U.S. patent application number 10/269842 was filed with the patent office on 2003-07-17 for methods and compositions for the inhibition of cancer metastasis mediated by endothelial adhesion molecules.
This patent application is currently assigned to John L. Magnani. Invention is credited to Berg, Ellen L., Butcher, Eugene C., Magnani, John L..
Application Number | 20030133937 10/269842 |
Document ID | / |
Family ID | 27104142 |
Filed Date | 2003-07-17 |
United States Patent
Application |
20030133937 |
Kind Code |
A1 |
Magnani, John L. ; et
al. |
July 17, 2003 |
Methods and compositions for the inhibition of cancer metastasis
mediated by endothelial adhesion molecules
Abstract
Methods and compositions are disclosed for the inhibition of
cancer metastases mediated by endothelial adhesion molecules. The
present invention discloses that sialyl Le.sup.a and di-sialyl
Le.sup.a, which are expressed at the surface of cancer cells,
function as a binding partner for LEC-CAMs, such as ELAM-1, which
are expressed at the surface of endothelial cells. The present
invention also discloses that LEC-CAMs, such as ELAM-1, involved in
cancer metastasis share a carbohydrate domain common to both sialyl
Le.sup.a and sialyl Le.sup.x. Antibodies, saccharides,
glycoconjugates, enzyme inhibitors and other compounds may be used
in the methods of the present invention to inhibit the binding of
malignant cells to endothelial cells for a variety of purposes in
vivo and in vitro.
Inventors: |
Magnani, John L.;
(Gaithersburg, MD) ; Butcher, Eugene C.; (Portola
Valley, CA) ; Berg, Ellen L.; (Palo Alto,
CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
John L. Magnani
Rockville
MD
|
Family ID: |
27104142 |
Appl. No.: |
10/269842 |
Filed: |
October 10, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10269842 |
Oct 10, 2002 |
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09447532 |
Nov 23, 1999 |
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6465434 |
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09447532 |
Nov 23, 1999 |
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08238684 |
May 5, 1994 |
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6121233 |
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08238684 |
May 5, 1994 |
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07721771 |
Jun 25, 1991 |
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07721771 |
Jun 25, 1991 |
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07688037 |
Apr 19, 1991 |
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Current U.S.
Class: |
424/155.1 ;
514/19.1; 514/19.8; 514/20.9; 514/54 |
Current CPC
Class: |
C07K 14/70564 20130101;
A61K 47/56 20170801; C07H 13/04 20130101; A61K 38/47 20130101; C07K
16/30 20130101 |
Class at
Publication: |
424/155.1 ;
514/8; 514/54 |
International
Class: |
A61K 038/17; A61K
039/395; A61K 031/739 |
Claims
1. A method for inhibiting within a biological preparation the
binding of malignant cells expressing sialyl Le.sup.a or di-sialyl
Le.sup.a, to endothelial cells expressing a LEC-CAM, comprising:
incubating the biological preparation with at least one agent that
inhibits the binding of malignant cells expressing sialyl Le.sup.a
or di-sialyl Le.sup.a, to endothelial cells expressing a
LEC-CAM.
2. The method of claim 1 wherein the agent is a saccharide, a
glycoconjugate or an antibody that inhibits the binding of sialyl
Le.sup.a or di-sialyl Le.sup.a to a LEC-CAM.
3. A method for inhibiting within a biological preparation the
binding of malignant cells expressing sialyl Le.sup.a or di-sialyl
Le.sup.a, to endothelial cells expressing ELAM-1, comprising:
incubating the biological preparation with at least one agent that
inhibits the binding of malignant cells expressing sialyl Le.sup.a
or di-sialyl Le.sup.a, to endothelial cells expressing ELAM-1.
4. The method of claim 3 wherein the agent is a saccharide, a
glycoconjugate or an antibody that inhibits the binding of sialyl
Le.sup.a or di-sialyl Le.sup.a to ELAM-1.
5. A method for inhibiting within a biological preparation the
binding of malignant cells expressing sialyl Le.sup.a or di-sialyl
Le.sup.a, to endothelial cells expressing a LEC-CAM, comprising:
incubating said malignant cells with at least one enzyme inhibitor
that inhibits the biosynthesis of sialyl Le.sup.a or di-sialyl
Le.sup.a by said malignant cells.
6. A method for inhibiting within a biological preparation the
binding of malignant cells expressing sialyl Le.sup.a or di-sialyl
Le.sup.a, to endothelial cells expressing ELAM-1, comprising:
incubating said malignant cells with at least one enzyme inhibitor
that inhibits the biosynthesis of sialyl Le.sup.a or di-sialyl
Le.sup.a by said malignant cells.
7. A compound having the formula: 4wherein x, y and z are
independently selected from saccharides or y or z or both are not
present, and R is H, OH, lipid, ceramide, or one or more amino
acids, with the proviso that x, y and z are not present in the
combination wherein x is GlcNAc, y is Gal and z is Glc.
8. A compound having the formula: 5wherein x, y and z are
independently selected from saccharides or y or z or both are not
present, and R is H, OH, lipid, ceramide, or one or more amino
acids, with the proviso that x, y and z are not present in the
combination wherein x is GlcNAc, y is Gal and z is Glc.
9. A method for inhibiting in a warm-blooded animal the spread of
malignant cells expressing sialyl Le.sup.a or di-sialyl Le.sup.a,
to secondary sites, comprising: administering to a warm-blooded
animal an effective amount of at least one agent that inhibits the
binding of malignant cells expressing sialyl Le.sup.a or di-sialyl
Le.sup.a, to endothelial cells expressing a LEC-CAM.
10. The method of claim 9 wherein the agent is a saccharide, a
glycoconjugate or an antibody that inhibits the binding of sialyl
Le.sup.a or di-sialyl Le.sup.a to a LEC-CAM.
11. A method for inhibiting in a warm-blooded animal the spread of
malignant cells expressing sialyl Le.sup.a or di-sialyl Le.sup.a,
to secondary sites by hematogenous metastases, comprising:
administering to a warm-blooded animal an effective amount of at
least one agent that inhibits the binding of malignant cells
expressing sialyl Le.sup.a or di-sialyl Le.sup.a, to endothelial
cells expressing ELAM-1.
12. The method of claim 11 wherein the agent is a saccharide, a
glycoconjugate or an antibody that inhibits the binding of sialyl
Le.sup.a or di-sialyl Le.sup.a to ELAM-1.
13. A method for inhibiting in a warm-blooded animal the spread of
malignant cells expressing sialyl Le.sup.a or di-sialyl Le.sup.a,
to secondary sites, comprising: administering to a warm-blooded
animal an effective amount of at least one enzyme inhibitor that
inhibits the biosynthesis of sialyl Le.sup.a or di-sialyl Le.sup.a
by said malignant cells.
14. A method for inhibiting in a warm-blooded animal the spread of
malignant cells expressing sialyl Le.sup.a or di-sialyl Le.sup.a,
to secondary sites by hematogenous metastases, comprising:
administering to a warm-blooded animal an effective amount of at
least one enzyme inhibitor that inhibits the biosynthesis of sialyl
Le.sup.a or di-sialyl Le.sup.a by said malignant cells.
15. A method for inhibiting within a biological preparation the
binding of malignant cells expressing sialyl Le.sup.a, di-sialyl
Le.sup.a or sialyl Le.sup.x, to endothelial cells expressing a
LEC-CAM, comprising: incubating the biological preparation with at
least one agent capable of reacting with both sialyl Le.sup.a and
sialyl Le.sup.x.
16. A method for inhibiting within a biological preparation the
binding of malignant cells expressing sialyl Le.sup.a, di-sialyl
Le.sup.a or sialyl Le.sup.x, to endothelial cells expressing
ELAM-1, comprising: incubating the biological preparation with at
least one agent capable of reacting with both sialyl Le.sup.a and
sialyl Le.sup.x.
17. A method for inhibiting in a warm-blooded animal the spread of
malignant cells expressing sialyl Le.sup.a, di-sialyl Le.sup.a or
sialyl Le.sup.x, to secondary sites, comprising: administering to a
warm-blooded animal an effective amount of at least one agent
capable of reacting with both sialyl Le.sup.a and sialyl
Le.sup.x.
18. A method for inhibiting in a warm-blooded animal the spread of
malignant cells expressing sialyl Le.sup.a, di-sialyl Le.sup.a or
sialyl Le.sup.x, to secondary sites by hematogenous metastases,
comprising: administering to a warm-blooded animal an effective
amount of at least one agent capable of reacting with both sialyl
Le.sup.a and sialyl Le.sup.x.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part to Ser. No.
688,037, filed Apr. 19, 1991, which is incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention is generally directed toward the
inhibition of cancer metastasis mediated by endothelial adhesion
molecules, and more specifically, toward such inhibition through
the use of saccharides, glycoconjugates, antibodies, enzyme
inhibitors, and other agents which disrupt such binding of cancer
cells to endothelia.
BACKGROUND OF THE INVENTION
[0003] Despite enormous investments of financial and human
resources, cancer remains one of the major causes of death. Current
cancer therapies cure only about fifty percent of the patients who
develop a malignant tumor. In most human malignancies, metastasis
is the major cause of death.
[0004] Metastasis is the formation of a secondary tumor colony at a
distant site. It is a multistep process of which tumor invasion is
an early event. Tumor cells locally invade host tissue barriers,
such as the epithelial basement membrane, to reach the interstitial
stroma, where they gain access to blood vessels ("hematogenous
metastasis") or lymphatic channels for further dissemination. After
invading the endothelial layer of a vessel wall, the circulating
tumor cells are dislodged into the circulation and arrest in the
precapillary venules of the target organ by adherence to
endothelial cell lumenal surfaces, or exposed basement membranes.
The tumor cells again invade the vascular wall to enter the organ
parenchyma. Finally, the extravasated tumor cell grows in a tissue
different from where it originated.
[0005] Most cancer cells fail to survive in the circulation and it
appears that normally the lining of blood vessels acts as a barrier
to tumor cell extravasation. Endothelial injury or perturbation
increases tumor metastasis. In addition, certain factors, such as
cytokines, have been shown to substantially increase the adhesion
of cancer cells to treated endothelium in vitro. Interleukin 1
(IL-1) and tumor necrosis factor (TNF), which are cytokines, each
stimulate the biosynthesis and expression of a cell surface
receptor called ELAM-1 (endothelial leukocyte adhesion molecule)
ELAM-1 is a member of a family of calcium-dependent cell adhesion
receptors, known as LEC-CAMs or selectins, which includes LECAM-1
and GMP-140 (also known as PADGEM or CD62). During an inflammatory
response, ELAM-1 on endothelial cells functions as a "homing
receptor" for leukocytes. Recently, ELAM-1 on endothelial cells was
shown to mediate the increased adhesion of colon cancer cells to
endothelium treated with cytokines (Rice and Bevilacqua, Science
246:1303-1306, 1989).
[0006] In most human malignancies, distant metastases are often too
small to be detected at the time the primary tumor is treated.
Furthermore, widespread initiation of metastatic colonies usually
occurs before clinical symptoms of metastatic disease are evident.
The size and age variation in metastases, their dispersed
anatomical location, and their heterogeneous composition are all
factors that hinder surgical removal and limit the concentration of
anticancer drugs that can be delivered to the metastatic colonies.
It has been estimated, for example, that in 1991 there will be over
60,000 deaths and over 150,000 new cases from just colorectal
cancer in the U.S. alone.
[0007] Due to the difficulties in the current approaches to the
treatment and prevention of metastases, there is a need in the art
for improved methods and compositions for inhibiting metastasis
mediated by endothelial adhesion molecules. The present invention
fills this need, and further provides other related advantages.
SUMMARY OF THE INVENTION
[0008] Briefly stated, the present invention provides methods and
compositions for the inhibition of cancer metastasis mediated by
endothelial adhesion molecules. In one aspect, the present
invention provides methods for inhibiting, within a biological
preparation, the binding of malignant cells expressing sialyl
Le.sup.a or di-sialyl Le.sup.a, to endothelial cells. In one
embodiment, the method comprises incubating the biological
preparation with at least one agent that inhibits the binding of
malignant cells expressing sialyl Le.sup.a or di-sialyl Le.sup.a,
to endothelial cells expressing a LEC-CAM. In another embodiment,
the method comprises incubating the biological preparation with at
least one agent that inhibits the binding of malignant cells
expressing sialyl Le.sup.a or di-sialyl Le.sup.a, to endothelial
cells expressing ELAM-1. In another embodiment, the method
comprises incubating the malignant cells with at least one enzyme
inhibitor that inhibits the biosynthesis of sialyl Le.sup.a or
di-sialyl Le.sup.a by the malignant cells.
[0009] In another aspect of the present invention, methods are
provided for inhibiting the spread of malignant cells expressing
sialyl Le.sup.a or di-sialyl Le.sup.a, to secondary sites in a
warm-blooded animal. In one embodiment, the method comprises
administering to a warm-blooded animal an effective amount of at
least one agent that inhibits the binding of malignant cells
expressing sialyl Le.sup.a or di-sialyl Le.sup.a, to endothelial
cells expressing a LEC-CAM. In another embodiment involving
hematogenous metastasis, the method comprises administering to a
warm-blooded animal an effective amount of at least one agent that
inhibits the binding of malignant cells expressing sialyl Le.sup.a
or di-sialyl Le.sup.a, to endothelial cells expressing ELAM-1. In
another embodiment, the method comprises administering to a
warm-blooded animal an effective amount of at least one enzyme
inhibitor that inhibits the biosynthesis of sialyl Le.sup.a or
di-sialyl Le.sup.a by the malignant cells.
[0010] In a related aspect, methods are provided for inhibiting
within a biological preparation the binding of malignant cells
expressing sialyl Le.sup.a, di-sialyl Le.sup.a or sialyl Le.sup.x,
to endothelial cells. In one embodiment, the method comprises
incubating a biological preparation, containing endothelial cells
expressing a LEC-CAM, with at least one agent capable of reacting
with both sialyl Le.sup.a and sialyl Le.sup.x. In another
embodiment, the method comprises incubating a biological
preparation, containing endothelial cells expressing ELAM-1, with
at least one agent capable of reacting with both sialyl Le.sup.a
and sialyl Le.sup.x.
[0011] In another related aspect, methods are provided for
inhibiting the spread of malignant cells expressing sialyl
Le.sup.a, di-sialyl Le.sup.a or dialyl Le.sup.x, to secondary sites
in a warm-blooded animal. In one embodiment, the method comprises
administering to a warm-blooded animal an effective amount of at
least one agent capable of reacting with both sialyl Le.sup.a and
sialyl Le.sup.x. In another embodiment involving hematogenous
metastasis, the method comprises administering to a warm-blooded
animal an effective amount of at least one agent capable of
reacting with both sialyl Le.sup.a and sialyl Le.sup.x.
[0012] These and other aspects of the present invention will become
evident upon reference to the following detailed description and
attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 describes pictorially a cell binding assay used to
assess binding of ELAM-1 transfected cells to neoglycoproteins.
[0014] FIG. 2 graphically illustrates the relative binding of
ELAM-1 transfected cells to certain neoglycoproteins.
[0015] FIG. 3 graphically illustrates the relative binding of
ELAM-1 transfected cells to certain neoglycoproteins.
DETAILED DESCRIPTION OF THE INVENTION
[0016] Prior to setting forth the invention, it may be helpful to
an understanding thereof to set forth definitions of certain terms
to be used hereinafter.
[0017] Antibody--as used herein, includes both monoclonal and
polyclonal antibodies and may be an intact molecule, a fragment
thereof, or a functional equivalent thereof. The antibody may be
genetically engineered. Examples of antibody fragments include
F(ab').sub.2, Fab', Fab and Fv.
[0018] Saccharide--as used herein, includes oligosaccharides, and
may be naturally derived, synthetically prepared, portions of
either, and derivatives of any of the foregoing.
[0019] Glycoconiugate--as used herein, includes a saccharide which
is coupled to a non-saccharide molecule, e.g., a lipid or a
polypeptide.
[0020] As noted above, the present invention is generally directed
towards methods and compositions for the inhibition of cancer
metastasis mediated by endothelial adhesion molecules. More
specifically, the disclosure of the present invention shows that
antibodies, saccharides, glycoconjugates therefrom or enzyme
inhibitors may be used to inhibit the binding of malignant cells to
endothelial cells for a variety of purposes in vivo and in
vitro.
[0021] As described above, metastasis is a multistep process.
During metastasis, cancer cells circulate through the microvascular
and lymph systems and then migrate through the walls of the blood
or lymph vessels to establish a new and aggressive tumor at a
secondary organ site. A critical step in the metastasis process is
the adherence of circulating cancer cells to the endothelial lining
of blood vessel or lymph vessel walls. As disclosed within the
present invention, the carbohydrates sialyl Le.sup.a and di-sialyl
Le.sup.a, which are expressed at the surface of certain cancer
cells, function as a ligand (i.e., binding partner) for LEC-CAMs,
such as ELAM-1, which are expressed at the surface of endothelial
cells. Therefore, for those cancer cells, metastasis involves the
adherence of cancer cells to the endothelial cells via the binding
of sialyl Le.sup.a and/or di-sialyl Le.sup.a on the cancer cells to
adhesion molecules on endothelial cells. Other cancer cells express
predominantly sialyl Le.sup.x, or sialyl Le.sup.x and sialyl
Le.sup.a (and/or di-sialyl Le.sup.a). The present invention
discloses that LEC-CAMs, such as ELAM-1, share a carbohydrate
domain common to both sialyl Le.sup.a and sialyl Le.sup.x on
malignant cells, and therefore agents can be produced which are
capable of binding to both. Inhibition of the initial binding event
between LEC-CAMs and sialylated structures by the methods of the
present invention prevents the adhesion of metastatic cells to the
endothelial lining of blood or lymph vessel walls, thereby
eliminating the spread of metastatic cells to secondary organs.
Suitable blocking agents include those which inhibit the binding of
malignant cells expressing sialyl Le.sup.a, di-sialyl Le.sup.a, or
sialyl Le.sup.x, to endothelial cells expressing LEC-CAM adhesion
molecules such as ELAM-1. Representative agents include antibodies,
saccharides and glycoconjugates therefrom.
[0022] The antibodies employed in the present invention may be
polyclonal or monoclonal antibodies. Briefly, polyclonal antibodies
may be produced by immunization of an animal and subsequent
collection of its sera. Immunization is accomplished, for example,
by a systemic administration, such as by subcutaneous, intrasplenic
or intramuscular injection, into a rabbit, rat or mouse. It is
generally preferred to follow the initial immunization with one or
more booster immunizations prior to sera collection. Such
methodology is well known and described in a number of
references.
[0023] Monoclonal antibodies (MAbs) suitable within the present
invention include those of murine or human origin, or chimeric
antibodies such as those which combine portions of both human and
murine antibodies (i.e., antigen binding region of murine antibody
plus constant regions of human antibody). Human and chimeric
antibodies may be produced using methods known by those skilled in
the art. Human antibodies and chimeric human-mouse antibodies are
advantageous because they are less likely than murine antibodies to
cause the production of anti-antibodies when administered
clinically.
[0024] MAbs may be generally produced by the method of Kohler and
Milstein (Nature 256:495-497, 1975; Eur. J. Immunol. 6:511-519,
1976). Briefly, the lymph nodes and/or spleens of an animal
immunized with sialyl Le.sup.a or di-sialyl Le.sup.a are fused with
myeloma cells to form hybrid cell lines ("hybridomas" or "clones").
Each hybridoma secretes a single type of immunoglobulin and, like
the myeloma cells, has the potential for indefinite cell division.
It may be desirable to couple such molecules to a carrier to
increase their immunogenicity. Suitable carriers include keyhole
limpet hemocyanin, thyroglobulin, bovine serum albumin and
derivatives thereof. An alternative to the production of MAbs via
hybridomas is the creation of MAb expression libraries using
bacteriophage and bacteria (e.g., Sastry et al., Proc. Natl. Acad.
Sci USA 86:5728, 1989; Huse et al., Science 246:1275, 1989).
Selection of antibodies exhibiting appropriate specificity may be
performed in a variety of ways which will be evident to those
skilled in the art. Typically, such antibodies will selectively
bind with an affinity of about 10.sup.7 liters/mol or higher.
[0025] Representative examples of MAbs suitable within the present
invention include N-19-9 and HECA-452 for sialyl Le.sup.a, and FH-7
for di-sialyl Le.sup.a. MAb N-19-9 is available from ATCC (American
Type Tissue Collection, Rockville, Md.) as ATCC HB 8059 or may be
produced as described in U.S. Pat. No. 4,471,057 (and Somatic Cell
Genet. 5:957-971, 1979; J. Biol. Chem. 257:14365, 1982). MAb
HECA-452 may be produced according to Duijvestijn et al., Am. J.
Path. 130:147-155, 1988. FH-7 may be produced according to Nudelman
et al., J. Biol. Chem. 261:5487, 1986.
[0026] In addition to antibodies which are capable of binding to
sialyl Le.sup.a, di-sialyl Le.sup.a or sialyl Le.sup.x, saccharides
and glycoconjugates therefrom may also inhibit the binding of
metastatic cells expressing sialyl Le.sup.a, di-sialyl Le.sup.a or
sialyl Le.sup.x, to endothelia. As used herein, the terms "sialyl
Le.sup.a" and "di-sialyl Le.sup.a" represent structures I and II,
respectively, as follows: 1
[0027] Neu5Ac represents sialic acid; Gal represents galactose;
GlcNAc represents N-acetyl-glucosamine; Fuc represents fucose and R
is typically a ceramide (with a glucose residue interposed) or a
protein. Sialyl Le.sup.x is an isomer of sialyl Le.sup.a wherein
the Gal-GlcNAc linkage is .beta.1-4 and the Fuc-GlcNAc linkage is
.alpha.1.fwdarw.3. Saccharides suitable within the present
invention include the carbohydrate portion of sialyl Le.sup.a or
di-s-ialyl Le.sup.a (i.e., formula I or II minus R), and
derivatives of either, including those which cross-react with both
sialyl Le.sup.a and sialyl Le.sup.x. Derivatives of these compounds
include substitution of individual saccharide residues with other
saccharide residues and/or with non-saccharide molecules such as
hexyl rings without hydroxyl groups. For example, the internal
GlcNAc may be replaced with another saccharide residue such as a
glucose (Glc). Alternatively (or in addition to substitutions), the
carbohydrate portion of sialyl Le.sup.a, di-sialyl Le.sup.a, or
derivatives thereof, may be truncated by deletion of one or more
saccharide residues. For example, a tetrasaccharide may be created
with the structure: 2
[0028] Given the teachings described herein, it will be evident to
those skilled in the art that other saccharides will be suitable
within the present invention.
[0029] A saccharide may be coupled to a non-saccharide molecule to
form a glycoconjuqate. For example, a saccharide may be linked to a
polyacrylamide. Alternatively, a saccharide may be linked to a
lipid. Typical lipids include ceramide, i.e., sphingolipid bases
which are acylated on the amine with a fatty acid. For example,
sialyl Le.sup.a, di-sialyl Le.sup.a, or a saccharide cross-reaction
with sialyl Le.sup.a and sialyl Le.sup.x may be linked to a
ceramide. Alternatively, a saccharide may be bonded to an amino
acid or an amino acid-containing molecule, such as a peptide, a
polypeptide or a protein. Saccharides are naturally linked to an
amino acid or amino acid-containing molecule via the hydroxyl group
of a serine or threonine amino acid residue, but can also be linked
through other groups such as an amino group.
[0030] Saccharides and glycoconjugates provided by the present
invention may be represented by structures III and IV as follows:
3
[0031] R includes H, OH, lipid, ceramide, or one or more amino
acids; x, y and z are independently selected from saccharides, or
either y or z or both may be absent.
[0032] Numerous methods for preparing saccharides and
glycoconjugates are well known to those skilled in the art.
Saccharides may be prepared synthetically using chemical, and/or
enzymatic, reagents and techniques. For example, sialyl Le.sup.a
saccharides have been prepared by enzymatic synthesis (e.g., Palcic
et al., Carbohydr. Res. 190:1-11, 1989). Glycoconjugates may be
prepared, for example, through reductive amination. The method of
Zopf et al. (Meth. Enzymol. 50:171-175, 1978; Jeffrey et al.,
Biochem. Biophys. Res. Commun. 62:608-613, 1975) involves
4-aminophenethylamine derivatives of saccharides via reductive
amination using sodium borohydride. In brief, sugars are first
reacted with the amino reagent by dissolving them in the neat
reagent for 15 hours. Sodium borohydride in ethanol is then added.
After 5 hours, the product is separated from the reagent by gel
filtration and ion exchange chromatography. The derivatives may
then be coupled to a molecule containing a group which is reactive
with amines. The same amine derivative may be coupled to
saccharides using sodium cyanoborohydride. (Svensson et al., J.
Immunol. Meth. 25:323-335, 1979). In brief, a sugar is dissolved in
water, and the same volume of amine (a 170-fold molar excess) is
added together with sodium cyanoborohydride (a ten-fold molar
excess). The reduction is performed at pH 8 for 48 hours, and the
product purified by gel chromatography. Coupling to different
molecules, such as proteins, may be performed by the isothiocyanate
coupling method.
[0033] Another example of a reagent suitable for preparing
glycoconjugates by reductive amination is
p-trifluoroacetamidoaniline (TFAN). The reductive amination
reaction is carried out in aqueous solution overnight at pH 5-6
with sodium cyanoborohydride as the reducing agent. Typically, a
5-fold excess of TFAN is used. TFAN-derivatized saccharides are
generally protected from oxidation by N-acetylation, e.g., by
treatment with methanolic acetic anhydride, to yield
TFAc-derivatives. Prior to conjugation, the N-trifluoroacetamido
protective group is removed by treatment of the TFAc derivative
with aqueous ammonia or 0.5 M sodium hydroxide for 3 hours.
Conjugation of the derivatives to molecules, for example to
proteins such as bovine serum albumin (BSA), may be achieved by
isothiocyanate coupling methods. Other examples of suitable
reagents and reactions include p-tetradecylaniline derivatives of
saccharides and the preparation of aminoalditols by oxidation of
saccharide TFAN derivates with cerium ammonium sulfate (Lindenberg
et al., J. Reprod. Fert. 89:431-439, 1990).
[0034] The inhibition of the binding of cancer cells expressing
sialyl Le.sup.a, di-sialyl Le.sup.a or sialyl Le.sup.x, to
endothelia has a variety of in vitro and in vivo uses. Sialyl
Le.sup.a and di-sialyl Le.sup.a are type 1 carbohydrate chains
(i.e., have a Gal.beta.1.fwdarw.3GlcNAc polylactosamine unit
structure) and sialyl Le.sup.x is a type 2 carbohydrate chain
(i.e., has a Gal.beta.1.fwdarw.4 GlcNAc polylactosamine unit
structure. A number of cancer cells, such as colorectal and
pancreatic, have a prevalence of type 1 carbohydrate chains
including sialyl Le.sup.a and di-sialyl Le.sup.a. Other cancer
cells, such as breast, lung and ovarian, have a prevalence of type
2 carbohydrate chains including sialyl Le.sup.x.
[0035] Regarding in vitro aspects, as noted above, the present
invention provides methods for inhibiting the binding of cancer
cells to endothelia in a biological preparation. Representative
examples of biological preparations include blood vessel and/or
lymph vessel endothelia in combination with a malignancy. The
endothelia and the malignancy may be in the form of tissue or cells
removed from an organism, or cultured cells. In one embodiment, the
method comprises incubating a biological preparation, which
contains malignant cells expressing sialyl Le.sup.a, di-sialyl
Le.sup.a or sialyl Le.sup.x and endothelial cells expressing a
LEC-CAM, with an effective amount of at least one agent, such as an
antibody, saccharide or glycoconjugate as described above. In
another embodiment, the method comprises incubating malignant cells
with at least one enzyme inhibitor that inhibits the biosynthesis
of sialyl Le.sup.a or di-sialyl Le.sup.a by the cells. Suitable
enzyme inhibitors include inhibitors of glycosyltransferases.
Representative examples of inhibitors for glycosyltransferases
include inhibitors for fucosyltransferases (e.g., as described by
Palcic et al., J. Biol. Chem. 264:17174-17181, 1989), for
N-acetylglucosaminyltransferases (e.g., as described by Palcic et
al., J. Biol. Chem. 265:6759-6769, 1990), and for
sialyltransferases (e.g., as described by Broquet et al., J.
Neurochem. 54:388-394, 1990; Karaivanova et al., Cancer Biochem.
Biophys. 11:311-315, 1990).
[0036] The present invention also provides methods for inhibiting
metastasis in a warm-blooded animal such as a human. In one
embodiment, the method comprises administering to a warm-blooded
animal an effective amount of at least one agent, such as an
antibody, saccharide or glycoconjugate as described above. In
another embodiment, the method comprises administering to a
warm-blooded animal an effective amount of at least one enzyme
inhibitor (as described above) that inhibits the biosynthesis of
sialyl Le.sup.a or di-sialyl Le.sup.a by malignant cells. It will
be evident to those skilled in the art how to determine the optimal
effective dose for a particular agent or enzyme inhibitor, e.g.,
based upon in vitro and in vivo studies in non-human animals. A
variety of routes of administration may be used. Typically,
administration will be intravenous, intracavitory (e.g., in pleural
or peritoneal cavities), or in the bed of a resected tumor.
[0037] An agent may be administered as a composition, i.e., in
combination with a pharmaceutically acceptable carrier or diluent,
such as physiological saline. It will be recognized by those
skilled in the art that an agent and a composition may be prepared
in a sterile form. Moreover, an agent may be administered in
combination with an immunotherapeutic or chemotherapeutic agent.
When such a combination is desired, each substance may be
administered sequentially, simultaneously, or combined and
administered as a single composition. Diagnostic techniques, such
as CAT scans for tumors, may be performed prior to and subsequent
to administration to confirm effectiveness.
[0038] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1
Glycoconjugates and Assays
[0039] Synthetic Glycoproteins (Neoglycoproteins)
[0040] Neoglycoproteins were produced by BioCarb AB (Lund, Sweden)
by chemically coupling 10-20 moles of a specific oligosaccharide to
1 mole of nonglycosylated albumin, bovine (BSA) or human (HSA). The
resulting synthetic glycoprotein (neoglycoprotein) contains
multiple copies of the identical carbohydrate sequence, thereby
producing a well characterized, mutivalent glycoconjugate which is
extremely effective for studying carbohydrate-protein interactions.
Depending on the size of the oligosaccharide, three different
chemical spacer arms were used to couple the oligosaccharides to
proteins 1) p-aminophenyl (PAP); 2) aminophenylethyl (APE); and 3)
acetyl phenylene diamine were used to couple the shorter
oligosaccharides to albumin since they will retain the anomeric
configuration of the reducing sugars which may be involved in a
potential binding site. APD was used to couple the larger sugars to
protein by reductive amination, which converts the reducing sugar
to an aminoalditol.
[0041] Direct Binding of Antibodies to Synthetic Glycoproteins
(Neoglycoproteins)
[0042] Synthetic glycoproteins were coated onto microtiter plates
by filling each well with 100 ng of the neoglycoprotein in 100
.mu.l of 0.15 M sodium chloride, 0.01 M sodium phosphate, 0.1%
sodium azide, pH 7.4, (PBS-azide) overnight at 4.degree. C.
Standard enzyme-linked immunoassays (ELISA) were then performed on
the solid phase carbohydrate structures using the appropriate
antibody diluted to 10 .mu.g/ml.
[0043] Production of ELAM-1 cDNA Transfected Cell Lines
[0044] L1-2/pMRB107 cells (L1-2.sup.ELAM-1) were prepared by
transfecting the ELAM-1 gene into the murine pre-B cell line L1-2
(Gallatin et al., Nature 304:30-34, 1983). A cDNA clone encoding
ELAM-1 was obtained from a cDNA library made from activated human
umbilical vein endothelial cell cultures by polymerase chain
reaction (PCR) amplification. The ELAM-1 gene was inserted
downstream of the hCMV promoter in pMRB101 [a derivative of EE6
which contains the E. coli gpt gene (Mulligan and Berg, Proc.
Nat'l. Acad. Sci. USA 78:2072, 1981; Stephens and Corbett, N.A.R.
17:7110, 1989)]. DNA was introduced into L1-2 cells by
electroporation and the cells selected for resistance to
mycophenolic acid. A population of cells staining brightly for
ELAM-1 were selected by FACS and cloned by limiting dilution. These
cells are ELAM-1.sup.hi LFA-1.sup.mod CD45.sup.hi CD44.sup.neg
LECAM-1.sup.neg, differing from the parent cell line or control
vector transfectants only in their expression of ELAM-1.
L1-2/pMRB101 (L1-2.sup.vector) cells are a similarly transformed
derivative of L1-2 transfected with pMRB101 and lacking ELAM-1
expression.
[0045] Cell Binding Assays
[0046] One hundred microliter samples of each synthetic
glycoconjugate in phosphate buffered saline (PBS), pH 7.2, were
absorbed onto glass wells of 8-chamber slides (LabTek) for two
hours at RT. For some experiments glass slides were pre-coated with
rabbit anti-human serum albumin (Sigma) at 200 .mu.g/ml overnight
at 4.degree. C. and washed with PBS prior to the addition of the
glycoconjugate. After blocking with 5% NBS/10 mM HEPES/Dulbecco's
Modified Eagles Medium (DMEM), pH 7.0 (CM), L1-2.sup.ELAM-1 or
L1-2.sup.vector cells were applied to each well
(1.5.times.10.sup.6/0.15 ml in CM). After a 25 minute incubation at
RT on a rotating shaker at 50 rpm, the tops of the wells were
removed and the slides washed 3.times. in DMEM and then fixed by
incubation in 1.5% glutaraldehyde (Kodak)/DMEM. Three to six
100.times. fields were counted for each data point.
[0047] Inhibition of Binding of ELAM-1 Containing Cells by
Compounds
[0048] One hundred and twenty nanograms of Sialyl Le.sup.a-HSA or
Sialyl Le.sup.x-HSA dissolved in 100 .mu.l of phosphate-buffered
saline were absorbed per well of an 8 chambered glass (LabTek)
slide for 2 hours at room temperature. During this period,
L1-2.sup.ELAM-1 cells were pre-incubated for 20 minutes on ice
with-- increasing concentrations of Sialyl Le.sup.a-HSA at 10.sup.7
cells/ml. After washing and blocking the wells in Complete Medium
(CM, 5% normal bovine serum, 10 mM HEPES, pH 7.0, DMEM),
L1-2.sup.ELAM-1 cells pre-incubated with compounds were added
(1.times.10.sup.7 cells/ml) and incubated at room temperature while
rotating at 50 rpm. After 25 minutes, slides were washed 3 times in
Dulbecco's Modified Eagles Medium (DMEM) and then fixed in 1.5%
glutaraldehyde/DMEM.
Example 2
Carbohydrate Structure Recognized by ELAM-1
[0049] The sensitive binding assay described in Example 1 uses
cells permanently transfected with ELAM-1 cDNA. The mouse pre-B
cell line, L1-2, transfected with ELAM-1 cDNA (L1-2.sup.ELAM-1),
but not vector control cDNA, L1-2.sup.vector expresses very high
levels of ELAM-1. The ELAM-1 expressed by these cells is functional
as L1-2.sup.ELAM-1 cells are adhesive for neutrophils and this
adhesion is blocked by anti-ELAM-1 monoclonal antibodies. When
added to glass slides coated with various synthetic
glycoconjugates, L1-2.sup.ELAM-1 cells bound selectively to Sialyl
Le.sup.a and Sialyl Le.sup.x neoglycoproteins, but not to a number
of other glycoconjugates. L1-2.sup.ELAM-1 cells also bound, albeit
more weakly, to Le.sup.a neoglycoprotein. The binding to Le.sup.a
is significant as L1-2.sup.ELAM-1 cells bound poorly to Le.sup.x
and not at all to the glycoconjugates prepared with the structural
analogs such as LNF I. That L1-2.sup.ELAM-1 cells did not bind
other monosialylated carbohydrates, such as 3'SL, 6'SL, LSTa or
LSTc demonstrates that the binding to Sialyl Le.sup.a and Sialyl
Le.sup.x is not due to non-specific charge effects, but rather
reflects specific structural features of these oligosaccharides.
The low level of binding of ELAM-1 transfectants to Le.sup.a is
consistent with an essential role of fucose in recognition, but
shows that neuraminic acid (also known as sialic acid) also plays a
key role.
[0050] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0051] From the foregoing, it will be evident that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modification may be made
without deviating from the spirit and scope of the invention.
* * * * *