U.S. patent application number 13/002180 was filed with the patent office on 2012-02-16 for glycopeptide and uses thereof.
This patent application is currently assigned to University of Georgia Research Foundation, Inc.. Invention is credited to Geert-Jan Boons, Therese Buskas, Alex J. Harvey, Sampat Ingale, Robert Lance Wells, Margaretha Wolfert.
Application Number | 20120039984 13/002180 |
Document ID | / |
Family ID | 41466515 |
Filed Date | 2012-02-16 |
United States Patent
Application |
20120039984 |
Kind Code |
A1 |
Boons; Geert-Jan ; et
al. |
February 16, 2012 |
GLYCOPEPTIDE AND USES THEREOF
Abstract
A glycolipopeptide comprising a carbohydrate component, a
peptide component and a lipid component, for use as a therapeutic
or prophylactic vaccine. Also provided are monoclonal and
polyclonal antibodies that recognize the glycolipopeptide of the
invention, as well as uses thereof.
Inventors: |
Boons; Geert-Jan; (Athens,
GA) ; Buskas; Therese; (Athens, GA) ; Harvey;
Alex J.; (Athens, GA) ; Ingale; Sampat; (San
Diego, CA) ; Wolfert; Margaretha; (Athens, GA)
; Wells; Robert Lance; (Athens, GA) |
Assignee: |
University of Georgia Research
Foundation, Inc.
Athens
GA
|
Family ID: |
41466515 |
Appl. No.: |
13/002180 |
Filed: |
July 2, 2009 |
PCT Filed: |
July 2, 2009 |
PCT NO: |
PCT/US2009/003944 |
371 Date: |
August 5, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12217376 |
Jul 3, 2008 |
7820797 |
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13002180 |
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61197386 |
Oct 27, 2008 |
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Current U.S.
Class: |
424/450 ;
424/141.1; 424/147.1; 424/148.1; 424/149.1; 424/150.1; 424/151.1;
424/155.1; 424/159.1; 424/160.1; 424/161.1; 424/164.1; 424/174.1;
424/175.1; 424/193.1; 424/196.11; 424/197.11; 435/331; 435/7.1;
530/322; 530/388.9; 530/389.8; 530/391.3 |
Current CPC
Class: |
A61P 31/18 20180101;
G01N 33/92 20130101; A61K 39/0011 20130101; C07K 16/3076 20130101;
A61K 2039/6075 20130101; A61P 31/12 20180101; A61K 2039/545
20130101; A61K 9/127 20130101; A61P 33/02 20180101; A61P 31/14
20180101; A61K 2039/55555 20130101; C07K 2317/34 20130101; A61K
39/0008 20130101; A61K 47/646 20170801; A61P 35/00 20180101; G01N
2400/02 20130101; G01N 2800/2821 20130101; A61K 2039/6018 20130101;
C12N 2740/16011 20130101; A61K 2039/57 20130101; A61K 2039/55511
20130101; C12N 2740/16134 20130101; C07K 9/00 20130101; C07K 16/44
20130101; A61K 39/00117 20180801; A61P 31/04 20180101; C07K 14/4727
20130101; A61K 38/00 20130101; C07K 14/22 20130101; G01N 2800/042
20130101 |
Class at
Publication: |
424/450 ;
530/322; 424/193.1; 424/141.1; 424/175.1; 424/196.11; 424/197.11;
424/147.1; 424/150.1; 424/151.1; 424/159.1; 424/164.1; 424/148.1;
424/149.1; 424/160.1; 424/161.1; 424/155.1; 424/174.1; 530/388.9;
530/389.8; 530/391.3; 435/331; 435/7.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 39/395 20060101 A61K039/395; A61K 9/127 20060101
A61K009/127; A61K 39/12 20060101 A61K039/12; A61K 39/02 20060101
A61K039/02; A61K 39/42 20060101 A61K039/42; A61K 39/40 20060101
A61K039/40; A61K 39/002 20060101 A61K039/002; C07K 16/00 20060101
C07K016/00; C07K 17/00 20060101 C07K017/00; A61P 35/00 20060101
A61P035/00; A61P 31/12 20060101 A61P031/12; A61P 33/02 20060101
A61P033/02; A61P 31/04 20060101 A61P031/04; A61P 31/14 20060101
A61P031/14; A61P 31/18 20060101 A61P031/18; C12N 5/16 20060101
C12N005/16; G01N 33/53 20060101 G01N033/53; C07K 17/02 20060101
C07K017/02 |
Goverment Interests
STATEMENT OF GOVERNMENT RIGHTS
[0002] This invention was made with government support under a
grant from the National Cancer Institute of the National Institute
of Health (Grant No RO1 CA88986). The U.S. Government has certain
rights in this invention.
Claims
1-51. (canceled)
52. A glycolipopeptide comprising: at least one carbohydrate
component comprising a B-epitope; at least one peptide component
comprising a T-epitope; and at least one lipid component; wherein
the carbohydrate component and the peptide component are
heterologous with respect to each other.
53. The glycolipopeptide of claim 52 wherein the B-epitope is from
a microorganism.
54. The glycolipopeptide of claim 53 wherein the microorganism is
selected from the group consisting of a virus, a bacterium, a
fungus, and a protozoan.
55. The glycolipopeptide of claim 53 wherein the microorganism is a
human immunodeficiency virus or a hepatitis C virus.
56. The glycolipopeptide of claim 52 wherein the B epitope is
overexpressed on a cancer cell.
57. The glycolipopeptide of claim 52 wherein the carbohydrate
component comprises a self-antigen.
58. The glycolipopeptide of claim 57 wherein the self-antigen
comprises a MUC-1 glycopeptide.
59. The glycolipopeptide of claim 52 wherein the carbohydrate
component comprises a viral antigen.
60. The glycolipopeptide of claim 59 wherein the viral antigen is
from human immunodeficiency virus or a hepatitis C virus.
61. The glycolipopeptide of claim 52 wherein the carbohydrate
component comprises a glycoconjugate selected from the group
consisting of a glycosylated protein, a glycosylated peptide, a
glycosylated lipid, a glycosylated amino acid, a DNA and an
RNA.
62. The glycolipopeptide of claim 61 wherein the glycosylated
peptide or glycosylated protein comprises a
.beta.-N-acetylglucosamine (.beta.-O-GlcNAc) modified peptide.
63. The glycolipopeptide of claim 52 comprising a glycosylated
peptide, wherein the glycosylated peptide comprises the B-epitope
and the T-epitope.
64. The glycolipopeptide of claim 52 wherein the carbohydrate
component comprises a heparan sulfate fragment.
65. The glycolipopeptide of claim 52 wherein the lipid component
comprises a Toll-like receptor (TLR) ligand.
66. The glycolipopeptide of claim 52 wherein the lipid component
facilitates internalization of the glycolipopeptide by a target
cell.
67. The glycolipopeptide of claim 52 wherein the T-epitope
comprises a helper T-epitope.
68. A pharmaceutical composition comprising: at least one of the
glycolipopeptide of claim 52 or a monoclonal or polyclonal antibody
that binds thereto; and a pharmaceutically acceptable carrier.
69. The pharmaceutical composition of claim 68 further comprising a
liposome.
70. The pharmaceutical composition of claim 69 wherein the
glycolipopeptide is covalently or noncovalently incorporated into
the liposome.
71. The pharmaceutical composition of claim 70 wherein a plurality
of glycolipopeptides are covalently or noncovalently incorporated
in the liposome.
72. The pharmaceutical composition of claim 71 wherein at least two
of the plurality of glycolipopeptides contain different
B-epitopes.
73. The pharmaceutical composition of claim 71 wherein at least two
of the plurality of glycolipopeptides contain the same
B-epitopes.
74. The pharmaceutical composition of claim 68 which does not
contain an external adjuvant.
75. The pharmaceutical composition of claim 68 further comprising
an external adjuvant.
76. The pharmaceutical composition of claim 75 wherein the external
adjuvant comprises QS-21.
77. The pharmaceutical composition of claim 68 formulated for use
as a vaccine.
78. A method for treating or preventing an infection, disease or
disorder in a subject comprising administering to the subject the
pharmaceutical composition of claim 68.
79. The method of claim 78 wherein the pharmaceutical composition
includes an adjuvant comprising QS-21, and wherein the inclusion of
QS-21 skews the immune response of the subject toward a Th1
response, compared to a comparable pharmaceutical composition that
does not include QS-21.
80. The method of claim 78 wherein the infection, disease or
disorder is caused by a microorganism.
81. The method of claim 80 wherein the microorganism is selected
from the group consisting of a virus, a bacterium, a fungus, and a
protozoan.
82. The method of claim 78 wherein the infection, disease or
disorder is caused by a human immunodeficiency virus or a hepatitis
C virus.
83. The method of claim 78 wherein the disease is cancer, a
precancerous condition, or an autoimmune disease.
84. A polyclonal or monoclonal antibody that binds to the
glycolipopeptide of claim 52.
85. The polyclonal or monoclonal antibody of claim 84 which is an
IgG antibody.
86. The polyclonal or monoclonal antibody of claim 85 which has
broad selectivity for O-GlcNAc modified proteins.
87. A hybridoma that produces a monoclonal antibody that binds to
the glycolipopeptide of claim 52.
88. A method for making a glycolipopeptide comprising: contacting a
candidate compound with a target cell containing a Toll-like
receptor (TLR); determining whether the candidate compound binds to
the TLR so as to identify a TLR ligand; and covalently linking the
identified TLR ligand to a carbohydrate component comprising a
B-epitope and a peptide component comprising a T-epitope to yield a
glycolipopeptide comprising the carbohydrate component, the peptide
component, and the TLR ligand.
89. The method of claim 88 further comprising determining whether
the candidate compound is internalized by the target cell.
90. The method of claim 88 wherein the candidate compound comprises
a lipid.
91. A glycolipopeptide made according to the method of claim
88.
92. A kit comprising: a monoclonal antibody that binds to the
glycolipopeptide of claim 52; packaging; and instructions for
use.
93. The kit of claim 92 wherein the monoclonal antibody is
conjugated to a detectable label.
94. The kit of claim 92 further comprising a second antibody that
binds to the monoclonal antibody.
95. The kit of claim 94 wherein the second antibody is conjugated
to a detectable label.
96. The kit of claim 92 formulated for diagnostic or laboratory
research.
97. A method for detecting, diagnosing, or monitoring an infection,
disease or disorder in a subject, the method comprising: contacting
a biological sample from the subject with an antibody that binds to
the glycolipopeptide of claim 52; and detecting binding of the
antibody to a component in the biological sample; wherein binding
of the antibody to a sample component is indicative of the presence
of the infection, disease or disorder in the subject.
98. The method of claim 97 further comprising: quantitating the
level of antibody binding to the sample component; quantitating the
level of antibody binding to components in a comparable
non-diseased sample; and comparing the binding levels; wherein a
change in antibody binding in the biological sample compared to the
non-diseased sample is indicative of the presence of the infection,
disease or disorder in the subject.
99. A method for detecting a glycosylated protein comprising:
contacting a biological sample with an antibody that binds to a
glycolipopeptide of claim 52; and detecting binding of the antibody
to the protein.
100. The method of claim 99 further comprising identifying the
protein.
101. A method for identifying a protein associated with a disease
state comprising: contacting a first biological sample associated
with a disease state with an antibody that binds to a
glycolipopeptide of claim 52; contacting a second biological sample
associated with different disease state or no disease with said
antibody; detecting binding of said antibody to glycosylated
proteins in the first and second samples; and identifying
glycosylated proteins that are enriched in one sample compared to
the other; wherein a difference in the amount of a glycosylated
protein in the two samples is indicative of a protein associated
with a disease state.
102. The method of claim 101 further comprising identifying the
protein associated with a disease state.
103. A glycolipopeptide comprising: at least one carbohydrate
component comprising a B-epitope; at least one peptide component
comprising a T-epitope; and at least one lipid component; wherein
the carbohydrate component comprises a saccharide selected from the
group consisting of N-acetylglucosamine (GlcNAc),
N-acetylgalactoseamine (GalNAc), mannose, or a glycosaminoglycan or
fragment thereof.
104. The glycolipopeptide of claim 103 wherein the saccharide is
O-linked, S-linked or N-linked to the glycolipopeptide.
105. The glycolipopeptide of claim 103 wherein the saccharide
comprises .beta.-N-acetylglucosamine (.beta.-O-GlcNAc).
106. The glycolipopeptide of claim 103 wherein the
glycosaminoglycan is selected from the group consisting of heparin,
heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan
sulfate and hyaluronan.
107. The glycolipopeptide of claim 103 wherein the carbohydrate
component comprises a glycopeptide comprising the saccharide.
108. The glycolipopeptide of claim 103 wherein the carbohydrate
component comprises a self-antigen.
109. The glycolipopeptide of claim 103 wherein the lipid component
comprises a Toll-like receptor (TLR) ligand.
110. A polyclonal or monoclonal antibody that binds to the
glycolipopeptide of claim 103.
111. The polyclonal or monoclonal antibody of claim 110 which is an
IgG antibody.
112. The polyclonal or monoclonal antibody of claim 111 which binds
a broad spectrum of glycoproteins.
113. A hybridoma that produces a monoclonal antibody that binds to
the glycolipopeptide of claim 103.
114. The polyclonal or monoclonal antibody of claim 110 that binds
to glycolipopeptide 52.
115. A pharmaceutical composition comprising: the glycolipopeptide
of claim 103, or a polyclonal or monoclonal antibody that binds
thereto; and a pharmaceutically acceptable carrier.
116. The pharmaceutical composition of claim 115 comprising a
monoclonal antibody.
117. A kit comprising: a monoclonal antibody that binds to the
glycolipopeptide of claim 103; packaging; and instructions for
use.
118. The kit of claim 117 wherein the antibody is conjugated to a
detectable label.
119. The kit of claim 117 further comprising a second antibody that
binds to the first antibody.
120. The kit of claim 119 wherein the second antibody is conjugated
to a detectable label.
121. The kit of claim 117 formulated for diagnostic or laboratory
research use.
122. A method for detecting, diagnosing or monitoring an infection,
disease or disorder in a subject, the method comprising: contacting
a biological sample from the subject with an antibody that binds to
the glycolipopeptide of claim 103; and detecting binding of the
antibody to a component in the biological sample; wherein binding
of the antibody to a sample component is indicative of the presence
of the infection, disease or disorder in the subject.
123. The method of claim 122 further comprising: quantitating the
level of antibody binding to the sample component; quantitating the
level of antibody binding to components in a comparable
non-diseased sample; and comparing the binding levels; wherein a
change in antibody binding in the biological sample compared to the
non-diseased sample is indicative of the presence of the infection,
disease or disorder in the subject.
124. A method for detecting a glycosylated protein comprising:
contacting a biological sample with an antibody that binds to a
glycolipopeptide of claim 103; and detecting binding of the
antibody to the protein.
125. The method of claim 124 further comprising identifying the
protein.
126. A method for identifying a protein associated with a disease
state comprising: contacting a first biological sample associated
with a disease state with an antibody that binds to a
glycolipopeptide of claim 103; contacting a second biological
sample associated with different disease state or no disease with
said antibody; detecting binding of said antibody to glycosylated
proteins in the first and second samples; and identifying
glycosylated proteins that are enriched in one sample compared to
the other; wherein a difference in the amount of a glycosylated
protein in the two samples is indicative of a protein associated
with a disease state.
127. A polyclonal or monoclonal pan-specific O-GlcNAc antibody.
128. The antibody of claim 127 which in an IgG antibody.
129. The antibody of claim 127 which binds to the glycolipopeptide
of claim 103.
130. The method of claim 126 further comprising identifying the
protein associated with a disease state.
131. The method of claim 126 wherein the antibody is a pan-specific
O-GlcNAc antibody.
132. The kit of claim 117 wherein the antibody is a pan-specific
O-GlcNAc antibody.
133. A method for analyzing a biological sample comprising
different glycoproteins, the method comprising: contacting the
biological sample with a pan-specific O-GlcNAc antibody that binds
to the glycolipopeptide claim 103; and detecting binding of the
antibody to different glycoproteins in the biological sample.
134. The method of claim 133 further comprising determining the
O-GlcNAc level in the biological sample.
135. The method of claim 134 wherein O-GlcNAc levels are determined
before and after a treatment intervention, over time to monitor
progression of disease, or to compare normal samples with samples
from patients suspected of suffering from a disease, infection or
disorder characterized by changes in protein glycosylation.
136. The method of claim 133 further comprising isolating,
identifying and/or characterizing a glycoprotein bound by the
pan-specific O-GlcNAc antibody.
137. The method of claim 136 wherein the glycoprotein has altered
glycosylation in a disease state.
138. The method of claim 135 wherein the disease is Alzheimer's
disease or Type II diabetes.
139. The method of claim 133 wherein the different glycoproteins
recognized by the pan-specific antibodies share a substantially
similar or identical glycopeptide sequence and/or a substantially
similar secondary or tertiary structure at the glycosylation
site.
140. The method of claim 133 wherein the pan-specific O-GlcNAc
antibody has a broad selectivity for O-GlcNAc modified proteins
and/or recognizes a broad spectrum of glycoproteins.
141. The method of claim 133 further comprising determining the
site localization for an O-GlcNAc modification.
Description
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 61/197,386, filed Oct. 27, 2008, and is
continuation-in-part of U.S. application Ser. No. 12/217,376, filed
on Jul. 3, 2008, which claims the benefit of U.S. Provisional
Application Ser. No. 61/127,710, filed May 15, 2008, and is also a
continuation-in-part of International Application
PCT/US2007/000158, with an international filing date of Jan. 3,
2007, which in turn claims the benefit of U.S. Provisional
Application Ser. Nos. 60/755,881, filed Jan. 3, 2006; 60/796,769,
filed May 2, 2006; and 60/809,272, filed May 30, 2006; each of
which is incorporated herein by reference in its entirety.
BACKGROUND
[0003] A large number of tumor-associated carbohydrate antigens
(TACA) are expressed on human cancer cells in the form of
glycolipids and glycoproteins. A common feature of oncogenic
transformed cells is the over-expression of oligosaccharides, such
as Globo-H, Lewis.sup.Y, and Tn antigens. Numerous studies have
shown that this abnormal glycosylation can promote metastasis and
hence it is strongly correlated with poor survival rates of cancer
patients.
[0004] The differential expression that is characteristic of these
tumor-associated carbohydrate antigens renders them attractive
targets for immunotherapy and the development of cancer vaccines.
Recently, several elegant studies have attempted to capitalize on
the differential expression of tumor-associated carbohydrates for
the development of cancer vaccines (e.g., Raghupathi, Cancer
Immunol. 1996, 43, 152-157; Musselli et al., J. Cancer Res. Clin.
Oncol. 2001, 127, R20-R26; Sabbatini et al., Int. J. Cancer, 2000,
87, 79-85; Lo-Man et al., Cancer Res. 2004, 64, 4987-4994; Kagan et
al., Immunol. Immunother. 2005, 54, 424-430).
[0005] Carbohydrate antigens are also abundant on the surface the
human immunodeficiency virus (HIV), the causative agent of acquired
immune deficiency syndrome (AIDS). Hepatitis C virus (HCV) is also
known to contain carbohydrate antigens.
[0006] For most immunogens, including carbohydrates, antibody
production depends on the cooperative interaction of two types of
lymphocytes, B-cells and helper T-cells. Carbohydrates alone,
however, cannot activate helper T-cells and therefore are
characterized by poor immunogenicity. The formation of low affinity
IgM antibodies and the absence of IgG antibodies manifest this
limited immunogenicity. It has proven difficult to overcome the
immunotolerance that characterizes these antigens.
[0007] In an effort to activate helper T cells, researchers have
conjugated carbohydrate antigens to a foreign carrier protein, e.g.
keyhole limpet hemocyanin (KLH) or detoxified tetanus toxoid (TT).
The carrier protein enhances the presentation of the carbohydrate
to the immune system and supplies T-epitopes (typically peptide
fragments of 12-15 amino acids) that can activate T-helper
cells.
[0008] However, conjugation of carbohydrates to a carrier protein
poses several new problems. The conjugation chemistry is difficult
to control, resulting in conjugates, with ambiguities in
composition and structure that may affect the reproducibility of an
immune response. In addition, the foreign carrier protein may
elicit a strong B-cell response, which in turn may lead to the
suppression of an antibody response against the carbohydrate
epitope. The latter is particularly a problem when self-antigens
are employed such as tumor-associated carbohydrates. Also, linkers
employed for conjugating carbohydrates to proteins can themselves
be immunogenic, leading to epitope suppression. See also McGeary et
al., for a review of lipid and carbohydrate based adjuvant/carriers
in vaccines (J. Peptide Sci. 9 (7): 405-418, 2003).
[0009] Not surprisingly, several clinical trials with
carbohydrate-protein conjugate cancer vaccines failed to induce
sufficiently strong helper T-cell responses in all patients.
Therefore, alternative strategies need to be developed for the
presentation of tumor associated carbohydrate epitopes that will
result in a more efficient class switch to IgG antibodies. These
strategies may prove useful as well for the development of vaccines
based on other carbohydrate epitopes, particularly those from
pathogenic viruses such as HIV and HCV.
SUMMARY OF THE INVENTION
[0010] The present invention provides a glycolipopeptide, also
referred to herein as a lipidated glycopeptide, for use in
immunotherapy, as well as pharmaceutical compositions containing
such glycolipopeptide and methods of making and using such
glycolipopeptide. In a preferred embodiment, the glycolipopeptide
of the invention is fully synthetic.
[0011] The glycolipopeptide preferably contains at least 2
epitopes: a B-epitope and a T-epitope, as well as a lipid
component. The glycolipopeptide is thus able to elicit both a
humoral response to the B-epitope and a cellular immune response to
T-epitope. In a preferred embodiment, the glycolipopeptide of the
invention advantageously combines features from a B-epitope glycan
or glycopeptide and a T-epitope peptide derived from glycoproteins
of mammalian (preferably human or murine) or microbial origin.
[0012] Lipidation confers several additional advantages to the
glycolipopeptide. It helps the glycolipopeptides self assemble into
vesicles, and may also facilitate the incorporation of the
immunogen into a liposome which in turn can improve the
presentation of the immunogen to the immune system. Additionally,
the lipid component serves as a built-in adjuvant. Cellular uptake
of the glycopeptide is also facilitated by the lipidation. Cytokine
production is also enhanced by inclusion of the lipid
component.
[0013] Accordingly, in one aspect, the invention provides a
glycolipopeptide containing at least one carbohydrate component
that includes all or part of a B-epitope; at least one peptide
component that includes all or part of a T-epitope; and at least
one lipid component. The carbohydrate component and the peptide
component may be heterologous with respect to each other or they
may be homologous with respect to each other. The glycolipopeptide
of the invention may include a glycopeptide that includes all or
part of both the B-epitope and the T-epitope.
[0014] The carbohydrate component of the glycolipopeptide can
include a glycoconjugate, for example, a glycosylated protein, a
glycosylated peptide (also referred to herein as a glycopeptide) a
glycosylated lipid, a glycosylated amino acid, a DNA or an RNA. The
B-epitope of the carbohydrate component may be from a
microorganism, such as a virus, a bacterium, a fungus, and a
protozoan. Exemplary viruses as sources for the B-epitope include
human immunodeficiency virus and hepatitis C virus, without
limitation. The B-epitope can therefore constitute all or part of a
viral antigen, such as a viral antigen from human immunodeficiency
virus or hepatitis C virus. Alternatively or additionally, the
B-epitope can constitute all or part of a self-antigen. For
example, the B-epitope can be one that is overexpressed on a cancer
cell. An exemplary self-antigen is MUC-1 glycopeptide. Another
example of a glycopeptide that can constitute the carbohydrate
component of the glycolipopeptide of the invention is a
.beta.-N-acetylglucosamine (.beta.-O-GlcNAc) modified peptide. In
another embodiment, the carbohydrate component of the
glycolipopeptide includes a heparin fragment or a heparan sulfate
fragment.
[0015] The peptide component of the glycolipopeptide, which
includes a T-epitope, preferably includes a helper T-epitope.
[0016] The lipid component of the glycolipopeptide is preferably an
antigenic, immunogenic, or otherwise immunostimulatory lipid. For
example, the lipid component can include a Toll-like receptor (TLR)
ligand, such as a PamCys-type lipid. Examples of a PamCys-type
lipid include Pam.sub.2Cys, Pam.sub.3Cys, Pam.sub.2CysSK.sub.n and
Pam.sub.3CysSK.sub.n, wherein n=0, 1, 2, 3, 4 or 5. A particularly
preferred lipid component includes Pam.sub.3CysSK.sub.4. In another
preferred embodiment, the lipid component binds to a Toll-like
receptor and facilitates internalization of the glycolipopeptide by
a target cell. Exemplary lipid components can be found, for
example, in Scheme 8 hereinbelow. The lipid may serve as an
internal (covalently linked) adjuvant. Preferably, the lipid
component includes a TLR agonist, i.e., a TLR ligand that has a
stimulatory effect on a Toll-like receptor.
[0017] Optionally, the glycolipopeptide of the invention includes
at least one linker component. The linker component may link one or
more of the carbohydrate component, peptide component and/or lipid
component to each other or to a different component or
structure.
[0018] A particularly preferred embodiment of the glycolipopeptide
is one that contains at least one carbohydrate component that
includes a self-antigen having a B-epitope, for example a MUC-1
glycopeptide; at least one peptide component comprising a
T-epitope, preferably a helper T-epitope; and at least one lipid
component, for example a Toll-like receptor ligand (TLR ligand). In
another particularly preferred embodiment, the glycolipopeptide of
the invention includes at least one carbohydrate component that has
a B-epitope; at least one peptide component that has a helper
T-epitope; and at least one lipid component that binds to a
Toll-like receptor and facilitates uptake of the glycolipopeptide
by a target cell that includes the Toll-like receptor; wherein the
carbohydrate component and the peptide component are heterologous
with respect to each other. In another particularly preferred
embodiment, the glycolipopeptide includes at least one carbohydrate
component that includes a self-antigen that has a B-epitope; at
least one peptide component that has a helper T-epitope; and at
least one lipid component that binds to a Toll-like receptor, i.e.,
a TLR ligand. Advantageously, the TLR ligand may facilitate uptake
of the glycolipopeptide by a target cell that includes the
Toll-like receptor.
[0019] In another aspect, the invention provides a pharmaceutical
composition. In one embodiment, the pharmaceutical composition
includes a glycolipopeptide of the invention, without limitation.
Optionally, the pharmaceutical composition contains plurality of
glycopeptides, which may include glycolipopeptides having different
or the same B-epitopes, having different or the same T-epitopes
and/or having different or the same lipid components. In another
embodiment, the pharmaceutical composition includes an antibody
against a glycolipopeptide of the invention, without limitation.
The antibody can be a monoclonal or polyclonal antibody, and may be
a humanized antibody. Techniques for humanizing antibodies are well
known in the art.
[0020] Optionally, the pharmaceutical composition contains a
liposome. Formulations with liposomes, micelles, or other lipid
vesicles may facilitate delivery of the glycopeptide to a subject
in need thereof. The glycolipopeptide may be covalently or
noncovalently incorporated into the liposome, micelle or other
lipid vesicle.
[0021] The pharmaceutical composition preferably includes a
pharmaceutically acceptable carrier. In one embodiment, the
pharmaceutical composition does not contain an external adjuvant.
In another embodiment, the pharmaceutical composition contains an
external adjuvant. An example of an external adjuvant is QS-21.
[0022] Advantageously, the pharmaceutical composition of the
invention can be used as a vaccine, for example to treat or prevent
an infection, disease or disorder. Additionally, the
glycolipopeptide of the invention can be used for the manufacture
of a medicament to treat or prevent an infection, disease or
disorder.
[0023] Accordingly, in another aspect, the invention provides
method for treating or preventing an infection, disease or disorder
in a subject that involves administering a pharmaceutical
composition of the invention to a subject in need thereof.
Inclusion of QS-21 as an external adjuvant may skew the immune
response of the subject toward a Th1 response, compared to a
comparable pharmaceutical composition that does not include QS-21.
The infection, disease or disorder that is treated or prevented may
be one that is caused by a microorganism, such as a virus, a
bacterium, a fungus, and a protozoan. Viral infections that can be
treated or prevented include, without limitation, those caused a
human immunodeficiency virus or a hepatitis C virus. Alternatively,
the infection, disease or disorder that is treated or prevented can
include cancer, a precancerous condition, or an autoimmune disease,
such as diabetes type II.
[0024] In another aspect, the invention includes a method for
making the glycolipopeptide of the invention. The carbohydrate
component, the peptide component and the lipid component are
synthetically linked, for example by using chemical or in vitro
enzymatic methods.
[0025] In yet another aspect, the invention provides a method for
identifying a Toll-like receptor (TLR) ligand. A Toll-like receptor
ligand is useful for inclusion in a glycolipopeptide vaccine of the
invention. The method includes contacting a candidate compound with
a target cell containing a TLR, and determining whether the
candidate compound binds to the TLR. Optionally, the method also
includes determining whether the candidate compound is internalized
by the target cell. In a preferred embodiment, the candidate
compound includes a lipid, and the TLR ligands thus identified are
useful as the lipid component for the glycolipopeptide of the
invention. Accordingly, another embodiment of the glycolipopeptide
of the invention includes at least one carbohydrate component
having a B-epitope; at least one peptide component having a helper
T-epitope; and at least one lipid component identified using the
method of identifying a TLR ligand as described herein.
[0026] In another embodiment, the glycolipopeptide of the invention
includes at least one carbohydrate component comprising a
B-epitope; at least one peptide component comprising a T-epitope;
and at least one lipid component; wherein the carbohydrate
component comprises a saccharide selected from the group consisting
of N-acetylglucosamine (GlcNAc) or N-acetylgalactoseamine (GalNAc)
or mannose. In one embodiment, the saccharide is O-linked, S-linked
or N-linked to the glycolipopeptide. Preferably, the saccharide
comprises .beta.-N-acetylglucosamine (.beta.-O-GlcNAc). In another
embodiment, carbohydrate component of the glycolipopeptide includes
a saccharide that includes a glycosaminoglycan or fragment thereof.
Examples of glycosaminoglycans include heparin, heparan sulfate,
chondroitin sulfate, dermatan sulfate, keratan sulfate and
hyaluronan. The carbohydrate component may include a glycopeptide
comprising the saccharide. The carbohydrate component may include a
self-antigen. The lipid component of the glycolipopeptide may
include a Toll-like receptor (TLR) ligand.
[0027] In another aspect, the invention provides a polyclonal or
monoclonal antibody against the glycolipopeptide of the invention,
as well as hybridoma cells and cell lines that produce said
antibody. Humanized antibodies are encompassed by the invention.
Exemplary hybridoma cell lines include hybridoma 1F5.D6, hybridoma
9D1.E4, hybridoma 5H11.H6, and hybridoma 18B10.C7. The monoclonal
antibodies produced by these hybridoma cell lines are also included
in the invention.
[0028] Also included in the invention is a polyclonal or monoclonal
antibody that competes with a monoclonal antibody described herein
for binding to glycolipopeptide 52, as well as a polyclonal or
monoclonal antibody that binds to glycolipopeptide 52.
[0029] Also included in the invention is a method for making an
antibody of the invention. In one embodiment, a glycolipopeptide of
the invention is injected into a mammal, and at least one antibody
that binds to the glycolipopeptide is isolated from the mammal.
Alternatively, cells, for example spleen or lymph node cells, can
be isolated from the mammal and fused with myeloma cells to form
hybridomas. At least one hybridoma that produces an antibody that
binds to the glycolipopeptide is selected, and the antibody is
isolated. Antibodies made using any of the methods of the invention
are also included in the invention, as is use of a glycolipopeptide
of the invention to produce a polyclonal or monoclonal antibody
that binds the glycolipopeptide.
[0030] Also included in the invention is an antibody produced by
immunizing a mammal or a mammalian cell with an immunogenic
glycopeptide of the invention. Antibodies to the glycopeptide of
the invention can also be produced using commonly available
techniques such as phage display. Preferably, the antibody produced
is an IgG antibody that binds a broad spectrum of glycoproteins.
The carbohydrate component of the glycolipopeptide used to immunize
the mammal may include, without limitation, a saccharide selected
from the group consisting of N-acetylglucosamine (GlcNAc) or
N-acetylgalactoseamine (GalNAc) or mannose, or a saccharide
comprising a glycosaminoglycan or fragment thereof. The lipid
component of the glycolipopeptide used to immunize the mammal may
include a Toll-like receptor (TLR) ligand.
[0031] Preferably, the monoclonal or polyclonal antibody of the
invention is an IgG antibody; more preferably, it is an antibody
that binds a broad spectrum of glycoproteins.
[0032] The invention further provides for use of a glycolipopeptide
or antibody of the invention, without limitation, for the
manufacture of a medicament to treat or prevent an infection,
disease or disorder.
[0033] In another aspect, the invention provides a kit, for example
a diagnostic kit or a kit for laboratory research use, that
includes an antibody of the invention, for example a monoclonal
antibody that binds to a glycolipopeptide of the invention, without
limitation, along with packaging and instructions for use. The kit
optionally also includes a second antibody that binds to the
primary antibody. Either or both of the primary or secondary
antibodies is optionally conjugated to a detectable label.
[0034] The invention further provides a method for detecting,
diagnosing or monitoring an infection, disease or disorder in a
subject. A biological sample, such as a body fluid or tissue from
the subject, is contacted with an antibody of the invention; and
binding of the antibody to a component in the biological sample is
detected. The antibody selected for use in the method can be one
that is known to bind to a biomolecule that is associated with
infection, disease or disorder. Binding of the antibody to a sample
component is indicative of the presence of the infection, disease
or disorder in the subject. Optionally the method further includes
quantitating the level of antibody binding to the sample component;
quantitating the level of antibody binding to components in a
comparable non-diseased sample; and comparing the binding levels;
wherein a change in antibody binding in the biological sample
compared to the non-diseased sample is indicative of the presence
of the infection, disease or disorder in the subject.
[0035] The invention further includes a method for detecting a
glycosylated protein. A biological sample is contacted with an
antibody of the invention, for example a monoclonal antibody that
binds to a glycolipopeptide of the invention; and binding of the
antibody to the protein is detected. Optionally the method includes
identifying the protein.
[0036] The invention further includes method for identifying a
protein associated with a disease state. A first biological sample
associated with a disease state is contacted with an antibody of
the invention; and a second biological sample associated with
different disease state or no disease is also contacted with the
antibody. Binding of said antibody to glycosylated proteins in the
first and second samples is detected, and glycosylated proteins
that are enriched in one sample compared to the other are detected.
A difference in the amount of a glycosylated protein in the two
samples is indicative of a protein associated with a disease state.
Optionally the method includes identifying the protein associated
with a disease state.
[0037] Unless otherwise specified, "a," "an," "the," and "at least
one" are used interchangeably and mean one or more than one.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 shows an exemplary glycolipopeptide of the
invention.
[0039] FIG. 2 shows flow cytometry analysis for specific anti-MUC-1
antibodies. Reactivity was tested on MCF-7 (A) and SK-MEL-28 (B)
cells. Fluorescence intensity of serum (1:50 diluted) was assessed
before (serum control; open peak) and after immunization with 3
(filled peak).
[0040] FIG. 3 shows TNF-.alpha. production by murine macrophages
after stimulation with LPS and synthetic compounds. Murine RAW
.gamma.NO(-) cells were incubated for 5.5 hours with increasing
concentrations of E. coli LPS (.box-solid.), 1 ( ),
Pam.sub.2CysSK.sub.4 (), 2 (.diamond-solid.), Pam.sub.3CysSK.sub.4
(.tangle-solidup.), or 3 (.quadrature.) as indicated.
[0041] FIG. 4 shows the effect of TLR ligand on cellular
uptake.
[0042] FIG. 5 shows the chemical structures of synthetic
antigens.
[0043] FIG. 6 shows TNF-.alpha. and IFN-.beta. production by murine
macrophages after stimulation with synthetic compounds 21-24, E.
coli LPS, and E. coli lipid A. Murine 264.7 RAW .gamma.NO(-) cells
were incubated for 5.5 h with increasing concentrations of 21-24,
E. coli LPS, or E. coli lipid A as indicated. TNF-.alpha. (A) and
IFN-.beta. (B) in cell supernatants were measured using ELISAs.
Data represent mean values.+-.SD (n=3).
[0044] FIG. 7 shows cell recognition analysis for specific
anti-MUC1 antibodies. Reactivity of sera was tested on MCF7 cells.
Serial dilutions of serum samples after 4 immunizations with 21
(A), 22/23 (B), or 22/24 (C) were incubated with MCF7 cells. After
incubation with FITC-labeled anti-mouse IgG antibody, the
fluorescence intensity was assessed in cell lysates. No
fluorescence over background was observed with pre-immunization
sera and incubation of the serum samples with control SK-MEL-28
cells (shown in FIG. 9). AU indicates arbitrary fluorescence
units.
[0045] FIG. 8 shows ELISA anti-MUC1 and anti-T-epitope antibody
titers after 4 immunizations with 21, 22, 22/23, 22/24 and 25/26.
ELISA plates were coated with BSA-MI-MUC-1 conjugate (A-F) or
neutravidin-biotin-T-epitope (G) and titers were determined by
linear regression analysis, plotting dilution vs. absorbance.
Titers were defined as the highest dilution yielding an optical
density of 0.1 or greater over that of normal control mouse sera.
Each data point represents the titer for an individual mouse after
4 immunizations and the horizontal lines indicate the mean for the
group of five mice.
[0046] FIG. 9 shows cell recognition analysis for specific
anti-MUC-1 antibodies. Reactivity of sera was tested on MCF7 and
SK-MEL-28 cells. Serum samples (1:30 diluted) after 4 immunizations
with 21, 22/23, or 22/24 were incubated with MCF7 and SK-MEL-28
cells. After incubation with FITC-labeled anti-mouse IgG antibody,
the fluorescence intensity was assessed in cell lysates. Also shown
are media, conjugate, and mouse (normal control mouse sera)
controls. Data represent mean values.+-.SD. AU indicates arbitrary
fluorescence units.
[0047] FIG. 10 shows compound 22.
[0048] FIG. 11 shows compound 23.
[0049] FIG. 12 shows compound 25.
[0050] FIG. 13 shows compound 26.
[0051] FIG. 14 shows compound 27.
[0052] FIG. 15 shows the structure of fully synthetic
three-component immunogens.
[0053] FIG. 16 shows competitive inhibition of monoclonal antibody
binding to GSTPVS(.beta.-O-GlcNAc)SANM (68) by the corresponding
glycopeptide, peptide and sugar. ELISA plates were coated with
BSA-MI-CGSTPVS(.beta.-O-GlcNAc)SANM (BSA-MI-66) conjugate. MAbs,
diluted to obtain in the absence of an inhibitor an OD of
approximately 1 in the ELISA, were first mixed with (a)
glycopeptide 68 (GSTPVS(.beta.-O-GlcNAc)SANM), (b) peptide 69
(GSTPVSSANM; SEQ ID NO:11) or (c) sugar 70 (.beta.-O-GlcNAc-Ser)
(0-500 .mu.M final concentration) and then applied to the coated
microtiter plate. OD values were normalized for the OD values
obtained with monoclonal antibody alone (0 .mu.M inhibitor,
100%).
[0054] FIG. 17 shows Western blots of cell lysates and
immunoprecipitated samples. HEK293TN cells were transiently
transfected with an OGT plasmid or mock transfected. (a) Cell
lysates of mock transfected cells (lanes 1, 6, 10 and 15) and OGT
overexpressing cells (lanes 2, 7, 11 and 16) and immunoprecipitated
samples using rabbit polyclonal CK2II alpha antibody of mock
transfected cell lysates (lanes 3, 8, 12 and 17) and OGT
overexpressing cells lysates (lanes 4, 9, 13 and 18) were resolved
by SDS-PAGE (10%), transferred to PVDF membranes and probed with
cell culture supernatants (1:10 diluted) of monoclonal antibody
clones 9D1.E4(10) (lanes 1-4), 18B10.C7(3) (lanes 6-9), 1F5.D6(14)
(lanes 10-13 and 5H11.H6(4) (lanes 15-18). As secondary antibody an
anti-mouse IgG antibody linked to peroxidase was used. (b) The
blots of (a) were stripped and reprobed with rabbit polyclonal
anti-CKII antibody and an anti-rabbit IgG antibody linked to
peroxidase as secondary antibody was used. Blots were visualized
with ECL substrate by exposing on film.
[0055] FIG. 18 shows a large-scale immunoprecipitation of O-GlcNAc
modified proteins by Mab3, 10 and 14 as well as CTD110.6 from
HEK29T cells treated with PUGNAc. Following Lys-C digestion,
samples were subject to ESI (CID-pseudo neutral loss) analysis.
Results were filtered at 1% false recovery rate and proteins
appeared in mock IP were subtracted from the final list.
[0056] FIG. 19 shows the structures of fully synthetic
three-component immunogens 52 and 53 and the reagents 63-65 for
their preparation. Compounds 66-70 were employed for ELISA and
inhibition ELISA.
[0057] FIG. 20 shows ELISA anti-GSTPVS(.beta.-O-GlcNAc)SANM (68)
antibody titers after 4 immunizations with 52 and 53. ELISA plates
were coated with BSA-MI-GSTPVS(.beta.-O-GlcNAc) SANM (BSA-MI-66)
conjugate and (a) IgG total, (b) IgG1, (c) IgG2a, (d) IgG2b, (e)
IgG3 and (f) IgM titers were determined by linear regression
analysis, plotting dilution vs. absorbance. Titers were defined as
the highest dilution yielding an optical density of 0.1 or greater
over that of normal control mouse sera. Each data point represents
the titer for an individual mouse after 4 immunizations and the
horizontal lines indicate the mean for the group of five mice.
[0058] FIG. 21 shows immunoblotting of three monoclonal antibodies.
(a) CKII .alpha. subunit was immunoprecipitated from HEK293T cells
with or without OGT overexpression. Eluates were resolved by
SDS-PAGE and immunoblotted with MAbs 18B10.C7(3), 9D1.E4(10) and
1F5.D6(14). A band corresponding to CKII .alpha. subunit was
detected with signal intensity correlated with O-GlcNAc status. All
blots were stripped and reprobed with antibody against CKII .alpha.
subunit (only one representative blot is shown here). Also, equal
amount of CKII .alpha. subunit was present in the input regardless
of the status of O-GlcNAc levels. (b) HEK293T lysates with low (OGA
overexpression), median (Mock transfection) and high (OGT
overexpression) levels of O-GlcNAc modification were exposed to
MAbs 18B10.C7(3), 9D1.E4(10) and 1F5.D6(14) respectively. The
signals obtained mirror the corresponding O-GlcNAc status in each
sample. Immunoblots against OGT, OGA and tubulin are also shown.
While equal loading of tubulin was detected in all samples, higher
OGA and OGT protein levels were detected with lysates from OGA and
OGT transfection. Note: Endogenous OGT and OGA levels do appear
after longer exposure. (c) O-GlcNAc proteins were
immunoprecipitated from HEK293T cells treated with PUGNAc (an OGA
inhibitor), resolved by SDS-PAGE and subjected to CTD110.6 (an IgM
isotype O-GlcNAc specific antibody) blotting. Cross-reactivity of
MAbs 18B10.C7(3), 9D1.E4(10) and 1F5.D6(14) with CTD110.6, albeit
distinct in pattern, were detected.
[0059] FIG. 22 shows application of MAbs for O-GlcNAc-omics. (a)
Number of O-GlcNAc modified proteins pulled down with different
MAbs. 254 proteins were assigned to be O-GlcNAc modified in the
combination of all MAbs, where 191 appeared to be novel. (b)
Distribution of O-GlcNAc modified proteins based on their
biological process categorized in HPRD.
[0060] FIG. 23 shows compound 52.
[0061] FIG. 24 shows compound 53.
[0062] FIG. 25 shows compound 63.
[0063] FIG. 26 shows compound 64.
[0064] FIG. 27 shows compound 65.
[0065] FIG. 28 shows compound 66.
[0066] FIG. 29 shows compound 67; SEQ ID NO: 12.
[0067] FIG. 30 shows compound 68.
[0068] FIG. 31 shows compound 69; SEQ ID NO: 11.
[0069] FIG. 32 shows compound 70.
[0070] FIG. 33 shows Western blots in rat liver samples 24 hours
after trauma-hemorrhage and resuscitation. Protein samples were
prepared using T-PER lysis, Laemmli buffer, and 5%
.beta.-mercaptoethanol. 25 .mu.g protein was loaded in each lane
and antibody binding was visualized using enhanced
chemiluminescence. Livers subjected to trauma-hemorrhage and
resuscitation demonstrated significantly lower overall hepatic
O-GlcNAc levels 24 hrs compared to sham controls.
[0071] FIG. 34 shows show the distribution of the identified
O-GlcNAc proteins according to function. In the control (sham)
group, 96 O-GlcNAc modified proteins were identified. In the in
trauma-hemorrhage and resuscitated group, 30 different O-GlcNAc
modified proteins were identified.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0072] The glycolipopeptide of the invention includes at least one
B-epitope, at least one T-epitope, and a lipid component. In a
preferred embodiment, the glycolipopeptide consists essentially of
three main components: at least one carbohydrate component that
contains a B-epitope; at least one peptide component that contains
a helper T-epitope; and at least one lipid component. Exemplary
carbohydrate, peptide and lipid components are described herein and
also, for example, in references cited herein, including Koganty et
al., US Patent Publication 20060069238, published Mar. 30, 2006;
see also Koganty et al., Drug Disc. Today, 1 (5): 190-198, 1996.
The three components are covalently linked, either directly or
indirectly, to form a single glycolipopeptide molecule. Indirect
linkage involves the use of an optional linker component "L" to
link two or more of the main components together. The three main
components can be linked together (directly or indirectly) in any
order. For example, the lipid and carbohydrate component can each
be covalently linked to the peptide component to form the
glycolipopeptide. Alternatively, the lipid component and the
peptide component can each be covalently linked to the carbohydrate
component. Likewise, the carbohydrate component and the peptide
component can each be covalently linked to the lipid component. Or,
all three components can be linked such that each of the three
components is covalently linked to each of the other two
components. Intermolecular crosslinking is also possible, as
described in more detail below.
[0073] In a preferred embodiment, the glycolipopeptide of the
invention contains one carbohydrate component, one peptide
component, and one lipid component. In another embodiment, the
glycolipopeptide contains a plurality of carbohydrate components,
which may be the same, or may be different. Likewise, in another
embodiment, the glycolipopeptide contains a plurality of peptide
components, which may be the same, or may be different. Further, in
another embodiment, the glycolipopeptide contains a plurality of
lipid components, which may be the same, or may be different. Thus,
various embodiments of the glycolipopeptide of the invention may
contain one or more carbohydrate components, one or more peptide
components, and/or one or more lipid components. For example, the
concept of "multiple antigenic glycopeptides" (Bay et al., U.S.
Pat. No. 6,676,946, Jan. 13, 2004, Bay et al.; WO 98/43677,
published Oct. 8, 1998, Bay et al.) can be adapted for use in the
present invention. High antigen density can be achieved using a
core, for example a poly-lysine core, to which extended peptidic
"arms" (the peptide component of the glycolipopeptide of the
invention) are attached, which peptidic arms display the
carbohydrate antigen components of the glycolipopeptide in
clustered presentation. The lipid component of the glycolipopeptide
can likewise extend from the lysine core, particularly in
embodiments wherein the peptide component is attached to the lysine
core via a nonterminal amino acid. High antigen density can also be
achieved by using a liposome as a delivery vehicle, as exemplified
in Examples II and III. Additionally or alternatively, the
glycolipopeptides can be optionally cross-linked to form a
multi-molecular complex, thereby increasing the antigen
density.
[0074] The various carbohydrate, peptide and lipid components of
the glycolipopeptide can be structurally derived from or based on,
and/or can mimic, those found in naturally occurring biological
molecules. The glycolipopeptide components preferably contain
molecular structures or parts of structures (including epitopes)
that are identical to or similar to those found in a living
organism. Typically, while the components of the glycolipopeptide
are derived from, are structurally based on, and/or mimic naturally
occurring structures, they are prepared synthetically, using
chemical or in vitro enzymatic methods, for example. In some
embodiments, epitopes that are formed in the naturally occurring
antigen from molecular elements that are close in space but distant
from each other in terms of chemical bonding can be formed in the
glycolipopeptide of the invention by a different chemical structure
(with different bonding order or pattern) that forms the same or a
similar epitope.
[0075] The three component glycolipopeptide of the invention can be
viewed as cassette, wherein the carbohydrate component, the peptide
component, and the lipid component are each independently selected
for inclusion in the glycolipopeptide. Any combination (i.e.,
mixing and matching) of carbohydrate component, peptide component
and lipid component as described herein to form a glycolipopeptide
is encompassed by the invention.
Carbohydrate Component
[0076] The carbohydrate component of the glycolipopeptide can be
any component that contains a carbohydrate. Examples of suitable
carbohydrate components include oligosaccharides, polysaccharides
and monosaccharides, and glycosylated biomolecules
(glycoconjugates) such as glycoproteins, glycopeptides,
glycolipids, glycosylated amino acids, DNA, or RNA. Glycosylated
peptides (glycopeptides) and glycosylated amino acids, which
contain one or more carbohydrate moieties as well as a peptide or
amino acid, are particularly preferred as the carbohydrate
component of the glycolipopeptide of the invention. An example of a
glycopeptide is CD52, which is expressed on virtually all human
lymphocytes and believed to play an important role in the human
immune system. An example of a glycosylated amino acid is the Tn
antigen. It should be understood that when the carbohydrate
component is a glycopeptide, the peptide part of the glycopeptide
optionally includes a T-epitope as well as a B-epitope and thus may
serve as a peptide component of the glycolipopeptide. A
glycopeptide that contains both a T-epitope and a B-epitope is
sometimes referred to as possessing a "B-T" epitope or a "T-B"
epitope. The B-epitope and the T-epitope present on the
glycolipopeptide of the invention may or may not overlap.
[0077] The carbohydrate component of the glycolipopeptide of the
invention includes a carbohydrate that contains one or more
saccharide monomers. For example, the carbohydrate can include a
monosaccharide, a disaccharide or a trisaccharide; it can include
an oligosaccharide or a polysaccharide. An oligosaccharide is a
oligomeric saccharide that contains two or more saccharides and is
characterized by a well-defined structure. A well-defined structure
is characterized by the particular identity, order, linkage
positions (including branch points), and linkage stereochemistry
(.alpha., .beta.) of the monomers, and as a result has a defined
molecular weight and composition. An oligosaccharide typically
contains about 2 to about 20 or more saccharide monomers. A
polysaccharide, on the other hand, is a polymeric saccharide that
does not have a well defined structure; the identity, order,
linkage positions (including brand points) and/or linkage
stereochemistry can vary from molecule to molecule. Polysaccharides
typically contain a larger number of monomeric components than
oligosaccharides and thus have higher molecular weights. The term
"glycan" as used herein is inclusive of both oligosaccharides and
polysaccharides, and includes both branched and unbranched
polymers. When the carbohydrate component contains a carbohydrate
that has three or more saccharide monomers, the carbohydrate can be
a linear chain or it can be a branched chain. In a preferred
embodiment, the carbohydrate component contains less than about 15
saccharide monomers; more preferably in contains less than about 10
saccharide monomers.
[0078] The carbohydrate component of the glycolipopeptide includes
a carbohydrate that contains a B-epitope. It should be understood
that the carbohydrate may be coextensive with the B-epitope, or the
carbohydrate may be inclusive of the B-epitope, or the carbohydrate
may include only part of the B-epitope (i.e., the B-epitope may
additionally encompass other parts of the glycolipopeptide such as
the peptide component, the lipid component, and/or the linker
component). An example of a glycopeptide that includes a B-epitope
is the glycosylated peptide MUC-1. Thus, a carbohydrate or
carbohydrate component that "comprises" a B-epitope is to be
understood to mean a carbohydrate or carbohydrate component that
encompasses all or part of a B-epitope that is present on the
glycolipopeptide.
[0079] The B-epitope can be a naturally occurring epitope or a
non-naturally occurring epitope. Preferably, two or more saccharide
monomers of the carbohydrate interact to form a conformational
epitope that serves as the B-epitope. A B-epitope is an epitope
recognized by a B cell. Any antigenic carbohydrate that contains a
B-epitope can be used as the carbohydrate component, without
limitation.
[0080] Non-naturally occurring carbohydrates that can be used as
components of the glycolipopeptide of the invention include
glycomimetics, which are molecules that mimic the shape and
features of a sugar such as a monosaccharide, disaccharide or
oligosaccharide (see, e.g., Barchi, Current Pharmaceutical Design,
6(4):485-501 (March 2000); Martinez-Grau et al., Chemical Society
Reviews, 27(2):155-162 (1998); Schweizer, Angewandte
Chemie-International Edition, 41(2):230-253 (2002)). Glycomimetics
can be engineered to supply the desired B-epitope and potentially
provide greater metabolic stability.
[0081] In another embodiment, the carbohydrate component contains
all or part of a self-antigen. Self-antigens are antigens that are
normally present in an animal's body. They can be regarded as
"self-molecules," e.g., the molecules present in or on the animal's
cells, or proteins like insulin that circulate in the animal's
blood. An example of a self-antigen is a carbohydrate-containing
component derived from a cancer cell of the animal, such as a
tumor-associated carbohydrate antigen (TACA). Typically, such
self-antigens exhibit low immunogenicity. Examples include
tumor-related carbohydrate B-epitope such as Le.sup.y antigen (a
cancer related tetrasaccharide; e.g.,
Fuc.alpha.(1,2)-Gal.beta.(1,4)-[Fuc.alpha.(1,3)]-GlcNAc); Globo-H
antigen (e.g.,
L-Fuc.alpha.(1,2)-Gal.beta.(1,3)-GalNAc.beta.(1,3)-Gal.alpha.(1,4)-
-Gal.beta.(1,4)-Glu); T antigen (e.g.,
Gal.beta.(1,3)-GalNAc.alpha.-O-Ser/Thr); STn antigen (sialyl Tn,
e.g., NeuAc.alpha.(2,6)-GalNAc.alpha.-O-Ser/Thr); and Tn antigen
(e.g., .alpha.-GalNAc-O-Ser/Thr). Another example of a self-antigen
is a glycopeptide derived from the tandem repeat of the breast
tumor-associated MUC-1 of human polymorphic epithelial mucin (PEM),
an epithelial mucin (Baldus et al., Crit. Rev. Clin. Lab. Sci.,
41(2):189-231 (2004)). A MUC-1 glycopeptide comprises at least one
Tn and/or sialyl Tn (sialyl .alpha.-6 GalNAc, or "STn") epitope;
preferably linked to a threonine (T-Tn or T-STn).
[0082] Structures of exemplary tumor-associated carbohydrate
antigens (TACA) that can be used as a component of the
glycolipopeptide include, without limitation, the structures shown
in Schemes 1 and 2.
##STR00001## ##STR00002##
It should be noted that the Tn, STn, and TF structures shown in
Scheme 1 (monomeric, trimeric, clustered) are all shown with a
threonine residue. The corresponding serine analogues are also
suitable structures. In the case of Tn3, STn3, TF3 and their
respective clusters, all possible homo- and hetero-analogues with
differences in the threonine/serine composition of the backbone are
included.
##STR00003## ##STR00004##
[0083] Another self-antigen for use in the carbohydrate component
of the glycolipopeptide is a glycopeptide that includes an amino
acid or peptide covalently linked to a monosaccharide. Preferably
the monosaccharide is N-acetylglucosamine (GlcNAc) or
N-acetylgalactoseamine (GalNAc). A preferred glycopeptide
self-antigen is a .beta.-N-acetylglucosamine (.beta.-O-GlcNAc)
modified peptide. Preferably the monosaccharide is O-linked to a
serine or a threonine of the polypeptide. Also suitable for use as
a self-antigen are the related thiol (S-linked) and amine
(N-linked) analogues, some examples of which are described in
Example VIII. The monosaccharide is preferably linked to the
peptide via a beta (.beta.) linkage but it may be an alpha
(.alpha.) linkage. In a particularly preferred embodiment, the
carbohydrate component of the glycolipopeptide of the invention
(which may be coextensive with the peptide component when
formulated as a glycopeptide) contains a TPVSS (SEQ ID NO:10) amino
acid sequence modified by O-GlcNAc. Examples of a carbohydrate that
contains a .beta.-GlcNAc modified glycopeptide as a B-epitope are
shown as compounds 52 (O-linked) and 53 (S-linked) in FIG. 15; see
Example VIII.
[0084] In another embodiment, the carbohydrate component contains
all or part of a carbohydrate antigen (typically a glycan) from a
microorganism, preferably a pathogenic microorganism, such as a
virus (e.g., a carbohydrate present on gp120, a glycoprotein
derived from the HIV virus), a Gram-negative or Gram-positive
bacterium (e.g., a carbohydrate derived from Haemophilus
influenzae, Streptococcus pneumoniae, or Neisseria meningitides), a
fungus (e.g., a 1,3-.beta.-linked glucan) a parasitic protozoan
(e.g., a GPI-anchor found in protozoan parasites such as Leishmania
and Trypanosoma brucei), or a helminth. Preferably, the
microorganism is a pathogenic microorganism.
[0085] An exemplary glycan from viral pathogens, Man9 from HIV-1
gp120, is shown in Scheme 3.
##STR00005##
[0086] Exemplary HIV carbohydrate and glycopeptide antigens are
described in Wang et al., Current Opinion in Drug Disc. &
Develop., 9(2): 194-206 (2006), and include both naturally
occurring HIV carbohydrates and glycopeptides, as well as synthetic
carbohydrates and glycopeptides based on naturally occurring HIV
carbohydrates and glycopeptides.
[0087] Exemplary HCV carbohydrate and glycopeptide antigens are
described in Koppel et al. Cellular Microbiology 2005; 7(2):157-165
and Goffard et al. J. of Virology 2005; 79(13):8400-8409, and
include both naturally occurring HCV carbohydrates and
glycopeptides, as well as synthetic carbohydrates and glycopeptides
based on naturally occurring HCV carbohydrates and
glycopeptides.
[0088] Exemplary glycans from bacterial pathogens are shown in
Scheme 4.
##STR00006## ##STR00007##
[0089] Exemplary glycans from protozoan pathogens are shown in
Scheme 5.
##STR00008##
[0090] An exemplary glycan from a fungal pathogen is shown in
Scheme 6.
##STR00009##
[0091] An exemplary glycan from helminth pathogen is shown in
Scheme 7.
##STR00010##
It will be appreciated by one of skill in the art that while
numerous antigenic carbohydrate structures are known, many more
exist, since only a small fraction of the antigenic or immunogenic
carbohydrates have been identified thus far. Examples of the many
carbohydrate antigens discovered thus far can be found in Kuberan
et al., Curr. Org. Chem, 4, 653-677 (2000); Ouerfelli et al.,
Expert Rev. Vaccines 4(5):677-685 (2005); Hakomori et al., Chem.
Biol. 4, 97-104 (1997); Hakomori, Acta Anat. 161, 79-90 (1998);
Croce and Segal-Eiras, Drugs of Today 38(11):759-768 (2002);
Mendonca-Previato et al., Curr Opin. Struct. Biol. 15(5):499-505
(2005); Jones, Anais da Academia Brasileira de Ciencias
77(2):293-324 (2005); Goldblatt, J. Med. Microbiol. 47(7):563-567
(1998); Diekman et al., Immunol. Rev., 171: 203-211, 1999; Nyame et
al., Arch. Biochem. Biophys., 426 (2): 182-200, 2004; Pier, Expert
Rev. Vaccines, 4 (5): 645-656, 2005; Vliegenthart, FEBS Lett., 580
(12): 2945-2950, Sp. Iss., 2006; Ada et al., Clin. Microbiol. Inf.,
9 (2): 79-85, 2003; Fox et al., J. Microbiol. Meth., 54 (2):
143-152, 2003; Barber et al., J. Reprod. Immunol., 46 (2): 103-124,
2000; and Sorensen, Persp. Drug Disc. Design, 5: 154-160, 1996. Any
antigenic carbohydrate derived from a mammal or from an infectious
organism can be used as the carbohydrate component of the
glycolipopeptide of the invention, without limitation.
Peptide Component
[0092] The peptide component of the glycolipopeptide includes a
T-epitope, preferably a helper T epitope. The peptide component can
be any peptide-containing structure, and can contain naturally
occurring and/or non-naturally occurring amino acids and/or amino
acid analogs (e.g., D-amino acids). The peptide component may be
from a microorganism, such as a virus, a bacterium, a fungus, and a
protozoan. The T-epitope can therefore constitute all or part of a
viral antigen. Alternatively or additionally, the T-epitope can be
from a mammal, and optionally constitutes all or part of a
self-antigen. For example, the T-epitope can be part of a
glycopeptide that is overexpressed on a cancer cell. When the
peptide component of the glycolipopeptide of the invention is a
glycopeptide, the peptide component may also include all or part of
the B-epitope, as described elsewhere herein. More generally, it
should be understood that the peptide component of the
glycolipopeptide may be coextensive with the T-epitope, or the
peptide component may be inclusive of the T-epitope, or the peptide
component may include only part of the T-epitope (i.e., the
T-epitope may additionally encompass other parts of the
glycolipopeptide such as the carbohydrate component, the lipid
component, and/or the linker component). Thus, a peptide or peptide
component that "comprises" a T-epitope is to be understood to mean
a peptide or peptide component that encompasses all or part of a
T-epitope that is present on the glycolipopeptide.
[0093] Preferably peptide component contains fewer than about 20
amino acids and/or amino acid analogs. Examples of peptide
components include the universal helper T peptide, QYIKANSKFIGITEL
("QYI") (SEQ ID NO:1), the universal helper T peptide
YAFKYARHANVGRNAFELFL ("YAF") (SEQ ID NO:2), the murine helper T
peptide KLFAVWKITYKDT ("KLF") (SEQ ID NO:3) derived from polio
virus, and pan-DR binding (PADRE) peptides (PCT WO 95/07707;
Alexander et al., Immunity 1:751-761 (1994); Alexander et al., J.
Immunol. 2000 Feb. 1; 164(3):1625-33; U.S. Pat. No. 6,413,935
(Sette et al., Jul. 2, 2002)).
[0094] Preferred immunogenic peptide components for use in the
glycolipopeptide of the invention include universal (degenerate or
"promiscuous") helper T-cell peptides, which are peptides that are
immunogenic in individuals of many major histocompatibility complex
(MHC) haplotypes. Numerous universal helper T-cell peptide
structures are known; however, it should be understood that
additional universal T-epitopes, including some with similar or
even higher potency, will be identified in the future, and such
peptides are well-suited for use as the peptide component the
glycolipopeptide of the invention.
[0095] Exemplary T-cell peptides for use in the glycolipopeptide
include, without limitation:
[0096] Synthetic, nonnatural PADRE peptide,
DAla-Lys-Cha-Val-Ala-Ala-Trp-Thr-Leu-Lys-Ala-Ala-DAla, including
all the analogues described by J Alexander et al. in Immunity, Vol.
1, 751-761, 1994;
[0097] Peptides derived from tetanus toxin, e.g., (TT830-843)
QYIKANSKFIGITEL (SEQ ID NO:1), (TT1084-1099) VSIDKFRIFCKANPK (SEQ
ID NO:4), (TT1174-1189) LKFIIKRYTPNNEIDS (SEQ ID NO:5),
(TT1064-1079) IREDNNITLKLDRCNN (SEQ ID NO:6), and (TT947-967)
FNNFTVSFWLRVPKVSASHLE (SEQ ID NO:7);
[0098] Peptides derived from polio virus, e.g., KLFAVWKITYKDT (SEQ
ID NO:3);
[0099] Peptides derived from Neisseria meningitidis, e.g.,
YAFKYARHANVGRNAFELFL (SEQ ID NO:8); and
[0100] Peptides derived from P. falsiparum CSP, e.g.,
EKKIAKMEKASSVFNVNN (SEQ ID NO:9).
[0101] The peptide component of the glycolipopeptide contains a
T-epitope. A T-epitope is an epitope recognized by a T cell. The
T-epitope can elicit a CD4+ response, thereby stimulating the
production of helper T cells; and/or it can elicit a CD8+ response,
thereby stimulating the production of cytotoxic lymphocytes.
Preferably, the T-epitope is an epitope that stimulates the
production of helper T cells (i.e., a helper T-cell epitope or
Th-epitope), which in turn makes possible a humoral response to the
B-epitope supplied by the carbohydrate component of the
glycolipopeptide of the invention.
[0102] It should be understood that the glycolipopeptide of the
invention can contain multiple T-epitopes, which may be the same or
different. Further, T-epitopes may be present on the carbohydrate
component and/or the lipid component (e.g., in embodiments that
include glycopeptides and/or glycolipids as the carbohydrate and/or
lipid components) in addition to, or in place of, the peptide
component.
[0103] In one embodiment, the B-epitopes and the T-epitopes are
homologous; that is, they are derived from the same organism. For
example, in a glycolipopeptide suitable for use as a vaccine
against a microbial pathogen, the T-epitope in addition to the
B-epitope may be epitopes that are present in the microbial
pathogen. In another embodiment, the B-epitopes and the T-epitopes
are heterologous; that is, they are not derived from the same
organism. For example, a glycolipopeptide suitable for use as an
anti-cancer vaccine may have a B-cell epitope from a cancer cell,
but a T-cell epitope from a bacterium or virus.
Lipid Component
[0104] It was originally postulated that a glycopeptide having just
two main components, i.e., a carbohydrate component and a peptide
component, would be effective to elicit an immune response in an
animal. The helper T-cell epitope was expected to induce a T-cell
dependent immune response, resulting in the production of IgG
antibodies against a tumor-related carbohydrate B-epitope such as
Le.sup.y and Tn. However, in some applications, the two component
vaccine was not found to be very effective. It was postulated that
the B-cell and helper T-cell epitopes lack the ability to provide
appropriate "danger signals" for dendritic cell (DC) maturation. To
remedy this problem, a lipid component was included in the
compound, resulting in the glycolipopeptide of the invention.
[0105] The lipid component can be any lipid-containing component,
such as a lipopeptide, fatty acid, phospholipid, steroid, or a
lipidated amino acids and glycolipids such as Lipid A derivatives.
Preferably, the lipid component is non-antigenic; that is, it does
not elicit antibodies directed against specific regions of the
lipid component. However, the lipid component may and preferably
does serve as an immunoadjuvant. The lipid component can serve as a
carrier or delivery system for the multi-epitopic glycolipopeptide.
It assists with incorporation of the glycolipopeptide into a
vesicle or liposome to facilitate delivery of the glycolipopeptide
to a target cell, and it enhances uptake by target cells, such as
dendritic cells. Further, the lipid component stimulates the
production of cytokines.
[0106] One class of preferred lipid components for use in the
glycolipopeptide of the invention comprises molecular ligands of
the various Toll-like receptors (TLRs). There are many known
subclasses of Toll-like receptors (e.g., TLR1, TLR2, TRL3, TLR4,
TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TLR13, TLR14,
TLR15 and TLR16). See Roach et al., PNAS 2005, 102:9577-9582, for a
review of the relationships between and evolution of Toll-like
receptors; and Duin et al., TRENDS Immunol., 2006, 27:49-55, for a
discussion of TLR signaling in vaccination. Particularly preferred
are lipid components that interact with TLR2 and TLR4. TLR2 is
involved in the recognition of a wide array of microbial molecules
from Gram-positive and Gram-negative bacteria, as well as
mycoplasma and yeast. TLR2 ligands include lipoglycans,
lipopolysaccharides, lipoteichoic acids and peptidoglycans. TLR4
recognizes Gram-negative lipopolysaccharide (LPS) and lipid A, its
toxic moiety. TLR ligands are widely available commercially, for
example from Apotech and InvivoGen. Preferably, the lipid component
is a TLR ligand that facilitates uptake of the glycolipopeptide by
antigen presenting cells (see Example III).
[0107] Suitable lipids for use as the lipid component of the
glycolipopeptide of the invention include PamCys-type lipid
structures, such as those derived from Pam.sub.3Cys
(S--[(R)-2,3-dipalmitoyloxy-propyl]-N-palmitoyl-(R)-cysteine) and
Pam.sub.2Cys (S--[(R)-2,3-dipalmitoyloxy-propyl]-(R)-cysteine),
which lacks the N-palmitoyl group of Pam.sub.3Cys. Pam.sub.3Cys and
Pam.sub.2Cys are derived from the immunologically active N-terminal
sequence of the principal lipoprotein of Escherichia coli. This
class of lipids also includes Pam.sub.3CysSK.sub.4
(N-palmitoyl-S--[(R)-2,3-bis(palmitoyloxy)-propyl]-(R)-cysteinyl-(S)-sery-
l-(S)-lysine-(S)-lysine-(S)-lysine-(S)-lysyne) and
Pam.sub.2CysSK.sub.4
(S--[(R)-2,3-bis(palmitoyloxy)-propyl]-(R)-cysteinyl-(S)-seryl-(S)-lysine-
-(S)-lysine-(S)-lysine-(S)-lysyne), which lacks the N-palmitoyl
group of Pam.sub.3CysSK4; it should be understood that the number
of lysines in these structures can be 0, 1, 2, 3, 4, 5 or more
(i.e., K.sub.n where n=0, 1, 2, 3, 4, 5 or more).
[0108] Another preferred class of lipids includes Lipid A (LpA)
type lipids, such as Lipid As derived from E. coli, S. typhimurium
and Neisseria meningitidis. The Lipid As can be attached to the
carbohydrate component (containing a B-epitope) of the
glycolipopeptide and/or to the peptide component (containing a
T-epitope) through a linker that is connected, for example, to the
anomeric center or anomeric phosphate, the C-4' phosphate or the
C-6' position. The phosphates can be modified, for example, to
include one or more phosphate ethanolamine diesters. Exemplary
Lipid A derivatives are described in, for example, Caroff et al.,
Microbes Infect. 4, 915-926 (2002); Raetz et al., Annu. Rev.
Biochem. 71, 635-700 (2002); and Dixon et al., J. Dent. Res. 84,
584-595 (2005).
[0109] Below, in Scheme 8, are exemplary immunogenic lipids for the
incorporation into the glycolipopeptide of the invention. The first
structure in the first row is Pam.sub.3CysSK.sub.n; the second
structure in the first row is Pam.sub.2CysSK.sub.n; and the last 4
structures are Lipid A derivatives.
##STR00011## ##STR00012##
[0110] Lipids that are structurally based on Pam.sub.3Cys are
particularly preferred for use as the lipid component. Pam.sub.3Cys
is derived from the immunologically active N-terminal sequence of
the principal lipoprotein of Escherichia coli. These lipopeptides
are powerful immunoadjuvants. Recent studies have shown that
Pam.sub.3Cys exerts its activity through the interaction with
Toll-like receptor-2 (TLR2).
[0111] Without being bound by theory, it is believed that
interaction between the lipid component and a TLR results in the
production of pro-inflammatory cytokines and chemokines, which, in
turn, stimulates antigen-presenting cells (APCs), and thus,
initiating helper T cell development and activation. Covalent
attachment of the TLR ligand to the B- and T-epitopes ensures that
cytokines are produced at the site where the vaccine interacts with
immune cells. This leads to a high local concentration of cytokines
facilitating maturation of relevant immune cells. The lipopeptide
promotes selective targeting and uptake by antigen presenting cells
and B-lymphocytes. Additionally, the lipopeptide facilitates the
incorporation of the glycolipopeptide into liposomes. Liposomes
have attracted interest as vectors in vaccine design due to their
low intrinsic immunogenicity, thus, avoiding undesirable
carrier-induced immune responses.
Optional Linker
[0112] One or more linkers ("L") are optionally used for assembly
of the three components of the glycolipopeptide. In one embodiment,
the linker is a bifunctional linker that has functional groups in
two different places, preferably at a first and second end, in
order to covalently link two of the three components together. A
bifunctional linker can be either homofunctional (i.e., containing
two identical functional groups) or heterofunctional (i.e.,
containing two different functional groups). In another embodiment,
the linker is trifunctional (hetero- or homo-) and can link all
three components of the glycolipopeptide together. A suitable
functional group has reactivity toward or comprises any of the
following: amino, alcohol, carboxylic acid, sulfhydryl, alkene,
alkyne, azide, thioester, ketone, aldehyde, or hydrazine. An amino
acid, e.g., cysteine, can constitute a linker.
[0113] Bifunctional linkers are exemplified in Scheme 9.
##STR00013##
[0114] FIG. 1 shows an exemplary fully synthetic glycolipopeptide
of the invention containing a carbohydrate-based B-epitope, a
peptide T-epitope and a lipopeptide. The compound shown in FIG. 1
contains a L-glycero-D-manno-heptose sugar that acts as a
B-epitope, the peptide sequence YAFKYARHANVGRNAFELFL (SEQ ID NO:2)
that has been identified as a MHC class II restricted recognition
site for human T-cells and is derived from an outer-membrane
protein of Neisseria meningitidis, and the lipopeptide
S-2-3[dipalmitoyloxy]-(R/S)-propyl-N-palmitoyl-R-Cysteine
(Pam.sub.3Cys). As noted elsewhere herein, lipopeptide Pam.sub.3Cys
and the related compound Pam.sub.3CysSK.sub.4 are highly potent
B-cell and macrophage activators.
[0115] Methods of making the glycolipopeptide, as exemplified in
the Examples, are also encompassed by the invention. Preferably,
the method for making the glycolipopeptide utilizes chemical
synthesis, resulting in a fully synthetic glycolipopeptide. In
embodiments that make use of one or more linkers, the optional
linker component is functionalized so as to facilitate covalent
linkage of one of the main components to another of the main
components. For example, the linker can be functionalized at each
end with a thiol-reactive group, such as maleimide or bromoacetyl,
and the components to be joined are modified to include reactive
thiols. Other options for ligation chemistry include Native
Chemical Ligation, the Staudinger Ligation and Huisgen ligation
(also known as "Click Chemistry"). Example II illustrates how the
carbohydrate component, in that case an oligosaccharide, and the
peptide component can be functionalized with a thiol-containing
linker. Preferably, the linker component, if used, is
nonantigenic.
[0116] The glycolipopeptide of the invention is capable of
generating an immune response in a mammal. The glycolipopeptide is
antigenic, in that it can generate a humoral response, resulting in
the activation of B cells and production of antibodies
(immunoglobulins) such as IgM. Additionally, the glycolipopeptide
is immunogenic, in that it can generate a cellular response; for
example, it facilitates the activation of T cells, particularly
helper T cells which are also instrumental in the generation of a
more complex antibody response that includes the production of IgG.
Ultimately, the immune response elicited in the animal includes the
production of anti-carbohydrate antibodies.
[0117] In another aspect, the glycolipopeptide of the invention is
used to produce a polyclonal or monoclonal antibody that recognizes
either or both of the carbohydrate component and the peptide
component. The invention encompasses the method of making said
antibodies, as well as the antibodies themselves and hybridomas
that produce monoclonal antibodies of the invention.
[0118] The immunogenic glycolipopeptide of the invention for use in
the production an antibody can contain any carbohydrate component
described herein, without limitation. Preferably it contains, as
its carbohydrate component, a glycopeptide. The glycopeptide
includes a glycosylated peptide sequence that includes a
carbohydrate moiety, such as a saccharide. The saccharide can be a
monosaccharide, an oligosaccharide or a polysaccharide. Preferably,
the carbohydrate component of the glycolipopeptide used to generate
the antibodies contains a self-antigen as described above.
Advantageously, even if carbohydrate component, e.g., the
glycopeptide, is poorly antigenic (such as a self-antigen),
covalent attachment of the carbohydrate component to the peptide
component and the lipid component produces a remarkably immunogenic
glycolipopeptide.
[0119] Antibodies of the invention that bind to the
glycolipopeptide preferably bind to a B-epitope that includes the
saccharide moiety and, in a preferred embodiment, at least part of
the peptide that forms the glycopeptide. A preferred antibody binds
to the glycopeptide used as the carbohydrate component, but does
not bind to the deglycosylated peptide or to the saccharide residue
alone. The selectivity of an antibody for the glycopeptide can be
determined using, for example, the methods set forth in Examples
VIII and X.
[0120] When used to generate antibodies, the glycolipopeptide of
the invention successfully generates high affinity IgG antibodies.
This is especially surprising and unexpected for embodiments of the
glycolipopeptide having a poorly antigenic carbohydrate component,
such as a self-antigen. The polyclonal or monoclonal antibody is
thus preferably an IgG isotype antibody. Without being bound by
theory, it is believed that the glycolipopeptide of the invention
is a superior antigen (compared to the non-lipidated glycopeptide)
because it stimulates local production of cytokines, upregulates
co-stimulatory proteins, enhances uptake by macrophages and
dendritic cells and/or avoids epitope suppression.
[0121] Antibodies of the invention include but are not limited to
those that recognize B-epitopes that contain O-GlcNAc, O-GalNAc,
O-mannose, or other saccharide modifications. Other B-epitopes that
may be recognized by the antibodies of the invention include those
that contain fragments of glycosaminoglycans such as heparin,
heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan
sulfate, hyaluronan, and generally any glycosaminoglycan. In the
case of a glycosaminoglycan formed by repeating disaccharide units,
the B-epitope may contain one or more disaccharide unit. B-epitopes
recognized by the antibodies of the invention may contain pentose,
hexose or other sugar moieties including acids, including but not
limited to glucuronic acid, iduronic acid, hyaluronic acid,
glucose, galactose, galactosamine, glucosamine and the like. The
antibodies of the invention are preferably produced using, as an
immunogen, the glycolipopeptide of the invention wherein the
carbohydrate component contains the B-epitope of interest.
Analogues of naturally occurring B-epitopes, such as those
containing N-linked or S-linked structures or glycomimetics, can be
used as the carbohydrate component, for example to make the
glycolipopeptide immunogen more metabolically stable.
[0122] The antibodies produced using the glycolipopeptide of the
invention advantageously include high affinity IgG antibodies that
recognize a broad spectrum of glycoproteins. Thus, even though
antibodies produced using the glycolipopeptide of the invention as
an immunogen are specific for the glycopeptide used as the
carbohydrate component, they may bind to a broad spectrum of
glycoproteins. An antibody with relatively broad selectivity for
glycosylated peptides or proteins containing a B-epitope component
of interest is referred to herein as a "pan-specific" antibody. A
polyclonal or monoclonal antibody of the invention may be either
pan-specific or site-specific. An antibody that is pan-specific, as
the term is used herein, is one that specifically recognizes a
selected B-epitope, for example a B-epitope that contains O-GlcNAc,
but that has a relatively broad selectivity for proteins and
peptides containing the B-epitope. A pan-specific antibody is thus
able to bind multiple different glycosylated proteins or peptides
that contain the B-epitope of interest, although it does not
necessarily bind all glycosylated proteins or peptides that contain
the selected B-epitope. See, for example, Tables 8-11 in Examples
IX and X which show binding selectivity for several pan-specific
monoclonal antibodies, including the monoclonal antibodies produced
by hybridoma cell lines 1F5.D6 (Mab14), 9D1.E4 (Mab10), 18B10.C7
(Mab3) and the commercially available monoclonal IgM antibody
CTD110.6 (Covance Research Products, Inc.). A site-specific
antibody, on the other hand, typically shows greater selectivity
for a particular individual glycosylated protein or peptide.
[0123] Without intending to be being bound by theory, the different
glycoproteins recognized by the pan-specific antibodies of the
invention may share a substantially similar or identical
(glyco)peptide sequence (i.e., primary sequence) or a substantially
similar secondary or tertiary structure at the glycosylation site,
thereby resulting in a broad spectrum of binding targets being
recognized by the antibody. A secondary or tertiary epitope
structure shared by the O-GlcNAc modified glycoproteins to which an
antibody binds may advantageously be maintained in the
glycolipopeptide immunogen, as evidenced by the successful
production of IgG antibodies that recognize the broad spectrum of
glycoproteins.
[0124] Preferably, the antibody of the invention binds to a
plurality of glycosylated proteins or peptides having an epitope
comprising O-GlcNAc, O-GalNAc, or other saccharide modifications,
but does not detectably bind a protein or peptide that does not
contain the saccharide. More preferably, the antibody binds to a
protein or peptide having an epitope comprising O-GlcNAc, O-GalNAc,
or other saccharide modifications, but does not detectably bind the
same protein or peptide that does not contain O-GlcNAc, O-GalNAc,
or other saccharide modifications.
[0125] An example of a preferred polyclonal or monoclonal antibody
is one that binds to a glycopeptide that contains an O-GlcNAc
monosaccharide residue. In a particularly preferred embodiment, the
antibody has a relatively broad selectivity for O-GlcNAc modified
proteins. For example, many proteins of interest have a TPVSS (SEQ
ID NO:10) sequence modified by O-GlcNAc, and a preferred monoclonal
antibody recognizes this and/or similar glycosylated peptide
sequences. Examples of preferred monoclonal antibodies specific for
O-GlcNAc modified sequences include the monoclonal antibodies
produced by hybridoma cell lines 1F5.D6, 9D1.E4, 18B10.C7 and
5H11.H6. These monoclonal antibodies were produced using compounds
52 and/or 53 as an immunogen. Thus, in one embodiment, the antibody
of the invention binds to the carbohydrate component of compound 52
or of compound 53. Hybridoma cell lines 1F5.D6, 9D1.E4 and 18B10.C7
were deposited with the American Type Culture Collection (ATCC),
10801 University Blvd., Manassas, Va., 20110-2209, USA, on Jul. 1,
2008, and assigned ATCC deposit numbers PTA-9339, PTA-9340, and
PTA-9341, respectively. The invention encompasses the hybridoma
cell lines as well as the monoclonal antibodies they produce.
[0126] Another example of a preferred polyclonal or monoclonal
antibody is one that binds to a heparan sulfate fragment.
[0127] It is to be understood that any carbohydrate or glycopeptide
of clinical significance or interest can be incorporated as the
carbohydrate and/or peptide component of the glycolipopeptide of
the invention and used to generate polyclonal and monoclonal
antibodies according to the method of the invention. Such
carbohydrates and peptides include those of medical and veterinary
interest, as well as those with other commercial or research
applications. It should be understood that the monoclonal and
polyclonal antibodies of the invention are not limited to those
that recognize any particular ligand but include, without
limitation and by way of example only, antibodies against any type
of tumor associated carbohydrate antigen (TACA) and against any
saccharides derived from any microorganism.
[0128] To recapitulate, use of the glycolipopeptide of the
invention to make monoclonal antibody of the invention is
surprisingly effective in producing monoclonal IgG antibodies
having high affinity for their carbohydrate or glycopeptide
antigen, even when the antigens are poorly antigenic. This opens
the door for the creation of antibodies useful to study, diagnose
and treat immune-related diseases or diseases having autoimmune or
inflammatory components including cancer, diabetes type II,
allergies, asthma, Crohn's disease, Alzheimer's disease, muscular
dystrophy, microbial infections and the like. Monoclonal antibodies
of the invention that recognize O-GlcNAc-modified glycoproteins,
for example, are far superior to commercially available antibodies
such CTD110.6 (Covance Research Products, Inc.; see Examples VIII,
IX and X). The glycolipopeptide of the invention can be assembled
using a modular synthesis, wherein the lipid, peptide and
carbohydrate component are selected according to the desired
application. Moreover, the glycolipopeptide of the invention is a
remarkably effective antigen for use in producing pan-specific
antibodies, particularly pan-specific monoclonal IgG antibodies
that recognize glycosylated peptides and proteins that contain an
O-linked monosaccharide such as O-GlcNAc.
[0129] The antibodies of the invention and those created by the
method of the invention are important research tools for the
identification and characterization of proteins, peptides and other
biomolecules associated with various disease states. For example,
the pan-specific antibodies of the invention can be used to pull
down glycoproteins from complex biological samples, as shown in
Examples VIII through XI below. This method can be used to detect
and identify proteins not heretofore known to be identified with a
particular disorder or disease state, thereby identifying potential
therapeutic or diagnostic targets. In one embodiment, an antibody
of the invention can be contacted with a biological sample under
conditions that enable the antibody to bind to a plurality of
glycosylated proteins or glycosylated peptides and detecting
antibody-protein binding. Optionally the method may include
isolating the glycosylated proteins or glycosylated peptides. The
method may further include identifying one or more of the proteins
or peptides within the plurality of glycosylated proteins or
glycosylated peptides. The identification of glycosylated proteins
and peptides may provide an opportunity to explore the role of
glycosylation and its biological implications in various biological
processes. For example, glycosylation of proteins or peptides may
be involved in a number of biological processes including, but not
limited to, transcription, translation, signal transduction, the
ubiquitin pathway, anterograde trafficking of intracellular
vesicles and post-translational modifications (e.g. SUMOylation and
phosphorylation). Methods for identifying a protein or peptide are
well known in the art and may include, without limitation,
techniques such as mass spectrometry and Edman degradation.
Examples of proteins or peptides that may be glycosylated are
exemplified in Tables 8-11, and 13.
[0130] The pan-specific antibody of the invention may also be used
to identify proteins or peptides having altered glycosylation in a
disease state. O-GlcNAc modifications are associated with a variety
of disease states. For example, an increase of O-GlcNAc
modifications in skeletal muscle and pancreas glycopeptides
correlates with development of Type II Diabetes while a reduction
in O-GlcNAc modifications in neural glycopeptides correlates with
the onset of Alzheimer's disease (Dias and Hart; Mol. BioSyst.
3:766-772 (2007)). Therefore, detection of changes in the levels of
O-GlcNAc modifications may be used as a diagnostic or prognostic
tool. Additionally, the glycosylation state of the proteins or
peptides listed in Tables 8-11 and 13 may be correlated with
disease state. A method for identifying proteins or peptides having
altered glycosylation that is correlated with disease state
includes incubating an antibody of the present invention with a
first biological sample of a known disease state and incubating the
antibody with a second biological sample of a non-diseased state
under conditions enabling the antibody to bind to a plurality of
glycosylated proteins and peptides within the first sample and to a
plurality of glycosylated proteins and peptides within the second
sample, independently isolating the glycosylated proteins and
glycosylated peptides from the samples, and identifying the
glycosylated proteins and glycosylated peptides. The method may
further include comparing the identified glycosylated proteins and
glycosylated peptides in the first sample to the glycosylated
proteins and glycosylated peptides in the second sample wherein a
protein or peptide that demonstrates a change in glycosylation
state between first and second samples is indicative of the
glycosylated protein or a glycosylated peptide being associated
with a disease state. Correlations between glycosylation and
disease state include the disease state having increased or
decreased glycosylation relative to the non-diseased state. In
addition, the disease state may exhibit glycosylation while the
non-disease state shows complete absence of glycosylation or
conversely, the disease state may show complete absence of
glycosylation while the non-disease exhibits the presence of
glycosylation. In each example, the protein or peptide is
considered to have differential or altered glycosylation in the
disease state. Methods of using the antibody of the invention to
detect the presence or overexpression glycosylation and to detect
changes in the level of glycosylation have been previously
described.
[0131] The antibodies of the invention are broadly useful in
diagnostic or therapeutic applications as described in more detail
elsewhere herein. Comparative analysis can be done on two or more
different biological samples. For example, large scale
immunoprecipitation can be performed on samples before and after a
treatment intervention, or over time to monitor the progression of
disease, or to compare normal samples with samples from patients
suspected of suffering from a disease, infection or disorder
characterized by changes in protein glycosylation.
[0132] In one embodiment, the present invention includes methods to
diagnose the presence of a disease state in a subject. The method
includes incubating a biological sample from the subject with an
antibody of the present invention and detecting binding of the
antibody to a protein or peptide having differential glycosylation
in the disease state. Methods of detecting antibody binding have
been previously described. In cases where glycosylation is
completely absent in the disease state, a lack of binding of the
antibody to the protein or peptide is indicative of subject having
the disease state. In cases where glycosylation is present in the
disease state but completely absent in the non-disease state,
binding of the antibody to the protein or peptide is indicative of
the presence of the disease state in the subject. Optionally, the
method may further include incubating a second, non-diseased,
biological sample with an antibody of the invention, detecting
binding of the antibody to a protein or peptide, and comparing
antibody binding in the first and second samples.
[0133] Additionally, for protein and peptides where glycosylation
is present in both the disease state and the non-disease state, but
is altered (i.e. increased or decreased) in the disease state, the
method may further include quantitating the level of antibody
binding in the first sample, quantitating the level of antibody
binding in the second, non-diseased sample, and comparing the
binding levels. A change in antibody binding in the first sample
compared to the non-diseased sample is indicative of the presence
of the infection, disease or disorder in the subject.
[0134] For preparation of an antibody of the present invention, any
technique which provides for the production of antibody molecules
by continuous cell lines in culture may be used. For example, the
hybridoma technique originally developed by Kohler and Milstein
(256 Nature 495-497 (1975)) may be used. See also Ausubel et al.,
Antibodies: a Laboratory Manual, (Harlow & Lane eds., Cold
Spring Harbor Lab. 1988); Current Protocols in Immunology,
(Colligan et al., eds., Greene Pub. Assoc. & Wiley Interscience
N.Y., 1992-1996).
[0135] The present invention also provides for a hybridoma cell
line that produces a monoclonal antibody, preferably one that has a
high degree of specificity and affinity toward its antigen. The
present invention further includes variants and mutants of the
hybridoma cell lines, such as those described in Example VIII. Such
cell lines can be produced artificially using known methods and
still have the characteristic properties of the starting material.
For example, they may remain capable of producing the antibodies
according to the invention or derivatives thereof, and secreting
them into the surrounding medium. Optionally, the hybridoma cell
lines may occur spontaneously. Clones and sub-clones of hybridoma
cell lines are to be understood as being hybridomas that are
produced from the starting clone by repeated cloning and that still
have the main features of the starting clone.
[0136] Antibodies can be elicited in an animal host by immunization
with the glycolipopeptide of the invention, or can be formed by in
vitro immunization (sensitization) of immune cells. The antibodies
can also be produced in recombinant systems in which the
appropriate cell lines are transformed, transfected, infected or
transduced with appropriate antibody-encoding DNA. Alternatively,
the antibodies can be constructed by biochemical reconstitution of
purified heavy and light chains.
[0137] Once an antibody molecule has been produced by an animal,
chemically synthesized, or recombinantly expressed, it may be
purified by any method known in the art for purification of an
immunoglobulin molecule, for example, by chromatography (e.g., ion
exchange, affinity, particularly by affinity for the specific
antigen after Protein A, and sizing column chromatography),
centrifugation, differential solubility, or by any other standard
technique for the purification of proteins. In addition, the
antibodies of the present invention or fragments thereof can be
fused to heterologous polypeptide sequences known in the art to
facilitate purification.
[0138] In a preferred embodiment, the monoclonal antibody
recognizes and/or binds to an antigen present on the carbohydrate
component or the peptide component of the glycolipopeptide of the
invention. In a particularly preferred embodiment, the monoclonal
antibody binds to an antigen present on a selected feature of the
carbohydrate component.
[0139] An example of a selected feature would include the
modification on a glycopeptide such as O-GlcNAc. Other
modifications include, but are not limited to, GalNAc and other
saccharide modifications.
[0140] The term "antibody" is used in the broadest sense and
specifically covers monoclonal antibodies (including full length
monoclonal antibodies) and antibody fragments so long as they
exhibit the desired biological activity. "Antibody fragments"
comprise a portion of a full length antibody, generally the antigen
binding or variable region thereof. Examples of antibody fragments
include, but are not limited to Fab, Fab', and Fv fragments;
diabodies; linear antibodies; and single-chain antibody molecules.
The term "monoclonal antibody" as used herein refers to antibodies
that are highly specific, being directed against a single antigenic
site. The term "antibody" as used herein also includes naturally
occurring antibodies 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. 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 as described by Huse et al. (Science
246:1275-1281 (1989)). These and other methods of making functional
antibodies are well known to those skilled in the art (Winter and
Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature
341:544-546 (1989); Harlow and Lane, supra, 1988); Hilyard et al.,
Protein Engineering: A practical approach (IRL Press 1992);
Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press
1995)).
[0141] In all mammalian species, antibody peptides contain constant
(i.e., highly conserved) and variable regions, and, within the
latter, there are the complementarity determining regions (CDRs)
and the so-called "framework regions" made up of amino acid
sequences within the variable region of the heavy or light chain
but outside the CDRs. Preferably the monoclonal antibody of the
present invention has been humanized. As used herein, the term
"humanized" antibody refers to antibodies in which non-human
(usually from a mouse or a rat) CDRs are transferred from heavy and
light variable chains of the non-human immunoglobulin into a
variable region designed to contain a number of amino acid residues
found within the framework region in human IgG. Similar conversion
of mouse/human chimeric antibodies to a humanized antibody has been
described before. General techniques for cloning murine
immunoglobulin variable domains are described, for example, by the
publication of Orlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833
(1989), which is incorporated by reference in its entirety.
Techniques for producing humanized MAbs are described, for example,
by Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature
332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), and
Singer et al., J. Immun. 150: 2844 (1993), each of which is hereby
incorporated by reference.
[0142] Methods of using the monoclonal antibody that recognizes
and/or binds to a component of the glycolipopeptide are also
encompassed by the invention. Uses for the monoclonal antibody of
the invention include, but are not limited to, diagnostic,
therapeutic, and research uses. In a preferred embodiment, the
monoclonal antibody can be used for diagnostic purposes. Because
O-GlcNAc modifications are associated with a variety of disease
states, detection of changes in the levels of O-GlcNAc
modifications may be interpreted as early indicators of the onset
of such diseases. For example, an increase in O-GlcNAc
modifications in skeletal muscle and pancreas glycopeptides
correlates with development of Type II Diabetes while a reduction
in O-GlcNAc modifications in neural glycopeptides correlates with
the onset of Alzheimer's disease (Dias and Hart, Mol. BioSyst.
3:766-772 (2007); Lefebvre et al., Exp. Rev. Proteomics
2(2):265-275 (2005)). Therefore, identifying an increase in the
amount of O-GlcNAc in a sample of skeletal muscle tissue relative
to a non-disease control sample may be indicative of development of
Type II Diabetes.
[0143] It should be understood that the monoclonal and polyclonal
antibodies of the invention are not limited to those that recognize
any particular ligand but include, without limitation and by way of
example only, antibodies against any type of tumor associated
carbohydrate antigen (TACA) and against any saccharides derived
from any microorganism. The antibodies of the invention are broadly
useful in diagnostic or therapeutic applications.
[0144] Antibodies of the invention can be used to detect the
presence or overexpression of a specific protein or a specific
modification. Techniques for detection are known to the art and
include but are not limited to Western blotting, dot blotting,
immunoprecipitation, agglutination, ELISA assays, immunoELISA
assays, tissue imaging, mass spectrometry, immunohistochemistry,
and flow cytometry on a variety of tissues or bodily fluids, and a
variety of sandwich assays. See, for example, U.S. Pat. No.
5,876,949, hereby incorporated by reference.
[0145] In order to detect changes in the level of O-GlcNAc modified
glycopeptides, monoclonal antibodies of the invention may be
labeled covalently or non-covalently with any of a number of known
detectable labels, such as fluorescent, radioactive, or enzymatic
substances, as is known in the art. Alternatively, a secondary
antibody specific for the monoclonal antibody of the invention is
labeled with a known detectable label and used to detect the
O-GlcNAc-specific antibody in the above techniques.
[0146] Preferred detectable labels include chromogenic dyes. Among
the most commonly used are 3-amino-9-ethylcarbazole (AEC) and
3,3'-diaminobenzidine tetrahydrochloride (DAB). These can be
detected using light microscopy. Also preferred are fluorescent
labels. Among the most commonly used fluorescent labeling compounds
are fluorescein isothiocyanates (e.g. FITC and TRITC),
Idotricarbocyanines (e.g. Cy5 and Cy7), rhodamine, phycoerythrin,
phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
Chemiluminescent and bioluminescent compounds such as luminol,
isoluminol, theromatic acridinium ester, imidazole, acridinium
salt, oxalate ester, luciferin, luciferase, and aequorin may also
be used. When the fluorescent-labeled antibody is exposed to light
of the proper wavelength, its presence can be detected due to its
fluorescence. Also preferred are radioactive labels. Radioactive
isotopes which are particularly useful for labeling the antibodies
of the present invention include .sup.3H, .sup.125I, .sup.131I,
.sup.35S, .sup.32P, and .sup.14C. The radioactive isotope can be
detected by such means as the use of a gamma counter, a
scintillation counter, or by autoradiography. Enzymes which can be
used to detectably label antibodies and which can be detected, for
example, by spectrophotometric, fluorometric, or visual means
include, but are not limited to, malate dehydrogenase,
staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol
dehydrogenase, alpha-glycerophosphate dehydrogenase, triose
phosphate isomerase, horseradish peroxidase, alkaline phosphatase,
asparaginase, glucose oxidase, beta-galactosidase, ribonuclease,
urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase,
and acetylcholinesterase. Other methods of labeling and detecting
antibodies are known in the art and are within the scope of this
invention.
[0147] Because it is antigenic and immunogenic, the
glycolipopeptide of the invention is well-suited for use in an
immunotherapeutic pharmaceutical composition. The invention thus
includes pharmaceutical compositions that include a
glycolipopeptide of the invention as well as a pharmaceutically
acceptable carrier. In a preferred embodiment, the pharmaceutical
composition contains liposomes, for example phospholipid-based
liposomes, and the glycolipopeptide is incorporated into liposomes
as a result of noncovalent interactions such as hydrophobic
interactions. Alternatively, the glycolipopeptide can be covalently
linked to a component of the liposome. The liposome formulation can
include glycolipopeptides that have the same or different
B-epitopes; the same or different T-cell epitopes; and/or the same
or different lipid components.
[0148] The glycolipopeptide of the invention is readily formulated
as a pharmaceutical composition for veterinary or human use. The
pharmaceutical composition optionally includes excipients or
diluents that are pharmaceutically acceptable as carriers and
compatible with the glycolipopeptide. The term "pharmaceutically
acceptable carrier" refers to a carrier(s) that is "acceptable" in
the sense of being compatible with the other ingredients of a
composition and not deleterious to the recipient thereof or to the
glycolipopeptide. Suitable excipients include, for example, water,
saline, dextrose, glycerol, ethanol, or the like and combinations
thereof. In addition, if desired, the pharmaceutical composition
may contain minor amounts of auxiliary substances such as wetting
or emulsifying agents, pH buffering agents, salts, and/or adjuvants
which enhance the effectiveness of the immune-stimulating
composition. For oral administration, the glycolipopeptide can be
mixed with proteins or oils of vegetable or animal origin. Methods
of making and using such pharmaceutical compositions are also
included in the invention.
[0149] The pharmaceutical composition of the invention can be
administered to any subject including humans and domesticated
animals (e.g., cats and dogs). In a preferred embodiment, the
pharmaceutical composition is useful as a vaccine and contains an
amount of glycolipopeptide effective to induce an immune response
in a subject. Dosage amounts, schedules for vaccination and the
like for the glycolipopeptide vaccine of the invention are readily
determinable by those of skill in the art. The vaccine can be
administered to the subject using any convenient method, preferably
parenterally (e.g., via intramuscular, intradermal, or subcutaneous
injection) or via oral or nasal administration. The useful dosage
to be administered will vary, depending on the type of animal to be
vaccinated, its age and weight, the immunogenicity of the
attenuated virus, and mode of administration.
[0150] Inclusion of an adjuvant, such as alum or QS-21, in the
pharmaceutical composition is optional. However, it has been found
that as long as the three main components of the glycolipopeptide
are covalently linked, an adjuvant is not needed in order to
effectively generate an immune response in an animal. Moreover, the
inclusion of QS-21 may skew the immune response, resulting in a
change in the relative amounts Th1 and Th2 T cells produced (see
Example III). QS-21 can be included as an adjuvant in the
pharmaceutical composition when, for example, a shift toward a Th1
response is desired, as opposed to a bias toward a Th2 response
that is observed in the absence of QS-21.
[0151] As noted, the pharmaceutical composition is useful as a
vaccine. The vaccine can be a prophylactic or protective vaccine,
administered before or after contact with a pathogen but prior to
the development of infection or disease. Likewise, the vaccine can
be a therapeutic vaccine, administered after infection with a
pathogen, or the development of a disease or disorder such as
cancer, precancerous conditions, or autoimmune disease. Thus
vaccines that include a glycolipopeptide as described herein,
including antimicrobial (e.g., anti-viral or anti-bacterial) and
anti-cancer vaccines, are encompassed by the present invention.
Cancers that can be effectively treated or prevented include, but
are not limited to, prostate cancer, bladder cancer, colon cancer
and breast cancer.
[0152] The glycolipopeptide of the invention can also be used in
passive immunization methods. For example, the glycolipopeptide can
be administered to a host animal such as a rabbit, mouse, rat,
chicken or goat to generate antibody production in the host animal.
Protocols for raising polyclonal antibodies in host animals are
well known. The T-epitope or T-epitopes included in the
glycolipopeptide optionally are selected to be the same as or
similar to the corresponding T-epitope of the host animal in which
the antibody is raised. The antibodies are isolated from the
animal, then administered to a mammalian subject, preferably a
human subject, prophylactically or therapeutically to treat or
prevent disease or infection. Monoclonal antibodies against the
glycolipopeptide of the invention can be isolated from hybridomas
prepared in accordance with standard laboratory protocols; they can
also be produced using recombinant techniques such as phage
display. Such antibodies are also useful for passive immunization.
Optionally, the anti-glycolipopeptide monoclonal antibodies are
human antibodies or humanized antibodies. The B-epitope or
B-epitopes included in the glycolipopeptide used to create the
polyclonal or monoclonal antibodies is selected with reference to
the intended purpose of treatment. The invention encompasses
polyclonal and monoclonal anti-glycolipopeptide antibodies, as well
as methods for making and using them.
[0153] Accordingly, also provided by the invention is a
pharmaceutical composition that includes the monoclonal or
polyclonal antibody of the invention as well as a pharmaceutically
acceptable carrier. Preferably the monoclonal antibody is a
humanized antibody. Humanized antibodies are more preferable for
use in therapies of human diseases or disorders because the
humanized antibodies are much less likely to induce an immune
response, particularly an allergic response, when introduced into a
human host. As noted, the pharmaceutical composition optionally
includes excipients or diluents that are pharmaceutically
acceptable as carriers and are compatible with the monoclonal
antibody and can be administered to any subject including humans
and domesticated animals (e.g. cats and dogs). Methods of making
and using such a pharmaceutical composition are also included in
this invention.
[0154] A common feature of oncogenic transformed cells is the
over-expression of oligosaccharides, such as Globo-H, Lewis.sup.Y,
and Tn antigens. Optionally, the pharmaceutical composition of the
invention that includes the monoclonal or polyclonal antibody of
the invention as well as a pharmaceutically acceptable carrier may
be useful in targeting a tumor comprising oncogenic transformed
cells over-expressing such oligosaccharides. For example, an
antibody conjugated to a chemotherapeutic molecule may be used to
deliver the chemotherapeutic molecule to the tumor.
[0155] Another pharmaceutical composition of the invention may
include a compound (e.g. an antibody, ligand, small molecule, or
peptide) that can affect the activity of a protein listed in Tables
8-11 or 13 as well as a pharmaceutically acceptable carrier. The
effect of the compound on the protein may include, without
limitation, agonizing, antagonizing, inhibiting, or enhancing the
normal biological process of the protein. Preferably, the compound
is an antibody than binds to an epitope on the protein that
includes an O-glycosylation site. Preferably, the O-glycosylation
site is an O-GlcNAc site. Numerous studies have shown that this
abnormal glycosylation can promote metastasis and hence it is
strongly correlated with poor survival rates of cancer patients.
Thus, the ability to affect the activity of an abnormally
glycosylated protein may enable the prevention of the abnormal
activity.
[0156] As noted elsewhere herein, it has been surprisingly found
that covalent attachment of a Toll-like receptor (TLR) ligand to a
glycopeptide comprising a carbohydrate component (containing a B
epitope) and a peptide component (containing a T-epitope) enhances
uptake and internalization of the glycopeptide by a target cell
(see Example III). TLR ligands thus identified that are
characterized as lipids are preferred lipid components for use in
the glycolipopeptide of the invention. The invention thus further
provides a method for identifying TLR ligands, preferably lipid
ligands, that includes contacting a candidate compound with a
target cell containing a Toll-like receptor (TLR), and determining
whether the candidate compound binds to the TLR (i.e., is a TLR
ligand). Preferably, the candidate compound is internalized by the
target cell through the TLR. Lipid-containing TLR ligands
identified by binding to a TLR and, optionally, by internalization
into the target cell are expected to be immunogenic and are
well-suited for use as the lipid component of the glycolipopeptide
of the invention. The invention therefore also encompasses
glycolipopeptides which include, as the lipid component(s), one or
more lipid-containing TLR ligands identified using the method of
the invention.
[0157] The present invention also includes a diagnostic kit. The
kit provided by the invention can contain an antibody of the
invention, preferably a monoclonal antibody, and a suitable buffer
(such as Tris, phosphate, carbonate, etc.), thus enabling the kit
user to identify O-GlcNAc modifications. The user can then
detectably label the antibodies as desired. Alternatively, the kit
provided by the invention can contain the antibody in solution,
preferably frozen in a quenching buffer, or in powder form (as by
lyophilization). The antibody, which may be conjugated to a
detectable label, or unconjugated, is included in the kit with
buffers that may optionally also include stabilizers, biocides,
inert proteins, e.g., serum albumin, or the like. Generally, these
materials will be present in less than 5% wt. based on the amount
of active antibody, and usually present in total amount of at least
about 0.001% wt. based again on the antibody concentration.
Optionally, the kit may include an inert extender or excipient to
dilute the active ingredients, where the excipient may be present
in from about 1% to 99% wt. of the total composition. In a
preferred embodiment, the antibody provided by the kit is
detectably labeled such that bound antibody is detectable. The
detectable label can be a radioactive label, an enzymatic label, a
fluorescent label, or the like. Optionally, the kit may contain an
unconjugated monoclonal antibody of the invention and further
contain a secondary antibody capable of binding to the primary
antibody. Where a secondary antibody capable of binding to the
primary antibody is employed in an assay, this will usually be
present in a separate vial. The secondary antibody is typically
conjugated to a detectable label and formulated in an analogous
manner with the antibody formulations described above. The kit will
generally also include packaging and a set of instructions for
use.
EXAMPLES
[0158] The present invention is illustrated by the following
examples. It is to be understood that the particular examples,
materials, amounts, and procedures are to be interpreted broadly in
accordance with the scope and spirit of the invention as set forth
herein.
Example I
Towards a Fully Synthetic Carbohydrate-Based Anti-Cancer Vaccine:
Synthesis and Immunological Evaluation of a Lipidated Glycopeptide
Containing the Tumor-Associated Tn-Antigen
[0159] In this Example, a fully synthetic candidate cancer vaccine,
composed of a tumor associated Tn-antigen, a peptide T-epitope and
the lipopeptide Pam.sub.3Cys was prepared by a combination of
polymer-supported and solution phase chemistry. Incorporation of
the glycolipopeptide into liposomes gave a formulation that was
able to elicit a T-cell dependent antibody response in mice.
[0160] A common feature of oncogenic transformed cells is the
over-expression of oligosaccharides, such as Globo-H, Lewis.sup.Y,
and Tn antigens (Lloyd, Am. J Clin. Pathol. 1987, 87, 129; Feizi et
al., Trends in Biochem. Sci. 1985, 10, 24-29; Springer, J. Mol.
Med. 1997, 75, 594-602; Hakomori, Acta Anat. 1998, 161, 79-90).
Numerous studies have shown that this abnormal glycosylation can
promote metastasis (Sanders et al., Mol. Pathol. 1999, 52, 174-178)
and hence its expression is strongly correlated with poor survival
rates of cancer patients.
[0161] Several elegant studies have exploited the differential
expression of tumor-associated carbohydrates for the development of
cancer vaccines (Ragupathi, Cancer Immunol. 1996, 43, 152-157;
Musselli et al., J Cancer Res. Clin. Oncol. 2001, 127, R20-R26).
The inability of carbohydrates to activate helper T-lymphocytes has
complicated, however, their use as vaccines (Kuberan et al.,
Current Organic Chemistry 2000, 4, 653-677). For most immunogens,
including carbohydrates, antibody production depends on the
cooperative interaction of two types of lymphocytes, B-cells and
helper T-cells (Jennings et al., Neoglycoconjugates, preparation
and application, Academic, San Diego, 1994). Saccharides alone
cannot activate helper T-cells and therefore have a limited
immunogenicity. The formation of low affinity IgM antibodies and
the absence of IgG antibodies manifest this limited
immunogenicity.
[0162] In order to overcome the T-cell independent properties of
carbohydrates, past research has focused on the conjugation of
saccharides to a foreign carrier protein (e.g. Keyhole Limpet
Hemocyanin (KLH) detoxified tetanus toxoid). In this approach, the
carrier protein enhances the presentation of the carbohydrate to
the immune system and provides T-epitopes (peptide fragments of
12-15 amino acids) that can activate T-helper cells.
[0163] However, the conjugation of carbohydrates to a carrier
protein poses several problems. In general, the conjugation
chemistry is difficult to control, resulting in conjugates with
ambiguities in composition and structure, which may affect the
reproducibility of an immune response (Anderson et al., J. Immunol.
1989, 142, 2464-2468). In addition, the foreign carrier protein can
elicit a strong B-cell response, which may lead to the suppression
of an antibody response against the carbohydrate epitope. The
latter is a greater problem when self-antigens are employed such as
tumor-associated carbohydrates. Also linkers for the conjugation of
carbohydrates to proteins can be immunogenic, leading to epitope
suppression (Buskas et al., Chem. Eur. J. 2004, 10, 3517-3523). Not
surprisingly, several clinical trials with carbohydrate-protein
conjugate cancer vaccines failed to induce sufficiently strong
helper T-cell responses in all patients (Sabbatini et al., Int. J.
Cancer 2000, 87, 79-85). Therefore, alternative strategies need to
be developed for the presentation of tumor associated carbohydrate
epitopes that will result in a more efficient class switch to IgG
antibodies (Keil et al., Angew. Chem. Int. Ed. 2001, 40, 366-369;
Angew. Chem. 2001, 113, 379-382; Toyokuni et al., Bioorg. &
Med. Chem. 1994, 2, 1119-1132; Lo-Man et al., Cancer Res. 2004, 64,
4987-4994; Kagan et al., Cancer Immunol. Immunother. 2005, 54,
424-430; Reichel et al., Chem. Commun. 1997, 21, 2087-2088).
[0164] Here we report the synthesis and immunological evaluation of
a structurally well-defined fully synthetic anti-cancer vaccine
candidate (compound 9) that constitutes the minimal structural
features required for a focused and effective T-cell dependent
immune response. The vaccine candidate is composed of the
tumor-associated Tn-antigen, the peptide T-epitope
YAFKYARHANVGRNAFELFL (YAF) (SEQ ID NO:2), and the lipopeptide
S--[(R)-2,3-dipalmitoyloxy-propyl]-N-palmitoyl-(R)-cysteine
(Pam.sub.3Cys). The Tn-antigen, which will serve as a B-epitope, is
over-expressed on the surface of human epithelial tumor-cells of
breast, colon, and prostate. This antigen is not present on normal
cells, and thus rendering it an excellent target for immunotherapy.
To overcome the T-cell independent properties of the carbohydrate
antigen, the YAF peptide was incorporated. This 20 amino acid
peptide sequence is derived from an outer-membrane protein of
Neisseria meningitides and has been identified as a MHC class II
restricted site for human T-cells (Wiertz et al., J. Exp. Med.
1992, 176, 79-88). It was envisaged that this helper T-cell epitope
would induce a T-cell dependent immune response resulting in the
production of IgG antibodies against the Tn-antigen. The combined
B-cell and helper T-cell epitope lacks the ability to provide
appropriate "danger signals" (Medzhitov et al., Science 2002, 296,
298-300) for dendritic cell (DC) maturation. Therefore, the
lipopeptide Pam.sub.3Cys, which is derived from the immunologically
active N-terminal sequence of the principal lipoprotein of
Escherichia coli (Braun, Biochim. Biophys. Acta 1975, 415,
335-377), was incorporated. This lipopeptide has been recognized as
a powerful immunoadjuvant (Weismuller et al., Physiol. Chem. 1983,
364, 593) and recent studies have shown that it exerts its activity
through the interaction with Toll-like receptor-2 (TLR-2)
(Aliprantis et al., Science 1999, 285, 736-73). This interaction
results in the production of pro-inflammatory cytokines and
chemokines, which, in turn, stimulates antigen-presenting cells
(APCs), and thus, initiating helper T cell development and
activation (Werling et al., Vet. Immunol. Immunopathol. 2003, 91,
1-12). The lipopeptide also facilitates the incorporation of the
antigen into liposomes. Liposomes have attracted interest as
vectors in vaccine design (Kersten et al., Biochim. Biophys. Acta
1995, 1241, 117-138) due to their low intrinsic immunogenicity,
thus, avoiding undesirable carrier-induced immune responses.
[0165] The synthesis of target compound 9 requires a highly
convergent synthetic strategy employing chemical manipulations that
are compatible with the presence of a carbohydrate, peptide and
lipid moiety. It was envisaged that 9 could be prepared from spacer
containing Tn-antigen 7, polymer-bound peptide 1, and
S-[2,3-bis(palmitoyloxy)propyl]-N-Fmoc-Cys (Pam.sub.2FmocCys, 2,
(Metzger et al., Int. J. Peptide Protein Res. 1991, 38, 545-554)).
The resin-bound peptide 1 was assembled by automated solid-phase
peptide synthesis using Fmoc protected amino acids in combination
with the hyper acid-sensitive HMPB-MBHA resin and
2-(1H-benzotriazole-1-yl)-oxy-1,1,3,3-tetramethyluronium
hexafluorophosphate/1-hydroxybenzotriazole (HBTU/HOBt) (Knorr et
al., Tetrahedron Lett. 1989, 30, 1927-1930) as the activation
cocktail (Scheme 10). The HMPB-MBHA resin was selected because it
allows the cleavage of a compound from the resin without
concomitant removal of side-chain protecting groups. This feature
was important because side-chain functional groups of aspartic
acid, glutamic acid and lysine would otherwise interfere with the
incorporation of the Tn-antigen derivative 7. Next, the
Pam.sub.2FmocCys derivative 2 was manually coupled to the
N-terminal amine of peptide 1 using PyBOP (Martinez et al., J. Med.
Chem. 1988, 28, 1874-1879) and HOBt in the presence of DIPEA in a
mixture of DMF and dichloromethane to give the resin-bound
lipopeptide 3. The Fmoc group of 3 was removed under standard
conditions and the free amine of the resulting compound 4 was
coupled with palmitic acid in the presence of PyBOP and HOBt to
give the fully protected and resin-bound lipopeptide 5. The amine
of the Pam.sub.2Cys moiety was palmitoylated after coupling with 1
to avoid racemization of the cysteine moiety. Cleavage of compound
5 from the resin was achieved with 2% TFA in dichloromethane
followed by the immediate neutralization with 5% pyridine in
methanol. After purification by LH-20 size exclusion
chromatography, the C-terminal carboxylic acid of lipopeptide 6 was
coupled with the amine of Tn-derivative 7, employing DIC/HOAt/DIPEA
(Carpino, J. Am. Chem. Soc 1993, 115, 4397-4398) as coupling
reagents to give, after purification by Sephadex LH-20
size-exclusion chromatography, fully protected lipidated
glycopeptide 8 in a yield of 79%. Mass spectrometric analysis by
MALDI-TOF showed signals at m/z 5239.6 and 5263.0, corresponding to
[M+H].sup.+ and [M+Na].sup.+, respectively. Finally, the side-chain
protecting groups of 8 were removed by treatment with 95% TFA in
water using 1,2-ethanedithiol (EDT) as a scavenger. It was found
that the alternative use of triisopropyl silane (TIS) resulted in
the formation of unidentified by-products. The target compound 9
was purified by size-exclusion chromatography followed by RP-HPLC
using a Synchropak C4 column. MALDI mass analysis of 9 showed a
signal at m/z 3760.3 corresponding to [M+Na].sup.+.
##STR00014## ##STR00015##
[0166] Next, the compound 9 was incorporated into
phospholipid-based liposomes. Thus, after hydration of a lipid-film
containing 9, cholesterol, phosphatidylcholine and
phosphatidylethanolamine, small uni-lamellar vesicles (SUVs) were
prepared by extrusion through 100 nm Nuclepore.RTM. polycarbonate
membranes. Transmission electron microscopy (TEM) by negative stain
confirmed that the liposomes were uniformly sized with an expected
diameter of approximately 100 nm (see FIG. 1 of Buskas et al.,
Angew. Chem. Int. Ed. 2005, 44, 5985-5988). The liposome
preparations were analyzed for N-acetyl galactosamine content by
hydrolysis with TFA followed by quantification by high pH anion
exchange chromatography. Concentrations of approximately 30
.mu.g/mL of GalNAc were determined, which corresponded to an
incorporation of approximately 10% of the starting compound 9.
[0167] Groups of five female BALB/c mice were immunized
subcutaneously at weekly intervals with freshly prepared liposomes
containing 0.6 .mu.g carbohydrate. To explore the adjuvant
properties of the built-in lipopeptide Pam.sub.3Cys, the
antigen-containing liposomes were administered with or without the
potent saponin immuno-adjuvant QS-21 (Antigenics Inc., Lexington,
Mass.). Anti-Tn antibody titers were determined by coating
microtiter plates with a BSA-Tn conjugate and detection was
accomplished with anti-mouse IgM or IgG antibodies labeled with
alkaline phosphatase. As can be seen in Table 1, the mice immunized
with the liposome preparations elicited IgM and IgG antibodies
against the Tn-antigen (Table 1, entries 1 and 2). The presence of
IgG antibodies indicated that the helper T-epitope peptide of 9 had
activated helper T-lymphocytes. Furthermore, the observation that
IgG antibodies were raised by mice which were only immunized with
liposomes (group 1) indicated that the built-in adjuvant
Pam.sub.3Cys had triggered appropriate signals for the maturation
of DCs and their subsequent activation of helper T-cells. However,
the mice which received the liposomes in combination with QS-21
(group 2), elicited higher titers of anti Tn-antibodies. This
stronger immune response may be due to a shift from a mixed Th1/Th2
to a Th1 response (Moore et al., Vaccine 1999, 17, 2517-2527).
TABLE-US-00001 TABLE 1 ELISA anti-Tn antibody titers.sup.[a] after
4 immunizations with the glycolipopeptide/liposome formulation.
Entry Group IgM Titers IgG Titers 1. 1. Pam.sub.3Cys-YAF-Tn 250
1410 2. 2. Pam.sub.3Cys-YAF-Tn + QS-21 170 2675 .sup.[a]ELISA
plates were coated with a BSA-BrAc-Tn conjugate. All titers are
means for a group of five mice. Titers were determined by
regression analysis, plotting log.sub.10 dilution vs. absorbance.
The titers were calculated to be the highest dilution that gave 0.1
or higher than the absorbance of normal saline mouse sera diluted
1:100.
[0168] The results presented herein provide, for the first time, a
proof-of-principle for the use of lipidated glycopeptides as a
minimal subunit vaccine. It is to be expected that several
improvements can be made. For example, it has been found that a
clustered presentation of the Tn-antigen is a more appropriate
mimetic of mucins, and hence antibodies raised against this
structure recognize better Tn-antigens expressed on cancer cells
(Nakada et al., J. Biol. Chem. 1991, 266, 12402-12405; Nakada et
al., Proc. Natl. Acad. Sci. USA 1993, 90, 2495-2499; Reddish et
al., Glycoconj. J. 1997, 14, 549-560; Reis et al., Glycoconj. J.
1998, 15, 51-62). The Th-epitope employed in this study is known to
be a MHC class II restricted epitope for humans. Thus, a more
efficient class-switch to IgG antibodies may be expected when a
murine Th-epitope is employed. On the other hand, compound 9 is a
more appropriate vaccine candidate for use in humans. A recent
report indicated that Pam.sub.2Cys is a more potent immunoadjuvant
than Pam.sub.3Cys (Jackson et al., Proc. Nat. Acad. Sci. USA 2004,
101, 15440-15445). It has also been suggested that the Pam.sub.2Cys
adjuvant has improved solubility properties (Zeng et al., J.
Immunol. 2002, 169, 4905-4912), which is a problematic feature of
compound 9. Studies addressing these issues are ongoing.
[0169] This work is reported in Buskas et al., Angew. Chem. Int.
Ed. 2005, 44, 5985-5988.
Supporting Information
[0170] Reagents and general experimental procedures. Amino acids
and resins were obtained from Applied Biosystems and NovaBiochem;
DMF from EM science; and NMP from Applied Biosystems.
Phosphatidylethanolamine (PE), cholesterol, phosphatidylcholine
(PC; egg yolk), and phosphatidylglycerol (PG; egg yolk) were from
purchased from Sigma-Aldrich and Fluka. All other chemicals were
purchased from Aldrich, Acros, and Fluka and used without further
purification. All solvents employed were of reagent grade and dried
by refluxing over appropriate drying agents. TLC was performed
using Kieselgel 60 F.sub.254 (Merck) plates, with detection by UV
light (254 nm) and/or by charring with 8% sulfuric acid in ethanol
or by ninhydrine. Column chromatography was performed on silica gel
(Merck, mesh 70-230). Size exclusion column chromatography was
performed on Sephadex LH-20. Extracts were concentrated under
reduced pressure at .ltoreq.40.degree. C. (water bath). An Agilent
1100 series HPLC system equipped with an autosampler, UV-detector
and fraction-collector and a Synchropak C4 column 100.times.4.6 mm
RP with a flow rate of 1 mL/min was used for analysis and
purifications. Positive ion matrix assisted laser desorption
ionization time of flight (MALDI-TOF) mass spectra were recorded
using an HP-MALDI instrument using gentisic acid as a matrix.
.sup.1H NMR and .sup.13C NMR spectra were recorded on a Varian
Inova300 spectrometer, a Varian Inova500 spectrometer, and a Varian
Inova600 spectrometer all equipped with Sun workstations. .sup.1H
spectra recorded in CDCl.sub.3 were referenced to residue
CHCl.sub.3 at 7.26 ppm or TMS, and .sup.13C spectra to the central
peak of CDCl.sub.3 at 77.0 ppm. Assignments were made using
standard 1D experiments and gCOSY/DQCOSY, gHSQC and TOCSY 2D
experiments.
[0171] Lipopeptide 6. Compound 1 was synthesized on HMPB-MBHA resin
(maximum loading, 0.1 mmol). The synthesis of peptide 1 was carried
out on an ABI 433A peptide synthesizer equipped with a UV-detector
using Fmoc-protected amino acids and
2-(1H-benzotriazole-1-yl)-oxy-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU)/1-hydroxybenzotriazole (HOBt) as the
coupling reagents. Single coupling steps were performed with
conditional capping as needed. After completion of the synthesis of
peptide 1, the remaining steps were performed manually.
N-Fluorenylmethoxycarbonyl-S-(2,3-bis(palmitoyloxy)-(2R-propyl)-(R)-cyste-
ine 2 (120 mg, 0.13 mmol) was dissolved in DMF (5 mL) and PyBOP
(0.13 mmol), HOBt (0.13 mmol), and DIPEA (0.27 mmol) were added.
After premixing for 2 min., DCM (1 mL) was added and the mixture
was added to the resin. The coupling step was performed twice. Upon
completion of the coupling, as determined by the Kaiser test, the
N-Fmoc group was cleaved using 20% piperidine in DMF (5 mL).
Palmitic acid (77 mg, 0.3 mmol) was coupled to the free amine as
described above using PyBop (0.3 mmol), HOBt (0.3 mmol) and DIPEA
(0.6 mmol) in DMF. The resin was thoroughly washed with DMF and DCM
and dried under vacuum for 4 h. The fully protected lipopeptide 6
was released from the resin by treatment with 2% trifluoroacetic
acid in DCM (2.5 mL) for 2 min. The mixture was filtered into 5%
pyridine in methanol solution (5 mL). The procedure was repeated
and fractions containing the lipopeptide were pooled and
concentrated to dryness. The crude product was purified by
size-exclusion chromatography (LH-20, DCM/MeOH, 1:1) to give
lipo-peptide 6 (275 mg, 0.057 mmol) as a white solid: R.sub.f=0.57
(DCM/MeOH 9:1); selected NMR data (CDCl.sub.3/CD.sub.3OD 1/1 v/v
600 MHz): .sup.1H, .delta. 0.48-0.90 (m, 27H, Pam CH.sub.3, Leu
CH.sub.3, Val CH.sub.3), 0.96-1.61 (m, Leu CH.sub.2, Leu CH, Lys
CH.sub.2, .sup.tBu CH.sub.3, Boc CH.sub.3, Ala CH.sub.3, Arg
CH.sub.2), 1.18 (br s, 72H, Pam CH.sub.2), 1.95, 1.99 (s,
4.times.3H, Pbf CH.sub.3C), 2.36, 2.41, 2.44 (s, 6.times.3H, Pbf
CH.sub.3), 2.48 (s, 2.times.2H, Pbf CH.sub.2) 2.65-2.73 (m, 6H,
S--CH.sub.2-glyceryl, His CH.sub.2, Cys.sup..beta.), 3.47 (m, 2H,
Gly.sup..alpha.), 3.57 (m, 2H, Gly.sup..alpha.), 4.06 (m, 1H,
S-glyceryl-CH.sub.2.sup.bO), 4.32 (m, 1H,
S-glyceryl-CH.sub.2.sup.aO), 3.65-4.39 (m, 17H, Phe.sup..alpha.,
Ala.sup..alpha., His.sup..alpha., Lys.sup..alpha., Val.sup..alpha.,
Asn.sup..alpha., Glu.sup..alpha., Tyr.sup..alpha.,
Arg.sup..alpha.), 4.45 (m, 1H, Cys.sup..alpha.), 5.06 (m, 1H,
S-glyceryl-CH), 6.72-7.39 (m, 70H, His CH, Tyr aromat, Phe aromat,
Trt aromat), 7.48-8.29 (m, NH). MALDI-MS calcd for
C.sub.269H.sub.373N.sub.33O.sub.42S.sub.3 [M+Na] m/z=4860.22: found
4860.31.
[0172] Protected glycolipopeptide 8. A solution of lipopeptide 6
(22 mg, 4.6 .mu.mol), HOAt (6.3 mg, 46 .mu.mol), and DIC (7 .mu.L,
46 .mu.mol) in DCM/DMF (2/1 v/v, 1.5 mL) was stirred under argon
atm. at ambient temperature for 15 min. Compound 7 (8 mg, 19
.mu.mol) and DIPEA (14 .mu.L, 92 .mu.mol) in DMF (1.5 mL) was added
to the stirred mixture of lipopeptide and the reaction was kept at
room temperature for 18 h. The mixture was diluted with toluene and
concentrated to dryness under reduced pressure. Purification of the
residue by size-exclusion chromatography (LH-20, DCM/MeOH 1:1) gave
compound 8 (19 mg, 79%) as a white solid: selected NMR data
(CDCl.sub.3/CD.sub.3OD 1/1 v/v 600 MHz): .sup.1H, 0.60-0.90 (m,
27H, Pam CH.sub.3, Leu CH.sub.3, Val CH.sub.3), 0.96-1.61 (m, Leu
CH.sub.2, Leu CH, Lys CH.sub.2, .sup.tBu CH.sub.3, Boc CH.sub.3,
Ala CH.sub.3, Arg CH.sub.2), 1.18 (br s, 72H, Pam CH.sub.2), 1.94,
1.98, 1.99, 2.00 (s, 6.times.3H, Pbf CH.sub.3C, HNAc CH.sub.3),
2.36, 2.41, 2.45 (s, 6.times.3H, Pbf CH.sub.3), 2.48 (s,
2.times.2H, Pbf CH.sub.2), 3.42-4.31 (m, Phe.sup..alpha., Ala, Lys,
Val, Asp, Glu, Tyr, Arg, Gly, Leu, His, Asn CH.sub.2, Tyr CH.sub.2,
Phe CH.sub.2, Arg .sup.-CH.sub.2), 3.71 (H-3), 3.88 (H-4) 4.06
(S-glyceryl-CH.sub.2.sup..beta.O), 4.20 (t, 1H, H-2), 4.32 (m, 1H,
S-glyceryl-CH.sub.2.sup..alpha.O), 4.42 (m, 1H, Cys.sup..alpha.),
4.82 (d, 1H, H-1, J=3.68 Hz), 5.06 (m, 1H, S-glyceryl-CH),
6.72-7.39 (m, 70H, His CH, Tyr aromat, Phe aromat, Trt aromat),
7.48-8.29 (m, NH). MALDI-MS calcd for
C.sub.286H.sub.403N.sub.37O.sub.49S.sub.3 [M+Na] m/z=5262.67: found
5262.99.
[0173] Glycolipopeptide 9. Compound 8 (12 mg, 2.3 .mu.mol) in a
deprotection cocktail of TFA/H.sub.2O/ethane-1,2-dithiol
(95:2.5:2.5, 3 mL) was stirred at room temperature for 1 h. The
solvents were removed under reduced pressure and the crude compound
was first purified by a short size-exclusion LH-20 column (DCM/MeOH
1:1) and the then by HPLC using a gradient of 0-100% acetonitrile
in H.sub.2O (0.1% TFA) to give, after lyophilization, compound 9
(6.8 mg, 79%) as a white solid: selected NMR data
(CDCl.sub.3/CD.sub.3OD 600 MHz): .sup.1H, .delta. 0.74-0.96 (m,
27H, Pam CH.sub.3, Leu CH.sub.3, Val CH.sub.3), 1.11-2.35 (Leu
CH.sub.2, Leu CH, sp CH.sub.2, Lys CH.sub.2, Glu CH.sub.2, Ala
CH.sub.3, Val CH, Asp CH.sub.2), 1.29 (br S, 72H, Pam CH.sub.2),
2.43-3.87 (Ala.sup..alpha., Gly.sup..alpha., S-glyceryl-OCH.sub.2,
Cys.sup..beta., H-2, H-3, H-4, H-5, H-6), 4.05-4.73 (m,
Cys.sup..alpha., Phe.sup..alpha., Tyr.sup..alpha., His.sup..alpha.,
Leu.sup..alpha., Lys.sup..alpha., Asp.sup..alpha., Val.sup..alpha.,
Arg.sup..alpha., Glu.sup..alpha., H-1), 5.12 (m, 1H,
S-glyceryl-CH), 6.64-6.71 (dd+dd, 2H, His CH, NH), 6.86-7.12 (dd+dd
2H, His CH, NH) 7.16-8.23 (m, Tyr aromat, Phe aromat, NH).
HR-MALDI-MS calcd for C.sub.186H.sub.297N.sub.37O.sub.41S [M+Na]
m/z=3760.1911: found 3760.3384.
##STR00016##
[0174] Tn derivative 11. Compound 10 was dissolved in DMF (10 mL)
and di-isopropylcarbodiimide (DIC) (82 .mu.L, 0.53 mmol) and HOAt
(216 mg, 1.58 mmol) were added. After stirring for 15 min.,
3-(N-(tert. butyloxycarbonyl)-amino)propanol (111 mg, 0.63 mmol)
was added and the reaction was kept at ambient temperature for 15
h. The mixture was concentrated to dryness under reduced pressure
and the residue was purified by silica gel column chromatography
(0-5% MeOH in DCM) and LH-20 size-exclusion chromatography
(DCM/MeOH 1:1) to give compound 11 (363 mg, 83%). R.sub.f=0.63
(DCM/MeOH 9:1); [.alpha.].sub.D+4.4 (c 1.0 mg/mL,
CH.sub.2Cl.sub.2); NMR data (CDCl.sub.3, 500 MHz): .sup.1H, .delta.
1.27 (d, 3H, CH.sub.3 Thr), 1.43 (s, 9H, .sup.tBu CH.sub.3),
1.46-1.61 (m, 2H, CH.sub.2), 1.99 (s, 3H, CH.sub.3 Ac), 2.05 (s,
6H, CH.sub.3 Ac), 2.06 (s, 3H, CH.sub.3 Ac), 2.17 (s, 3H, CH.sub.3
Ac), 3.17-3.27 (m, 3H, CH.sub.2, CH.sub.2a), 3.48-3.50 (m, 1H,
CH.sub.2b), 4.07-4.28 (m, 6H, H-6, H-5, Thr.sup..alpha.,
Thr.sup..beta., CH Fmoc), 4.43-4.51 (m, 2H, CH.sub.2 Fmoc), 4.62
(dd, 1H, H-2), 4.89 (br t, 1H, NH), 5.04-5.11 (m, 2H, H-1, H-3),
5.41 (d, 1H, H-4), 5.75 (br d, 1H, NH T), 6.81 (br d, 1H, NH
GalNAc), 7.17-7.79 (m, 8H, aromatic H); .sup.13C (CDCl.sub.3, 75
MHz) .delta.17.19, 20.92, 20.99, 21.09, 23.30, 28.55, 30.69, 35.87,
36.92, 47.43, 47.77, 58.57, 62.36, 67.47, 68.68, 77.46, 80.08,
99.88, 120.25, 125.34, 127.35, 128.00, 128.76, 129.13, 141.55,
143.94, 144.01, 156.51, 157.52, 169.68, 170.66, 170.94, 170.99.
[0175] HR-MALDI-MS calcd for C.sub.41H.sub.54N.sub.4O.sub.14 [M+Na]
m/z=849.3535: found 849.3391.
##STR00017##
[0176] Tn derivative 7. A solution of compound 11 (194 mg, 0.24
mmol) in 20% piperidine in DMF (5 mL) was stirred at ambient
temperature for 1 h. The mixture was concentrated to dryness and
the residue was treated with pyridine/acetic anhydride (3:1, 5 mL)
for 2 h. The reaction mixture was diluted with toluene and
concentrated to dryness. The residue was dissolved in
dichloromethane and washed with 1M HCl and sat. aq. NaHCO.sub.3,
dried with MgSO.sub.4, filtered and concentrated. Purification of
the residue by size-exclusion chromatography (LH-20, DCM/MeOH 1:1)
furnished compound 12 (167 mg, 91%): NMR data (CDCl.sub.3, 300
MHz): .sup.1H, .delta. 1.24 (d, 1H, Thr CH.sub.3), 1.42 (s, 9H,
.sup.tBu CH.sub.3), 1.55-1.59 (m, 2H,
NHCH.sub.2CH.sub.2CH.sub.2NH), 1.95, 2.02, 2.03, 2.12, 2.14 (s,
15H, CH.sub.3 Ac), 3.13-3.23 (m, 3H, CH.sub.2+CH.sub.2a), 3.36-3.41
(m, 1H, CH.sub.2b), 4.03-4.12 (m, 2H), 4.19-4.23 (m, 2H,
Thr.sup..beta.), 4.54-4.61 (m, H-2, Thr.sup..alpha.), 4.88 (m, 1H,
NH), 4.96 (s, 1H, J=3.57 Hz, H-1), 5.07 (dd, 1H, H-3), 5.35 (d, 1H,
H-4), 6.43 (br S, 1H, NH), 6.72 (br S, 1H, NH). MALDI-MS calcd for
C.sub.28H.sub.46N.sub.4O.sub.13 [M+Na] m/z=669.296: found 669.323.
Compound 12 was deprotected by stirring with 5% hydrazine-hydrate
in methanol (5 mL) at room temperature for 35 min. The reaction
mixture was diluted with toluene and concentrated. The residue was
co-evaporated twice with toluene. Purification by silica gel column
chromatography (DCM/MeOH 5:1) yielded 13 (119 mg, 89%): NMR data
(CD.sub.3OD, 300 MHz): .sup.1H, .delta. 1.26 (d, 3H, Thr CH.sub.3),
1.43 (s, 9H, .sup.tBu CH.sub.3), 1.57-1.63 (m, 2H,
NHCH.sub.2CH.sub.2CH.sub.2NH), 2.06, 2.10 (s, 2.times.3H NHAc),
2.12-3.09 (m, 2H, CH.sub.2), 3.15 (m, 2H, CH.sub.2), 3.31 (br s,
2H, H-6), 3.68-3.76 (m, 2H, H-3, H-5), 3.88 (d, 1H, H-4), 4.22-4.26
(m, 2H, H-2, Thr.sup..beta.), 4.46 (m, 1H, Thr.sup..alpha.), 4.84
(d, 1H, H-1), 6.60 (br m, 1H, NH), 7.50 (br d, 1H, NH). MALDI-MS
calcd. for C.sub.22H.sub.40N.sub.4O.sub.10 [M+Na] m/z=543.264:
found 543.301. A solution of 13 in trifluoro acetic acid (4 mL) was
stirred under an argon atmosphere at ambient temperature for 45
min. The reaction mixture was then diluted with DCM and
concentrated to dryness. The crude product was purified by column
chromatography (Iatro beads, EtOAc/MeOH/H.sub.2O
2:2:1.fwdarw.MeOH/H.sub.2O 1:1). After concentration of the pooled
fractions, the solid was lyophilized from H.sub.2O to give compound
7 (91 mg, 0.21 mmol, 95%) as a white powder. R.sub.f=0.17
(EtOAc/MeOH/H.sub.2O 6:3:1); [.alpha.].sub.D-37 (c 1.0 mg/mL,
H.sub.2O); NMR data (D.sub.2O, 300 MHz): .sup.1H, .delta. 1.15 (d,
3H, J=6.3 Hz, Thr CH.sub.3), 1.73-1.77 (m, 2H, CH.sub.2), 1.95 (s,
3H, NHAc), 2.04 (s, 3H, NHAc), 2.82-2.87 (m, 2H, CH.sub.2),
3.11-3.15 (m, 1H, CH.sub.2a), 3.22-3.26 (m, 1H, CH.sub.2b), 3.65
(m, 2H, H-6), 3.76 (dd, 1H, J=2.9, 11.2 Hz, H-3), 3.87 (d, 1H,
J=2.9 Hz, H-4), 3.92 (t, 1H, H-5), 3.99 (dd, 1H, J=3.41, 11.2 Hz,
H-2), 4.28-4.30 (m, 1H, Thr.sup..beta.), 4.32 (d, 1H, J=2.4 Hz,
Thr.sup..alpha.) 4.78 (d, 1H, J=3.56 Hz, J=3.9 Hz, H-1), 7.97 (br
d, 1H, NH), 8.17 (br t, 1H, NH), 8.27 (br d, 1H, NH); .sup.13C
(D.sub.2O, 75 MHz), .delta. 18.17 Thr CH.sub.3), 21.93, 22.33
(2.times.NAc) 26.98 (CH.sub.2), 36.55 (CH.sub.2), 37.22 (CH.sub.2),
49.98 (C-6), 58.30 (C-3), 61.46 (C-4), 67.76 (C-5), 68.65 (C-2),
71.54 (C-Thr.sup..beta.), 74.60 (C-Thr.sup..alpha.), 98.60 (C-1),
172.09, 174.37, 175.18 (3.times.C.dbd.O, NHAc). HR-MALDI-MS calcd
for C.sub.17H.sub.32N.sub.4O.sub.8 [M+Na] m/z=443.2118: found
443.2489.
[0177] Liposome preparation. Liposomes were prepared from PC, PG,
cholesterol, and the glycolipopeptide 9 (15 .mu.mol, molar ratio
65:25:50:10). The lipids were dissolved in DCM/MeOH (3/1, v/v)
under an atmosphere of argon. The solvent was then removed by
passing a stream of dry nitrogen gas, followed by further drying
under high vacuum for one hour. The resulting lipid film was
suspended in 1 mL 10 mM Hepes buffer, pH 6.5, containing 145 mM
NaCl. The solution was vortexed on a shaker (250 rpm), under Ar
atmosphere at 41.degree. C. for 3 hours. The liposome suspension
was extruded ten-times through 0.6 .mu.m, 0.2 .mu.m and 0.1 .mu.m
polycarbonate membranes (Whatman, Nuclepore.RTM., Track-Etch
Membrane) at 50.degree. C. to obtain SUV.
[0178] Immunizations. Groups of five mice (female BALB/c, 6 weeks)
were immunized subcutaneously on days 0, 7, 14 and 21 with 0.6
.mu.g of carbohydrate-containing liposomes and 10 .mu.g of the
adjuvant QS-21 in each boost. The mice were bled on day 28
(leg-vein) and the sera were tested for the presence of
antibodies.
[0179] ELISA. 96-well plates were coated over night at 4.degree. C.
with Tn-BSA, (2.5 .mu.g mL.sup.-1) in 0.2 M borate buffer (pH 8.5)
containing 75 mM sodium chloride (100 .mu.L) per well). The plates
were washed three times with 0.01 M Tris buffer containing 0.5%
Tween 20% and 0.02% sodium azide. Blocking was achieved by
incubating the plates 1 h at room temperature with 1% BSA in 0.01 M
phosphate buffer containing 0.14 M sodium chloride. Next, the
plates were washed and then incubated for 2 h at room temperature
with serum dilutions in phosphate buffered saline. Excess antibody
was removed and the plates were washed three times. The plates were
incubated with rabbit anti-mouse IgM and IgG Fc.gamma. fragment
specific alkaline phosphatase conjugated antibodies (Jackson
ImmunoResearch Laboratories Inc., West Grove, Pa.) for 2 h at room
temperature. Then, after the plates were washed, enzyme substrate
(p-nitrophenyl phosphate) was added and allowed to react for 30 min
before the enzymatic reaction was quenched by addition of 3 M
aqueous sodium hydroxide and the absorbance read at dual
wavelengths of 405 and 490 nm. Antibody titers were determined by
regression analysis, with log.sub.10 dilution plotted against
absorbance. The titers were calculated to be the highest dilution
that gave two times the absorbance of normal mouse sera diluted
1:120.
Example II
Non-Covalently Linked Diepitope Liposome Preparations
[0180] In a first set of experiments, the tumor-related
carbohydrate B-epitope and the universal T-epitope peptide were
incorporated separately into preformed liposomes to form a
diepitopic construct. Additionally, the lipopeptide Pam.sub.3Cys
was incorporated into the liposome with the expectation that it
would function as a built-in adjuvant, and thus circumvent the
necessity of using an additional external adjuvant, such as
QS-21.
[0181] The liposomes were prepared from lipid anchors carrying two
different thiol-reactive functionalities, maleimide and
bromoacetyl, at their surface. The Pam.sub.3Cys adjuvant was also
incorporated into the preformed liposome and included a maleimide
functionality. Conveniently, the maleimide and the bromoacetyl
group show a marked difference in their reactivity towards
sulfhydryl groups. The maleimide reacts rapidly with a sulfhydryl
compound at pH 6.5, whereas the bromoacetyl requires slightly
higher pH 8-9 to react efficiently with a thiol compound.
[0182] By exploiting this difference in reactivity, a diepitope
liposome construct carrying the cancer related Le.sup.y
tetrasaccharide and the universal T helper peptide QYIKANSKFIGITEL
(QYI) (SEQ ID NO:1) was prepared (Scheme 11). For the conjugation
to the thiol-reactive anchors, both the oligosaccharide and the
peptide were functionalized with a thiol-containing linker. The
two-step consecutive conjugation to preformed liposomes has a great
advantage: it is a very flexible approach that makes it easy to
prepare liposomes carrying an array of different carbohydrate
B-epitopes. The yield of conjugation, as based on quantitating the
carbohydrate and peptide covalently coupled to the vesicles, was
high, 70-80% for the oligosaccharide and 65-70% for the peptide,
and the results were highly reproducible.
[0183] It is important to note that in these first diepitope
liposome constructs, the carbohydrate B-epitope and peptide
T-epitope are not themselves joined together by covalent linkages,
but rather are held in proximity by their respective lipid anchors
to which they are conjugated, and by hydrophobic interactions. It
has been shown in several reports in the literature regarding
vaccine candidates with pathogen-related peptide B-epitopes that
this approach is successful leading to good titers of both IgM and
specific IgG antibodies. These studies also indicate that the
built-in adjuvant Pam.sub.3Cys is sufficient to induce a proper
immune response.
[0184] However, in our study with the tumor-related carbohydrate
B-epitope Le.sup.y, immunizations of mice using the non-covalently
linked diepitope liposome preparation described in this Example
resulted in only very low titers of IgM antibodies. No IgG
anti-Le.sup.y antibodies were detected. Even more surprising,
co-administering the liposomal vaccine candidate with the powerful
external adjuvant, QS-21, did not improve the outcome.
Additionally, it was found that mice that had been immunized with
an un-coated liposome control, i.e. a liposome that carried nothing
but the maleimide and bromoacetyl functional groups on the surface,
elicited high titers of IgG antibodies as detected by ELISA. More
detailed ELISA studies of the anti-sera from this group of mice
using a variety of protein conjugates revealed that the mice had
responded to and elicited antibodies towards the maleimide linker.
Also the anti-sera from the mice immunized with the liposomes
coated with the Le.sup.y antigen and the QYI peptide were screened
for anti-linker antibodies and it was found that also these mice
had elicited IgG antibodies towards the maleimide linker.
##STR00018##
[0185] Due to its high reactivity at near neutral pH, the maleimide
linker is widely used in conjugation chemistry to reach glyco- and
peptide-protein conjugates that are further used in immunization
studies. There are commercially available protein conjugation kits
(Pierce Endogen Inc.) that utilize the maleimide linker both for
the antigenic conjugate and the detection conjugate. Our data show
that using these kits can lead to false positive results,
especially when working with antigens of low immunogenicity (See T.
Buskas, Y. Li and G-J. Boons, Chem. Eur. J., 10:3517-3523,
2004).
[0186] To test whether the highly immunogenic maleimide linker
suppressed the immune response towards the Le.sup.y
tetrasaccharide, we prepared the non-covalent diepitope liposome
using only the bromoacetyl linker. In this experiment, the
thiol-containing Le.sup.y tetrasaccharide and the universal T
helper peptide were conjugated, in separate reactions, to lipids
containing the bromoacetyl linker. The conjugated lipids were then
mixed together to form lipid vesicles. Administering this new
liposome formulation to mice, with or without the external adjuvant
QS-21, raised only low titers of anti-Le.sup.y antibodies. Thus,
the lack of an effective immune response toward the Le.sup.y
tetrasaccharide was not due solely to the immunogenic maleimide
linker.
[0187] Since the tumor-associated Le.sup.y tetrasaccharide is known
to be only weakly immunogenic, we prepared another diepitope
liposomal construct where the more immunogenic Tn(cluster) antigen
was used as a target B-epitope. However, the same negative results
were obtained with this antigen. Again, immunizations of mice
resulted in only very low titers of anti-Tn(c) IgM antibodies.
Co-administering with QS-21 as an external adjuvant did nothing to
enhance the immune response.
[0188] From these results we concluded that the non-covalently
linked diepitope liposome approach that has proven successful for a
range of peptide antigens failed when a tumor-associated
carbohydrate antigen of low immunogenicity was used as a B-epitope.
Thus, we reasoned that the tumor-associated carbohydrate B-epitope
and the helper T-epitope needed to be presented differently to the
immune system to evoke a T-cell dependent immune response.
Example III
Covalently Linked Diepitope Liposome Preparations
[0189] We speculated that in order to achieve a better presentation
of the carbohydrate B-epitope and peptide T-epitope, perhaps they
needed to be covalently linked together. To test this idea we
synthesized construct 1 (Scheme 12), a structurally well-defined
anti-cancer vaccine candidate containing the structural features
needed for a focused and effective T-cell dependent immune
response. The vaccine candidate is composed of the tumor-associated
Tn-antigen, the peptide T-epitope YAFKYARHANVGRNAFELFL (YAF) (SEQ
ID NO:2) (Neisseria meningitides) and the lipopeptide Pam.sub.3Cys.
Due to difficulties in the synthesis using the original helper
T-epitope peptide QYI, a different universal T-epitope (YAF) that
displayed better solubility properties was used in this study.
[0190] Compound 1 was synthesized in a highly convergent manner by
a combination of solid-phase and solution phase synthesis.
##STR00019##
[0191] The construct was then incorporated into phospholipid-based
liposomes. Compound 1 suffers from low solubility in a range of
solvents, which probably is the main reason the incorporation into
the liposomes was only 10%.
[0192] Mice were immunized with the construct at weekly intervals.
To explore the adjuvant properties of the built-in lipopeptide
Pam.sub.3Cys, the antigen-containing liposomes were administered
with (group 2) or without (group 1) the adjuvant QS-21.
[0193] As can be seen in Table 1 (Example I), the mice immunized
with the liposome preparations elicited both IgM and IgG antibodies
against the Tn-antigen (Table 1, entries 1 and 2). The presence of
IgG antibodies indicated that the helper T-epitope peptide of 1 had
activated helper T-lymphocytes. Furthermore, the observation that
IgG antibodies were raised by mice which were immunized with
liposomes in the absence of the external adjuvant QS-21 (group 1)
indicated that the built-in adjuvant Pam.sub.3Cys had triggered
appropriate signals for the maturation of DCs and their subsequent
activation of helper T-cells. However, the mice which received the
liposomes in combination with QS-21 (group 2) elicited higher
titers of anti Tn-antibodies. This stronger immune response may be
due to a shift from a mixed Th1/Th2 to a Th1 skewed response.
[0194] The results provide, for the first time, a
proof-of-principle for the use of a lipidated glycopeptide that
contains a carbohydrate B-epitope, a helper T-cell epitope and a
lipopeptide adjuvant as a minimal, self-contained subunit vaccine.
It was also concluded that to evoke a T-cell dependent immune
response toward the tumor-associated carbohydrate antigen, it is
not enough that the carbohydrate B-epitope and the peptide
T-epitope are presented together in a non-covalent manner on the
surface of a adjuvant-containing liposome; rather, the entities are
preferably covalently joined together. Finally, it was observed
that an external adjuvant (QS-21) was not needed when the three
components (carbohydrate B-epitope, helper T-cell epitope and
lipopeptide) are covalently linked to form the lipidated
glycopeptides.
Alternative Glycolipopeptide Components
[0195] Several improvements can be made to compound 1. For example,
it has been found that antibodies elicited against the Tn-antigen
poorly recognize cancer cells. However, clustering (Nakada et al.,
Proc. Natl. Acad. Sci. USA 1993, 90, 2495-2499; Reddish et al.,
1997, 14, 549-560; Zhang et al., Cancer Res. 1995, 55, 3364-3368;
Adluri et al., Cancer Immunol. Immunother 1995, 41, 185-192) or
presenting the Tn antigen as part of the MUC-1 glycopeptide elicits
antibodies with improved binding characteristics (Snijdewint et
al., Int. J. Cancer 2001, 93, 97-106 The T-epitope employed in
compound 1 is a MHC class II restricted epitope for humans. Thus, a
more efficient class-switch to IgG antibodies may be expected when
a murine T-epitope is used. Furthermore, it has been found that the
lipopeptide Pam.sub.2Cys or Pam.sub.3CysSK.sub.4 are more potent
immunoadjuvants than Pam.sub.3Cys (Spohn et al., Vaccine 2004, 22,
2494-2499). However, it was not known whether attachment of
Pam.sub.2Cys or Pam.sub.3CysSK.sub.4 to the T- and B-epitope would
affect their efficacies and potencies. Thus, based on these
considerations, compounds 2 and 3 (Scheme 12) were designed, which
contain the MUC-1 glycopeptide as a B-epitope, the well-documented
murine helper T-cell epitope KLFAVWKITYKDT (KLF) (SEQ ID NO:3)
derived from Polio virus (Leclerc et al., J. Virol. 1991, 65,
711-718) as the T-epitope, and the lipopeptide Pam.sub.2Cys or
Pam.sub.3CysSK.sub.4, respectively.
[0196] Glycolipopeptides 2 and 3 were incorporated into
phospholipid-based liposomes as described for compound 1.
Surprisingly, the solubility problems that plagued compound 1 were
not an issue for compounds 2 and 3. Female BALB/c mice were
immunized four times at weekly intervals with the liposome
formulations with or without the external adjuvant QS-21 (Kensil et
al., J. Immunol. 1991, 146, 431-437). Anti-Muc1 antibody titers
were determined by coating microtiter plates with
CTSAPDT(.alpha.GalNAc)RPAP conjugated to BSA and detection was
accomplished with anti-mouse IgG antibodies labeled with alkaline
phosphatase. The results are summarized in Tables 2 and 3.
TABLE-US-00002 TABLE 2 ELISA anti-MUC-1 antibody titers* after 4
immunizations with the glycolipopeptide/liposome formulations.
Entry Group IgG1 1. 1. Pam.sub.2Cys-MUC-1 24,039 2. 2.
Pam.sub.2Cys-MUC-1 + QS-21 36,906 3. 3. Pam.sub.3Cys-MUC-1 183,085
4. 4. Pam.sub.3Cys-MUC-1 + QS-21 450,494 *ELISA plates were coated
with a BSA-BrAc-MUC-1 conjugate. Anti-MUC1 antibody titers are
presented as means of groups of five mice. Titers are defined as
the highest dilution yielding an optical density of 0.1 or greater
over background of blank mouse sera.
TABLE-US-00003 TABLE 3 ELISA anti-MUC-1 antibody titers* after 4
immunizations with the glycolipopeptide/liposome formulations.
Entry Group IgG1 IgG2a IgG2b IgG3 1. 1. Pam.sub.2Cys-MUC-1 74,104
3,599 5,515 17,437 2. 2. Pam.sub.2Cys- 126,754 22,709 5,817 20,017
MUC-1 + QS-21 3. 3. Pam.sub.3Cys-MUC-1 448,023 57,139 61,094
115,131 4. 4. Pam.sub.3Cys- 653,615 450,756 70,574 305,661 MUC-1 +
QS-21 *ELISA plates were coated with a BSA-BrAc-MUC-1 conjugate.
Anti-MUC1 antibody titers are presented as means of groups of five
mice. Titers are defined as the highest dilution yielding an
optical density of 0.1 or greater over background of blank mouse
sera.
[0197] As can be seen in Table 2, mice immunized with liposomal
preparations of compounds 2 and 3 elicited high titers of
anti-MUC-1 IgG antibodies. Surprisingly, mice that were immunized
with the Pam.sub.3CysSK.sub.4-based vaccine elicited higher titers
of antibodies than mice immunized with Pam.sub.2Cys derivative.
These results are contradictory to reports that have compared
adjuvancy of Pam.sub.2Cys and Pam.sub.3CysSK.sub.4. Sub-typing of
the IgG antibodies (IgG1, IgG2a, IgG2b and IgG3) indicated a bias
towards a Th2 immune response (entries 1 and 3, Table 3).
Co-administering of the adjuvant QS-21 did not lead to a
significant increase of IgG antibody, however, in these cases a
mixed Th1/Th2 response was observed (entries 2 and 4, Table 3).
[0198] To ensure that the mouse sera were able to recognize native
MUC-1 glycopeptide present on cancer cells, the binding of the sera
to the MUC-1 expressing MCF-7 human breast cancer cell line was
examined. Thus, the cells were treated with a 1:50 diluted sera for
30 minutes after which goat anti-mouse IgG antibodies labeled with
FITC was added. The percentage of positive cells and mean
fluorescence was determined by flow cytometry analysis. As can be
seen in (FIG. 2), the anti-sera reacted strongly with the MUC-1
positive tumor cells whereas no binding was observed for sera
obtained from naive mice. Furthermore, no binding was observed when
SK-MEL 28 cell were employed, which do not express the MUC-1
glycopeptide. These results demonstrate that anti-MUC-1 antibodies
induced by 3 recognize the native antigen on human cancer cells.
Further ELISA studies showed that titers against the T-epitope were
very low, showing that no significant epitope suppression had
occurred.
[0199] The lipopeptide moiety of the three-component vaccine is
required for initiating the production of necessary cytokines and
chemokines (danger signals) (Bevan, Nat. Rev. Immunol. 2004, 4,
595-602; Eisen et al., Curr. Drug Targets 2004, 5, 89-105; Akira et
al., Nat. Immunol. 2001, 2, 675-680; Pasare et al., Immunity 2004,
21, 733-741; Dabbagh et al., Curr. Opin. Infect. Dis. 2003, 16,
199-204; Beutler, Mol. Immunol. 2004, 40, 845-859). The results of
recent studies indicate that the lipopeptide initiates innate
immune responses by interacting with the Toll-like receptor 2 on
the surface of mononuclear phagocytes. After activation, the
intracellular domain of TLR-2 recruits the adaptor protein MyD88,
resulting in the activation of a cascade of kinases leading to the
production of a number of cytokines and chemokines. On the other
hand, lipopolysaccharides induce cellular responses by interacting
with the Toll-like receptor 4 (TLR4)/MD2, which results in the
recruitment of the adaptor proteins MyD88 and TRIF leading to a
more complex pattern of cytokine. TNF-.alpha. secretion is the
prototypical measure for activation of the MyD88-dependent pathway,
whereas secretion of IFN-.beta. is commonly used as an indicator of
TRIF-dependent cellular activation (Akira et al., Nat. Immunol.
2001, 2, 675-680; Beutler, Mol. Immunol. 2004, 40, 845-859).
[0200] To examine whether attachment of a glycopeptide containing a
T epitope and a B epitope to the TLR ligand affects cytokine
production, the efficacy (EC.sub.50) and potency (maximum
responsiveness) of TNF-.alpha. and IFN-.beta. secretion induced by
compounds 1, 2 and 3 was determined and the results compared with
those of Pam.sub.2CysSK.sub.4, Pam.sub.3CysSK.sub.4 and LPS. Thus,
RAW NO.sup.- mouse macrophages were exposed over a wide range of
concentrations to compounds 1, 2 and 3, Pam.sub.2CysSK.sub.4,
Pam.sub.3CysSK.sub.4 and E. coli 055:B5 LPS. After 5 hours, the
supernatants were harvested and examined for mouse TNF-.alpha. and
IFN-.beta. using commercial or in-house developed capture ELISA
assays, respectively.
TABLE-US-00004 TABLE 4 EC.sub.50 and E.sub.max values of
concentration-response curves of E. coli LPS and synthetic
compounds for TNF-.alpha. production by mouse macrophages (RAW
.gamma.NO(-) cells). EC.sub.50 (nM)* E.sub.max (pg/mL)* E. coli LPS
0.002 2585 1 10.230 363 Pam.sub.2CysSK.sub.4 0.003 631 2 0.223 622
Pam.sub.3CysSK.sub.4 3.543 932 3 2.151 802 *Values of EC50 and Emax
are reported as best-fit values according to Prism (GraphPad
Software, Inc). Concentration-response data were analyzed using
nonlinear least-squares curve fitting in Prism.
[0201] As can be seen in FIG. 3 and Table 4, glycolipopeptide 3 and
Pam.sub.3CysSK.sub.4 induced the secretion of TNF-.alpha. with
similar efficacies and potencies indicating that attachment of the
B-epitope and T-epitope had no effect on cytokine and chemokine
responses. Surprisingly, attachment of the B-epitope and the
T-epitope to Pam.sub.2CysSK.sub.4 led to a significant reduction in
potency and thus in this case the attachment of the B-epitope and
the T-epitope led to a reduction in activity. Compound 1 which
contains the Pam.sub.3Cys moiety is significantly less active than
the compounds 2 and 3, which may explain the poor antigenicity of
compound 1. Compounds 1, 2 and 3 did not induce the production of
INF-.beta.. Surprisingly, E. coli 055:B5 displayed much larger
potencies and efficacies for TNF-.alpha. induction compared to
compounds 1, 2, 3, and Pam.sub.3CysSK.sub.4. In addition, it was
able to stimulate the cells to produce INF-.beta.. E. coli LPS is
too active resulting in over-activation of the innate immune
system, leading to symptoms of septic shock.
[0202] It was speculated that in addition to initiating the
production of cytokines and chemokines, the lipopeptide may
facilitate selective targeting and uptake by antigen presenting
cells in a TLR2 dependent manner. To test this hypothesis,
compounds 4, which contains a fluorescence label, was administered
to RAW NO.sup.- mouse macrophages and after 30 minutes the cells
were harvested, lysed and the fluorescence measured. To account for
possible cell surface binding without internalization, the cells
were also trypsinized before lyses and then examination for
fluorescence. As can be seen in FIG. 4, a significant quantity of
the 4 was internalized whereas a small amount was attached to the
cell surface. To determine whether the uptake was mediated by TLR2,
the uptake studies were repeated using native HEK297 cell and
HEK297 cell transfected with either TLR2 or TLR4/MD2. Importantly,
significant uptake was only observed when the cells were
transfected with TLR2 indicating that uptake is mediated by this
receptor. These studies show that TLR2 facilitates the uptake of
antigen, which is an important step in antigen processing and
immune responses.
Example IV
Covalent Attachment of the Lipid Component
[0203] To establish the importance of covalent attachment of the
TLR ligand to the vaccine candidate, compound 5 (Scheme 13) which
only contains the B-epitope and the T-epitope was designed and
synthesized. Mice were immunized four times at weekly intervals
with this compound in the presence of PAM.sub.3CysSK.sub.4.
Interestingly, the mixture of glycopeptide 5 and the adjuvant
Pam.sub.3CysSK.sub.4 elicited no- or very low titers of IgG
antibodies, demonstrating that covalent attachment of
Pam.sub.3CysSK.sub.4 to the B-epitope and T-epitope is critical for
strong immune responses.
##STR00020##
Example V
Lipid Component
[0204] To determine the importance of lipidation with a ligand of a
Toll like receptor, compound 6 (Scheme 14) was designed and
synthesized. This compound is composed of the B-epitope and
T-epitope linked to non-immunogenic lipidated amino acids. Mice
were immunized with a liposomal preparation of compound 6, similar
to the procedure employed for compound 1 and 2. Liposomes
containing compound 6 induced titers that were significantly lower
than those elicited by compound 3, demonstrating that a TLR ligand
of the three-component vaccine is important for optimal immune
responses.
##STR00021##
Conclusions
[0205] The three-component carbohydrate-based vaccine has a number
of distinctive advantages over a traditional conjugate vaccine. For
example, the minimal subunit vaccine does not suffer from epitope
suppression, a characteristic of carbohydrate-protein conjugates.
Apart from providing danger signals, the lipopeptide
Pam.sub.3CysSK.sub.4 also facilitates the incorporation of the
antigen into liposomes. A liposomal formulation is attractive
because it presents efficiently the antigen to the immune system. A
unique feature of the vaccine is that Pam.sub.3CysSK.sub.4 promotes
selective targeting and uptake by antigen presenting cells,
T-helper cells and B-lymphocytes, which express Toll loll like
receptors (Example III). Finally, a fully synthetic compound has as
an advantage that it can be fully characterized, which facilitates
its production in a reproducible manner.
Example VI
Increasing the Antigenicity of Synthetic Tumor-Associated
Carbohydrate Antigens by Targeting Toll-Like Receptors
[0206] In this Example, a number of fully synthetic vaccine
candidates have been designed, chemically synthesized, and
immunologically evaluated to establish strategies to overcome the
poor immunogenicity of tumor-associated carbohydrates and
glycopeptides and to study in detail the importance of TLR
engagement for antigenic responses. Covalent attachment of a TLR2
agonist, a promiscuous peptide T-helper epitope, and a
tumor-associated glycopeptide, gives a compound that elicits in
mice exceptionally high titers of IgG antibodies which recognize
cancer cells expressing the tumor-associated carbohydrate.
[0207] The over-expression of oligosaccharides, such as Globo-H,
LewisY, and Tn antigens is a common feature of oncogenic
transformed cells (Springer, Mol. Med. 1997, 75, 594-602; Hakomori,
Acta Anat. 1998, 161, 79-90; Dube, Nat. Rev. Drug Discov. 2005, 4,
477-488). Numerous studies have shown that this abnormal
glycosylation can promote metastasis (Sanders, J. Clin. Pathol.
Mol. Pathol. 1999, 52, 174-178) and hence the expression of these
compounds is strongly correlated with poor survival rates of cancer
patients. A broad and expanding body of preclinical and clinical
studies demonstrates that naturally acquired, passively
administered or actively induced antibodies against
carbohydrate-associated tumor antigens are able to eliminate
circulating tumor cells and micro-metastases in cancer patients
(Livingston, Cancer Immunol. 1997, 45, 10-19; Ragupathi, Cancer
Immunol. 1996, 43, 152-157; von Mensdorff-Pouilly, Int. J. Cancer
2000, 86, 702-712; Finn, Nat. Rev. Immunol. 2003, 3, 630-641).
[0208] Traditional cancer vaccine candidates composed of a
tumor-associated carbohydrate (Globo-H, Lewis.sup.Y, and Tn)
conjugated to a foreign carrier protein (e.g. KLH and BSA) have
failed to elicit sufficiently high titers of IgG antibodies in most
patients. It appears that the induction of IgG antibodies against
tumor-associated carbohydrates is much more difficult than
eliciting similar antibodies against viral and bacterial
carbohydrates. This observation is not surprising because tumor
associated saccharides are self-antigens and consequently tolerated
by the immune system. The shedding of antigens by the growing tumor
reinforces this tolerance. In addition, a foreign carrier protein
such as KLH can elicit a strong B-cell response, which may lead to
the suppression of an antibody response against the carbohydrate
epitope. The latter is a greater problem when self-antigens such as
tumor-associated carbohydrates are employed. Also, linkers that are
utilized for the conjugation of carbohydrates to proteins can be
immunogenic leading to epitope suppression (Buskas, Chem. Eur. J.
2004, 10, 3517-3524; Ni, Bioconjug. Chem. 2006, 17, 493-500). It is
clear that the successful development of a carbohydrate-based
cancer vaccine requires novel strategies for the more efficient
presentation of tumor-associated carbohydrate epitopes to the
immune system, resulting in a more efficient class switch to IgG
antibodies (Reichel, J. Chem. Commun. 1997, 21, 2087-2088;
Alexander, J. Immunol. 2000, 164, 1625-1633; Kudryashov, Proc.
Natl. Acad. Sci. U.S.A. 2001, 98, 3264-3269; Lo-Man, J. Immunol.
2001, 166, 2849-2854; Jiang, Curr. Med. Chem. 2003, 10, 1423-1439;
Jackson, Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15440-5; Lo-Man,
Cancer Res. 2004, 64, 4987-4994; Buskas, Angew. Chem. Int. Ed.
2005, 44, 5985-5988 (Example I); Dziadek, Angew. Chem. Int. Ed.
2005, 44, 7630-7635; Krikorian, Bioconjug. Chem. 2005, 16, 812-819;
Pan, J. Med. Chem. 2005, 48, 875-883).
[0209] Advances in the knowledge of the cooperation of innate and
adaptive immune responses (Pasare, Semin. Immunol. 2004, 16, 23-26;
Pashine, Nat. Med. 2005, 11, S63-S68; Akira, Nat. Rev. Immunol.
2004, 4, 499-511; O'Neill, Curr Opin Immunol 2006, 18, 3-9; Lee,
Semin Immunol 2007, 19, 48-55; Ghiringhelli, Curr Opin Immunol
2007, 19, 224-31) are offering new avenues for vaccine design for
diseases such as cancer, for which traditional vaccine approaches
have failed. The innate immune system responds rapidly to families
of highly conserved compounds, which are integral parts of
pathogens and perceived as danger signals by the host. Recognition
of these molecular patterns is mediated by sets of highly conserved
receptors, such as Toll-like receptors (TLRs), whose activation
results in acute inflammatory responses such as direct local attack
against invading pathogens and the production of a diverse set of
cytokines. Apart from antimicrobial properties, the cytokines and
chemokines also activate and regulate the adaptive component of the
immune system (Lin, J Clin Invest 2007, 117, 1175-83). In this
respect, cytokines stimulate the expression of a number of
co-stimulatory proteins for optimum interaction between T-helper
cells and B- and antigen presenting cells (APC). In addition, some
cytokines and chemokines are responsible for overcoming suppression
mediated by regulatory T-cells. Other cytokines are important for
directing the effector T-cell response towards a T-helper-1 (Th-1)
or T-helper-2 (Th-2) phenotype (Dabbagh, Curr. Opin. Infect. Dis.
2003, 16, 199-204).
[0210] Recently, we described a fully synthetic three-component
vaccine candidate (compound 21, FIG. 5) composed of a
tumor-associated MUC-1 glycopeptide B-epitope, a promiscuous helper
T-cell epitope and a TLR2 ligand (Buskas, Angew. Chem. Int. Ed.
2005, 44, 5985-5988 (Example I); Ingale, Nat. Chem. Biol. 2007, 3,
663-667; Ingale, J. Org. Lett. 2006, 8, 5785-5788; Bundle, Nat.
Chem. Biol. 2007, 3, 604-606). The exceptional antigenic properties
of the three-component vaccine were attributed to the absence of
any unnecessary features that are antigenic and may induce immune
suppression. It contains, however, all the mediators required for
eliciting relevant IgG immune responses. Furthermore, attachment of
the TLR2 agonist Pam.sub.3CysSK.sub.4 to the B- and T-epitopes
ensures that cytokines are produced at the site where the vaccine
interacts with immune cells. This leads to a high local
concentration of cytokines facilitating maturation of relevant
immune cells. Apart from providing danger signals, the lipopeptide
Pam.sub.3CysSK.sub.4 facilitates the incorporation of the antigen
into liposomes and promotes selective targeting and uptake by
antigen presenting cells and B-lymphocytes.
[0211] To establish the optimal architecture of a fully synthetic
three-component cancer vaccine and to study in detail the
importance of TLR engagement for antigenic responses, we have
chemically synthesized, and immunologically evaluated a number of
fully synthetic vaccine candidates. It has been found that a
liposomal preparation of compound 22, which is composed of an
immunosilent lipopeptide, a promiscuous peptide T-helper epitope,
and a MUC-1 glycopeptide, is significantly less antigenic than
compound 21, which is modified with a TLR2 ligand
(Pam.sub.3CysSK.sub.4). However, liposomal preparations of compound
22 with Pam.sub.3CysSK.sub.4 (23) or monophosphoryl lipid A (24),
which are TLR2 and TLR4 agonists, respectively, elicited titers
comparable to compound 21. However, the antisera elicited by
mixtures of 22 and 23 or 24 had an impaired ability to recognize
cancer cells. Surprisingly, a mixture of compounds 25 and 26, which
are composed of a MUC-1 glycopeptide B-epitope linked to lipidated
amino acids and the helper T-epitope attached to
Pam.sub.3CysSK.sub.4, did not raise antibodies against the MUC-1
glycopeptide. Collectively, the results demonstrate that TLR
engagement is not essential but greatly enhanced antigenic
responses against the tumor-associated glycopeptide MUC-1. Covalent
attachment of the TLR agonist to the B- and helper T-epitope is
important for antibody maturation for improved cancer cell
recognition.
Results and Discussion.
Chemical Synthesis.
[0212] Compound 21 (FIG. 5), which contains as B-epitope a
tumor-associated glycopeptide derived from MUC-1 (Berzofsky, Nat.
Rev. Immunol. 2001, 1, 209-219; Baldus, Crit. Rev. Clin. Lab. Sci.
2004, 41, 189-231; Apostolopoulos, Curr. Opin. Mol. Ther. 1999, 1,
98-103; Hang, Bioorg. Med. Chem. Lett. 2005, 13, 5021-5034), the
well-documented murine MHC class II restricted helper T-cell
epitope KLFAVWKITYKDT (SEQ ID NO:3) derived from the Polio virus
(Leclerc, J. Virol. 1991, 65, 711-718), and the lipopeptide
Pam.sub.3CysSK.sub.4 (TLR2 agonist) (Spohn, Vaccine 2004, 22,
2494-2499), was previously shown to elicit exceptionally high
titers of IgG antibodies in mice (Ingale, Nat. Chem. Biol. 2007, 3,
663-667). Compound 22 has a similar architecture as 21, however,
the TLR2 ligand has been replaced by lipidated amino acids (Toth,
Tetrahedron Lett. 1993, 34, 3925-3928). The lipidated amino acids
do not induce production of cytokines, however, they enable
incorporation of the compound into liposomes. Thus,
glycolipopeptide 22 is ideally suited to establish the importance
of TLR engagement for antigenic responses against tumor-associated
glycopeptides. To determine the importance of covalent attachment
of the TLR ligand, liposomal preparations of compound 22 and
Pam.sub.3CysSK.sub.4 (23) or monophosphoryl lipid A (24), which are
TLR2 and TRL4 agonists, respectively were employed (Spohn, Vaccine
2004, 22, 2494-2499; Chow, J. Biol. Chem. 1999, 274, 10689-10692).
Finally, compounds 25 and 26, which are composed of a MUC-1
glycopeptide B-epitope linked to lipidated amino acids and the
helper T-epitope attached to Pam.sub.3CysSK.sub.4, were employed to
establish the importance of covalent linkage of the B- and helper
T-epitope. Compound 21 was prepared as described previously
(Ingale, Nat. Chem. Biol. 2007, 3, 663-667; Ingale, Org. Lett.
2006, 8, 5785-5788). Compound 22 was synthesized by SPPS using a
Rink amide resin, Fmoc protected amino acids,
Fmoc-Thr-(AcO.sub.3-.alpha.-D-GalNAc) (Cato, J. Carbohydr. Chem.
2005, 24, 503-516) and Fmoc protected lipidated amino acid
(Gibbons, Liebigs Ann. Chem. 1990, 1175-1183; Koppitz, Helv. Chim.
Acta 1997, 80, 1280-1300). The standard amino acids were introduced
using 2-(1H-benzotriazole-1-yl)-oxy-1,1,3,3-tetramethyl
hexafluorophosphate (HBTU)/N-hydroxybenzotriazole (HOBO (Knorr,
Tetrahedron Lett. 1989, 30, 1927-1930) as an activating reagent,
the glycosylated amino acid was installed with
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HATU)/1-hydroxy-7-azabenzotriazole (HOAt), and
the lipidated amino acids with
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate (PyBOP)/HOBt. After completion of the assembly
of the glycolipopeptide, the N-terminal Fmoc protecting group was
removed using standard conditions and the resulting amine capped by
acetylation with acetic anhydride and diisopropylethyl amine
(DIPEA) in N-methylpyrrolidone (NMP). Next, the acetyl esters of
the saccharide moiety were cleaved with 60% hydrazine in MeOH and
treatment with reagent B (TFA, H.sub.2O, phenol, triethylsilane,
88/5/5/2, v/v/v/v) resulted in removal of the side chain protecting
groups and release of the glycopeptide from the solid support.
[0213] Pure compound 22 was obtained after purification of the
crude product by precipitation with ice-cold diethyl ether followed
by HPLC on a C-4 semi-preparative column. A similar protocol was
used for the synthesis of compound 25. Derivative 26 was
synthesized by SPPS on a Rink amide resin and after assembly of the
peptide, the resulting product was coupled manually with
N-fluorenylmethoxycarbonyl-R-(2,3-bis(palmitoyloxy)-(2R-propyl)-(R)-cyste-
ine (Fmoc-Pam.sub.2Cys-OH) (Metzger, Int. J. Pept. Protein Res.
1991, 38, 545-554). The N-Fmoc group of the product was removed
with 20% piperidine in DMF and the resulting amine was coupled with
palmitic acid using and PyBOB, HOBt and DIPEA in DMF. The
lipopeptide was treated with reagent B to cleavage it from the
resin and to remove side chain protecting groups. The crude product
was purified by precipitation with ice-cold diethyl ether followed
by HPLC on a C-4 semi-preparative column.
Immunizations and Immunology.
[0214] Compounds 21 and 22 were incorporated into
phospholipid-based small uni-lamellar vesicles (SUVs) by hydration
of a thin film of egg phosphatidylcholine (PC),
phosphatidylglycerol (PG), cholesterol (Chol), and compound 21 or
22 (molar ratios: 65/25/50/10) in a HEPES buffer (10 mM, pH 6.5)
containing NaCl (145 mM) followed by extrusion through 100 nm
Nuclepore.RTM. polycarbonate membrane. Groups of five female BALB/c
mice were immunized subcutaneously four times at weekly intervals
with liposomes containing 3 .mu.g of saccharide. Furthermore,
similar liposomes were prepared of a mixture of glycopeptide 22
with 23 or 24 (molar ratios: PC/PG/Chol/22/23 or 24, 65/25/5/5/5)
in HEPES buffer and administered four times at weekly intervals
prior to sera harvesting. Finally, mice were immunized with a
liposomal preparation of compound 25 and 26 (molar ratios:
PC/PG/Chol/25/26, 65/25/5/5/5) employing standard procedures.
[0215] Anti-MUC-1 antibody titers of anti-sera were determined by
coating microtiter plates with the MUC-1 derived glycopeptide
TSAPDT(.alpha.-D-GalNAc)RPAP conjugated to BSA and detection was
accomplished with anti-mouse IgM or IgG antibodies labeled with
alkaline phosphatase. Mice immunized with 21 elicited exceptionally
high titers of anti-MUC-1 IgG antibodies (Table 5). Sub-typing of
the IgG antibodies (IgG1, IgG2a, IgG2b, and IgG3) indicated a bias
towards a Th2 immune response. Furthermore, the observed high IgG3
titer is typical of an anti-carbohydrate response. Immunizations
with glycolipopeptide 22, which contains lipidated amino acids
instead of a TLR2 ligand, resulted in significantly lower titers of
IgG antibodies demonstrating that TLR engagement is very important
for optimum antigenic responses. However, liposomal preparations of
compound 22 with Pam.sub.3CysSK.sub.4 (23) or monophosphoryl lipid
A (24) elicited IgG (total) titers similar to 21. In the case of
the mixture of 22 with 23, the immune response was biased towards a
Th2 response as evident by high IgG1 and low IgG2a,b titers. On the
other hand, the use of monophosphoryl lipid A led to significant
IgG1 and IG2a,b responses, and thus this preparation elicited a
mixed Th1/Th2 response. Finally, liposomes containing compound 25
and 26 did not induce measurable titers of anti MUC-1 antibodies
indicating that the B- and T epitope need to be covalent linked for
antigenic responses.
TABLE-US-00005 TABLE 5 ELISA anti-MUC1 and anti-T-epitope antibody
titers.sup.a after 4 immunizations with various preparations. IgG
total IgG1 IgG2a IgG2b IgG3 IgM IgG total Immunization.sup.b MUC1
MUC1 MUC1 MUC1 MUC1 MUC1 T-epit. 21 177,700 398,200 49,200 37,300
116,200 7,200 23,300 22 13,300 44,700 300 1,800 18,600 1,300 100
22/23 160,500 279,800 36,200 52,500 225,600 11,000 700 22/24
217,400 359,700 161,900 106,000 131,700 33,400 100 25/26 12,800
12,700 4,800 10,100 34,400 29,000 7,600 .sup.aAnti-MUC1 and
anti-T-epitope antibody titers are presented as the median for
groups of five mice. ELISA plates were coated with BSA-MI-MUC1
conjugate for anti-MUC1 antibody titers or
neutravidin-biotin-T-epitope for anti-T-epitope antibody titers.
Titers were determined by linear regression analysis, plotting
dilution vs. absorbance. Titers are defined as the highest dilution
yielding an optical density of 0.1 or greater over that of normal
control mouse sera. .sup.bLiposomal preparations were employed.
Individual anti-MUC1 titers for IgG total, IgG1, IgG2a, IgG2b, IgG3
and IgM, and anti-T-epitope for IgG total are reported in FIG.
8.
[0216] Next, possible antigenic responses against the helper
T-epitope were investigated. Thus, streptavidin coated microtiter
plates were treated with the helper T-epitope modified with biotin.
After the addition of serial dilutions of sera, detection was
accomplished with anti-mouse IgM or IgG antibodies labeled with
alkaline phosphatase. Interestingly, compound 21 elicited low
whereas mixtures of 22 with 23 or 24 elicited no antibodies against
the helper T-epitope.
[0217] Pam.sub.3CysSK.sub.4 or monophosphoryl lipid A are employed
for initiating the production of cytokines by interacting with TLR2
or TLR4, respectively, on the surface of mononuclear phagocytes
(Kawai, Semin. Immunol. 2007, 19, 24-32). After activation with
Pam.sub.3CysSK.sub.4, the intracellular domain of TLR2 recruits the
adaptor protein MyD88 resulting in the activation of a cascade of
kinases leading to the production of a number of cytokines and
chemokines. On the other hand, lipopolysaccharides (LPS) and lipid
As induce cellular responses by interacting with the TLR4/MD2
complex, which results in the recruitment of the adaptor proteins
MyD88 and TRIF leading to the induction of a more complex pattern
of cytokine. TNF-.alpha. secretion is the prototypical measure for
activation of the MyD88-dependent pathway, whereas secretion of
IFN-.beta. is commonly used as an indicator of TRIF-dependent
cellular activation.
[0218] To examine cytokine production, mouse macrophages (RAW
.gamma.NO(-) cells) were exposed over a wide range of
concentrations to compounds 21-24, E. coli 055:B5 LPS and
prototypic E. coli bisphosphoryl lipid A (Zhang, J. Am. Chem. Soc.
2007, 129, 5200-5216). After 5.5 h, the supernatants were harvested
and examined for mouse TNF-.alpha. and IFN-.beta. using commercial
or in-house developed capture ELISAs, respectively (FIG. 6).
Potencies (EC.sub.50, concentration producing 50% activity) and
efficacies (maximal level of production) were determined by fitting
the dose-response curves to a logistic equation using PRISM
software. Glycolipopeptide 21 and Pam.sub.3CysSK.sub.4 (23) induced
secretion of TNF-.alpha. with similar efficacies and potencies,
indicating that attachment of the B- and T-epitopes had no effect
on cytokine responses. As expected, none of the compounds induced
the production of INF-.beta.. Furthermore, compound 22 did not
induce TNF-.alpha. and IFN-.beta. secretion, indicating that its
lipid moiety is immunosilent. Compound 24 stimulated the cells to
produce TNF-.alpha. and INF-.beta. but its potency was much smaller
than that of E. coli 055:B5 LPS. It displayed a much larger
efficacy of TNF-.alpha. production compared to compounds 21 and 23.
The reduced efficacy of compounds 21 and 23 is probably a
beneficial property, because LPS can over-activate the innate
immune system leading to symptoms of septic shock.
[0219] Next, the ability of the mouse antisera to recognize native
MUC-1 antigen present on cancer cells was established. Thus, serial
dilutions of the serum samples were added to MUC-1 expressing MCF-7
human breast cancer cells (Horwitz, Steroids 1975, 26, 785-95) and
recognition was established using a FITC-labeled anti-mouse IgG
antibody. As can be seen in FIG. 7, anti-sera obtained from
immunizations with the three-component vaccine 1 displayed
excellent recognition of MUC-1 tumor cell whereas no binding was
observed when SK-MEL 28 cells, which do not express the MUC-1
antigen, were employed (FIG. 9).
[0220] Although sera obtained from mice immunizations with a
mixture of lipidated T-B epitope (22) and Pam.sub.3CysSK.sub.4 (23)
elicited equally high IgG antibody titers as 21 (table 5), a
much-reduced recognition of MCF-7 cells was observed. This result
indicates that covalent attachment of the adjuvant PamsCysSK.sub.4
(23) to the B-T epitope is important for proper antibody maturation
leading to improved cancer cell recognition. Immunizations with a
mixture of compound 22 and monophosphoryl lipid A (24) led to
variable results and two mice displayed excellent, and three
modest, recognition of MCF-7 cells.
Discussion
[0221] Most efforts aimed at developing carbohydrate-based cancer
vaccines have focused on the use of chemically synthesized
tumor-associated carbohydrates linked through an artificial linker
to a carrier protein (Springer, Mol. Med. 1997, 75, 594-602; Dube,
Nat. Rev. Drug Discov. 2005, 4, 477-488; Ouerfelli, Expert Rev.
Vaccines 2005, 4, 677-685; Slovin, Immunol. Cell Biol. 2005, 83,
418-428). It has been established that the use of KLH as a carrier
protein in combination with the powerful adjuvant QS-21 gives the
best results. However, a drawback of this approach is that KLH is a
very large and cumbersome protein that can elicit high titers of
anti-KLH-antibodies (Cappello, Cancer Immunol Immunother 1999, 48,
483-492), leading to immune suppression of the tumor-associated
carbohydrate epitope. Furthermore, the conjugation chemistry is
often difficult to control as it results in conjugates with
ambiguities in composition and structure, which may affect the
reproducibility of immune responses. Also, the linker moiety can
elicit strong B-cell responses (Buskas, Chem. Eur. J. 2004, 10,
3517-3524; Ni, Bioconjug. Chem. 2006, 17, 493-500). Not
surprisingly, preclinical and clinical studies with
carbohydrate-protein conjugates have led to results of mixed merit.
For example, mice immunized with a trimeric cluster of Tn-antigens
conjugated to KLH (Tn(c)-KLH) in the presence of the adjuvant QS-21
elicited modest titers of IgG antibodies (Kuduk, J. Am. Chem. Soc.
1998, 120, 12474-12485). Examination of the vaccine candidate in a
clinical trial of relapsed prostate cancer patients gave low median
IgG and IgM antibody titer (Slovin, J. Clin. Oncol. 2003, 21,
4292-4298).
[0222] The studies reported herein show that a three-component
vaccine, in which a MUC-1 associated glycopeptide B-epitope, a
promiscuous murine MHC class II restricted helper T-cell epitope,
and a TLR2 agonist (21) are covalently linked, can elicit robust
IgG antibody responses. Although covalent attachment of the TLR2
ligand to the T-B glycopeptide epitope was not required for high
IgG antibody titers, it was found to be very important for optimal
cancer cell recognition. In this respect, liposomes containing
compounds 21 or a mixture compound 22 and TLR2 agonist 23 elicited
similar high anti-MUC-1 IgG antibody titers. However, antisera
obtained from immunizations with 21 recognized MUC-1 expressing
cancer cells at much lower sera dilutions than antisera obtained
from immunizations with a mixture of 22 and 23. It appears that
immunizations with three-component vaccine 21 lead to more
efficient antibody maturation resulting in improved cancer cell
recognition.
[0223] Differences in antigenic responses against the helper
T-epitope were also observed. Thus, 21 elicited low titers of IgG
antibodies against the helper T-epitope whereas mixtures of 22 with
23 induced no antigenic responses against this part of the
candidate vaccine. Thus, the covalent attachment of the TLR2 ligand
makes compound 21 more antigenic resulting in low antibody
responses against the helper T-epitope.
[0224] It was observed that a mixture of compound 22 with 23 or 24
induced similar high titers of total IgG antibodies. However, a
bias towards a Th2 response (IgG1) was observed when the TLR2
agonist Pam.sub.3CysSK.sub.4 (23) was employed whereas mixed
Th1/Th2 responses (IgG2a,b) was obtained when the TLR4 agonist
monophosphoryl lipid A (24) was used. The difference in
polarization of helper T-cells is probably due to the induction of
different patterns of cytokines by TLR2 or TLR4. In this respect,
it was previously observed that Pam.sub.3Cys induces lower levels
of Th1 inducing cytokines I1-12(p70) and much higher levels of
Th2-inducing IL-10 than E. coli LPS (Dillon, B. J Immunol 2004,
172, 4733-43). The differences are likely due to the ability of
TLR4 to recruit the adaptor proteins MyD88 and Trif whereas TLR2
can only recruit MyD88. The results indicate that the immune system
can be tailored in a particular direction by proper selection of an
adjuvant, which is significant since different IgG isotypes perform
different effector functions.
[0225] The results described herein also show that compound 22
alone, which contains an immuno-silent lipopeptide, elicits much
lower IgG titers compared to compound 21, which is modified by a
TLR2 ligand. In particular, the ability of compound 22 to elicit
IgG2 antibodies was impaired. Recent studies employing mice
deficient in TLR signaling have cast doubt about the importance of
these innate immune receptors for adaptive immune responses
(Blander, Nature 2006, 440, 808-812; Gavin, Science 2006, 314,
1936-1938; Meyer-Bahlburg, J Exp Med 2007, 204, 3095-101;
Pulendran, N Engl J Med 2007, 356, 1776-8). In this respect,
studies with MyD88 deficient mice showed that IgM and IgG1 are
largely, but not completely, dependent of TLR signaling whereas the
IgG2 isotype is entirely TLR-dependent (Blander, Nature 2006, 440,
808-812). These observations, which are in agreement with the
results reported here, were attributed to a requirement of TLR
signaling for B-cell maturation. However, another study found that
MyD88.sup.-/-/Trif.sup.lps/lps double knockout mice elicited
similar titers of antibodies as wild type mice when immunized with
trinitrophenol-hemocyanin (TNP-Hy) or TNP-KLH in the presence or
absence of several adjuvants (Gavin, Science 2006, 314, 1936-1938).
It was concluded that it might be desirable to exclude TLR agonists
from adjuvants. It has been noted that the importance of an
adjuvant may depend on the antigenicity of the immunogen
(Meyer-Bahlburg, J Exp Med 2007, 204, 3095-101; Pulendran, N Engl J
Med 2007, 356, 1776-8). In this respect, proteins conjugates of TNP
are highly antigenic and may not require an adjuvant for optimal
responses. However, self-antigens such as tumor-associated
carbohydrates have low intrinsic antigenicity and the results
reported here clearly show that much more robust antibody responses
are obtained when a TLR ligand is co-administered. In addition, it
is demonstrated here that the architecture of a candidate vaccine
is very important for optimal antigenic responses and in particular
covalent attachment of a TLR ligand to a T-B epitope led to
improved cancer cell recognition.
[0226] The failure of a mixture of compounds 25 and 26 to elicit
anti-MUC-1 glycopeptide antibodies indicates that covalent
attachment of the T- to the B-epitope is needed to elicit antigenic
responses. In this respect, activation of B-cells by helper T-cells
requires a similar type of cell-cell interaction as for helper
T-cell activation by antigen presenting cells. Thus, a protein or
peptide-containing antigen needs to be internalized by B-cells for
transport to endosomal vesicles, where proteases will digest the
protein and some of the resulting peptide fragments will be
complexed with class II MHC protein. The class II MHC-peptide
complex will then be transported to the cell surface of the
B-lymphocyte to mediate an interaction with helper T-cell resulting
in a class switch from low affinity IgM to high affinity IgG
antibody production. Unlike antigen presenting cells, B-cells have
poor phagocytic properties and can only internalize molecules that
bind to the B-cell receptor. Therefore, it is to be expected that
internalization of the helper T-epitope is facilitated by covalent
attachment to the B-epitope (MUC-1 glycopeptide) and as a result
covalent attachment of the two epitopes will lead to more robust
antigenic responses.
[0227] In conclusion, it has been demonstrated that antigenic
properties of a fully synthetic cancer vaccine can be optimized by
structure-activity relationship studies. In this respect, it has
been established that a three-component vaccine in which a
tumor-associated MUC-1 glycopeptide B-epitope, a promiscuous helper
T-cell epitope and a TLR2 ligand are covalently linked can elicit
exceptionally high IgG antibody responses, which have an ability to
recognize cancer cells. It is very important that the helper
T-epitope is covalently linked to the B-epitope, probably since
internalization of the helper T-epitope by B-cells requires the
presence of a B-epitope. It has also been shown that incorporation
of a TLR agonist is important for robust antigenic responses
against tumor associated glycopeptide antigens. In this respect,
cytokines induced by the TLR2 ligand are important for maturation
of immune cells leading to robust antibody responses. A surprising
finding was that improved cancer cell recognition was observed when
the TLR2 epitope was covalently attached to the glycopeptide T-B
epitope. The result presented here provides important information
of the optimal constitution of three-component vaccines and will
guide successful development of carbohydrate-based cancer
vaccines.
Experimental
[0228] Peptide synthesis: Peptides were synthesized by established
protocols on an ABI 433A peptide synthesizer (Applied Biosystems),
equipped with a UV-detector using N.sup..alpha.-Fmoc-protected
amino acids and 2-(1H-benzotriazole-1-yl)-oxy-1,1,3,3-tetramethyl
hexafluorophosphate (HBTU)/N-hydroxybenzotriazole (HOBt) (Knorr,
Tetrahedron Lett. 1989, 30, 1927-1930) as the activating reagents.
Single coupling steps were performed with conditional capping. The
following protected amino acids were used:
N.sup..alpha.-Fmoc-Arg(Pbf)-OH,
N.sup..alpha.-Fmoc-Asp(O.sup.tBu)-OH,
N.sup..alpha.-Fmoc-Asp-Thr(.PSI..sup.Me,Mepro)-OH,
N.sup..alpha.-Fmoc-Ile, Thr(.PSI..sup.Me,Mepro)-OH,
N.sup..alpha.-Fmoc-Lys(Boc)-OH,
N.sup..alpha.-Fmoc-Ser(.sup.tBu)-OH,
N.sup..alpha.-Fmoc-Thr(.sup.tBu)-OH, and
N.sup..alpha.-Fmoc-Tyr(.sup.tBu)-OH. The coupling of glycosylated
amino acid N.sup..alpha.-Fmoc-Thr-(AcO.sub.3-.alpha.-D-GalNAc) 1S
(Cato, J. Carbohydr. Chem. 2005, 24, 503-516) was carried out
manually using
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HATU)/1-hydroxy-7-azabenzotriazole (HOAt) as a
coupling agent. The coupling of N.sup..alpha.-Fmoc-lipophilic amino
acid (N.sup..alpha.-Fmoc-D,L-tetradeconic acid) 2S (Gibbons,
Liebigs Ann. Chem. 1990, 1175-1183; Koppitz, Helv. Chim. Acta 1997,
80, 1280-1300) and
N.sup..alpha.-Fmoc-S-(2,3-bis(palmitoyloxy)-(2R-propyl)-(R)-cysteine
3S (Metzger, Int. J. Pept. Protein Res. 1991, 38, 545-554; Roth,
Bioconj. Chem. 2004, 15, 541-553), which was prepared from
(R)-glycidol, were carried out using
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate (PyBOP)/HOBt as coupling agent (See Supporting
Information). Progress of the manual couplings was monitored by
standard Kaiser test (Kaiser, Anal. Biochem. 1970, 34, 595).
[0229] Liposome preparation: Egg phosphatidylcholine (PC),
phosphatidylglycerol (PG), cholesterol (Chol) and compound 21 or 22
(15 mmol, molar ratios 65:25:50:10) or PC/PG/Chol/22/23 or 24 (15
mmol, molar ratios 60:25:50:10:5) or PC/PG/Chol/25/26 (15 mmol,
molar ratios 65:25:50:5:5) were dissolved in a mixture of
trifluoroethanol and MeOH (1:1, v/v, 5 mL). The solvents were
removed in vacuo to give a thin lipid film, which was hydrated by
shaking in HEPES buffer (10 mM, pH 6.5) containing NaCl (145 mM) (1
mL) under argon atmosphere at 41.degree. C. for 3 h. The vesicle
suspension was sonicated for 1 min and then extruded successively
through 1.0, 0.4, 0.2, and 0.1 .mu.m polycarbonate membranes
(Whatman, Nuclepore.RTM. Track-Etch Membrane) at 50.degree. C. to
obtain SUVs. The GalNAc content was determined by heating a mixture
of SUVs (50 .mu.L) and aqueous TFA (2 M, 200 .mu.L) in a sealed
tube for 4 h at 100.degree. C. The solution was then concentrated
in vacuo and analyzed by high-pH anion exchange chromatography
using a pulsed amperometric detector (HPAEC-PAD; Methrome) and
CarboPac columns PA-10 and PA-20 (Dionex).
[0230] Dose and immunization schedule: Groups of five mice (female
BALB/c, age 8-10 weeks; Jackson Laboratories) were immunized four
times at weekly intervals. Each boost included 3 .mu.g of
saccharide in the liposome formulation. Serum samples were obtained
before immunization (pre-bleed) and one week after the final
immunization. The final bleeding was done by cardiac bleed.
[0231] Serologic assays: Anti-MUC-1 IgG, IgG1, IgG2a, IgG2b, IgG3,
and IgM antibody titers were determined by enzyme-linked
immunosorbent assay (ELISA), as described previously (Buskas, Chem.
Eur. J. 2004, 10, 3517-3524). Briefly, ELISA plates (Thermo
Electron Corp.) were coated with a conjugate of the MUC-1
glycopeptide conjugated to BSA through a maleimide linker
(BSA-MI-MUC-1). Serial dilutions of the sera were allowed to bind
to immobilized MUC-1. Detection was accomplished by the addition of
phosphate-conjugated anti-mouse IgG (Jackson ImmunoResearch
Laboratories Inc.), IgG1 (Zymed), IgG2a (Zymed), IgG2b (Zymed),
IgG3 (BD Biosciences Pharmingen), or IgM (Jackson ImmunoResearch
Laboratories Inc.) antibodies. After addition of p-nitrophenyl
phosphate (Sigma), the absorbance was measured at 405 nm with
wavelength correction set at 490 nm using a microplate reader (BMG
Labtech). Antibody titers against the T (polio)-epitope were
determined as follows. Reacti-bind NeutrAvidin coated and
pre-blocked plates (Pierce) were incubated with biotin-labeled
T-epitope (10 .mu.g/mL) for 2 h. Next, serial dilutions of the sera
were allowed to bind to immobilized T-epitope. Detection was
accomplished as described above. The antibody titer was defined as
the highest dilution yielding an optical density of 0.1 or greater
over that of normal control mouse sera.
[0232] Cell culture: RAW 264.7 .gamma.NO(-) cells, derived from the
RAW 264.7 mouse monocyte/macrophage cell line, were obtained from
ATCC. The cells were maintained in RPMI 1640 medium with
L-glutamine (2 mM), adjusted to contain sodium bicarbonate (1.5 g
L.sup.-1), glucose (4.5 g L.sup.-1), HEPES (10 mM) and sodium
pyruvate (1.0 mM) and supplemented with penicillin (100 u
mL.sup.-1)/streptomycin (100 .mu.g mL.sup.-1; Mediatech) and FBS
(10%; Hyclone). Human breast adenocarcinoma cells (MCF7) (Horwitz,
Steroids 1975, 26, 785-95), obtained from ATCC, were cultured in
Eagle's minimum essential medium with L-glutamine (2 mM) and
Earle's BSS, modified to contain sodium bicarbonate (1.5 g
L.sup.-1), non-essential amino acids (0.1 mM) and sodium pyruvate
(1 mM) and supplemented with bovine insulin (0.01 mg mL.sup.-1;
Sigma) and FBS (10%). Human skin malignant melanoma cells
(SK-MEL-28) were obtained from ATCC and grown in Eagle's minimum
essential medium with L-glutamine (2 mM) and Earle's BSS, adjusted
to contain sodium bicarbonate (1.5 g L.sup.-1), non-essential amino
acids (0.1 mM) and sodium pyruvate (1 mM) and supplemented with FBS
(10%). All cells were maintained in a humid 5% CO.sub.2 atmosphere
at 37.degree. C.
[0233] TNF-.alpha. and IFN-.beta. assays. RAW 264.7 .gamma.NO(-)
cells were plated on the day of the exposure assay as
2.times.10.sup.5 cells/well in 96-well plates (Nunc) and incubated
with different stimuli for 5.5 h in the presence or absence of
polymyxin B. Culture supernatants were collected and stored frozen
(-80.degree. C.) until assayed for cytokine production.
Concentrations of TNF-.alpha. were determined using the TNF-.alpha.
DuoSet ELISA Development kit from R&D Systems. Concentrations
of IFN-.beta. were determined as follows. ELISA MaxiSorp plates
were coated with rabbit polyclonal antibody against mouse
IFN-.beta. (PBL Biomedical Laboratories). IFN-.beta. in standards
and samples was allowed to bind to the immobilized antibody. Rat
anti-mouse IFN-.beta. antibody (USBiological) was then added,
producing an antibody-antigen-antibody "sandwich". Next,
horseradish peroxidase (HRP) conjugated goat anti-rat IgG (H+L)
antibody (Pierce) and a chromogenic substrate for HRP
3,3',5,5'-tetramethylbenzidine (TMB; Pierce) were added. After the
reaction was stopped, the absorbance was measured at 450 nm with
wavelength correction set to 540 nm. Concentration-response data
were analyzed using nonlinear least-squares curve fitting in Prism
(GraphPad Software, Inc.). These data were fit with the following
four parameter logistic equation:
Y=E.sub.max/(1+(EC.sub.50/X).sup.Hill slope), where Y is the
cytokine response, X is the concentration of the stimulus,
E.sub.max is the maximum response and EC.sub.50 is the
concentration of the stimulus producing 50% stimulation. The Hill
slope was set at 1 to be able to compare the EC.sub.50 values of
the different inducers. All cytokine values are presented as the
means.+-.SD of triplicate measurements, with each experiment being
repeated three times.
[0234] Evaluation of materials for contamination by LPS: To ensure
that any increase in cytokine production was not caused by LPS
contamination of the solutions containing the various stimuli,
avidly binds to the lipid A region of LPS, thereby preventing
LPS-induced cytokine production (Tsubery, Biochemistry 2000, 39,
11837-44). TNF-.alpha. and IFN-.beta. concentrations in
supernatants of cells preincubated with polymyxin B (30 .mu.g
mL.sup.-1; Bedford Laboratories) for 30 min before incubation with
E. coli O55:B5 LPS for 5.5 h showed complete inhibition, whereas
preincubation with polymyxin B had no effect on TNF-.alpha.
synthesis by cells incubated with the synthetic compounds 21 and
23. Therefore, LPS contamination of the latter preparations was
inconsequential.
[0235] Cell recognition analysis by fluorescence measurements:
Serial dilutions of pre- and post-immunization sera were incubated
with MCF7 and SK-MEL-28 single-cell suspensions for 30 min on ice.
Next, the cells were washed and incubated with goat anti-mouse IgG
.gamma.-chain specific antibody conjugated to fluorescein
isothiocyanate (FITC; Sigma) for 20 min on ice. Following three
washes and cell lysis, cell lysates were analyzed for fluorescence
intensity (485 ex/520 em) using a microplate reader (BMG Labtech).
Data points were collected in triplicate and are representative of
three separate experiments.
Example VII
Synthesis of Compounds
[0236] General methods: Fmoc-L-amino acid derivatives and resins
were purchased from NovaBioChem and Applied Biosystems; peptide
synthesis grade N,N-dimethylformamide (DMF) from EM Science; and
N-methylpyrrolidone (NMP) from Applied Biosystems. Egg
phosphatidylcholine (PC), phosphatidylglycerol (PG), cholesterol
(Chol), and monophosphoryl lipid A (MPL-A) were obtained from
Avanti Polar Lipids. EZ-Link.RTM. NHS-Biotin reagent
(succinimidyl-6-(biotinamido)hexanoate) was obtained from Pierce.
All other chemical reagents were purchased form Aldrich, Acros,
Alfa Aesar, and Fisher Scientific and used without further
purification. All solvents employed were reagent grade. Reversed
phase high performance liquid chromatography (RP-HPLC) was
performed on an Agilent 1100 series system equipped with an
auto-injector, fraction-collector, and UV-detector (detecting at
214 nm) using an Agilent Zorbax Eclipse.TM. C8 analytical column (5
.mu.m, 4.6.times.150 mm) at a flow rate of 1 mL/min, Agilent Zorbax
Eclipse.TM. C8 semi preparative column (5 .mu.m, 10.times.250 mm)
at a flow rate of 3 mL/min or Phenomenex Jupiter.TM. C4 semi
preparative column (5 .mu.m, 10.times.250 mm) at a flow rate of 2
mL/min. All runs were performed using a linear gradient of 0-100%
solvent B over 40 min (solvent A=5% acetonitrile, 0.1%
trifluoroacetic acid (TFA) in water, solvent B=5% water, 0.1% TFA
in acetonitrile). Matrix assisted laser desorption ionization time
of flight mass spectrometry (MALDI-ToF) mass spectra were recorded
on a ABI 4700 proteomic analyzer.
[0237] Synthesis of glycolipopeptide 22: The synthesis 22 was
carried out on a Rink amide resin (28, 0.1 mmol) as described under
peptide synthesis in the experimental. The first four amino acids,
Arg-Pro-Ala-Pro were coupled on the peptide synthesizer using a
standard protocol to obtain 29. After the completion of the
synthesis, a manual coupling of 1S (0.2 mmol, 134 mg) was carried
out. N.sup..alpha.-Fmoc-Thr-(AcO.sub.3-.alpha.-D-GalNAc) 1S (Cato,
J. Carbohydr. Chem. 2005, 24, 503-516) was dissolved in NMP (5 mL)
and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HATU; 0.2 mmol, 76 mg),
1-hydroxy-7-azabenzotriazole (HOAt; 0.2 mmol, 27 mg), and
diisopropylethylamine (DIPEA; 0.4 mmol, 70 .mu.L) were added to the
solution and the resulting mixture was added to the resin. The
coupling reaction was monitored by standard Kaiser test. After 12
h, the resin was washed with NMP (6 mL) and methylene chloride
(DCM; 6 mL), and resubjected to the same coupling conditions to
ensure complete coupling. The glycopeptide 30 was then elongated on
the peptide synthesizer. After the completion of the synthesis, the
resin was thoroughly washed with NMP (6 mL), DCM (6 mL) and
methanol (MeOH; 6 mL) and dried in vacuo. The resin was then
swelled in DCM (5 mL) for 1 h and the rest of the couplings were
carried out manually. Next, N.sup..alpha.-Fmoc-lipophilic amino
acid (N.sup..alpha.-Fmoc-D,L-tetradeconic acid) 2S (Gibbons,
Liebigs Ann. Chem. 1990, 1175-1183; Koppitz, Helv. Chim. Acta 1997,
80, 1280-1300) (0.3 mmol, 139 mg) dissolved in NMP (5 mL),
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate (PyBOP; 0.3 mmol, 156 mg), HOBt (0.3 mmol, 40
mg) and DIPEA (0.4 mmol, 67 .mu.L) were premixed for 2 min., and
then added to the resin. The coupling reaction was monitored by the
Kaiser test and was complete after standing for 8 h. The
N.sup..alpha.-Fmoc group was cleaved using piperidine (20%) in DMF
(6 mL). N.sup..alpha.-Fmoc-Gly-OH (0.3 mmol, 90 mg) dissolved in
NMP (5 mL), PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg), and
DIPEA (0.4 mmol, 67 .mu.L) were premixed for 2 min, and were then
added to the resin. The coupling reaction was monitored by Kaiser
test and was complete after standing for 4 h. The
N.sup..alpha.-Fmoc group was cleaved using piperidine (20%) in DMF
(6 mL). One more cycle of coupling of 2S (0.3 mmol, 139 mg) was
carried out as described above using PyBOP (0.3 mmol, 156 mg), HOBt
(0.3 mmol, 40 mg), and DIPEA (0.4 mmol, 67 .mu.L) in NMP (5 mL).
Finally, the N.sup..alpha.-Fmoc group was cleaved using piperidine
(20%) in DMF (6 mL) and the resulting free amino group was
acetylated by treatment of the resin with Ac.sub.2O (10%) and DIPEA
(5%) in NMP (5 mL) for 10 min. The resin was washed thoroughly with
NMP (5 mL.times.2), DCM (5 mL.times.2), and MeOH (5 mL.times.2),
and dried in vacuo. The resin was swelled in DCM (5 mL) for 1 h,
treated with hydrazine (60%) in MeOH.sup.4,5 (10 mL) for 2 h,
thoroughly washed with NMP (5 mL.times.2), DCM (5 mL.times.2), and
MeOH (5 mL.times.2), and dried in vacuo. The resin was swelled in
DCM (5 mL) for 1 h and then treated with reagent B (TFA (88%),
water (5%), phenol (5%), and TIS (2%), 10 mL) for 2 h. The resin
was filtered, washed with neat TFA (2 mL), and the filtrate was
then concentrated in vacuo to approximately 1/3 of its original
volume. The glycolipopeptide was precipitated using diethyl ether
(0.degree. C., 40 mL) and recovered by centrifugation at 3,000 rpm
for 15 min. The crude glycolipopeptide was purified by RP-HPLC on a
semi preparative C-4 column using a linear gradient of 0-95%
solvent B in A over 40 min, and the appropriate fractions were
lyophilized to afford 22 (FIG. 10) (57 mg, 16%).
C.sub.165H.sub.267N.sub.37O.sub.44, MALDI-ToF MS: observed, [M+]
3473.4900 Da; calculated, [M+] 3473.1070 Da.
##STR00022##
[0238] Synthesis of lipopeptide 23: The synthesis of 23 was carried
out on a Rink amide resin (28, 0.1 mmol) as described under peptide
synthesis in the experimental. After coupling of the first five
amino acids, the lipid portion of the molecule was coupled
manually.
N.sup..alpha.-Fmoc-S-(2,3-bis(palmitoyloxy)-(2R-propyl)-(R)-cysteine,
3S (Metzger, Int. J. Pept. Protein Res. 1991, 38, 545-554; Roth,
Bioconj. Chem. 2004, 15, 541-553) (0.3 mmol, 267 mg) was dissolved
in DMF (5 mL) and PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg),
and DIPEA (0.4 mmol, 67 .mu.L) were added to the solution. After 2
min the reaction mixture was added to the resin. The coupling
reaction was monitored by the Kaiser test and was complete after
standing for 12 h. Next, the N.sup..alpha.-Fmoc group was cleaved
using piperidine (20%) in DMF (6 mL) to obtain 36. Palmitic acid
(0.3 mmol, 77 mg) was coupled to the free amine of 36 as described
above using PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg), and
DIPEA (0.4 mmol, 67 .mu.L) in DMF. The resin was washed thoroughly
with DMF (5 mL.times.2), DCM (5 mL.times.2), and MeOH (5
mL.times.2) and then dried in vacuo. The resin was swelled in DCM
(5 mL) for 1 h and then treated with TFA (95%), water (2.5%), and
TIS (2.5%) (10 mL) for 2 h at room temperature. The resin was
filtered and washed with neat TFA (2 mL). The filtrate was then
concentrated in vacuo to approximately 1/3 of its original volume.
The lipopeptide was precipitated using diethyl ether (0.degree. C.;
30 mL) and recovered by centrifugation at 3000 rpm for 15 min. The
crude lipopeptide was purified by RP-HPLC on a semi preparative C-4
column using a linear gradient of 0 to 95% solvent B in solvent A
over a 40 min period and the appropriate fractions were lyophilized
to afford 23 (FIG. 11) (40 mg, 26%).
C.sub.81H.sub.156N.sub.11O.sub.12S, MALDI-ToF MS: observed [M+Na],
1531.2240 Da; calculated [M+Na], 1531.1734 Da.
##STR00023##
[0239] Synthesis of glycolipopeptide 25: The synthesis 25 was
carried out on a Rink amide resin (28, 0.1 mmol) as described under
peptide synthesis in the experimental. The first four amino acids,
Arg-Pro-Ala-Pro were coupled on the peptide synthesizer using a
standard protocol to obtain 29. After the completion of the
synthesis, a manual coupling was carried out using 1S (0.2 mmol,
134 mg). 1S was dissolved in NMP (5 mL) and HATU (0.2 mmol, 76 mg),
HOAt (0.2 mmol, 27 mg), and DIPEA (0.4 mmol, 70 were added and the
resulting mixture was added to the resin. The coupling reaction was
monitored by standard Kaiser test. After 12 h, the resin was washed
with NMP (6 mL) and DCM (6 mL), and re-subjected to the same
coupling conditions to ensure complete coupling. Glycopeptide 30
was then elongated on the peptide synthesizer. After the completion
of the synthesis, the resin was thoroughly washed with NMP (6 mL),
DCM (6 mL), and MeOH (6 mL) and dried in vacuo. The resin was then
swelled in DCM (5 mL) for 1 h and the rest of the peptide sequence
was completed manually. 2S (0.3 mmol, 139 mg) was dissolved in NMP
(5 mL) and PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg), and
DIPEA (0.4 mmol, 67 .mu.L) were added to the solution. After 2 min,
the mixture was added to the resin. The coupling reaction was
monitored by standard Kaiser test and was complete after standing
for 8 h. Next, the N.sup..alpha.-Fmoc group was cleaved using
piperidine (20%) in DMF (6 mL). N.sup..alpha.-Fmoc-L-glycine (0.3
mmol, 90 mg) was dissolved in NMP (5 mL) and premixed with PyBOP
(0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg), and DIPEA (0.4 mmol, 67
.mu.L) for 2 min before the reaction mixture was added to the
resin. The coupling reaction was monitored by Kaiser test and was
complete after standing for 4 h. The N.sup..alpha.-Fmoc group was
cleaved using piperidine (20%) in DMF (6 mL). One more cycle of
coupling of 2S (0.3 mmol, 139 mg) was carried out as described
above using PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg), and
DIPEA (0.4 mmol, 67 .mu.L) in NMP (5 mL) Finally, the
N.sup..alpha.-Fmoc group was cleaved using piperidine (20%) in DMF
(6 mL) and the resulting free amino group was acetylated using
Ac.sub.2O (10%) and DIPEA (5%) in NMP (5 mL) for 10 min. The resin
was washed thoroughly with NMP (5 mL.times.2), DCM (5 mL.times.2),
and MeOH (5 mL.times.2), and dried in vacuo. The resin was swelled
in DCM (5 mL) for 1 h, treated with hydrazine (60%) in MeOH (10 mL)
for 2 h, washed thoroughly with NMP (5 mL.times.2), DCM (5
mL.times.2) and MeOH (5 mL.times.2) and dried in vacuo. The resin
was swelled in DCM (5 mL) for 1 h after which it was treated with
reagent B (TFA (88%), water (5%), phenol (5%), and TIS (2%), 10 mL)
for 2 h. The resin was filtered, washed with neat TFA (2 mL) and
the filtrate was then concentrated in vacuo to approximately 1/3 of
its original volume. The glycolipopeptide was precipitated using
diethyl ether (0.degree. C.; 40 mL) and recovered by centrifugation
at 3,000 rpm for 15 min. The crude glycolipopeptide was purified by
RP-HPLC on a semi preparative C-4 column using a linear gradient of
0-95% solvent B in A over 40 min, and the appropriate fractions
were lyophilized to afford 5 (FIG. 12) (35 mg, 19%).
C.sub.84H.sub.145N.sub.19O.sub.25, MALDI-ToF MS: observed, [M+]
1821.1991 Da; calculated, [M+] 1821.1624 Da.
##STR00024##
[0240] Synthesis of lipopeptide 26: The synthesis of 26 was carried
out on a Rink amide resin (28, 0.1 mmol). After the assembly of the
peptide by using standard SPPS, the lipid portion of the molecule
was coupled manually. 3S (0.3 mmol, 267 mg) was dissolved in DMF (5
mL) and PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg), and DIPEA
(0.4 mmol, 67 .mu.L) were added to the solution. After activation
of 3S for 2 min the reaction mixture was added to the resin. The
coupling reaction was monitored by the Kaiser test and was complete
after standing for 12 h. The N-Fmoc group was cleaved using
piperidine (20%) in DMF (6 mL) to obtain 43. Palmitic acid (77 mg,
0.3 mmol) was coupled to the free amine of 43 as described above
using PyBOP (0.3 mmol, 156 mg), HOBt (0.3 mmol, 40 mg), and DIPEA
(0.4 mmol, 67 .mu.L) in DMF. The resin was washed thoroughly with
DMF (5 mL.times.2), DCM (5 mL.times.2), and MeOH (5 mL.times.2) and
then dried in vacuo. The resin was swelled in DCM (5 mL) for 1 h,
treated with reagent B (TFA (88%), water (5%), phenol (5%), and TIS
(2%), 10 mL) for 2 h, filtered and washed with neat TFA (2 mL). The
filtrate was then concentrated in vacuo to approximately 1/3 of its
original volume, and the lipopeptide was precipitated using diethyl
ether (0.degree. C.; 30 mL) and recovered by centrifugation at 3000
rpm for 15 min. The crude lipopeptide was purified by RP-HPLC on a
semi preparative C-4 column using a linear gradient of 0-95%
solvent B in A over a 40 min., and the appropriate fractions were
lyophilized to afford 26 (FIG. 13) (57 mg, 18%).
C.sub.162H.sub.278N.sub.29O.sub.31S, MALDI-ToF MS: observed, [M+]
3160.9423 Da; calculated, [M+] 3160.1814 Da.
##STR00025##
[0241] Synthesis of biotin-T-epitope peptide 27: The synthesis of
27 was carried out on a Rink amide resin (28, 0.1 mmol) as
described in the general method. After the completion of synthesis
the resin was washed thoroughly with DMF (5 mL.times.2), DCM (5
mL.times.2), and MeOH (5 mL.times.2) and then dried in vacuo. The
resin was swelled in DCM (5 mL) for 1 h. Next, a mixture of
EZ-Link.RTM. NHS-Biotin reagent
(succinimidyl-6-(biotinamido)hexanoate) (0.2 mmol, 90 mg) and DIPEA
(0.2 mmol, 36 .mu.L) in DMF (5 mL) was added to the resin. The
coupling was monitored by standard Kaiser test and was complete
within 8 h. The resin was washed thoroughly with DMF (5
mL.times.2), DCM (5 mL.times.2), and MeOH (5 mL.times.2) and then
dried in vacuo. The resin was swelled in DCM (5 mL) for 1 h and
treated with reagent B (TFA (88%), water (5%), phenol (5%), and TIS
(2%), 15 mL) for 2 h at room temperature. The resin was filtered
and washed with neat TFA (2 mL). The filtrate was concentrated in
vacuo to approximately 1/3 of its original volume. The peptide was
precipitated using diethyl ether (0.degree. C.; 30 mL) and
recovered by centrifugation at 3,000 rpm for 15 min. The crude
peptide was purified by RP-HPLC on a semi preparative C-8 column
using a linear gradient of 0 to 95% solvent B in solvent A over a
40 min period and the appropriate fractions were lyophilized to
afford 27 (FIG. 14) (60% based on resin loading capacity).
C.sub.95H.sub.147N.sub.21O.sub.21S, MALDI-ToF MS: observed [M+],
1951.2966 Da; calculated [M+], 1951.3768 Da.
##STR00026##
Example VIII
Monoclonal Antibodies Against Carbohydrates and Glycopeptides by
Using Fully Synthetic Three-Component Immunogens
[0242] Glycoconjugates are the most functionally and structurally
diverse molecules in nature and it is now well established that
protein- and lipid-bound saccharides play essential roles in many
molecular processes impacting eukaryotic biology and disease.
Examples of such processes include fertilization, embryogenesis,
neuronal development, hormone activities, the proliferation of
cells and their organization into specific tissues. Remarkable
changes in the cell-surface carbohydrates occur with tumor
progression, which appears to be intimately associated with
metastasis. Furthermore, carbohydrates are capable of inducing a
protective antibody response and this immunological reaction is a
major contributor to the survival of the organism during
infection.
[0243] The inability of saccharides to activate helper
T-lymphocytes has complicated their development as vaccines. For
most immunogens, including carbohydrates, antibody production
depends on the cooperative interaction of two types of lymphocytes,
the B-cells and helper T-cells (Jennings, Neoglyconjugates:
Preparation and Applications 325-371 (Academic Press, Inc., 1994);
Kuberan, Curr. Org. Chem. 2000, 4, 653-677). Saccharides alone
cannot activate helper T-cells and therefore have a limited
immunogenicity as manifested by low affinity IgM antibodies and the
absence of IgG antibodies. In order to overcome the T-cell
independent properties of carbohydrates, past research has focused
on the conjugation of saccharides to a foreign carrier protein
(e.g. Keyhole Limpet Hemocyanin (KLH) detoxified tetanus toxoid)
(Jennings, Neoglyconjugates: Preparation and Applications 325-371
(Academic Press, Inc., 1994); Kuberan, Curr. Org. Chem. 2000, 4,
653-677; Jones, An. Acad. Bras. Cienc. 2005, 77, 293-324). In this
approach, the carrier protein enhances the presentation of the
carbohydrate to the immune system and provides T-epitopes (peptide
fragments of 12-15 amino acids) that can activate T-helper cells.
As a result, a class switch from low affinity IgM to high affinity
IgG antibodies is accomplished. This approach has been successfully
applied for the development of a conjugate vaccine to prevent
infections with Haemophilus influenzae.
[0244] Carbohydrate-protein conjugate candidate vaccines composed
of more demanding carbohydrate antigens, such as tumor associated
carbohydrate and glycopeptides, have failed to elicit high titers
of IgG antibodies. These results are not surprising because
tumor-associated saccharides are of low antigenicity, because they
are self-antigens and consequently tolerated by the immune system.
The shedding of antigens by the growing tumor reinforces this
tolerance. In addition, foreign carrier proteins such as KLH and
BSA and the linker that attach the saccharides to the carrier
protein can elicit strong B-cell responses, which may lead to the
suppression of antibody responses against the carbohydrate epitope
(Buskas, Chem. Eur. J. 2004, 10, 3517-3524; Ni, Bioconjug. Chem.
2006, 17, 493-500). It is clear that the successful development of
carbohydrate-based cancer vaccines requires novel strategies for
the more efficient presentation of tumor-associated carbohydrate
epitopes to the immune system, resulting in a more efficient class
switch to IgG antibodies (Reichel, Chem. Commun. 1997, 21,
2087-2088; Alexander, J. Immunol. 2000, 164, 1625-1633; Kudryashov,
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 3264-3269; Lo-Man, J.
Immunol. 2001, 166, 2849-2854; Jiang, Curr. Med. Chem. 2003, 10,
1423-1439; Jackson, Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
15440-5; Lo-Man, Cancer Res. 2004, 64, 4987-4994; Buskas, Angew.
Chem. Int. Ed. 2005, 44, 5985-5988 (Example I); Dziadek, Angew.
Chem. Int. Ed. 2005, 44, 7624-7630; Krikorian, Bioconjug. Chem.
2005, 16, 812-819; Pan, J. Med. Chem. 2005, 48, 875-883).
[0245] We have found that a three-component vaccine composed of a
TLR2 agonist, a promiscuous peptide T-helper epitope and a
tumor-associated glycopeptide, can elicit in mice exceptionally
high titers of IgG antibodies that can recognize cancer cells
expressing the tumor-associated carbohydrate (see compound 21, FIG.
5, Example VI and compound 51, FIG. 15) (Ingale, Nat. Chem. Biol.
2007, 3, 663-667). The superior properties of the vaccine candidate
are attributed to the local production of cytokines, upregulation
of co-stimulatory proteins, enhanced uptake by macrophages and
dendritic cells and avoidance of epitope suppression.
[0246] We expect that the three-component immunogen technology of
the invention can be used to generate monoclonal antibodies (MAbs)
for poorly antigenic carbohydrates and glycopeptides. We have
initially focused on MAbs against .beta.-N-acetylglucosamine
(.beta.-O-GlcNAc) modified peptides (Wells, Science 2001, 291,
2376-2378; Whelan, Methods Enzymol. 2006, 415, 113-133; Zachara,
Biochim. Biophys. Acta, 2006, 1761, 599-617; Dias and Hart, Mol.
Biosyst. 2007, 3, 766-772; Hart, Nature 2007, 446, 1017-1022;
Lefebvre, Exp. Rev. Proteomics 2005, 2, 265-275). Myriad nuclear
and cytoplasmic proteins in metazoans are modified on Ser and Thr
residues by the monosaccharide .beta.-O-GlcNAc. The rapid and
dynamic change in O-GlcNAc levels in response to extracellular
stimuli suggests a key role for O-GlcNAc in signal transduction
pathways. Modulation of O-GlcNAc levels has profound effects on the
functioning of cells, in part mediated through a complex interplay
between O-GlcNAc and O-phosphate. Recently, O-GlcNAc has been
implicated in the etiology of type II diabetes, the regulation of
stress response pathways and in the regulation of the proteasome.
Progress in this exciting field of research is seriously hampered
by the lack of reagents such as appropriate MAbs. In this respect,
only one poorly performing IgM MAb with relative broad specificity
(Corner, Anal. Biochem. 2001, 293, 169-177) is commercially
available (Covance Research Products Inc).
[0247] We have designed and synthesized compound 52 (FIG. 15),
which contains as a B-epitope a 13-GlcNAc modified glycopeptide
derived from casein kinase II (CKII) (Kreppel, J. Biol. Chem. 1999,
274, 32015-32022), the well-documented murine MHC class II
restricted helper T-cell epitope KLFAVWKITYKDT (SEQ ID NO:3)
derived from the polio virus and the inbuilt adjuvant
Pam.sub.3CysSK.sub.4. In addition, compound 53 was prepared which
has an artificial thio-linked GlcNAc moiety, which was expected to
have better metabolic stability. Compounds 52 and 53 were
incorporated into phospholipid-based small uni-lamellar vesicles
(SUVs) by hydration of a thin film of the synthetic compounds, egg
phosphatidylcholine, phosphatidylglycerol and cholesterol in a
HEPES buffer (10 mM, pH 6.5) containing NaCl (145 mM) followed by
extrusion through a 100 nm Nuclepore.RTM. polycarbonate membrane.
Groups of five female BALB/c mice were immunized intra-peritoneal
four times at weekly intervals with liposomes containing 3 .mu.g of
saccharide.
[0248] Anti-glycopeptide antibody titers were determined by coating
microtiter plates with CGSTPVS(.beta.-O-GlcNAc)SANM conjugated to
maleimide (MI) modified BSA and detection was accomplished with
anti-mouse IgG antibodies labeled with alkaline phosphatase. As can
be seen in Table 6, compounds 52 and 53 elicited excellent titers
of anti-MUC1 IgG antibodies. Furthermore, no significant difference
in titer was observed between the O- and S-linked saccharide
derivatives.
TABLE-US-00006 TABLE 6 ELISA anti-GSTPVS(.beta.-O-GlcNAc)SANM(68)
titers.sup.a after 4 immunizations with two different preparations
Im- muni- zation.sup.b IgG total IgG1 IgG2a IgG2b IgG3 IgM O-
76,500 61,400 33,200 12,500 69,400 81,900 GlcNAc 52.sup.c S-
151,600 111,800 55,600 21,300 111,700 21,900 GlcNAc 53.sup.d
.sup.aAnti-GSTPVS(.beta.-O-GlcNAc)SANM (68) antibody titers are
presented as the mean of groups of five mice. ELISA plates were
coated with BSA-MI-GSTPVS(.beta.-O-GlcNAc)SANM (BSA-MI-66)
conjugate and titers were determined by linear regression analysis,
plotting dilution vs. absorbance. Titers are defined as the highest
dilution yielding an optical density of 0.1 or greater over that of
normal control mouse sera. .sup.bLiposomal preparations were
employed. .sup.cO-GlcNAc 52;
Pam.sub.3CysSK.sub.4G-C-KLFAVWKITYKDT-G-GSTPVS(.beta.-O-GluNAc)SANM.
.sup.dS-GlcNAc 53;
Pam.sub.3CysSK.sub.4G-C-KLFAVWKITYKDT-G-GSTPVS(.beta.-S-GluNAc)SANM.
A statistically significant difference was observed between 52
versus 53 for IgM titers (P = 0.0327). Individual titers for IgG
total, IgG1, IgG2a, IgG2b, IgG3 and IgM are reported in FIG.
20.
[0249] Next, spleens of two mice immunized with the O-linked
glycolipopeptide 52 were harvested and standard hybridoma culture
technology gave seven IgG1, seven IgG2a, two IgG2b and fourteen
IgG3 producing hybridoma cell lines (Table 7). The ligand
specificity of the resulting MAbs was investigated using ELISA and
inhibition ELISA. All MAbs recognized CGSTPVS(.beta.-O-GlcNAc)SANM
linked to BSA whereas only a small number recognized the peptide
CGSTPVSSANM (SEQ ID NO:12) conjugated to BSA. Furthermore, the
interaction of nineteen MAbs with
BSA-MI-CGSTPVS(.beta.-O-GlcNAc)SANM could be inhibited with the
glycopeptide GSTPVS(.beta.-O-GlcNAc)SANM.
TABLE-US-00007 TABLE 7 Monoclonal antibodies against
GSTPVS(.beta.-O-GlcNAc)SANM. Inhibition with ELISA coating: Titer
O-GlcNAc ELISA coating: Fusion Cell Line glycopeptide.sup.a Isotype
Isotype.sup.b glycopeptide.sup.c peptide.sup.d Mouse 1D3.D6(1) +
IgG1 38,000 - - #1 3C1.E8(2) + 38,000 ++ - 18B10.C7(3) + 19,000 +++
- 5H11.H6(4) + 6,000 +++ + 6G3.A5(5) + IgG2a 17,000 +++ -
7A3.G8.F7(6) + 9,000 - - 13F10.G6(7) + IgG2b NA.sup.e - +
11D6.C1(8) + IgG3 29,000 +++ - 1H2.F2(27) + NA.sup. +++ + Mouse
7B8.F5(9) + IgG1 38,000 + - #4 9D1.E4(10) + 38,000 +++ -
16B9.F1(11) + 38,000 ++ - 1D5.C1(12) + IgG2a 3,000 +++ - 1E5.H3(13)
+ <500 - 1F5.D6(14) + 4,000 +++ - 8G11.D6(22) + 2,000 +++ -
14D9.D4(23) + 17,000 + - 3G5.A2(15) + IgG2b 15,000 + - 1E9.E3(16) +
IgG3 14,000 +++ - 2A8.F3(17) + 7,000 + - 2D5.E6(18) + 7,000 - -
5F6.G4(19) + 14,000 +++ - 7B3.A3(20) + 14,000 +++ - 8C3.H2(24) +
7,000 - - 11C6.E5(25) + 14,000 + - 16E2.A3(26) + 14,000 - -
6B5.A8(21) + <500 + 1D7.B4(28) + <500 + 6A5.H1.C6(29) +
<500 + 8F12.A6.C5(30) + 14,000 +++ + .sup.aELISA plates were
coated with BSA-MI-CGSTPVS(.beta.-O-GlcNAc)SANM (BSA-MI-66)
conjugate and supernatants of the different cell lines were
screened undiluted. .sup.bELISA plates were coated with
BSA-MI-CGSTPVS(.beta.-O-GlcNAc)SANM (BSA-MI-66) conjugate and
titers were determined by linear regression analysis, plotting
dilution vs. absorbance. Titers are defined as the highest dilution
yielding an optical density of 0.1 or greater over that of
background. .sup.cELISA plates were coated with
BSA-MI-CGSTPVS(.beta.-O-GlcNAc)SANM (BSA-MI-66) conjugate and
inhibition by GSTPVS(.beta.-O-GlcNAc)SANM (68) was determined: -,
+, ++ and +++ indicate no inhibition, weak inhibition, inhibition
approximately 50% at 500 .mu.M and complete inhibition at 500
.mu.M, respectively. .sup.dELISA plates were coated with
BSA-MI-CGSTPVSSANM (BSA-MI-67) conjugate and supernatants of the
different cell lines were screened undiluted. .sup.eNA indicates
not analyzed.
[0250] Hybridoma cell lines 1F5.D6, 9D1.E4 and 18B10.C7 were
deposited with the American Type Culture Collection (ATCC), 10801
University Blvd., Manassas, Va., 20110-2209, USA, on Jul. 1, 2008,
and assigned ATCC deposit numbers PTA-9339, PTA-9340, and PTA-9341,
respectively. It is nonetheless to be understood that the written
description herein is considered sufficient to enable one skilled
in the art to fully practice the present invention. Moreover, the
deposited embodiment is intended as a single illustration of one
aspect of the invention and is not to be construed as limiting the
scope of the claims in any way.
[0251] Four hybridomas (18B10.C7(3), 5H11.H6(4), 9D1.E4(10),
1F5.D6(14)) were cultured at a one-liter scale and the resulting
antibodies purified by saturated ammonium sulfate precipitation
followed by Protein G column chromatography to yield, in each case,
approximately 10 mg of IgG. The selectivity of the MAbs was
investigated by inhibition ELISA using microtiter plates coated
with BSA-MI-CGSTPVS(.beta.-O-GlcNAc)SANM and glycopeptide, peptide
and .beta.-O-GlcNAc-Ser as inhibitors. As can be seen in FIG. 16,
each MAb was strongly inhibited by the glycopeptide whereas no or
very little inhibition was observed with peptide and
.beta.-O-GlcNAc-Ser. These results show that the MAbs require
carbohydrate and peptide (glycopeptide) for binding.
[0252] Although CKII is an abundant protein, only a small portion
is glycosylated with O-GlcNAc. Therefore, HEK293 cells were
transfected with O-GlcNAc-transferase (OGT, enzyme that adds
O-GlcNAc) and cell lysates analyzed by Western blotting using the
four MAbs as primary antibody and anti-mouse IgG labeled with HRP
as secondary antibody and the results were compared with
mock-transfected HEK293 cells. Furthermore, CKII was
immuno-precipitated with a rabbit polyclonal CKII alpha antibody
followed by analysis by Western blotting using the four MAbs (FIG.
17a). In addition, the blots were stripped and examined with a
commercial anti-CKII antibody (FIG. 17b). In the case of MAbs
9D1.E4(10), 18B10.C7(3) and 1F5D6(14), CKII (a band at .about.42
kDa) was detected after immuno-precipitation and as expected a
stronger response was measured in samples transfected with OGT
(lanes 3 vs. 4, 8 vs. 9 and 12 vs. 13). Interestingly, multiple
bands were observed in cell lysates developed with MAbs 9D1.E4(10),
18B10.C7(3) and 1F5D6(14) (lanes 1, 6 and 10), which were more
pronounced in lysates of cells over-expressing OGT (lanes 2, 7 and
11). Furthermore, additional bands were observed when OGT was
overexpressed. Thus, it appears that these MAbs have a relatively
broad selectivity for O-GlcNAc modified proteins. Although no
consensus sequence for O-GlcNAc has been identified, many proteins
have a TPVSS (SEQ ID NO:10) sequence modified by O-GlcNAc and it is
probable that the MAbs recognize this or similar glycosylated
peptide sequence.
Example IX
Identification of O-GlcNAc Modified Proteins
[0253] Large-scale immunoprecipitation (IP) was performed using
monoclonal IgG antibodies Mab3, Mab10, and Mab14 produced by
hybridomas 18B10.C7, 9D1.E4, and 1F5.D6, respectively, as well as
the commercially available monoclonal IgM antibody CTD110.6 that
was isolated from HEK29T cells treated with PUGNAc (Covance
Research Products, Inc.). The establishment of the hybridomas and
characterization of the antibodies derived therefrom are described
in Example VIII. Following Lys-C digestion, samples were subjected
to electron spray ionization (ESI) mass spectrometry (Collision
induced dissociation (CID)-pseudo neutral loss) analysis. Results
were filtered at 1% false recovery rate and proteins that appeared
in mock IP control experiments were subtracted from the final list
(FIG. 18; Table 8). As shown in Table 8, monoclonal IgG antibodies
demonstrate much higher affinity for O-GlcNAc than the commercially
available IgM antibody.
TABLE-US-00008 TABLE 8 O-GlcNAc modified proteins identified by
immunoprecipitation pulled down with Protein Abbreviation CTD110.6
Mab3 Mab10 Mab14 Acyl-CoA-binding domain-containing protein 7 ACBD7
1 Apoptotic chromatin condensation inducer 1 (Apoptotic chromatin
ACIN1 1 condensation inducer in the nucleus) Actin-like protein 6A
ACTL6A 1 Adenosylhomocysteinase (S-adenocylhomocysteine hydrolase)
AHCY 1 Aldolase A, Fructose-bisphosphate (Fructose-bisphosphate
aldolase A) ALDOA 1 Archaelysin family metallopeptidase 2
(Archaemetzincin-2) AMZ2 1 Annexin A1 ANXA1 1 Apolipoprotein D APOD
1 AT-rich interactive domain-containing protein 1A (SWI-like)
(Chromatin ARID1A 1 remodeling factor p250) Additional sex combs
like 1 ASXL1 1 Additional sex combs like 2 (KIAA1685) ASXL2 1
Atrophin 1 ATN1 1 Ataxin-2 ATXN2 1 Ataxin-2-like protein ATXN2L 1 1
1 HLA-B associated transcript 2 (Large proline-rich protein BAT2)
BAT2 1 BAT2 domain containing 1 (BAT2-iso) BAT2D1 1 1 1 1 Protein
Chromosome 14 open reading frame 166 (CGI-99) C14orf166 1 1 1
Protein Chromosome 14 open reading frame 166 (CGI-99) C14orf166 1 1
Calmodulin-like protein 5 CALML5 1 Capping protein (actin filament)
muscle Z-line, beta CAPZB 1 Coactivator-associated arginine
methyltransferase 1(Histone-arginine CARM1 1 methyltransferase 1)
Cell cycle association protein 1 (Caprin-1; Cytoplasmic CARPIN1 1 1
activation/proliferation-associated protein-1) Cell division cycle
and apoptosis regulator protein 1 CCAR1 1 1 Cysteine conjugate-beta
lyase 2 (RNA-binding motif protein X-linked-like CCBL2 1 1 1)
Cyclin-K CCNK 1 Chaperonin containing TCP1, subunit 8 (theta) CCT8
1 Cofilin-1 CFL1 1 1 Protein capicua homolog CIC 1 Cold-inducible
RNA-binding protein (A18hnRNP) CIRBP 1 1 1 Clathrin light chain B
CLTB 1 Cdc2-related kinase, arginine/serine rich (Cell division
cycle 2-related CRKRS 1 protein kinase 7) Cold shock
domain-containing E1, RNA binding (N-ras upstream gene CSDE1 1
protein) Casein kinase II subunit alpha' CSNK2A2 1 Casein kinase 2,
beta polypeptide CSNK2B 1 Aspartyl-tRNA synthetase, cytoplasmic
DARS 1 Dermcidin precursor DCD 1 DEAD (Asp-Glu-Ala-Asp) box
poplypeptide 1 (ATP-dependent RNA DDX1 1 1 helicase DDX1) DEAD
(Asp-Glu-Ala-Asp) box poplypeptide 5 (Probable ATP-dependent DDX5 1
RNA helicase DDX5) DEAD (Asp-Glu-Ala-Asp) box polypeptide 21
(Nucleolar RNA helicase DDX21 1 2) Death-inducer obliterator 1
DIDO1 1 DnaJ (Hsp40) homolog subfamily A member 1 DNAJA1 1 1 1 DnaJ
(Hsp40) homolog subfamily A member 2 DNAJA2 1 1 Dopey family member
1 DOPEY1 1 Histone-lysine N-methyltransferase, H3 lysine-79
specific (DOT1-like) DOT1L 1 Destrin (actin depolymerizing factor)
DSTN 1 Eukaryotic translation initiation factor 3 subunit G EIF3G 1
Eukaryotic translation initiation factor 3 subunit I (subunit 2)
EIF3I 1 Eukaryotic translation initiation factor 3 subunit J
(subunit 1) EIF3J 1 Glutamyl-prolyl-tRNA synthetase (EPRS protein)
EPRS 1 Endoplasmic reticulum protein ERp29 precursor ERP29 1 1
Ewing sarcoma breakpoint region 1 (RNA-binding protein EWS) EWSR1 1
1 1 Exosome component 1 (3'-5' exoribonuclease CSL4 homolog;
Exosomal EXOSC1 1 core protein CSL4) Fatty acid-binding protein,
brain FABP7 1 Family with sequence similarity 98, member B (Protein
FAM98) FAM98B 1 1 Four and a half LIM domains 1 FHL1 1 Four and a
half LIM domains protein 2 - FHL2 1 Far upstream element-binding
protein 1 FUBP1 1 Ras GTPase-activating protein-binding protein
(SH3 domain) 1 G3BP1 1 Ras GTPase-activating protein-binding (SH3
domain) protein 2 G3BP2 1 Guanine nucleotide-binding protein
subunit beta 2-like 1 (Proliferation- GNB2L1 1 inducing gene 21)
Glutathione S-transferase P GSTP1 1 1 1 Glycogenin-1 GYG1 1 Histone
H1.5 (Histone cluster 1, H1b) H1B 1 Histone H1x H1FX 1 H2A histone
family, member J H2AFJ 1 Host cell factor C1 HCFC1 1 1 1 1
Histidine triad nucleotide-binding protein 1 (Protein kinase
C-interacting HINT1 1 protein 1) High-mobility group box 1 - Homo
sapiens (Human) HMGB1 1 Heterogeneous nuclear ribonucleoprotein A0
hnRNPA0 1 1 Heterogeneous nuclear ribonucleoprotein D (AU-rich
element RNA HNRNPD 1 1 1 1 binding protein 1, 37 kDa) Heterogeneous
nuclear ribonucleoprotein L HNRNPL 1 1 Hypoxanthine-guanine
phosphoribosyltransferase 1 (Lesch-Nyhan HPRT1 1 syndrome) HIV-1
Rev binding protein (Nucleoporin-like protein RIP) HRB 1 Heat shock
70 kDa protein 1 HSP70.1 1 1 1 1 Heat shock protein 90 kDa alpha
(cytosolic), class B member ** HSP90AB2P 1 pseudogene (Heat shock
protein 90Bb) Heat shock 70 kDa protein 4 HSPA4 1 1 60 kDa heat
shock protein, mitochondrial precursor HSPD1 1 Hest shock 10 kDa
protein 1 (Chaperonin 10; 10 kDa heat shock protein, HSPE1 1
mitochondrial) Interleukin enhancer-binding factor 3, 90 kDa ILF3 1
1 1 1 Inosine-5'-monophosphate dehydrogenase 2 IMPDH2 1
Isochorismatase domain-containing protein 1 ISOC1 1 Uncharacterized
protein KIAA1310 KIAA1310 1 Importin subunit beta-1 (Karyopherin)
KPNB1 1 Lipocalin 2 (25 kDa alpha-2-microglobulin-related subunit
of MMP-9) LCN2 1 Lymphocyte cytosolic protein 1 (L-plastin) LCP1 1
L-lactate dehydrogenase A chain LDHA 1 1 L-lactate dehydrogenase B
LDHB 1 1 1 LIN-54 homolog LIN54 1 Protein LSM12 homolog LSM12 1 1
Microtubule-associated protein 4 MAP4 1 MBTD1 protein MBTD1 1
Myeloid/lymphoid or mixed-lineage leukemia (Zinc finger protein
HRX; MLL (HRX 1 Lysine N-methyltransferase 2A)
Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 (C-1-
MTHFD1 1 tetrahydrofolate synthase, cytoplasmic) v-myb
myeloblastosis viral oncogene homolog (avian)-like 2(Myb-related
MYBL2 1 protein B; B-Myb) Myosin, heavy chain 9, non-muscle MYH9 1
1 Myosin, heavy chain 10 (Myosin-10) MYH10 1 1 N-acetyltransferase
13 NAT13 1 1 Nucleolin NCL 1 1 1 Nuclear factor related to
kappa-.beta.-binding protein NFRKB 1 Nucleophosmin (Nucleolar
phosphorprotein B23, numatrin) NPM1 1 1 Nuclear fragile X mental
retardation-interacting protein 2 (FMRP- NUFIP2 1 interacting
protein2) Nucleoporin 153 kDa (Nuclear pore complex protein Nup153)
Nup153 1 1 1 1 Nucleoporin 214 kDa (Nuclear pore complex protein
Nup214) Nup214 1 1 1 1 Nucleoporin 54 kDa NUP54 1 1 1 Nucleoporin
62 kDa (Nuclear pore glycoprotein p62) NUP62 1 1 1 1 Nucleoporin 98
kDa (Nuclear pore complex protein Nup98-Nup96 NUP98 1 1 1
precursor) Nucleoporin like 1 (Nucleoporin p58/p45) NUPL1 1 1 1
O-linked N-acetylglucosamine (GlcNAc) transferase (UDP-N- OGT 1
acetylglucosamine:peptide N-acetylglucosaminyltransferase 110 kDa
subunit) Poly(A) binding protein, cytoplasmic 1
(Polyadenylate-binding protein 1) PABPC1 1 Poly(A) binding protein,
cytoplasmic 4 (inducible form) PABPC4 1
Phosphoribosylaminoimidazole carboxylase, PAICS 1
phosphoribosylaminoimidazole succinocarboxamide synthetase
(Multifunctional protein ADE2) Poly [ADP-ribose] polymerase 1 PARP1
1 1 Protein-L-isoaspartate(D-aspartate) O-methyltransferase PCMT1 1
1 Phosphatidylethanolamine-binding protein 1 PEBP1 1 1 Profilin-1
PFN1 1 1 1 Polyhomeotic-like protein 3 PHC3 1 PHD finger protein 12
(PHD zinc finger transciption factor) PHF12 1 Pyruvate kinase,
muscle (Pyruvate kinase isozymes M1/M2) PK 1 POM121 membrane
glycoprotein(Nuclear envelope pore membrane POM121 1 protein POM
121) Peptidyl-prolyl cis-trans isomerase A (Cyclophilin A;
Cyclosporin A- PPIA 1 1 1 binding protein) Peptidyl-prolyl
cis-trans isomerase B (Cyclophilin B) PPIB 1 Peptidyl-prolyl
cis-trans isomerase F (Cyclophilin F) PPIF 1 Protein phosphatase 1
regulatory subunit 12A PPP1R12A 1 Peroxiredoxin-1 PRDX1 1 1 1
Proteasome 26S subunit, non-ATPase 1 (26S proteasome non-ATPase
PSMD1 1 regulatory subunit) Polypyrimidine tract-binding protein
1(Heterogeneous nuclear PTBP1 1 1 ribonucleoprotein1) Glutamine and
serine-rich protein 1 QSER1 1 1 RAE1 RNA export 1 homolog (mRNA
export factor; mRNA-associated RAE1 1 protein mrnp 41) RAN, member
RAS oncogene family(GTP-binding nuclear protein Ran) RAN 1 Ran
GTPase-activating protein 1 RANGAP1 1 Putative RNA-binding protein
15 RBM15 1 1 RNA-binding protein 26 RBM26 1 RNA binding motif
protein 27 (RNA-binding protein 27) RBM27 1 1 RNA binding motif
protein, X-linked (Heterogeneous nuclear RBMX 1 ribonucleoprotein
G) Ringer finger protein 2 (E3 ubiquitin-protein ligase RING2) RNF2
1 RNA(guanine-7-)methyltransferase (mRNA cap guanine-N7 RNMT 1
methyltransferase) Replication protein A 70 kDa RPA1 1 60S
ribosomal protein L3 RPL3 1 Ribosomal protein L9 RPL9 1 1 1 60S
ribosomal protein L10 RPL10 1 60S ribosomal protein L17 RPL17 1
Ribosomal protein L18a RPL18A 1 60S ribosomal protein L23 RPL23 1 1
60S ribosomal protein L23a RPL23A 1 1 1 60S ribosomal protein L24
RPL24 1 1 60S ribosomal protein L26 RPL26 1 1 60S ribosomal protein
L27a RPL27A 1 Ribosomal protein L28 variant RPL28 1 1 60S ribosomal
protein L29 RPL29 1 1 1 60S ribosomal protein L31 RPL31 1 1 60S
ribosomal protein L36a RPL36A 1 1 Ribosomal protein, large P2 (60S
acidic ribosomal protein P2) RPLP2 1 40S ribosomal protein S6 RPS6
1 1 40S ribosomal protein S11 - Homo sapiens (Human) RPS11 1 1 40S
ribosomal protein S18 RPS18 1 40S ribosomal protein S19 RPS19 1 40S
ribosomal protein S20 RPS20 1 1 40S ribosomal protein S23 RPS23 1 1
1 Ribosomal protein S27 RPS27 1 1 Ribosomal RNA processing 1
homolog (RRP1-like protein B) RRP1B 1 RuvB-like 1 (49 kDa TATA box
binding protein-interacting protein) RUVBL1 1 RuvB-like 2 (48 kDa
TATA box-binding protein-interacting protein) RUVBL2 1 1 S100
calcium binding protein A7(Protein S100-A7) S100A7 1 S100 calcium
binding protein A8 (Protein S100-A8) S100A8 1 Protein S100-A9
S100A9 1 Scaffold attachment factor B (HSP27 estrogen response
element-TATA SAFB 1 box-binding protein) Protein SEC13 homolog
SEC13 1 Sec23 homolog A (Protein transport protein Sec23A) SEC23A 1
Sec23 homolog B (Protein transport protein Sec23B) SEC23B 1
SEC23-interacting protein SEC23IP 1 1 SEC 24 related gene family,
member C (Protein transport protein Sec24C) SEC24C 1 Protein
transport protein Sec31A (SEC31 homolog A) SEC31A 1 SET domain
containing 1A (Histone-lysine N-methyltransferase, H3 SETD1A 1
lysine-4 specific SET1) Splicing factor 1 SF1 1 Splicing factor,
proline/glutamine-rich (polypyrimidine tract binding SFPQ 1 protein
associated) Splicing factor, arginine/serine-rich 3 SFRS3 1 SIN3
homolog, transcription regulator (Paired amphipathic helix protein
SIN3B 1
Sin3b) SWI/SNF-related matrix-associated actin-dependent regulator
of SMARCC1 1 chromatin subfamily C member 1 Sp1 transcription
factor Sp1 1 Snf2-related CREBBP activator protein (KIAA0309
protein) SRCAP 1 Signal recognition particle 14 kDa protein SRP14 1
1 1 Sjogren syndrome antigen B (Lupus La protein; autoantigen La)
SSB 1 Serine-threonine kinase receptor-associated protein STRAP 1 1
Transcription elongation regulator 1 TCERG1 1 TRK-fused gene
protein (TRKT3 oncogene) TFG 1 Triosephosphate isomerase TPI1 1
Thioredoxin TXN 1 1 1 Ubiquitin-associated protein 2 UBAP2 1 1 1 1
Ubiquitin-associated protein 2-like (Protein NICE-4) UBAP2L 1 1 1 1
Vimentin VIM 1 1 WD repeat protein 5 WDR5 1 WD repeat protein 35
WDR35 1 Serine/threonine-protein kinase WNK1(WNK lysine deficient
protein WNK1 (p65) 1 1 1 1 kinase 1; Erythrocyte 65 kDa protein)
WNK lysine deficient protein kinase 3 (Serine/threonine-protein
kinase WNK3 1 1 1 WNK3) Y box binding protein 1(Nuclease sensitive
element-binding protein 1) YBX1 1 1 YEATS domain-containing protein
2 YEATS2 1 14-3-3 protein epsilon (tyrosine
3-monooxygenase/tryptophan 5- YWHAE 1 monooxygenase activation,
epsilon polypeptide) Zinc finger RNA-binding protein (M-phase
phosphoprotein homolog) ZFR 1 Zyxin ZYX 1 Zinc finger
ZZ-type-containing protein 3 ZZZ3 1
Example X
Generation of O-GlcNAc Specific Monoclonal Antibodies Using a Novel
Synthetic Immunogen
[0254] Combining a fully synthetic three-component immunogen with
hybridoma technology led to the generation of O-GlcNAc-specific IgG
MAbs having a broad spectrum of binding targets. Large-scale
shotgun proteomics led to the identification of 254 mammalian
O-GlcNAc modified proteins, including a large number of novel
glycoproteins. The data imply a role of O-GlcNAc in
transcriptional/translational regulation, signal transduction, the
ubiquitin pathway, anterograde trafficking of intracellular
vesicles and post-translational modification.
[0255] O-glycosylation of serine and threonine of nuclear and
cytoplasmic proteins by a single .beta.-N-acetyl-D-glucosamine
moiety (.beta.-GlcNAc) is a ubiquitous post-translational
modification that is highly dynamic and fluctuates in response to
cellular stimuli through the action of the cycling enzymes,
O-linked GlcNAc transferase (OGT) and O-GlcNAcase (OGA). This type
of glycosylation has been implicated in many cellular processes,
frequently via interplay with phosphorylation that can occur on the
same amino acid residue1. Importantly, alteration of O-GlcNAc
levels has been linked to the etiology of prevalent human diseases
including type II diabetes and Alzheimer's disease (Hart et al.,
2007 Nature 446, 1017-1022).
[0256] Unlike phosphorylation for which a wide range of pan- and
site-specific phospho-antibodies are available, studies of O-GlcNAc
modification are hampered by a lack of effective tools for its
detection, quantification, and site localization. In particular,
only two pan-O-GlcNAc specific antibodies have been described: an
IgM pan-O-GlcNAc antibody (CTD 110.6; Comer et al., 2001 Anal.
Biochem. 293, 169-177), and an IgG antibody raised against O-GlcNAc
modified components of the nuclear pore (RL-2; Snow et al., 1987 J.
Cell Biol. 104, 1143-1156) that shows restricted cross-reactivity
with O-GlcNAc modified proteins. In fact, multiple studies have
shown that O-GlcNAc modified glycoconjugates do not elicit relevant
IgG isotype antibodies and thus, the challenge to elicit O-GlcNAc
specific IgG antibodies is considerable.
[0257] We reasoned that O-GlcNAc-specific antibodies can be
elicited by employing a three-component immunogen (compound 52,
FIG. 19) composed of an O-GlcNAc containing peptide, which in this
study is derived from casein kinase II (CKII) .alpha. subunit,
(Kreppel and Hart, 1999 J. Biol. Chem. 274, 32015-32022) a
well-documented murine MHC class II restricted helper T-cell
epitope and a Toll-like receptor-2 (TLR2) agonist as an in-built
adjuvant. Such a compound is expected to circumvent immune
suppression caused by a carrier protein or linker region of a
classical conjugate vaccine; yet it contains all mediators required
for eliciting a strong and relevant IgG immune response (Ingale et
al., 2007 Nat. Chem. Biol. 3, 663-667). In addition, compound 53
was prepared that has an artificial thio-linked GlcNAc moiety,
which has an improved metabolic stability compared to its O-linked
counter-part thereby providing additional opportunities to
eliciting O-GlcNAc specific antibodies.
[0258] Compounds 52 and 53 were readily obtained by
liposome-mediated native chemical ligations (Ingale et al., 2006
Org. Lett. 8, 5785-5788) of C-terminal lipopeptide thioester 63
with glycopeptides 64 and 65, respectively (FIG. 19). The starting
thioester 63 was assembled on a sulfonamide "safety-catch" linker
followed by release by alkylation with iodoacetonitrile and
treatment with benzyl mercaptan to give a compound that was
deprotected using standard conditions. Compounds 64 and 65 were
prepared employing a Rink amide resin, Fmoc protected amino acids
and Fmoc-Ser-(AcO3-.alpha.-D-GluNAc) or
Fmoc-Ser-(1-thio-AcO3-.alpha.-D-GluNAc), respectively. After
completion of the assembly, the acetyl esters were cleaved by
treatment with 60% hydrazine in MeOH and the resulting compounds
were cleaved from the resin by treatment with reagent K and
purified by reverse phase HPLC. Compounds 52 and 53 were
incorporated into phospholipid based small unilamellar vesicles
(SUVs) followed by extrusion through a 100 nm Nuclepore.RTM.
polycarbonate membrane. Groups of five female BALB/c mice were
immunized intra-peritoneal four times at two-weekly intervals with
liposomes containing 3 .mu.g of saccharide. Antiglycopeptide
antibody titers were determined by coating microtiter plates with
CGSTPVS(.beta.-O-GlcNAc) SANM (66) conjugated to maleimide (MI)
modified BSA and detection was accomplished with anti-mouse IgG
antibodies labeled with alkaline phosphatase. Compounds 52 and 53
elicited excellent titers of IgG antibodies (Table 6; FIG. 20).
Furthermore, no significant difference in IgG titers was observed
between the O- and S-linked saccharide derivatives, and therefore
further attention was focused on mice immunized with 52.
[0259] Spleens of two mice immunized with 52 were harvested and
standard hybridoma culture technology gave seven IgG1, seven IgG2a,
two IgG2b and fourteen IgG3 producing hybridoma cell lines (Table
7). The ligand specificity of the resulting MAbs was investigated
by ELISA. All MAbs recognized CGSTPVS(.beta.-O-GlcNAc)SANM linked
to BSA (BSA-MI-66) whereas only a small number recognized the
peptide CGSTPVSSANM (SEQ ID NO:12) conjugated to BSA (BSA-MI-67).
Furthermore, the interaction of twenty MAbs could be inhibited with
the glycopeptide GSTPVS(.beta.-O-GlcNAc)SANM (68), but not with
peptide GSTPVSSANM (SEQ ID NO: 13) (69) or .beta.-O-GlcNAc-Ser (70)
demonstrating glycopeptide specificity.
[0260] Three hybridomas (18B10.C7(3), 9D1.E4(10), 1F5.D6(14)) were
cultured at a one-liter scale and the resulting antibodies purified
by saturated ammonium sulfate precipitation followed by Protein G
column chromatography to yield, in each case, approximately 10 mg
of IgG. Inhibition ELISA confirmed that the MAbs require
carbohydrate and peptide (glycopeptide) for binding (FIG. 16).
[0261] To establish the usefulness of the MAbs for immuno
detection, CKII .alpha. subunit was immunoprecipitated from HEK293T
lysates with or without exogenous overexpression of OGT and the
eluates were subjected to standard immunoblotting procedures. While
equal amounts of CKII .alpha. subunit were pulled down, the
monoclonal antibodies showed cross-reactivity towards a band
corresponding to CKII .alpha. subunit with an increased signal for
the OGT overexpressed sample supporting GlcNAc-dependence of
recognition (FIG. 21a). The latter was supported by the absence of
detection when a glycosylated recombinant CKII .alpha. subunit
expressed in E. coli was employed (data not shown). The specificity
of the MAbs was further evaluated in mammalian cell crude extracts
by genetically manipulating OGA or OGT levels. Importantly, three
distinct global O-GlcNAc levels were observed, in which lysates
with OGA, mock and OGT transfection yielded lowest, median and
highest modification status, which is in good agreement with the
expression levels of the cycling enzymes (FIG. 21b). The results
imply that although the epitope was derived from a single protein,
the MAbs have a broad spectrum of binding targets. We also compared
the immunoblotting profiles of the new MAbs with CTD110.6 (a
commercially available pan-O-GlcNAc IgM antibody) after enrichment
of O-GlcNAc modified proteins with each of the MAbs (FIG. 21c). The
data clearly illustrate that each of the antibodies enrich for
CTD110.6 cross-reactive proteins. Immunopurification with CTD110.6
also enriched for proteins that cross-reacted with the three new
antibodies upon immunoblotting (data not shown).
[0262] Although one gene encodes for OGT and another for OGA in
mammals, no obvious primary consensus sequence for O-GlcNAc
modification has been identified. A recently reported crystal
structure of an OGT homolog (Martinez-Fleites et al., 2008 Nat.
Struct. Mol. Biol. 15, 764-765) showed a large groove near the
active site and it has been proposed that it may accommodate a
diverse set of polypeptide substrates and/or a particular secondary
structure. The crystal structure data in conjunction with our
findings that all characterized antibodies were pan-O-GlcNAc
antibodies indicates that the O-GlcNAc modified regions of
polypeptide chains may share a limited number of conserved
secondary structures.
[0263] Finally, the MAbs were employed for large-scale enrichment
of O-GlcNAc modified proteins for shotgun proteomics. Agarose
covalently conjugated MAbs were mixed with nucleocytoplasmic
proteins extracted from HEK293T cells cultured in the presence of
the OGA inhibitor, PUGNAc (Haltiwanger et al., 1998 J. Biol. Chem.
273, 3611-3617). The released proteins were subjected to Lys-C
digestion and the recovered peptides and glycopeptides were
analyzed by LC-MS/MS on an LTQ-XL. Protein assignments and
false-discovery rates (1% at the protein level) were calculated
using TurboSequest and ProteoIQ. Proteins were excluded that
appeared in control experiments (mixture of Protein AJG PLUS
agarose and anti-Mouse IgM agarose) and localization was confirmed
with the aid of Human Protein Reference Database (HPRD) and
UniProt. Using the three MAbs generated in this study, we
identified 254 O-GlcNAc modified proteins, 134 of which are novel
(FIG. 22a, Tables 9 and 10). This represents the largest single set
of putative O-GlcNAc modified proteins reported to date. A large
number of previously characterized O-GlcNAc modified proteins, such
as SP1, OGT and nuclear pore protein p62 were found adding
confidence to proper assignment and further supports the
selectivity of the antibodies for O-GlcNAc modification. However,
at this point the possibility that some proteins may have been
co-purified due to tight association to O-GlcNAc modified proteins
can not be excluded.
TABLE-US-00009 TABLE 9 List of enriched known O-GlcNAc proteins.
SwissProt Gene Number Name Protein Name 18B10.C7(3) 9D1.E4(10)
1F5.D6(14) CTD110.6 P18621 RPL17 60S ribosomal protein L17 Q9H4A3
WNK1 Serine/threonine-protein kinase WNK1 (Protein kinase, lysine
deficient 1) Q12771 HNRNPS P37 AUF1 (heterogeneous nuclear
ribonucleoprotein D; AU-rich element RNA binding protein 1, 37 kDa)
P09651 HNRPA1 Heterogeneous nuclear ribonucleoprotein A1 P37198
NUP62 Nuclear pore glycoprotein p62 Q53H29 -- Nucleoporin 54 kDa
variant P62937 PPIA Peptidyl-prolyl cis-trans isomerase A
(cyclophilin A) Q14157 UBAP2L Ubiquitin-associated protein 2-like
(Protein NICE-4, KIAA0144) P09211 GSTP1 Glutathione S-transferase P
P11940 PABPC1 Polyadenylate-binding protein 1 P10599 TXN
Thioredoxin P63244 GNB2L1 Guanine nucleotide binding protein
subunit beta 2-like 1 (RACK1) P32119 PRDX2 Peroxiredoxin-2 P35580
MYH10 Myosin-10 P60709 ACTB Actin, cytoplasmic 1 P34932 HSPA4 Heat
shock 70 kDa protein 4 P14866 HNRPL Heterogeneous nuclear
ribonucleoprotein L P38159 RBMX Heterogeneous nuclear
ribonucleoprotein G P19338 NCL Nucleolin P04075 ALDOA
Fructose-bisphosphate aldolase A P12268 IMPDH2
Inosine-5'-monophosphate dehydrogenase 2 P23246 SFPQ Splicing
factor, proline- and glutamine- rich Q17RM7 EMSY EMSY protein
Q15436 SEC23A Protein transport protein Sec23A Q15437 SEC23B
Protein transport protein Sec23B P35579 MYH9 Myosin-9 P13639 EEF2
Elongation factor 2 P23526 AHCY Adenosylhomocysteinase (S-
adenosylhomocysteine hydrolase) Q92499 DDX1 ATP-dependent RNA
helicase DDX1 (DEAD box protein retinoblastoma) Q15393 SF3B3
Splicing factor 3B subunit 3 P50990 CCT8 T-complex protein 1
subunit theta P61604 HSPE1 10 kDa heat shock protein, mitochondrial
P23528 CFL1 Cofilin-1 A4UCT1 GAPDH Glyceraldehyde 3-phosphate
dehydrogenase P08238 HSP90AB1 Heat shock protein HSP 90-beta P60174
TPI1 Triosephosphate isomerase P26641 EEF1G Elongation factor
1-gamma O43390 HNRPR Heterogeneous nuclear ribonucleoprotein R
P04083 ANXA1 Annexin A1 Q14974 KPNB1 Importin subunit beta-1
(Karyopherin beta11) P14618 PKM2 Pyruvate kinase, isozymes M1/M2
P00338 LDHA L-lactate dehydrogenase A chain Q6P4R8 NFRKB Nuclear
factor related to kappa-B- binding protein Q9BYJ9 YTHDF1 YTH domain
family protein 1 (DACA-1 homolog) Q96KR1 ZFR Zinc finger
RNA-binding protein A0AVA9 EPRS Glutamyl-prolyl-tRNA synthetase
Q59EJ3 -- Heat shock 70 kDa protein 1A variant Q9P2J5 LARS
Leucyl-tRNA synthetase, cytoplasmic P11831 SRF Serum response
factor P08047 SP1 Transcription factor Sp1 P23396 RPS3 40S
ribosomal protein S3 P07910 HNRPC Heterogeneous nuclear
ribonucleoproteins C1/C2 P19784 CSNK2A2 Casein kinase II subunit
alpha' P67870 CSNK2B Casein kinase II subunit beta Q5SP16 HSPA1A
Heat shock 70 kDa protein 1A Q9BYG9 NPM1 Nucleophosmin/B23.2 O15294
OGT UDP-N-acetylglucosamine-peptide N-
acetylglucosaminyltransferase 110 kDa subunit Q8TE73 DNAH5 Dynein,
axonemal, heavy polypeptide 5 Q02447 SP3 Transcription factor Sp3
P36578 RPL4 60S ribosomal protein L4 P49368 CCT3 T-complex protein
1 subunit gamma P10809 HSPD1 60 kDa heat shock protein,
mitochondrial precursor P13807 GYS1 Glycogen [starch] synthase,
muscle SwissProt Biological Primary Alternate Number Process
Localization Localization Previously Identified Method (Ref) P18621
Tl R No Click-chemistry-based tagging enrichment, LC-MS/MS(1)
Q9H4A3 S C Galactose-ketone-biotin enrichment, LC- MS/MS(2) Q12771
G C N Azido-biotin enrichment, LC-MS/MS(3) P09651 G N C, No
CTD110.6 immunopurify, LC-MS/MS(4) P37198 Tp N CTD110.6
immunopurify, LC-MS/MS(5) Q53H29 Tp N C CTD110.6 immunopurify,
LC-MS/MS(5) P62937 Tl C No CTD110.6 immunopurify, LC-MS/MS(5)
Q14157 U N CTD110.6 immunopurify, LC-MS/MS(5) P09211 M C N
Azido-biotin enrichment, LC-MS/MS(6) P11940 G C N CTD110.6
immunopurify, LC-MS/MS(4) P10599 M C N Azido-biotin enrichment,
LC-MS/MS(3) P63244 S C N, PM Azido-biotin enrichment, LC-MS/MS(3)
P32119 M C PM Azido-biotin enrichment, LC-MS/MS(3) P35580 S C
Lectin affinity chromatography, LC-MS/MS(7) P60709 S C G, PM, No
Azido-biotin enrichment, LC-MS/MS(3) P34932 Tl C Azido-biotin
enrichment, LC-MS/MS(3) P14866 G N No, C 2DE, CTD110.6
immunoblotting, LC-MS/MS(8) P38159 G N No Hot labeling, HPLC(9)
P19338 G No N, C, PM CTD110.6 immunopurify, LC-MS/MS(4) P04075 M C
Lectin affinity chromatography, LC-MS/MS(7) P12268 M U Azido-biotin
enrichment, LC-MS/MS(3) P23246 G N No CTD110.6 immunopurify,
LC-MS/MS(4) Q17RM7 U U Lectin weak affinity chromatography, LC-
MS/MS(10) Q15436 Tp C ER CTD110.6 immunopurify, LC-MS/MS(5) Q15437
Tp ER G, C CTD110.6 immunopurify, LC-MS/MS(5) P35579 S C No Lectin
affinity chromatography, LC-MS/MS(7) P13639 Tl C No
Click-chemistry-based tagging enrichment, LC-MS/MS(1) P23526 M C
Azido-biotin enrichment, LC-MS/MS(3) Q92499 G N No Azido-biotin
enrichment, LC-MS/MS(3) Q15393 G N C Azido-biotin enrichment,
LC-MS/MS(3) P50990 Tl C Azido-biotin enrichment, LC-MS/MS(3) P61604
Tl M No CTD110.6 immunopurify, LC-MS/MS(4) P23528 S C N, PM
CTD110.6 immunopurify, LC-MS/MS(5) A4UCT1 M C CTD110.6
immunopurify, LC-MS/MS(5) P08238 S C CTD110.6 immunopurify,
LC-MS/MS(5) P60174 M C Lectin affinity chromatography, LC-MS/MS(7)
P26641 Tl U Azido-biotin enrichment, LC-MS/MS(3) O43390 G N No
CTD110.6 immunopurify, LC-MS/MS(4) P04083 S PM C, N CTD110.6
immunopurify, LC-MS/MS(5) Q14974 Tp C N CTD110.6 immunopurify,
LC-MS/MS(5) P14618 M C CTD110.6 immunopurify, LC-MS/MS(5) P00338 M
C No Lectin affinity chromatography, LC-MS/MS(7) Q6P4R8 G N
Galactose-ketone-biotin enrichment, LC- MS/MS(2) Q9BYJ9 U U
Galactose-ketone-biotin enrichment, LC- MS/MS(2) Q96KR1 G N No
Galactose-ketone-biotin enrichment, LC- MS/MS(2) A0AVA9 Tl C
Azido-biotin enrichment, LC-MS/MS(3) Q59EJ3 U U Azido-biotin
enrichment, LC-MS/MS(3) Q9P2J5 M C Azido-biotin enrichment,
LC-MS/MS(3) P11831 G N Hot labeling, HPLC, Gas chromatography,
Edman degradation(11) P08047 G N Hot labeling, HPLC. Gas
chromatography, Edman degradation(12) P23396 Tl C N CTD110.6
immunopurify, LC-MS/MS(4) P07910 G N C, No CTD110.6 immunopurify,
LC-MS/MS(4) P19784 S N C, PM, No CTD110.6 immunopurify, LC-MS/MS(5)
P67870 S N C CTD110.6 immunopurify, LC-MS/MS(5) Q5SP16 Tl U
CTD110.6 immunopurify, LC-MS/MS(5) Q9BYG9 Tl N CTD110.6
immunopurify, LC-MS/MS(5) O15294 Tl N C, M CTD110.6 immunopurify,
LC-MS/MS(5) Q8TE73 S C CTD110.6 immunopurify, LC-MS/MS(5) Q02447 G
N O-GlcNAc immunoblotting(13) P36578 Tl R No Azido-biotin
enrichment, LC-MS/MS(3) P49368 Tl C No Azido-biotin enrichment,
LC-MS/MS(3) P10809 Tl 2DE, CTD110.6 immunoblotting, LC-MS/MS(8)
P13807 M C N O-GlcNAc immunoblotting(14) * Abbreviations: G, Gene
expression/Transcription; M, Metabolism; S, Signal transduction;
Tl, Translation; Tp, Transport: U, Unknown; C, Cytoplasm; N,
Nucleus; No, Nucleolus; ER, Endoplasmic reticulum; G, Golgi
apparatus; Ex, Extracellular. References 1. Gurcel et al., (2008)
Anal Bioanal Chem, 390: 2089-2097. 2. Khidekel et al., (2004) PNAS.
101: 13132-13137. 3. Nandi, et al., (2006) Anal Chem, 78: 452-458.
4. Wang et al., (2007) MCP, 6: 1365-1379. 5. Wells et al., (2002)
MCP. 1: 791-804. 6. Sprung et al., (2005) J Proteome Res, 4:
950-957. 7. Cieniewski-Bernard et al., (2004) MCP, 3: 577-585. 8.
Park et al., (2007) JBMB, 40: 1058-1068. 9. Soulard et al., (1993)
Nucl Acids Res, 21: 4210-4217. 10. Vosseller et al., (2006) MCP, 5:
923-934. 11. Reason et al., (1992) JBC, 267: 16911-16921. 12. Roos
et al., (1997) MCB, 17(11): 6472-6480. 13. Yao, et al., (2007) JBC:
282(42): 1038-1045. 14. Parker et al., (2003) JBC, 278:
10022-10027.
TABLE-US-00010 TABLE 10 List of enriched novel O-GlcNAc proteins.
SwissProt Number Gene Name Protein Name P35658 NUP214 Nuclear pore
complex protein Nup214 P49790 NUP153 Nuclear pore complex protein
Nup153 Q06587 RING1 E3 ubiquitin-protein ligase RING1 Q5T6F2 UBAP2
Ubiquitin-associated protein 2 Q9Y520 BAT2D1 BAT2-iso (BAT2
domain-containing protein 1; HBxAg transactivated protein 2) Q9Y6Y8
SEC23IP SEC23-interacting protein Q13151 HNRPA0 Heterogeneous
nuclear ribonucleoprotein A0 Q9GZZ1 NAT13 N-acetyltransferase 13
(Mak3) Q14011 CIRBP Cold-inducible RNA-binding protein O75821
EIF3S4 Eukaryotic translation initiation factor 3 subunit 4 P16402
HIST1H1D Histone H1.3 P26373 RPL13 60S ribosomal protein L13 Q2KHR3
QSER1 Glutamine and serine-rich protein 1 (FLJ21924) Q52LJ0 FAM98B
Protein FAM98B Q8NC51 SERBP1 Plasminogen activator inhibitor 1
RNA-binding protein (SERPINE1 mRNA binding protein 1) P16401
HIST1H1B Histone H1.5 P52948 NUP98 Nuclear pore complex protein
Nup98-Nup96 precursor [Contains: Nuclear pore complex protein Nup98
P78406 RAE1 mRNA export factor (MRNP41) Q05BK6 TFG TFG protein
(TRK-fused gene protein) Q5JRG1 NUPL1 Nucleoporin like 1 Q9P2N5
RBM27 RNA-binding protein 27 P07737 PFN1 Profilin-1 P32969 RPL9 60S
ribosomal protein L9 Q9Y3F4 STRAP Serine-threonine kinase
receptor-associated protein Q8IYH5 ZZZ3 Zinc finger
ZZ-type-containing protein 3 Q92522 H1FX Histone H1x A2A3R5 RPS6
Ribosomal protein S6, isoform CRA_a P30050 RPL12 60S ribosomal
protein L12 P46779 RPL28 60S ribosomal protein L28 P54652 HSPA2
Heat shock-related 70 kDa protein 2 P63220 RPS21 40S ribosomal
protein S21 Q15717 ELAVL1 ELAV-like protein 1 (Embryonic lethal
abnormal vision like 1) A1L431 PPIAL4 Peptidyl-prolyl cis-trans
isomerase A-like 4 (Cyclophilin LC) P55735 SEC13 Protein SEC13
homolog Q86X55 CARM1 Histone-arginine methyltransferase CARM1
(Coactivator associated arginine methyltransferase 1) Q8IX12 CCAR1
Cell division cycle and apoptosis regulator protein 1 O60884 DNAJA2
DnaJ homolog subfamily A member 2 O14776 TCERG1 Transcription
elongation regulator 1 Q24JQ7 ATXN2 ATXN2 protein Q8NCA5 FAM98A
DKFZP564F0522 protein Q8WWM7 ATXN2L Ataxin-2-domain protein Q96HA1
POM121 POM121 membrane glycoprotein (Nuclear envelop pore membrane
protein POM121) Q9NX58 LYAR Cell growth-regulating nucleolar
protein A2A3N6 PIPSL Novel protein similar to
phosphatidylinositol-4-phosphate 5-kinase, type I, alpha (Putative
PIP5K1A and PSMD4-like protein) A2A3R7 RPS6 Ribosomal protein S6
A5JHP3 DCD Dermcidin isoform 2 O43148 RNMT mRNA cap guanine-N7
methyltransferase O75534 CSDE1 Cold shock domain-containing protein
E1 (Upstream of NRAS) P00492 HPRT1 Hypoxanthine-guanine
phosphoribosyltransferase P02795 MT2A Metallothionein-2 P05387
RPLP2 60S acidic ribosomal protein P2 P09429 HMGB1 High mobility
group protein B1 P22234 PAICS Multifunctional protein ADE2
[Includes: Phosphoribosylaminoimidazole- succinocarboxamide
synthase P27694 RPA1 Replication protein A 70 kDa DNA-binding
subunit P30086 PEBP1 Phosphatidylethanolamine-binding protein 1
(Raf kinase inhibitor protein) P31689 DNAJA1 DnaJ homolog subfamily
A member 1 P35125 USP6 Ubiquitin carboxyl-terminal hydrolase 6
P43487 RANBP1 Ran-specific GTPase-activating protein P48634 BAT2
Large proline-rich protein BAT2 (HLA-B associated transcript 2)
P61927 RPL37 60S ribosomal protein L37 P62633 CNBP Cellular nucleic
acid-binding protein (Zinc finger protein 9) Q09028 RBBP4
Histone-binding protein (Retinoblastoma binding protein 4) Q13310
PABPC4 Polyadenylate-binding protein 4 Q13347 EIF3S2 Eukaryotic
translation initiation factor 3 subunit 2 Q14192 FHL2 Four and a
half LIM domains protein 2 Q14444 GPIAP1 Caprin-1 Q14684 KIAA0179
RRP1-like protein B Q2M2Y6 ZNF615 Zinc finger protein 615 Q5JXK1
FAM135A Protein FAM135A Q5RLJ0 -- CLE Q6IBH5 PPIB Peptidyl-prolyl
cis-trans isomerase B (Cyclophilin B) Q6ZUI0 FAM79B Protein FAM79B
9Family with sequence similarity 79, member B) Q7LBC6 JMJD1B JmjC
domain-containing histone demethylation protein 2B (Jumonji domain
containing 1B) Q96AE4 FUBP1 Far upstream element-binding protein 1
Q9UJV9 DDX41 Probable ATP-dependent RNA helicase DDX41 (DEAD
Asp-Glu-Ala-Asp box polypeptide 41) A1L3W5 SUMO4 SUMO4 (SMT3
suppressor of mif two 3 homolog 4) A2A305 UBAP2 Ubiquitin
associated protein 2 O60333 KIF1B Kinesin-like protein KIF1B O60506
SYNCRIP Heterogeneous nuclear ribonucleoprotein Q O95259 KCNH1
Potassium voltage-gated channel subfamily H member 1 P05109 S100A8
S100 calcium binding protein A8 P06702 S100A9 S100 calcium binding
protein A9 P31151 S100A7 S100 calcium binding protein A7 Q01469
FABP5 Fatty acid-binding protein, epidermal Q69YU5 -- Putative
uncharacterized protein DKFZp547P055 Q8IVL0 NAV3 Neuron navigator 3
Q99567 NUP88 Nuclear pore complex protein Nup88 A0AVI3 H2BFS H2B
histone family, member S A4D1M5 LOC401404 Similar to ribosomal
protein S14 O14497 ARIDIA AT-rich interactive domain-containing
protein 1A O14974 PPP1R12A Protein phosphatase 1 regulatory subunit
12A O15026 SRCAP Helicase SRCAP (Transcription activator SRCAP)
O15047 SETD1A Histone-lysine N-methyltransferase, H3 lysine-4
specific SET1 O60907 TBL1X F-box-like/WD repeat protein TBL1X
(SMAP55) O75182 SIN3B Paired amphipathic helix protein Sin3b O75528
TADA3L Transcriptional adapter 3-like O75822 EIF3S1 Eukaryotic
translation initiation factor 3 subunit 1 O75937 DNAJC8 DnaJ
homolog subfamily C member 8 O94979 SEC31A Protein transport
protein Sec31A (SEC31 like 1) P14174 MIF Macrophage migration
inhibitory factor P14859 POU2F1 Octamer binding transcription
factor 1 (POU domain, class 2, transcription factor 1) P14868 DARS
Aspartyl-tRNA synthetase, cytoplasmic P15822 HIVEP1 Zinc finger
protein 40 (Major histocompatibility complex binding protein 1)
P26599 PTBP1 Polypyrimidine tract-binding protein 1 (hnRNPI) P49750
YLPM1 YLP motif-containing protein 1 (Nuclear protein ZAP) P49792
RANBP2 E3 SUMO-protein ligase RanBP2 P49916 LIG3 DNA ligase 3
P51532 SMARCA4 Probable global transcription activator SNF2L4
P52594 HRB Nucleoporin-like protein RIP P53992 SEC24C Protein
transport protein Sec24C P54198 HIRA Protein HIRA P54259 ATN1
Atrophin-1 P61964 WDR5 WD repeat protein 5 P61981 YWHAG 14-3-3
protein gamma P82914 MRPS15 28S ribosomal protein S15,
mitochondrial precursor Q02878 RPL6 60S ribosomal protein L6 Q03164
MLL Zinc finger protein HRX (MLL, Histone-lysine
N-methyltransferase HRX) Q12830 BPTF Nucleosome-remodeling factor
subunit (Fetal Alzheimer antigen) Q13185 CBX3 Chromobox protein
homolog 3 (Modifier 2 protein) Q13283 G3BP1 Ras GTPase-activating
protein-binding protein 1 Q13547 HDAC1 Histone deacetylase 1 Q14119
VVEZF1 Vascular endothelial zinc finger 1 Q14978 NOLC1 Nucleolar
phosphoprotein p130 Q15046 KARS Lysyl-tRNA synthetase Q32M68 LIN54
Protein lin54-homolog Q504R3 -- Putative uncharacterized protein
Q58EY4 SMARCC1 SWI/SNF related, matrix associated, actin dependent
regulator of chromatin, subfamily c, member 1 Q59FT6 --
CS0DA006YC23 variant Q59GV3 -- SWI/SNF-related matrix-associated
actin-dependent regulator of chromatin c2 isoform b variant Q5H9F2
BCORL1 BCL6 co-repressor-like 1 Q5JRC2 WNK3 WNK lysine deficient
protein kinase 3 Q5RKT7 RPS27A Ribosomal protein S27a Q5T0K1 TAF8
Transcription initiation factor TFIID subunit 8 Q5T8P6 RBM26
RNA-binding protein 26 (Cutaneous T cell lymphoma tumor antigen
se70-2) Q5TBM7 HSPH1 Heat shock 105 kDa/110 kDa protein 1 Q5VU77
UBAP2L Ubiquitin associated protein 2-like [Fragement] Q69YQ9 CMYA5
Cardiomyopathy-associated protein 5 Q6UVJ0 SASS6 Spindle assembly
abnormal protein 6 homolog (DKFZp761A078) Q6ZU65 KIAA2030
Uncharacterized protein KIAA2030 Q7Z3Z3 PIXIL3 Piwi-like protein 3
Q7Z417 NUFIP2 Nuclear fragile X mental retardation-interacting
protein 2 Q7Z739 YTHDF3 YTH domain family protein 3 Q8IVW4 CDKL3
Cyclin-dependent kinase-like 3 Q8IWZ2 hCG_204590 Multiple ankyrin
repeats single KH domain protein isoform 2 Q8N6V5 NUP50 Nucleoporin
50 kDa (Variant1) Q8NDX5 PHC3 Polyhomeotic-like protein 3 Q8TB57
RAD54L2 Helicase ARIP4 (RAD54-like 2) Q96QT6 PHF12 PHD finger
protein 12 Q96RK0 CIC Protein capicua homolog Q96T37 RBM15 Putative
RNA-binding protein 15 Q99496 RNF2 E3 ubiquitin-protein ligase
RING2 Q99700 ATXN2 Ataxin-2 Q9BQG0 MYBBP1A Myb-binding protein 1A
(P160) Q9BTC0 DIDO1 Death-inducer obliterator 1 (Death associated
transcription factor 1) Q9C005 DPY30 Protein dpy-30 homolog Q9GZR7
DDX24 ATP-dependent RNA helicase DDX24 Q9HB23 -- Lysyl-tRNA
synthetase Q9NYV4 CDKRS Cell division cycle 2-related protein
kinase 7 Q9P2N6 KIAA1310 Uncharacterized protein KIAA1310;
Hypothetical protein FLJ10081 Q9UBL3 ASH2L Set1/Ash2 histone
methyltransferase complex subunit ASH2 Q9UKX7 NUP50 Nucleoporin 50
kDa Q9ULM3 YEATS2 YEATS domain-containing protein 2 Q9UQC1 HSP70-1
Heat shock protein 72 Q9Y2N3 POM121 Nuclear envelope pore membrane
protein POM 121 Q9Y3S1 WNK2 Serine/threonine-protein kinase WNK2
(Protein kinase, lysine deficient 2) Q9Y5G6 PCDHGA7 Protocadherin
gamma A7 precursor A2RUN2 AR Androgen receptor O00193 C11orf58
Small acidic protein O95757 HSPA4L Heat shock 70 kDa protein 4L
P09234 SNRPC U1 small nuclear ribonucleoprotein C P42677 RPS27 40S
ribosomal protein S27 P46013 MKI67 Antigen KI-67 P46776 RPL27A 60S
ribosomal protein L27a P46976 GYG1 Glycogenin-1 P61353 RPL27A 60S
ribosomal protein L27 P62316 SNRPD2 Small nuclear ribonucleoprotein
Sm D2 P62318 SNRPD3 Small nuclear ribonucleoprotein Sm D3 P62753
RPS6 40S ribosomal protein S6 P84098 RPL19 60S ribosomal protein
L19 Q13765 NACA Nascent polypeptide-associated complex subunit
alpha Q14247 CTTN Src substrate cortactin Q15637 SF1 Splicing
factor 1 Q5JRC6 PHF6 PHD finger protein 6 Q5SW79 CEP170 Centrosomal
protein of 170 kDa Q5T2J2 C20orf117 Novel protein C20orf117 Q5THK1
C22orf30 Uncharacterized protein C22orf30 Q6ZQN2 -- CDNA FLJ46846
fis, clone UTERU3004635, moderately similar to Neuroblast
differentiation associated protein AHNAK. Q7Z6E9 RBBP6
Retinoblastoma-binding protein 6 Q8WUR7 C15orf40 UPF0235 protein
C15orf40 Q96MX3 ZNF553 Zinc finger protein 553 Q9UNX3 RPL26L1 60S
ribosomal protein L26-like 1 SwissProt Biological Primary Alternate
Number 18B10.C7(3) 9D1.E4(10) 1F5.D6(14) CTD110.6 Process
localization localization P35658 Tp N P49790 Tp N Q06587 G N No
Q5T6F2 U U Q9Y520 U C Q9Y6Y8 Tp ER C, N, G Q13151 G N No
Q9GZZ1 U C Q14011 S N O75821 Tl C P16402 G N P26373 Tl R No Q2KHR3
U U Q52LJ0 U U Q8NC51 G C N P16401 G N No P52948 Tp N C P78406 G N
C Q05BK6 S C Q5JRG1 U U N Q9P2N5 U C N P07737 S C Ex P32969 Tl R No
Q9Y3F4 S C Q8IYH5 G N Q92522 G N No A2A3R5 Tl R P30050 Tl N No
P46779 Tl R No P54652 Tl N C, No P63220 Tl R Q15717 G N C, No
A1L431 U C P55735 Tp ER N, C Q86X55 M N Q8IX12 S C O60884 Tl C N, M
O14776 G N No Q24JQ7 U U Q8NCA5 U U Q8WWM7 U N Q96HA1 Tp N ER
Q9NX58 G No A2A3N6 U C A2A3R7 Tl R A5JHP3 U U O43148 G C N O75534 G
C P00492 M C P02795 U N C P05387 Tl C No P09429 G N C, PM P22234 M
C P27694 G N P30086 S C PM P31689 Tl C N, No, G P35125 Tl N P43487
Tp C N P48634 U U P61927 Tl R P62633 G C ER Q09028 G N No Q13310 G
C Q13347 Tl R Q14192 G N C, M Q14444 Tp PM Q14684 U No Q2M2Y6 G N
Q5JXK1 U U Q5RLJ0 U U Q6IBH5 Tl Q6ZUI0 U U Q7LBC6 U N C, No Q96AE4
G N C Q9UJV9 G No A1L3W5 Tl N A2A305 U U O60333 S C M O60506 G C
ER, R, No O95259 Tp PM P05109 S C PM, Ex P06702 S C PM P31151 S C
ER, N, PM Q01469 Tp C ER Q69YU5 U U Q8IVL0 U N Q99567 Tp N A0AVI3 G
N A4D1M5 Tl R O14497 G C N O14974 M C PM O15026 G N O15047 S N
O60907 S N O75182 G N O75528 G N O75822 Tl C O75937 Tl No O94979 Tp
ER C, G P14174 S Ex C P14859 S N P14868 M C P15822 G N P26599 G N
C, No P49750 U N P49792 S N C P49916 G N M P51532 G N No P52594 G N
C, No P53992 Tp ER C P54198 G N P54259 S N C P61964 S C No P61981 S
C ER, G P82914 Tl M R Q02878 Tl No Q03164 G N Q12830 G N C Q13185 G
N No Q13283 S C C, N, PM Q13547 G N No Q14119 G N Q14978 G No C
Q15046 Tl C M Q32M68 S N Q504R3 U N Q58EY4 G N Q59FT6 U U Q59GV3 G
N Q5H9F2 G N Q5JRC2 S C Q5RKT7 Tl R Q5T0K1 G N C Q5T8P6 G N Q5TBM7
Tl C Q5VU77 U U Q69YQ9 U C Q6UVJ0 U U Q6ZU65 U U Q7Z3Z3 U U Q7Z417
G N C Q7Z739 U C Q8IVW4 S C Q8IWZ2 U U Q8N6V5 Tp N Q8NDX5 U N
Q8TB57 G N Q96QT6 G N Q96RK0 G U Q96T37 G U Q99496 Tl C Q99700 G C
Q9BQG0 G No Q9BTC0 S N C Q9C005 G N Q9GZR7 Tp N No Q9HB23 Tl C
Q9NYV4 S N Q9P2N6 U U Q9UBL3 G N Q9UKX7 Tp N C Q9ULM3 U U Q9UQC1 Tl
U Q9Y2N3 Tp N ER Q9Y3S1 M U Q9Y5G6 S PM A2RUN2 S N C O00193 U U
O95757 Tl C P09234 G N No P42677 G C N, No P46013 S No P46776 Tl R
No P46976 M U P61353 Tl C No P62316 G N P62318 G N No P62753 Tl R
No P84098 Tl R Q13765 Tl N C, R Q14247 S C PM Q15637 G N Q5JRC6 G
No N Q5SW79 S N C Q5T2J2 U U Q5THK1 U U Q6ZQN2 U U Q7Z6E9 Tl U
Q8WUR7 U U Q96MX3 G U Q9UNX3 U R No * Abbreviations: G, Gene
expression/Transcription; M, Metabolism; S, Signal transduction;
Tl, Translation; Tp, Transport; U, Unknown: C, Cytoplasm; N,
Nucleus; No, Nucleolus; ER, Endoplasmic reticulum; G, Golgi
apparatus; Ex, Extracellular. indicates data missing or illegible
when filed
[0264] The extensive list of O-GlcNAc modified proteins made it
possible to assign biological functions using HPRD (FIG. 22b). A
large number of identified proteins are involved in
transcriptional/translational regulation and signal transduction
(Tables 9-11), which is consistent with recent reports that
functionally implicates O-GlcNAc modification on insulin signaling
and transcriptional control (Vosseller et al., 2002 Proc. Natl.
Acad. Sci. U.S.A. 99, 5313-5318; Dentin et al., 2008 Science 319,
1402-1405; Housley et al., 2008 J. Biol. Chem. 283, 16283-16292).
Of particular interest is that several of the glycoproteins are
involved in the ubiquitin pathway. A role for O-GlcNAc has already
been established for regulation of the proteasome (Zhang et al.,
2003 Cell 115, 715-725) but our data indicate that O-GlcNAc may
also be actively involved in earlier steps of the degradation
cascade. SEC23 components and interacting proteins were also
captured by multiple antibodies suggesting a possible role for
O-GlcNAc modification in anterograde trafficking of intracellular
vesicles. Finally, several ribosomal proteins were observed, which
is in agreement with the recent finding that O-GlcNAc modification
of ribosomal proteins plays a role in stress granule and processing
body assembly (Ohn et al., 2008 Nat. Cell Biol. 10, 1224-1231).
[0265] Several interesting examples of newly identified O-GlcNAc
proteins were identified by only one of the antibodies and included
proteins participating in other types of post-translational
modifications such as WNK2 and WNK3 for phosphorylation and RanBP2
and SUMO4 for SUMOylation. Also, a range of proteins that modulate
gene expression at the chromatin levels such as SMARCC1 and CARM1
were identified. This indicates that the different MAbs recognize
subtly different sequential and/or structural epitopes that all
include an O-GlcNAc modified residue.
[0266] In conclusion, the three-component immunogen methodology has
been successfully employed to generate a panel of pan-GlcNAc
specific MAbs, which offer powerful new tools for exploring the
biological implications of this type of protein glycosylation. The
newly identified O-GlcNAc modified proteins open new avenues to
explore the importance of this type of posttranslational for a
variety of biological processes. It is to be expected that the
three-component immunization technology will find wide application
for the generation of MAbs for other forms of protein
glycosylation.
TABLE-US-00011 TABLE 11 Proteins enriched by more than one of the
antibodies SwissProt Biological Primary Alternate Number Gene Name
Protein Name Mab3 Mab10 Mab14 CTD110.6 Process localization
localization Q9Y520 BAT2D1 BAT2-iso (BAT2 domain-containing U C
protein 1; HBxAg transactivated protein 2) Q06587 RING1 E3
ubiquitin-protein ligase RING1 G N No P49790 NUP153 Nuclear pore
complex protein Nup153 Tp N P35658 NUP214 Nuclear pore complex
protein Nup214 Tp N Q9Y6Y8 SEC23IP SEC23-interacting protein Tp ER
C, N, G Q5T6F2 UBAP2 Ubiquitin-associated protein 2 U U Q13151
HNRPA0 Heterogeneous nuclear ribonucleoprotein A0 G N No Q9GZZ1
NAT13 N-acetyltransferase 13 (Mak3) U C P16401 HIST1H1B Histone
H1.5 G N No P78406 RAE1 mRNA export factor (MRNP41) G N C P52948
NUP98 Nuclear pore complex protein Tp N C Nup98-Nup96 precursor
[Contains: Nuclear pore complex protein Nup98 Q9P2N5 RBM27
RNA-binding protein 27 U C N Q05BK6 TFG TFG protein (TRK-fused gene
protein) S C Q14011 CIRBP Cold-inducible RNA-binding protein S N
P26373 RPL13 60S ribosomal protein L13 Tl R No O75821 EIF3S4
Eukaryotic translation initiation factor 3 Tl C subunit 4 Q2KHR3
QSER1 Glutamine and serine-rich protein 1 U U (FLJ21924) P16402
HIST1H1D Histone H1.3 G N Q52LJ0 FAM98B Protein FAM98B U U Q8NC51
SERBP1 Plasminogen activator inhibitor 1 G C N RNA-binding protein
(SERPINE1 mRNA binding protein 1) Q5JRG1 NUPL1 Nucleoporin like 1 U
U N Q8IX12 CCAR1 Cell division cycle and apoptosis S C regulator
protein 1 Q86X55 CARM1 Histone-arginine methyltransferase CARM1 M N
(Coactivator associated arginine methyltransferase 1) A1L431 PPIAL4
Peptidyl-prolyl cis-trans isomerase U C A-like 4 (Cyclophilin LC)
P55735 SEC13 Protein SEC13 homolog Tp ER N, C Q92522 H1FX Histone
H1x G N No Q8IYH5 ZZZ3 Zinc finger ZZ-type-containing protein 3 G N
P32969 RPL9 60S ribosomal protein L9 Tl R No P07737 PFN1 Profilin-1
S C Ex Q9Y3F4 STRAP Serine-threonine kinase receptor- S C
associated protein Abbreviations: G, Gene expression/Transcription;
M, Metabolism; S, Signal transduction; Tl, Translation; Tp,
Transport; U, Unknown; C, Cytoplasm; N, Nucleus; No, Nucleolus; ER,
Endoplasmic reticulum; G, Golgi apparatus; Ex, Extracellular.
Methods
[0267] Reagents and general procedures for synthesis. Fmoc-L-Amino
acid derivatives and resins were purchased from NovaBioChem and
Applied Biosystems, peptide synthesis grade N,N-dimethylformamide
(DMF) from EM Science and N-methylpyrrolidone (NMP) from Applied
Biosystems. Egg phosphatidylcholine (PC), egg phosphatidylglycerol
(PG), cholesterol, monophosphoryl lipid A (MPL-A) and dodecyl
phosphocholine (DPC) were obtained from Avanti Polar Lipids. All
other chemical reagents were purchased from Aldrich, Acros, Alfa
Aesar and Fischer and used without further purification. All
solvents employed were reagent grade. Reversed phase high
performance liquid chromatography (RP-HPLC) was performed on an
Agilent 1100 series system equipped with an auto-injector,
fraction-collector and UV-detector (detecting at 214 nm) using an
Agilent ZorbaxEclipse.TM. C8 analytical column (5 .mu.m,
4.6.times.150 mm) at a flow rate of 1 ml Agilent Zorbax Eclipse.TM.
C8 semi preparative column (5 .mu.m, 10.times.250 mm) at a flow
rate of 3 ml min.sup.-1 or Phenomenex Jupiter.TM. C4 semi
preparative column (5 .mu.m, 10.times.250 mm) at a flow rate of 2
ml min.sup.-1. All runs were performed using a linear gradients of
0 to 100% solvent B over 40 min. (solvent A=5% acetonitrile, 0.1%
trifluoroacetic acid (TFA) in water, solvent B=5% water, 0.1% TFA
in acetonitrile). Matrix assisted laser desorption ionization time
of flight mass spectrometry (MALDI-ToF) mass spectra were recorded
on an ABI 4700 proteomic analyzer.
[0268] General methods for solid-phase peptide synthesis (SPPS).
Peptides were synthesized by established protocols on an ABI 433A
peptide synthesizer (Applied Biosystems) equipped with UV-detector
using N.sup..alpha.-Fmoc-protected amino acids and
2-(1H-benzotriazole-1-yl)-oxy-1,1,3,3-tetraethyl
hexafluorophosphate (HBTU)/1-hydroxybenzotriazole (HOBt; Knorr et
al., 1989 Tetrahedron Lett. 30, 1927-1930) as the activating
reagents. Single coupling steps were performed with conditional
capping. The following protected amino acids were used:
N.sup.o-Fmoc-Arg(Pbf)-OH, N.sup..alpha.-Fmoc-Asp(O.sup.tBu)-OH,
N.sup..alpha.-Fmoc-Asp-Thr(.psi..sup.Me,Mepro)-OH,
N.sup..alpha.-Fmoc-Ile-Thr(.psi..sup.Me,Mepro)-OH,
N.sup..alpha.-Fmoc-Lys(Boc)-OH,
N.sup..alpha.-Fmoc-Ser(.sup.tBu)-OH,
N.sup..alpha.-Fmoc-Thr(.sup.tBu)-OH, N.sup..alpha.-Fmoc-Tyr(Bu)-OH.
The coupling of the glycosylated amino acid
N.sup..alpha.-FmocSer-(AcO3-.alpha.-D-O-GlcNAc)OH,
N.sup..alpha.-FmocSer-(AcO3-.alpha.-D-S-GlcNAc)OH, was carried out
manually using
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HATU)/1-hydroxy-7-azabenzotriazole (HOAt) as a
coupling agent. The coupling of
N.sup..alpha.-Fmoc-S-(2,3-bis(palmitoyloxy)-(2R-propyl)-(R)-cysteine
(Metzger et al., 1991 Int. J. Pept. Protein Res. 38, 545-554; Roth
et al., 2004 Bioconjugate Chem. 15, 541-553) which was prepared
from (R)-glycidol were carried out using
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate (PyBOP)/HOBt as coupling agent. Progress of the
manual couplings was monitored by standard Kaiser test (Kaiser et
al., 1970 Anal. Biochem. 34, 595).
[0269] Synthesis of lipopeptide 63. The synthesis of 63 was carried
out on a H-Gly-sulfamylbutyryl Novasyn TG resin as described in the
general method section for peptide synthesis. After coupling of the
first five amino acids, the remaining steps were performed
manually.
N-.alpha.-Fmoc-S-(2,3-bis(palmitoyloxy)-(2R-propyl)-(R)-cysteine
(267 mg, 0.3 mmol) was dissolved in DMF (5 ml) and PyBOP (156.12
mg, 0.3 mmol), HOBt (40 mg, 0.3 mmol) and DIPEA (67 .mu.l, 0.4
mmol) were premixed for 2 min, and was added to the resin. The
coupling reaction was monitored by the Kaiser test and was complete
after standing for 12 h. Upon completion of the coupling, the
N-Fmoc group of was cleaved using 20% piperidine in DMF (6 ml) and
palmitic acid (77 mg, 0.3 mmol) was coupled to the free amine of as
described above using PyBOP (156.12 mg, 0.3 mmol), HOBt (40 mg, 0.3
mmol) and DIPEA (67 .mu.l, 0.4 mmol) in DMF. The resin was
thoroughly washed with DMF (10 ml), DCM (10 ml) and MeOH (10 ml)
and then dried in vacuo. The resin was swelled in DCM (5 ml) for 1
h and treated with DIPEA (0.5 ml, 3 mmol), iodoacetonitrile (0.36
ml, 5 mmol) in NMP (6 ml). It is important to note that the
iodoacetonitrile was filtered through a plug of basic alumina
before addition to the resin. The resin was agitated under the
exclusion of light for 24 h, filtered and washed with NMP (5
ml.times.4), DCM (5 ml.times.4) and THF (5 ml.times.4). The
activated N-acyl sulfonamide resin was swollen in DCM (5 ml) for 1
h, drained and transferred to a 50 ml round bottom flask. To the
resin-containing flask was added THF (4 ml), benzyl mercaptan (0.64
ml, 5 mmol) and sodium thiophenate (27 mg, 0.2 mmol). After
agitation for 24 h, the resin was filtered and washed with hexane
(5 ml.times.2). The combined filtrate and washings were collected
and concentrated in vacuo to approximately 1/3 of its original
volume. The crude product was then precipitated by the addition of
tert-butyl methyl ether (0.degree. C.; 60 ml) and recovered by
centrifugation at 3000 rpm for 15 min, and after the decanting of
the ether the peptide precipitate was dissolved in mixture DCM and
MeOH (1.5 ml/1.5 ml). The thiol impurity present in the peptide
precipitate was removed by passing it through a LH-20 size
exclusion column. The fractions containing product were collected
and solvents removed to give the fully protected peptide thioester.
The protected peptide was treated with a reagent B (TFA 88%, phenol
5%, H2O 5%, TIS 2%; 5 ml) for 4 h at room temperature. The TFA
solution was then added dropwise to a screw cap centrifuge tube
containing ice cold tert-butyl methyl ether (40 ml) and the
resulting suspension was left overnight at 4.degree. C., after
which the precipitate was collected by centrifugation at 3000 rpm
(20 min), and after the decanting of the ether the peptide
precipitate was re-suspended in ice cold tert-butyl methyl ether
(40 ml) and the process of washing was repeated twice. The crude
peptide was purified by HPLC on a semi preparative C-4 reversed
phase column using a linear gradient of 0 to 100% solvent B in A
over a 40 min, and the appropriate fractions were lyophilized to
afford 63 (110 mg, 65%). C.sub.90H.sub.165N.sub.11O.sub.13S.sub.2,
MALDI-ToF MS: observed, [M+Na] 1695.2335 Da; calculated, [M+Na]
1695.4714 Da (FIG. 25).
[0270] Synthesis of glycopeptide 64. SPPS was performed on Rink
amide resin (0.1 mmol) as described in the general procedures. The
first four amino acids, Ser-Ala-Asn-Met, were coupled on the
peptide synthesizer using a standard protocol. After the completion
of the synthesis, a manual coupling was carried out using
N.alpha.-FmocSer-(AcO.sub.3-.alpha.-D-O-GlcNAc)OH (0.2 mmol, 131
mg), with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HATU; 0.2 mmol, 76 mg),
1-hydroxy-7-azabenzotriazole (HOAt; 0.2 mmol, 27 mg) and
diisopropylethylamine (DIPEA; 0.4 mmol, 70 .mu.l) in NMP (5 ml) for
12 h. The coupling reaction was monitored by standard Kaiser test.
The resin was then washed with NMP (6 ml) and methylene chloride
(DCM; 6 ml), and resubjected to the same coupling conditions to
ensure completion of the coupling. The glycopeptide was then
elongated on the peptide synthesizer after which the resin was
thoroughly washed with NMP (6 ml), DCM (6 ml) and MeOH (6 ml) and
dried in vacuo. The resin was swelled in DCM (5 ml) for 1 h and
then treated with hydrazine (60%) in MeOH (10 ml) for 2 h and
washed thoroughly with NMP (5 ml.times.2), DCM (5 ml.times.2) and
MeOH (5 ml.times.2) and dried in vacuo. The resin was swelled in
DCM (5 ml) for 1 h, after which it was treated with reagent K (TFA
(81.5%), phenol (5%), thioanisole (5%), water (5%), EDT (2.5%), TIS
(1%)) (30 ml) for 2 h at room temperature. The resin was filtered
and washed with neat TFA (2 ml). The filtrate was then concentrated
in vacuo to approximately 1/3 of its original volume. The peptide
was precipitated using diethyl ether (0.degree. C.) (30 ml) and
recovered by centrifugation at 3000 rpm for 15 min. The crude
peptide was purified by RP-HPLC on a semi preparative C-8 column
using a linear gradient of 0 to 100% solvent B in solvent A over a
40 min period and the appropriate fractions were lyophilized to
afford 64 (118 mg, 40%). C.sub.129H.sub.204N.sub.32O.sub.40S.sub.2,
MALDI-ToF MS: observed [M+], 2907.5916 Da; calculated [M+],
2905.4354 Da (FIG. 26).
[0271] Synthesis of glycopeptide 65. SPPS was performed on Rink
amide resin (0.1 mmol) as described in the general procedures. The
first four amino acids, Ser-Ala-Asn-Met, were coupled on the
peptide synthesizer using a standard protocol. After the completion
of the synthesis, a manual coupling was carried out using
N.alpha.-FmocSer-(AcO.sub.3-.alpha.-D-S-GlcNAc)OH (0.2 mmol, 134
mg), with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HATU; 0.2 mmol, 76 mg),
1-hydroxy-7-azabenzotriazole (HOAt; 0.2 mmol, 27 mg) and
diisopropylethylamine (DIPEA; 0.4 mmol, 70 .mu.l) in NMP (5 ml) for
12 h. The coupling reaction was monitored by standard Kaiser test.
The resin was then washed with NMP (6 ml) and methylene chloride
(DCM; 6 ml), and resubjected to the same coupling conditions to
ensure complete coupling. The resulting glycopeptide was then
elongated on the peptide synthesizer. After the completion of the
synthesis, the resin was thoroughly washed with NMP (6 ml), DCM (6
ml) and MeOH (6 ml) and dried in vacuo. The resin was swelled in
DCM (5 ml) for 1 h and then treated with hydrazine (60%) in MeOH
(10 ml) for 2 h and washed thoroughly with NMP (5 ml.times.2), DCM
(5 ml.times.2) and MeOH (5 ml.times.2) and dried in vacuo. The
resin was swelled in DCM (5 ml) for 1 h, after which it was treated
with TFA (81.5%), phenol (5%), thioanisole (5%), water (5%), EDT
(2.5%), TIS (1%) (30 ml) for 2 h at room temperature. The resin was
filtered and washed with neat TFA (2 ml). The filtrate was then
concentrated in vacuo to approximately 1/3 of its original volume.
The peptide was precipitated using diethyl ether (30 ml, 0.degree.
C.) and recovered by centrifugation at 3000 rpm for 15 min. The
crude peptide was purified by RP-HPLC on a semi preparative C-8
column using a linear gradient of 0 to 100% solvent B in solvent A
over a 40 min period and the appropriate fractions were lyophilized
to afford 65 (95 mg, 34%).
C.sub.129H.sub.204N.sub.32O.sub.39S.sub.3, MALDI-ToF MS: observed
[M+], 2923.6716 Da; calculated [M+], 2923.3861 Da (FIG. 27).
[0272] Synthesis of glycolipopeptide 52. The lipopeptide thioester
63 (4.3 mg, 2.5 .mu.mol), glycopeptide 64 (5.0 mg, 1.7 .mu.mol) and
dodecyl phosphocholine (6.0 mg, 17.0 .mu.mol) were dissolved in a
mixture of trifluoroethanol and CHCl.sub.3 (2.5 ml/2.5 ml). The
solvents were removed under reduced pressure to give a
lipid/peptide film, which was hydrated for 4 h at 37.degree. C.
using 200 mM phosphate buffer (pH 7.5, 3 ml) in the presence of
tris(carboxyethyl)phosphine (2% w/v, 40.0 .mu.g) and EDTA (0.1%
w/v, 20.0 .mu.g). The mixture was ultrasonicated for 1 min. To the
vesicle suspension was added sodium 2-mercaptoethane sulfonate (2%
w/v, 40.0 .mu.g) to initiate the ligation reaction. The reaction
was carried out in an incubator at 37.degree. C. and the progress
of the reaction was periodically monitored by MALDI-ToF, which
showed disappearance of glycopeptide 64 within 2 h. The reaction
was then diluted with 2-mercaptoethanol (20%) in ligation buffer (2
ml) and the crude peptide was purified by semi preparative C-4
reversed phase column using a linear gradient of 0 to 100% solvent
B in A over a 40 min, and lyophilization of the appropriate
fractions afforded 52 (4.3 mg, 57%).
C.sub.212H.sub.360N.sub.43O.sub.53S.sub.3, MALDI-ToF MS: observed,
4461.9177 Da, calculated, 4455.578 Da (FIG. 23).
[0273] Synthesis of glycolipopeptide 53. Lipopeptide thioester 63
(2.5 mg, 1.5 .mu.mol), glycopeptide 65 (3.0 mg, 1.0 .mu.mol) and
dodecyl phosphocholine (3.5 mg, 10 .mu.mol) were dissolved in a
mixture of trifluoroethanol and CHCl.sub.3 (2.5 ml/2.5 ml). The
solvents were removed under reduced pressure to give a
lipid/peptide film, which hydrated for 4 h at 37.degree. C. using
200 mM phosphate buffer (pH 7.5, 2 ml) in the presence of
tris(carboxyethyl)phosphine (2% w/v, 40.0 .mu.g) and EDTA (0.1%
w/v, 20.0 .mu.g). The mixture was ultrasonicated for 1 min. To the
vesicle suspension was added sodium 2-mercaptoethane sulfonate (2%
w/v, 40.0 .mu.g) to initiate the ligation reaction. The reaction
was carried out in an incubator at 37.degree. C. and the progress
of the reaction was periodically monitored by MALDI-ToF, which
showed disappearance of glycopeptide within 2 h. The reaction was
then diluted with 2-mercaptoethanol (20%) in ligation buffer (2
ml). The crude peptide was purified by semi preparative C-4
reversed phase column using a linear gradient of 0 to 100% solvent
B in A over a 40 min, and lyophilization of the appropriate
fractions afforded 53 (2.8 mg, 64%).
C.sub.212H.sub.360N.sub.43O.sub.52S.sub.4, MALDI-ToF MS: observed,
4469.9112 Da, calculated, 4471.6437 Da (FIG. 24).
[0274] Compounds 66-70 were prepared as described in the standard
procedures section on Rink amide resin (0.1 mmol). Glycopeptide 66
(78 mg, 61%); C.sub.48H.sub.82N.sub.14O.sub.21S.sub.2, MALDI-ToF
MS: observed [M+Na], 1277.4746 Da; calculated [M+Na], 1277.5220 Da
(FIG. 29). Peptide 67 (89 mg, 83%);
C.sub.40H.sub.69N.sub.13O.sub.16S.sub.2, MALDI-ToF MS: observed
[M+Na], 1074.4789 Da; calculated [M+Na], 1074.4427 Da (FIG. 30).
Glycopeptide 68 (57 mg, 48%); C.sub.45H.sub.77N.sub.13O.sub.20S,
MALDI-ToF MS: observed [M+Na], 1174.4740 Da; calculated [M+Na],
1174.5129 Da (FIG. 31). Peptide 69 (76 mg, 78%).
C.sub.37H.sub.64N.sub.12O.sub.15S, MALDI-ToF MS: observed [M+Na],
969.8162 Da; calculated [M+Na], 970.8657 Da (FIG. 32). Glycosylated
amino acid 70 (12 mg, 33%), C.sub.14H.sub.25N.sub.3O.sub.8,
MALDI-ToF MS: observed [M+Na], 386.2749 Da; calculated [M+Na]
386.3636 Da (FIG. 32).
[0275] General procedure for the conjugation to BSA-MI. The
conjugations were performed as instructed by Pierce Endogen Inc. In
short, the purified (glyco)peptide 66 or 67 (2.5 equiv. excess to
available MI-groups on BSA) was dissolved in the conjugation buffer
(sodium phosphate, pH 7.2 containing EDTA and sodium azide; 100
.mu.l) and added to a solution of maleimide activated BSA (2.4 mg)
in the conjugation buffer (200 .mu.l). The mixture was incubated at
room temperature for 2 h and then purified by a D-Salt.TM. dextran
de-salting column (Pierce Endogen, Inc.), equilibrated and eluted
with sodium phosphate buffer, pH 7.4 containing 0.15 M sodium
chloride. Fractions containing the conjugate were identified using
the BCA protein assay. Carbohydrate content was determined by
quantitative monosaccharide analysis by HPAEC/PAD.
[0276] General procedure for the preparation of liposomes. Egg PC,
egg PG, cholesterol, MPL-A and compound 52 or 53 (15 .mu.mol, molar
ratios 60:25:50:5:10) were dissolved in a mixture of
trifluoroethanol and MeOH (1:1, v/v, 5 ml). The solvents were
removed in vacuo to produce a thin lipid film, which was hydrated
by suspending in HEPES buffer (10 mM, pH 6.5) containing NaCl (145
mM; 1 ml) under argon atmosphere at 41.degree. C. for 3 h. The
vesicle suspension was sonicated for 1 min and then extruded
successively through 1.0, 0.6, 0.4, 0.2 and 0.1 .mu.m polycarbonate
membranes (Whatman, Nucleopore Track-Etch Membrane) at 50.degree.
C. to obtain SUVs. The sugar content of liposomes was determined by
heating a mixture of SUVs (50 .mu.l) and aqueous TFA (2 M, 200
.mu.l) in a sealed tube for 4 h at 100.degree. C. The solution was
then concentrated in vacuo and analyzed by high-pH anion exchange
chromatography using a pulsed ampherometric detector (HPAEC-PAD;
Methrome) and CarboPac columns PA-10 and PA-20 (Dionex).
[0277] Dose and immunization schedule. Groups of five mice (female
BALB/c, age 8-10 weeks, from Jackson Laboratories) were immunized
four times at two-week intervals. Each boost included 3 .mu.g of
saccharide in the liposome formulation. Serum samples were obtained
before immunization (pre-bleed) and 1 week after the final
immunization. The final bleeding was done by cardiac bleed.
[0278] Hybridoma culture and antibody production. Spleens of two
mice immunized with 52 were harvested and standard hybridoma
culture technology gave 30 IgG producing hybridoma cell lines.
Three hybridomas (18B10.C7(3), 9D1.E4(10), 1F5.D6(14)) were
cultured at a one-liter scale and the resulting antibodies were
purified by saturated ammonium sulfate precipitation followed by
Protein G column chromatography to yield, in each case,
approximately 10 mg of IgG.
[0279] Reagents for biological experiments. Protease inhibitor
cocktail was obtained from Roche (Indianapolis, Ind.). PUGNAc
O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenyl
carbamate was ordered from Toronto Research Chemicals, Inc
(Ontario, Canada). Mouse IgM anti-O-GlcNAc (CTD110.6; Corner et
al., 2001 Anal. Biochem. 293, 169-177) and rabbit polyclonal
anti-OGT (AL28) antibodies were previously generated in Dr. Gerald
W. Hart's laboratory (Johns Hopkins University School of Medicine,
Baltimore, Md.). Rabbit polyclonal anti-OGA antibody was a kind
gift from Dr. Sidney W. Whiteheart (University of Kentucky College
of Medicine). Rabbit polyclonal anti-CKII alpha antibodies
(NB100-377 for immunoblotting and NB100-378 for
immunoprecipitation) were purchased from Novus Biologicals
(Littleton, Colo.). Mouse monoclonal antibody against
.alpha.-tubulin and anti-Mouse IgM chain)-agarose was obtained from
Sigma (St. Louis, Mo.). Normal rabbit IgG agarose, normal rabbit
IgG agarose and Protein A/G PLUS agarose were ordered from Santa
Cruz Biotechnology, Inc. (Santa Cruz, Calif.).
[0280] Serologic assays. Anti-GSTPVS(.beta.-O-GlcNAc)SANM (68) IgG,
IgG1, IgG2a, IgG2b, IgG3 and IgM antibody titers were determined by
enzyme-linked immunosorbent assay (ELISA), as described previously
(Buskas and Boons, 2004 Chem. Eur. J. 10, 3517-3524; Ingale et al.,
2007 Nat. Chem. Biol. 3, 663-66). Briefly, Immulon II-HB flat
bottom 96-well microtiter plates (Thermo Electron Corp.) were
coated overnight at 4.degree. C. with 100 .mu.l per well of a
conjugate of the glycopeptide conjugated to BSA through a maleimide
linker (BSA-MI-GSTPVS(.beta.-O-GlcNAc) SANM; BSA-MI-66) at a
concentration of 2.5 .mu.g ml-1 in coating buffer (0.2 M borate
buffer, pH 8.5 containing 75 mM sodium chloride). Serial dilutions
of the sera or MAb containing cell supernatants were allowed to
bind to immobilized GSTPVS(.beta.-O-GlcNAc)SANM for 2 h at room
temperature. Detection was accomplished by the addition of alkaline
phosphatase-conjugated anti-mouse IgG (Jackson ImmunoResearch
Laboratories Inc.), IgG1 (Zymed), IgG2a (Zymed), IgG2b (Zymed),
IgG3 (BD Biosciences Pharmingen) or IgM (Jacksons ImmunoResearch
Laboratories) antibodies. After addition of p-nitrophenyl phosphate
(Sigma), the absorbance was measured at 405 nm with wavelength
correction set at 490 nm using a microplate reader (BMG Labtech).
The antibody titer was defined as the highest dilution yielding an
optical density of 0.1 or greater over that of background.
[0281] To explore competitive inhibition of the binding of MAbs to
GSTPVS(.beta.-O-GlcNAc)SANM (68) by the corresponding glycopeptide,
peptide and sugar, MAbs were diluted in diluent buffer in such a
way that, without inhibitor, expected final OD values were
approximately 1. For each well 60 .mu.l of the diluted MAbs were
mixed in an uncoated microtiter plate with 60 .mu.l diluent buffer,
glycopeptide 68 (GSTPVS(.beta.-O-GlcNAc)SANM), peptide 69
(GSTPVSSANM; SEQ ID NO: 11) or sugar 70 (.beta.-O-GlcNAc-Ser) in
diluent buffer with a final concentration of 0-500 .mu.M. After
incubation at room temperature for 30 min, 100 .mu.l of the
mixtures were transferred to a plate coated with
BSA-MICGSTPVS(.beta.-O-GlcNAc)SANM (BSA-MI-66). The microtiter
plates were incubated and developed as described above using the
appropriate alkaline phosphatase-conjugated detection antibody.
[0282] Plasmids construction. The human OGT and OGA cDNA were PCR
amplified in a two-step manner to introduce an attB1 site and a HA
epitope at the 5' end as well as an attB2 site at the 3' end to
facilitate Gateway cloning strategy (Invitrogen, Carlsbad, Calif.).
The primers include (1) Sense primer for first PCR to incorporate
HA epitope into ogt after the start codon:
5'-CCCCATGTATCCATATGACGTCCCAGACTATGCCGCGTCTTCCGTGGGCAACGT-3' (SEQ
ID NO: 13); (2) Sense primer containing an attB1 site for using
HA-ogt PCR product as the template:
5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTGGATGATGTATCCATATGACGTCC
CAGACTATGCCGCGTCTTCCG-3' (SEQ ID NO: 14); (3) Antisense primer with
3' attB2 site for both ogt PCR:
5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTCTATGCTGACTCAGTGACTTCAA
CGGGCTTAATCATGTGG-3' (SEQ ID NO: 15); (4) Sense primer for first
PCR to incorporate HA epitope into oga after the start codon:
5'-CCCCATGTATCCATATGACGTCCCAGACTATGCCGTGCAGAAGGA GAGTCAAGC-3' (SEQ
ID NO: 16); (5) Sense primer containing an attB1 site for using
HA-oga PCR product as the template:
5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTGGATGATGTATCCATATG
ACGTCCCAGACTATGCCGTGCAGAAGG-3' (SEQ ID NO: 17); (6) Antisense
primer with 3' attB2 site for both oga PCR:
5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTCACAGGCTCCGACCAA GTAT-3' (SEQ ID
NO: 18). The purified DNA fragments were then subjected to Gateway
cloning according to manufacturer's instruction yielding final
expression constructs, pDEST26/HA-OGT and pDEST26/HA-OGA.
[0283] Cell culture, transfection and treatment. HEK 293T cells
were obtained from ATCC (Manassas, Va.) and maintained in
Dulbecco's modified Eagle's medium (4.5 g l-1 glucose,
Cellgro/Mediatech, Inc., Herndon, Va.) supplemented with 10% fetal
bovine serum (GIBCO/Invitrogen, Carlsbad, Calif.) in 37.degree. C.
incubator humidified with 5% CO.sub.2. Transfection was performed
with 8 .mu.g of DNA and Lipofectamine 2000 reagent (Invitrogen
Carlsbad, Calif.) per 10 cm plate of cells according to
manufacturer's instruction. Mock transfection was performed in the
absence of DNA. Cells were harvested 48 h post-transfection. For
immunoprecipitation experiments, cells were washed of the plates
with ice-cold PBS and store as a pellet at -80.degree. C. until
used. For immunoblotting experiments, cells were washed twice with
ice-cold PBS and scraped in lysis buffer (10 mM Tris-HCl, pH 7.5,
150 mM NaCl, 1% Igepal CA-630, 0.1% SDS, 4 mM EDTA, 1 mM DTT, 0.1
mM PUGNAc, Protease inhibitor cocktail) and the lysates were
clarified in a microfuge with 16,000 g, for 25 min at 4.degree. C.
The protein concentration was quantified with Bradford protein
assay with standard procedure (Bio-Rad, Hercules, Calif.) and
boiled in sample buffer for 5 min. For mass spectrometry
experiment, 2.times.15 cm plates of 293T cells were treated with 50
.mu.M of PUGNAc for 24 h, cells were pellet and stored as
above.
[0284] Immunoprecipitation and Western blotting. To prepare the
nucleocytosolic fraction for CKII immunoprecipitation, HEK293T cell
pellets with mock or OGT transfection were resuspended in 4 volumes
of hypotonic buffer (5 mM Tris-HCl, pH 7.5, Protease inhibitor
cocktail) and transferred into a 2 ml homogenizer. After incubating
on ice for 10 min, the cell suspension was subjected to dounce
homogenization followed by another 5 min incubation on ice. One
volume of hypertonic buffer (0.1 M Tris-HCl, pH 7.5, 2 M NaCl, 5 mM
EDTA, 5 mM DTT, Protease inhibitor cocktail) was then added to the
lysate. The lysate was incubated on ice for 5 min followed by
another round of dounce homogenization. The resulting lysates were
transferred to microfuge tubes containing PUGNAc (final
concentration 10 .mu.M) and centrifuged at 18,000 g for 25 min at
4.degree. C. Protein concentration was determined using Bradford
protein assay (Bio-Rad, Hercules, Calif.). Prior to IP, the lysates
were supplemented with 1% Igepal CA-630 and 0.1% SDS, and
precleared with a mixture of normal rabbit or mouse IgG AC and
protein A/G PLUS agarose at 4.degree. C. for 30 min. Following
clarification, the precleared supernatant was incubated at
4.degree. C. in the presence of antibodies of interested for 4 at
4.degree. C. After adding protein A/G PLUS agarose, the samples
were incubated for another 2 h at 4.degree. C. and extensively
washed with IP wash buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1%
Igepal CA-630, 0.1% SDS). Finally, SDSPAGE sample buffer was added
into the IP complex and boiled for 3 min. Supernatant was resolved
by a 10% or 4-15% Tris-HCl precast minigel (Bio-Rad, Hercules,
Calif.), and transferred to Immobilon-P transfer membrane
(Millipore, Bedford, Mass.). The membranes were blocked with either
3% BSA (O-GlcNAc blots) or 5% milk (protein blots) in TBST (TBS
with 0.1% Tween 20), and probed with each antibody (1:1000 dilution
for O-GlcNAc blots, 1:8000 dilution for CKII, OGT and OGA blots,
and 1:10,000 dilution for .alpha.-tubulin blot) at 4.degree. C. for
overnight followed by incubating with secondary antibodies
conjugated to HRP at room temperature for 2 h. The final detection
of HRP activity was performed using SuperSignal chemiluminescent
substrates (Thermo Scientific, Rockford, Ill.) as followed: MAbs
18B10.C7(3), 9D1.E4(10) and 1F5.D6(14) used Femto; CKII, OGT, OGA
and tubulin used PICO. The films were exposed to CL-XPosure film
(Thermo Scientific, Rockford, Ill.). After developing the image on
the film, the blot was then stripped with 0.1 M glycine (pH 2.5) at
room temperature for 1 h, wash with ddH2O and reprobed for loading
control (CKII or .alpha.-tubulin) as described above.
[0285] Conjugation of MAbs to agarose and sample preparation for
LC-MS/MS analysis. MAbs 18B10.C7(3), 9D1.E4(10) and 1F5.D6(14) or
CTD110.6 were covalently conjugated to Protein A/G PULS agarose or
anti-Mouse IgM agarose via disuccinimidyl substrate (DSS, Thermo
Scientific, Rockford, Ill.) according to manufacturer's
instruction. PUGNAc treated HEK293T nucleocytosolic fraction was
prepared as above in larger scale, incubated with antibody
conjugated agarose, and washed as above. To elute proteins off the
agarose, 0.1 M of glycine (pH 2.5) was added and the eluates were
immediately neutralized with 1 M Tris-HCl (pH 8.0). The samples
were then reduced and alkylated as previously described8 and
subjected to LysC digestion at 37.degree. C. for overnight. After
digestion, the samples were processes as previously described (Lim
et al., 2008 J. Proteome Res. 7, 1251-1263).
[0286] Mass spectrometry. The samples were resuspended with 19.5
.mu.l of 0.1% formic acid (in water) and 0.5 .mu.l of 80%
acetonitrile/0.1% formic acid (in water) and filtered with 0.2
.mu.m filters (Nanosep, PALL). Samples were then loaded off-line
onto a nanospray C18 column and separated with a 160-min linear
gradient as previously described (Lim et al., 2008 J. Proteome Res.
7, 1251-1263) using Finnigan LTQ/XL mass spectrometer
(ThermoFisher, San Jose, Calif.). Each sample was subjected to 3
runs with different settings: (1) ETD (electron transferred
dissociation) mode, where a full MS spectrum was collected followed
by 6 MS/MS spectra following ETD (enabled supplemental activation)
of the most intense peaks. The dynamic exclusion was set at 1 for
30 sec of duration. (2) CID-NL (collision induced
dissociation-pseudo neutral loss) mode, where a full MS spectrum
was collected followed by 8 MS/MS spectra following CID of the most
intense peaks. Upon encountering a pseudo neutral loss event (a
loss of GlcNAc, 203.08), a MS8 spectrum will be created based of
the MS/MS spectrum. The dynamic exclusion has the same setting as
ETD method. (3) DDNL-ETD (Data dependent neutral loss MS8 under CID
followed by ETD activation upon every neutral loss event), where
MS/MS spectra from top 5 peaks of each full MS scan were collected
with CID (35% normalized collision energy) and monitored for a
neutral loss of 203.08 during which a MS8 spectrum will be created.
A repeat scan event with neutral loss will be performed using ETD
enabled with supplemental activation. The dynamic exclusion was
also set the same as above.
[0287] Data analysis and validation. MS spectra were searched
against the human (Homo sapiens, 32876 entries, Aug. 13, 2007
released) forward and reverse databases extracted from the
Swiss-Prot human proteome database using the TurboSequest algorithm
(Bioworks 3.3, Thermo Finnigan). The DTA files were generated for
spectra with a threshold of 15 ions and a TIC of 1e3. Dynamic mass
increases of 15.99, 57.02 and 203.08 Da were considered for
oxidized methione, alkylated cysteine and O-GlcNAc modified
serine/threonine respectively. The resulting OUT files each samples
obtained forward and reversed databases searched were further
parsed with ProtoeIQ (Bioinquire) and filtered with 1% FDR (metric
used: F-value) and starting peptide coverage for ProFDR at 5.
[0288] Statistical analysis. Statistical significance between
groups was determined by two-tailed, unpaired Student's t test.
Differences were considered significant when P<0.05.
Example XI
O-GlcNAc Modified Proteins in Rat Liver
[0289] Changes in cell and tissue levels of O-GlcNAc have typically
been associated with a range of chronic pathophysiological
conditions including aging (Fulop et al., Am J Physiol Cell Physiol
2007; 292:C1370-8; Rex-Mathes et al., Biochimie 2001; 83:583-90;
Fulop et al., Biogerontology 2008; 9:139-51), cancer (Chou et al.,
J Biol Chem 1995; 270:18961-5; Shaw et al., Oncogene 1996;
12:921-30; Donadio et al., J Cell Biochem 2008; 103:800-11),
neurodegenerative disorders (Hanover, FASEB J. 2001; 15:1865-187;
Wells et al., Biochem Biophys Res Commun 2003; 302:435-41; Love and
Hanover, Sci STKE 2005; 2005:re13; Dias et al., Mol Biosyst 2007;
3:766-72) as well as diabetes and diabetic complications (Buse, Am
J Physiol Endocrinol Metab 2006; 290:E1-E8; Copeland et al., Am J
Physiol Endocrinol Metab 2008; 295:E17-28). However, a number of
studies have recently demonstrated that acute augmentation of
O-GlcNAc levels is associated with increased tolerance of cells to
stress and conversely, inhibition of O-GlcNAc formation decreases
cell survival (Champattanachai et al., Am J Physiol Cell Physiol
2007; 292:C178-87; Champattanachai et al., Am J Physiol Cell
Physiol 2008; 294:C1509-20; Zachara et al., J Biol Chem 2004;
279:30133-30142). We have also shown in a rat model of
trauma-hemorrhage that increasing O-GlcNAc synthesis with
glucosamine or inhibiting O-GlcNAc degradation with PUGNAc during
resuscitation leads to improved organ function, decreased tissue
injury, reduced inflammatory responses and lower mortality (Not et
al., Faseb J 2008; 22:1227; Not et al., SHOCK 2007; 28:345-351;
Yang et al., Shock 2006; 25:600-607; Zou et al., Shock 2007;
27:402-408). Surprisingly, however, it has been found that
resuscitation results in marked loss of overall O-GlcNAc levels in
multiple tissues, which was sustained for up to 24 hrs and that
treatment with either glucosamine or PUGNAc prevented this loss
(Not et al., Faseb J 2008; 22:1227; Zou et al., Shock 2007;
27:402-408). Moreover, significant correlations have been shown
between the overall level of tissue O-GlcNAc levels and indices of
tissue injury (Not et al., Faseb J 2008; 22:1227; Liu et al., Am J
Physiol Heart Circ Physiol 2007; 293:H1391-9); however, to date
identification of specific proteins that exhibit changes in
O-GlcNAc modification in response to trauma-hemorrhage and
resuscitation has not been examined. In FIG. 33, we show that
similar to CTD110.6, all three MAbs generated in this study
demonstrated significantly lower overall hepatic O-GlcNAc levels 24
hrs following trauma-hemorrhage and resuscitation compared to sham
controls.
TABLE-US-00012 TABLE 12 Monoclonal O-GlcNAc antibodies. Antibody
Type Conc. Primary Secondary CTD 110.6 IgM 1:5000 1:10000 #3
(18B10.C7) IgG1 0.86 mg/mL 1:1000 1:2500 #10 (9D1.E4) IgG1 0.59
mg/mL 1:1000 1:2500 #14 (1F5.D6) IgG2a 0.97 mg/mL 1:1000 1:2500
[0290] To provide insight into proteins whose O-GlcNAc status is
modified, the antibodies (Table 12) were employed for
immuno-precipitation of O-GlcNAc modified proteins from livers
samples of rats subjected to trauma-hemorrhage and resuscitated and
sham controls (FIG. 33). Thus, agarose covalently conjugated MAbs
were mixed with liver extracts and subjected to Lys-C digestion and
the recovered peptides and glycopeptides were analyzed by LC-MS/MS
on an LTQ-XL. Protein assignments and false-discovery rates (1% at
the protein level) were calculated using TurboSequest and ProteoIQ.
Proteins were excluded that appeared in control experiments
(mixture of Protein A/G PLUS agarose and anti-Mouse IgM agarose)
and localization was confirmed with the aid of Human Protein
Reference Database (HPRD) and UniProt. In the control (sham) group,
we identified 69 O-GlcNAc modified proteins, whereas in
trauma-hemorrhage and resuscitated group, 30 different O-GlcNAc
modified proteins were identified (FIG. 34, Table 13). These
results demonstrate that the antibodies can be employed to identify
O-GlcNAc modified proteins from tissue samples.
TABLE-US-00013 TABLE 13 O-GlcNAc modified proteins identified in
rat liver. UniProt Gene Sham Total THR Total Biological Primary
Alternate ID Name Sequence Name Peptides Peptides Process
Localization Localization P07756 Cps1 carbamoyl-phosphate 20 7 M
Mitochondrion synthetase 1 P63039 Hspd1 heat shock protein 1 7 0 S
Mitochondrion Cytoplasm; (chaperonin) ER; Golgi; Nucleolus Q9WVK7
Hadh L-3-hydroxyacyl- 4 0 M Mitochondrion Coenzyme A dehydrogenase
P04785 P4hb prolyl 4-hydroxylase, beta 5 1 M ER Nucleus;
polypeptide Extracellular; Nucleolus -- -- PREDICTED: similar to 8
14 G Nucleus Cytoplasm host cell factor C1 P22791 Hmgcs2
hydroxymethylglutaryl- 6 2 M Mitochondrion CoA synthase 2 Q66HT1
Aldob aldolase B, fructose- 4 3 M Cytoplasm bisphosphate P48500
Tpi1 triosephosphate isomerase 1 0 4 M Cytoplasm Q9JIH7 Wnk1 WNK
lysine deficient 6 12 S Cytoplasm protein kinase 1 O09171 Bhmt
betaine-homocysteine 3 3 M Cytoplasm methyltransferase P13437 Acaa2
acetyl-Coenzyme A 4 2 M Mitochondrion acyltransferase 2 -- --
PREDICTED: similar to 5 0 M Cytoplasm Plasma heat shock protein 8
Membrane O08658 Nup88 nucleoporin 88 kDa 0 3 Tp Nucleus Q5XFW8
Sec13 SEC13 homolog 5 4 Tp ER Nucleus; Cytoplasm P05197 Eef2
eukaryotic translation 3 1 Tl Cytoplasm Nucleus; elongation factor
2 Cytosol P52759 Hrsp12 heat-responsive protein 12 3 0 Tl Cytoplasm
Plasma (Ribonuclease UK114; membrane; 14.5 kDa translational Golgi,
ER; inhibitor protein) Nucleus Q66HA5 Cc2d1 coiled-coil and C2
domain 0 3 G, S Cytoplasm Nucleus containing 1A O35077 Gpd1
glycerol-3-phosphate 3 0 M Cytoplasm dehydrogenase 1 (soluble)
P10860 Glud1 glutamate dehydrogenase 1 4 0 M Mitochondrion -- --
PREDICTED: similar to 3 1 M Cytoplasm Plasma Alpha-enolase (2-
membrane; phospho-D-glycerate Nucleus; hydro-lyase) (Non-neural
Extracellular enolase) (NNE) (Enolase 1) -- -- PREDICTED: similar
to 2 4 U Cytoplasm HBxAg transactivated protein 2 -- -- PREDICTED:
similar to 3 0 M Mitochondrion aldehyde dehydrogenase 4 family,
member A1 P16638 Acly ATP citrate lyase isoform 2 2 0 M Cytoplasm
Q3T1I4 Prrc1 proline-rich coiled-coil 1 0 3 U Golgi Cytoplasm
P15999 Atp5a1 ATP synthase, H+ 3 0 M, Tp Mitochondrion
transporting, mitochondrial F1 complex, alpha subunit 1, cardiac
muscle -- -- ubiquitin-associated 0 2 U Cytoplasm protein 2 B6DYP7
Gsta2 glutathione S-transferase 3 0 M Cytoplasm alpha 2 P06761
Hspa5 heat shock protein 5 (BIP; 3 2 M ER Plasma Heat shock 70 kDa
protein membrane; 5; 78 kDa glucose- Cytoplasm; regulated protein)
Nucleolus B5DFC3 Sec23a SEC23 homolog A 4 2 Tp Cytoplasm ER;
Cytoplasmic vesicle Q6AYR1 Tfg Trk-fused 0 2 S Cytoplasm P02692
Fabp1 fatty acid binding protein 1 3 0 Tp Cytoplasm Nucleus P67779
Phb PREDICTED: prohibitin 2 0 S Mitochondrion Plasma membrane;
Nucleus; Nucleolus; Cytoplasm; Extracellular Q02974 Khk
ketohexokinase 2 0 M Cytoplasm? P56558 Ogt O-linked N- 0 2 M, S, G
Nucleus Cytoplasm; acetylglucosamine Mitochondrion transferase --
-- PREDICTED: similar to 3 7 Tp Nucleus nucleoporin 214 kDa P06757
Adh1 alcohol dehydrogenase 1 2 1 M Cytoplasm Q6P6R2 Dld
dihydrolipoamide 2 0 M Mitochondrion dehydrogenase Q9JM53 Aifm1
apoptosis-inducing factor, 2 0 S Mitochondrion Nucleus;
mitochondrion-associated 1 Cytoplasm -- -- PREDICTED: similar to 2
0 Str Cytoplasm Actin, cytoplasmic 2 (Gamma-actin) -- -- granulin
isoform a 0 1 S Extracellular Cytoplasm Q9WVK3 Pecr peroxisomal
trans-2- 2 0 M Peroxisome enoyl-CoA reductase P11884 Aldh2
mitochondrial aldehyde 3 2 M Mitochondrion Cytoplasm dehydrogenase
2 B2RYJ5 Tmprss13 transmembrane protease, 0 1 M Transmembrane
serine 13 O88764 Dapk3 Death-associated protein 0 1 S Nucleus
Cytoplasm kinase 3 P70581 Nupl1 nucleoporin like 1 3 3 U Nucleus
(Nucleoporin p58/p45) P25093 Fah fumarylacetoacetate 2 0 M
Cytoplasm Extracellular hydrolase B0BMW2 Hsd17b10 hydroxysteroid
(17-beta) 1 0 M Mitochondrion ER; Plasma dehydrogenase 10 membrane
P07824 Arg1 arginase 1 2 0 M Cytoplasm; Extracellular Plasma
Membrane P11232 Txn thioredoxin 2 0 M Cytoplasm Nucleus;
Extracellular B5DF65 Blvrb biliverdin reductase B 1 1 M Cytoplasm
(flavin reductase (NADPH)) -- -- filamin, beta 1 0 B0BN46 Grhpr
glyoxylate 1 0 M Cytoplasm reductase/hydroxypyruvate reductase
Q9Z2Q1 Sec31a SEC31 homolog A 2 0 Tp ER Cytoplasm; Golgi P63245
Gnb2l1 guanine nucleotide 1 0 S Cytoplasm Nucleus; binding protein,
beta Plasma polypeptide 2-like 1 membrane (RACK1) -- -- PREDICTED:
similar to 2 0 M Cytoplasm Nucleolus; L-lactate dehydrogenase A
Cytosol chain (LDH-A) (LDH muscle subunit) (LDH-M) Q66HF1 Ndufs1
NADH dehydrogenase 1 0 M Mitochondrion (ubiquinone) Fe--S protein
1, 75 kDa -- -- PREDICTED: similar to 1 0 M U aldehyde
dehydrogenase family 7, member A1 P38918 Akr7a3 aldo-keto reductase
family 1 0 M Cytoplasm 7, member A3 (aflatoxin aldehyde reductase)
P14173 Ddc dopa decarboxylase 1 0 M Cytoplasm (aromatic L-amino
acid decarboxylase) P14141 Ca3 carbonic anhydrase III 2 0 M
Cytoplasm Extracellular -- -- PREDICTED: similar to 2 3 Tp ER
Cytoplasm SEC24 related gene family, member C isoform 5 P45953
Acadvl acyl-Coenzyme A 1 0 M Mitochondrion dehydrogenase, very long
chain -- -- PREDICTED: similar to 1 0 M Mitochondrion solute
carrier family 25, member 5 -- -- PREDICTED: similar to 1 0 M
Mitochondrion Glycine cleavage system H protein, mitochondrial
precursor P13697 Me1 malic enzyme 1 (NADP- 1 0 M Cytoplasm
dependent malic enzyme) P29147 Bdh1 3-hydroxybutyrate 1 0 M
Mitochondrial dehydrogenase, type 1 membrane -- -- PREDICTED:
similar to 1 0 M Mitochondrion hydroxyacyl-Coenzyme A
dehydrogenase/3-ketoacyl- Coenzyme A thiolase/enoyl-Coenzyme A
hydratase (trifunctional protein), alpha subunit Q4V8I9 Ugp2
UDP-glucose 1 0 M Cytoplasm pyrophosphorylase 2 Q9JKB7 Gda guanine
deaminase 1 0 M Cytoplasm Plasma membrane Q5U2T3 Spats2l
hypothetical protein 2 0 U U LOC316426 (SPATS2- like protein)
P00786 Ctsh cathepsin H 1 0 M Lysosome Microsome, Nucleus,
Mitochondrion Q02253 Aldh6a1 aldehyde dehydrogenase 1 0 M
Mitochondrion family 6, subfamily A1 P19112 Fbp1 fructose-1,6- 1 0
M Cytoplasm biphosphatase 1 P07632 Sod1 superoxide dismutase 1, 1 0
M Cytoplasm Peroxisome; soluble Nucleus; Extracellular Q68FZ8 Pccb
propionyl Coenzyme A 1 0 M Mitochondrion carboxylase, beta
polypeptide B6ID08 Mt2A metallothionein 2A 1 0 U U -- -- PREDICTED:
similar to 1 0 Tp Mitochondrion Calcium-binding mitochondrial
carrier protein Aralar2 (Mitochondrial aspartate glutamate carrier
2) (Solute carrier family 25 member 13) (Citrin) P13803 Etfa
electron-transfer- 1 0 M, Tp Mitochondrion flavoprotein, alpha
polypeptide -- -- PREDICTED: similar to 1 0 M Mitochondrion
Cytochrome P450 2C7 (CYPIIC7) (P450F) (PTF1) -- -- PREDICTED:
similar to 1 5 U Nucleus Nice-4 protein homolog isoform 1 Q5SGE0
Lrpprc leucine-rich PPR-motif 1 0 G Nucleus Cytoplasm; containing
protein Plasma membrane * Abbreviations: G, gene
expression/transcription; M, metabolism; S, signal transduction;
Tl, translation; Tp, transport; U, unknown.
[0291] The complete disclosure of all patents, patent applications,
and publications, and electronically available material (including,
for example, nucleotide sequence submissions in, e.g., GenBank and
RefSeq, and amino acid sequence submissions in, e.g., SwissProt,
PIR, PRF, PDB, and translations from annotated coding regions in
GenBank and RefSeq) cited herein are incorporated by reference. The
foregoing description and examples have been given for clarity of
understanding only. No unnecessary limitations are to be understood
therefrom. The invention is not limited to the exact details shown
and described, for variations obvious to one skilled in the art
will be included within the invention defined by the claims.
Sequence CWU 1
1
19115PRTartificialhelper T peptide 1Gln Tyr Ile Lys Ala Asn Ser Lys
Phe Ile Gly Ile Thr Glu Leu1 5 10 15220PRTartificialhelper T
peptide 2Tyr Ala Phe Lys Tyr Ala Arg His Ala Asn Val Gly Arg Asn
Ala Phe1 5 10 15Glu Leu Phe Leu 20313PRTartificialhelper T peptide
3Lys Leu Phe Ala Val Trp Lys Ile Thr Tyr Lys Asp Thr1 5
10415PRTartificialtetanus toxin peptide 4Val Ser Ile Asp Lys Phe
Arg Ile Phe Cys Lys Ala Asn Pro Lys1 5 10 15516PRTartificialtetanus
toxin peptide 5Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu
Ile Asp Ser1 5 10 15616PRTartificialtetanus toxin peptide 6Ile Arg
Glu Asp Asn Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn1 5 10
15721PRTartificialtetanus toxin peptide 7Phe Asn Asn Phe Thr Val
Ser Phe Trp Leu Arg Val Pro Lys Val Ser1 5 10 15Ala Ser His Leu Glu
20820PRTNeisseria meningitidis 8Tyr Ala Phe Lys Tyr Ala Arg His Ala
Asn Val Gly Arg Asn Ala Phe1 5 10 15Glu Leu Phe Leu
20918PRTPlasmodium falsiparum 9Glu Lys Lys Ile Ala Lys Met Glu Lys
Ala Ser Ser Val Phe Asn Val1 5 10 15Asn Asn105PRTartificialamino
acid sequence modified by O-GlcNac 10Thr Pro Val Ser Ser1
51110PRTartificialpeptide component of glycopeptide 11Gly Ser Thr
Pro Val Ser Ser Ala Asn Met1 5 101211PRTartificialpeptide
conjugated to BSA 12Cys Gly Ser Thr Pro Val Ser Ser Ala Asn Met1 5
101310PRTartificialpeptide component of glycopeptide 13Gly Ser Thr
Pro Val Ser Ser Ala Asn Met1 5 101454DNAartificialoligonucleotide
primer 14ccccatgtat ccatatgacg tcccagacta tgccgcgtct tccgtgggca
acgt 541574DNAartificialoligonucleotide primer 15ggggacaagt
ttgtacaaaa aagcaggctg gatgatgtat ccatatgacg tcccagacta 60tgccgcgtct
tccg 741670DNAartificialoligonucleotide primer 16ggggaccact
ttgtacaaga aagctgggtt ctatgctgac tcagtgactt caacgggctt 60aatcatgtgg
701754DNAartificialoligonucleotide primer 17ccccatgtat ccatatgacg
tcccagacta tgccgtgcag aaggagagtc aagc
541874DNAartificialoligonucleotide primer 18ggggacaagt ttgtacaaaa
aagcaggctg gatgatgtat ccatatgacg tcccagacta 60tgccgtgcag aagg
741950DNAartificialoligonucleotide primer 19ggggaccact ttgtacaaga
aagctgggtt cacaggctcc gaccaagtat 50
* * * * *