U.S. patent application number 14/378805 was filed with the patent office on 2015-01-22 for xenoantigen-displaying anti-cancer vaccines and method of making.
This patent application is currently assigned to THE UNIVERSITY OF TOLEDO. The applicant listed for this patent is THE UNIVERSITY OF TOLEDO. Invention is credited to Sourav Sarkar, Steven J. Sucheck, Katherine A. Wall.
Application Number | 20150024037 14/378805 |
Document ID | / |
Family ID | 48984725 |
Filed Date | 2015-01-22 |
United States Patent
Application |
20150024037 |
Kind Code |
A1 |
Sucheck; Steven J. ; et
al. |
January 22, 2015 |
Xenoantigen-Displaying Anti-Cancer Vaccines and Method of
Making
Abstract
Compositions, methods of making, and methods of using,
xenoantigen-displaying anticancer vaccines are described. In
another broad aspect, there is provided herein a method of
synthesizing an alkynefunctionalized composition of claim 1,
comprising: deprotecting an ester comprising a Fmoc moiety to form
a free acid; coupling the free acid of step (a) with an amine; and,
removing the Fmoc moiety, and coupling the remaining moiety with
palmitic acid to yield an alkyne-functionalized composition.
Inventors: |
Sucheck; Steven J.; (Maumee,
OH) ; Wall; Katherine A.; (Toledo, OH) ;
Sarkar; Sourav; (Toledo, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNIVERSITY OF TOLEDO |
Toledo |
OH |
US |
|
|
Assignee: |
THE UNIVERSITY OF TOLEDO
Toledo
OH
|
Family ID: |
48984725 |
Appl. No.: |
14/378805 |
Filed: |
February 15, 2013 |
PCT Filed: |
February 15, 2013 |
PCT NO: |
PCT/US13/26271 |
371 Date: |
August 14, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61599925 |
Feb 16, 2012 |
|
|
|
Current U.S.
Class: |
424/450 ;
424/185.1; 530/322; 530/327; 530/329; 530/350; 530/395;
554/101 |
Current CPC
Class: |
A61K 2039/55555
20130101; A61K 39/0011 20130101; C07K 7/06 20130101; C07K 16/1275
20130101; A61K 47/6925 20170801; C07K 14/4727 20130101; A61K
39/00117 20180801; A61K 2039/55516 20130101; A61K 9/0019 20130101;
A61K 47/543 20170801; C07K 14/705 20130101; A61K 2039/55572
20130101; C07K 16/3092 20130101; A61K 39/001169 20180801; C07K
2317/34 20130101; A61K 39/39 20130101; A61K 39/0012 20130101; C07C
323/60 20130101; C07K 7/08 20130101; A61K 39/001172 20180801; A61K
2039/6018 20130101; A61K 2039/627 20130101; A61K 9/127
20130101 |
Class at
Publication: |
424/450 ;
554/101; 530/350; 530/329; 530/327; 530/395; 424/185.1;
530/322 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 14/705 20060101 C07K014/705; A61K 47/48 20060101
A61K047/48; C07K 7/08 20060101 C07K007/08; C07K 14/47 20060101
C07K014/47; A61K 9/127 20060101 A61K009/127; C07C 323/60 20060101
C07C323/60; C07K 7/06 20060101 C07K007/06 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] The invention was made with U.S. Government support under
Grant Number GM094734 awarded by the National Institutes of Health.
The United States Government has certain rights in the invention.
Claims
1. A composition comprising a first lipid (lipid.sub.a) moiety and
an alkyne amide moiety having a Formula IV: ##STR00007##
2. The composition of claim 1, wherein the first lipid
(lipid.sub.a) moiety comprises a Toll-like receptor (TLR) agonist
ligand selected from one or more of: TLR2, TLR1, TLR3, TLR4, TLR5,
TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TLR13, TLR14, TLR15
and TLR16.
3. The composition of claim 1, wherein the lipid moiety comprises:
dipalmitoyl-S-glyceryl-cys-(Pam.sub.2Cys-);
tripalmitoyl-S-glyceryl-cys-(Pam.sub.3Cys-);
dipalmitoyl-S-glyceryl-cys-ser-lys-lys-lys-lys
(Pam.sub.2Cys-Ser-(Lys).sub.4) [SEQ ID NO: 2];
tripalmitoyl-S-glyceryl-cys-ser-lys-lys-lys-lys
(Pam.sub.3Cys-Ser-(Lys).sub.4) [SEQ ID NO: 3]; or MALP-2
dipalmitoyl-S-glyceryl-cys-gly-asn-asn-asp-glu-ser-asn-ile-ser-phe-lys-gl-
u-lys (Pam.sub.2CGNNDESNISFKEK)] [SEQ ID NO: 4].
4. The composition of claim 1, wherein Formula IV comprises an
alkyne-functionalized Pam.sub.3Cys amide composition (6)
comprising: ##STR00008## wherein Pam is a
dipalmitoyl-S-glyceryl-moiety.
5. A method of synthesizing an alkyne-functionalized composition of
claim 1, comprising: a) deprotecting an ester comprising a Fmoc
moiety to form a free acid; b) coupling the free acid of step (a)
with an amine; and, c) removing the Fmoc moiety, and coupling the
remaining moiety with palmitic acid to yield an
alkyne-functionalized composition.
6. A method of synthesizing an alkyne-functionalized Pam.sub.3Cys
amide composition (6) of claim 4, comprising: a) deprotecting
0-palmitoylated Fmoc L-cystine tert-butyl ester (4) to form a free
acid; b) coupling the free acid of step (a) with propargyl amine in
presence of benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate (PyBOP), 1-hydroxy-benzotriazole (HOBt) and
N,N-diisopropylethylamine (DIPEA) to yield composition (5); and, c)
removing a Fmoc group of composition (5) by treatment with a
mixture of acetonitrile-dichloromethane-diethyl amine, followed by
subsequent palmitoylation by coupling with palmitic acid, PyBOP,
HOBt and DIPEA to yield the alkyne-functionalized Pam.sub.3Cys
amide composition (6).
7. A lipidated glycopeptide composition comprising: a first lipid
(lipid.sub.a) moiety, a first linker (linker.sub.a) moiety, and an
antigen moiety, having a Formula II: ##STR00009##
8. (canceled)
9. The composition of claim 7, wherein the first lipid
(lipid.sub.a) moiety comprises:
dipalmitoyl-S-glyceryl-cys-(Pam.sub.2Cys-);
tripalmitoyl-S-glyceryl-cys-(Pam.sub.3Cys-);
dipalmitoyl-S-glyceryl-cys-ser-lys-lys-lys-lys
(Pam.sub.2Cys-Ser-(Lys).sub.4) [SEQ ID NO: 2];
tripalmitoyl-S-glyceryl-cys-ser-lys-lys-lys-lys
(Pam.sub.3Cys-Ser-(Lys).sub.4) [SEQ ID NO: 3]; or MALP-2
dipalmitoyl-S-glyceryl-cys-gly-asn-asn-asp-glu-ser-asn-ile-ser-phe-lys-gl-
u-lys (Pam.sub.2CGNNDESNISFKEK)] [SEQ ID NO: 4].
10-12. (canceled)
13. The composition of claim 9 comprising mucin 1 (MUC1) variable
number tandem repeats (VNTRs) conjugated to tumor-associated
carbohydrate antigens (TACA).
14. The composition of claim 9, wherein the antigen is a TACA,
selected from the group consisting of TF, Tn, sialyl Tn (sTn), or
sialyl Lewis a (sLe.sup.a) antigens, having the formulae:
##STR00010##
15. (canceled)
16. The composition of claim 9, wherein the antigen comprises MUC1
VNTR having one of the following amino acid sequences:
PDTRPAPGST(Tn)APPAHGVTSA [SEQ ID NO: 1]; TSAPDTRPAPGSTAPPAHGV [SEQ
ID NO: 5]; or TSAPDT(Tn)RPAPGSTAPPAHGV [SEQ ID NO: 6].
17. (canceled)
18. The composition of claim 7, wherein the first linker
(linker.sub.a) comprises a dialkyl-substituted heteroaryl C.sub.1-n
alkyl of Formula VI ##STR00011## wherein the "A" group comprises: a
chain of C.sub.1-n alkyl, dialkyl substituted aryl C.sub.1-n alkyl,
or --CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.n--; and n is a
positive integer.
19. (canceled)
20. A composition of claim 7, comprising the compound (9):
##STR00012##
21. A composition of claim 7, comprising the compound (17):
##STR00013##
22. A composition of claim 7, comprising the compound (21):
##STR00014##
23-26. (canceled)
27. A composition comprising a second lipid (lipid.sub.b) moiety, a
second linker (linker.sub.b) moiety, and a xenoantigen moiety,
having the Formula VII: ##STR00015##
28. The composition of claim 27, wherein the second lipid
(lipid.sub.b) moiety contains a structure of the Formula IX:
##STR00016##
29. The composition of claim 27, wherein the second linker
(linker.sub.b) comprises: a chain of C.sub.1-n alkyl, dialkyl
substituted aryl C.sub.1-n alkyl, or
--CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.n--; wherein n is a
positive integer.
30-35. (canceled)
36. The composition of the claim 27, wherein the xenoantigen moiety
contains a structure comprising: an .alpha.- or .beta.-linked
L-rhamnose epitope, a .beta.-linked .alpha.-Gal disaccharide
epitope, or an .alpha.- or .beta.-linked Forssmann disaccharide
epitope: ##STR00017##
37. A vaccine composition, comprising: 1) an antigen composition
comprising: a first lipid (lipid.sub.a) moiety, a first linker
(linker.sub.a) moiety, and an antigen moiety; 2) a xenoantigen
composition comprising: a second lipid (lipid.sub.b) moiety, a
second linker (linker.sub.b) moiety, and a xenoantigen moiety; and
3) at least one liposomal formulation.
38-40. (canceled)
41. A vaccine composition of claim 37, wherein: the antigen
composition comprises a Pam.sub.3Cys-MUC1 VNTR-TACA conjugate; the
second linker (linker.sub.b) moiety comprises a tetraethyleneglycol
(TEG) portion; and the xenoantigen moiety comprises .alpha.- or
.beta.-linked L-rhamnose.
42-59. (canceled)
60. A method for improving immunogenicity of vaccines, the method
comprising incorporating at least one .alpha.- or .beta.-linked
L-Rha epitope by direct conjugation or by non-covalent association
with at least one liposomal vaccine formulation to increase the
immunogenicity of the vaccine by a NA-dependant antigen uptake
mechanism.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 61/599,925 filed Feb. 16, 2012, the entire
disclosure of which is expressly incorporated herein by
reference.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted via EFS-web and is hereby incorporated by
reference in its entirety. The ASCII copy, created on Feb. 12,
2013, is named 420.sub.--53665_SEQ_LIST_D2012-14.txt, and is 6,494
bytes in size.
TECHNICAL FIELD AND INDUSTRIAL APPLICABILITY OF THE INVENTION
[0004] The invention relates to compositions having a xenoantigen
that is incorporated onto a target antigen and method of making the
same. In particular, there is provided herein a vaccine composition
comprised of a lipid-linked antigen in non-covalent association
with a lipid-linked xenoantigen, where both the lipid-linked
antigen and the lipid-linked xenoantigen are independently embedded
in liposomes in a liposomal formulation.
BACKGROUND
[0005] The use of vaccines against a variety of agents is an
important objective for disease control worldwide. It is understood
that the protective immunity afforded by vaccines against specific
antigen is achieved by humoral, cellular and mucosal immune
responses.
[0006] For example, humoral or antibody responses are important in
pathogen neutralization and can be very effective in some
conditions such as cancer and infectious diseases. These
therapeutic cancer vaccines are a new class of active specific
immunotherapy agents that trigger a targeted immune response
against cancer. It would be useful to have efficient methods for
the synthesis of such vaccines in addition to those presently
available.
SUMMARY
[0007] In a first aspect, there is provided herein a composition
comprising a first lipid (lipid.sub.a) moiety and an alkyne amide
moiety having a Formula IV.
[0008] In certain embodiments, the first lipid (lipid.sub.a) moiety
comprises a Toll-like receptor (TLR) agonist ligand selected from
one or more of: TLR2, TLR1, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,
TLR9, TLR10, TLR11, TLR12, TLR13, TLR14, TLR15 and TLR16.
[0009] In certain embodiments, the TLR2 ligand comprises:
dipalmitoyl-S-glyceryl-cys-(Pam.sub.2Cys-);
tripalmitoyl-S-glyceryl-cys-(Pam.sub.3Cys-);
dipalmitoyl-S-glyceryl-cys-ser-lys-lys-lys-lys
(Pam.sub.2Cys-Ser-(Lys).sub.4) [SEQ ID NO: 2];
tripalmitoyl-S-glyceryl-cys-ser-lys-lys-lys-lys
(Pam.sub.3Cys-Ser-(Lys).sub.4) [SEQ ID NO: 3]; or MALP-2
dipalmitoyl-S-glyceryl-cys-gly-asn-asn-asp-glu-ser-asn-ile-ser-phe-lys-gl-
u-lys (Pam.sub.2CGNNDESNISFKEK)] [SEQ ID NO: 4].
[0010] In certain embodiments, the Formula IV comprises an
alkyne-functionalized Pam.sub.3Cys amide composition (6), wherein
Pam is a dipalmitoyl-S-glyceryl-moiety.
[0011] In another broad aspect, there is provided herein a method
of synthesizing an alkyne-functionalized composition of claim 1,
comprising: deprotecting an ester comprising a Fmoc moiety to form
a free acid; coupling the free acid of step (a) with an amine; and,
removing the Fmoc moiety, and coupling the remaining moiety with
palmitic acid to yield an alkyne-functionalized composition.
[0012] In another broad aspect, there is provided herein a method
of synthesizing an alkyne-functionalized Pam3Cys amide composition
(6), comprising:
[0013] a) deprotecting 0-palmitoylated Fmoc L-cystine tert-butyl
ester (4) to form a free acid;
[0014] b) coupling the free acid of step (a) with propargyl amine
in presence of benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate (PyBOP), 1-hydroxy-benzotriazole (HOBt) and
N,N-diisopropylethylamine (DIPEA) to yield composition (5);
and,
[0015] c) removing a Fmoc group of composition (5) by treatment
with a mixture of acetonitrile-dichloromethane-diethyl amine,
followed by subsequent palmitoylation by coupling with palmitic
acid, PyBOP, HOBt and DIPEA to yield the alkyne-functionalized
Pam3Cys amide composition (6).
[0016] In another broad aspect, there is provided herein a
lipidated glycopeptide composition comprising: a first lipid
(lipida) moiety, a first linker (linkera) moiety, and an antigen
moiety having a Formula II.
[0017] In certain embodiments, the first lipid (lipida) moiety
comprises a Toll-like receptor (TLR) agonist ligand selected from
one or more of: TLR2, TLR1, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,
TLR9, TLR10, TLR11, TLR12, TLR13, TLR14, TLR15 and TLR16.
[0018] In certain embodiments, the first lipid (lipida) moiety
comprises a TLR2 ligand comprising:
TABLE-US-00001 dipalmitoyl-S-glyceryl-cys-(Pam2Cys-);
tripalmitoyl-S-glyceryl-cys-(Pam3Cys-); [SEQ ID NO: 2]
dipalmitoyl-S-glyceryl-cys-ser-lys-lys-lys-lys
(Pam2Cys-Ser-(Lys)4); [SEQ ID NO: 3]
tripalmitoyl-S-glyceryl-cys-ser-lys-lys-lys-lys
(Pam3Cys-Ser-(Lys)4); or [SEQ ID NO: 4] MALP-2
dipalmitoyl-S-glyceryl-cys-gly-asn-asn-
asp-glu-ser-asn-ile-ser-phe-lys-glu-lys (Pam2CGNNDESNISFKEK)].
[0019] In certain embodiments, the the antigen moiety comprises: an
anti-cancer composition, an anti-bacterial composition, an
anti-viral composition, a protein, an isolated DNA, an isolated
RNA, an isolated carbohydrate, or an isolated lipid.
[0020] In certain embodiments, the the antigen moiety comprises a
TACA that interacts with B-cell receptors.
[0021] In certain embodiments, the the TACA comprises one or more
glycoproteins and glycolipids on a cancer cell.
[0022] In certain embodiments, the composition comprises mucin 1
(MUC1) variable number tandem repeats (VNTRs) conjugated to
tumor-associated carbohydrate antigens (TACA).
[0023] In certain embodiments, the the TACA comprises: TF, Tn,
sialyl Tn (sTn), or sialyl Lewis a (sLea) antigens.
[0024] In certain embodiments, the the TACA comprises: an
autologous or heterologous helper T-cell epitope, wherein the
autologous or heterologous helper T-cell epitope comprises a
sequence expressed on a tumor cell.
[0025] T In certain embodiments, the he autologous or heterologous
helper T-cell epitope comprises MUC1 VNTR having one of the
following amino acid sequences:
TABLE-US-00002 [SEQ ID NO: 1] PDTRPAPGST(Tn)APPAHGVTSA; [SEQ ID NO:
5] TSAPDTRPAPGSTAPPAHGV; or, [SEQ ID NO: 6]
TSAPDT(Tn)RPAPGSTAPPAHGV.
[0026] In certain embodiments, the the threonine in the sequence
GST or PDT is synthetically modified to incorporate
.alpha.-GalNAc-O-Thr (Tn) TACA.
[0027] In certain embodiments, the the first linker (linkera)
comprises a dialkyl-substituted heteroaryl C1-n alkyl of Formula
VI, wherein the "A" group comprises: a chain of C1-n alkyl, dialkyl
substituted aryl C1-n alkyl, or --CH2CH2(OCH2CH2)n-; and n is a
positive integer.
[0028] In certain embodiments, the the "A" group comprises a C1-5
alkyl chain.
[0029] In another broad aspect, there is provided herein a
composition comprising the compound (9).
[0030] In another broad aspect, there is provided herein a
composition comprising the compound (17).
[0031] In another broad aspect, there is provided herein a
composition comprising the compound (21).
[0032] In another broad aspect, there is provided herein a method
of synthesizing a lipidated glycopeptide composition, comprising
reacting a composition of Formula IV, with a composition Formula
V.
[0033] In another broad aspect, there is provided herein a method
of synthesizing a Pam3Cys-MUC-1 VNTR-TACA conjugate, comprising:
synthesizing a 20-amino acid tandem repeat of MUC1; and modifying a
glycopeptide with a terminal azido group to make a `click`
conjugation to a Pam3Cys alkyne.
[0034] In certain embodiments, the the 20-amino acid tandem repeat
of MUC1 comprises a GS(.alpha.-GalNAc-O-T)A epitope.
[0035] In certain embodiments, the the 20-amino acid tandem repeat
of MUC1 comprises a PD(.alpha.-GalNAc-O-T)R epitope.
[0036] In another broad aspect, there is provided herein a
composition comprising a second lipid (lipidb) moiety, a second
linker (linkerb) moiety, and a xenoantigen moiety, having the
Formula VII.
[0037] In certain embodiments, the the second lipid (lipidb) moiety
contains a structure of the Formula IX.
[0038] In certain embodiments, the the second linker (linkerb)
comprises: a chain of C1-n alkyl, dialkyl substituted aryl C1-n
alkyl, or --CH2CH2(OCH2CH2)n-; wherein n is a positive integer.
[0039] In certain embodiments, the the second linker (linkerb)
comprises a tetraethyleneglycol (TEG) of Formula VIII.
[0040] In certain embodiments, the the xenoantigen moiety comprises
a xenoantigen linked to a lipid.
[0041] In certain embodiments, the the xenoantigen binds a natural
antibody (NA).
[0042] In certain embodiments, the NA comprises an autoantigen that
is present in blood.
[0043] In certain embodiments, the the NA comprises IgM, IgG, or
IgA isotypes in human blood.
[0044] In certain embodiments, the the NA comprises an
anti-carbohydrate NA in human serum.
[0045] In certain embodiments, the the xenoantigen moiety contains
a structure comprising: an .alpha.- or .beta.-linked L-rhamnose
epitope, a .beta.-linked .alpha.-Gal disaccharide epitope, or an
.alpha.- or .beta.-linked Forssmann disaccharide epitope:
[0046] In another broad aspect, there is provided herein a vaccine
composition, comprising: an antigen composition comprising:
[0047] 1) a first lipid (lipida) moiety, a first linker (linkera)
moiety, and an antigen moiety;
[0048] 2) a xenoantigen composition comprising: a second lipid
(lipidb) moiety, a second linker (linkerb) moiety, and a
xenoantigen moiety; and,
[0049] 3) at least one liposomal formulation.
[0050] In another broad aspect, there is provided herein a vaccine
composition, wherein a lipid-linked antigen is in non-covalent
association with a lipid-linked xenoantigen, and wherein both the
lipid-linked antigen and the lipid-linked xenoantigen are
independently embedded in liposomes in a liposomal formulation.
[0051] In certain embodiments, there is no chemically synthesized
complex xenoantigen-antigen conjugate molecule.
[0052] In certain embodiments, the the liposomal formulation
comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and
cholesterol in a ratio of from about 80:20 to about 70:30,
respectively.
[0053] In another broad aspect, there is provided herein a vaccine
composition of claim 37, wherein: the antigen composition comprises
a Pam3Cys-MUC1 VNTR-TACA conjugate; the second linker (linkerb)
moiety comprises a tetraethyleneglycol (TEG) portion; and the
xenoantigen moiety comprises .alpha.- or .beta.-linked
L-rhamnose.
[0054] In another broad aspect, there is provided herein an
anti-cancer vaccine comprising a vaccine composition as described
herein
[0055] In another broad aspect, there is provided herein an
anti-viral vaccine comprising a vaccine composition as described
herein.
[0056] In another broad aspect, there is provided herein an
anti-bacterial vaccine comprising a vaccine composition as
described herein.
[0057] In certain embodiments, the vaccine composition further
includes at least one immunologic adjuvant.
[0058] In certain embodiments, the immunologic adjuvant comprises
one or more of: a saponin, monophosphoryl lipid A, 3'-O-deacylated
monophosphoryl lipid A, and interleukin 12.
[0059] In another broad aspect, there is provided herein a method
for eliciting an immune response to a cancer cell surface antigen
in a subject with cancer, comprising administering to the subject
an antigen-xenoantigen-liposome composition of claim 37 in
sufficient dose to elicit the immune response to the cancer cell
surface antigen, wherein the antigen-liposome-xenoantigen
composition comprises the cancer cell surface antigen, and wherein
the immune response is sufficient for treating the cancer.
[0060] In certain embodiments, the both the lipid-linked antigen
and the lipid-linked xenoantigen are independently embedded in
liposomes in the liposomal formulation.
[0061] In certain embodiments, the cancer cell surface antigen is
expressed only in cancer cells in the subject.
[0062] In certain embodiments, the cancer cell surface antigen is
expressed only by cancer cells.
[0063] In certain embodiments, the cancer cell surface antigen
comprises a peptide epitope.
[0064] In certain embodiments, the cell surface antigen comprises
MUC1 mucin.
[0065] In certain embodiments, the subject is a human or non-human
mammal.
[0066] In certain embodiments, the antigen-liposome-xenoantigen
composition is administered intranasally, intramuscularly,
subcutaneously, intravenously, or orally to the subject.
[0067] In certain embodiments, the method further comprises
measuring humoral and/or cellular immune responses to the cancer
cell surface antigen, and administering a further dose to elicit
the immune response, if necessary.
[0068] In another broad aspect, there is provided herein a method
for selectively killing cancer cells expressing a cancer cell
surface antigen in a subject in need thereof, comprising
administering to the subject an antigen-liposome-xenoantigen
composition of claim 27, under conditions that result in production
in the subject of antibodies against the cancer cell surface
antigen, wherein the antibodies produced bind the cancer cell
surface antigen on cancer cells in the subject, thereby killing
cancer cells that express the cancer cell surface antigen.
[0069] In certain embodiments, both the lipid-linked antigen and
the lipid-linked xenoantigen are independently embedded in
liposomes in the liposomal formulation.
[0070] In certain embodiments, the cancer cell surface antigen is
expressed only in cancer cells in the subject.
[0071] In certain embodiments, the cancer cell surface antigen is
expressed only by cancer cells.
[0072] In another broad aspect, there is provided herein a method
for improving immunogenicity of vaccines, the method comprising
incorporating at least one .alpha.- or .beta.-linked L-Rha epitope
by direct conjugation or by non-covalent association with at least
one liposomal vaccine formulation to increase the immunogenicity of
the vaccine by a NA-dependant antigen uptake mechanism.
[0073] In another broad aspect, there is provided herein a method
of synthesizing an alkyne functionalized composition (IV),
comprising: deprotecting an ester having a Fmoc moiety to form a
free acid; coupling the free acid with an amine; and, removing the
Fmoc moiety, and coupling the remaining moiety with palmitic acid
to yield the alkyne functionalized composition.
[0074] In certain embodiments, the antigen moiety comprises: an
anti-cancer composition, an anti-bacterial composition, or an
anti-viral composition.
[0075] In certain embodiments, the antigen moiety comprises: a TACA
that interacts with B-cell receptors. In certain embodiments, the
TACA comprises an autologous helper T-cell epitope.
[0076] In one broad aspect, there is provided herein a method of
synthesizing component 1) above, comprising reacting an
alkyne-functionalized amide derivative with a modified peptide
antigen.
[0077] In another broad aspect, there is provided herein the
composition (VII), where the xenoantigen binds a natural antibody
(NA). In certain embodiment, the xenoantigen contains a structure
comprised of: .alpha.- or .beta.-linked L-rhamnose, a .beta.-linked
.alpha.-Gal epitope, or an .alpha.- or .beta.-linked Forssmann
disaccharide epitope.
[0078] In another broad aspect, there is provided herein a method
for eliciting an immune response to a cancer cell surface antigen
in a subject with cancer, comprising administering to the subject
an antigen-liposome-xenoantigen composition described herein, in
sufficient dose to elicit the immune response to the cancer cell
surface antigen.
[0079] Other systems, methods, features, and advantages of the
present invention will be or will become apparent to one with skill
in the art upon examination of the following drawings and detailed
description. It is intended that all such additional systems,
methods, features, and advantages be included within this
description, be within the scope of the present invention, and be
protected by the accompanying claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0080] The patent or application file may contain one or more
drawings executed in color and/or one or more photographs. Copies
of this patent or patent application publication with color
drawing(s) and/or photograph(s) will be provided by the Patent
Office upon request and payment of the necessary fee.
[0081] FIG. 1: Schematic representation of Fc-Fc.gamma. receptor
interaction in the in vivo generated immune complex and APC leading
to enhanced antigen uptake and presentation on MHC I or MHC II
(APC=antigen presenting cells, e.g., a dendritic cell).
[0082] FIG. 2: Schematic illustration of a liposomal vaccine
composition comprised of: an antigen composition comprised of a
first lipid (lipid.sub.a) moiety, a first linker (linker.sub.a)
moiety, and an antigen moiety; and, a second lipid (lipid.sub.b)
moiety, a second linker (linker.sub.b) moiety, and a xenoantigen
moiety; at least one liposome forming lipid; and, optionally, one
or more adjuvants.
[0083] FIG. 3: Schematic illustration of a lipid-linked antigen
component.
[0084] FIG. 4: Schematic illustration of a dialkyl-substituted
heteroaryl C.sub.1-n alkyl component.
[0085] FIG. 5: Schematic illustration of a composition IV reacting
with a composition V to form a composition II.
[0086] FIG. 6: Schematic illustration of a linker.sub.a
component.
[0087] FIG. 7: Schematic illustration of a lipid-linked xenoantigen
component.
[0088] FIG. 8: TEG composition used in a linker.sub.b
component.
[0089] FIG. 9: Schematic illustration of a structure contained in a
lipid.sub.b portion of a lipid-linked xenoantigen component.
[0090] FIG. 10: Scheme 1: Schematic illustration of synthesis of a
L-Rhamnose-TEG-Cholesterol (3).
[0091] FIG. 11: Scheme 2: Schematic illustration of synthesis of an
alkyne functionalized Pam.sub.3Cys composition (6).
[0092] FIG. 12: Scheme 3: Schematic illustration of synthesis of a
Pam.sub.3Cys-MUC-1 VNTR-TACA conjugate (9) [SEQ ID NOS: 8, 9 and
7], respectively, in order of appearance.
[0093] FIGS. 13A-13C: Size characterization of liposomes at
1/10,000 dilution: FIG. 13A--Batch 1 liposomes; FIG. 13B--Batch 2
liposomes; and FIG. 13C--Batch 3 liposomes.
[0094] FIGS. 14A-14B: Photographs showing size characterization of
liposomes: SEM images at 5 kV acceleration voltage. FIG. 14A: Batch
1 liposomes under 50,000.times. magnification. FIG. 14B: Batch 1
liposomes under 250,000.times. magnification.
[0095] FIGS. 15A-15C: Fluorescence microscope images with Batch 1
liposomes under 60.times. magnification. FIG. 15A: Images with
control antibodies (antibodies isolated from preimmunization serum)
1st, 2nd and 3rd images: brightfield, FITC and overlay. FIG. 15B:
Images with anti-rhamnose antibodies, 1st, 2nd and 3rd images:
brightfield, FITC and overlay. FIG. 15C: Images with anti-MUC 1
antibodies, 1st, 2nd and 3rd images: brightfield, FITC and
overlay.
[0096] FIG. 16: .sup.1H NMR of
(5-Cholesten-3.alpha.-yloxy)-3n.sup.3.sub.9-trixaundecanyl-2,3,4-tri-O-ac-
etyl-.alpha.-L-Rhamnopyranoside (2).
[0097] FIG. 17: .sup.13C NMR of
(5-Cholesten-3.alpha.-yloxy)-3n.sup.3.sub.9-trixaundecanyl-2,3,4-tri-O-ac-
etyl-.alpha.-L-Rhamnopyranoside (2).
[0098] FIG. 18: .sup.1H-gCosy of
(5-Cholesten-3.alpha.-yloxy)-3n.sup.3.sub.9-trixaundecanyl
2,3,4-tri-O-acetyl-.alpha.-L-Rhamnopyranoside (2).
[0099] FIG. 19: HRMS of
(5-Cholesten-3.alpha.-yloxy)-3n.sup.3.sub.9-trixaundecanyl
2,3,4-tri-O-acetyl-.alpha.-L-Rhamnopyranoside (2).
[0100] FIG. 20: .sup.1H NMR of
(5-Cholesten-3.alpha.-yloxy)-3n.sup.3.sub.9-trixaundecanyl
Rhamnopyranoside (3).
[0101] FIG. 21: .sup.13C NMR of
(5-Cholesten-3.alpha.-yloxy)-3n.sup.3.sub.9-trixaundecanyl
Rhamnopyranoside (3).
[0102] FIG. 22: .sup.1H-gCosy of
(5-Cholesten-3.alpha.-yloxy)-3n.sup.3.sub.9-trixaundecanyl
Rhamnopyranoside (3).
[0103] FIG. 23: HRMS of
(5-Cholesten-3.alpha.-yloxy)-3n.sup.3.sub.9-trixaundecanyl
Rhamnopyranoside (3).
[0104] FIG. 24: .sup.1H NMR of N-Propargyl Pam.sub.2FmocCys Amide
Derivative (5).
[0105] FIG. 25: .sup.13C NMR of N-Propargyl Pam.sub.2FmocCys Amide
Derivative (5).
[0106] FIG. 26: .sup.1H gCosy of N-Propargyl Pam.sub.2FmocCys Amide
Derivative (5).
[0107] FIG. 27: HRMS of N-Propargyl Pam.sub.2FmocCys Amide
Derivative (5).
[0108] FIG. 28: .sup.1H NMR of N-Propargyl Pam.sub.3Cys Amide
Derivative (6).
[0109] FIG. 29: .sup.13C NMR of N-Propargyl Pam.sub.3Cys Amide
Derivative (6).
[0110] FIG. 30: .sup.1H-gCosy of N-Propargyl Pam.sub.3Cys Amide
Derivative (6).
[0111] FIG. 31: HRMS of N-Propargyl Pam.sub.3Cys Amide Derivative
(6).
[0112] FIG. 32: HR-MALDI-TOF of Glycopeptide Azide (9) [SEQ ID
NO:8].
[0113] FIG. 33: HR-MALDI-TOF of Glycopeptide Azide (8) [SEQ ID NO:
10].
[0114] FIG. 34: HR-MALDI-TOF of Lipopeptide (9) [SEQ ID NO:7].
[0115] FIG. 35: Scheme showing synthetic route of
2-Aminoethyle-.alpha.-L-Rhamnopyranoside (13).
[0116] FIG. 36: Anti-Rha Antibody Isotype Titers after 4th Boost
with Rha-Ficoll (group A) or Rha-OVA (group B) at 1/5 or 1/500
Serum Dilutions respectively.
[0117] FIG. 37: T-Cell Proliferation in BALB/c mice with MUC1-Tn
(10) Peptide in Presence and Absence of Dendritic Cells.
[0118] FIGS. 38A-38: FIG. 38A) T-cell proliferation measured by
[.sup.3H]thymidine incorporation in T-cells from mice spleens
primed with MUC1-Tn (8) and challenged with Pam.sub.3Cys-MUC1-Tn
(9) +Rha liposomes in the presence of anti-Rha antibodies (abs) or
control abs [anti Rha(OVA) and anti Rha(Ficoll) abs are the
antibodies isolated from the serum of Rha-OVA and Rha-Ficoll
immunized mice, respectively]. FIG. 38B) Stepwise immunization
plan. Groups A1, A2, B1, and B2 each represent four groups of
female BALB/c mice. Stage I: groups A2 and B2 were immunized with
Rha-Ficoll/Alum whereas groups A1 and B1 were non-immunized. Stage
II: vaccination; groups A1 and A2 vaccinated and boosted with
Pam.sub.3Cys-MUC1-Tn liposomes whereas groups B2 and B2 were
vaccinated with Pam.sub.3Cys-MUC1-Tn+Rha liposomes.
[0119] FIGS. 39A-39B: FIG. 39A) Group average of anti-Rha antibody
titers after fourth boost with Rha-Ficoll/Alum. FIG. 39B) Group
average of anti-MUC1-Tn antibody titers after first boost with
Pam.sub.3Cys-MUC1-Tn liposomes or Pam.sub.3Cys-MUC1-Tn+Rha
liposomes.
[0120] FIG. 40: Anti-MUC1-Tn Antibody Isotype Titers after first
boost with Pam.sub.3Cys-MUC1-Tn liposomes or
Pam.sub.3Cys-MUC1-Tn+Rha liposomes at 1/50 Serum Dilutions.
[0121] FIGS. 41A-41B: FIG. 41A) Competitive binding of anti-MUC1-Tn
antibodies with bound MUC1-Tn in presence of free MUC1-Tn (8). FIG.
41B) Groups average of anti-Tn antibody titer after first boost
with Pam.sub.3Cys-MUC1-Tn liposomes or Pam.sub.3Cys-MUC1-Tn+Rha
liposomes.
[0122] FIGS. 42A-42B: Binding of anti-MUC1-Tn antibodies to human
leukemia U266 cells. FIG. 42A) Second antibody alone and with mouse
anti-human MUC1 antibodies; FIG. 42B) with 1/5 dilution of
non-immunized mouse serum, and with 1/5 dilution of group B2 mouse
serum.
[0123] FIG. 43: .sup.1H NMR of
2-Azidoethyl-2,3,4-Tri-O-acetyl-.alpha.-L-rhamnopyranoside
(11).
[0124] FIG. 44: .sup.13C NMR of
2-Azidoethyl-2,3,4-Tri-O-acetyl-.alpha.-L-rhamnopyranoside
(11).
[0125] FIG. 45: .sup.1H-gCosy of
2-Azidoethyl-2,3,4-Tri-O-acetyl-.alpha.-L-rhamnopyranoside
(11).
[0126] FIG. 46: HRMS of
2-Azidoethyl-2,3,4-Tri-O-acetyl-.alpha.-L-rhamnopyranoside
(11).
[0127] FIG. 47: .sup.1H NMR of 2-Azidoethyl
.alpha.-L-rhamnopyranoside (12).
[0128] FIG. 48: .sup.13C NMR of 2-Azidoethyl
.alpha.-L-rhamnopyranoside (12).
[0129] FIG. 49: .sup.1H-gCosy of 2-Azidoethyl
.alpha.-L-rhamnopyranoside (12).
[0130] FIG. 50: HRMS of 2-Azidoethyl .alpha.-L-rhamnopyranoside
(12).
[0131] FIG. 51: ESI MS of 2-Aminoethyl .alpha.-L-rhamnopyranoside
(13).
[0132] FIG. 52: Scheme showing the synthesis of Pam.sub.3Cys-MUC-1
VNTR conjugate (17) [SEQ ID NOS: 11, 11 and 12], respectively, in
order of appearance.
[0133] FIG. 53: HRMS (MALDI-TOF) spectrum of peptide (16).
[0134] FIG. 54: HPLC trace of peptide (16).
[0135] FIG. 55: HRMS (MALDI-TOF) spectrum of lipopeptide (17).
[0136] FIG. 56: Synthesis of Pam.sub.3Cys-MUC-1 VNTR conjugate (21)
[SEQ ID NOS: 13, 14 15], respectively, in order of appearance.
[0137] FIG. 57: HRMS (MALDI-TOF) of glycopeptide (19).
[0138] FIG. 58: HPLC trace of glycopeptide (19).
[0139] FIG. 59: HRMS (MALDI-TOF) of glycopeptide (20).
[0140] FIG. 60: HRMS (MALDI-TOF) of Pam.sub.3Cys-MUC-1 VNTR
conjugate (21).
DETAILED DESCRIPTION
[0141] Throughout this disclosure, various publications, patents
and published patent specifications are referenced by an
identifying citation. The disclosures of these publications,
patents and published patent specifications are hereby incorporated
by reference into the present disclosure to more fully describe the
state of the art to which this invention pertains.
GENERAL DEFINITIONS
[0142] The terms "a," "an," and "the" include the plural referents
unless the context clearly dictates otherwise.
[0143] The term "antibody" broadly refers to monoclonal antibodies
(including full length monoclonal antibodies) and "antibody
fragments" that exhibit a desired biological activity. "Antibody
fragments" can comprise a portion of a full length antibody,
generally the antigen binding or variable region thereof.
Non-limiting examples of antibody fragments include Fab, Fab', and
Fv fragments; diabodies; linear antibodies; and single-chain
antibody molecules.
[0144] The term "monoclonal antibody" broadly refers to antibodies
that are highly specific, being directed against a single antigenic
site.
[0145] The term "antibody" also broadly includes naturally
occurring antibodies (NAs) as well as non-naturally occurring
antibodies, including, for example, single chain antibodies,
chimeric, bifunctional and humanized antibodies, as well as
antigen-binding fragments thereof. For example, such non-naturally
occurring antibodies can be constructed using solid phase peptide
synthesis, can be produced recombinantly or can be obtained, for
example, by screening combinatorial libraries consisting of
variable heavy chains and variable light chains.
[0146] The term "subject/s" generally refers to any animal that
generates an adaptive immune response and can include mammals,
birds and reptiles. Examples of subjects can include, but are not
limited to, humans, non-human primates, dogs, cats, horses, cows,
goats, guinea pigs, mice, rats and rabbits, as well as any other
domestic or commercially valuable animal.
[0147] The term/s "nucleic acid/s" generally encompass both RNA and
DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically
synthesized) DNA and chimeras of RNA and DNA. The nucleic acid can
be double-stranded or single-stranded. Where single-stranded, the
nucleic acid can be a sense strand or an antisense strand. The
nucleic acid can be synthesized using oligonucleotide analogs or
derivatives (e.g., inosine or phosphorothioate nucleotides). Such
oligonucleotides can be used, for example, to prepare nucleic acids
that have altered base-pairing abilities or increased resistance to
nucleases.
[0148] It is to be further understood that the term "C.sub.1-n
alkyl" can be any linear or branched alkyl group containing 1 to n
carbon atoms. For example, the term"C.sub.1-6 alkyl" can comprise
groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl,
sec-butyl, tert-butyl, pentyl, isopentyl(3-methylbutyl),
neopentyl(2,2-dimethylpropyl), hexyl, isohexyl(4-methylpentyl) and
the like.
[0149] The term "C.sub.2-n alkyl" is to be understood to mean any
linear or branched alkyl group containing 2 to n carbon atoms. For
example, the term "C.sub.2-6 alkyl" can comprise groups such as
ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,
pentyl, isopentyl(3-methylbutyl), neopentyl(2,2-dimethylpropyl),
hexyl, isohexyl(4-methylpentyl) and the like.
[0150] The term "dialkyl-substituted aryl C.sub.1-n alkyl" is to be
understood to mean an aryl group substituted at two positions
wherein the substituents are composed of a C.sub.1-n alkyl group,
as defined above. The aryl group may be optionally substituted with
at least one substituent selected from the group consisting of
hydroxyl, C.sub.1-2 alkoxy and halogen. Examples of
dialkyl-substituted aryl C.sub.1-n alkyl groups include, but are
not limited to:
##STR00001##
[0151] The term "dialkyl-substituted heteroaryl C.sub.1-n alkyl" is
to be understood to mean a heteroaryl group substituted at two
positions wherein the substituents are composed of a C.sub.1-n
alkyl group, as defined above. The aryl group may be optionally
substituted with at least one substituent selected from the group
consisting of hydroxyl, C.sub.1-2 alkoxy and halogen. Examples of
substituted heteroaryl C.sub.1-n alkyl groups include, but are not
limited to:
##STR00002##
[0152] Accordingly, the term "xenoantigen" is to be understood to
mean at least one foreign antigen capable of binding natural
antibodies. Examples of xenoantigens include, but are not limited
to, the .alpha.-gal epitope, L-Rha, and the Forssman
disaccharide.
[0153] The term "peptide" may be any peptide comprising natural or
non-natural amino acids; and if chiral, in its L or D
configurations or as racemate. Examples of non-natural amino acids
include, but are not limited to: homocysteine, homoarginine,
cylcohexylalanine, ornithine, and
C.sup..alpha..alpha.-dibenzylglycine.
[0154] As used herein, the term "lipid" is defined to include any
of a broad range of substances that is characteristically insoluble
in water and extractable with an organic solvent. This broad class
of compounds is known to those of skill in the art, and as the term
"lipid" is used herein, it is not limited to any particular
structure. Examples include compounds which contain long-chain
aliphatic hydrocarbons and their derivatives. A lipid may be
naturally occurring or synthetic (i.e., designed or produced by
man). Biological lipids are well known in the art and include, for
example, neutral fats, phospholipids, phosphoglycerides, steroids,
terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides,
lipids with ether and ester-linked fatty acids and polymerizable
lipids, and combinations thereof. Of course, compounds other than
those specifically described herein that are understood by one of
skill in the art as lipids are also encompassed by the compositions
and methods described herein.
GENERAL DESCRIPTION
[0155] In a first broad aspect, there is provided herein a
composition having xenoantigen that is incorporated onto a target
antigen.
[0156] In another broad aspect, there is provided herein a vaccine
composition comprising a lipid-linked antigen in non-covalent
association with a lipid-linked xenoantigen, where both the
lipid-linked antigen and the lipid-linked xenoantigen are
independently embedded in liposomes in a liposomal formulation.
[0157] In another broad aspect, there is provided herein
tumor-associated carbohydrate antigens (TACAs) that are on the
heavily glycosylated glycoprotein mucin 1 (MUC1).
[0158] In another aspect, the tumor-associated carbohydrate
antigens (TACAs) that are on the heavily glycosylated glycoprotein
mucin 1 (MUC1) are useful as targets for anticancer active
immunotherapy.
[0159] In another aspect, there is provided herein the non-covalent
association of the L-Rhamnose xenoantigen in a liposome with the
other components of a vaccine independently embedded in the
liposome. This non-covalent association eliminates the need to
chemically synthesize a complex xenoantigen-antigen conjugate
molecule.
[0160] The peptide sequences described herein act as an autologous
helper T-cell epitope. The use of foreign carrier proteins and
peptides as conjugates provide helper T-cell epitopes unrelated to
the tumor and act as heterologous helper T-cell epitopes.
[0161] In one embodiment, the MUC1 sequence described herein is
shown to be a potent candidate for generating antibodies.
[0162] The glycoconjugates on the surface of cancer cells are
expressed in abnormal quantities and show modifications in the
structure of the carbohydrate moieties linked to the peptide or
lipid components of the conjugates. These aberrant carbohydrate
epitopes are specifically known as tumor associated carbohydrate
antigens (TACAs). TACAs demonstrate potential as markers for cancer
detection and disease progression and therefore are under intense
investigation for their role in the development of anti-cancer
vaccines.
[0163] The TACAs found on the heavily glycosylated glycoprotein
mucin 1 (MUC1) are useful targets for anticancer active
immunotherapy. In most cancers of epithelial origin, e.g., breast,
colorectal, and prostate, this glycoprotein becomes highly over
expressed and loses its apical distribution, becoming expressed
over the entire cell surface. In the transformed state, the glycan
chains of MUC1 are typically shorter and show increased sialylation
relative to normal. A number of TACAs have been identified from
MUC1. TACAs includes TF, Tn, STn, sLe.sup.a antigens:
##STR00003##
[0164] In certain embodiments, the above TACAs (which are found on
tumor cells) are useful as the "B-cell epitopes" in the design of
the vaccines described herein.
[0165] The MUC1 glycoprotein is also shed into the serum and is a
tumor marker for cancer. For example, patients possessing naturally
occurring anti-MUC1 antibodies demonstrate better disease-specific
survival. It is believed that anti-MUC1 antibodies control
hematogenous tumor dissemination and outgrowth. It is also now
believed that that TACAs present on MUC1 variable number tandem
repeat (VNTR) domain may help to break the immune system's self
tolerance to MUC1, at least in human MUC1-expressing transgenic
mice while unglycosylated human MUC1 VNTRs generated a weaker
immune response. While not wishing to be bound by theory, it is now
also believed that this is because the glycopeptide is possibly
seen by the immune system as a more foreign epitope in comparison
to the unglycosylated MUC1 which is more self-like.
[0166] Xenoantigens:
[0167] Many carbohydrate antigens are not highly immunogenic. One
approach to increasing the immunogenicity of antigens is to target
the antigens to antigen presenting cells (APCs). The effectiveness
of vaccines can be increased by the incorporation of a xenoantigen.
For example, the xenoantigen Gal.alpha.1-3Gal.beta.1 (.alpha.Gal)
can be used as an in vivo generated immune complex between the
.alpha.Gal epitopes and the natural antibodies (NA) against
.alpha.Gal in serum. The Fc portion on this immune complex is
recognized by the Fc.gamma. receptors on the APCs, thereby leading
to the overall internalization of the antigen facilitating
presentation by the major histocompatibility complex (MHC) on APCs.
NA can also enhance uptake by APCs using other receptors such as
complement receptors. Also, there are a variety of NAs that can
bind antigens, including autoantigens, normally present in the
blood.
[0168] The NAs, which can be of the IgM, IgG, or IgA isotype in
humans, exhibit specific binding to a variety of different
antigens, including proteins, DNA, numerous neutral and anionic
phospholipids, carbohydrates, and a variety of other lipids,
including neutral and anionic glycolipids, cholesterol, and
squalene. For example, a large fraction of NAs-to-phospholipid is
circulating in vivo as immune complexes to phospholipids. Thus, not
only can the xenoantigens complex NAs, but also the lipids and
phospholipids present in liposomes can complex NAs as well.
[0169] In addition, there are other, and highly abundant,
anti-carbohydrate NAs in human serum. The most abundant NA
detectable was specific for .beta.-linked L-rhamnose (L-Rha) with
the fourth most abundant being against .alpha.-linked L-Rha; for
example:
##STR00004##
[0170] Antibodies against both .alpha./.beta. L-Rha are more
prevalent than those against .alpha.-Gal.
[0171] In one aspect, there is described herein a method where the
incorporation of .alpha./.beta.-linked L-Rha epitopes into vaccines
(by direct conjugation or by non-covalent association with
liposomal vaccine formulations) increases immunogenicity by
NA-dependant antigen uptake mechanism.
[0172] In another aspect, there is described herein a vaccine
composition that incorporates the non-covalent association of the
L-Rha xenoantigen in a liposome with one or more other components
of the vaccine composition that are independently embedded in the
liposome. This incorporation eliminates the need to chemically
synthesize a complex xenoantigen-antigen conjugate molecule. Thus,
any lipidated antigen can be introduced into the vaccine
composition. One non-limiting example is the L-Rha motif which
binds anti-L-Rha NAs.
[0173] The Fc portion of the NA of an in vivo generated immune
complex is recognized by the Fc.gamma. receptors located on the
surface of the APCs or by complement and complement receptors,
thereby leading to the better internalization of the vaccine.
[0174] Also, the T-cell proliferation is enhanced when the antigen
and xenoantigen are co-localized on a liposome in the presence of
APCs and anti L-Rha antibodies.
[0175] In one embodiment described herein, the xenoantigen is
conjugated to a lipid (such as cholesterol) by means of a linker
(such as tretraethylene glycol (TEG)), which allows the xenoantigen
to be displayed on the surface of the liposome along with the
lipidated-tumor, bacterial or viral antigen.
[0176] In such embodiments, the linker between the xenoantigen and
the cholesterol lipid allows for a xenoantigen-NA binding
interaction.
[0177] Antigens:
[0178] For the development of a vaccine (e.g., an anti-cancer
vaccine, an anti-viral vaccine, or an anti-bacterial vaccine), an
antigen is required. Broadly, an antigen is defined as an entity or
foreign molecule that, when introduced into the body, triggers the
production of an antibody by the immune system. The term "antigen"
also refers to any molecule or molecular fragment that can be bound
by a major histocompatibility complex (MHC) and presented to a
T-cell receptor.
[0179] Tumors of epithelial origin provide a variety of TACAs that
can act as antigens by interacting with B-cell receptors. These
include TACAs identified on the glycoproteins and glycolipids that
decorate the cancer cells. Non-limiting examples of TACAs include:
the TF, Tn, sialyl Tn (sTn) and sialyl Lewis a (sLe.sup.a)
antigens, whose structures are shown above. These structures as
well as many other TACAs can produce a considerable humoral immune
response.
[0180] In addition, peptides and glycopeptides can interact with
B-cell receptors to produce antibodies.
[0181] One of the major disadvantages of using TACAs alone as
cancer vaccines, however, is their weak immunogenicity. Generation
of high amounts of high affinity IgG antibodies against the cancer
antigen depends upon the combined interaction of the B-cells and
the helper T-cells, and therefore requires the vaccine to contain a
peptide epitope which can be displayed on the major
histocompatibility complex (MHC) class-II or class-I molecules that
are present on the surface of APCs, such as DCs. In general, TACAs
alone cannot activate the helper T-cells, resulting in poor
immunogenicity. Previous efforts to counteract this problem
involved the conjugation of TACAs to large carrier proteins like
keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), TLR
agonists and zwitterionic polysaccharides. However, one drawback to
the protein conjugation strategy is that the major immune response
is towards the carrier protein, thus resulting in a suppressed
immune response for the cancer antigen.
[0182] The protein conjugates provide the peptide sequences, which
protein sequences can then be displayed as helper T-cell epitopes
on MHC class-II molecules. The resulting complexes activate
CD4.sup.+ helper T-cells which then interact with
antibody-producing B-cells allowing the B-cells to produce higher
affinity antibodies.
[0183] For example, smaller 20-amino acid peptides which can bind
MHC class-II can be conjugated to TACAs. These conjugates can
produce high amounts of high affinity IgG anti-TACA antibodies.
This strategy provides an advantage over the use of large carrier
proteins as helper T-cell epitope, as the immune system can now
focus more resources on the available TACA antigens rather than
producing antibodies to the carrier protein.
[0184] In one aspect, the use of peptide sequences acts as an
"autologous helper T-cell epitope." Such autologous helper T-cell
epitopes can be sequences expressed on the tumor. The use of
foreign carrier proteins and peptides as conjugates thus provide
helper-T cell epitopes unrelated to the tumor, and thus act as
"heterologous helper T-cell epitopes."
[0185] One non-limiting example of an autologous helper T-epitope
is the MUC1 VNTR consisting of the 20 amino acid sequence
PDTRPAPGST(Tn)APPAHGVTSA [SEQ ID NO: 1]. In a particular example,
the threonine in the sequence GST is synthetically modified to
incorporate the .alpha.-GalNAc-O-Thr (Tn) TACA. This region in the
MUC1 sequence is a more potent candidate for generating high
antibody titers against Tn than the unglycosylated MUC1 VNTR.
[0186] Lipid Attachment to Antigen
[0187] Glycopeptides formed by the conjugation of B-cell epitopes
and helper T-cell epitopes often show moderate immunogenicity. A
major reason for such moderate immunogenicity is the inappropriate
maturation of the DCs.
[0188] Described herein is a method which overcomes this problem,
where a lipid component is incorporated into a vaccine capable of
imparting self adjuvanating properties. Toll-like receptor (TLR)
agonists interact with TLRs to facilitate the maturation of
dendritic cells. The maturation of the dendritic cells can be
achieved by the involvement of a variety of TLR subclasses, as for
example: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,
TLR10, TLR11, TLR12, TLR13, TLR14, TLR15 and TLR16.
[0189] Non-limiting examples of TLR2 interactive lipopeptides
include: dipalmitoyl-S-glyceryl-cys-(Pam.sub.2Cys);
tripalmitoyl-S-glyceryl-cys-(Pam.sub.3Cys-);
dipalmitoyl-S-glyceryl-cys-ser-lys-lys-lys-lys
(Pam.sub.2Cys-Ser-(Lys).sub.4) [SEQ ID NO: 2],
tripalmitoyl-S-glyceryl-cys-ser-lys-lys-lys-lys
(Pam.sub.3Cys-Ser-(Lys).sub.4) [SEQ ID NO: 3], and MALP-2
dipalmitoyl-S-glyceryl-cys-gly-asn-asn-asp-glu-ser-asn-ile-ser-phe-lys-gl-
u-lys (Pam.sub.2CGNNDESNISFKEK)] [SEQ ID NO: 4].
[0190] Non-limiting examples of palmitoylated lipids [SEQ ID NOS:
3, 2 and 4], respectively, in order of appearance, that may
interact with TLR2 thus include:
##STR00005##
[0191] Pam.sub.2Cys-Ser-(Lys).sub.4 [SEQ ID NO:2], for example,
acts as TLR2/TLR6 agonists, while Pam.sub.3Cys-Ser-(Lys).sub.4 [SEQ
ID NO:3] acts as a TLR2/1 agonist. Lipopeptide MALP-2 acts a TLR2/6
ligand. Tripalmitoyl-S-glyceryl-cys (Pam.sub.3Cys), and
dipalmitoyl-S-glyceryl-cys (Pam.sub.2Cys), are minimal structural
epitopes common to palmitoylated TLR2 ligands.
[0192] Synthesis of Two-component Vaccine
[0193] Lipid Components
[0194] In certain embodiments, the lipid component of a vaccine
composition serves three purposes: first, the lipid component acts
as a vaccine adjuvant capable of interacting with Toll-Like
receptors; second, the lipid component facilitates the formulation
of the glycolipopeptide into liposomes anchoring the glycopeptide
epitope to the inner and outer surfaces of the liposomes; and
third, the lipidation facilitates cross-presentation of peptide
antigens by APCs.
[0195] Xenoantigen Component
[0196] Another aspect is the ability to bind natural antibodies
(NAs) specific for a desired target epitope (e.g., the xenoantigen
L-Rha epitope). The bound NAs thus facilitate the opsonization of
the liposomal vaccine by APCs. Covalent attachment of xenoantigens
to lipids through the use of a suitable linker thus facilitates the
incorporation of these epitopes into the liposomes.
[0197] Linker Components
[0198] In some embodiments, the xenoantigen is covalently attached
to a lipid through the use of a suitable linker. Further, in
particular embodiments, the length of the linker between the
xenoantigen and lipid can be optimized to achieve a desired
xenoantigen-NA interaction. The conjugation of a lipid to the
xenoantigen allows for the display of multiple xenoantigens on the
surface of the liposomes, thereby facilitating multivalent
NA-antibody binding to the liposome.
[0199] The Fc portion of the NA in the in vivo generated immune
complex is thus recognized by the Fc.gamma. receptors on APCs,
thereby leading to opsonization of the vaccine by APCs.
[0200] Vaccine Compositions
[0201] In certain embodiments, the composition of the vaccine
contains non-covalently associated components: i) a lipid-linked
antigen; ii) a composition of lipids capable of forming liposomes;
iii) a lipid-linked xenoantigen; and, optionally, iv) one or more
independent adjuvant molecules. These components form a well
defined liposomal vaccine composition I, as shown in FIG. 2. The
lipid-linked antigen is comprised of an antigen linked to a first
lipid (also called lipid.sub.a herein). In certain embodiments, the
antigen can be a peptide or glycopeptide of 1 to 100 amino acids in
length. The antigen may also include a B-cell epitope conjugated to
a peptide or glycopeptide sequence that is capable of providing a
T-cell epitope. The peptide, glycopeptide, peptide-B-cell, or
glycopeptides-B-cell epitope conjugate is linked to
lipid.sub.a.
[0202] The generalized composition of the lipid.sub.a-linked
antigen component is the structure of composition II, shown in FIG.
3.
[0203] In one embodiment, the antigen contains a glycopeptide
having the 20 amino acid sequence PDTRPAPGST(Tn)APPAHGVTSA [SEQ ID
NO:1]. The T(Tn) in the sequence is the .alpha.-GalNAc-O-T (Tn)
TACA. In another embodiment, the antigen contains a glycopeptide
having the 20 amino acid sequence TSAPDTRPAPGSTAPPAHGV [SEQ ID NO:
5]. In another embodiment, the antigen contains a glycopeptides
having the 20 amino acid sequence TSAPDT(Tn)RPAPGSTAPPAHGV [SEQ ID
NO: 6]. The T(Tn) in the sequence is the .alpha.-GalNAc-O-T (Tn)
TACA.
[0204] The linker.sub.a component can be: a chain of C.sub.2-n
alkyl, dialkyl-substituted aryl C.sub.1-n alkyl,
dialkyl-substituted heteroaryl C.sub.1-n alkyl, or
--CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.n--, where n is a
positive integer. In one embodiment, the linker.sub.a component
comprises a dialkyl-substituted heteroaryl C.sub.1-n alkyl of
formula III, as shown in FIG. 4. In certain embodiments, the
lipid.sub.a portion of the formula III contains a lipid of one of
the following structures: Pam.sub.2Cys-Ser-(Lys).sub.4,
Pam.sub.3Cys-Ser-(Lys).sub.4, MALP-2, Pam.sub.3Cys, or
Pam.sub.2Cys. In particular embodiments, the first lipid
(lipid.sub.a) component of the formula III can contain a lipid
having the structure Pam.sub.3Cys, or the structure
Pam.sub.2Cys.
[0205] Synthesis of Toll-Receptor Ligands
[0206] Another aspect is the synthesis of a composition IV (shown
in FIG. 5) which, when reacted with an appropriate catalyst in the
presence of a modified peptide antigen of composition V, forms a
composition of composition II.
[0207] In certain embodiments, the lipid.sub.a moiety of the
composition IV can be a structure of the Formulae [SEQ ID NOS: 3, 2
and 4], respectively, in order of appearance:
##STR00006##
[0208] Referring again to FIG. 5 and the composition V, the "A"
group therein represents a linker. In certain embodiments, the
linker can be comprised of: a chain of C.sub.1-n alkyl, dialkyl
substituted aryl C.sub.1-n alkyl, or
--CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.n--; n is a positive
integer. In a particular embodiment, A comprises a C.sub.1-5 alkyl
chain.
[0209] Further, in composition V, the antigen moiety can be a
peptide or glycopeptide that is covalently bonded at the N-terminus
to a carbonyl group on the linker, thus forming an amide bond. In a
particular embodiment, the antigen contains a glycopeptide having a
20 amino acid sequence PDTRPAPGST(Tn)APPAHGVTSA [SEQ ID NO:1],
where the T(Tn) in the sequence GSTA is the .alpha.-GalNAc-O-Thr
TACA. In another embodiment, the antigen contains a glycopeptide
having the 20 amino acid sequence TSAPDTRPAPGSTAPPAHGV [SEQ ID NO:
5]. In another embodiment, the antigen contains a glycopeptide
having the 20 amino acid sequence TSAPDT(Tn)RPAPGSTAPPAHGV [SEQ ID
NO: 6]. The T(Tn) in the sequence is the .alpha.-GalNAc-O-T (Tn)
TACA.
[0210] Upon reaction of composition IV with composition V in the
presence of an appropriate catalyst, a composition II is formed. In
certain embodiments, the linker.sub.a moiety can be a
dialkyl-substituted heteroaryl C.sub.1-n alkyl of formula VI, as
shown in FIG. 6.
[0211] In formula VI, the "A" group can be a linker comprised of: a
chain of C.sub.1-n alkyl, dialkyl substituted aryl C.sub.1-n alkyl,
or --CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.n--; n is a positive
integer. In a particular embodiment, the "A" group comprises a
C.sub.1-5 alkyl chain.
[0212] Referring now to FIG. 7, the lipid-linked xenoantigen can be
a xenoantigen linked to a lipid. The xenoantigen can be any
structure which binds NAs. The xenoantigen is linked to a second
lipid (lipid.sub.b) through a second linker (linker.sub.b). The
generalized composition of the lipid-linked xenoantigen component
is structure of composition VII as shown in FIG. 7.
[0213] In one embodiment, the xenoantigen contains a structure
comprised of: alpha- or beta-linked L-rhamnose, a beta-linked
alpha-gal epitope, or an alpha- or beta-linked Forssmann
disaccharide epitope. In a particular embodiment, the xenoantigen
contains a structure consisting of alpha- or beta-linked
L-rhamnose.
[0214] The linker.sub.b component can be comprised of: a chain of
C.sub.1-n alkyl, dialkyl substituted aryl C.sub.1-n alkyl, or
--CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.n--; where n is a
positive integer. In a particular embodiment, the linker.sub.b
component has a tetraethyleneglycol (TEG) of formula VIII, as shown
in FIG. 8.
[0215] In a particular embodiment, the lipid.sub.b portion can
contain a structure of the formula IX, as shown in FIG. 9. In one
particular embodiment, the liposomal forming lipids can be
comprised of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and
cholesterol in a ratio of from about 80:20 to about 70:30,
respectively.
[0216] It is to be understood that, in certain embodiments, an
immunologic adjuvant may or may not be included as part of the
composition of the vaccine. For example, in certain embodiments,
the immunologic adjuvants may be, but are not limited to: a saponin
(e.g., QS21), monophosphoryl lipid A, 3'-O-deacylated
monophosphoryl lipid A, or interleukin 12.
[0217] Pharmaceutical Preparations
[0218] Pharmaceutical compositions of the present invention
comprise an effective amount of a compound(s) or composition(s)
disclosed herein, and/or additional agents, dissolved or dispersed
in a pharmaceutically acceptable carrier. The phrases
"pharmaceutical" or "pharmacologically acceptable" refers to
molecular entities and compositions that produce no adverse,
allergic or other untoward reaction when administered to an animal,
such as, for example, a human. The preparation of a pharmaceutical
composition that contains at least one compound or additional
active ingredient will be known to those of skill in the art in
light of the present disclosure, as exemplified by Remington's
Pharmaceutical Sciences, 2003, incorporated herein by reference.
Moreover, for animal (e.g., human) administration, it will be
understood that preparations should meet sterility, pyrogenicity,
general safety and purity standards as required by FDA Office of
Biological Standards.
[0219] A vaccine composition disclosed herein may comprise
different types of carriers depending on whether it is to be
administered in solid, liquid or aerosol form, and whether it need
to be sterile for such routes of administration as injection.
Vaccine compositions disclosed herein can be administered
intravenously, intradermally, transdermally, intrathecally,
intraarterially, intraperitoneally, intranasally, intravaginally,
intrarectally, topically, intramuscularly, subcutaneously,
mucosally, in utero, orally, topically, locally, via inhalation
(e.g., aerosol inhalation), by injection, by infusion, by
continuous infusion, by localized perfusion bathing target cells
directly, via a catheter, via a lavage, in cremes, in lipid
compositions (e.g., liposomes), or by other method or any
combination of the forgoing as would be known to one of ordinary
skill in the art (see, for example, Remington's Pharmaceutical
Sciences, 2003, incorporated herein by reference).
[0220] The actual dosage amount of a composition disclosed herein
administered to an animal or human patient can be determined by
physical and physiological factors such as body weight, severity of
condition, the type of disease being treated, previous or
concurrent therapeutic interventions, idiopathy of the patient and
on the route of administration. Depending upon the dosage and the
route of administration, the number of administrations of a
preferred dosage and/or an effective amount may vary according to
the response of the subject. The practitioner responsible for
administration will, in any event, determine the concentration of
active ingredient(s) in a composition and appropriate dose(s) for
the individual subject.
[0221] In certain embodiments, pharmaceutical compositions may
comprise, for example, at least about 0.1% of an active compound.
In other embodiments, an active compound may comprise between about
2% to about 75% of the weight of the unit, or between about 25% to
about 60%, for example, and any range derivable therein. Naturally,
the amount of active compound(s) in each therapeutically useful
composition may be prepared is such a way that a suitable dosage
will be obtained in any given unit dose of the compound. Factors
such as solubility, bioavailability, biological half-life, route of
administration, product shelf life, as well as other
pharmacological considerations will be contemplated by one skilled
in the art of preparing such pharmaceutical formulations, and as
such, a variety of dosages and treatment regimens may be
desirable.
[0222] In other non-limiting examples, a dose may also comprise
from about 1 microgram/kg/body weight, about 5 microgram/kg/body
weight, about 10 microgram/kg/body weight, about 50
microgram/kg/body weight, about 100 microgram/kg/body weight, about
200 microgram/kg/body weight, about 350 microgram/kg/body weight,
about 500 microgram/kg/body weight, about 1 milligram/kg/body
weight, about 5 milligram/kg/body weight, about 10
milligram/kg/body weight, about 50 milligram/kg/body weight, about
100 milligram/kg/body weight, about 200 milligram/kg/body weight,
about 350 milligram/kg/body weight, about 500 milligram/kg/body
weight, to about 1000 mg/kg/body weight or more per administration,
and any range derivable therein. In non-limiting examples of a
derivable range from the numbers listed herein, a range of about 5
mg/kg/body weight to about 100 mg/kg/body weight, about 5
microgram/kg/body weight to about 500 milligram/kg/body weight,
etc., can be administered, based on the numbers described
above.
[0223] In certain embodiments, a vaccine composition herein and/or
additional agents is formulated to be administered via an
alimentary route. Alimentary routes include all possible routes of
administration in which the composition is in direct contact with
the alimentary tract. Specifically, the pharmaceutical compositions
disclosed herein may be administered orally, buccally, rectally, or
sublingually. As such, these compositions may be formulated with an
inert diluent or with an assimilable edible carrier, or they may be
enclosed in hard- or soft-shell gelatin capsules, or they may be
compressed into tablets, or they may be incorporated directly with
the food of the diet.
[0224] In further embodiments, a vaccine composition described
herein may be administered via a parenteral route. As used herein,
the term "parenteral" includes routes that bypass the alimentary
tract. Specifically, the pharmaceutical compositions disclosed
herein may be administered, for example but not limited to,
intravenously, intradermally, intramuscularly, intraarterially,
intrathecally, subcutaneous, or intraperitoneally (U.S. Pat. Nos.
6,753,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and
5,399,363 are each specifically incorporated herein by reference in
their entirety).
[0225] Solutions of the vaccine compositions disclosed herein as
free bases or pharmacologically acceptable salts may be prepared in
water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions may also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms. The pharmaceutical forms suitable for injectable
use include sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions (U.S. Pat. No. 5,466,468, specifically
incorporated herein by reference in its entirety). In all cases the
form must be sterile and must be fluid to the extent that easy
injectability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms, such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (i.e., glycerol, propylene glycol,
and liquid polyethylene glycol, and the like), suitable mixtures
thereof, and/or vegetable oils. Proper fluidity may be maintained,
for example, by the use of a coating, such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. The prevention of the action of
microorganisms can be brought about by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption such as, for example, aluminum
monostearate or gelatin.
[0226] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous, and
intraperitoneal administration. In this connection, sterile aqueous
media that can be employed will be known to those of skill in the
art in light of the present disclosure. For example, one dosage may
be dissolved in 1 ml of isotonic NaCl solution and either added to
1000 ml of hypodermoclysis fluid or injected at the proposed site
of infusion, (see for example, "Remington's Pharmaceutical
Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some
variation in dosage will necessarily occur depending on the
condition of the subject being treated. The person responsible for
administration will, in any event, determine the appropriate dose
for the individual subject. Moreover, for human administration,
preparations should meet sterility, pyrogenicity, general safety
and purity standards as required by FDA Office of Biologics
standards.
[0227] Sterile injectable solutions are prepared by incorporating
the vaccine compositions in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
compositions into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, some methods of
preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof. A
powdered composition is combined with a liquid carrier such as,
e.g., water or a saline solution, with or without a stabilizing
agent.
[0228] In other embodiments, the vaccine compositions may be
formulated for administration via various miscellaneous routes, for
example, topical (i.e., transdermal) administration, mucosal
administration (intranasal, vaginal, etc.) and/or via
inhalation.
[0229] Pharmaceutical compositions for topical administration may
include the vaccine compositions formulated for a medicated
application such as an ointment, paste, cream or powder. Ointments
include all oleaginous, adsorption, emulsion and water-soluble
based compositions for topical application, while creams and
lotions are those compositions that include an emulsion base only.
Topically administered medications may contain a penetration
enhancer to facilitate adsorption of the active ingredients through
the skin. Suitable penetration enhancers include glycerin,
alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram.
Possible bases for compositions for topical application include
polyethylene glycol, lanolin, cold cream and petrolatum as well as
any other suitable absorption, emulsion or water-soluble ointment
base. Topical preparations may also include emulsifiers, gelling
agents, and antimicrobial preservatives as necessary to preserve
the vaccine composition and provide for a homogenous mixture.
Transdermal administration of the vaccine compositions may also
comprise the use of a "patch." For example, the patch may supply
one or more vaccine compositions at a predetermined rate and in a
continuous manner over a fixed period of time.
[0230] In certain embodiments, the vaccine compositions may be
delivered by eye drops, intranasal sprays, inhalation, and/or other
aerosol delivery vehicles. Methods for delivering compositions
directly to the lungs via nasal aerosol sprays has been described
in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically
incorporated herein by reference in their entirety). Likewise, the
delivery of drugs using intranasal microparticle resins (Takenaga
et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat.
No. 5,725,871, specifically incorporated herein by reference in its
entirety) are also well-known in the pharmaceutical arts and could
be employed to deliver the vaccine compositions described herein.
Likewise, transmucosal drug delivery in the form of a
polytetrafluoroetheylene support matrix is described in U.S. Pat.
No. 5,780,045 (specifically incorporated herein by reference in its
entirety), and could be employed to deliver the vaccine
compositions described herein.
[0231] It is further envisioned the vaccine compositions disclosed
herein may be delivered via an aerosol. The term aerosol refers to
a colloidal system of finely divided solid or liquid particles
dispersed in a liquefied or pressurized gas propellant. The typical
aerosol for inhalation consists of a suspension of active
ingredients in liquid propellant or a mixture of liquid propellant
and a suitable solvent. Suitable propellants include hydrocarbons
and hydrocarbon ethers. Suitable containers will vary according to
the pressure requirements of the propellant. Administration of the
aerosol will vary according to subject's age, weight and the
severity and response of the symptoms.
EXAMPLES
[0232] The present invention is further defined in the following
Examples, in which all parts and percentages are by weight and
degrees are Celsius, unless otherwise stated. It should be
understood that these Examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only. From the above discussion and these Examples, one skilled in
the art can ascertain the essential characteristics of this
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions. All publications,
including patents and non-patent literature, referred to in this
specification are expressly incorporated by reference. The
following examples are intended to illustrate certain embodiments
of the invention and should not be interpreted to limit the scope
of the invention as defined in the claims, unless so specified.
[0233] General Methods
[0234] All fine chemicals such as L-rhamnose, cholesterol,
p-toluene sulfonyl chloride, copper sulfate, etc., and anhydrous
solvents, such as anhydrous methanol, were purchased from Acros
Organics. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was
obtained from Avanti Polar Lipids Inc. (Alabaster, Ala.). Boron
trifluoride etherate was obtained from Aldrich. The chemicals were
used without further purification. All solvents were obtained from
Fisher and used as received except dichloromethane, which was dried
and distilled following the standard procedures. Silica (230-400
mesh) for flash column chromatography was obtained from Sorbent
Technologies; thin-layer chromatography (TLC) precoated plates were
from EMD. TLCs (silica gel 60, f.sub.254) were visualized under UV
light or by charring (5% H.sub.2SO.sub.4-MeOH). Flash column
chromatography was performed on silica gel (230-400 mesh) using
solvents as received.
[0235] .sup.1H NMR was recorded either on a Varian VXRS 400 MHz or
an INOVA 600 MHz spectrometer in CDCl.sub.3 or CD.sub.3OD using
residual CHCl.sub.3 and CHD.sub.2OH as internal references,
respectively.
[0236] .sup.13C NMR was recorded on a Varian VXRS 100 MHz or an
INOVA 150 MHz in CDCl.sub.3 using the triplet centered at .delta.
77.23 or CD.sub.3 OD using the septet centered at .delta. 49.0 as
internal reference. High resolution mass spectrometry (HRMS) was
performed on a TOF mass spectrometer. The peptide was synthesized
on a Omega 396 synthesizer (Advanced ChemTech, Louisville, Ky.).
Tris [(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA),
preloaded Fmoc-L-Ala-Wang resin and all other Fmoc-L-amino acids
were procured from Anaspec (San Jose, Calif.). FITC goat anti-mouse
IgG/IgM and purified mouse anti-human CD227 (anti-human MUC1) were
obtained from BD-biosciences (San Jose, Calif.). Scanning electron
microscope imaging was done on a JEOL JSM-7500F field scanning
electron microscope. Dynamic light scattering measurements was done
DynaPro Titan temperature controlled microsampler (Wyatt Technology
Corporation). Fluorescence microscopy was done on a Nikon TiU
microscope.
Example 1
Synthesis of L-Rhamnose-TEG-Cholesterol (3)
[0237] Cholesterol tetraethylene glycol (1) was glycosylated with
peracetyl L-rhamnose in presence of boron trifluoride etherate to
afford peracetyl rhamnose-TEG-cholesterol (2) (32%) which was
deacetylated under Zemplen conditions to generate
L-rhamnose-TEG-cholesterol (3) (85%). See FIG. 10, illustrating
Scheme 1. Compound (3) anchors the Rha epitopes on the surface of
the liposomes thereby facilitating anti-Rha antibody binding.
(5-Cholesten-3.alpha.-yloxy)-3n.sup.3.sub.9-trixaundecanyl-2,3,4-tri-O-ace-
tyl-.alpha.-L-Rhamnopyranoside (2)
[0238] To a solution of 1,2,3,4-tetra-O-acetyl rhamnopyranose (0.64
g, 1.92 mmol) in CH.sub.2Cl.sub.2 (3 mL) was added
(5-cholesten-3.alpha.-yloxy)-3n.sup.3.sub.9-trixoundecan-1-ol (1)
(1.30 g, 2.30 mmol) in CH.sub.2Cl.sub.2 and the mixture was cooled
to 0.degree. C. BF.sub.3.OEt.sub.2 (486 mL, 3.84 mmol) was added
dropwise to the reaction mixture and the resulting solution was
stirred at ambient temperature under N.sub.2 atmosphere. The
reaction was monitored by TLC (EtOAc:hexanes=1:1) and appeared
complete after 18 h. The reaction mixture was diluted with
CH.sub.2Cl.sub.2 (25 mL) and washed with saturated NaHCO.sub.3 (25
mL), water (25 mL) and brine (25 mL) after which the organic layer
was dried over anhydrous Na.sub.2SO.sub.4. Excess solvent was
evaporated under reduced pressure and the residue was purified by
silica gel flash column chromatography using 30% EtOAc in hexanes
as solvent to afford (2) as a light yellow solid (0.51 g, 32%).
.sup.1H NMR (600 MHz, CDCl.sub.3): .delta. 0.67 (s, 3H,
cholesterol), 0.85-1.15 (23H, cholesterol), 1.21 (d, 3H, J=6 Hz,
C-5 CH.sub.3), 1.24-1.52 (12H, cholesterol), 1.80-1.95 (5H,
cholesterol), 1.98 (s, 3H, COCH.sub.3), 2.05 (s, 3H, COCH.sub.3),
2.15 (s, 3H, COCH.sub.3), 3.17 (m, 1H, --OCH-cholesterol),
3.63-3.66 (16H, --CH.sub.2--CH.sub.2O-TEG), 3.92 (m, 1H, H-5), 4.77
(d, 1H, T=1.8 Hz, H-1), 5.06 (t, 1H, J=10.2 Hz, H-4), 5.26 (dd, 1H,
J=1.8, 3.6 Hz, H-2), 5.30 (dd, 1H, J=4.2, 9.9 Hz, H-3), 5.33 (m,
1H, --C.dbd.CH-cholesterol). .sup.13C NMR (100.56 MHz, CDCl.sub.3):
.delta. 12.04, 17.61, 18.90, 19.58, 20.95, 21.03, 21.14, 21.25,
22.76, 23.02, 24.01, 24.48, 28.21, 28.43, 28.53, 29.90, 32.07,
32.13, 35.97, 36.37, 37.07, 37.42, 39.23, 39.70, 39.96, 42.50,
50.36, 53.63, 56.32, 56.96, 66.46, 67.30, 67.46, 69.30, 70.03,
70.24, 71.35, 79.68, 97.74 (C-1), 121.74 (C.dbd.C), 141.15
(C.dbd.C), 170.18 (COCH.sub.3), 170.25 (COCH.sub.3), 170.32
(COCH.sub.3). HRMS [M+Na] m/z: calcd for
C.sub.47H.sub.78NaO.sub.12, 857.5391. Found, 857.5396.
(5-Cholesten-3.alpha.-yloxy)-3n.sup.3.sub.2-trixaundecanyl
Rhamnopyranoside (3)
[0239] To a solution of (2) (0.45 g, 0.54 mmol) in MeOH (10 mL) was
added metallic sodium (0.03 g) and the resulting solution was
stirred at ambient temperature under N.sub.2 atmosphere. The
reaction was monitored by TLC (5% MeOH in CH.sub.2Cl.sub.2) and
appeared complete after 1 h. The solution was neutralized by
Amberlite H.sup.+ exchange resin. Excess solvent was evaporated
under reduced pressure and the residue was purified by silica gel
flash column chromatography using 5% MeOH in CH.sub.2Cl.sub.2 as
solvent to afford (3) as a yellowish white solid (0.32 g, 85%).
.sup.1H NMR (600 MHz, CDCl.sub.3): .delta. 0.68 (s, 3H,
cholesterol), 0.86-1.25 (24H, cholesterol), 1.32 (d, 3H, J=6 Hz,
C-5 CH.sub.3), 1.44-1.53 (16H, cholesterol), 2.83 (s, 1H, C-4 OH),
3.08 (d, 1H, T=3 Hz, H-1), 3.20 (m, 1H, --O--CH-cholesterol), 3.43
(t, 1H, T=9.6 Hz, H-4), 3.62-3.71 (16H, --CH.sub.2--CH.sub.2O-TEG),
3.73 (m, 1H, H-5), 3.83 (dd, 1H, T=3, 6.9 Hz, H-3), 3.98 (s, 1H,
H-2), 4.87 (s, 1H, C-2 OH), 5.31 (s, 1H, C-3 OH), 5.35 (m, 1H,
--C.dbd.CH-cholesterol). .sup.13C NMR (100.56 MHz, CDCl.sub.3):
.delta. 12.07, 17.83, 18.92, 19.60, 21.27, 22.78, 23.04, 24.03,
24.50, 28.23, 28.44 (2), 32.08, 32.15, 35.99, 36.39, 37.06, 37.39,
39.09, 39.73, 39.97, 42.53, 50.36, 56.34, 56.97, 66.74, 67.32,
68.07, 70.48, 70.63, 70.75, 70.81, 70.93, 70.97, 71.05, 71.80,
73.81, 79.87, 99.98 (C-1), 121.96 (C.dbd.C-cholesterol), 140.98
(C.dbd.C-cholesterol). HRMS [M+Na] m/z: calcd for
C.sub.41H.sub.72NaO.sub.9, 731.5074. Found, 731.5090.
Example 2
Synthesis of Alkyne Functionalized Pam.sub.3Cys (6)
[0240] O-palmitoylated Fmoc L-cystine tert-butyl ester (4) was
deprotected by a brief treatment with trifluoroacetic acid (TFA).
The free acid was coupled with propargyl amine in the presence of
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
(PyBOP), 1-hydroxy-benzotriazole (HOBt) and
N,N-diisopropylethylamine (DIPEA) to yield (5) (66% over 2 steps).
Fmoc group in compound (5) was removed by treatment with a mixture
of acetonitrile-dichloromethane-diethyl amine (2:1:2) followed by
subsequent palmitoylation by coupling with palmitic acid, PyBOP,
HOBt and DIPEA to afford our target alkyne functionalized
Pam.sub.3Cys amide derivative (6) (80% over 2 steps), as shown in
FIG. 11, Scheme 2. Compound (6), which is a TLR-2 agonist, serves
the purpose of an immunoadjuvant for a vaccine, and also anchors
the MUC1-Tn conjugate on the surface of the liposome.
N-propargyl Pam.sub.2FmocCys Amide Derivative (5)
[0241] Pam.sub.2FmocCys tertiary butyl ester (0.30 g, 0.32 mmol)
was dissolved in minimum volume of neat TFA (1 mL) and stirred at
ambient temperature under N.sub.2 atmosphere. TLC
(EtOAc:hexanes=1:4) indicated the completion of the reaction after
1 h. The reaction mixture was evaporated to dryness under vacuum
and the residue was dissolved in CH.sub.2Cl.sub.2 (3 mL). PyBOP
(198 m g, 0.38 mmol), HOBt (58 mg, 0.38 mmol), DIPEA (78 .mu.L,
0.47 mmol) and 4 .ANG. mol. sieves (2-3 beads) were added
sequentially and the mixture was stirred for 5 minutes at room
temperature followed by the addition of propargyl amine (25 .mu.L,
0.38 mmol) and stirred at ambient temperatures under N.sub.2
atmosphere. The reaction was monitored by TLC (EtOAc:hexanes=1:4)
and appeared complete after 4 h. The reaction mixture was filtered,
washed with phosphate buffer (10 mL) and extracted with
CH.sub.2Cl.sub.2 (3.times.10 mL). The organic layer was dried over
anhydrous Na.sub.2SO.sub.4 and concentrated. The residue was
purified by silica gel flash column chromatography using
EtOAc-hexanes (1:4) as solvent to afford (5) as a white solid (192
mg, 66%). .sup.1H NMR (600 MHz, CDCl3): .delta. 0.87 (t, 6H, J=7.2
Hz, Pam-CH.sub.3), 1.14-1.65 (m, 52H, Pam-CH.sub.2), 1.68 (s, 1H,
alkyne-CH), 2.18-2.35 (m, 4H, COCH.sub.2), 2.83 (m, 1H, Cys-CHH),
2.89 (dd, 1H, J=7.2, 14.4 Hz, S-glyceryl-OCHH), 3.01 (dd, 1H, J=6,
14.4 Hz, cys-CHH), 4.06 (dd, 1H, J=3, 4.8 Hz, S-glycerylO-CHH),
4.08 (s, 2H, CO--NH--CH2), 4.18 (dd, 1H, J=6, 11.4 Hz,
S-glyceryl-O--CHH), 4.23 (t, 1H, J=7.2 Hz, Fmoc-CH), 4.39 (m, 1H,
NH--CH--CO), 4.42 (m, 2H, Fmoc-CH2), 5.12 (m, 1H,
S-glyceryl-O--CH), 5.73 (d, 1H, J=7.8 Hz, Pam-NH), 6.89 (s, 1H,
CO--NHCH2), 7.31-7.81 (m, 8H, Fmoc-ArH). .sup.13C NMR (150.84 MHz,
CDCl3): .delta. 14.35-36.70 (30C, Pam-C), 47.29 (2), 53.32, 63.58,
67.48, 70.61, 71.78, 72.07, 79.09, 79.85, 120.22, 125.30, 127.30
(2), 127.97 (2) 141.49, 141.50, 143.85, 143.89 (Aromatic-C),
170.07, 173.66 (2), 174.04 (Cys-CO). HRMS [M+Na] m/z: calcd for
C.sub.56H.sub.86N.sub.2NaO.sub.7S, 953.6053. Found, 953.6073.
N-propargyl Pam.sub.3Cys Amide Derivative (6)
[0242] Composition (5) (192 mg, 0.21 mmol) was dissolved in a
mixture of CH.sub.3CN--CH.sub.2Cl.sub.2-Et.sub.2NH (2:1:2, 2.50 mL)
and stirred at ambient temperature under N.sub.2 atmosphere. TLC
(EtOAc:hexanes=1:4) indicated the complete deprotection of the Fmoc
group after 2 h. The reaction mixture was evaporated to dryness
under vacuum. Palmitic acid (64 mg, 0.25 mmol), PyBOP (128 mg, 0.25
mmol), HOBt (38 mg, 0.25 mmol) were dissolved in CH.sub.2Cl.sub.2
(3 mL) followed by the addition of DIPEA (51 .mu.L, 0.31 mmol). The
mixture was stirred for 5 minutes and added to the residue of the
Fmoc deprotected product from compound (5) containing 4 A mol.
sieves (2-3 beads). The reaction mixture was stirred at ambient
temperature under N.sub.2 atmosphere. The reaction was monitored by
TLC (EtOAc:hexanes=1:4) and appeared complete after 4 h. The
reaction mixture was diluted with CH.sub.2Cl.sub.2 (15 mL),
filtered and evaporated to dryness. The residue was purified by
silica gel flash column chromatography using EtOAC-hexanes (1:4) as
solvent to afford (6) as a pale yellow solid (156 mg, 80%). .sup.1H
NMR (600 MHz, CDCl.sub.3): .delta. 0.88 (t, 12H, J=6.6 Hz,
Pam-CH.sub.3), 1.10-1.63 (m, 78H, Pam-CH.sub.2), 2.23 (s, 1H,
Alkyne-CH), 2.24-2.36 (m, 6H, COCH.sub.2), 2.71 (dd, 1H, J=7.8,
14.4 Hz, Cys-CHH), 2.86 (m, 6H, COCH.sub.2), 2.95 (dd, 1H, J=6, 14
Hz, Cys-CHH), 4.06 (m, 2H, CO--NH--CH.sub.2), 4.18 (dd, 1H, J=6.6,
12 Hz, S-glyceryl-O--CHH), 4.40 (dd, 1H, J=3, 12 Hz,
S-glyceryl-O--CHH), 4.64 (q, 1H, J=6 Hz, NH--CH--CO), 5.12 (m, 1H,
S-flyceryl-OCH), 6.64 (d, 1H, J=8.4 Hz, Pam-NH), 7.02 (t, 1H, J=5.4
Hz, CO--NH--CH.sub.2). .sup.13C NMR (150.84 MHz, CDCl.sub.3):
.delta. 14.35-42.19 (48C, Pam-C, Cys-C.sub.R, S-glyceryl-C, NH--C),
51.30 (Cys-Ca, 63.65, 70.61, 71.98, 79.08, 170.40 (Cys-CO), 173.70,
173.86, 174.06 (Pam-CO). HRMS [M+Na] m/z: calcd for
C.sub.57H.sub.106N.sub.2NaO.sub.6S, 969.7669. Found, 969.7682.
Example 3
Synthesis of Pam.sub.3Cys-MUC-1 VNTR-TACA Conjugate (9)
[0243] A 20-amino acid tandem repeat of MUC1 which included the
GS(.alpha.-GalNAc-O-T)A epitope was synthesized. The glycopeptide
was modified with a terminal azido group in order to make a `click`
conjugation to the Pam.sub.3Cys alkyne. The glycopeptide azide was
synthesized by Fmoc strategy on an Omega 396 synthesizer (Advanced
ChemTech, Louisville, Ky.) starting from preloaded Fmoc-L-Ala Wang
resin using solid-phase chemistry, as shown in FIG. 12, Scheme
3.
[0244] The peptide synthesis was performed by coupling amino acid
esters of HOBt using DIC as the coupling agent. A six-fold excess
of N.sup..alpha.-Fmoc amino acid esters of HOBt in NMP were used in
the synthesis. A 1:1 ratio of amino acid to DIC was used in all the
coupling reactions. Deprotection of N.sup..alpha.-Fmoc group was
accomplished by treatment with piperidine in DMF. After the
synthesis was complete, the peptide was cleaved from the solid
support and deprotected using a modified reagent K cocktail
consisting of TFA-thioanisole-ethanedithiol-water-phenol
(88:3:5:2:2). The cocktail mixture was filtered through a Quick
Snap column, purified by C18 reverse phase HPLC and lyophilized to
afford composition (7b).
[0245] The acetyl groups in composition (7b) were deprotected by
treatment with 6 mmol sodium methoxide in methanol. The product was
purified by Bio-Gel (P-2, fine 45-90 .mu.m) size exclusion
chromatography using deionized water as solvent. Lyophilization of
the fractions afforded composition (8) (100%) as a white
powder.
[0246] Conjugation of the composition (8) (1 eqv) with the
composition (6) (3 eqv) in the presence of copper sulfate
pentahydrate (12 eqv), Tris
[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (12 eqv) and
sodium ascorbate (12 eqv) in H.sub.2O-MeOH-THF (1:1:2) as solvent
at ambient temperatures thus afforded the target, Pam.sub.3Cys-MUC1
VNTR-TACA conjugate (9), after 40 h.
[0247] Composition (9) was purified by LH20 using
MeOH-dichloromethane (1:1) as solvent. The eleutants were
lyophilized to afford composition (9) as a white solid (100%).
[0248] Synthesis of Azide (7b)
[0249] The glycopeptide azide was synthesized by Fmoc strategy on
an Omega 396 synthesizer (Advanced ChemTech, Louisville, Ky.) using
solid phase chemistry. The peptide synthesis was performed by
coupling amino acid esters of HOBt using DIC as the coupling agent
starting with a preloaded Fmoc-alanyl-Wang resin. A six-fold excess
of N.sup..alpha.-Fmoc amino acid esters of HOBt in NMP were used in
the synthesis. A 1:1 ratio of amino acid to DIC was used in all the
coupling reactions. Deprotection of N.sup..alpha.-Fmoc group was
accomplished by treatment with 25% piperidine in dimethylformamide
twice; first for 5 minutes and then a second time for 25 minutes to
afford composition (7a).
[0250] After the synthesis was complete, the peptide was cleaved
from the solid support and deprotected using a modified reagent K
cocktail consisting of 88% TFA, 3% thioanisole, 5% ethanedithiol,
2% water and 2% phenol. 4 mL of cleavage cocktail was added to the
dried peptide-resin in a 15 mL glass vial blanketed with nitrogen.
Cleavage was carried out for 2.5 hrs with gentle magnetic stirring.
At the end cleavage time, the cocktail mixture was filtered on a
Quick-Snap column. The filtrate was collected in 20 mL ice-cold
butane ether. The peptide was allowed to precipitate for an hour at
-200.degree. C., centrifuged, and washed twice with ice-cold
methyl-t-butyl ether. The precipitate was dissolved in 25%
acetonitrile and lyophilized to complete dry powder affording
composition (7b). Quality of peptides was analyzed by analytical
reverse phase HPLC and MALDI-TOF (matrix assisted laser desorption
ionization time-of-flight) mass spectrometer, model 4800 from
Applied Biosystems. HR-MALDI-MS: [M+H] m/z calcd for
C.sub.100H.sub.155N.sub.29O.sub.37, 2355.1172. Found,
2355.1753.
[0251] Synthesis of azide (8)
[0252] Composition (7b) (5 mg, 2.24 .mu.mol) was dissolved in 2 mL
of dry methanol and 12 .mu.L of freshly prepared 1 M sodium
methoxide was added and the reaction mixture was stirred at ambient
temperature under N.sub.2 atmosphere for 2 h. The reaction mixture
was neutralized with solid carbon dioxide. The reaction mixture was
concentrated and purified by Bio-Gel (P-2, fine 45-90 .mu.m) size
exclusion chromatography using deionized water as solvent.
Lyophilization of the eleutants afforded composition (8) as a white
powder (4.7 mg, 100%). HR-MALDI-MS: [M+H] m/z calcd for
C.sub.94H.sub.149N.sub.29O.sub.34, 2229.0895. Found 2229.0959.
[0253] Synthesis of Glycolipopeptide (9)
[0254] CuSO4.5H.sub.2O (134 .mu.g, 0.54 .mu.mol) and TBTA (2.14 mg,
4.04 .mu.mol) were dissolved in H.sub.2O-THF (1:1, 0.40 mL) and to
it Na-ascorbate (0.80 mg, 4.04 .mu.mol) was added and stirred for 5
minutes. Composition (6) (1.27 mg, 1.35 .mu.mol) in THF (0.40 mL)
was added to the reaction mixture and stirred for 15 minutes
followed by the addition of a solution of composition (8) (1 mg,
0.45 .mu.mol) in H.sub.2O-MeOH (1:3, 0.4 mL). The reaction mixture
was stirred at ambient temperature under N.sub.2 atmosphere for 40
h. The reaction mixture was concentrated, dissolved in
CH.sub.2Cl.sub.2-MeOH (1:1) and purified by a short LH 20 size
exclusion column using CH.sub.2Cl.sub.2-MeOH (1:1) as solvent.
Lyophilization of the eleutants afforded composition (9) as a white
solid (1.9 mg, 100%). HR-MALDI-MS: [M+H] m/z calcd for
C.sub.151H.sub.255N.sub.31O.sub.40S, 3175.593. Found 3175.425. A
mass peak corresponding to a protonated methyl ester of the product
was also observed.
Example 4
Synthesis of a Liposomal Vaccine of Composition I and Control
Compositions
[0255] The liposomes were formulated by the extrusion method in a
total lipid concentration of 30 mM. For the preparation of the
liposomes, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was
used. Lipid stock solutions were prepared by dissolving each lipid
into chloroform inside glass vials. Aliquots of the stock solutions
were mixed in proportions in another small glass vial to give a
solution with a total lipid concentration of 30 mM in a total
volume of 2 mL.
[0256] Batch 1: DPPC 80%, Cholesterol 10%, Rha-cholesterol 10% and
Pam.sub.3cys-MUC1-Tn (9) 2 nM.
[0257] Batch 2: DPPC 80%, cholesterol 20%, Pam.sub.3cys-MUC1-Tn (9)
2 nM.
[0258] Batch 3: DPPC 80%, cholesterol 20%.
[0259] Chloroform was removed by subjecting the lipid solutions to
a constant stream of nitrogen. The resulting lipid films were dried
under vacuum overnight. The dried lipid films were hydrated with 2
mL of HEPES buffer (pH=7.4). The suspensions of the lipids in the
buffer were agitated at 43.degree. C. for 40 mins. The suspensions
were subjected to 10 freeze-thaw cycles (dry ice/acetone and water
at 40.degree. C.). Final liposomes were prepared by extrusion (21
times) using a LipoFast Basic fitted with a 100 nm polycarbonate
membrane to control the liposome size.
[0260] General Characterization of Liposomal Vaccine of Composition
I and Control Compositions
[0261] The homogeneity, stability as well as size characterization
of the liposomes were evaluated by scanning electron microscope
(SEM) imaging (FIGS. 14A-14B) and dynamic light scattering (DLS)
measurements (FIGS. 13A-13C).
[0262] All batches of liposomes were found to be stable at
4.degree. C. for 2 days and were around 100 nm in diameter.
Antibody binding study showed positive binding of the Batch 1
liposomes with anti-rhamnose antibodies as well as mouse
anti-human-MUC1 (CD 227, BD Biosciences, San Jose, Calif.)
antibodies using FITC goat anti-mouse IgG/IgM secondary antibodies
and fluorescence imaging of the coated liposomes, as shown in FIG.
15.
[0263] The binding assay proved that the L-rhamnose and the MUC1
VNTR-TACA conjugate were displayed on the surface of the liposomes.
No such antibody binding (both anti-rhamnose and mouse
anti-human-MUC1) were observed for the control Batch 3
liposomes.
[0264] Batch 2 liposomes only demonstrated mouse anti-human-MUC1
antibody binding.
Example 5
Liposome Characterization
[0265] Size Characterization
[0266] Size determination of the liposomes was done by scanning
electron microscope (SEM) imaging and dynamic light scattering
(DLS) measurements. For SEM characterization the liposome samples
were diluted 1000 times with HEPES buffer (pH=7.4) and freeze dried
over copper studs fitted with a carbon conducting tape and the
images recorded at an acceleration voltage of 5 kV. DLS
measurements were done after dilution of the liposome samples 10000
times with HEPES buffer (pH=7.4).
[0267] Anti-Rha and Anti-MUC1 Antibody Binding to Surface Exposed
Rha and MUC1 Epitopes on Liposomes
[0268] 10.sup.6 liposomes from each batch in 50 .mu.L 1.times.PBS
(pH=7.4) were added separately into a 1.5 mL Eppendrof tube
followed by 50 .mu.L of primary antibody solution in deionized
water containing 5-50 .mu.g/mL of antibodies [either control
antibodies (isolated from the serum of non-immunized mice) or
anti-Rha antibodies or mouse anti-human CD227 antibodies
(anti-human MUC1)] and incubated on ice for 30 mins. 1 mL PBS-0.1%
Tween was added to each tube and vortexed. Liposomes were spun at
14000 rpm in Eppendrof centrifuge at 4.degree. C. for 5 mins. The
supernatants were carefully discarded and the washing and
centrifugation steps were repeated 2 more times for a total of 3
washes. Liposomes were then resuspended in 50 .mu.L of PBS-0.1%
Tween. 50 .mu.L of diluted FITC goat anti-mouse IgG/IgM secondary
antibody were added (2-30 .mu.g/mL) to the tubes, mixed and covered
with aluminum foil to protect from light and incubated on ice for
30 mins. After washing 3 times with PBS-0.1% Tween and
centrifugation, the supernatants were removed and pellets were
resuspended in 1 mL PBS-0.1% Tween. 10 .mu.L of the resuspended
solutions were put on glass slides and imaged under a fluorescent
microscope.
Example 6
[0269] NMR spectra and HRMS data of compositions (2), (3), (5) and
(6) are shown in FIG. 16 through FIG. 31.
[0270] FIG. 16: .sup.1H NMR of
(5-Cholesten-3.alpha.-yloxy)-3n.sup.3.sub.9-trixaundecanyl-2,3,4-tri-O-ac-
etyl-.alpha.-L-Rhamnopyranoside (2).
[0271] FIG. 17: .sup.13C NMR of
(5-Cholesten-3.alpha.-yloxy)-3n.sup.3.sub.9-trixaundecanyl-2,3,4-tri-O-ac-
etyl-.alpha.-L-Rhamnopyranoside (2).
[0272] FIG. 18: .sup.1H-gCosy of
(5-Cholesten-3.alpha.-yloxy)-3n.sup.3.sub.9-trixaundecanyl
2,3,4-tri-O-acetyl-.alpha.-L-Rhamnopyranoside (2).
[0273] FIG. 19: HRMS of
(5-Cholesten-3.alpha.-yloxy)-3n.sup.3.sub.9-trixaundecanyl
2,3,4-tri-O-acetyl-.alpha.-L-Rhamnopyranoside (2).
[0274] FIG. 20: .sup.1H NMR of
(5-Cholesten-3.alpha.-yloxy)-3n.sup.3.sub.9-trixaundecanyl
Rhamnopyranoside (3).
[0275] FIG. 21: .sup.13C NMR of
(5-Cholesten-3.alpha.-yloxy)-3n.sup.3.sub.9-trixaundecanyl
Rhamnopyranoside (3).
[0276] FIG. 22: .sup.1H-gCosy of
(5-Cholesten-3.alpha.-yloxy)-3n.sup.3.sub.9-trixaundecanyl
Rhamnopyranoside (3).
[0277] FIG. 23: HRMS of
(5-Cholesten-3.alpha.-yloxy)-3n.sup.3.sub.9-trixaundecanyl
Rhamnopyranoside (3).
[0278] FIG. 24: .sup.1H NMR of N-Propargyl Pam.sub.2FmocCys Amide
Derivative (5).
[0279] FIG. 25: .sup.13C NMR of N-Propargyl Pam.sub.2FmocCys Amide
Derivative (5).
[0280] FIG. 26: .sup.1HgCosy of N-Propargyl Pam.sub.2FmocCys Amide
Derivative (5).
[0281] FIG. 27: HRMS of N-Propargyl Pam.sub.2FmocCys Amide
Derivative (5).
[0282] FIG. 28: .sup.1H NMR of N-Propargyl Pam.sub.3Cys Amide
Derivative (6).
[0283] FIG. 29: .sup.13C NMR of N-Propargyl Pam.sub.3Cys Amide
Derivative (6).
[0284] FIG. 30: .sup.1H-gCosy of N-Propargyl Pam.sub.3Cys Amide
Derivative (6).
[0285] FIG. 31: HRMS of N-Propargyl Pam.sub.3Cys Amide Derivative
(6).
[0286] The MALDI-TOF spectra data for compositions (7b), (8) and
(9) are shown in FIG. 32 through FIG. 34.
[0287] FIG. 32: HR-MALDI-TOF of Glycopeptide Azide (7b).
[0288] FIG. 33: HR-MALDI-TOF of Glycopeptide Azide (8).
[0289] FIG. 34: HR-MALDI-TOF of Lipopeptide (9).
Example 7
2-Azidoethyl-2,3,4-Tri-O-Acetyl-.alpha.-L-Rhamnopyranoside (11)
[0290] To a solution of 1,2,3,4-tetra-O-acetylrhamnopyranoside (10)
(2.00 g, 6.02 mmol) in CH.sub.2Cl.sub.2 (5.00 mL) were added
2-azidoethanol (0.79 g, 9.03 mmol) and BF.sub.3.OEt.sub.2 (1.53 mL,
12.04 mmol) at 0.degree. C. and the resulting solution was stirred
at ambient temperature under N.sub.2 atmosphere. The reaction was
monitored by TLC and appeared to be completed after 12 h. The
reaction mixture was diluted with CH.sub.2Cl.sub.2 (15 mL) and
washed with water (2.times.20 mL), saturated NaHCO.sub.3
(2.times.20 mL) and brine (20 mL), after which the organic layer
was dried over anhydrous Na.sub.2SO.sub.4. Excess solvent was
removed under reduced pressure and the crude material was purified
by silica gel flash column chromatography (3.3.times.8.5 cm).
Elution with 1:5 EtOAc/hexanes afforded (11) as a colorless solid
(1.78 g, 83%). .sup.1H NMR (600 MHz, CDCl.sub.3): .delta. 1.24 (d,
3H, J=6.6 Hz, C-5 CH.sub.3), 1.99 (s, 3H, COCH.sub.3), 2.06 (s, 3H,
COCH.sub.3), 2.16 (s, 3H, COCH.sub.3), 3.42 (m, 1H,
--CHH--N.sub.3), 3.48 (m, 1H, --CHH--N.sub.3), 3.64 (m, 1H,
--O--CHH), 3.87 (m, 1H, --O--CHH), 3.93 (m, 1H, H-5), 4.79 (d, 1H,
J=1.8 Hz, H-1), 5.09 (t, 1H, J=10.2 Hz, H-4), 5.27 (dd, 1H, J=1.2,
3.3 Hz, H-2), 5.31 (dd, 1H, J=3.3, 9.9 Hz, H-3). .sup.13C NMR (100
MHz, CDCl.sub.3): .delta. 17.66 (CH.sub.3), 20.93, 21.03, 21.13,
50.58, 66.91, 66.99, 69.08, 69.87, 71.09, 97.79 (C-1), 170.09
(C.dbd.O), 170.24 (C.dbd.O), 170.30 (C.dbd.O). HRMS [M+Na] m/z:
calcd for C.sub.14H.sub.21N.sub.3O.sub.8, 382.1226. Found,
382.1215.
2-Azidoethyl-.alpha.-L-Rhamnopyranoside (12)
[0291] To a solution of (11) (1.53 g, 4.26 mmol) in MeOH (5 mL) was
added metallic Na (0.01 g) and the resulting solution was stirred
at ambient temperature under N.sub.2 atmosphere. The reaction was
monitored by TLC and appeared complete after 2 h. Excess solvent
was removed under reduced pressure and the crude material was
purified by silica gel flash column chromatography (3.3.times.8.5
cm). Elution with 2:23 MeOH/CH.sub.2Cl.sub.2 yielded (12) as a
colorless solid (0.85 g, 86%). .sup.1H NMR (600 MHz, CDCl.sub.3):
.delta. 1.34 (d, 3H, J=6.6 Hz, C-5 CH.sub.3), 3.41 (m, 2H,
--CH.sub.2--N.sub.3), 3.49 (t, 1H, J=9.3 Hz, H-4), 3.63 (m, 1H,
--O--CHH), 3.69 (m, 1H, H-5), 3.81 (dd, 1H, J=3.3, 9.3 Hz, H-3),
3.89 (m, 1H, --O--CHH), 3.99 (q, 1H, J=1.6 Hz, H-2), 4.83 (d, 1H,
J=1.2 Hz, H-1). .sup.13C NMR (100 MHz, CDCl.sub.3): .delta. 17.75
(CH.sub.3), 50.71, 66.72, 68.49, 70.98, 71.76, 73.27, 100.02 (C-1).
HRMS [M+Na] m/z: calc for C.sub.8H.sub.15N.sub.3O.sub.5, 256.0909.
Found, 256.0906.
2-Aminoethyl-.alpha.-L-Rhamnopyranoside (13)
[0292] To a solution of (12) (0.42 g, 1.82 mmol) in MeOH (3 mL) was
added activated Pd/charcoal (0.025 g) and the resulting solution
was stirred at ambient temperature under H.sub.2 atmosphere. The
reaction was monitored by TLC and appeared to be complete after 12
h. The reaction mixture was diluted with MeOH (2 mL), filtered
through Celite and concentrated under reduced pressure to yield
(13) as a colorless gel (0.46 g, quantitative) which was used
without further purification for subsequent reactions. ESIMS [M+H]
m/z: calcd for C.sub.8H.sub.17NO.sub.5, 208.2243. Found,
208.30.
2-Aminoethyl-.alpha.-L-Rhamnopyranoside-Ficoll Conjugate (14)
[0293] Ficoll 400 (1.00 g, 0.0025 mmol) was dissolved in acetate
buffer (10 mL, pH 4.7) and NaIO.sub.4 (0.01 g, 0.047 mmol) was
added and the reaction mixture was stirred at ambient temperature
for 2 h in the dark. Excess NaIO.sub.4 was removed by dialysis
against the acetate buffer (pH 4.7) through dialysis tubing with a
molecular weight cutoff value of 10000 Da with six to seven changes
of the buffer at 4.degree. C. The oxidized Ficoll 400 was
transferred to a round bottom flask and excess solvent was
evaporated to dryness under reduced pressure. The residue was
dissolved in borate buffer (20 mL, pH 8.0) followed by the addition
of (13) (0.05 g, 0.25 mmol) and stirred at ambient temperature for
2 h. To the reaction mixture was added NaBH.sub.3CN (0.094 g, 1.50
mmol) and the resulting solution was incubated overnight at
4.degree. C. The mixture was dialyzed through dialysis tubing with
a molecular weight cutoff of 10000 Da with six to seven changes in
buffer at 4.degree. C. to afford (14). The epitope ratio of (14)
was calculated to be 9.44 (Rha:Ficoll) by hydrolysis of (14)
followed by derivatization with
4-amino-N-[2-(diethylamino)ethyl]benzamide (DEAEAB) and comparison
of the UV-HPLC peak area with a standard curve obtained from DEAEAB
derivative of (12) by the methods described by Dalpathado and
coworkers. Briefly, the standard curve was generated by refluxing
compound (12) (0.007 g, 0.031 mmol) with 1 M HCl at 100.degree. C.
for 4 h and the reaction mixture was evaporated to dryness. The
residue was dissolved in tetrahydrofuran (2 mL). DEAEAB (0.011 g,
0.037 mmol) and Et.sub.3N (0.007 mL, 0.046 mmol) were added and the
resulting solution was refluxed for 2 h. The reaction mixture was
evaporated to dryness and the residue was dissolved in MeOH (2 mL)
followed by the addition of NaB(OAc).sub.3H, and the resulting
solution refluxed for 8 h. The solution was evaporated to dryness
and the residue was dissolved in MeOH (2 mL) and filtered through a
syringe filter. Serial dilutions from this stock solution were
prepared and the components were separated on a reverse phase HPLC
using a C18 column. Water containing 0.1% TFA (A) and 95%
ACN/H.sub.2O (B) was used as the mobile phases using a linear
gradient (5-20% B in 20 min) at the flow rate of 1 mL/min.
Absorbances were recorded at 289 nm. The standard curve was
generated by plotting the UV-HPLC peak area against the
concentration in mmol of DEAEAB derivative of (12).
[0294] NMR spectra and HRMS data of compositions (11), (12), and
(13) are shown in FIG. 43 through FIG. 51.
[0295] FIG. 43: .sup.1H NMR of
2-Azidoethyl-2,3,4-Tri-O-acetyl-.alpha.-L-rhamnopyranoside
(11).
[0296] FIG. 44: .sup.13C NMR of
2-Azidoethyl-2,3,4-Tri-O-acetyl-.alpha.-L-rhamnopyranoside
(11).
[0297] FIG. 45: .sup.1H-gCosy of
2-Azidoethyl-2,3,4-Tri-O-acetyl-.alpha.-L-rhamnopyranoside
(11).
[0298] FIG. 46: HRMS of
2-Azidoethyl-2,3,4-Tri-O-acetyl-.alpha.-L-rhamnopyranoside
(11).
[0299] FIG. 47: .sup.1H NMR of 2-Azidoethyl
.alpha.-L-rhamnopyranoside (12).
[0300] FIG. 48: .sup.13C NMR of 2-Azidoethyl
.alpha.-L-rhamnopyranoside (12).
[0301] FIG. 49: .sup.1H-gCosy of 2-Azidoethyl
.alpha.-L-rhamnopyranoside (12).
[0302] FIG. 50: HRMS of 2-Azidoethyl .alpha.-L-rhamnopyranoside
(12).
[0303] FIG. 51: ESI MS of 2-Aminoethyl .alpha.-L-rhamnopyranoside
(13).
Example 8
[0304] A 20-amino acid (TSAPDTRPAPGSTAPPAHGV [SEQ ID NO: 5]) tandem
repeat of MUC1 was synthesized. The peptide was modified with a
terminal azido group in order to make a `click` conjugation to the
Pam.sub.3Cys alkyne with a terminal azido group. The peptide was
synthesized by a Fmoc strategy on an Omega 396 synthesizer
(Advanced ChemTech, Louisville, Ky.) starting from preloaded
Fmoc-L-Val Wang resin using solid-phase chemistry, as shown in FIG.
52.
[0305] The peptide synthesis was performed by coupling amino acid
esters of HOBt using DIC as the coupling agent. A six-fold excess
of N.sup..alpha.-Fmoc amino acid esters of HOBt in NMP were used in
the synthesis. A 1:1 ratio of amino acid to DIC was used in all the
coupling reactions. Deprotection of N.sup..alpha.-Fmoc group was
accomplished by treatment with piperidine in DMF. After the
synthesis was complete, the peptide was cleaved from the solid
support and deprotected using a modified reagent K cocktail
consisting of TFA-thioanisole-ethanedithiol-water-phenol
(88:3:5:2:2). The cocktail mixture was filtered through a Quick
Snap column, purified by C18 reverse phase HPLC, and lyophilized to
afford composition (16) as a white powder.
[0306] Conjugation of the composition (16) (1 eqv) with the
composition (6) (3 eqv) in the presence of copper(I) iodide (12
eqv), tris [(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (3
eqv), diisopropyl ethyl amine (DIEA) (12 eqv) and sodium ascorbate
(12 eqv) in H.sub.2O-THF-DMF (1:1:2) as solvent at 20.degree. C.
thus afforded the target, Pam3Cys-MUC1 VNTR conjugate (17), after
16 h.
[0307] Composition (17) was purified by neutralization by 7.5%
citric acid solution followed by extraction with chloroform.
Evaporation of the chloroform extract afforded the composition (17)
as a white solid.
[0308] Synthesis of Azide (16)
[0309] The peptidazide was synthesized by Fmoc strategy on an Omega
396 synthesizer (Advanced ChemTech, Louisville, Ky.) using solid
phase chemistry. The peptide synthesis was performed by coupling
amino acid esters of HOBt using DIC as the coupling agent starting
with a preloaded Fmoc-valinyl-Wang resin. A six-fold excess of
N.sup..alpha.-Fmoc amino acid esters of HOBt in NMP were used in
the synthesis. A 1:1 ratio of amino acid to DIC was used in all the
coupling reactions. Deprotection of N.sup..alpha.-Fmoc group was
accomplished by treatment with 25% piperidine in dimethylformamide
twice; first for 5 minutes and then a second time for 25 minutes to
afford composition (15).
[0310] After the synthesis was complete, the peptide was cleaved
from the solid support and deprotected using a modified reagent K
cocktail consisting of 88% TFA, 3% thioanisole, 5% ethanedithiol,
2% water, and 2% phenol. 4 mL of cleavage cocktail was added to the
dried peptide-resin in a 15 mL glass vial blanketed with nitrogen.
Cleavage was carried out for 2.5 hrs with gentle magnetic stirring.
At the end cleavage time, the cocktail mixture was filtered on a
Quick-Snap column. The filtrate was collected in 20 mL ice-cold
butane ether. The peptide was allowed to precipitate for an hour at
-200.degree. C., centrifuged, and washed twice with ice-cold
methyl-t-butyl ether. The precipitate was dissolved in 25%
acetonitrile and lyophilized to complete dry powder affording
composition (16). Quality of peptides was analyzed by analytical
reverse phase HPLC and MALDI-TOF (matrix assisted laser desorption
ionization time-of-flight) mass spectrometer, model 4800 from
Applied Biosystems. HR-MALDI-MS: [M+H] m/z calcd for
C.sub.86H.sub.136N.sub.28O.sub.29, 2026.17. Found, 2026.137.
[0311] Synthesis of Lipopeptide (17)
[0312] CuI (134 .mu.g, 0.54 .mu.mol) and TBTA (0.857 mg, 1.62
.mu.mol) were dissolved in H.sub.2O-THF (1:1, 0.40 mL) and to it
sodium ascorbate (0.80 mg, 4.04 .mu.mol) was added and stirred for
5 minutes. Composition (6) (1.27 mg, 1.35 .mu.mol) in THF (0.40 mL)
was added to the reaction mixture and stirred for 15 minutes
followed by the addition of a solution of composition (16) (1 mg,
0.49 .mu.mol) in H.sub.2O-DMF (1:3, 0.4 mL). The reaction mixture
was stirred at 20.degree. C. under N.sub.2 atmosphere for 16 h. The
reaction mixture was concentrated, dissolved in CHCl.sub.3, washed
with 7.5% aqueous citric acid solution, dried over sodium sulfate,
and the solvent was evaporated to afford composition (17) as a
white solid (1.47 mg, 100%). HR-MALDI-MS: [M+H] m/z calcd for
C.sub.143H.sub.242N.sub.30O.sub.35S, 2972.78. Found 2972.828.
[0313] FIGS. 53 and 54 show the HRMS (MALDI-TOF) spectrum and HPLC
trace, respectively, of peptide (16).
[0314] FIG. 55 shows the HRMS (MALDI-TOF) spectrum of lipopeptide
(17).
Example 9
[0315] A 20-amino acid (TSAPDT(Tn)RPAPGSTAPPAHGV [SEQ ID NO: 6])
tandem repeat of MUC1 which included the PD(.alpha.-GalNAc-O-T)R
epitope was synthesized. The glycopeptides was modified with a
terminal azido group in order to make a `click` conjugation to the
Pam.sub.3Cys alkyne. The glycopeptides azide was synthesized by
Fmoc strategy on an Omega 396 synthesizer (Advanced ChemTech,
Louisville, Ky.) starting from preloaded Fmoc-L-Val Wang resin
using solid-phase chemistry, as shown in FIG. 56.
[0316] The peptide synthesis was performed by coupling amino acid
esters of HOBt using DIC as the coupling agent. A six-fold excess
of N.sup..alpha.-Fmoc amino acid esters of HOBt in NMP were used in
the synthesis. A 1:1 ratio of amino acid to DIC was used in all the
coupling reactions. Deprotection of N.sup..alpha.-Fmoc group was
accomplished by treatment with piperidine in DMF. After the
synthesis was complete, the peptide was cleaved from the solid
support and deprotected using a modified reagent K cocktail
consisting of TFA-thioanisole-ethanedithiol-water-phenol
(88:3:5:2:2). The cocktail mixture was filtered through a Quick
Snap column, purified by C18 reverse phase HPLC and lyophilized to
afford composition (19).
[0317] The acetyl groups in composition (19) were deprotected by
treatment with 6 mmol sodium methoxide in methanol. The product was
purified by Bio-Gel (P-2, fine 45-90 .mu.m) size exclusion
chromatopgraphy using deionized water as solvent. Lyophilization of
the fractions afforded composition (20) (100%) as a white powder.
Conjugation of the composition (19) (1 eqv) with the composition
(6) (3 eqv) in the presence of copper(I) iodide (12 eqv), tris
[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (3 eqv),
diisopropyl ethyl amine (DIEA) (12 eqv) and sodium ascorbate (12
eqv) in H.sub.2O-THF-DMF (1:1:2) as solvent at 20.degree. C. thus
afforded the target, Pam.sub.3Cys-MUC1 VNTR-TACA conjugate (21),
after 16 h. Composition (21) was purified by neutralization by 7.5%
citric acid solution followed by extraction with chloroform.
Evaporation of the chloroform extract afforded the composition (21)
as light yellow solid.
[0318] Synthesis of Azide (20)
[0319] Composition (19) (5 mg, 2.24 .mu.mol) was dissolved in 2 mL
of dry methanol. 12 .mu.L of freshly prepared 1 M sodium methoxide
was added to the mixture and the reaction mixture was stirred at
ambient temperature under N.sub.2 atmosphere for 3 h. The reaction
mixture was neutralized with solid carbon dioxide. The reaction
mixture was concentrated and purified by Bio-Gel (P-2, fine 45-90
.mu.m) size exclusion chromatography using deionized water as
solvent. Lyophilization of the eleutants afforded composition (20)
as a white powder (4.7 mg, 100%). HR-MALDI-MS: [M+H] m/z calcd for
C.sub.94H.sub.149N.sub.29O.sub.34, 2229.09. Found 2026.336.
[0320] Synthesis of Glycolipopeptide (21)
[0321] CuI (134 .mu.g, 0.54 .mu.mol) and TBTA (0.857 mg, 1.62
.mu.mol) were dissolved in H.sub.2O-THF (1:1, 0.40 mL) and to it
sodium ascorbate (0.80 mg, 4.04 .mu.mol) was added and stirred for
5 minutes. Composition (6) (1.27 mg, 1.35 .mu.mol) in THF (0.40 mL)
was added to the reaction mixture and stirred for 15 minutes
followed by the addition of a solution of composition (20) (1 mg,
0.45 .mu.mol) in H.sub.2O-DMF (1:3, 0.4 mL). The reaction mixture
was stirred at 20.degree. C. under N.sub.2 atmosphere for 16 h. The
reaction mixture was concentrated, dissolved in CHCl.sub.3, washed
with 7.5% aqueous citric acid solution, dried over sodium sulfate,
and the solvent was evaporated to afford composition (21) as a
light yellow solid (1.9 mg, 100%). HR-MALDI-MS: [M+H] m/z calcd for
C.sub.151H.sub.255N.sub.31O.sub.40S, 3175.86. Found 3175.809.
[0322] FIGS. 57 and 58 show the HRMS (MALDI-TOF) spectrum and HPLC
trace of glycopeptide (19).
[0323] FIG. 59 is the HRMS (MALDI-TOF) spectrum of glycopeptides
(20).
[0324] FIG. 60 is the HRMS (MALDI-TOF) spectrum of
Pam.sub.3Cys-MUC-1 VNTR conjugate (21).
[0325] Methods of Use
[0326] Also provided herein is a method for eliciting an immune
response against a cancer cell surface antigen in a subject in need
thereof. The method comprises administering to the subject an
antigen-liposome-xenoantigen vaccine composition in sufficient dose
to elicit the immune response to the cancer cell surface antigen,
wherein the antigen-liposome-xenoantigen vaccine composition
comprises the cancer cell surface antigen and a liposome.
[0327] In certain embodiments, the cancer cell surface antigen is
expressed only in cancer cells in the subject, such that the immune
response specific for the antigen would not attack healthy
tissues/organs of the subject. In certain embodiments, the antigen
that is expressed on the cancer cell surface is a mutant protein
present only on cancer cells, and the immune response is specific
for the mutant protein epitope comprising the mutation. The
mutation can be a protein mutation, or abnormal sugar or lipid
structures on cancer cell surface.
Example 10
T-Cell Proliferation Study
Immunization
[0328] One female BALB/c mouse (6-8 weeks old, The Jackson
Laboratory) was primed (day 0) and boosted three time (days 14, 28,
and 42) with 100 .mu.L subcutaneous injections of an equivolume
emulsion of the MUC1-Tn conjugate (8) (prepared in phosphate buffer
saline-PBS) and sigma adjuvant system (SAS) (50 .mu.g of peptide
per mouse, each injection).
[0329] Preparation of Anti-Rha Antibodies
[0330] The Rha-Ficoll and the Rha-OVA immunized mice were bled on
day 66 and the sera was pooled. IgG fractions from each pool were
prepared by precipitation at 40% saturation of ammonium sulfate.
The mixtures were incubated overnight and centrifuged at
10000.times.g for 10 minutes and then resuspended in 0.5 mL water.
The antibody solutions were concentrated and buffer was changed
twice with PBS using an Ultrafree 0.5 centrifugal filter device
(Millipore, Billerica, Mass.) having a molecular cut off of 50000
D. Absorbances of the antibody solutions were recorded at 280 nm to
calculate the concentrations and the anti-Rha antibody solutions
generated and isolated from the Rha-Ficoll and the Rha-OVA
immunized mice were each diluted to 1.0 mg/mL.
[0331] Preparation of Spleen Cell Suspensions and Assay Setup
[0332] On day 49, the mouse was sacrificed and the spleen was
removed and placed in 5 mL of freshly prepared spleen cell culture
medium (DMEM with 10% fetal calf serium). Single cell suspension
was prepared using modified sterile glass homogenizers. The cells
were washed three times with culture medium and brought to
5.times.10.sup.6 cells/mL. 100 .mu.L of the spleen cell suspensions
were added to 96 well plates (5.times.10.sup.5 cells per well). The
dendritic cell (DC) suspension cultured from the bone marrow of a
BALB/c mouse was pulsed with the antigen by incubating with the
Rha-displaying MUC1-Tn liposomes at antigen concentrations of
8.8.times.10.sup.-3-1.1 .mu.g/mL at 37.degree. C. for 4 h together
with anti-Rha antibodies generated from either Rha-Ficoll or
Rha-OVA immunized mice sera (5 .mu.Ci/mL, 25 .mu.L per well) and
incubated overnight at 37.degree. C. The cells were harvested on
glass-fiber filters and incorporation was determined by
measurements on a Top Count scintillation counter.
[0333] Immunizations
[0334] The 20 female BALB/c mice used for this study were divided
into four groups A1, A2, B1, and B2, containing 5 mice each. Groups
A1 and B1 served as the control groups and were not immunized.
Groups A2 and B2 were injected subcutaneously (day 0) with a 100
.mu.L subcutaneous injections Rha-Ficoll/Alum on days 14, 28, 42,
and 56 (100 .mu.g of Rha-Ficoll per mouse, each boost). The mice in
each group A1, A2, B1, and B2 were bled on day 66 and the collected
sera were tested for anti-Rha antibodies.
[0335] ELISA for Measuring Anti-Rha Antibody Titers
[0336] 96 well plates (Immulon 4 HBX) were coated with Rha-BSA
conjugate (6) (2 .mu.g/mL) in 0.01 M PBS and incubated overnight at
4.degree. C. The plates were washed 5 times with PBS containing
0.1% Tween-20. Blocking was achieved by incubating the plates for 1
h at room temperature with BSA in 0.01 M PBS (1 mg/mL). The plates
were then washed 5 times and incubated for 1 h with serum dilutions
in PBS. Unbound antibody in the serum was removed by washing and
the plates were incubated for 1 h at room temperature with
Horseradish Peroxidase (HRP) goat anti-mouse IgG+IgM (Jackson
Immunoresearch Laboratories) diluted 5000 times in PBS/BSA. The
plates were washed with TMB (3,3',5,5'-tetramethylbenzidine). One
component HRP microwell substrate (Bio FX, Owings Mills, Md.) was
added and allowed to react for 10 mins. Absorbances were recorded
at 620 nm and were plotted against log.sub.10 [1/serum
dilution].
[0337] Vaccinations
[0338] Vaccinations were performed on day 77. Two separate
liposomal formulations were prepared with DPPC (80%), cholesterol
(20%) and Pam.sub.3Cys-MUC1-Tn (9) (2 nmol) (Pam.sub.3Cys-MUC1-Tn
liposomes) and DPPC (80%), cholesterol (10%), Rha-TEG-cholesterol 3
(10%) and Pam.sup.3Cys-MUC1-Tn (9) (2 nmol)
(Pam.sub.3Cys-MUC1-Tn+Rha liposomes) in total lipid concentrations
of 30 mmol. Groups A1 and A2 were cavvinated with 100 .mu.L
subcutaneous injections of the Pam.sub.3Cys-MUC1-Tn liposomes (2
nmol of peptide per mouse) and groups B1 and B2 were vaccinated
with 100 .mu.L subcutaneous injections of the
Pam.sub.3Cys-MUC1-Tn+Rha liposomes (2 nmol peptide per mouse). The
mice were boosted on day 91 with either the Pam.sub.3Cys-MUC1-Tn
liposomes (groups A1 and A2, 2 nmol peptide per mouse) or the
Pam.sub.3Cys-MUC1-Tn+Rha liposomes (groups B1 and B2). The mice
were bled on day 101 and the sera collected were tested for
anti-MUC1-Tn and anti-Tn antibodies.
[0339] ELISA for Measuring Anti-MUC1-Tn Antibody Titers
[0340] 96 Well plates (Immulon 4 HBX) were coated with MUC1-Tn
conjugate (8) (15 .mu.L/mL) in 0.01 M PBS and incubated overnight
at 4.degree. C. The ELISA was continued as described above.
[0341] ELISA for Measuring Anti-Tn Antibody Titers
[0342] 96 Well plates (Immulon 4 HBX) were coated with Tn-BSA
conjugate (15 .mu.L/mL) in 0.01 M PBS and incubated overnight at
4.degree. C. The ELISA was continued as described above.
[0343] Anti-MUC1-Tn Antibody Subclass Identification
[0344] 96 Well plates (Immulon 4 HBX) were coated with MUC1-Tn
conjugate (8) (15 .mu.L/mL) in 0.01 M PBS and incubated overnight
at 4.degree. C. The plates were washed 4 times with PBS containing
0.1% Tween-20. Blocking was achieved by incubating the plates for 1
h at room temperature with BSA in 0.01 M PBS (1 mg/mL). The plates
were then washed 4 times and incubated for 1 h with 1/100 serum
dilution in PBS. Unbound antibody in the serum was removed by
washing and the plates were incubated overnight at 4.degree. C.
with subclass specific (IgG1, IgG2s, IgG2b, IgG3, 1gA, and IgM)
rabbit anti-mouse antibody (Zymed Laboratories mouse monoAb-ID
kit). The plates were washed and incubated with HRP-goat
anti-rabbit IgG (H+L) for 1 h at room temperature. The plates were
washed and ABTS substrate buffer (diluted 50 times) was added and
allowed to react for 30 min. Absorbances were recorded at 405 nm
and compared for each antibody subclass in each group.
[0345] ELISA for Competitive Binding with Free MUC1-Tn
[0346] A 96-well plate (Immulon 4 HBX) was coated with MUC1-Tn
conjugate (8) (15 .mu.L/mL) in PBS and incubated overnight at
4.degree. C. The plate was washed 5 times with PBS containing 0.1%
Tween-20. Blocking was achieved by incubating the plate for 1 h at
room temperature with BSA in M PBS (1 mg/mL). The plate was then
washed 5 times and incubated for 1 h with serum dilutions of 1/100
in PBS with or without prior mixing with varying concentrations of
free MUC1-Tn (8) from 0, 10.sup.-5, 10.sup.-4, 10.sup.-3 M in PBS.
Unbound antibody in the serum was removed by washing and the plate
was incubated for 1 h at room temperature with Horseradish
Peroxidase (HRP) goat anti-mouse IgG+IgM (secondary antibody)
diluted 5000 times in PBS/BSA. The plate was washed and TMB 1
component HRP microwell substrate was added and allowed to react
for 10 mins. Absorbances were recorded at 620 nm and were plotted
against log.sub.10 [1/free Tn concentration].
[0347] Tumor Cell Staining
[0348] U266 cells (American Type Culture Collection, Manassas,
Va.), were cultured in RPMI 1640 with 15% fetal calf serum (FCS).
Cells were stained with purified mouse anti-human MUC1 antibodies
(CD227, 0.5 .mu.g), non-immune BALB/c mice serum (1/5 dilution) and
group B2 mice serum (1/5 dilution). The cells were then stained
with FITC-conjugated goat anti-mouse IgG+IgM (0.5 .mu.g) and
fluorescence was quantified with a BD FACS Calibur.
[0349] Comparison of Anti-Rha Antibody Titers Generated Against
Rha-Ficoll and Rha-OVA
[0350] Two groups of five female BALB/c mice each were immunized on
day 0 with Rha-Ficoll/Alum adjuvant (group A) or Rha-OVA/complete
Freund's adjuvant (CFA) (group B). The mice were boosted three more
times on days 14, 28, and 42 with either Rha-Ficoll/Alum (group A)
or rha-OVA/incomplete Freund's adjuvant (ICF) (group B). Sera were
collected separately from groups A and B after the third boost and
the anti-Rha antibodies in the sera from the two groups of mice
were isotyped by screening against Rha-BSA. See FIG. 36. The
results demonstrated the anti-Rha antibody titers in the Rha-OVA
immunized micro groups were 100-fold higher than those from the
Rha-Ficoll immunized mice. However, the isotype distribution
confirmed that Rha-Ficoll and Rha-OVA produced the anti-Rha
antibody subclasses in different proportions. Anti-Rha antibodies
from Rha-OVA immunization were dominated by IgG1 (65%) while
Rha-Ficoll immunization produced antibodies which comprised mainly
IgG3 (48%) and IgM (25%). IgG1 and IgG3 act similarly in that they
both stimulate high affinity Fc.gamma.RI receptors which trigger
responses from macrophages. However, IgG1 also stimulates low
affinity Fc.gamma.RIIB receptors which inhibit the signals from the
Fc.gamma.RI and B cell receptors thereby diminishing B-cell
activity and immunogenicity of macrophages. The anti-Rha antibody
isotypes from Rha-Ficoll immunized mice serum resembled those
naturally occurring in the human serum which is presumed to be
generated through a T-independent response.
[0351] T-Cell Proliferation Study
[0352] T-cell proliferation assays were performed to determine if
the combination of anti-Rha antibodies and Rha-modified liposomal
vaccine would potentiate a T-cell proliferative response. In the
first part of the study, the proliferation assay conditions were
optimized. BALB/c mice were immunized (day 0) and boosted (days 14,
28, and 42) with 100 .mu.L emulsions of MUC1-Tn (8)/Sigma adjuvant
system (SAS) (50 .mu.g peptide per mouse, each injection). The mice
were sacrificed (day 49), the spleens were removed, and single cell
suspensions were prepared and incubated with MUC1-Tn
(8.8.times.10.sup.-3-1.1 .mu.g/mL) alone or with syngeneic bone
marrow dendritic cells (DCs) previously pulsed with the same doses
of antigen. DCs showed enhanced proliferation, as shown in FIG. 37.
To test the ability of anti-Rha antibodies to enhance antigen
presentation, spleen cells from BALB/c mice immunized as above were
prepared. DCs from BALB/c bone marrow were pulsed with the antigen
by incubating with Pam.sub.3Cys-MUC1-Tn+Rha liposomes at antigen
concentrations of 8.8.times.10.sup.-3-0.22 .mu.g/mL together with
antibodies isolated from either Rha-Ficoll or Rha-OVA immunized
mice or nonimmune mice. The pulsed DCs were added to the spleen
cells and proliferation assessed after 3 days. The spleen T-cells
proliferated better in the presence of anti-Rha antibodies (from
both Rha-Ficoll and Rha-OVA immunized mice serum) than in the
presence of control serum antibodies over the antigen concentration
range of 8.8.times.10.sup.-3-0.22 .mu.g/mL. See FIG. 38. Also, the
T-cell proliferation was higher in the presence of anti-Rha
antibodies generated against Rha-Ficoll (6328, 6045, and 6521
counts per minute (cpm) at antigen concentrations of
8.8.times.10.sup.-3, 0.044, and 0.22 .mu.g/mL) than those against
Rha-OVA (5018, 4926, and 4880 cpm at antigen concentrations of
8.8.times.10.sup.-3, 0.044, and 0.22 .mu.g/mL), even though the
titer of anti-Rha antibodies was higher in the serum of Rha-OVA
immunized mice. The results strongly suggest that the Rha-modified
antigen was more effectively internalized and presented by the APCs
in the presence of anti-Rha antibodies, particularly those less
inhibitory isotypes characteristic of natural antibodies and
generated by Rha-Ficoll immunization. Therefore, BALB/c mice in
which anti-Rha antibodies are generated with Rha-Ficoll (14)
immunization are an appropriate model for the immunogenicity of the
Rha-conjugated MUC1-Tn liposomes.
[0353] Anti-Rha Antibody Generation
[0354] Four groups of five female BALB/c mice each (groups A1, A2,
B1, and B2) (6-8 weeks old) were used for this vaccination study.
Groups A2 and B2 were immunized (day 0) and boosted (days 14, 28,
42, and 56) with 100 .mu.L equivolume emulsion of Rha-Ficoll
(prepared in PBS) and alum adjuvant. Groups A1 and B1 served as the
control groups and were deprived of the Rha-Ficoll/Alum
immunization. See FIG. 38B. The mice were bled on day 66 and the
ELISA performed by screening the sera from the different groups
against Rha-BSA showed that the anti-Rha antibody titers in groups
A2 and B2 were 25-fold higher than the control groups. See FIG. 39.
Thus, immunization with Rha-Ficoll confirmed the generation of
anti-Rha antibodies in the experimental groups of mice.
[0355] Vaccination with Rha and non-Rha-Displaying MUCA1-Tn
Liposomes
[0356] Two separate liposomal formulations were prepared. The first
contained DPPC, cholesterol and Pam.sub.3Cys-MUC1-Tn (9) (2 nmol)
(Pam.sub.3Cys-MUC1-Tn liposomes) and the second contained DPPC,
cholesterol, Rha-TEG-cholesterol (3) and Pam.sub.3Cys-MUC1-Tn (9)
(2 nmol) (Pam.sub.3CysMUC1-Tn+Rha liposomes). In both formulations
the total lipid concentration was 30 mmol. The vaccination was
performed on day 77. Groups A1 and A2 were given 100 .mu.L
subcutaneous injections of the Pam.sub.3Cys-MUC1-Tn liposomes (2
nmol of peptide per mouse) and groups B1 and B2 were given 100
.mu.L subcutaneous injections of the Pam.sub.3CysMUC1-Tn+Rha
liposome (2 nmol peptide per mouse). The mice were boosted on day
91 with either the Pam.sub.3CysMUC1-Tn liposome (groups A1 and A2,
2 nmol peptide per mouse) or the Pam.sub.3CysMUC1-Tn+Rha liposome
(groups B1 and B2). The mice were bled on day 101 and the sera
evaluated for anti-MUC1-Tn and anti-Tn antibodies. See FIG.
39B.
[0357] Anti-MUC1-Tn antibody titers were determined by screening
the sera against the MUC1-Tn conjugate (8). (FIG. 39B). The data
showed that groups A1, A2, and B1 had similar absorbance at 1/25,
1/50, 1/100, and 1/200 serum dilutions. This proved that prior
immunization with Rha-Ficoll does not affect the response to a
non-Rha conjugated vaccine (groups A1 and A2). In addition, the Rha
epitopes on the vaccine do not alter the inherent immunogenicity of
the MUC1-Tn epitopes on the vaccine (groups A1 and B1). The
anti-MUC1-Tn titers for group B2 showed an 8-fold increase compared
to groups A1, A2, and B1, which was mediated by the anti-Rha
antibody-dependent antigen-uptake. Group B2 had an anti-MUC1-Tn
titer of approximately 1/300, where titer is defined as the highest
dilution giving a signal >0.1 above background. The anti-Muc1-Tn
antibodies from each group were isotyped, resulting in group B2
showing an increase in IgG1, IgG2a, IgG2b, and IgM isotypes
relative to the other three groups. See FIG. 40. The specificity of
the antibodies towards MUC1-Tn antigen was determined by a
competitive binding experiment. See FIG. 41A. Serum from every
group at 1/100 dilution was incubated with the MUC1-Tn conjugate
(8) at concentrations of 0, 10.sup.-5, 10.sup.-4, and 10.sup.-3 M
in 0.01 M PBS prior to addition in the ELISA plates coated with the
conjugate (8). The absorbances decreased uniformly with increasing
concentrations of free MUC1-Tn in the serum dilutions for each
group. As an example, the absorbances at 620 nm for the serum
dilution of group B2 at free MUC1-Tn concentrations of 0,
10.sup.-5, 10.sup.-4, and 10.sup.-3 M were 0.790, 0.601, 0.577, and
0.512, respectively. These results confirmed the specificity of the
anti-MUC1-Tn antibodies towards the respective antigen.
[0358] The antibody titer generated solely against the TACA was
determined by screening serum dilutions from every group against a
Tn-BSA conjugate. See FIG. 41B. A >8-fold increase in the
anti-Tn antibody titers for group B2 was also observed in
comparison to groups A1, A2, and B1. This was again attributed to
the better uptake of the antigen in the presence of the anti-Rha
antibodies by an antibody-dependent antigen-uptake mechanism. Also
observed in this study was that the anti-MUC1-Tn antibody titers
were higher than the corresponding anti-Tn antibody titers for the
same serum dilutions for every group, assuming similar levels of
antigen on the plate. For example, for the group B2, the
absorbances at 620 nm for the anti-MUC1-Tn and the anti-Tn
measurements at 1/100 serum dilutions were 0.922 and 0.509,
respectively. This observation demonstrates that the Rha-displaying
MUC1-Tn vaccine successfully generates antibodies against both the
MUC1 peptide and the TACA.
[0359] The ability of the anti-MUC1-Tn antibodies in the vaccinated
mice serum to bind to MUC1-Tn on human tumor cells was demonstrated
with U266 human leukemia cells. These cells express MUC1 on their
surface as shown by binding with mouse anti-human MUC1 antibodies
(CD 227). See FIG. 42A. Serum from group B2 mice also recognized
the MUC1 on the tumor cells with similar efficiency as the CD 227
antibodies relative to non-immunized mouse serum. See FIG. 42B.
This demonstrates that the antibodies generated against the
glycopeptides recognize the MUC1 protein in its native
environment.
[0360] Summary
[0361] As the examples herein describe, a fully synthetic
two-component vaccine containing the lipopeptide adjuvant
Pam.sub.3Cys appended to a 20-amino acid MUC1 peptide containing
the TACA GalNAc-O-Thr (Tn) was synthesized and was successfully
formulated into liposomes along with an Rha cholesterol conjugate.
The resulting liposomes were homogenous in size and were stable at
4.degree. C. for two days. Binding studies with both anti-Rha and
mouse anti-human MUC1 antibodies revealed that the Rha and the MUC1
glycopeptide epitopes were surface displayed on the liposomes. A
Rha-Ficoll conjugate (14) was synthesized for the generation of
anti-Rha antibodies in mice. The in vitro proliferation of MUC1-Tn
primed mice spleen T-cells showed increased proliferation to
Rha-liposomes in the presence of antibodies from Rha-Ficoll
immunized mice relative to nonimmune mice. Vaccination studies with
Rha- and non-Rha-displaying MUC1-Tn liposomes in mice either
non-immunized or immunized with Rha-Ficoll illustrated that
anti-MUC1-Tn and anti-Tn antibodies were >8-fold higher in the
groups of mice previously immunized with Rha-Ficoll and later
vaccinated with the Pam.sub.3Cys-MUC1-Tn+Rha liposomes. The
anti-MUC1-Tn antibodies in the serum of the vaccinated mice
recognized the aberrant MUC1 on human leukemia U266 cells. Overall,
this vaccine successfully triggered both T-cell and humoral
immunity enhanced by anti-Rha antibody dependant antigen uptake.
Because this vaccine uses separate rhamnose and anti-genic epitope
components, the vaccine can easily be targeted to different
antigens or epitopes by changing the peptide without having to
change the other components. For instance, the skilled practitioner
will appreciate that anti-viral or anti-bacterial vaccines can also
be made from them methods and compositions described herein.
[0362] While the invention has been described with reference to
various and preferred embodiments, it should be understood by those
skilled in the art that various changes may be made and equivalents
may be substituted for elements thereof without departing from the
essential scope of the invention. In addition, many modifications
may be made to adapt a particular situation or material to the
teachings of the invention without departing from the essential
scope thereof.
[0363] Therefore, it is intended that the invention not be limited
to the particular embodiment disclosed herein contemplated for
carrying out this invention, but that the invention will include
all embodiments falling within the scope of the claims.
[0364] The publication and other material used herein to illuminate
the invention or provide additional details respecting the practice
of the invention, are incorporated be reference herein, and for
convenience are provided in the following bibliography.
[0365] Citation of the any of the documents recited herein is not
intended as an admission that any of the foregoing is pertinent
prior art. All statements as to the date or representation as to
the contents of these documents is based on the information
available to the applicant and does not constitute any admission as
to the correctness of the dates or contents of these documents.
Sequence CWU 1
1
15120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro
Pro Ala His Gly 1 5 10 15 Val Thr Ser Ala 20 26PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Cys
Ser Lys Lys Lys Lys 1 5 36PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 3Cys Ser Lys Lys Lys Lys 1 5
414PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Cys Gly Asn Asn Asp Glu Ser Asn Ile Ser Phe Lys
Glu Lys 1 5 10 520PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 5Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
Gly Ser Thr Ala Pro Pro 1 5 10 15 Ala His Gly Val 20
620PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser
Thr Ala Pro Pro 1 5 10 15 Ala His Gly Val 20 721PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 7Cys
Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 1 5 10
15 Gly Val Thr Ser Ala 20 821PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 8Xaa Pro Asp Thr Arg Pro Ala
Pro Gly Ser Thr Ala Pro Pro Ala His 1 5 10 15 Gly Val Thr Ser Ala
20 921PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Xaa Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
Pro Pro Ala His 1 5 10 15 Gly Val Thr Ser Ala 20 1021PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 10Xaa
Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 1 5 10
15 Gly Val Thr Ser Ala 20 1121PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 11Xaa Thr Ser Ala Pro Asp Thr
Arg Pro Ala Pro Gly Ser Thr Ala Pro 1 5 10 15 Pro Ala His Gly Val
20 1221PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Cys Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly
Ser Thr Ala Pro 1 5 10 15 Pro Ala His Gly Val 20 1321PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 13Xaa
Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro 1 5 10
15 Pro Ala His Gly Val 20 1421PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 14Xaa Thr Ser Ala Pro Asp Thr
Arg Pro Ala Pro Gly Ser Thr Ala Pro 1 5 10 15 Pro Ala His Gly Val
20 1521PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Cys Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly
Ser Thr Ala Pro 1 5 10 15 Pro Ala His Gly Val 20
* * * * *