U.S. patent application number 11/081198 was filed with the patent office on 2005-08-18 for compositions and methods for treatment of cancer.
This patent application is currently assigned to The Wistar Institute of Anatomy and Biology. Invention is credited to Blaszczyk-Thurin, Magdalena, Kieber-Emmons, Thomas.
Application Number | 20050181987 11/081198 |
Document ID | / |
Family ID | 34840767 |
Filed Date | 2005-08-18 |
United States Patent
Application |
20050181987 |
Kind Code |
A1 |
Blaszczyk-Thurin, Magdalena ;
et al. |
August 18, 2005 |
Compositions and methods for treatment of cancer
Abstract
Compositions containing one or more peptido-mimetics or modified
peptido-mimetics of a carbohydrate ligand of an adhesion molecule
in a physiologically acceptable carrier are useful for methods of
reducing metastasis in a mammal and for inhibiting inflammatory
response in a mammal. Particularly useful are embodiments in which
the ligand is a Lewis antigen and/or the adhesion molecule is a
selectin, e.g., E-selectin. Methods are disclosed for identifying
peptido-mimetics of carbohydrate ligands, which may be involved in
binding of tumor cells to other cells, such as endothelial
cells.
Inventors: |
Blaszczyk-Thurin, Magdalena;
(Philadelphia, PA) ; Kieber-Emmons, Thomas;
(Newton Square, PA) |
Correspondence
Address: |
HOWSON AND HOWSON
ONE SPRING HOUSE CORPORATION CENTER
BOX 457
321 NORRISTOWN ROAD
SPRING HOUSE
PA
19477
US
|
Assignee: |
The Wistar Institute of Anatomy and
Biology
Philadelphia
PA
The Trustees of the University of Pennsylvania
Philadelphia
PA
|
Family ID: |
34840767 |
Appl. No.: |
11/081198 |
Filed: |
March 16, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11081198 |
Mar 16, 2005 |
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09831047 |
May 3, 2001 |
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09831047 |
May 3, 2001 |
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PCT/US99/26277 |
Nov 5, 1999 |
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60107478 |
Nov 6, 1998 |
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Current U.S.
Class: |
424/185.1 ;
514/19.1; 514/19.3; 514/21.6 |
Current CPC
Class: |
A61K 38/12 20130101;
A61K 38/177 20130101; A61K 38/10 20130101 |
Class at
Publication: |
514/008 ;
514/015 |
International
Class: |
A61K 038/16; A61K
038/14 |
Goverment Interests
[0002] This invention was supported in part by funds from the U.S.
Government (United States Army Grant DAMD 17-96-1-6232 and NIH
Grant No. A145133). The U.S. Government may therefore have certain
rights in the invention.
Claims
What is claimed is:
1. A composition comprising a peptido-mimetic of a carbohydrate
ligand of an adhesion molecule in a physiologically acceptable
carrier.
2. The composition according to claim 1, wherein said adhesion
molecule is a selectin.
3. The composition according to claim 1, wherein said ligand is a
Lewis antigen.
4. The composition according to claim 3, wherein the Lewis antigen
is selected from the group consisting of SA-Le.sup.a, SA-LeX, and
LeY.
5. The composition according to claim 2, wherein said adhesion
molecule is E-selectin and said ligand is SA-Le.sup.a or
SA-LeX.
6. The composition according to claim 5, wherein said
peptido-mimetic is selected form the group consisting of:
7 DLWDFVVGKPAG, SEQ ID NO:63 DLWDWVIGKPAG, SEQ ID NO:64
DLWDWVVAKPAG, SEQ ID NO:65 DLWDWVVSKPAG, SEQ ID NO:66 DLWDWVVEKPAG,
SEQ ID NO:67 DLWDWVVDKPAG, SEQ ID NO:68 DLWDWVVGEPAG, SEQ ID NO:69
DLWDWVVGDPAG, SEQ ID NO:70 DLWDWVVGKEAG, SEQ ID NO:71 DLWDWVVGKDAG,
SEQ ID NO:72 DLWDWVVGKPDG, SEQ ID NO:73 DLWDWVVGKPAD, SEQ ID NO:74
DLWDWVKEKPAG, SEQ ID NO:75 DLWDWVLAKPAG, SEQ ID NO:76 DLWDWVVGEDAG,
SEQ ID NO:77 DLWDWVVGKPEK SEQ ID NO:78 DLWDWVKEEPAG, SEQ ID NO:79
DLWDWVVGKDEK, SEQ ID NO:80 DLWDWVVGEDEK, SEQ ID NO:81 DLWDWVKEEDEK,
SEQ ID NO:82 TKRPDLIVDPIP, SEQ ID NO:98 DEVRPDLISTEE, SEQ ID NO:99
NLRPKYIXLDAD, SEQ ID NO:100 and TLIAFADLVDVI. SEQ ID NO:101
7. The peptide or polypeptide according to claim 6, which is
modified to enhance stability or enhance adhesion molecule
binding.
8. The peptide or polypeptide according to claim 6, which is a
multivalent peptide or polypeptide.
9. A peptide or polypeptide consisting of from 7 to 15 amino acids,
said peptide or polypeptide mimicking a carbohydrate ligand of an
adhesion molecule in a physiologically acceptable carrier, wherein
said ligand is a Lewis SA-Le.sup.a or SA-LeX antigen.
10. The peptide or polypeptide according to claim 9, which is
modified to enhance stability or enhance adhesion molecule
binding.
11. The peptide or polypeptide according to claim 9, which is a
multivalent peptide or polypeptide.
12. The peptide or polypeptide according to claim 9, which is
selected form the group consisting of:
8 DLWDFVVGKPAG, SEQ ID NO:63 DLWDWVIGKPAG, SEQ ID NO:64
DLWDWVVAKPAG, SEQ ID NO:65 DLWDWVVSKPAG, SEQ ID NO:66 DLWDWVVEKPAG,
SEQ ID NO:67 DLWDWVVDKPAG, SEQ ID NO:68 DLWDWVVGEPAG, SEQ ID NO:69
DLWDWVVGDPAG, SEQ ID NO:70 DLWDWVVGKEAG, SEQ ID NO:71 DLWDWVVGKDAG,
SEQ ID NO:72 DLWDWVVGKPDG, SEQ ID NO:73 DLWDWVVGKPAD, SEQ ID NO:74
DLWDWVKEKPAG, SEQ ID NO:75 DLWDWVLAKPAG, SEQ ID NO:76 DLWDWVVGEDAG,
SEQ ID NO:77 DLWDWVVGKPEK SEQ ID NO:78 DLWDWVKEEPAG, SEQ ID NO:79
DLWDWVVGKDEK, SEQ ID NO:80 DLWDWVVGEDEK, SEQ ID NO:81 DLWDWVKEEDEK,
SEQ ID NO:82 TKRPDLIVDPIP, SEQ ID NO:98 DEVRPDLISTEE, SEQ ID NO:99
NLRPKYIXLDAD, SEQ ID NO:100 and TLIAFADLVDVI. SEQ ID NO:101
13. A modified peptide or polypeptide mimicking a carbohydrate
ligand of an adhesion molecule in a physiologically acceptable
carrier, wherein said ligand is a Lewis antigen and wherein said
modification is selected from the group consisting of (i) use of
one or more D amino acids, (ii) insertion of a moiety which can
provide a net positive charge at the N-terminus of said peptide or
polypeptide, (iii) insertion of a spacer of greater than 3 amino
acids interposed between the N- and C-termini to cyclize the
peptide, (iv) insertion of a free hydroxyl on the C-terminus, (v)
insertion of an amide or imide on the C-terminus, and (vi)
insertion of a sequence of one or up to about 15 additional amino
acids on the C-terminus.
14. The peptide or polypeptide according to claim 13 which is
modified by cyclizing the N- and C-termini.
15. The peptide or polypeptide according to claim 13, wherein said
modification enhances stability or enhance adhesion molecule
binding.
16. The peptide or polypeptide according to claim 13, which is a
multivalent peptide or polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/831,047, filed May 3, 2001, which is a 371
of International Patent Application No. PCT/US99/26277, filed Nov.
5, 1999, which claims the benefit of the priority of U.S.
Provisional Patent Application No. 60/107,478, filed Nov. 6, 1998,
now abandoned.
BACKGROUND OF THE INVENTION
[0003] The invention relates generally to animal cell adhesion, and
more specifically to peptido-mimetics of carbohydrate ligands of
animal cell adhesion proteins and methods of using the same.
[0004] Tumor metastasis is a multistep process requiring detachment
of malignant cells from the primary tumor, penetration of blood or
lymph vessels and attachment to endothelium of distant organs and
formation of new tumors. The ability of disseminating cancer cells
to establish metastases in secondary organs is regulated by a
combination of factors, including access to the organ
microvasculature and specific host-tumor interactions [Radinsky and
Fidler, 1992, In Vivo 6:325-331]. Hematogenous dissemination brings
cancer cells into contact with leukocytes, platelets, and
endothelium [Nicolson, 1989, Curr. Opin. Cell. Biol. 1: 1009-1019;
Bevilaqua et al., 1989, Science 243:1160-1165]. The attachment of
circulating tumor cells to the vascular endothelium of the target
organ is thought to be a key step in the metastatic cascade. Once
tumor cells adhere to endothelial cells (EC), they penetrate
through the EC layer, moving into subendothelial tissues where
metastasis is established.
[0005] Studies of leukocyte transmigration have suggested that the
specificity of the interaction between circulating lymphocytes and
the microvascular endothelium may be determined by the outcome of a
series of sequential adhesion molecule-ligand interactions
involving complex carbohydrate structures on the surface of
leukocytes and adhesion molecules on the surface of endothelial
cells. It appears likely that the process of tumor extravasation is
mediated through a series of analogous adhesive interactions
[Brodt, 1996, In: Cell adhesion and invasion in cancer metastasis,
vol. 21 pp. 167-242, Brodt, ed., Landes, Austin and
Springer-Verlag, Berlin].
[0006] Prominent among the vascular endothelial cell adhesion
molecules implicated in both leukocyte and tumor cell
transmigration are members of the selectin family. This family of
adhesion molecules supports the adhesion of leukocytes to the
vessel wall through the recognition of specific carbohydrate
structures and thereby mediates critical cell-cell interactions in
processes such as leukocyte trafficking, thrombosis, acute and
chronic inflammation and ischemia reperfusion injury [Bucher and
Picker, 1996, Science 272:60-66; Lasky, 1995, Annu. Rev. Biochem.
64:113-139; Varki, 1994, Proc. Natl. Acad. Sci. USA 91:7390-7397;
Bevilaqua and Nelson, 1993, J. Clin. Invest. 91:379-387]. Selectins
recognize and bind specific carbohydrate antigens expressed on
tumor cell surfaces and mediate the initial interaction between
tumor cells and endothelium [Mannori et al., 1995, Cancer. Res.
55:4425-4431 (Mannori 1)].
[0007] Selectins are subdivided into E-, L-, and P-selectin
subgroups. Inflammatory mediators such as tumor necrosis factor
.alpha. (TNF-.alpha.) and interleukin 1.beta. (IL-1.beta.) induce
vascular endothelium to express E- and P-selectin. E-selectin is a
calcium-dependent molecule expressed by activated vascular
endothelium during the process of leucocyte recruitment. E-selectin
binds to glycoconjugates carrying a terminal tetrasaccharide Lewis
(Le) antigen, sialyl-LeX (SA-LeX),
[NeuAc.alpha.2,3Gal.alpha.1.beta.1,4(Fuc.alpha.1,3)
GlcNAc.beta.1,3Gal.beta.1,4Glc.beta.1-R], but displays higher
affinity for the SA-Le.sup.a structure
([NeuAc.alpha.2,3Gal.beta.1,3(Fuc.alpha.1,4- )
GlcNAc.beta.1,3Gal.beta.1,4Glc.beta.1-R], a positional isomer of
SA-LeX.
[0008] P-selectin is expressed by activated platelets and
endothelial cells. P-selectin has been shown to mediate adhesive
interactions of some colon adenocarcinoma cells with
thrombin-activated platelets [Mannori I, cited above]. During
metastatic dissemination, tumor-platelet adhesion may result in the
formation of neoplastic emboli that facilitate the arrest of tumor
cells in the microvasculature of organs [Karpatkin and Pearlstein,
1981, Ann. Intern. Med. 95: 636-641].
[0009] L-selectin is constitutively expressed by a majority of
leukocytes, including neutrophils, monocytes, natural killer cells
and most lymphocytes. L-selectin expressed on leukocytes can
support interaction with cancer cells, enhancing metastatic
potential [Welch et al., 1989, Proc. Natl. Acad. Sci. USA
86:5859-5863]. Other studies demonstrate that the engagement of
L-selectin on lymphocytes can stimulate their anti-tumor cell
cytotoxic activity [Seth et al., 1991, Proc. Natl. Acad. Sci. USA
88: 7877-7881].
[0010] Selectin recognition of carbohydrate ligands involves
primarily the N-terminal of C-type lectin domain, influenced by the
EGF-like domain, and to a lesser degree the short consensus repeats
[Kolbinger et al., 1988, Biochemistry 35:6385-6392; Revelle et al.,
1996, J. Biol. Chem. 271:16160-16170; Li et al., 1994, J. Biol.
Chem. 269:4431-4437]. Mutagenesis studies of the lectin domain of
the selectins [Erbe et al., 1993, J. Cell. Biol. 120:1227-1235] and
the crystal structure of the E-selectin lectin plus EGF-like domain
solved at 2.0 .ANG. resolution [Graves et al., 1994, Nature
367:532-538] have identified a number of positively charged
residues involved in the binding to the SA-LeX carbohydrate ligand.
Most of these residues are identical in all selectins and they are
likely to recognize essential carbohydrate moieties such as sialic
acid and fucose of SA-LeX. The identified residues are derived from
noncontiguous sequences at both the N- and C-terminus of the lectin
domain.
[0011] Although adhesion pathways utilized by different tumors
exhibit considerable diversity, metastasis apparently involves the
interaction of at least one member of the selectin family of
adhesion molecules with the antigens SA-Le.sup.a and/or SA-LeX.
These ligands may be involved in tumor metastasis by mediating
binding of blood-borne tumor cells via E- and/or P-selectin to
vascular endothelium [Giavazzi et al., 1993, J. Clin. Invest.
92:3038-3044; Dejana et al., 1992, Lab. Invest. 66:324-3130; Takada
et al., 1993, Cancer. Res. 53:354-361; Sawada et al., 1994, J.
Biol. Chem. 269:1425-1431]. Cancer cells that express both
SA-Le.sup.a and SA-LeX undergo SA-Le.sup.a-mediated adhesion almost
exclusively, possibly due to the higher affinity for the
SA-Le.sup.a structure, or differential presentation of this
oligosaccharide determinant. Thus, SA-Le.sup.a might play a major
role as a ligand in the E-selectin dependent adhesion to EC in
vivo. Indeed, SA-Le.sup.a specific monoclonal antibodies (MAbs)
were inhibitory for adhesion of colon carcinoma cells to human
umbilical cord vein endothelial cells (HUVEC).
[0012] In vivo studies have provided further evidence of the
potential importance of the carbohydrate ligand/E-selectin
interaction in tumor metastasis [Brodt et al., 1997, Int. J. Cancer
71:612-619; Mannori et al., 1997, Am. J. Pathol. 151:233-243
(Mannori II); Biancone et al., 1996, J. Exp. Med. 183:581-587].
[0013] Alternatively, some carcinoma cells do not express these
carbohydrate determinants (i.e., SA-LeX and SA-Le.sup.a) and yet
they can attach to EC prior to activation. Further, this adhesion
is not augmented by cytokine treatment, suggesting
E-selectin-independent adhesion [Iwai et al., 1993, Int. J. Cancer
54:972-977; Tozeren et al., 1995, Int. J. Cancer 60:426-431; Miyake
et al., 1992, New Eng. J. Med. 327:14-18; Garrigues et al., 1992,
J. Cell. Biol. 125:129-142].
[0014] Studies have also demonstrated the role of oligosaccharides
in inflammatory responses. Neutrophil extravasation is enabled by a
multistep process initiated by the selectin family [Kansas, 1996,
Blood 88: 3259-3287]. Neutrophil-endothelial cell interaction
mediated via the selectins in the context of vascular shear flow,
are characterized by transient tethering of the neutrophils,
followed by rolling of the neutrophil along the endothelial surface
of the vessel wall. Studies in vivo and in vitro indicate that
selectin-dependent neutrophil rolling is essential to subsequent
events in the transmigration process. Neutrophils are exposed to
endothelial cell derived IL-8, platelet-activating factor and other
neutrophil-activating molecules [Lowe, 1997, In: The selectins:
Inhibitors of leukocyte endothelial adhesion, pp. 143-177,
Vestweber, ed., Harwood Academic Publishers, Reading, UK], which in
turn promote activation of neutrophil P2 integrins, leading to
integrin-dependent firm adhesion to the integrin receptor ICAM-1,
and finally to neutrophil extravasation, possibly via homophilic
interaction of platelet/endothelial cell adhesion molecule 1.
[0015] The expression of ligands for selectins, particularly
E-selectin, by both neutrophils and carcinoma cells raises the
possibility that metastases are equivalent to the inflammatory
process in which tumor cells, particularly carcinoma cells, use the
same molecular mechanism(s) for cancer cell-EC interaction as
lymphocytes, through the adhesion interaction of the endothelial
cell selectins with the tumor-associated carbohydrate ligands,
e.g., SA-LeX, SA-Le.sup.a, and LeY.
[0016] In addition to their role in cell adhesion, carbohydrate
structures also play a role in angiogenesis. Aberrant angiogenesis
can occur in a variety of pathologic conditions. Neovascularization
of tumors occurs by aberrant stimulation of normally quiescent
endothelial cells to migrate, proliferate and form new capillary
blood vessels [Ingber and Folkman, 1989, J. Cell. Biol.
109:3317-3330]. The experimental evidence suggests that E-selectin
and its ligand SA-LeX function in angiogenesis [Nguyen et al.,
1982, J. Biol. Chem. 267:26157-26165]. Thus, proliferating
microvascular endotheliurn presents a potential target for
anti-cancer and anti-angiogenic therapies through the inhibition of
E-selectin-dependent carbohydrate-mediated interactions [Folkman,
1995, N. Engl. J. Med. 333:1757-1763].
[0017] Although recent studies suggest the importance of
carbohydrate ligand-cell adhesion interactions in tumor metastasis,
angiogenesis and inflammatory responses, the complex nature of the
carbohydrate ligands involved has long hampered studies of these
processes. The difficult chemical or enzymatic synthesis required
by these complex carbohydrate ligands and the technical complexity
involved in analyzing the functional/structural interactions of
these ligands with selectins at the molecular level have severely
hindered the development of anti-adhesion therapeutics for
treatment of these disease processes for which there is no
effective treatment.
[0018] Many peptide mimics of carbohydrate structures have been
described in the literature [see, e.g., Agadjanyan, M. et al, 1997,
Nature Biotechnol. 15: 547-551, among others] including those
binding with high affinity to E-selectin [Tsukida, T. et al, 1998,
J. Med. Chem. 41: 4279-4287]. A peptide that mimics the GD1
ganglioside, also involved in cell adhesion and metastasis of
melanoma cells, has been recently described [Ishikawa, D. et al,
1998, FEBS Lett. 441: 20-24]. This peptide isolated from a peptide
phage display library using an anti-GD1 antibody inhibits
metastasis in an in vivo model.
[0019] Thus, there remains a long-felt and acute need for the
development of techniques and probes for the study of complex
carbohydrate ligand-cell adhesion molecule interactions, and for
the development of anti-tumor, anti-inflammatory and
angiogenesis-blocking therapeutics based on the selective
inhibition of these interactions.
SUMMARY OF THE INVENTION
[0020] The present invention meets the above-stated needs by
providing novel peptido-mimetics of carbohydrate structures and
uses therefor in affecting animal cell adhesion mediated by
carbohydrate ligand-cellular lectin protein receptor
interactions.
[0021] In one aspect, the invention includes a composition
comprising a peptido-mimetic of a carbohydrate ligand of an
adhesion molecule in a physiologically acceptable carrier. In one
embodiment, the adhesion molecule is a selectin, particularly
E-selectin. In another embodiment, the ligand is a Lewis antigen.
Particularly desirable are peptidomimetics of the Lewis antigens
SA-Le.sup.a, SA-LeX, and LeY. A variety of specific peptido-memtics
are recited in the detailed disclosure such peptido-mimetics may be
modified as described herein.
[0022] In still another aspect, the invention provides a method of
modulating binding of an adhesion molecule to a carbohydrate
ligand. The method comprises contacting the adhesion molecule
(e.g., a selectin) with a peptido-mimetic of the carbohydrate
ligand, so that binding of the adhesion molecule to the
carbohydrate ligand is modified or altered in a therapeutically
effective manner.
[0023] In a further aspect, the invention provides a method of
modulating adhesion of a tumor cell to an adhesion molecule located
on an endothelial cell. The method comprises contacting the tumor
cell with a peptido-mimetic of a carbohydrate ligand. The
peptido-mimetic modulates adhesion of the tumor cell to the
endothelial cell. The adhesion molecule may be, e.g., a selectin.
The ligand is preferably a Lewis antigen.
[0024] In still another aspect, the invention provides a method of
treating cancer in a mammal by administering an effective amount of
a peptido-mimetic of a carbohydrate ligand to the mammal. The
ligand is preferably a Lewis antigen. Administration of the
peptido-mimetic reduces adhesion of tumor cells to endothelial
cells in the mammal, thereby reducing metastasis of the cancer.
[0025] In yet a further aspect, the invention provides a method of
inhibiting an inflammatory response in a mammal by contacting an
endothelial cell with an effective amount of a peptido-mimetic of a
carbohydrate ligand. The ligand is preferably a Lewis antigen. The
specific peptido-mimetics described herein can be used in this
method.
[0026] As another aspect, the invention provides additional methods
of identifying a variety of peptido-mimetics of carbohydrate
ligands. The peptido-mimetics inhibit the normal binding between
the ligand and its natural binding partner. In one embodiment, the
binding partner is an adhesion molecule, such as a selectin. In
another embodiment, carbohydrate ligand is located on the surface
of a tumor cell and is a Lewis antigen. In a further embodiment of
this method, the carbohydrate ligand is located on the surface of a
tumor cell and it normally binds to an adhesion molecule on human
umbilical cord vein endothelial cells (HUVEC). In another
embodiment, the carbohydrate ligand affects capillary tube
formation or angiogenesis. In still another embodiment, the
peptido-mimetic affects adhesion of a selected cell, e.g., a
neutrophil or tumor cell, to an adhesion molecule located on an
endothelial cell. In another embodiment, the carbohydrate ligand
affects neutrophil recruitment. The steps of these methods are
described in detail below.
[0027] In yet another aspect, the invention provides a method of
producing peptido-mimetics of Lewis antigens, particularly
peptido-mimetics not including APWLYAGP [SEQ ID NO: 83]. This
method comprises the steps of screening a random peptide library,
in which the peptides are expressed as fusion proteins on the
surface of bacterial clones, with antibodies specific for the Lewis
antigens and/or an E-selectin immunoglobulin fusion protein; and
selecting clones which bind the antibodies and/or the fusion
protein. The selected clones produce peptido-mimetics of the Lewis
antigens.
[0028] Other aspects and advantages of the present invention are
described further in the following detailed description of the
preferred embodiments thereof.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0029] FIG. 1A is a diagram depicting the reduction of neutrophil
influx upon administration of DLWDWVVGKPAG [SEQ ID NO: 1] mimicking
SA-Le.sup.a carbohydrate in mice with chemically induced
peritonitis. The neutrophil count results were obtained from four
experiments (three mice in each group). Unrelated peptide was
administered in control mice. Statistical analysis of the data
using nonparametric unpaired t-test yielded P values<0.001 for
the data shown in FIG. 1A.
[0030] FIG. 1B is a diagram illustrating the myeloperoxidase
activity in the collected neutrophils of the animals of FIG. 1A.
Statistical analysis of the data using nonparametric unpaired
t-test yielded p values<0.005 for this data.
[0031] FIG. 2A is a bar graph demonstrating the
E-selectin-independent adhesion of human mammary adenocarcinoma
cells SkBr5 to the human endothelioma cell line ECV-304. SkBr5 and
HUVEC cells were allowed to adhere for 15 minutes in the continuous
presence of MAbs and MAb F(ab).sub.2 fragments (marked in the
graph) at 40 pg/ml. Control anti-influenza hemagglutinin MAb H24135
was used at the same concentration. The results represent the
percentage (%) of adherent cells in the presence of specific MAb as
compared to control anti-influenza hemagglutinin MAb H24135.
Results are reported as means.+-.standard error (SE) of five
independent experiments.
[0032] FIG. 2B is a graph depicting the representative data of the
expression of various antigens on the surface of cells in the
absence (-) or presence (+) of the inflammatory cytokine,
Interleukin-1, as determined by MAbs binding in radioimmunoassay
(RIA) with monolayer ECV-304 cells. The presence of the following
antigens was detected using the MAbs indicated:
SA-Le.sup.a(MAbNS19-9), CD63 (MAb C029 and ME491), E-selectin
(specific MAb anti-ELAM-1, British Bio-Technology, UK), and
anti-influenza virus hemagglutinin negative control (MAb
H24135).
[0033] FIG. 3 is a graph illustrating the effect of Trp5
substitution with Phe in peptide DLWDWVVGKPAG [SEQ ID NO: 1]
resulting in peptide DLWDFVVGKPAG [SEQ ID NO: 63] on binding of
SA-Le.sup.a specific MAb NS 19-9. Constant amounts of MAb were
incubated with increasing amounts of peptides and binding of free
antibody to carbohydrate SA-Le.sup.a was measured by enzyme linked
immunosorbent assay. Results show competitive inhibition of MAb
binding to solid phase SA-Le.sup.a polyacrylamide matrix
(SA-Le.sup.a-PAA) by 12-mer peptides DLWDWVVGKPAG (.box-solid.)
[SEQ ID NO: 1] and DLWDFVVGKPAG (.tangle-solidup.) [SEQ ID NO: 63]
with respect to the MAb binding without peptide (100% of binding)
and a negative control unrelated peptide (.smallcircle.).
[0034] FIG. 4 is a circular dichroism (CD) spectra comparing
dodecapeptides DLWDWVVGKPAG (solid line) [SEQ ID NO: 1] and
DLWDFVVGKPAG (---) [SEQ ID NO:63]. The spectra were recorded at
0.51 mg/ml for both peptides.
[0035] FIG. 5 is a graph illustrating the inhibition of lung
experimental metastases with peptide DLWDFVVGKPAG [SEQ ID NO: 63].
Tumor cells were admixed with the specific or unrelated peptide
solution (1 mg per mouse) and animals were inoculated with
1.times.10.sup.5 B16F10FtIII tumor cells in 200 .mu.l volume of PBS
via tail vein. Results are from 4 experiments (5 mice in each
group) are shown. Each dot represents enumerated tumor nodules in
one lung in experimental group of C57B1/6 mice treated with the
peptide (panel B), control group of C57B1/6 mice treated with
unrelated peptide (panel A) and E-selectin knock-out (KO) mice of
C57B1/6 background (panel C). Statistical analysis using a
nonparametric unpaired t test gave a two-tailed p values<0.008
and 0.009 for animals treated with peptide and E-selectin KO,
respectively, as compared to control group. The horizontal bars
represent median values and vertical bars denote standard
deviation.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The invention is based on the discovery that
peptido-mimetics of complex carbohydrate structures block adhesion
of tumor cells and leukocytes to endothelial cells. The production
of small peptide molecules which mimic complex carbohydrate
structures are useful for blocking carbohydrate ligand-cell
adhesion molecule interactions involved in metastasis,
angiogenesis, and inflammatory responses. Thus, these
peptido-mimetics are useful as anti-adhesion therapeutics. The
evaluation of complex carbohydrate ligand-adhesion molecule
interactions at the molecular level enables the design of
peptido-mimetics and possibly other compounds which block these
adhesion interactions thereby disrupting the disease processes
mediated by them. Further, the ability to generate peptide
mimotopes of complex carbohydrate structures enables the use of
recombinant peptide library display technology in discovering novel
blocking agents for important therapeutic targets which bind
carbohydrate ligands. The compositions and methods of this
invention are also useful in designing additional therapeutics to
inhibit the adhesion interactions required for disease
processes.
[0037] As used herein, the term "peptido-mimetic" means a peptide
or polypeptide that mimics complex carbohydrate conformations and
structures. A subset of peptido-mimetics are referred to as
"mimotopes". Mimotopes are small peptido-mimetics, generally from 7
to about 15 amino acids in length which mimic complex carbohydrate
structures, including the carbohydrate ligands for endothelial
adhesion molecules. Mimotypes are also capable of blocking the
ligand-adhesion molecule interaction. Throughout this
specification, these term are used interchangeably. These small
peptides also facilitate the study of complex
structural/conformational relationships between these ligands and
cellular lectins.
[0038] A. Peptides of the Invention
[0039] A composition of this invention comprises at least one
peptido-mimetic of a carbohydrate ligand of an adhesion molecule in
a physiologically acceptable carrier. In one embodiment, the
adhesion molecule is a selectin, such as E-selectin. In another
embodiment, the ligand is a Lewis antigen. Particularly desirable
are peptido-mimetics of the Lewis antigens SA-Le.sup.a, SA-LeX, and
LeY. The illustrative peptides described below may be modified, as
described in more detail herein, to improve anti-adhesion
properties and to increase their stability to degradation in
vivo.
[0040] Desirable compositions of this invention contain one or more
of the following peptido-mimetics which mimic the topography of the
E-selectin ligand: ASAVNLYIPTQE [SEQ ID NO:84], VYLAPGRISRDY [SEQ
ID NO: 85], VYLAPGRFSRDY [SEQ ID NO:86], CTSHWGVLSQRR [SEQ ID
NO:87], RVLSPESYLGPS [SEQ ID NO:88], RVLSPESYLGPA [SEQ ID NO:89],
VGNGVLMGRRG [SEQ ID NO:90], RVLSPESYLGPA [SEQ ID NO:92],
GNCRYIGLRQFG [SEQ ID NO:93], DIRVEPGGGYTH [SEQ ID NO:94],
APIHTYTGRARG [SEQ ID NO:96], and RHTCVRSCGHDR [SEQ ID NO:97].
Similarly, exemplary peptido-mimetics of SA-Lea include, without
limitation, VGIWSVVSEGSR [SEQ ID NO:102], RCSVGVPFTMES [SEQ ID
NO:103], QDGVWEHVLEGG, [SEQ ID NO: 104], DLWDWVVGKPAG [SEQ ID
NO:1], VELSGRGGLCTW [SEQ ID NO:105], VIGAASHDEDVD [SEQ ID NO:106],
TEPVLAEMFMG [SEQ ID NO:107], DKETFELGLFDR [SEQ ID NO:108],
FSGVRGVYESRT [SEQ ID NO:109], PDDAPMHSTRVE [SEQ ID NO:110],
STGLMVDFLEPG [SEQ ID NO: 91], AKTFGLEHGCEA [SEQ ID NO: 95],
GGTVEVWSIKGG [SEQ ID NO:115], DHFSQAGSSNHH [SEQ ID NO:116];
DDPVTPVIDFGK [SEQ ID NO:117], and RDGLIDFVVAGT [SEQ ID NO:118].
[0041] As described in the examples below, families of mimics of
carcinoma-associated antigens that represent SA-Le.sup.a, in
particular, were identified from a combinatorial peptide library
using MAb NS 19-9 specific for this carbohydrate structure. One of
the peptides, DLWDWVVGKPAG [SEQ ID NO:1], was selected that
specifically competes for binding of MAb for SA-Le.sup.a. This
peptide displays an ability to partially inhibit neutrophil
recruitment in an acute inflammation model in vivo. As described
below, this peptide was analyzed by systematic amino acid
replacements to identify optimal conformationally stabilized
SA-Le.sup.a mimics with higher affinity using a solid phase peptide
array library. Comparison of signal intensities revealed
significant differences in MAb binding as a result of
substitutions, in particular at the N-terminus. Substitution
analysis allowed for delineation of key residues that were
sensitive to replacement. MAb NS 19-9 discriminated against
multiple amino acid substitutions affecting its recognition.
Specific residues within this peptide were identified that may
contribute to the mimicry of carbohydrate structure by the
peptide.
[0042] On the other hand MAb NS 19-9 could tolerate replacement of
the lead peptide sequence by a variety of amino acids. These
substitutions did not abrogate binding, suggesting that they did
not affect the structural specificity required for MAb recognition.
Different amino acids in themselves can act as structural mimics
within an identified peptide and bind through non-specific
interactions. The different consensus sequences among the families
of peptides identified with the same MAb from the random peptide
library or sequences without an obvious consensus characterized in
previous studies support the notion that indeed different amino
acid residues provide structural similarity. Alternatively,
different consensus sequences mimic different topographies of the
carbohydrate epitope recognized by the antibody.
[0043] The identification of several cross-reactive peptides
displaying higher NS 19-9 binding further delineate specific
residues that may improve upon peptide mimicry of the carbohydrate
structure. Several substitutions within the C-terminus, in
particular with amino acids containing carboxyl groups that
increased MAb binding, were identified suggesting an important role
of polar interactions in binding affinity. The strongest signal
however resulted from the single amino acid substitution of Phe for
the Trp at position 5, creating the SA-Le.sup.a peptidomimetic
DLWDFVVGKPAG [SEQ ID NO: 63].
[0044] The present invention demonstrates that peptides mimicking
SA-Le.sup.a are able to bind surfaces of proteins specific for
these structures and thus they can act as antagonists for the
recognition of the cognate carbohydrate antigen or ligand. In vivo
oligosaccharide dependent reduction of metastasis formation may be
a function of the interruption of these interactions. Antagonists
interfere with the metastatic process at the level of cellular
adhesion and blood vessel formation since E-selectin and SA-LeX are
expressed on actively growing blood vessels. Alternatively, peptide
mimics act on signal transduction events mediated by selectins and
their ligands and the in vivo consequences of selectin-ligand
antagonism to the complex signal transduction processes associated
with selectin-dependent cell adhesion.
[0045] Certain exemplary peptidomimetics of LeY include the
peptides TKRPDLIVDPIP [SEQ ID NO:98], DEVRPDLISTEE [SEQ ID NO: 99],
NLRPKYIXLDAD [SEQ ID NO:100], and TLIAFADLVDVI [SEQ ID NO:101].
[0046] Peptides mimicking carbohydrate antigens retain
conformational properties of cognate carbohydrate structures and
can block recognition of cells expressing such ligands in vivo.
Thereby, they can mediate anti-metastatic functions as demonstrated
by blocking of experimental metastasis. Thus, the peptides
identified above which mimic the topography of the E-selectin
ligand and/or the other Lewis antigens can be employed in
pharmaceutical compositions. Such peptido-mimetics are useful in
pharmaceutical compositions directed toward the treatment of
cancer, and the prevention and/or inhibition of metastases (see,
e.g., Example 14). Such peptides may also be used in pharmaceutical
treatments to block selectin-dependent interactions, e.g.
diminishing the inflammatory response (see, e.g., Example 8).
[0047] The above peptido-mimetics and others which may be
identified by use of the assays referred to below may be further
modified to increase or enhance the stability of such peptides for
in vivo use or to enhance the binding abilities of these
peptido-mimetics. Peptido-mimetics according to this invention
includes peptido-mimetics such as those identified specifically
above, which are modified to increase their stability in vivo.
[0048] For example, the incorporation of unnatural amino acids
(e.g., D configuration amino acids) at the N or C termini, the most
frequent peptide degradation sites, may improve the pharmacological
properties of the peptides, without loss of the binding efficacy.
Another modification involves incorporating onto the N-terminus of
the peptide a moiety which can provide a net positive charge on the
N-terminus of the peptide. Such moieties can include straight
chain, branched, cyclic or heterocyclic alkyl groups, straight
chain, branched, cyclic or heterocyclic alkanoyl groups, a
positively charged reporter group; and/or one or up to 15
additional amino acids independently selected from L-configuration
or D-configuration amino acids, optionally substituted with a
straight chain, branched, cyclic or heterocyclic alkyl group, a
straight chain, branched, cyclic or heterocyclic alkanoyl group, or
a reporter group. The amino acids may be naturally occurring amino
acids or unnatural amino acids, such as D configuration amino
acids, or amino acids which have been cyclized by the insertion of
modifying sugars, imide groups and the like. One specific
embodiment of an N-terminal moiety is the positively charged
1-aminocyclo-hexane carboxylic acid. Another is a single positively
charged amino acid such as L-Val- or D-Val-. In still other
embodiments, such additional amino acids are modified by an acetyl
group, providing that a net positive charge results. In still other
embodiments of modified peptides, the N-terminal group is a
positively charged moiety which can function as a reporter group
for detection purposes.
[0049] These peptides may also be modified to cyclize the peptide
by joining the N- and C-termini of the peptide. Additional amino
acids or spacers may be introduced into the peptides also form
spacers, which may be needed to cyclize the peptide by bridging
between the N- and C-termini of the peptide. Spacers are sequences
of greater than 3 amino acids which are interposed between the
normal N-terminus and C-terminus of the modified peptido-mimetic.
These spacers permit linkage therebetween without imposing any
adverse restraint upon the molecular structure. Spacers may also
contain restriction endonuclease cleavage sites to enable
separation of the sequences, where desired. Desirably, spacers
duplicate a portion of the peptide. Suitable spacers or linkers are
known and may be readily designed and selected by one of skill in
the art.
[0050] Similarly, the C terminus of a peptido-mimetic of this
invention may be a free hydroxyl, an amide, an imide, a sugar, or a
sequence of one or up to about 15 additional amino acids,
optionally substituted with a free hydroxyl, an amide, an imide or
a sugar. The C-termini may also be modified in the same manner as
the modified N-termini, described above. As one specific
embodiment, the C terminus of the a peptido-mimetic may be modified
with 2-acetamido-2-deoxyglucose. Another specific embodiment is the
addition to the C-terminal amino acid of the peptido-mimetic of
triacetyl 2-acetamido-2-deoxyglucose. In another embodiment of a
modified peptides of this invention, the C-terminal amino acid is
modified by the addition of a .beta.-acetyl-2,3-diamino propionic
acid group.
[0051] Still another modification of the peptides of this invention
includes the addition into the peptides of two adjacent amino acids
which are resistant to cleavage by endopeptidases. Still another
modification involves replacing conventional inter-residue amide
bonds by bonds resistant to proteases, such as, a thioamide bond or
a reduced amide bond. Such modifications of the bonds between amino
acids may change the conformation of the peptide. A variety of
methods for producing non-natural amino acids are known and insert
modified inter-residue bonds, and/or cyclize the peptides may be
selected by one of skill in the art. Other backbone-modifications
of these peptides are also anticipated to improve proteolytic
stability and yield analogs with slightly modified activity
spectrum. The specific peptide described above may also be readily
modified by replacing one or more amino acids with another, by
substituting individual amino acids with chemical components or
labels or by other peptide modification methods known to those of
skill in the art. Such modifications may be made to enhance
stability of the peptide in a pharmaceutical composition or to
enhance the binding ability of the peptido-mimetic to the desired
ligand [See, e.g., Example 13]. Such modifications include peptide
modifications disclosed in J. E. Oh et al, J. Peptide Res.,
54:129-136 (1999), incorporated herein by reference.
[0052] Peptide multivalency is another modification that should
result in higher affinity binding as compared to low affinity
interaction with monovalent peptides. Multivalency should increase
peptide/EC interaction and lower concentration of multiple antigen
peptides needed to block it. The generation of high affinity
ligands might require formation of a "clustered oligosaccharide
patch" or a "clustered anionic patch" or a combination of
polypeptide backbone and modifications such as sulfation. Thus,
these peptides may be employed individually, or presented in a
multivalent form, such as a multiple antigenic peptide ("MAP", also
referred to as an octameric lysine core peptide) construct. Such a
construct may be designed employing the MAP system described by
Tam, Proc. Natl. Acad. Sci. USA, 85:5409-5413 (1988); D. Posnett et
al., J. Biol. Chem., 263(4):1719-1725 (1988); J. Tam, Vaccine
Research and Developments, Vol. 1, ed. W. Koff and H. Six, pp.
51-87 (Marcel Deblau, Inc., New York 1992)]. In any multivalent
form, each peptide may be optionally separated by amino acid
spacers, which are defined above.
[0053] One may generate homologous amino acid or polynucleotide
sequences which share significant homology with the
above-identified peptido-mimetics or with other peptido-mimetics
identified by the methods of this invention. Sequence homology for
polypeptides, which is also referred to as sequence identity, is
typically measured using sequence analysis software. See, e.g., the
Sequence Analysis Software Package of the Genetics Computer Group
(GCG), University of Wisconsin Biotechnology Center, 910 University
Avenue, Madison, Wis. 53705. Protein analysis software matches
similar sequences using a measure of homology assigned to various
substitutions, deletions and other modifications, including
conservative amino acid substitutions. For instance, GCG contains
programs such as "Gap" and "Bestfit" which can be used with default
parameters to determine sequence homology or sequence identity
between closely related polypeptides, such as homologous
polypeptides from different species of organisms or between a wild
type protein and a mutein thereof. A preferred algorithm when
comparing a specific polypeptide sequence to a database containing
a large number of sequences from different organisms is the
computer program BLAST, especially blastp or tblastn (Altschul et
al., 1997, herein incorporated by reference). Database searching
using amino acid sequences can be measured by algorithms other than
blastp known in the art, e.g., Fasta, a program in GCG Version 6.1.
The term "substantial homology" or "substantial similarity," when
referring to a nucleic acid or fragment thereof, indicates that,
when optimally aligned with appropriate nucleotide insertions or
deletions with another nucleic acid (or its complementary strand),
there is nucleotide sequence identity in at least about 60% of the
nucleotide bases, usually at least about 70%, more usually at least
about 80%, preferably at least about 90%, and more preferably at
least about 95-98% of the nucleotide bases, as measured by any
well-known algorithm of sequence identity, such as Fasta, as
discussed above.
[0054] B. Methods of Preparing Peptides of this Invention
[0055] The peptides of the invention may be prepared conventionally
by resort to known chemical synthesis techniques, e.g., solid-phase
chemical synthesis, such as described by Merrifield, J. Amer. Chem.
Soc., 85:2149-2154 (1963), and J. Stuart and J. Young, Solid Phase
Peptide Synthelia, Pierce Chemical Company, Rockford, Ill. (1984),
or detailed in the examples below. Chemical synthesis methods are
particularly desirable for large-scale production of such peptides.
A variety of methods for producing the above-identified
modifications of the peptides, e.g., non-natural amino acids, are
known and may be selected by one of skill in the art.
[0056] Alternatively, the peptides of this invention may be
prepared by known recombinant DNA techniques by cloning and
expressing within a host microorganism or cell a DNA fragment
carrying a nucleic acid sequence encoding one of the
above-described peptides [See, e.g., Sambrook et al., Molecular
Cloning. A Laboratory Manual., 2d Edit., Cold Spring Harbor
Laboratory, New York (1989)]. Conventional molecular biology
techniques, and site-directed mutagenesis may be employed to
provide desired modified peptide sequences.
[0057] The preparation or synthesis of the peptido-mimetics
disclosed herein is well within the ability of the person having
ordinary skill in the art using available material. The synthetic
methods are not a limitation of this invention.
[0058] C. Pharmaceutical Preparations of this Invention
[0059] The selected peptido-mimetics are desirably formulated into
pharmaceutical compositions suitable for administering to a
mammalian subject, preferably a human. Such formulations comprise
one or more of the peptides identified above, combined with a
pharmaceutically or physiologically acceptable carrier, such as
sterile water or sterile isotonic saline. Such sterile injectable
formulations may be prepared using a non-toxic
parenterally-acceptable diluent or solvent, such as water or
1,3-butanediol, for example. Other acceptable diluents and solvents
include, but are not limited to, Ringer's solution, isotonic sodium
chloride solution, and fixed oils such as synthetic mono- or
di-glycerides. Such formulations may further comprise one or more
additional ingredients including, but not limited to, suspending,
stabilizing, or dispersing agents. In one embodiment of a
formulation for parenteral administration, the peptide is provided
in dry (i.e., powder or granular) form for reconstitution with a
suitable vehicle (e.g., sterile pyrogen-free water) prior to
parenteral administration of the reconstituted composition.
[0060] Other parenterally-administrable formulations which are
useful include those which comprise the peptide in microcrystalline
form, in a liposomal preparation, or as a component of a
biodegradable polymer systems. Compositions for sustained release
or implantation may comprise pharmaceutically acceptable polymeric
or hydrophobic materials such as an emulsion, an ion exchange
resin, a sparingly soluble polymer, or a sparingly soluble salt.
The peptides of the present invention may be administered by oral,
intraperitoneal, intramuscular and other conventional routes of
pharmaceutical administration.
[0061] Pharmaceutical compositions of the present invention may be
administered either as individual therapeutic/prophylactic agents
or in combination with other agents. They can be administered
alone, but are generally administered with a pharmaceutical carrier
selected on the basis of the chosen route of administration and
standard pharmaceutical practice. One of skill in the
pharmaceutical arts may readily design acceptable pharmaceutical
formations for delivery of the peptido-mimetics of this invention
with recourse to well-known information on pharmacology and with
use of commercially available materials, in view of the teachings
herein.
[0062] The "effective amount" of the peptide of the invention
present in each effective dose is selected with regard to
consideration of the condition being treated, the patient's age,
weight, sex, general physical condition and the like. The amount of
active component required to induce a desired effect without
significant adverse side effects varies depending upon the
pharmaceutical composition employed and the optional presence of
other components. Generally, for the compositions containing
peptide, fusion protein, or MAP, each daily dose will comprise
between about 50 .mu.g to about 2 mg of the peptide per mL of a
sterile solution per kg body weight. Another desired daily dosage
of active ingredient can be about 0.0001 to 1 grams per kilogram of
body weight. Another desired dosage range can be about 0.1 to 100
milligrams per kilogram of body weight. Still another dosage is in
the range of 0.5 to 50 milligrams per kilogram of body weight, and
preferably 1 to 10 milligrams per kilogram per day. Other dosage
ranges may also be contemplated by one of skill in the art.
[0063] Initial doses may be optionally followed by repeated
administration for a duration selected by the attending physician.
Dosage frequency may also depend upon the factors identified above,
and may range from daily dosages to once or twice a week i.v. or
i.m., for a duration of about 6 weeks. The compositions of the
present invention can also be employed in treatments for chronic
inflammatory ailments or for long term treatment of cancer patients
for a period to be selected by the physician.
[0064] D. Methods of the Invention
[0065] The peptides described above and the pharmaceutical
compositions containing them may be employed in therapeutic methods
or in in vitro methods for the development of other pharmaceutical
agents. Each of the methods described employs the peptido-mimetic
to, interfere with, or inhibit, the binding of a desired
carbohydrate antigen with its normal receptor or adhesion molecule,
so as to prevent or inhibit an undesired therapeutic result. It is
anticipated that the therapeutic methods may be performed ex vivo
or in vivo.
[0066] Thus, one embodiment of the present invention provides a
method of modulating binding of an adhesion molecule to a
carbohydrate ligand. According to this method, an adhesion
molecule, such as a selectin, is contacted with a peptido-mimetic
which mimics the topography of the carbohydrate ligand. Binding of
the adhesion molecule by the peptido-mimetic in place of the normal
ligand thus modulates, or reduces the amount of binding between the
adhesion molecule and its carbohydrate ligand and thereby
suppresses or reduces the normal effect of such binding. Modulating
binding of an adhesion molecule, (e.g., E-selectin) to a
carbohydrate ligand refers to any reduction or increase in binding
of the selectin to the carbohydrate ligand wherein the difference
in binding results in a desired therapeutic effect. It is desirable
for this method that the carbohydrate ligands and adhesion
molecules are present on the surface of the cells in such a manner
as to allow binding of the adhesion molecule to the ligand and/or
to the peptido-mimetic of the ligand.
[0067] The peptido-mimetics of this invention may be used in
treatments for cancer. For example, one embodiment is a method of
modulating adhesion of a tumor cell to an adhesion molecule on an
endothelial cell, e.g., a selectin. As above, the method comprises
contacting the tumor cell with an effective amount of a
peptido-mimetic (or a pharmaceutical composition containing the
effective amount) of a carbohydrate ligand. By interfering with the
binding between the tumor cell and its naturally occurring selected
binding partner, this method provides a way of inhibiting or
otherwise modulating the biological activity mediated by the
natural adhesion of the tumor cell to the endothelial cell. This
method provides a therapy for cancer patients. The peptido-mimetics
described herein may be used in this method.
[0068] In another method of treating cancer in a mammal, an
effective amount of a peptido-mimetic of a carbohydrate ligand is
administered to the mammal. Such treatment also reduces the
adhesion of tumor cells to endothelial cells in the mammal, thereby
reducing metastasis of the cancer. An "adherence modulating dose"
refers to any amount of a peptido-mimetic, or any combination of
peptido-mimetics, of a carbohydrate ligand which affects the
adherence of a cell bearing the carbohydrate ligand to an adhesion
molecule on endothelial cells. By "adherence reducing dose" is any
amount of a peptido-mimetic of a carbohydrate ligand which, when
administered to a mammal, reduces the binding of an adhesion
molecule to a carbohydrate ligand. This dose reduces the adhesion
of certain cells which present carbohydrate ligands on their
surfaces (e.g., tumor cells and neutrophils) to endothelial cells,
compared with the adhesion of those same cells to endothelial cells
in the mammal prior to the administration of the
peptido-mimetic.
[0069] Preferred embodiments of this method are illustrated in the
examples below which demonstrate the use of a peptidomimic of a
carbohydrate tumor associated antigen to reduce the number of
experimental metastasis in vivo. SA-Le.sup.a provides the critical
carbohydrate ligand for the adhesion molecule, E-selectin, that
facilitates the initial steps involved in a cascade of tumor
cell-endothelial interactions leading to metastatic spread. The use
of the peptides of this invention which mimic the E-selectin
ligand, SA-Le.sup.a, inhibit metastasis of tumor cells expressing
this structure. The abrogated tumor growth in E-selectin knock-out
(KO) mice are demonstrated in the examples below, which provide
evidence that SA-Le.sup.a and E-selectin are important in
metastasis formation.
[0070] Briefly described, B16F10FTIII melanoma cells employed in a
metastasis model express SA-Le.sup.a carbohydrate antigen and form
lung tumors after i.v. inoculation through the tail vein. Tumor
colonization appears to be highly E-selectin dependent, as the
incidence of metastasis was completely abrogated in E-selectin KO
mice. The initial stages required for tumor colonization are
dependent not only on adhesion molecules inducible on endothelial
cells, but also on the ligands expressed on tumor cells. The
expression of E-selectin ligand, SA-Le.sup.a, is likely to
contribute to the metastasis of cells expressing this structure.
The 50% inhibition of tumor metastasis achieved upon administration
of the peptide antagonist of SA-Le.sup.a expressed on the B16F10
tumor cell surface can be explained by interruption of the initial
steps of cascade of inhibitory events initiated by tumor cell
adhesion with this conformational equivalent of the SA-Le.sup.a
structure. These findings suggest that expression of SA-Le.sup.a
leads to tumor specific colonization in vivo.
[0071] Gross histological examination of the lungs (not shown) did
not reveal significant differences in the appearance of the small
spherical tumor nodules between animals treated with peptide and
the control group. This observation suggests that the peptide
treatment may indeed block the initial stages of adhesion to lung
endothelium required for initiation of tumor cell migration into
the subendothelial space, resulting in reduced number of nodules
but not tumor growth after the micrometastases are established.
Although the reduced number of metastases was the prevalent effect
of peptide treatment as compared with the control group, large lung
tumor masses were observed in untreated animals.
[0072] The examples below provide evidence that members of all cell
adhesion molecule families and carbohydrate structures, SA-Le.sup.a
and SA-LeX, expression are associated with capillary tube formation
and neovascularization necessary to maintain metastasis. This
indicates that the anti-angiogenic mechanism of tumor growth
inhibition in peptide treated animals also takes place. Similarly,
the complete inability of tumor cell colonization in the lungs of
E-selectin KO animals may result from the lack of initial adhesion
steps mediated by this adhesion molecule, as well as impaired
angiogenesis, in which E-selectin is involved. E-selectin is one of
the few adhesion molecules truly restricted to activated
endothelium, thus E-selectin may be used to selectively target
activated and/or proliferating endothelium in vivo not only by
blocking adhesion of tumor cells to EC, but also by halting the
neovascularization processes. Consequently, proliferating
microvascular endothelium presents an unique and universal target
for anti-cancer therapy.
[0073] In still another therapeutic embodiment of this invention, a
method for inhibiting an inflammatory response in a mammal is
provided. This method involves contacting an endothelial cell with
an effective amount of a peptido-mimetic of a carbohydrate ligand,
thereby modulating adherence of neutrophils to endothelial cells.
By "modulating neutrophil adherence," as the term is used herein,
is meant any difference in the adherence of a neutrophil to an
endothelial cell in the presence of a peptido-mimetic of a
carbohydrate ligand compared with the adherence of a neutrophil to
an endothelial cell in the absence of the peptido-mimetic of a
carbohydrate ligand. By "inhibiting an inflammatory response," as
the term is used herein, is meant decreasing the inflammatory
response in a mammal. Inflammatory response refers to the
physiological response in an animal which includes, but is not
limited to, recruitment of neutrophils to the site of inflammation
and/or the adherence of neutrophils to endothelial cells. In an
exemplary embodiment, one inflammatory response which may be
treated by the present invention is peritonitis (i.e., an
irritation of the peritoneum). A substantial reduction in pathology
of inflammation is achieved upon 30% reduction of neutrophil
recruitment in the inflammatory model illustrated below. However,
this aspect of the invention is not limited to the manner in which
inflammation is caused or the tissue in which inflammation is
present and/or treated. The peptido-mimetics described herein are
particularly useful in this aspect of the invention.
[0074] Still other conditions mediated by interaction between
adhesion molecules and their carbohydrate ligands may be treated by
the use of peptidomimetics of either the antigen or the ligand in a
manner similar to that disclosed above.
[0075] E. Cells of the Invention
[0076] In a further aspect, the invention provides a cell (e.g.,
yeast, bacteria, phage, or mammalian) which is constructed to
display an exogenous peptido-mimetic of a Lewis antigen or a
selectin on the surface of the cell. More particularly, this
invention is directed to cells displaying the specific peptides
disclosed herein, either alone or as fusion proteins, on the cell
surface. One manner in which this may be obtained is described in
Example 1, through the use of a random peptide library. Analogous
cells, other than E. coli may be produced by similar methods. See
the cells described in Examples 10 and 14, which also provide
instruction on the provision of cells developed to express
peptido-mimetics.
[0077] F. Use of Peptides and Methods for Identification of Other
Useful Pharmaceutical Peptides or Compounds
[0078] Use of additional combinatorial synthetic chemistry
technologies will allow for improved antagonists of tumor cell
adhesion, leading to the further development of agents with greatly
enhanced therapeutic potential. Using a combinatorial approach
based on functional equivalence of chemically dissimilar molecules
sharing common surface topology instead of derivatized parental
structures is effective in developing antagonists of
physiologically important molecular interactions.
[0079] As one embodiment, the invention provides a method for
identifying an additional peptido-mimetic of a carbohydrate ligand
which affects the binding of the carbohydrate ligand to a binding
partner. The method comprises the steps of: (a) contacting the
binding partner with a peptido-mimetic and (b) comparing the
binding of the binding partner of (a) to the carbohydrate ligand
with the binding of the same binding partner which is not contacted
with the peptido-mimetic to the carbohydrate ligand. The level of
binding of the binding partner contacted with the peptido-mimetic
to the carbohydrate ligand is compared with the level of binding of
the binding partner not contacted with the peptido-mimetic with the
same carbohydrate ligand. Any significant change in these two
levels of binding is an indication that the peptido-mimetic affects
the binding of the carbohydrate ligand to the binding partner. In
one embodiment the binding partner is an adhesion molecule located
on an endothelial cell.
[0080] In another embodiment, the carbohydrate ligand is located on
the surface of a tumor cell and the adhesion molecule is a
selectin, e.g., E-selectin. Any change in the level of binding of
E-selectin or any other adhesion molecule contacted with the
peptido-mimetic to the carbohydrate ligand compared with the level
of binding of E-selectin or any other adhesion molecule not
contacted with the peptido-mimetic with the same carbohydrate
ligand is an indication that the peptido-mimetic affects the
binding of the tumor cell to the adhesion molecule.
[0081] Another method of identifying a peptido-mimetic of a
carbohydrate ligand which affects angiogenesis is provided by this
invention. The method involves contacting a primary capillary
endothelial cell with a peptido-mimetic of a carbohydrate ligand,
e.g., a Lewis antigen. Any difference between capillary tube
formation observed by the cell contacted with the peptido-mimetic
is compared to the capillary tube formation of the cell which is
not contacted with the peptido-mimetic. Any significant change in
capillary tube formation is an indication that the peptido-mimetic
affects angiogenesis. A number of assays exist in the art for the
determination of effects on angiogenesis, including microscope
examination of capillary tube formation in tissue [See, e.g.,
Nguyen et al, cited above]. It should be understood that this same
method may be employed using other means of determining changes in
angiogenesis caused by a similar use of peptido-mimetics.
[0082] Yet another method of identifying a peptido-mimetic which
affects adhesion of a selected cell to an endothelial cell involves
contacting an endothelial cell with a peptido-mimetic of a
carbohydrate ligand located on the endothelial cell. The binding of
the endothelial cell contacted with the peptido-mimetic to the
ligand is compared to the binding of an endothelial cell which has
not been contacted with the peptido-mimetic to the ligand. Any
change in the levels of binding is an indication that the
peptido-mimetic affects binding of the selected cell to the
endothelial cell. In one embodiment, the selected cell is a tumor
cell. In another embodiment, the selected cell is a neutrophil. In
still another embodiment, the carbohydrate ligand is located on the
surface of a tumor cell and the adhesion molecule of the ligand is
located on a human umbilical cord vein endothelial cell (HUVEC).
Any change in binding caused by the peptido-mimetic is an
indication that the peptido-mimetic affects the binding of the
tumor cell to HUVEC. This method is also not limited to the
identity of the selected cells, nor to the assays used to detect
differences in binding.
[0083] The invention further provides a method of identifying a
peptido-mimetic of a carbohydrate ligand which is involved in
inflammatory processes. That method involves administering an
inflammation-inducing substance (e.g., a peritonitis-inducing
substance) into a suitable tissue or organ of a mammal. Thereafter,
an effective amount of a peptido-mimetic of the carbohydrate ligand
is administered at the site or area of the inflammation. A control
mammal having a similar inflammation at the same site is not
treated. Any effect on inflammation which is observed in the
treated mammal vs. the control, such as neutrophil influx into the
site of inflammation, is an indication that the peptido-mimetic
affects inflammation. In one embodiment, the inflammation inducing
substance causes peritonitis and the site of inflammation is the
peritoneam. The levels of neutrophil influx in the peritoneum of
the treated mammal are observed to be lower that the levels of
neutrophil influx in the peritoneum of the control. This result is
an indication that the peptido-mimetic inhibits an inflammatory
response. This method is not limited by the method of inducing
inflammation or the site of inflammation or the indication of
inflammation that is assessed. For example, myeloperoxidase
activity could be assayed in place of neutrophil recruitment. The
site and method of administration of the peptido-mimetic and the
assayed indication may be selected by one of skill in the art.
[0084] In general, the present invention provides a method of
producing peptido-mimetics of Lewis antigens, particularly those
not including APWLYAGP [SEQ ID NO: 83]. This method comprises the
step of: (a) screening a random peptide library, the peptides
expressed as fusion proteins on the surface of bacterial clones,
with antibodies specific for the Lewis antigens and/or with
adhesion molecule constructs, e.g., molecules expressed as fusion
proteins. One such example is the E-selectin immunoglobulin fusion
protein used in Example 2. Other such fusion molecules may be used
similarly in this step, and (b) The clones which bind the
antibodies or adhesion molecule constructs. The clones which bind
the antibodies or adhesion molecule constructs produce
peptido-mimetics of the Lewis antigens. This method is exemplified
by Examples 1 and 2 below. Modifications of these methods are
contemplated in the performance of this method. Such modifications
are readily made by one of skill in the art.
[0085] The peptides and polynucleotide sequences of the present
invention may also be used in the screening and development of
chemical compounds, small molecules or proteins which mimic the
structure or activity of the carbohydrate ligands, and thus have
utility as therapeutic drugs for the treatment of cancer and
inflammation. Competition assays, such as the ELISA described in
Example 3, may be employed and readily designed for such use.
Additionally, a compound which has structural similarity to the
peptido-mimetic, or the binding of the peptide to the ligand may
also be computationally evaluated and designed by means of a series
of steps in which chemical entities or fragments are screened and
selected for their ability to associate with the peptides of this
invention.
[0086] Specialized computer programs that may also assist in the
process of selecting fragments or chemical entities similar to the
peptides, or entities which can interact with the peptides and thus
mimic the receptor, include the GRID program available from Oxford
University, Oxford, UK. [P. J. Goodford, "A Computational Procedure
for Determining Energetically Favorable Binding Sites on
Biologically Important Macromolecules", J. Med. Chem., 28:849-857
(1985)]; the MCSS program available from Molecular Simulations,
Burlington, Mass. [A. Miranker and M. Karplus, "Functionality Maps
of Binding Sites: A Multiple Copy Simultaneous Search Method",
Proteins: Structure, Function and Genetics, 11:29-34 (1991)]; the
AUTODOCK program available from Scripps Research Institute, La
Jolla, Calif. [D. S. Goodsell and A. J. Olsen, "Automated Docking
of Substrates to Proteins by Simulated Annealing", Proteins:
Structure, Function, and Genetics, 8:195-202 (1990)]; and the DOCK
program available from University of California, San Francisco,
Calif. [I. D. Kuntz et al, "A Geometric Approach to
Macromolecule-Ligand Interactions", J. Mol. Biol., 161:269-288
(1982)], software such as Quanta and Sybyl, followed by energy
minimization and molecular dynamics with standard molecular
mechanics force fields, such as CHARMM and AMBER. Additional
commercially available computer databases for small molecular
compounds include Cambridge Structural Database, Fine Chemical
Database, and CONCORD database [for a review see Rusinko, A., Chem.
Des. Auto. News, 8:44-47 (1993)].
[0087] Once suitable chemical entities or fragments have been
selected, they can be assembled into a single compound. Assembly
may proceed by visual inspection of the relationship of the
fragments to each other on the three-dimensional image displayed on
a computer screen in relation to the structure of the
peptido-mimetic of this invention. Useful programs to aid one of
skill in the art in connecting the individual chemical entities or
fragments include the CAVEAT program [P. A. Bartlett et al,
"CAVEAT: A Program to Facilitate the Structure-Derived Design of
Biologically Active Molecules", in Molecular Recognition in
Chemical and Biological Problems", Special Pub., Royal Chem. Soc.
78, pp. 182-196 (1989)], which is available from the University of
California, Berkeley, Calif.; 3D Database systems such as MACCS-3D
database (MDL Information Systems, San Leandro, Calif.) [see, e.g.,
Y. C. Martin, "3D Database Searching in Drug Design", J. Med.
Chem., 35:2145-2154 (1992)]; and the HOOK program, available from
Molecular Simulations, Burlington, Mass.
[0088] Compounds that mimic a peptide of this invention or a ligand
of the peptides may be designed as a whole or "de novo" using
either an empty active site or optionally including some portion(s)
of a known ligand(s). Suitable methods describing such methods
include the LUDI program [H.-J. Bohm, "The Computer Program LUDI: A
New Method for the De Novo Design of Enzyme Inhibitors", J. Comp.
Aid. Molec. Design, 6:61-78 (1992)], available from Biosym
Technologies, San Diego, Calif.; the LEGEND program [Y. Nishibata
and A. Itai, Tetrahedron, 47:8985 (1991)], available from Molecular
Simulations, Burlington, Mass.; and the LeapFrog program, available
from Tripos Associates, St. Louis, Mo. Other molecular modelling
techniques may also be employed in accordance with this invention.
See, e.g., N. C. Cohen et al, "Molecular Modeling Software and
Methods for Medicinal Chemistry", J. Med. Chem., 33:883-894 (1990).
See also, M. A. Navia and M. A. Murcko, "The Use of Structural
Information in Drug Design", Current Opinions in Structural
Biology, 2:202-210 (1992). For example, where the structures of
test compounds are known, a model of the test compound may be
superimposed over the model of the peptide of the invention.
Numerous methods and techniques are known in the art for performing
this step, any of which may be used. See, e.g., P. S. Farmer, Drug
Design, Ariens, E. J., ed., Vol. 10, pp 119-143 (Academic Press,
New York, 1980); U.S. Pat. No. 5,331,573; U.S. Pat. No. 5,500,807;
C. Verlinde, Structure, 2:577-587 (1994); and I. D. Kuntz, Science,
257:1078-1082 (1992). The model building techniques and computer
evaluation systems described herein are not a limitation on the
present invention.
[0089] Thus, using these computer evaluation systems, a large
number of compounds may be quickly and easily examined and
expensive and lengthy biochemical testing avoided. Moreover, the
need for actual syntheses of many compounds is effectively
eliminated. Once identified by the modelling techniques, the
proposed "new antibacterial" compound may be tested for bioactivity
using standard techniques, such as the assays of the examples
below.
[0090] The invention is further described in detail by reference to
the following, experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
EXAMPLE 1
Screening of a Random Peptide Library
[0091] To develop novel molecules to inhibit the adhesion of human
adenocarcinoma cells to EC and, ultimately, to inhibit metastasis
in vivo, peptides were derived from a 12-mer random peptide
library. A diverse library of random dodecapeptides, displayed as
flagellin-thioredoxin fusion proteins (FLITRX) on the surfaces of
E. Coli bacterial cells, was obtained from Invitrogen (Carlsbad,
Calif.) [LaVallie et al., 1993, Bio/Technology, 11:187-193]. This
library enables efficient isolation of bacteria displaying peptides
with affinity to immobilized antibodies or to other binding
proteins. The use of this library offered advantages over phage
display in which the level of expression of phage coat protein
genes is low and the selected peptides are usually unconstrained
molecules with many degrees of conformational freedom. Moreover, no
phage infection or isolation steps are necessary using, the FLITRX
fusion protein system. Such highly diverse peptide libraries offer
many distinct advantages over difficult chemical or enzymatic
synthesis of complex carbohydrates, providing for the inexpensive
and rapid identification and optimization of novel ligands.
[0092] Specifically, an aliquot of the FLITRX library containing at
least 2.times.10.sup.10 cells to ensure full representation of
peptides, was grown to saturation for 15 hours in IMC/amp100 medium
(M9 medium containing 1 mM MgCl.sub.2 supplemented with 0.5%
glucose, 0.2% casamino acids and 100 .mu.g/ml ampicillin). The
expression of thioredoxin with incorporated 12-mer peptide sequence
was induced by further 6 hours of incubation of a culture of
10.sup.10 cells, diluted 1:25 with fresh IMC/amp 100 medium
containing 100 .mu.g/ml tryptophan. The induced bacteria were
panned on a MAb-coated tissue culture (20 .mu.g/ml) plate followed
by blocking with 1% nonfat milk containing 150 mM NaCl and 1%
.alpha.-methyl mannoside for 1 hour. The bound cells were washed
gently. Eluted cells were collected by rinsing the plate with 10 ml
of fresh IMC/amp 100 medium and then incubated at 25.degree. C.
until reaching saturation. The entire selection process was
repeated four more times. Colonies of isolated bacteria were grown
on ampicillin-containing plates. Individual colonies were isolated
and grown as a small scale culture (2 ml) in IMC/amp 100 medium
with tryptophan for 6 hours.
EXAMPLE 2
Protein Expression, Isolation and Sequencing
[0093] In the final selection cycle described above, the clones
were tested for protein expression using sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by
Western blotting [Towbin et al., 1979, Proc. Natl. Acad. Sci. USA
76:4350-4354] as follows. Specifically, each bacterial pellet was
collected and dissolved directly in SDS-PAGE sample buffer
containing SDS and P-mercaptoethanol. Proteins were separated using
12% polyacrylamide gel and transferred into nitrocellulose filters
according to standard procedures. Expression of the FLITRX fusion
protein-containing peptide sequences mimicking carbohydrate
structures expressed by the bacterial clones was detected using one
of the following monoclonal antibodies (10 pg/ml):
[0094] (1) MAb BR15-6A, which is specific for the carbohydrate
ligand LeY [Rodeck et al., 1987, Hybridoma 6:389-401],
[0095] (2) MAb NS19-9, which is specific for the carbohydrate
ligand SA-Le.sup.a [Magnani et al., 1981, Science 212:55-56;
Bechtel et al, 1990, J. Biol. Chem., 265:2028-2037], and
[0096] (3) MAb FH6, which is specific for the carbohydrate ligand
SA-LeX [Fukushi et al., 1985, Cancer Res. 45:3711-3717].
[0097] E-selectin-immunoglobulin chimeric protein IgG
(E-selectin-IgG; Centocor, Inc., Malvern, Pa.) [Geng et al., 1992,
J. Biol. Chem. 267:19846-19853] was also used.
[0098] The expression and the location of thioredoxin on the
nitrocellulose filter was confirmed after incubation of parallel
filters with trxA-specific MAb (anti-Thio.TM.) (Invitrogen,
Carlsbad, Calif.). Following antibody binding, the filters were
washed, incubated with horseradish peroxidase (HRP)-conjugated goat
anti-mouse antibody, and then washed again. Finally, the filters
were exposed to a freshly prepared mixture of a solution comprising
luminol and oxidizing solution (1:1) for 1 minute, air-dried, and
exposed on Reflection.TM. (NEN.TM. Life Sciences Products, Boston,
Mass.). Only clones which displayed a strong signal on the Western
blot with the respective MAb were selected for sequence
identification. See Tables 1-3, discussed in detail in the Examples
below.
[0099] The DNA of each clone was sequenced as follows. DNA was
isolated from the selected bacteria using standard isopropanol
precipitation in the presence of potassium acetate using a
mini-column DNA purification column (Qiagen, Chatsworth, Calif.).
The nucleotide sequences of the DNA from the selected bacteria were
determined by the dideoxynucleotide chain termination method using
specific primers using standard methods [see, e.g., Sambrook et al.
1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, New York; Ausubel et al. 1997, Current Protocols in
Molecular Biology, Green & Wiley, New York]. The peptides
encoded by the sequenced DNA were either purchased commercially
(Research Genetics, Inc., Huntsville, Ala.) and/or were chemically
synthesized using FMOC-based, solid phase chemistry and were
purified to homogeneity on a C-18 reverse-phase HPLC column. The
structures were confirmed by fast-atom bombardment mass
spectrometry.
[0100] For each sequenced dodecapeptide, a highly conserved core
region was observed to be typically 3 to 5 amino acids long and the
positional preference of these "consensus" residues within the
dodecapeptide varied. Without wishing to be bound by theory, such
positional preference may be a reflection of structural constraints
of the inserted peptide imposed by thioredoxin which may limit the
position for the antibody binding. Further examination of the
sequences revealed that the peptides isolated by the BR15-6A and NS
19-9 MAbs and E-selectin-IgG fell into distinct consensus-sequence
groups that discriminated between the MAbs and the lectin. The
native carbohydrate antigens LeY and SA-Le.sup.a and the peptides
bound only to the specific MAb or lectin used for their isolation.
See, e.g., Tables 1-3 below.
[0101] Selected peptides were subject to assays for demonstrating
whether the isolated peptides retain conformational properties of
carbohydrates. Such assays include 1) direct binding to respective
MAbs and E-selectin-IgG in solid phase; 2) inhibition of MAbs and
E-selectin-IgG binding to the cognate carbohydrate ligands in solid
phase; and 3) kinetic characterization of peptide binding to
E-selectin using BIACORE methodology.
EXAMPLE 3
Competition Enzyme-Linked Immunosorbent Assay
[0102] The peptide competition/inhibition assays referred to
throughout the following examples are performed as follows: Test
peptides at concentrations ranging from 10 nM to 1 mM were
preincubated with 100 .mu.l of the MAb NS19-9 (5 .mu.g/ml) diluted
in 10% .mu.-globulin free horse serum/PBS at room temperature.
Fifty .mu.l of preincubated inhibition complex antibody-peptide is
added to each well of 96-well plate and incubated at 30-37.degree.
C. for 1 hour. After 1 hour of incubation, MAb/peptide complex
mixtures were transferred to wells precoated with a constant amount
of neoglycoprotein containing coupled multivalent carbohydrate
determinant (SA-Le.sup.a-polyacrylamide matrix (SA-LeX-PAA or
LeY-PAA) (5 .mu.g/well) and allowed to bind for 1 hour followed by
blocking with 10% .gamma.-globulin-free horse serum for 2 hours at
room temperature. Wells were washed with 100 .mu.l PBS four times.
Goat anti-mouse immunoglobulin G conjugated to horse radish
peroxidase (Boehringer-Manheim, Indianapolis, Ind.) was diluted
1000-fold with 10% .gamma.-globulin free horse serum/PBS and 100
.mu.l was added in each well and incubated at 30-37.degree. C. for
1 hour and washed with 100 .mu.l PBS five times. Ten mg substrate
for horse radish peroxidase, tetramethylbenzidine dichloride (TMB)
(Sigma Chemical Co., St. Louis, Mo.) was dissolved in 10 ml of 0.05
M phosphate-citrate buffer, pH 5.0, containing 0.03% sodium
perborate (Sigma Chemical Co.). One-hundred .mu.l substrate was
added in each well and incubated at room temperature for 10
minutes. The developed blue color was read at 450 nm after stopping
the reaction with 100 .mu.l 1M phosphoric acid. Fifty % inhibitory
concentration (IC.sub.50) was calculated by non-linear
least-squares regression to a four-parameter logistic equation. The
results of various competition assays performed as described are
detailed in the following examples.
EXAMPLE 4
Peptido-Mimetics of Lewis Y and SA-Le.sup.a
[0103] A. Le-Y
[0104] Peptides were isolated using MAb BR15-6A as described in
Example 2 above. BR15-6A inhibits adhesion of human tumor cells to
EC in E-selectin-independent mode. The family of peptides isolated
using BR15-6A (Table 1), which mimics the LeY carbohydrate, also
demonstrates the consensus sequence motif RPDL [SEQ ID NO:113].
This motif was also recognized by another LeY-specific MAb, BR55-2,
suggesting that the basis for this cross-reactivity is structural
mimicry.
[0105] The isolated sequences of the families of peptides mimicking
LeY can be compared with a putative peptide APWLYAGP [SEQ ID NO:83]
identified previously by phage display panning with another
anti-LeY antibody [Hoess et al., 1993, Gene 128:43-49]. This result
indicates that the same consensus sequences contribute to the
mimicry of the carbohydrate structure and are responsible for the
topological similarity with the carbohydrate antigen.
[0106] On the other hand, comparison of these three MAbs
demonstrated their different binding specificity for LeY
carbohydrate structure, suggesting that they recognize different
epitopes displayed by the hexasaccharide [Blaszczyk-Thurin et al.,
1987, J. Biol. Chem. 262:372-379; Rodeck et al., 1987, Hybridoma
6:389-401; Pastan et al., 1991, Cancer Res. 51:3781-3787]. Thus,
the peptide topological equivalents of the LeY carbohydrate very
likely represent overlapping epitopes common for all LeY-specific
MAbs tested. Examples of peptide sequence families mimicking LeY
structure are listed in Table 1 below.
1 TABLE 1 Peptide Sequence SEQ ID NO TKRPDLIVDPIP 98 DEVRPDLISTEE
99 NLRPKYIXLDAD 100 TLIAFADLVDVI 101 GLDLLGDVRIPVVRR 119
VGITGFVDPLPLRLL 120
[0107] Peptide SEQ ID NO: 99 from the family of LeY-mimicking
peptides was selected because it contained a consensus motif which
appeared in multiple bacterial isolates after five rounds of
panning with the BR15-6A MAb. This peptide was chemically
synthesized and its binding to BR15-6A MAb was evaluated.
Dose-dependent binding of the MAb to solid phase coated peptide at
concentrations of 1 nM to 1 mM was demonstrated using enzyme-linked
immunosorbent assay (ELISA). This indicates that the peptide was
recognized by the MAb and that the identified sequence DEVRPDLISTEE
[SEQ ID NO: 99] represents a putative epitope for the MAb. This
peptide was demonstrated to inhibit MAb BR15-6A binding to the
carbohydrate structure of LeY. It likely represents a
conformational mimic of the LeY structure. This peptide is further
evaluated in competition assays with native LeY containing
neoglycoprotein and LeY-specific MAbs BR15-6A and BR55-2.
[0108] B. SA-Le.sup.a
[0109] Two distinct consensus sequences GXWXXVLEG [SEQ ID NO:111]
and VVGXP [SEQ ID NO:112] were identified in families of peptides
isolated with MAb NS 19-9, which recognizes E-selectin ligand,
SA-Le.sup.a. The structural relevance of isolation of these two
motifs by a single MAb is unclear. Without wishing to be bound by
theory, these motifs indicate the following: peptides based on two
different motifs isolated with the same MAb can mimic different
structural topographies of the cognate SA-Le.sup.a carbohydrate.
These subsets of peptides likely represent non-overlapping surfaces
of cognate antigen, and peptide binding to MAb occurs at separate
sites. Peptide SEQ ID NO:1 from NS 19-9 family II mimicking
SA-Le.sup.a ligand was selected based on the presence of consensus
motif which appeared in multiple bacterial isolates after five
rounds of panning. Table 2 identifies peptide sequence families I
and II mimicking SA-Le.sup.a carbohydrate structure. Table 3
identifies peptide sequence family III mimicking SA-Le.sup.a
carbohydrate structure.
2TABLE 2 FAMILY I FAMILY II Peptide Sequence SEQ ID # Peptide SEQ
ID # VGIWSVVSEGSR 102 RCSVGVPFTMES 103 QDGVWEHVLEGG 104
DLWDWVVGKPAG 1 VELSGRGGLCTW 105 VIGAASHDEDVD 106 TIEPVLAEMFMG 107
DKETFELGLFDR 108 FSGVRGVYESRT 109 PDDAPMHSTRVE 110
[0110]
3 TABLE 3 Peptide Sequence SEQ ID NO STGLMVDFLEPG 91 AKTFGLEHGCEA
95 GGTVEVWSIKGG 115 DHFSQAGSSNHH 116 DDPVTPVIDFGK 117 RDGLIDFVVAGT
118
[0111] SEQ ID NO:1 demonstrated dose-dependent binding with MAb in
the ELISA of Example 2. This peptide was also inhibitory for MAb NS
19-9 binding to the synthetic multivalent
SA-Le.sup.a-polyacrylamide (PAA) matrix conjugate neoglycoprotein
(Glycotech, Inc., Rockville, Md.) which contains the cognate
oligosaccharide determinant. This implies that the sequence
DLWDWVVGKPAG [SEQ ID NO:1] represents a solvent-accessible epitope.
The 50% inhibitory concentration (IC.sub.50) value of SEQ ID NO:1
was calculated to be 700 .mu.M by competition ELISA [see, e.g.,
Blaszczyk et al, 1984, Arch. Biochem. Biophys. 233:161-168].
Control peptides containing the same amino acid composition but in
a scrambled sequence failed to block antibody binding, indicating
that the inhibitory effects of the native sequences are not due to
nonspecific effects. Further, although SEQ ID NO:1 exhibits
relatively low-affinity binding with the antibody, a strong binding
signal was observed on the Western blots when the peptide sequence
was expressed in the context of thioredoxin. Without wishing to be
bound by theory, this might be a reflection of structural
constraints of the inserted peptide imposed by thioredoxin as
compared with the conformation of the peptide in solution or in
solid phase.
EXAMPLE 5
Molecular Basis for Peptide Binding to Anti-Lewis Antigen
Antibodies
[0112] The molecular basis for peptide binding to anti-Lewis
antigen antibodies was determined using the LIGAND-DESIGN (LUDI)
program (Biosym Technologies, San Diego, Calif.) [Bohm 1992, J.
Comput. Aided Mol. Des. 6:593-606]. This program searches a
molecular library for fragments representative of the amino acids
in the target peptide sequence. The program then positions the
fragments within the combining site devoid of steric conflicts.
[0113] Previously, a molecular basis for interaction of LeY
tetrasaccharide and MAb BR55-2 binding site was elucidated using
molecular modeling [Blaszczyk-Thurin et al., 1996, Protein
Engineering 9:101-113]. Modeling studies of the APWLYAGP [SEQ ID
NO: 83] sequence mimicking LeY carbohydrate in the combining site
of the anti-LeY antibody B3 [Murali and Kieber-Emmons, 1997, J.
Molec. Rec., 10:269-276] indicate that the putative contact
residues APWLYA of SEQ ID NO: 83 adopt a turn conformation. Such a
conformation is also projected for residues displayed in the
constrained library.
[0114] In order to establish how the putative APWLY sequence of SEQ
ID NO: 83 mimics LeY binding to B3, the pentapeptide sequence was
"fitted" into the B3 combining site using the LUDI program. Using
this program, the APWLY sequence of SEQ ID NO: 83 was modeled such
that the Trp (W), Tyr (Y), Leu (L) and Ala (A) residues occupied
relative positions as the identified LUDI fragments. Judicious
positioning relied upon intermolecular interaction calculations in
which several potential binding modes of the peptide were ranked
according to the stability of the complex.
[0115] In the most stable conformation, the AP residues occupied a
similar position to the LeY GlcNAc residue. This positioning
indicates that the proline residue mimics the spatial position of
the glucose unit of GlcNAc, while the Ala methyl group is
positioned similarly to the terminal methyl group of GlcNAc's
N-acetyl. The Trp residue occupies a volume associated with the
Fuca 1,3 moiety, and the Leu residue occupies the volume of the
.beta. Gal moiety and has the hydrophobic interaction of P Gal. The
Tyr residue occupies a position not associated with LeY binding to
B3. The computer modeling disclosed that the low energy binding
mode conformation adopts a turn region similar to that observed for
the YPY motif in binding to ConA [Kaur et al., 1997, J. Biol. Chem.
272:5539-5543]. This conformation lends itself to the Tyr residue
of the peptide to potentially interact with several residues in
CDR2 of the heavy chain of B3 that include Asp H53, Ser H52, Ser
H55, or Ser H56. These residues are different in BR55-2, which does
not bind the monovalent APWLYGPA [SEQ ID NO: 121] peptide in a
series of ELISA assays.
[0116] More importantly, energy optimization of the positioned
peptide identified similar functional groups within the B3
combining site in contact with the peptide and carbohydrate
tetrasaccharide core of LeY. Therefore, this analysis provides a
strategy for determining the molecular basis for antigenic mimicry
of particular motifs, providing a unique perspective of how a
peptide sequence fits into the antibody or a receptor combining
site, competing with a native antigen. This approach also enables
the design of therapeutic compounds which more effectively compete
with the native carbohydrate ligand for binding to cell adhesion
molecules.
[0117] The above-disclosed approach was further extended to
determine the types of motifs that bind to BR55-2 and whether such
motifs could be isolated with BR55-2 from a peptide phage screen.
Identification of such peptides as motifs would be a first step in
to improving upon antigenic mimicry for LeY. Search of the LUDI
database using the modeled structure of BR55-2 identified the
motifs YPY, YRY and WRY, which are known to mimic various
carbohydrate subunits, as interacting with BR55-2 and also
identified a non planar-X-planar type motif, FSLLW [SEQ ID NO:114],
as possibly interacting. Thus, non-overlapping residue types were
identified using the LUDI program as in the B3 studies. A
computer-generated space filling model was generated which depicted
the identification and placement of an optimized "FSLLW" amino acid
motif in the combining site of anti-LeY monoclonal antibody BR55-2
in contrast to LeY positioned in the BR55-2 combining site. The
topological similarity is very good. This peptide competes with LeY
for BR55-2 binding. These data further demonstrate another method
to develop peptide mimotopes that are specific. This method may be
extended to identify motifs mimicking SA-LeX and SA-Le.sup.a using
the crystal structure of the lectin domain of E-selectin in
optimizing respective mimotopes.
EXAMPLE 6
Identification of Sequences Critical for MAb Binding and an
SA-Le.sup.a Mimic with Higher Antibody Binding Affinity
[0118] To analyze amino acid residues that are critical for NS 19-9
recognition, an array library of 163 unique peptides was generated
by systematic amino acid replacement in which each position of the
starting peptide DLWDWVVGKPAG [SEQ ID NO:1] was replaced by other
L-amino acids. In addition, peptides were synthesized with
simultaneous incorporation of multiple amino acids or with
truncation of specific regions.
[0119] The peptide array of 163 unique peptides was generated by
substituting all amino acids for each individual amino acid in lead
peptide (DLWDWVVGKPAG [SEQ ID NO:1] identified by combinatorial
library panning with MAb NS 19-9. An array of synthetic 12-mer
peptides was synthesized using 90.times.130 mm polyethylene
glycol-modified cellulose membrane functionalized with
approximately 4 nmole/mm.sup.2 amino groups, manufactured by Abimed
(Lagenfeld, Germany). Standard Fmoc chemistry was used according to
the manufacturer's instructions [Frank, R., 1992, Tetrahedron 48,
9217-9332]. The protected and activated amino acids were spotted
using an Abimed ASP 422 robotic arm. All washing, dyeing and
deprotection steps were done manually. The activated C-terminal
amino acids were spotted leaving 10 mm space in each direction, at
the concentration of 0.5 M in N-methylpyrrolidone. A volume of 0.5
ml provides spot of 7-8 mm in diameter. Activation of the amino
acids with dicyclohexyl-carbodiimide and N-hydroxy-benzotriazole
was done 30 minutes before spotting. After each coupling cycle, the
paper was washed with 12% acetic anhydride dissolved in
N,N'-dimethylformamide (DMF) twice for a total of 10 min to endcap
all unreacted amino groups. Repetitive removal of the Fmoc groups
was achieved by two treatments with 20% piperidine in DMF for 5 and
10 min, respectively. The second and consecutive amino acids were
coupled in a 1.1 molar excess, and were spotted 3-4 times depending
upon the outcome of the bromophenol blue assay of the
couplings.
[0120] After the coupling and deprotection steps, the membrane was
washed thoroughly with DMF and ethanol, dried and stained with
bromophenol blue dissolved in DMF. After successful coupling the
paper remains colorless; after successful deprotection steps the
peptide dots turn deep blue. The coupling steps were repeated until
all peptide spots remained colorless. The N-terminal amino acids at
the end of the syntheses remained uncapped. Final removal of the
side-chain protecting groups was performed by washing the paper
with a mixture of 12.7 ml trifluoroacetic acid, 3.7 ml m-cresol,
3.7 ml thioanisole, 3.7 ml water and 3.7 ml ethanedithiol. After
cleavage, the paper was washed several times with ethyl alcohol,
DMF, water and methyl alcohol, and dried.
[0121] The peptide array library was tested for binding of MAb NS
19-9. The cellulose filter was blocked with 5% non-fat milk in
phosphate buffered saline (PBS) for 1 hour at room temperature
followed by washing with PBS. Filters were incubated with goat
anti-mouse immunoglobulin G conjugated with horse radish peroxidase
(1 .mu.g/ml) for 1 hour. Filters were washed five times in PBS-T
(0.05% Tween in PBS, v/v) and developed using a chemiluminescence
reagent followed by autoradiography as described in Western
blot.
[0122] After probing of the membrane containing peptide spots with
NS 19-9 followed by chemiluminescence detection with a
peroxidase-labeled anti mouse immunoglobulin G antibody, the
peptide sequence was scanned by substitution of each amino acid
with other L-amino acids by spot synthesis. Resulting peptides were
tested for MAb NS 19-9 binding or a varying number of amino acids
were truncated. The number of peptides was 163 (6 raws, 27 spots
each). Spot analysis revealed a distinct pattern of key residues
important for binding and, therefore, sensitive to substitution
while other residues tolerated replacement by a variety of amino
acids.
[0123] Comparison of the signal intensities of the array scan
revealed that the critical residues for binding were clearly
identified within the N-terminal half of the DLWDWVVGKPAG peptide
[SEQ ID NO: 1] as determined by the lack of antibody binding to
substituted peptides (Table 4). In contrast, most of the
substitutions within the C-terminus were tolerated (amino acids 6
to 12), not influencing MAb binding. These results indicate that
the N-terminus is clearly involved in specific interaction with NS
19-9. Most substitutions of residues 2 to 5 abolished NS 19-9
binding, with Trp3 and Trp5 being the most critical. Identical sets
of amino acids (His, Tyr, Ala, Asp, Glu, Lys, Arg, Ser) were shown
to completely abolish antibody binding while Met significantly
decreased the signal upon replacement of either of these residues
(Trp 3 and 5). Similarly, substitutions of Leu2 with Ala, Asp, Tyr,
Glu, Lys, Arg, and Ser as well as substitution of Asp4 with Glu,
Ser, Pro, Val, Met, and Tyr completely abolished MAb binding. All
peptides generated by truncation of amino acids 2 to 8 from the
N-terminal of the peptide were no longer recognized by the
antibody. Furthermore, when the 2-6 amino acid segments covering
the key residues involved in antibody binding identified by the
single amino acid substitutions were incorporated into the peptide,
such a replacement always resulted in abolished binding. Most
substitutions upstream from position 5 (positions 6-9) allowed for
MAb binding with no evident preference for substituted amino acids.
Although substitution analysis failed to identify significant
differences in binding as a result of substitution of residues 9-12
within the C-terminal half of the peptide, MAb did not detect
truncated peptides within this region implying the importance of
the C-terminus for MAb binding. Table 4 lists the amino acid
substitutions within peptide DLWDWVVGKPAG [SEQ ID NO:1] that
significantly decrease or abolish binding of Mab NS 19-9 using the
peptide array.
4 TABLE 4 SEQ ID SEQ ID Peptide Sequence NO Peptide Sequence NO
DLWDWVVGKPAG 1 DLWDHVVGKPAG 25 DAWDWVVGKPAG 2 DLWDYVVGKPAG 26
DDWDWVVGKPAG 3 DLWDMVVGKPAG 27 DYWDWVVGKPAG 4 DLWDAVVGKPAG 28
DEWDWVVGKPAG 5 DLWDDVVGKPAG 29 DKWDWVVGKPAG 6 DLWDEVVGKPAG 30
DRWDWVVGKPAG 7 DLWDKVVGKPAG 31 DSWDWVVGKPAG 8 DLWDRVVGKPAG 32
DLHDWVVGKPAG 9 DLWDSVVGKPAG 33 DLYDWVVGKPAG 10 DLWDWLVGKPAG 34
DLFDWVVGKPAG 11 DLWDWYVGKPAG 35 DLMDWVVGKPAG 12 DLWDWAVGKPAG 36
DLADWVVGKPAG 13 DLWDWSVGKPAG 37 DLEDWVVGKPAG 14 DLWDWVLGKPAG 38
DLDDWVVGKPAG 15 DLWDWVAGKPAG 39 DLKDWVVGKPAG 16 DLWDWVDGKPAG 40
DLRDWVVGKPAG 17 DLWDWVDCKPAG 41 DLSDWVVGKPAG 18 DLWDWVDPKPAG 42
DLWEWVVGKPAG 19 DLWDWVDDYPAG 43 DLWSWVVGKPAG 20 DLWDWVDDFPAG 44
DLWPWVVGKPAG 21 DLHE 45 DLWVWVVGKPAG 22 DLWEHL 46 DLWMWVVGKPAG 23
LDWEWVVGKPAG 47 DLWMWVVGKPAG 24 DLDL 48 EIHDWVVGKPAG 49 WDWVVGKPAG
56 DLWEHL 50 DWVVGKPAG 57 LDDDWVVGKPAG 51 WVVGKPAG 58 EIHEWVVGKPAG
52 VVGKPAG 59 DLWDHLLA 53 VGKPAG 60 LDDLWVVGKPAG 54 GKPAG 61
EIHEHLVGKPAG 55 KPAG 62
[0124] Comparison of signal intensities on the peptide array
revealed that some substitutions led to enhanced NS 19-9 binding
allowing for identification of several peptides with increased
binding affinity to the antibody (Table 5). Improvement of peptide
binding was achieved mainly by substitution of residues 5 to 12
within the lead peptide, whereas no amino acid exchange at
N-terminus (residues 1-4) led to the increased binding. The
replacement of residues with amino acids containing polar groups
such as Glu and Asp showed clearly an enhancing effect at the
C-terminus but not the N-terminus.
[0125] Array analysis failed to reveal significant differences in
the binding intensities between the peptides substituted at
different positions, suggesting that single substitution at any
position in this region with carboxyl groups can enhance the
interaction with the MAb binding site. In addition, substitutions
with Ile, Ala and Ser also improved MAb binding. Similarly, the
simultaneous replacement of several residues with clusters of amino
acids upstream from position 6 demonstrated enhanced binding. The
highest intensity signal was, however, observed with peptide
DLWDFVVGKPAG [SEQ ID NO:63] containing a single substitution at
position 5 with Phe.
[0126] Table 5 lists the amino acid substitutions within peptide
DLWDWVVGKPAG [SEQ ID NO: 1] that increase the binding of MAb NS
19-9 using the peptide array.
5 TABLE 5 SEQ ID SEQ ID Peptide Sequence NO Peptide Sequence NO
DLWDFVVGKPAG 63 DLWDWVVGKPDG 73 DLWDWVIGKPAG 64 DLWDWVVGKPAD 74
DLWDWVVAKPAG 65 DLWDWVKEKPAG 75 DLWDWVVSKPAG 66 DLWDWVLAKPAG 76
DLWDWVVEKPAG 67 DLWDWVVGEDAG 77 DLWDWVVDKPAG 68 DLWDWVVGKPEK 78
DLWDWVVGEPAG 69 DLWDWVKEEPAG 79 DLWDWVVGDPAG 70 DLWDWVVGKDEK 80
DLWDWVVGKEAG 71 DLWDWVVGEDEK 81 DLWDWVVGKDAG 72 DLWDWVKEEDEK 82
[0127] A distinct pattern of substitutions that led to increased or
abolished signal intensities with respect to the C- and N-terminus
suggests that the region close to the N-terminus might contribute
to the specificity of the interaction with NS19-9. Amino acids
close to the C-terminus appear to add significantly to the affinity
of ligand binding.
EXAMPLE 7
Inhibition of Binding of MAb to the Carbohydrate with Synthetic
Peptides Mimicking SA-Le.sup.a Structure
[0128] DLWDWVVGKPAG [SEQ ID NO:1] and DLWDFVVGKPAG [SEQ ID NO:63]
were chemically synthesized and tested for their ability to compete
to immobilized synthetic SA-Le.sup.a-PAA neoglycoprotein. To
determine whether peptide SEQ ID NO:1 and peptide SEQ ID NO: 63 are
true mimics of SA-Le.sup.a, dose-response experiments were carried
out in order to determine the concentration of peptides required
for blocking of 50% of MAb binding to the native carbohydrate
antigen (IC.sub.50), as determined by competition ELISA (Example
3). Both peptides SEQ ID NOS: 1 and 63 blocked the binding of MAb
NS 19-9 to the constant amount of carbohydrate antigen in a
dose-dependent manner. The IC.sub.50 for peptide SEQ ID NO:1
blocking of MAb-SA-Le.sup.a binding was 700 .mu.M. Peptide SEQ ID
NO: 63 exhibited a more pronounced dose-dependent inhibition of the
MAb-SA-Le.sup.a binding as compared with peptide SEQ ID NO:1 as
demonstrated by the calculated IC.sub.50 value of 70 .mu.M for
peptide SEQ ID NO: 63.
[0129] Without wishing to be bound by theory, these data suggest
that the peptide sterically interferes with MAb binding to
carbohydrate antigen. The sequences DLWDWVVGKPAG [SEQ ID NO:1] and
DLWDFVVGKPAG [SEQ ID NO:63] represent solvent-accessible epitopes
and the peptides represent cognate determinants for the antibody.
No measurable blocking of anti-Le.sup.a MAb NS 19-9 binding was
found with non-related peptide, indicating that the inhibitory
effects of the native sequences are due to specific effects.
EXAMPLE 8
Inhibition of Neutrophil Recruitment in an Acute Inflammation Model
In Vivo by a Peptido-Mimetic of SA-Le.sup.a
[0130] The accumulation of neutrophils is a characteristic feature
of acute and chronic inflammatory disease, and early steps in the
recruitment of these cells to the site of inflammation depends upon
E-selectin-mediated interaction. Thus, inhibition of neutrophil
recruitment in vivo is an important test of the ability of
potential therapeutic agents to inhibit E-selectin-mediated events.
SA-Le.sup.a interaction with E-selectin is not relevant for
adhesion of neutrophils, since neutrophils do not express
SA-Le.sup.a on their surfaces. However, tumor cells have been
demonstrated to prefer SA-Le.sup.a over SA-LeX in mediating
E-selectin-dependent adhesion interactions with endothelial cells.
Because SA-Le.sup.a binds to E-selectin, the adhesion-blocking
ability of an SA-Le.sup.a peptido-mimetic was measured in a
neutrophil-recruitment in vivo model of acute inflammation. Thus,
this assay demonstrates that the administration of a SA-Le.sup.a
mimicking molecule inhibits neutrophil recruitment, i.e.,
diminishes the influx of neutrophils into chemically irritated
peritoneum in vivo.
[0131] The bioactivity of the 12-mer peptide SEQ ID NO:1 which
mimics the SA-Le.sup.a carbohydrate structure was determined by
administering Zymosan.TM. intraperitoneally (i.p.) into mice [see,
e.g., Martens et al. 1995, J. Biol. Chem. 270:21129-21136; Rao et
al. 1994, J. Biol. Chem. 269:19663-19666], followed three hours
later by an intravenous (i.v.) injection of peptide SEQ ID NO:1 (1
mg). Neutrophils were harvested and counted one hour later. As
shown in FIG. 1A, peptide treatment significantly (P<0.001)
reduced the number of neutrophils in peritoneal lavage fluids.
Control experiments using the same dose of "scrambled" peptide
sequence, which did not bind to NS19-9 MAb, exhibited no decrease
in neutrophil influx relative to PBS-injected mice.
[0132] To confirm these results, myeloperoxidase (MPO), activity,
which is an enzymatic marker for neutrophils, was measured
spectrophotometrically as an absorbance rate in the homogenates of
collected cells [Bradley et al., 1982, J. Invest. Derm.
78:206-209]. Significant reduction of enzymatic activity
(P<0.005) was observed in parallel with decreased neutrophil
numbers assessed by total neutrophil count (FIG. 1B). The reduction
in enzyme activity is apparently due to reduction in neutrophil
recruitment in mice treated with peptide mimicking E-selectin
ligand SA-Le.sup.a. This peptide inhibits E-selectin function in
vivo. Although peptide SEQ ID NO:1 exhibited relatively
low-affinity binding to MAb in vitro, IC.sub.50 data from in vitro
competition assays (Example 3) may not be relevant to the in vivo
situation since the E-selectin blocking effect was clearly observed
despite the low affinity demonstrated in vitro.
EXAMPLE 9
Peptido-Mimetics of E-Selectin Ligand
[0133] To identify peptides mimicking E-selectin ligand that may
involve other than carbohydrate epitopes of the natural ligand, the
12-mer FLITRX fusion library was screened directly using
E-selectin-IgG fusion protein, as described in Example 2. Among
isolated peptides, the motif VLSP was found in 7 of 16 clones
recovered. In addition, preferences for V/G and RR in the sites
flanking the 5-amino acid motif were also found. In another group
of peptides, a PGR sequence was found in 3 clones. As discussed
above, peptides with different sequence motifs may mimic different
topographical surfaces of the cognate carbohydrates or may bind to
different binding areas of E-selectin combining site. Family I
peptide sequences and Family II peptide sequences mimicking
E-selectin ligand are reported in Table 6.
6TABLE 6 FAMILY I FAMILY II Peptide SEQ ID # Peptide SEQ ID #
ASAVNLYIPTQE 84 DIRVEPGGGYTH 94 VYLAPGRISRDY 95 VYLAPGRISRDY 95
CTSHWGVLSQRR 87 APIHTYTGRARG 96 RVLSPESYLGPS 91 RHTCVRSCGHDR 97
VGNGVLMLGRRG 90 GNCRYIGLRQFG 93 RVLSPESYLGPA 92
[0134] In summary, these data demonstrate that the conformation of
small peptides can mimic the topography of the carbohydrate
epitopes recognized by MAbs and by selectin. Carbohydrate mimotopes
are effective in vivo in blocking selectin-dependent interactions,
which can diminish leukocyte recruitment in inflammatory processes.
The data demonstrate the feasibility of using small molecule
antagonists isolated from combinatorial peptide libraries to block
E-selectin function in vivo and to inhibit metastatic and
inflammatory processes.
EXAMPLE 10
Characterization of Carbohydrate Expression on Various Cell
Lines
[0135] To develop tissue culture models of metastasis for in vitro
and in vivo studies, phenotypes of various human and murine cell
lines were characterized with respect to the expression of various
carbohydrate structures implicated as ligands for selectins, e.g.,
SA-Le.sup.a and SA-LeX and also with respect to expression of LeY,
which is a carbohydrate tumor-associated antigen not recognized by
E-selectin. The expression of various carbohydrate antigens on
human colon carcinoma, breast, gastric, and prostate carcinoma cell
lines (originally obtained from ATCC, Rockville, Md.) was reported
previously [Magnani et al., 1981, Science 212:55-56; Steplewski et
al., 1985, Proc. Natl. Acad. Sci. USA 82:8653-8657;
Blaszczyk-Thurin et al., 1987, J. Biol. Chem. 262:372-379;
Blaszczyk et al., 1985, Proc. Natl. Acad. Sci. USA 82:3552-3556;
Hansson et al., 1983, J. Biol. Chem. 258:4091-4097; Blaszczyk et
al., 1985, Proc. Natl. Acad. Sci. USA 82:3552-3556;
Blaszczyk-Thurin et al., 1988, Biochem. Biophys. Res. Commun.
151:100-108].
[0136] This expression was re-examined and confirmed using
fluorescence-activated cell sorting (FACS) using specific MAbs
developed at the Wistar Institute (Philadelphia, Pa.). Expression
of surface carbohydrate ligands did not change as a function of
proliferation in vitro, confirming the stability of the phenotype.
Approximately 90-100% of cells obtained from human gastrointestinal
adenocarcinorna lines SW948, SW1116, SW480, S180, HT29, Colo 201
and KATOIII and mammary adenocarcinoma cells MCF-7, SkBr5, SkBr3
and BT20 uniformly expressed both SA-Le.sup.a and SA-LeX structures
at very high levels. Similarly, all cell lines demonstrated cell
surface expression of LeY determinant, although expression levels
varied among the cell lines.
[0137] Thus, these cell lines were the most suitable for the study
of adhesion to EC in vitro and for an orthotopic tumor model in
which immunodeficient mice may be inoculated with human tumor
cells. The expression of SA-LeX in SW620 cells was below the level
of detection, but the cells were uniformly and strongly stained for
SA-Le.sup.a. Thus, SW620 cells should be very useful in vivo
evaluation of the contribution of SA-Le.sup.a to tumor cell
colonizing ability in the absence of SA-LeX.
[0138] An autologous model of tumor metastasis is preferred for in
vivo studies in which murine tumor cells derived from the same
murine background may be inoculated to form metastases. Several
murine tumor cell lines such as MethA sarcoma, G1261 glioblastoma,
CT26 colon carcinoma, B16F1 and B16F10 melanoma clones obtained
from DCTDC Tumor Repository (NCI Frederick, Md.) and murine breast
adenocarcinoma 66.1, JC, 410.1 cell lines provided by Dr. Amy
Fulton (University of Maryland, Baltimore, Md.) were characterized
with respect to the expression of Lewis antigens. MethA clone #34
was isolated from the original MethA cell line cloned by limiting
dilution and was determined to express SA-LeX antigen on 100% of
cells as compared with only approximately 30% cells in the original
cell line. All cell lines expressed SA-LeX except B16F1 and B16F10
variants, whereas no SA-Le.sup.a expression was observed among any
cell lines. Therefore, MethA #34 is suitable to evaluate the role
of SA-LeX mimicking peptides in inhibiting adhesion of tumor cells
in vitro and in vivo.
[0139] A panel of tumor cells such as pancreatic PAN02, PAN03,
colon CT38, CA51, mammary EMT-6, E0771, 755, lung ASBXIV,
transitional bladder FCB and melanoma HP(Jax) (DCTDC Tumor
Repository) was assembled. These cells were continuously analyzed
with respect to the expression of SA-LeX, SA-Le.sup.a, and LeY
determinants. Moreover, expression vectors are available which
contain .alpha.1,3/4 fucosyltransferase, (.alpha.1,3/4FTIII) cDNA
[Seed, 1987, Proc. Natl. Acid. Sci. USA 84:8573-8577], and
.alpha.1,2 fucosyltransferase cDNA (.alpha.1,2FT) [Sun et al.,
1995, Proc. Natl. Acad. Sci. USA 92:5724-5728]. Tumor cell lines
were generated which express SA-Le.sup.a and LeY transfection,
respectively, using transfection procedures. Further,
1-2.times.10.sup.5 Meth A and B16 tumor cells were sufficient to
form countable lung metastasis within 2 to 3 weeks following
intravenous (i.v) tumor cell inoculation in Balb/c and C57B1/6
strains of mice, respectively.
EXAMPLE 11
E-Selectin-Independent Adhesion of Adenocarcinoma Cells
[0140] Tumor cell adhesion to EC monolayers was investigated in
vitro as described in Iwai et al., cited above, as a model for
metastatic invasion. The data disclosed herein demonstrate that
various mammary adenocarcinoma cells attach to TNF-.alpha.or
IL-1.beta. cytokine-activated human umbilical cord vein endothelial
cells (HUVEC) via an E-selectin-independent mode. Further, the data
demonstrate that human breast adenocarcinoma cell lines, SkBr5 and
SkBr3, but not colon adenocarcinoma cells LS 180, SW1116 and SW948,
adhere to TNF-.alpha.-stimulated EC and that this process is
mediated by E-selectin as determined by the inhibitory effect of
anti-E-selectin MAb as well as NS 19-9 MAb specific for SA-Le.sup.a
(FIG. 2A). Antibodies BR55-2 and BR15-6A directed to LeY
significantly decreased the adhesion in a dose-dependent fashion,
implicating the involvement of LeY in adhesion of these breast
carcinoma cells (FIG. 2A). These cells also adhered to the
spontaneously transformed human endothelioma cell line ECV-304 in
an LeY-dependent way. ECV-304 cells unlike primary HUVEC, did not
express E-selectin even in the presence of inflammatory cytokines
as assayed by radioimmunoassay [Blaszczyk et al. 1984, Cancer Res.
44:245-253] using anti-E-selectin antibody (FIG. 2B). These data
indicate that different human adenocarcinoma cells interacted with
EC via E-selectin-mediated and E-selectin-independent pathways. The
data disclosed are in agreement with previously published reports
that some invasive breast carcinoma cells do not interact with
TNF-.alpha.-stimulated EC under dynamic conditions, but adhere to
resting and stimulated EC via an E-selectin-independent mode
following static incubation [Iwai et al, cited above; Tozeren et
al., cited above; Miyake et al., cited above; Garrigues et al.,
cited above; Dettke and Loibner, cited above]. ECV-304 endothelioma
cells which have lost the ability to express E-selectin, are a
useful model for in vitro studies of E-selectin-independent
adhesion of tumor cells to EC and the role of this pathway in the
establishment of metastasis.
EXAMPLE 12
Competition of DLWDFVVGKPAG Peptide [SEQ ID NO:63] with SA-Le.sup.a
for MAb Binding
[0141] The lead peptide DLWDWVVGKPAG [SEQ ID NO:1] and the array
selected peptide DLWDFVVGKPAG [SEQ ID NO: 63] were synthesized
individually and their binding specificities to NS 19-9 MAb were
assayed by competitive solid phase enzyme linked immunosorbent
assay (Example 3), with the results as shown in FIG. 3. Both
peptides inhibited binding of MAb NS19-9 to a solid phase adsorbed
cognate carbohydrate antigen SA-Le.sup.a in a dose dependent
manner. IC.sub.50 of the substituted peptide DLWDFVVGKPAG [SEQ ID
NO: 63] was established at 70 .mu.M whereas the IC.sub.50 of the
lead peptide DLWDWVVGKPAG [SEQ ID NO: I] was 700 .mu.M (FIG. 3).
This 10-fold lower IC.sub.50 value for the array-selected peptide
reflects a higher binding affinity of this peptide as compared to
the lead peptide. These data suggest the DLWDFVVGKPAG peptide [SEQ
ID NO: 63] displays a better fit into MAb binding site as compared
with the original peptide. No significant binding of MAb NS 19-9
was observed with unrelated peptide in the concentration range up
to 5 mM (FIG. 3).
EXAMPLE 13
Secondary Structure of Peptides Mimicking Carbohydrate
[0142] Secondary structure prediction, based on a neutral net
algorithm [Chandonia, J. M., and Karplus, M., 1996, Protein Sci. 5:
768-774] indicated some propensity of both peptides DLWDWVVGKPAG
[SEQ ID NO:1] and DLWDFVVGKPAG [SEQ ID NO: 63] to assume extended
or helical structures centered at the mid-chain WVVG and FVVG
domains of SEQ ID NOS: 1 and 63, respectively (FIG. 4). The
presence of .beta.-pleated sheets was further supported by
calculations based on the Fasman-Chou probability values [Fasman,
G. D., 1985, J. Biosci. 8: 15-23], although these predictions
placed the extended structure closer to the amino termini, with the
C-terminal fragments showing reverse-turn conformations.
[0143] Low-resolution conformational analysis by CD supported these
calculations. CD spectra were taken on a Jasco J720 instrument at
room temperature in a 0.2 mm pathlength cell. Double distilled
water and spectroscopy grade trifluoroethanol were used as
solvents. The peptide concentration was 0.51 mg/ml, determined by
quantitative reversed-phase HPLC. The algorithms provided by JASCO
accomplished curve smoothing. Mean residue ellipticity is expressed
in degrees cm.sup.2/dmole by using a mean residue weight of 110.
Because the secondary structures of the peptides by the current
computer-assisted curve analyzing algorithms show a high error
rate, the CD spectra evaluations were based on comparison with
known peptide conformations [Woody, R. W. (1985) The Peptides, eds.
Hruby, V. J. (Academic Press, Orlando), pp. 15-114].
[0144] In water both peptides exhibited negative bands at 201-202
nm indicative of mostly unordered structures, common for
medium-sized peptides. However, the slight redshift from the
generally observed 197-198 nm band for entirely random coils
highlighted the presence of type I (III) .beta.-turns. Comparison
of the CD of the two peptides assigned an increased contribution of
reverse-turns to the conformational equilibrium for peptide SEQ ID
NO: 63, based on the minor redshift of both the 197 nm and the 202
nm bands. A negative shoulder, characteristic for turns, replaced
the small positive band between 220 and 230 nm (indicative for
random peptide structure). In contrast, peptide SEQ ID NO: 63 lacks
a negative shoulder between 210 and 220 nm, clearly present for
peptide SEQ ID NO:1. Peptides and proteins in .beta.-pleated sheets
or type II .beta.-turns exhibit negative bands in this wavelength
region. Considering the secondary structural prediction, the
presence of extended structure may be more likely than that of type
II .beta.-turns.
[0145] In the structure-inducing solvent 50% trifluoroethanol both
.pi..pi.* bands for both peptides were reshifted, indicating the
acquisition of more ordered structures, as expected (data not
shown). The 227 nm positive band of the random coils for peptide
SEQ ID NO:1 disappeared, but the 216 nm negative .beta.-pleated
sheet band remained intact as did the 228 nm negative shoulder for
peptide SEQ ID NO: 63, suggesting the random conformations were
merely replaced by the signature structures (i.e., extended for
peptide SEQ ID NO:1 and turn for peptide SEQ ID NO: 63).
[0146] Nevertheless, the conformational differences may not
represent casual correlations to antibody recognition. Both
peptides DLWDWVVGKPAG [SEQ ID NO:1] and DLWDFVVGKPAG [SEQ ID NO:
63] highlight the functional role played by the aromatic-X-aromatic
motif within the peptide. It is possible that these structure types
are realized within the antibody-combining site. This is consistent
with modeling and crystal analysis of this motif type which
suggested that these regions may adopt type I or type II turns
within the antibody-combining site. Turn conformations appear to
play an important role in E-selectin recognition based on structure
activity relations of modified Ser-Glu dipeptides that bind to
E-selectin. The increased binding of peptide SEQ ID NO: 63 with
substitution of Phe for Trp suggests that the Phe directly
contributes to MAb and that hydrophobic stacking interactions are
important for increased antibody binding and consequently antigenic
mimicry. This assertion is supported by X ray crystallographic and
molecular modeling studies of carbohydrate mimicking peptides.
EXAMPLE 14
Inhibition of Tumor Metastasis
[0147] SA-Le.sup.a appears to mediate the adhesion of many
carcinoma tumors to human umbilical vascular endothelial cells in
multiple in vitro studies. An in vivo experimental metastatic model
which permits investigation of SA-Le.sup.a supported adhesion of
tumor cells to lung endothelium is performed as follows:
[0148] B16F10 murine melanoma cells do not naturally express
E-selectin ligands SA-LeX or SA-Le.sup.a as demonstrated by FACS
analysis, and are syngeneic with C57B1/6 haplotype (American Type
Tissue Collection, Rockville, Md.). To manipulate these cells to
express the SA-Le.sup.a structure on the tumor cell surface, the
B16F10 cells were stably transfected with pCDNA-FTIII using
Effectene (Qiagen, Chatsworth, Calif.) as recommended by the
manufacturer. Plasmid pCDNA-FTIII is prepared by cloning HindIII
and NotI-digested .alpha.1-3/4-fucosyltransferase cDNA (FTIII)
obtained from the 7.pi.H3M vector containing FTIII cDNA (Brian
Seed, Massachusetts General Hospital, Boston, Mass.) was cloned
into pCDNA3(neo) vector. FTIII fucosyltransferase is specific for
both type 1 and 2 lactoseries oligosaccharide acceptor substrates
and thus is capable of synthesizing both SA-Le.sup.a and SA-LeX,
respectively. The resulting cell line, B16F10FTIII, expresses
SA-Le.sup.a carbohydrate structure as demonstrated by flow
cytometry analysis using MAb NS 19-9 as compared to the parental
B16F10, which did not show staining with this antibody.
[0149] The transfected cells were grown in the presence of G418
(500 .mu.g/ml) (Gibco-BRL, Grand Island, N.Y.) for 10 days. To
ensure the homogeneity of the transfected cells with respect to the
expression of SA-Le.sup.a, the cells were subjected to cell sorting
using SA-Le.sup.a specific MAb NS 19-9 followed by FITC-conjugated
goat anti-mouse immunoglobulin. The resulting cell line B16F10FTIII
appeared to express SA-Le.sup.a but not SA-LeX as assessed by FACS
(not shown), suggesting that type I but not type 2 acceptors were
available within the cells. Thus, the generated cell line made a
suitable model to determine the role of SA-Le.sup.a in the
metastatic process since the tumor cells are devoid of SA-LeX. The
tumorigenic dose for the C57B1/6 syngeneic tumor cells was
established by i.v. injection of various numbers of cells. A
1.times.10.sup.5 dose was chosen for further experiments as
countable lung metastases were observed after i.v. injection of
1.times.10.sup.5 of B16F10FTIII cells expressing SA-Le.sup.a after
21 days.
[0150] The role of tumor cell adhesion to vascular EC via
E-selectin and its ligand SA-Le.sup.a interaction in metastasis
formation was established in vivo in two ways. First, to directly
assess the role of E-selectin in tumor colonization in vivo, the
ability of B16F10 murine melanoma cells expressing SA-Le.sup.a to
colonize in the lung of E-selectin KO mice of C57B1/6 background
[Staite, N. D. et al, 1998, Blood, 88: 2973-2979, and kindly
provided by Dr. Daniel Bullard (University of Alabama at
Birmingham, Ala.)] was determined in parallel with wild-type six-
to 8-week-old C57B1/6 female mice (Jackson Laboratory, Bar Harbor,
Me.). KO mice lack E-selectin expression.
[0151] B16F10FTIII cells positive for SA-Le.sup.a were grown in
vitro in Iscove's culture medium supplemented with 10% FBS for 1
week before injecting into mice. Cells were collected and washed
twice in Iscove's medium without serum and suspended in PBS.
One.times.10.sup.5 tumor cells in a volume of 200 .mu.l in PBS were
inoculated by intravenous (i.v.) route via tail vein. To test the
effect of the peptide, animals were inoculated with a single dose
of peptide at the time of tumor challenge. One mg of peptide was
admixed with the tumor cells and together injected via i.v. route.
Control animals received injection of tumor cells admixed with the
same amount of unrelated peptide. Mice were euthanized after 3
weeks following tumor cells injection and lung and other organs
were examined under dissecting microscope for the presence of tumor
nodules. The lungs were excised and the number of nodules was
enumerated for each animal without fixation of the lungs. Data were
evaluated for statistical significance using a nonparametric
unpaired two-tailed t test. The results are shown in FIG. 5.
[0152] Mice of both strains received i.v. injection of
1.times.10.sup.5 B16F10FtIII tumor cells and mice were examined 3
weeks later. Only 20% of E-selectin deficient animals injected with
tumor cells developed small numbers of lung metastasis while the
rest of the E-selectin KO mice showed no detectable lung tumor
nodules. Statistical analysis gave a P values<0.009 for
E-selectin KO as compared to the control group (FIG. 4, A and C),
respectively. Small nodules were observed in a few E-selectin KO
mice that developed tumors whereas all animals in the control group
developed multiple metastasis and some of them died earlier than 3
weeks. The results demonstrate that lung metastasis of tumor
expressing SA-Le.sup.a antigen is completely abrogated in most of
the genetically manipulated mice that lack expression of
E-selectin, highlighting the critical role of E-selectin in
mediating carcinoma metastasis in vivo.
[0153] To further test the hypothesis that SA-Le.sup.a expression
supports adhesion of tumor cells to E-selectin on EC, the
inhibitory effect of peptide mimicking SA-Le.sup.a antigenic
structure DLWDFVVGKPAG [SEQ ID NO: 63] on lung colonization of
B16F10FTIII cells was tested. One.times.10.sup.5 tumor cells
expressing SA-Le.sup.a were admixed with a solution containing 1 mg
of the peptide DLWDFVVGKPAG [SEQ ID NO: 63], followed by
administration of the mixture to groups of mice. Because peptides
in general show rather short half-life in mouse serum, in the
peptide inhibition studies in vivo the peptide was admixed with the
tumor cells to sustain the highest transient concentration of
peptide at the time of tumor cell arrival into the lung capillary
system. Animals were euthanized after 21 days following tumor
challenge and the number of metastasis was enumerated in each lung
whereas, no metastatic growth was detected in the liver.
[0154] Administration of the peptide DLWDFVVGKPAG [SEQ ID NO: 63]
abrogated on average 50% lung colonization of the B16F10FTIII
induced tumor nodules developed in control animals; some mice being
completely devoid of tumor nodules (FIG. 4B). The injection of the
peptide 1 hour prior to tumor cells did not influence the rate of
metastases formation in comparison with the peptide administered
together with tumor cells. Animals treated with peptide showed
metastases ranging from 0 to 20 per lung (median 9.9), whereas,
animals in the control group developed multiple tumor nodules with
the number of metastases per mouse ranging from 3 to 40 per lung
(median 20.7) (FIGS. 4B and 4A), respectively). The difference was
highly statistically significant (p<0.008). In addition,
B16F10FtIII cells in C57B1/6 mice developed large tumor masses with
diffused infiltration of tumor cells and some mice died before the
termination of the experiment (median 16 days). Mice that developed
metastases, despite treatment with SA-Le.sup.a mimicking peptide,
showed the presence of small tumor nodules and all survived the 3
week observation time.
[0155] These results suggest that the synthetic structural
conformer mimicking SA-Le.sup.a antigen is able to significantly
block the adhesion of tumor cells to vascular endothelium at the
early stages of the multistep process, thus reducing tumor
metastases. This finding strongly suggests that the interaction of
SA-Le.sup.a carbohydrate tumor-associated antigen with E-selectin
expressed on vascular EC is an important step in establishing tumor
metastasis.
[0156] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety. While the invention has been
disclosed with reference to specific embodiments, it is apparent
that other embodiments and variations of this invention may be
devised by others skilled in the art without departing from the
true spirit and scope of the invention. The appended claims are
intended to be construed to include all such embodiments and
equivalent variations.
Sequence CWU 1
1
121 1 12 PRT peptido-mimetic of a Lewis antigen 1 Asp Leu Trp Asp
Trp Val Val Gly Lys Pro Ala Gly 1 5 10 2 12 PRT peptido-mimetic of
a Lewis antigen 2 Asp Ala Trp Asp Trp Val Val Gly Lys Pro Ala Gly 1
5 10 3 12 PRT peptido-mimetic of a Lewis antigen 3 Asp Asp Trp Asp
Trp Val Val Gly Lys Pro Ala Gly 1 5 10 4 12 PRT peptido-mimetic of
a Lewis antigen 4 Asp Tyr Trp Asp Trp Val Val Gly Lys Pro Ala Gly 1
5 10 5 12 PRT peptido-mimetic of a Lewis antigen 5 Asp Glu Trp Asp
Trp Val Val Gly Lys Pro Ala Gly 1 5 10 6 12 PRT peptido-mimetic of
a Lewis antigen 6 Asp Lys Trp Asp Trp Val Val Gly Lys Pro Ala Gly 1
5 10 7 12 PRT peptido-mimetic of a Lewis antigen 7 Asp Arg Trp Asp
Trp Val Val Gly Lys Pro Ala Gly 1 5 10 8 12 PRT peptido-mimetic of
a Lewis antigen 8 Asp Ser Trp Asp Trp Val Val Gly Lys Pro Ala Gly 1
5 10 9 12 PRT peptido-mimetic of a Lewis antigen 9 Asp Leu His Asp
Trp Val Val Gly Lys Pro Ala Gly 1 5 10 10 12 PRT peptido-mimetic of
a Lewis antigen 10 Asp Leu Tyr Asp Trp Val Val Gly Lys Pro Ala Gly
1 5 10 11 12 PRT peptido-mimetic of a Lewis antigen 11 Asp Leu Phe
Asp Trp Val Val Gly Lys Pro Ala Gly 1 5 10 12 12 PRT
peptido-mimetic of a Lewis antigen 12 Asp Leu Met Asp Trp Val Val
Gly Lys Pro Ala Gly 1 5 10 13 12 PRT peptido-mimetic of a Lewis
antigen 13 Asp Leu Ala Asp Trp Val Val Gly Lys Pro Ala Gly 1 5 10
14 12 PRT peptido-mimetic of a Lewis antigen 14 Asp Leu Glu Asp Trp
Val Val Gly Lys Pro Ala Gly 1 5 10 15 12 PRT peptido-mimetic of a
Lewis antigen 15 Asp Leu Asp Asp Trp Val Val Gly Lys Pro Ala Gly 1
5 10 16 12 PRT peptido-mimetic of a Lewis antigen 16 Asp Leu Lys
Asp Trp Val Val Gly Lys Pro Ala Gly 1 5 10 17 12 PRT
peptido-mimetic of a Lewis antigen 17 Asp Leu Arg Asp Trp Val Val
Gly Lys Pro Ala Gly 1 5 10 18 12 PRT peptido-mimetic of a Lewis
antigen 18 Asp Leu Ser Asp Trp Val Val Gly Lys Pro Ala Gly 1 5 10
19 12 PRT peptido-mimetic of a Lewis antigen 19 Asp Leu Trp Glu Trp
Val Val Gly Lys Pro Ala Gly 1 5 10 20 12 PRT peptido-mimetic of a
Lewis antigen 20 Asp Leu Trp Ser Trp Val Val Gly Lys Pro Ala Gly 1
5 10 21 12 PRT peptido-mimetic of a Lewis antigen 21 Asp Leu Trp
Pro Trp Val Val Gly Lys Pro Ala Gly 1 5 10 22 12 PRT
peptido-mimetic of a Lewis antigen 22 Asp Leu Trp Val Trp Val Val
Gly Lys Pro Ala Gly 1 5 10 23 12 PRT peptido-mimetic of a Lewis
antigen 23 Asp Leu Trp Met Trp Val Val Gly Lys Pro Ala Gly 1 5 10
24 12 PRT peptido-mimetic of a Lewis antigen 24 Asp Leu Trp Tyr Trp
Val Val Gly Lys Pro Ala Gly 1 5 10 25 12 PRT peptido-mimetic of a
Lewis antigen 25 Asp Leu Trp Asp His Val Val Gly Lys Pro Ala Gly 1
5 10 26 12 PRT peptido-mimetic of a Lewis antigen 26 Asp Leu Trp
Asp Tyr Val Val Gly Lys Pro Ala Gly 1 5 10 27 12 PRT
peptido-mimetic of a Lewis antigen 27 Asp Leu Trp Asp Met Val Val
Gly Lys Pro Ala Gly 1 5 10 28 12 PRT peptido-mimetic of a Lewis
antigen 28 Asp Leu Trp Asp Ala Val Val Gly Lys Pro Ala Gly 1 5 10
29 12 PRT peptido-mimetic of a Lewis antigen 29 Asp Leu Trp Asp Asp
Val Val Gly Lys Pro Ala Gly 1 5 10 30 12 PRT peptido-mimetic of a
Lewis antigen 30 Asp Leu Trp Asp Glu Val Val Gly Lys Pro Ala Gly 1
5 10 31 12 PRT peptido-mimetic of a Lewis antigen 31 Asp Leu Trp
Asp Lys Val Val Gly Lys Pro Ala Gly 1 5 10 32 12 PRT
peptido-mimetic of a Lewis antigen 32 Asp Leu Trp Asp Arg Val Val
Gly Lys Pro Ala Gly 1 5 10 33 12 PRT peptido-mimetic of a Lewis
antigen 33 Asp Leu Trp Asp Ser Val Val Gly Lys Pro Ala Gly 1 5 10
34 12 PRT peptido-mimetic of a Lewis antigen 34 Asp Leu Trp Asp Trp
Leu Val Gly Lys Pro Ala Gly 1 5 10 35 12 PRT peptido-mimetic of a
Lewis antigen 35 Asp Leu Trp Asp Trp Tyr Val Gly Lys Pro Ala Gly 1
5 10 36 12 PRT peptido-mimetic of a Lewis antigen 36 Asp Leu Trp
Asp Trp Ala Val Gly Lys Pro Ala Gly 1 5 10 37 12 PRT
peptido-mimetic of a Lewis antigen 37 Asp Leu Trp Asp Trp Ser Val
Gly Lys Pro Ala Gly 1 5 10 38 12 PRT peptido-mimetic of a Lewis
antigen 38 Asp Leu Trp Asp Trp Val Leu Gly Lys Pro Ala Gly 1 5 10
39 12 PRT peptido-mimetic of a Lewis antigen 39 Asp Leu Trp Asp Trp
Val Ala Gly Lys Pro Ala Gly 1 5 10 40 12 PRT peptido-mimetic of a
Lewis antigen 40 Asp Leu Trp Asp Trp Val Asp Gly Lys Pro Ala Gly 1
5 10 41 12 PRT peptido-mimetic of a Lewis antigen 41 Asp Leu Trp
Asp Trp Val Asp Cys Lys Pro Ala Gly 1 5 10 42 12 PRT
peptido-mimetic of a Lewis antigen 42 Asp Leu Trp Asp Trp Val Asp
Pro Lys Pro Ala Gly 1 5 10 43 12 PRT peptido-mimetic of a Lewis
antigen 43 Asp Leu Trp Asp Trp Val Asp Asp Tyr Pro Ala Gly 1 5 10
44 12 PRT peptido-mimetic of a Lewis antigen 44 Asp Leu Trp Asp Trp
Val Asp Asp Phe Pro Ala Gly 1 5 10 45 4 PRT peptido-mimetic of a
Lewis antigen 45 Asp Leu His Glu 1 46 6 PRT peptido-mimetic of a
Lewis antigen 46 Asp Leu Trp Glu His Leu 1 5 47 12 PRT
peptido-mimetic of a Lewis antigen 47 Leu Asp Trp Glu Trp Val Val
Gly Lys Pro Ala Gly 1 5 10 48 4 PRT peptido-mimetic of a Lewis
antigen 48 Asp Leu Asp Leu 1 49 12 PRT peptido-mimetic of a Lewis
antigen 49 Glu Ile His Asp Trp Val Val Gly Lys Pro Ala Gly 1 5 10
50 6 PRT peptido-mimetic of a Lewis antigen 50 Asp Leu Trp Glu His
Leu 1 5 51 12 PRT peptido-mimetic of a Lewis antigen 51 Leu Asp Asp
Asp Trp Val Val Gly Lys Pro Ala Gly 1 5 10 52 12 PRT
peptido-mimetic of a Lewis antigen 52 Glu Ile His Glu Trp Val Val
Gly Lys Pro Ala Gly 1 5 10 53 8 PRT peptido-mimetic of a Lewis
antigen 53 Asp Leu Trp Asp His Leu Leu Ala 1 5 54 12 PRT
peptido-mimetic of a Lewis antigen 54 Leu Asp Asp Leu Trp Val Val
Gly Lys Pro Ala Gly 1 5 10 55 12 PRT peptido-mimetic of a Lewis
antigen 55 Glu Ile His Glu His Leu Val Gly Lys Pro Ala Gly 1 5 10
56 10 PRT peptido-mimetic of a Lewis antigen 56 Trp Asp Trp Val Val
Gly Lys Pro Ala Gly 1 5 10 57 9 PRT peptido-mimetic of a Lewis
antigen 57 Asp Trp Val Val Gly Lys Pro Ala Gly 1 5 58 8 PRT
peptido-mimetic of a Lewis antigen 58 Trp Val Val Gly Lys Pro Ala
Gly 1 5 59 7 PRT peptido-mimetic of a Lewis antigen 59 Val Val Gly
Lys Pro Ala Gly 1 5 60 6 PRT peptido-mimetic of a Lewis antigen 60
Val Gly Lys Pro Ala Gly 1 5 61 5 PRT peptido-mimetic of a Lewis
antigen 61 Gly Lys Pro Ala Gly 1 5 62 4 PRT peptido-mimetic of a
Lewis antigen 62 Lys Pro Ala Gly 1 63 12 PRT peptido-mimetic of a
Lewis antigen 63 Asp Leu Trp Asp Phe Val Val Gly Lys Pro Ala Gly 1
5 10 64 12 PRT peptido-mimetic of a Lewis antigen 64 Asp Leu Trp
Asp Trp Val Ile Gly Lys Pro Ala Gly 1 5 10 65 12 PRT
peptido-mimetic of a Lewis antigen 65 Asp Leu Trp Asp Trp Val Val
Ala Lys Pro Ala Gly 1 5 10 66 12 PRT peptido-mimetic of a Lewis
antigen 66 Asp Leu Trp Asp Trp Val Val Ser Lys Pro Ala Gly 1 5 10
67 12 PRT peptido-mimetic of a Lewis antigen 67 Asp Leu Trp Asp Trp
Val Val Glu Lys Pro Ala Gly 1 5 10 68 12 PRT peptido-mimetic of a
Lewis antigen 68 Asp Leu Trp Asp Trp Val Val Asp Lys Pro Ala Gly 1
5 10 69 12 PRT peptido-mimetic of a Lewis antigen 69 Asp Leu Trp
Asp Trp Val Val Gly Glu Pro Ala Gly 1 5 10 70 12 PRT
peptido-mimetic of a Lewis antigen 70 Asp Leu Trp Asp Trp Val Val
Gly Asp Pro Ala Gly 1 5 10 71 12 PRT peptido-mimetic of a Lewis
antigen 71 Asp Leu Trp Asp Trp Val Val Gly Lys Glu Ala Gly 1 5 10
72 12 PRT peptido-mimetic of a Lewis antigen 72 Asp Leu Trp Asp Trp
Val Val Gly Lys Asp Ala Gly 1 5 10 73 12 PRT peptido-mimetic of a
Lewis antigen 73 Asp Leu Trp Asp Trp Val Val Gly Lys Pro Asp Gly 1
5 10 74 12 PRT peptido-mimetic of a Lewis antigen 74 Asp Leu Trp
Asp Trp Val Val Gly Lys Pro Ala Asp 1 5 10 75 12 PRT
peptido-mimetic of a Lewis antigen 75 Asp Leu Trp Asp Trp Val Lys
Glu Lys Pro Ala Gly 1 5 10 76 12 PRT peptido-mimetic of a Lewis
antigen 76 Asp Leu Trp Asp Trp Val Leu Ala Lys Pro Ala Gly 1 5 10
77 12 PRT peptido-mimetic of a Lewis antigen 77 Asp Leu Trp Asp Trp
Val Val Gly Glu Asp Ala Gly 1 5 10 78 12 PRT peptido-mimetic of a
Lewis antigen 78 Asp Leu Trp Asp Trp Val Val Gly Lys Pro Glu Lys 1
5 10 79 12 PRT peptido-mimetic of a Lewis antigen 79 Asp Leu Trp
Asp Trp Val Lys Glu Glu Pro Ala Gly 1 5 10 80 12 PRT
peptido-mimetic of a Lewis antigen 80 Asp Leu Trp Asp Trp Val Val
Gly Lys Asp Glu Lys 1 5 10 81 12 PRT peptido-mimetic of a Lewis
antigen 81 Asp Leu Trp Asp Trp Val Val Gly Glu Asp Glu Lys 1 5 10
82 12 PRT peptido-mimetic of a Lewis antigen 82 Asp Leu Trp Asp Trp
Val Lys Glu Glu Asp Glu Lys 1 5 10 83 8 PRT peptido-mimetic of a
Lewis antigen 83 Ala Pro Trp Leu Tyr Ala Gly Pro 1 5 84 12 PRT
peptido-mimetic of a Lewis antigen 84 Ala Ser Ala Val Asn Leu Tyr
Ile Pro Thr Gln Glu 1 5 10 85 12 PRT peptido-mimetic of a Lewis
antigen 85 Val Tyr Leu Ala Pro Gly Arg Ile Ser Arg Asp Tyr 1 5 10
86 12 PRT peptido-mimetic of a Lewis antigen 86 Val Tyr Leu Ala Pro
Gly Arg Phe Ser Arg Asp Tyr 1 5 10 87 12 PRT peptido-mimetic of a
Lewis antigen 87 Cys Thr Ser His Trp Gly Val Leu Ser Gln Arg Arg 1
5 10 88 12 PRT peptido-mimetic of a Lewis antigen 88 Arg Val Leu
Ser Pro Glu Ser Tyr Leu Gly Pro Ser 1 5 10 89 12 PRT
peptido-mimetic of a Lewis antigen 89 Arg Val Leu Ser Pro Glu Ser
Tyr Leu Gly Pro Ala 1 5 10 90 11 PRT peptido-mimetic of a Lewis
antigen 90 Val Gly Asn Gly Val Leu Met Gly Arg Arg Gly 1 5 10 91 12
PRT peptido-mimetic of a Lewis antigen 91 Ser Thr Gly Leu Met Val
Asp Phe Leu Glu Pro Gly 1 5 10 92 12 PRT peptido-mimetic of a Lewis
antigen 92 Arg Val Leu Ser Pro Glu Ser Tyr Leu Gly Pro Ala 1 5 10
93 12 PRT peptido-mimetic of a Lewis antigen 93 Gly Asn Cys Arg Tyr
Ile Gly Leu Arg Gln Phe Gly 1 5 10 94 12 PRT peptido-mimetic of a
Lewis antigen 94 Asp Ile Arg Val Glu Pro Gly Gly Gly Tyr Thr His 1
5 10 95 12 PRT peptido-mimetic of a Lewis antigen 95 Ala Lys Thr
Phe Gly Leu Glu His Gly Cys Glu Ala 1 5 10 96 12 PRT
peptido-mimetic of a Lewis antigen 96 Ala Pro Ile His Thr Tyr Thr
Gly Arg Ala Arg Gly 1 5 10 97 13 PRT peptido-mimetic of a Lewis
antigen 97 Arg His Thr Cys Val Arg Ser Cys Cys Gly His Asp Arg 1 5
10 98 12 PRT peptido-mimetic of a Lewis antigen 98 Thr Lys Arg Pro
Asp Leu Ile Val Asp Pro Ile Pro 1 5 10 99 12 PRT peptido-mimetic of
a Lewis antigen 99 Asp Glu Val Arg Pro Asp Leu Ile Ser Thr Glu Glu
1 5 10 100 12 PRT peptido-mimetic of a Lewis antigen MISC_FEATURE
(8)..(8) Xaa in position 8 can be any amino acid 100 Asn Leu Arg
Pro Lys Tyr Ile Xaa Leu Asp Ala Asp 1 5 10 101 12 PRT
peptido-mimetic of a Lewis antigen 101 Thr Leu Ile Ala Phe Ala Asp
Leu Val Asp Val Ile 1 5 10 102 12 PRT peptido-mimetic of a Lewis
antigen 102 Val Gly Ile Trp Ser Val Val Ser Glu Gly Ser Arg 1 5 10
103 12 PRT peptido-mimetic of a Lewis antigen 103 Arg Cys Ser Val
Gly Val Pro Phe Thr Met Glu Ser 1 5 10 104 12 PRT peptido-mimetic
of a Lewis antigen 104 Gln Asp Gly Val Trp Glu His Val Leu Glu Gly
Gly 1 5 10 105 12 PRT peptido-mimetic of a Lewis antigen 105 Val
Glu Leu Ser Gly Arg Gly Gly Leu Cys Thr Trp 1 5 10 106 12 PRT
peptido-mimetic of a Lewis antigen 106 Val Ile Gly Ala Ala Ser His
Asp Glu Asp Val Asp 1 5 10 107 12 PRT peptido-mimetic of a Lewis
antigen 107 Thr Ile Glu Pro Val Leu Ala Glu Met Phe Met Gly 1 5 10
108 12 PRT peptido-mimetic of a Lewis antigen 108 Asp Lys Glu Thr
Phe Glu Leu Gly Leu Phe Asp Arg 1 5 10 109 12 PRT peptido-mimetic
of a Lewis antigen 109 Phe Ser Gly Val Arg Gly Val Tyr Glu Ser Arg
Thr 1 5 10 110 12 PRT peptido-mimetic of a Lewis antigen 110 Pro
Asp Asp Ala Pro Met His Ser Thr Arg Val Glu 1 5 10 111 9 PRT
peptido-mimetic of a Lewis antigen MISC_FEATURE (2)..(2) Xaa in
position 2 can be any amino acid 111 Gly Xaa Trp Xaa Xaa Val Leu
Glu Gly 1 5 112 5 PRT peptido-mimetic of a Lewis antigen
MISC_FEATURE (4)..(4) Xaa in position 4 can be any amino acid 112
Val Val Gly Xaa Pro 1 5 113 4 PRT peptido-mimetic of a Lewis
antigen 113 Arg Pro Asp Leu 1 114 5 PRT peptido-mimetic of a Lewis
antigen 114 Phe Ser Leu Leu Trp 1 5 115 12 PRT peptido-mimetic of a
Lewis antigen 115 Gly Gly Thr Val Glu Val Trp Ser Ile Lys Gly Gly 1
5 10 116 12 PRT peptido-mimetic of a Lewis antigen 116 Asp His Phe
Ser Gln Ala Gly Ser Ser Asn His His 1 5 10 117 12 PRT
peptido-mimetic of a Lewis antigen 117 Asp Asp Pro Val Thr Pro Val
Ile Asp Phe Gly Lys 1 5 10 118 12 PRT peptido-mimetic of a Lewis
antigen 118 Arg Asp Gly Leu Ile Asp Phe Val Val Ala Gly Thr 1 5 10
119 15 PRT peptido-mimetic of a Lewis antigen 119 Gly Leu Asp Leu
Leu Gly Asp Val Arg Ile Pro Val Val Arg Arg 1 5 10 15 120 15 PRT
peptido-mimetic of a Lewis antigen 120 Val Gly Ile Thr Gly Phe Val
Asp Pro Leu Pro Leu Arg Leu Leu 1 5 10 15 121 8 PRT peptido-mimetic
of a Lewis antigen 121 Ala Pro Trp Leu Tyr Gly Pro Ala 1 5
* * * * *