlcCer),
Gal.alpha.1-6Gal.alpha.1-1'Cer, Gal.alpha.1-6Glc.alpha.1-1'Cer,
Gal.alpha.1-2Gal.alpha.1-1'Cer, Gal.beta.1-3Gal.alpha.1-1'Cer or a
derivative thereof, preferably a C-glycoside derivative thereof,
more preferably a C-glycoside derivative of .alpha.-GalCer.
[0136] In another preferred embodiment, said ligand has the
following formula
(2S,3S,4R)-1-O-(.alpha.-D-galactopyranosyl)-2-(N-hexa-cosanoylami-
no)-1,3,4-octadecanetriol.
[0137] In another embodiment, said ligand has the following formula
(2S,3S,4S)-1-O-(.alpha.-D-galactopyranosyloxy)-2-hexacosanolamino-3,4-oct-
adecanediol (KRN7000).
[0138] In another embodiment, said ligand is an .alpha.-GalCer
analog, called .alpha.-C-GalCer, as described in the patent
application US2004/0127429, said ligand having the following
formula:
##STR00019##
[0139] wherein X is O or NH; R1 is selected from the group
consisting of --(CH.sub.2).sub.11CH.sub.3,
--(CH.sub.2).sub.12CH.sub.3, --(CH.sub.2).sub.13CH.sub.3,
--(CH.sub.2).sub.9CH(CH.sub.3).sub.2,
--(CH.sub.2).sub.10CH(CH.sub.3).sub.2, --(CH.sub.2).sub.11
CH(CH.sub.3).sub.2 and
(CH.sub.2).sub.11CH(CH.sub.2)--C.sub.2H.sub.5;
[0140] R3 is OH or a monosaccharide and R4 is hydrogen, or R3 is
hydrogen and R4 is OH or a monosaccharide;
[0141] R5 is hydrogen or a monosaccharide;
[0142] Q.sub.1 is optionally present and is a C.sub.1-10 straight
or branched chain alkylene, alkenylene, or alkynylene;
[0143] X'' is optionally present and is O, S or NR.sup.8;
[0144] Q.sub.2 is optionally present and is a C.sub.1-10 straight
or branched chain alkylene, alkenylene or alkynylene;
[0145] X'' is optionally present and is O, S or NR.sup.8;
[0146] Q.sub.3 is a straight or branched chain C.sub.1-10 alkylene,
alkenylene or alkynylene, or is hydrogen, wherein each Q', Q.sup.2
or Q.sup.3 is optionally substituted with hydroxyl, halogen, cyano,
nitro, SO.sub.2 NHR.sup.8, or C(.dbd.O)--R.sup.9; and wherein
[0147] R8 is hydrogen, C.sub.1-5 alkyl, C.sub.1-5 alkoxy, halogen,
cyano, nitro, SO.sub.2 or C(.dbd.O)--R.sup.9;
[0148] R9 is hydrogen, C.sub.1-5 alkyl, C.sub.1-5 alkoxy or
NHR.sup.10;
[0149] R10 is hydrogen, C.sub.1-5 alkyl or C.sub.1-5 alkoxy;
[0150] and a pharmaceutically acceptable salt or ester thereof.
[0151] In another embodiment, said ligand is PBS-57, which
structure was described by Liu et al. Journal of Immunological
Methods, 312 (2006) 34-39.
[0152] In another embodiment, said ligand is a C-glycolipid, as
described in the patent application US2005/0222048, having the
following formula:
##STR00020##
[0153] wherein X is O or NH; R3 is OH or a monosaccharide and R4 is
hydrogen, or R3 is hydrogen and R4 is OH or a monosaccharide;
R.sup.5 is hydrogen or a monosaccharide; and pharmaceutically
acceptable salts or esters thereof.
[0154] In another embodiment of the present invention, said ligand
is an immunogenic compound as described in the patent application
US2006/0211856, having the following formula:
##STR00021##
[0155] wherein, R.dbd.COOR1 or CH.sub.2OR1;
[0156] R1=H or an alkyl group;
[0157] R2=H or SO.sub.3.sup.-;
[0158] R3=H or OH;
[0159] R.sub.3'=H or OH;
[0160] R.sub.4'=H, unsaturated or saturated, alkyl group;
unsaturated or saturated, alkyl group; and
[0161] R5=0H, acetamido or a halogen atom;
[0162] or a pharmaceutically acceptable salt thereof,
[0163] wherein if R.dbd.CH.sub.2OR.sub.1, R.sub.2.dbd.H, R.sub.3',
is OH and R.sub.3 is H, then R.sub.5=acetamido, halogen atom or OH
in an axial position or R.sub.4.dbd.H, unsaturated or saturated,
alkyl chain having 9 carbon atoms or fewer, or R.sub.4'=H,
unsaturated or saturated, alkyl chain having 20 carbon atoms or
fewer.
[0164] In a preferred embodiment of the present invention, said
ligand is selected from 3-O-sulfo-.alpha.-GalCer, .beta.-GalCer, an
OCH compound, .alpha.-C-GalCer.
[0165] The OCH compound is described in the patent application
US2006/0148723, and has the following formula:
##STR00022##
[0166] wherein, R1 is an aldopyranose group, R2 is a hydrogen atom
or a hydroxyl group, R3 is --CH.sub.2--, --CH(OH)--CH.sub.2-- or
--CH.dbd.CH--, R4 is a hydrogen atom or CH.sub.3, x is 0-35, y and
z represent integers satisfying y+z=0-3.
[0167] .alpha.-C-GalCer has been described above.
3-O-sulfo-.alpha.-GalCer is a sulfatide variant of .alpha.-GalCer
and has been described in the article Wu et al. (Wu et al. PNAS
2005, 102:1352-1356).
[0168] In another embodiment of the present invention, said ligand
is a microbe derived glycolipid.
[0169] In a preferred embodiment, said ligand is [0170] a
Sphingomonas species-derived glycosphingolipid selected in the
group consisting of GSL-1 and GSL'1 or [0171] a Borrelia species
derived glycolipid selected from the group consisting of BbGL-I and
BbGL-II or [0172] a Mycobacteria species derived phosphoglycolipid
PIM.
[0173] In one embodiment of the present invention, the composition
of the invention comprises the B subunit of Shiga toxin, or the
functional equivalent thereof, present in a universal polypeptidic
carrier having the formula STxB-Z(n)-Cys, wherein [0174] STxB is
the Shiga Toxin B subunit or a functional equivalent thereof,
[0175] Z is an amino-acid devoided of sulfydryl group, n being 0, 1
or a polypeptide, [0176] Cys is the amino-acid Cysteine.
[0177] This universal carrier has been described in the patent
application WO02/060937.
[0178] The STxB moiety of the universal carrier has the sequence
described in NA Strockbine et al. J Bacteriol 1988, 170, 1116-22,
or a functional equivalent thereof.
[0179] In a preferred embodiment, n is 0 and the universal carrier
has the following sequence
TABLE-US-00001 (SEQ ID NO: 1): COOH-MKKTLLIAASLSFFSASALATPDCVTGKVE
YTKYNDDDTFTVKVGDKELF
TNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFRC-NH2.
[0180] As a matter of fact, if the Z linker is too long, i.e. when
n is equal or greater than 2, some internal disulfide bridges might
occur, and prevent either the binding of STxB to Gb3 receptor.
[0181] In another embodiment of the invention, the antigen is to be
targeted to antigen presentating cells. Such cells are selected in
a group comprising T lymphocytes, dendritic cells, macrophages,
Langerhans cells and the like.
[0182] The coupling approaches for covalent binding of an antigen
to STxB-Z (n)-Cys can be any method or processes described or
carried out by a skilled person.
[0183] A first method that can be embodied is the use of SPDP
hetero-bi-functional cross-linker described par Carlsson et al (5).
However, SPDP is capable of being cleavable by serumthiolases that
is a cause of decreasing the yield of the reaction.
[0184] A second method for covalent coupling of STxB-Z (n)-Cys
peptides with an antigen is to produce bromoacetyl or maleimide
functions on the latter as described by P. Schelte et al (4).
[0185] Briefly, the antigen is chemically activated with
bromoacetate anhydride or by a maleimide group respectively. In
appropriate reaction conditions (pH, temperature, incubation
times), these groups are eliminated by cis-elimination, yielding
respectively to --S--S, --S--CH2-, to --S--CO- or S--NH-covalent
linkages.
[0186] As an example, the antigen to be coupled to the --SH moiety
the C-terminal Cysteine of the universal carrier, has its
N-terminus activated with bromoacetic anhydride following the
reaction scheme:
Br--CH2-CO--O--CO--CH2-Br+NH2-antigen=>Br--CH2-CO--NH-antigen+Br--CH2-
-COOH
[0187] The Bromoacetyl function has high chemoselectivity for
peptide thiol groups and the activated peptide can be reacted with
STxB-Cys as follows:
STxB-Cys-SH+Br--CH2-CO--NH-antigen=>STxB-Cys-S--CH2-CO--NH-antigen+HB-
r
[0188] The resulting thioether-linkage is stable to hydrolysis.
[0189] Another method for coupling an antigen to the universal
carrier of the invention is to use MBS
(m-Maleimidobenzoyl-N-hydroxysuccinimide ester).
[0190] This coupling allows the transport and processing of large
molecules such antigenic proteins or glycoproteins through MHC
class I and/or MHC class 11 pathways.
[0191] Thus, in one embodiment of the invention, the antigen is
covalently linked to the --S residue of the universal carrier by a
--S--S, or --S--CO, or S--CH.sub.2, or --S--NH linkage.
[0192] In another embodiment, the universal carrier according to
the present invention can be operably linked directly through a
covalent binding or indirectly through a linker.
[0193] The term "indirect binding" means that the universal carrier
is covalently linked through the sulfhydryl moiety of the
C-terminal Cysteine to a linker, said linker being operably linked
to an antigen to be internalized into Gb3 receptor bearing
cells.
[0194] This linkage might be a covalent binding or a non-covalent
binding, provided that the affinity between the linker and the
antigen is higher than 10.sup.-9 mole/1.
[0195] Thus, the universal carrier is covalently linked to an
oligopeptide or a polypeptide by a --S--S, or --S--CO, or
S--CH.sub.2, or --S--NH linkage, and the antigen to be targeted is
operably linked to the said oligopeptide or polypeptide. In a
preferred embodiment, the universal carrier is covalently linked to
a poly-lysine oligopeptide and the antigen to be targeted is
operably linked to the said poly-lysine moiety.
[0196] In one embodiment of the present invention, the antigen
complexed to the B subunit of Shiga toxin is a tumor antigen, a
viral antigen or a bacterial antigen.
[0197] In a preferred embodiment, the antigen is selected such that
it provides immunity against intracellular pathogens such as HIV,
tuberculosis, Chlamydia, HBV, HCV and influenza. Preferably, the
antigen is derived from HIV (such as gag or fragments thereof, such
as p24, tat, nef, envelope such as gp120 or gp160, or fragments of
any of these), human herpes viruses, such as gD or derivatives
thereof or Immediate Early protein such as ICP27 from HSV1 or HSV2,
cytomegalovirus ((esp Human) (such as gB or derivatives thereof),
Rotaviral antigen, Epstein Barr virus (such as gp350 or derivatives
thereof), Varicella Zoster Virus (such as gp1, 11 and 1E63), or
from a hepatitis virus such as hepatitis B virus (for example
Hepatitis B Surface antigen or a derivative thereof), or antigens
from hepatitis A virus, hepatitis C virus and hepatitis E virus, or
from other viral pathogens, such as paramyxoviruses: Respiratory
Syncytial virus (such as F G and N proteins or derivatives
thereof), parainfluenza virus, measles virus, mumps virus, human
papilloma viruses (for example HPV 6, 11, 16, 18,) flaviviruses
(e.g. Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis
virus, Japanese Encephalitis Virus) or Influenza virus purified or
recombinant proteins thereof, such as HA, NP, NA, or M proteins, or
combinations thereof), or derived from bacterial pathogens such as
Neisseria spp, including N. gonorrhea and N. meningitidis (for
example, transferrin-binding proteins, lactoferrin binding
proteins, PiIC, adhesins); S. pyogenes (for example M proteins or
fragments thereof, C5A protease,), S. agalactiae, S. mutans; H.
ducreyi; Moraxella spp, including M catarrhalis, also known as
Branhamella catarrhalis (for example high and low molecular weight
adhesins and invasins); Bordetella spp, including B. pertussis (for
example pertactin, pertussis toxin or derivatives thereof,
filamenteous hemagglutinin, adenylate cyclase, fimbriae), B.
parapertussis and B. bronchiseptica; Mycobacterium spp., including
M. tuberculosis (for example ESAT6, Antigen 85A, -B or -C), M.
bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis;
Legionella spp, including L. pneumophila; Escherichia spp,
including enterotoxic E. coli (for example colonization factors,
heat-labile toxin or derivatives thereof, heat-stable toxin or
derivatives thereof), enterohemorragic E. coli, enteropathogenic E.
coli Vibrio spp, including V. cholera (for example cholera toxin or
derivatives thereof); Shigella spp, including S. sonnei, S.
dysenteriae, S. flexnerii; Yersinia spp, including Y.
enterocolitica (for example a Yop protein), Y. pestis, Y.
pseudotuberculosis; Campylobacter spp, including C. jejuni (for
example toxins, adhesins and invasins) and C. coli; Salmonella spp,
including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis;
Listeria spp., including L. monocytogenes; Helicobacter spp,
including H. pylori (for example urease, catalase, vacuolating
toxin); Pseudomonas spp, including P. aeruginosa; Staphylococcus
spp., including S. aureus, S. epidermidis; Enterococcus spp.,
including E. faecalis, E. faecium; Clostridium spp., including C.
tetani (for example tetanus toxin and derivative thereof), C.
botulinum (for example botulinum toxin and derivative thereof), C.
difficile (for example clostridium toxins A or B and derivatives
thereof); Bacillus spp., including B. anthracis (for example
botulinum toxin and derivatives thereof); Corynebacterium spp.,
including C. diphtheriae (for example diphtheria toxin and
derivatives thereof); Borrelia spp., including B. burgdorferi (for
example OspA, OspC, DbpA, DbpB), B. garinii (for example OspA,
OspC, DbpA, DbpB), B. afzelii (for example OspA, OspC, DbpA, DbpB),
B. andersonii (for example OspA, OspC, DbpA, DbpB), B. hermsii;
Ehrlichia spp., including E. equi and the agent of the Human
Granulocytic Ehrlichiosis; Rickettsia spp, including R. rickettsii;
Chlamydia spp., including C. trachomatis (for example MOMP,
heparin-binding proteins), C. pneumoniae (for example MOMP,
heparin-binding proteins), C. psittaci; Leptospira spp., including
L. interrogans; Treponema spp., including T. pallidum (for example
the rare outer membrane proteins), T. denticola, T. hyodysenteriae;
or derived from parasites such as Plasmodium spp., including P.
falciparum; Toxoplasma spp., including T. gondii (for example SAG2,
SAGS, Tg34); Entamoeba spp., including E. histolytica; Babesia
spp., including B. microti; Trypanosoma spp., including T. cruzi;
Giardia spp., including G. lamblia; Leshmania spp., including L.
major; Pneumocystis spp., including P. carinii; Trichomonas spp.,
including T. vaginalis; Schisostoma spp., including S. mansoni, or
derived from yeast such as Candida spp., including C. albicans;
Cryptococcus spp., including C. neoformans.
[0198] Other preferred specific antigens for M. tuberculosis are
for example Tb Ra12, Tb H9, Tb Ra35, Tb38-1, Erd 14, DPV, MTI, MSL,
mTTC2 and hTCC1 (WO 99/51748).
[0199] Proteins for M. tuberculosis also include fusion proteins
and variants thereof where at least two, preferably three
polypeptides of M. tuberculosis are fused into a larger protein.
Preferred fusions include Ra12-TbH9-Ra35, Erd14-DPV-MTI,
DPV-MTI-MSL, Erd14-DPV-MTI-MSL-mTCC2, Erd14-DPV-MTI-MSL,
DPV-MTI-MSL-mTCC2, TbH9-DPV-MTI (WO 99/51748).
[0200] Most preferred antigens for Chlamydia include for example
the High Molecular Weight Protein (HMW) (WO 99/17741), ORF3 (EP 366
412), and putative membrane proteins (Pmps). Other Chlamydia
antigens of the vaccine formulation can be selected from the group
described in WO 99/28475.
[0201] Preferred bacterial antigens are derived from Streptococcus
spp, including S. pneumoniae (for example, PsaA, PspA,
streptolysin, choline-binding proteins) and the protein antigen
Pneumolysin (Biochem Biophys Acta, 1989, 67, 1007; Rubins et al.,
Microbial Pathogenesis, 25, 337-342), and mutant detoxified
derivatives thereof (WO 90/06951; WO 99/03884). Other preferred
bacterial vaccines comprise antigens derived from Haemophilus spp.,
including H. influenzae typeB, non typeable H. influenzae, for
example OMP26, high molecular weight adhesins, P5, P6, protein D
and lipoprotein D, and fimbrin and fimbrin derived peptides (U.S.
Pat. No. 5,843,464) or multiple copy varients or fusion proteins
thereof.
[0202] Derivatives of Hepatitis B Surface antigen are well known in
the art and include, inter alia, those PreS1, PreS2 S antigens set
forth described in European Patent applications EP-A-414 374;
EP-A-0304 578, and EP 198-474.
[0203] In another embodiment, the antigen is derived from the Human
Papilloma Virus (HPV) considered to be responsible for genital
warts (HPV 6 or HPV 11 and others), and the HPV viruses responsible
for cervical cancer (HPV16, HPV18. and others). Particularly
preferred forms of genital wart prophylactic, or therapeutic,
vaccine comprise L1 protein, and fusion 5, proteins comprising one
or more antigens selected from the HPV proteins E1, E2, E5, E6, E7,
L1, and L2. The most preferred forms of fusion protein are: L2E7 as
disclosed in WO 96/26277, and proteinD(1/3)-E7 disclosed in
WO99/10375. A preferred HPV cervical infection or cancer,
prophylaxis or therapeutic vaccine, composition may comprise HPV 16
or 18 antigens. Particularly preferred HPV 16 antigens comprise the
early proteins E6 or E7 in fusion with a protein D carrier to form
Protein D-E6 or E7 fusions from HPV 16, or combinations thereof; or
combinations of E6 or E7 with L2 (WO 96/26277). Alternatively the
HPV 16 or 18 early proteins E6 and E7, may be presented in a single
molecule, preferably a Protein D-E6/E7 fusion. Such vaccine may
optionally contain either or both E6 and E7 proteins from HPV 18,
preferably in the form of a Protein D-E6 or Protein D-E7 fusion
protein or Protein D E6/E7 fusion protein.
[0204] Antigens may also derived from parasites that cause Malaria,
for example, antigens from Plasmodia falciparum including
circumsporozoite protein (CS protein), RTS, S, MSP1, MSP3, LSA1,
LSA3, AMA1 and TRAP. RTS is a hybrid protein comprising
substantially all the C-terminal portion of the circumsporozoite
(CS) protein of P. falciparum linked via four amino acids of the
preS2 portion of Hepatitis B surface antigen to the surface (S)
antigen of hepatitis B virus. Its full structure is disclosed in
International Patent Application No. PCT/EP92/02591, published
under Number WO 93/10152 claiming priority from UK patent
application No. 9124390.7. When expressed in yeast RTS is produced
as a lipoprotein particle, and when it is co-expressed with the S
antigen from HBV it produces a mixed particle known as RTS, S. TRAP
antigens are described in International Patent Application No.
PCT/GB89/00895, published under WO 90/01496. Plasmodia antigens
that are likely candidates to be components of a multistage Malaria
vaccine are P. falciparum MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2,
Sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXP1, Pfs25,
Pfs28, PFS27/25, Pfs16, Pfs48/45, Pfs230 and their analogues in
Plasmodium spp.
[0205] Examples of tumor antigens include MAGE 1 and MAGE 3 or
other MAGE antigens (for the treatment of melanoma), PRAME, BAGE,
or GAGE (Robbins and Kawakami, 1996, Current Opinions in Immunology
8, pps 628-636; Van den Eynde et al., International Journal of
Clinical & Laboratory Research (submitted 1997); Correale et
al. (1997), Journal of the National Cancer Institute 89, p293.
Indeed these antigens are expressed in a wide range of tumour types
such as melanoma, lung carcinoma, sarcoma and bladder carcinoma.
Other tumour-specific antigens include, but are not restricted to
tumour-specific gangliosides, Prostate specific antigen (PSA) or
Her-2/neu, KSA (GA733), PAP, mammaglobin, MUC-1, carcinoembryonic
antigen (CEA). Other tumour-associated antigen comprise
Prostate-specific membrane antigen (PSMA), Prostate Stem Cell
Antigen (PSCA), tyrosinase, survivin, NY-ES01, prostase, PS108 (WO
98/50567), RAGE, LAGE, HAGE. Additionally said antigen may be a
self peptide hormone such as whole length Gonadotrophin hormone
releasing hormone (GnRH, WO 95/20600), a short 10 amino acid long
peptide, useful in the treatment of many cancers, or in
immunocastration.
[0206] In a preferred embodiment of the invention, the antigen
complexed to the B subunit of Shiga toxin is chosen among E6, E7,
antigens from the Mage family, Her2/neu, EGFRVIII, survivin,
telomerase, WT1 and ESAT6.
[0207] In one embodiment of the present invention, the composition
above described further comprises a pharmaceutically acceptable
carrier.
[0208] In one embodiment, the compositions of the present invention
are formulated as oral or parenteral dosage forms, such as uncoated
tablets, coated tablets, pills, capsules, powders, granulates,
dispersions or suspensions. In another embodiment, the compositions
of the present invention are formulated for intravenous
administration. In another embodiment, the compounds of the present
invention are formulated in ointment, cream or gel form for
transdermal administration. In another embodiment, the compounds of
the present invention are formulated as an aerosol or spray for
nasal application. In another embodiment, the compositions of the
present invention are formulated in a liquid dosage form. Examples
of suitable liquid dosage forms include solutions or suspensions in
water, pharmaceutically acceptable fats and oils, alcohols or other
organic solvents, including esters, emulsions, syrups or elixirs,
solutions and/or suspensions.
[0209] Suitable excipients and carriers may be, according to
embodiments of the invention, solid or liquid and the type is
generally chosen based on the type of administration being used.
Liposomes may also be used to deliver the composition. Examples of
suitable solid carriers include lactose, sucrose, gelatin and agar.
Oral dosage forms may contain suitable binders, lubricants,
diluents, disintegrating agents, coloring agents, flavoring agents,
flow-inducing agents, and melting agents. Liquid dosage forms may
contain, for example, suitable solvents, preservatives, emulsifying
agents, suspending agents, diluents, sweeteners, thickeners, and
melting agents Parenteral and intravenous forms should also include
minerals and other materials to make them compatible with the type
of injection or delivery system chosen. Of course, other excipients
may also be used.
[0210] There is also an object of the present invention to provide
a medicament comprising the composition of the invention.
[0211] Another object of the present invention is to provide a
vaccine comprising said composition.
[0212] The amount of antigen in each vaccine dose is selected as an
amount which induces an immunoprotective response without
significant, adverse side effects in typical vaccines.
[0213] Generally, it is expected that each human dose will comprise
0.1-1000 .mu.g of antigen, preferably 0.1-500 .mu.g, preferably
0.1-100 .mu.g, most preferably 0.1 to 50 .mu.g. An optimal amount
for a particular vaccine can be ascertained by standard studies
involving observation of appropriate immune responses in vaccinated
subjects. Following an initial vaccination, subjects may receive
one or several booster immunisations adequately spaced. Such a
vaccine formulation may be applied to a mucosal surface of a mammal
in either a priming or a boosting vaccination regime; or
alternatively be administered systemically, for example via the
transdermal, subcutaneous or intramuscular routes. Intramuscular
administration is preferred.
[0214] Another object of the present invention is a pharmaceutical
kit comprising:
[0215] a) a first container comprising a B subunit of Shiga toxin
or a functional equivalent thereof which is able to bind the Gb3
receptor, complexed with an antigen as described above and
[0216] b) at least a second container comprising at least one
ligand of CD1 capable of stimulating NK T cells as described
above.
[0217] Preferably, said ligand of CD1 is a ligand of CD1d. More
preferably, said ligand is chosen among all ligands cited
above.
[0218] Another object of the present invention is to provide a
process for the preparation of said composition wherein the B
subunit of Shiga toxin or a functional equivalent thereof which is
able to bind the Gb3 receptor, complexed with an antigen is mixed
with at least one ligand of CD 1 capable of stimulating NK T
cells.
[0219] The formulations of the present invention may be used for
both prophylactic and therapeutic purposes.
[0220] A further object of the present invention is therefore the
use of the composition as described above for the manufacture of a
pharmaceutical composition or a medicament, including a vaccine,
for treating an antigen-related condition in an individual, said
antigen-related condition being a tumor or an infection.
[0221] In a preferred embodiment, the composition as described
above is used for the manufacture of a pharmaceutical composition
or medicament, including a vaccine, for treating cancer.
[0222] In one embodiment, the present invention relates to the use
of said composition for treating an antigen-related state in an
individual in need thereof. Said pharmaceutical composition or
medicament is to be administrated to the individual in a
therapeutically effective amount for stimulating an immune response
against the antigen in said individual, thereby treating said
antigen-related condition in said individual.
[0223] In another embodiment, the present invention relates to the
use of said composition for treating an antigen-related condition
in an individual in need thereof, wherein the composition is to be
administered in combination with thalidomide or an analog thereof,
preferably lenalidomide.
[0224] Indeed, it was shown that lenalidomide and its analogues
enhance CD 1d-mediated presentation of glycolipid antigens, and
therefore enhance antigen-specific activation of NKT cells.
[0225] In another embodiment, the present invention relates to the
use of the kit as described above, wherein at least one ligand of
CD1 capable of stimulating NKT cells is to be administrated before,
simultaneously or after the B subunit of Shiga toxin or a
functional equivalent thereof which is able to bind the Gb3
receptor, complexed with an antigen.
[0226] According to the invention, the use of said composition
allows the delivery of the antigen into a Gb3 receptor expression
cells.
[0227] According to the invention, the stimulation of the immune
response induced by the use of the composition of the invention
comprises stimulating dendritic cells.
[0228] According to the invention, the stimulation of the immune
response induced by the use of the composition of the invention
comprises eliciting an antigen specific CD8+ response.
EXAMPLES
[0229] In the following description, all molecular biology
experiments for which no detailed protocol is given are performed
according to standard protocol.
[0230] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
[0231] I. Material and Methods.
[0232] Mice
[0233] Female C57BL/6 (H-2.sup.b) mice were purchased from Charles
River Laboratories (L'Arbresle, France). OT-I TCR transgenic (TG)
mice specific for the ovalbumin (OVA) peptide (SIINFEKL) in the
context of H-2K.sup.b were provided by Dr C Leclerc (Institut
Pasteur). OVA-TG mice were obtained from Dr P. Shrikant (Roswell
Park Cancer Institute, Buffalo, N.Y.) with the permission of Dr M.
K Jenkins (University of Minnesota). These mice present the C57BL/6
background and express low levels of the membrane-associated form
of chicken ovalbumin protein (OVA) in most actin-expressing cells.
Mice were kept under specific pathogen-free conditions and were
used between 6 and 8 weeks of age according to institutional
guidelines.
[0234] Chemical Reagents (Recombinant Proteins, Peptides,
Adjuvant)
[0235] Purified chicken ovalbumin (OVA) (grade V) was purchased
from Sigma (St Quentin Fallavier, France). Synthetic OVA-derived
peptide OVA.sub.257-264 (SIINFEKL) and HPV16-E7 derived peptide
E7.sub.49-57 (RAHYNIVTF) or E7.sub.43-57 (GQAEPDRAHYNIVTF) were
obtained from NeoMPS (Strasbourg, France) and stored in PBS.
[0236] STxB-OVA was obtained by chemical coupling, as previously
described (Haicheur, N., et al. 2003. Int Immunol 15:1-11).
Briefly, OVA was first activated via amino groups on lysine side
chains using the heterobifunctional cross-linker MBS (Pierce,
Rockford, Ill.). Activated OVA was then reacted with STxB-Cys, and
the reaction product was purified by gel filtration and
immunoaffinity chromatography. STxB-E7.sub.43-57 was produced using
a chemical coupling between the N-bromoacetylated E7.sub.43-57
peptide and the sulfhydril group of the STxB-Cys recombinant
protein as previously described (Mallard, F. et al. 2003. Methods
Mol Med 73:209-220). Removal of contaminating LPS was carried out
using Acticlean Etox columns (Biotech-IgG, Kobenhavn, Denmark).
After purification, endotoxin concentrations determined by the
Limulus assay test (Biowhittaker, Walkersville, Md.) were <0.5
EU/mg.
[0237] The iNKT cell ligand .alpha.-GalCer (KRN7000) was kindly
supplied by Kirin Brewery Co (Japan). Poly (I:C) and incomplete
Freund's adjuvant (IFA) were purchased from Sigma (St
Quentin-Fallavier, France). CpG ODN 1826 was purchased from Proligo
(Paris, France).
[0238] IFN.alpha. was kindly provided by Ion Gresser (Inserm,
France)
[0239] Cells
[0240] The mouse thymoma cell line EL4 (H-2.sup.b) was kindly
provided by K. Rock (University of Massachusetts Medical School,
Worcester, Mass.) and P Jeannin (Angers University Hospital,
France)
[0241] ELISPOT Assay
[0242] The functionality of the anti-OVA.sub.257-264/K.sup.b
CD8.sup.+ T cells induced after injection of STxB-OVA was
determined by ELISPOT according to the manufacturer's
recommendations (Diaclone, Besancon, France). Briefly, 10.sup.5
CD8.sup.+ T cells were transferred to anti-mouse IFN.gamma. mAb
precoated plates and cultured for 18 hours at 37.degree. C. with
10.sup.5 EL4 cells previously pulsed (or not) with the
OVA.sub.257-264 peptide and fixed with 1% paraformaldehyde. After
washings, the plates were further incubated with biotinylated
anti-mouse IFN.gamma. mAb. Finally, the plates were washed and
incubated for 30 min at 37.degree. C. with alkaline
phosphatase-labeled streptavidin. Spots were developed by adding
5-bromo-4-chloro-3-indolyl-phosphatase/nitroblue tetrazolium.
Positive controls included cells stimulated with phorbol myristate
acetate (100 ng/ml, Sigma) and ionomycin (500 ng/ml, Sigma).
Controls consisted of cells cultured in the absence of the
OVA.sub.257-264 peptide. IFN .gamma. spot-forming cells (SFCs) were
counted on a KS-ELISPOT system (Carl Zeiss, Munich, Germany).
[0243] Cytotoxicity Assay
[0244] Cytotoxicity was assessed on .sup.51Cr-labeled target cells
as previously described (Haicheur, N., et al. 2000. J Immunol
165:3301-3308)
[0245] Flow-Cytometry
[0246] To detect anti-OVA.sub.257-264/K.sup.b or
anti-E7.sub.49-57/D.sup.b specific CD8.sup.+ T cells, the cells
were stained with OVA.sub.257-264/K.sup.b or E7.sub.49.57/D.sup.b
tetramer according to the manufacturer's recommendations
(Beckman-Coulter Immunomics, Marseilles, France). Briefly, cells
were incubated with PE-labeled tetramer (45 min at 4.degree. C. in
the dark). After incubation and washes, labeled anti-CD8 mAbs
(ebioscience, San Diego, Calif.) were used to phenotype the
positive tetramer CD8.sup.+T cells. Irrelevant tetramers
recognizing a VSV derived peptide in the context of K.sup.b or
D.sup.b molecules were used in each experiment. Naive non immunized
mice were also included as control for these experiments.
[0247] Proliferation Assay to Detect Specific Anti-OVA CD4.sup.+T
Cells
[0248] CD4.sup.+T cells were purified from spleen and labeled with
CFSE (Molecular Probes, Eugene, Oreg.), used at 0.5 .mu.M for 30
min at 20.degree. C. Labeling was stopped by repeated washing with
ice-cold PBS supplemented with 5% FCS. Cells were then incubated
with T cell depleted splenocytes as APC pulsed or not with free
ovalbumin protein and cocultured for 5 days in AIM V serum free
medium. Proliferation in absence of APC sensitization were
subtracted as background from values obtained after ovalbumin
pulsing.
[0249] Ex Vivo Cross-Presentation Assay
[0250] Anti-OVA specific CD8.sup.+T cells derived from OT-1 mice
were labeled with CFSE. CD11c.sup.+ enriched dendritic cells
(10.sup.6) isolated as described (37) from mice vaccinated with
various vaccine formulations were co-cultured with OT-1 cells
(510.sup.5) for 72 hours. Dilution of CFSE detected by FACS
analysis was considered to be an indicator of OT-1 cell
proliferation after antigen recognition.
[0251] Serological Analysis
[0252] It was performed as previously described (Haicheur, N., F.
et al. 2003. Int Immunol 15:1-11).
[0253] Anti-Viral Protection Experiments
[0254] C57BL/6 mice (5 mice per group) were injected twice (d0 and
d21) intraperitoneally (200 .mu.l) with different vaccine
formulations and control mice were injected with PBS. Eight days
after the last injection, mice were challenged intraperitoneally
with 2.5.times.10.sup.6 PFU recombinant vaccina virus (rVV,
Westerns Reserve strains) expressing either the ovalbumin or the
HBx cDNA derived from Hepatitis B virus kindly provided, by Dr N
Etchart (INSERM, Lyon) and Dr Lone Yu Chun (Institut Pasteur),
respectively. After 4 days, the ovaries of the mice were harvested
and homogenized with a mechanical tissue grinder. The homogenates
were clarified by centrifugation at 4,000 g for 10 min, and the
number of rVV PFU in the resultant supernatant was enumerated by
infecting BHK 21 cell monolayers with 10-fold serial dilutions of
these fluids and plaques were counted after 2 days in culture at
37.degree. C. in a 5% CO.sub.2 environment as previously described
(52).
[0255] Results
[0256] .alpha.-GalCer Increased the Efficiency of STxB Coupled to
Antigen to Induce Specific CTL.
[0257] A first series of experiments tested the ability of various
well defined adjuvants to increase the efficiency of STxB-OVA to
trigger a specific CTL response.
[0258] As previously reported, when mice were immunized twice with
the STxB-OVA conjugate (50 .mu.g) alone, an induction of
anti-OVA.sub.257-264 CD8.sup.+T cells corresponding to 0.4% of
CD8.sup.+ T cells was demonstrated (FIGS. 1 and 2A). Except for
IFA, all other adjuvants (IFN.alpha., Poly (I:C), CpG and
.alpha.-GalCer) combined with STxB-OVA significantly enhanced the
frequency of anti-OVA.sub.257-264 CD8.sup.+T cells. However,
whereas, IFN.alpha., Poly (I:C) and CpG led to a modest increase of
anti-OVA.sub.257-264 CD8.sup.+T cells detected by specific tetramer
not exceeding 1% of total CD8.sup.+T cells, the glycolipid
.alpha.-GalCer elicited a dramatic increase of the percent of
specific anti-OVA CTL (FIG. 1). Indeed, after two immunizations
with STxB-OVA and .alpha.-GalCer, 4.6% of CD8.sup.+T cells stained
positively with OVA.sub.257-264/K.sup.b tetramer directly ex vivo
without any in vitro restimulation step (FIG. 1). We therefore
focused on analysis of this synergy between STxB and
.alpha.-GalCer. Since previous studies reported that repeated
administration of .alpha.-GalCer may lead to anergy and TH2
polarization (53-55) and no difference was observed when
.alpha.-GalCer was injected during both the first and second
immunizations or only at priming, we only combined this adjuvant
and STxB-OVA during the first immunization.
[0259] When different doses of the STxB-OVA conjugate were mixed
with .alpha.-GalCer, this adjuvant was found to significantly
induce the anti-OVA.sub.257-264 CTL response even at very low doses
of antigen (50 ng STxB-OVA corresponding to 25 ng of equivalent
ovalbumin antigen) (FIG. 2A). In contrast, free ovalbumin even at
high doses (200 .mu.g) combined with .alpha.-GalCer elicited a low
frequency of anti-OVA.sub.257-264 CTL not exceeding 1% of total
CD8.sup.+T cells (FIG. 2B) compared to 5-7% of anti-OVA.sub.257-264
specific CTL when ovalbumin was coupled to STxB and combined with
.alpha.-GalCer. In addition, no significant induction of
anti-OVA.sub.257-264 CD8.sup.+T cells was shown when less than 50
.mu.g of OVA was added to .alpha.-GalCer or when OVA alone was used
for immunization (FIG. 2B). The other adjuvants (IFN.alpha., Poly
(I:C), CpG) did not allow this priming of CTL when a low dose of
STxB-OVA conjugate was used reinforcing the significance of the
synergy between STxB and .alpha.-GalCer (data not shown).
[0260] The specific anti-OVA.sub.257-264 CD8.sup.+T cells induced
by the combination of .alpha.-GalCer and the STxB-OVA conjugate
were long-lasting as 2.49% of CD8.sup.+T cells stained positively
with OVA.sub.257-264/K.sup.b tetramer directly ex vivo 202 days
after the first immunization (FIG. 2C).
[0261] Since tetramer analysis does not discriminate between
anergic and functional CD8'T cells, two functional tests, an
Elispot assay and a cytotoxic assay on .sup.51Cr-labeled target
cells without an in vitro stimulation were performed. As shown in
FIG. 2D, after vaccination with STxB-OVA (1 .mu.g) mixed with
.alpha.-GalCer, large numbers of CD8.sup.+T cells produced
IFN.gamma. (mean 61/10.sup.5 cells) when co-incubated with
OVA.sub.257-264 peptide-pulsed EL4 cells (FIG. 2D left). Similarly,
as shown in FIG. 1D (right), spleen cells from mice immunized with
STxB-OVA mixed with .alpha.-GalCer efficiently lyzed EL4 target
cells loaded with the OVA.sub.257-264 peptide, whereas no
cytotoxicity was observed against EL4 alone. No ex vivo
cytotoxicity was demonstrated when mice were vaccinated twice with
STxB-OVA alone even when high doses (50 .mu.g) were used (data not
shown).
[0262] To check whether these results could be reproduced with
another more clinically relevant antigen, mice were immunized twice
with STxB coupled to a polypeptide derived from the HPV16-E7
protein (STxB-E7.sub.43-57) at a low dose (1 .mu.g). A marked
induction of anti-E7 CTL detectable ex vivo by the E7.sub.49-57 Db
tetramer (1.12% of CD8.sup.+T cells) was also demonstrated (FIG.
3). These CTL were functional (data not shown). In contrast, at
this dosage, only low levels of E7-specific CTL (0.12% of
CD8.sup.+T cells) were detected after immunization with
STxB-E7.sub.43-57 alone. The E7.sub.43-57 polypeptide mixed with
.alpha.-GalCer did not prime a CTL response (FIG. 3).
[0263] We then investigated whether the synergy observed between
.alpha.-GalCer and STxB was restricted to induction of CTL or could
be extended to other immune responses. Mice vaccinated with the
STxB-OVA conjugate mixed with .alpha.-GalCer displayed a more
potent anti-OVA CD4.sup.+T cell response compared to mice immunized
with STxB-OVA alone or free ovalbumin mixed with IFA (FIG. 4).
However, although .alpha.-GalCer increased the anti-OVA IgG2a
response compared to free ovalbumin with adjuvant, this humoral
response either for total IgG or the various isotypes was not
enhanced when STxB was admixed with .alpha.-GalCer compared to STxB
alone.
[0264] Analysis of Potential Mechanisms Leading to the Synergy
Between STxB and .alpha.-GalCer.
[0265] In a previous study, the adjuvant property of .alpha.-GalCer
was related to its ability to promote maturation of DC. A rapid
increase of costimulatory molecules (CD86 . . . ) and MHC class II
was observed, after administration of .alpha.-GalCer in mice (data
not shown). However, we also confirmed that other adjuvants used in
this study (CpG, Poly (I:C) . . . ) also activated dendritic cells
but their enhancing effect on the STxB immunogenicity remained
modest (FIG. 1 and data not shown).
[0266] Since the levels of CD1d may affect NKT cell activation, we
tested whether STxB could modulate the expression of CD1d. After
STxB administration in mice, no significant change was detected in
CD expression on APC (dendritic cells and B cells) derived from
splenocytes. Similarly, .alpha.-GalCer did not regulate the
membrane expression of Gb.sub.3 on dendritic and B cells (data not
shown).
[0267] As one striking consequence of the synergy between STxB and
.alpha.-GalCer is the ability to markedly reduce the efficient dose
of STxB vaccine, we analyzed the in vivo presentation of antigen
after vaccination. Dendritic cells derived from mice immunized with
low doses of STxB-OVA (1 .mu.g) combined with .alpha.-GalCer, 7
days earlier, more significantly presented the
OVA.sub.257-264/K.sup.b complex in vivo than mice vaccinated with
STxB alone (FIG. 5). This presentation was observed for both
dendritic cells derived from spleen or draining lymph node and
persisted for at least 12 days (data not shown). This increased
presentation could also be detected when STxB was mixed with other
adjuvants, but the levels of antigen presentation appeared to be
lower than those observed with .alpha.-GalCer (FIG. 5 and data not
shown). Although the Gb.sub.3 receptor and CD1d are also expressed
by B cells, addition of .alpha.-GalCer to STxB-OVA did not allow
these cells to express the OVA.sub.257-264/K.sup.b complex whose
expression remained restricted to dendritic cells after
immunization as previously reported (data not shown).
[0268] STxB Conjugate Mixed with .alpha.-GalCer Broke Tolerance
Against Self Antigens
[0269] It is often criticized that exogenous antigens such as
ovalbumin and viral proteins do not mimic the clinical situations
in which therapeutic vaccines will be developed because most tumor
antigens are self antigens and, during chronic infection, tolerance
to viral protein is already established. For these indications, a
potential vaccine is therefore expected to be endowed with the
ability to break tolerance against self antigen. For this purpose
we selected a novel transgenic mouse model that expresses ovalbumin
on the surface of all cells. When these mice were vaccinated with
STxB-OVA alone, no anti-OVA.sub.257-264 CD8.sup.+T cell induction
was observed in either blood or spleen at various times after
primary or secondary immunizations (FIG. 6 and data not shown). In
contrast, specific tetramer assay detected significant levels of
anti-OVA.sub.257-264 CD8.sup.+ T cells in 8 out of 11 mice
immunized with STxB-OVA combined with .alpha.-GalCer (FIG. 6). Some
of them (4 out of 7 vaccinated mice) were functional as they
produced IFN.gamma. using an Elispot assay. These specific
CD8.sup.+T cells seemed to be induced by the vaccine as they were
not present before immunization (data not shown). These anti-OVA
specific CD8.sup.+T cells were essentially found after primary
immunizations and rapidly disappeared from the blood and from the
spleen. It should be emphasized that this strain of mice expresses
membrane ovalbumin in all organs.
[0270] STxB Conjugate Mixed with .alpha.-GalCer Induces Anti-Viral
Protective Immunity.
[0271] To investigate the clinical relevance of the synergy
observed between STxB and .alpha.-GalCer, we challenged mice with
recombinant vaccinia virus encoding ovalbumin (VV-OVA), 7 days
after vaccination, and measured virus titers in the ovaries 5 days
later to assess protection. Vaccination of mice with STxB-OVA and
.alpha.-GalCer conferred potent protection against VV-OVA, with
virus titers in the ovaries reduced by 5 log (39210.sup.3 PFU)
compared to those of mice treated with PBS (82810.sup.8 PFU) (FIG.
7). Immunization with STxB-OVA alone conferred a statistically
significant reduction of virus titers in the ovary by >2 log but
this effect was dramatically amplified by the addition of
.alpha.-GalCer.
[0272] To exclude a direct role of .alpha.-GalCer in this
anti-viral protection, we showed that mice immunized with ovalbumin
and .alpha.-GalCer exhibited a slight reduction of infectious
virus, titers from 82810.sup.8 PFU in PBS-treated mice to
77310.sup.8 PFU corresponding to less that 1 log reduction of virus
titers (FIG. 7). The specificity of the protection was documented
by challenging STxB-OVA/.alpha.-GalCer immunized mice, with an
irrelevant vaccinia virus encoding hepatitis B.times.protein). No
significant reduction of viral load was observed (data not
shown).
[0273] No Adjuvant Effect of .alpha.-GalCer in Mice Deficient in
NKT Cells.
[0274] Ja18.sup.-/- mice were immunized with STxB-OVA (1 .mu.g) or
STxB-OVA+.alpha.-GalCer (2 .mu.g). 7 days after immunization, the
spleens of immunized and non-immunized mice were harvested and
stained with anti-CD8 antibody and OVA.sub.257-264/K.sup.b
tetramer. The results (FIG. 8) showed that almost no
anti-OVA.sub.257-264 specific CTL were observed in immunized
Ja18.sup.-/- mice with or without .alpha.-GalCer. This is to be
compared with the 6.43% of anti-OVA.sub.257-264 specific CTL
observed in immunized C57BL/6 mice with .alpha.-GalCer (FIG. 2A).
Therefore, no adjuvant effect of .alpha.-GalCer can be observed in
mice deficient in NKT cells, confirming the role of the activation
of NKT cells in the adjuvant mechanism of .alpha.-GalCer.
[0275] Vaccination with .alpha.GalCer Combined with STxB-Ova
Induces Protection Against Established Tumors
[0276] C57BL6 mice were grafted with EG7 tumor, a thymoma
transfected with cDNA encoding ovalbumin mice, and three days after
were treated with PBS, 5 .mu.g STx-B-Ova, 2 .mu.g .alpha.GalCer or
the combination of both (FIG. 9).
[0277] Mice vaccinated three days after subcutaneous graft of EG7
with the combination of STxB-Ova and .alpha.GalCer, showed a
significant regression of tumor. In contrast mice immunized with
STxB-Ova alone or .alpha.GalCer alone did not control the growth of
the tumor.
[0278] Vaccination with C-.alpha.GalCer Combined with STxB-Ova
Induces Protection Against Established Tumors
[0279] C57BL6 mice are grafted with EG7 tumor, a thymoma
transfected with cDNA encoding ovalbumin mice, and three days after
are treated with PBS, 5 .mu.g STx-B-Ova, 2 .mu.g C-.alpha.GalCer,
or the combination STx-B-Ova/C-.alpha.GalCer.
[0280] Mice vaccinated three days after subcutaneous graft of EG7
with the combination of STx-B-Ova/C-.alpha.GalCer will show a
significant regression of tumor. In contrast mice immunized with
STxB-Ova, or C-.alpha.GalCer alone will not control the growth of
the tumor.
Sequence CWU 1
1
1190PRTArtificial SequenceB subunit of Shiga toxin 1Met Lys Lys Thr
Leu Leu Ile Ala Ala Ser Leu Ser Phe Phe Ser Ala 1 5 10 15 Ser Ala
Leu Ala Thr Pro Asp Cys Val Thr Gly Lys Val Glu Tyr Thr 20 25 30
Lys Tyr Asn Asp Asp Asp Thr Phe Thr Val Lys Val Gly Asp Lys Glu 35
40 45 Leu Phe Thr Asn Arg Trp Asn Leu Gln Ser Leu Leu Leu Ser Ala
Gln 50 55 60 Ile Thr Gly Met Thr Val Thr Ile Lys Thr Asn Ala Cys
His Asn Gly 65 70 75 80 Gly Gly Phe Ser Glu Val Ile Phe Arg Cys 85
90
* * * * *