While protein--protein interactions in cell recognition have been recognized for some time, only recently has the role of carbohydrates in physiologically relevant recognition been widely considered (see Brandley, B. K., and Schnaar, R. L., J. Leuk. Biol. (1986) 40:97; and Sharon, N., and Lis, H., Science (1989) 246:227). Oligosaccharides are well positioned to act as recognition molecules due to their cell surface location and structural diversity. Many oligosaccharide structures can be created through the differential activities of a smaller number of glycosyltransferases. Their diverse structures, then, can be generated with relatively few gene products, suggesting a plausible mechanism for establishing the information necessary to direct a wide range of cell--cell interactions. Examples of differential expression of cell surface carbohydrates and putative carbohydrate binding proteins (lectins) on interacting cells have been described (see Dodd, J., and Jessel, T. M., J. Neurosci. (1985) 5:3278; Regan, L. J., et al., Proc. Natl. Acad. Sci. USA (1986) 83:2248; Constantine-Paton, M., et al., Nature (1986) 324:459; and Tiemeyer, M., et al., J. Biol. Chem. (1989) 263:1671). Further, the question has been raised as to the nature of the leukocyte receptor for ELAM-1 (see Bevilacqua et al. Proc Natl. Acad. Sci. USA (1987) 84:9238).
Tumor associated glycolipids have been reported in fetal tissue and a variety of human cancers, including CML cells (Fukuda, M. N., et al., J. Biol. Chem. (1986) 261:2376; Magnani, J. L., et al., J. Biol. Chem. (1982) 257:14365; Hakomori, S., et al., Biochem. Biophys. Res. Comm. (1983) 113:791). This has led to the hypothesis that these structures may be important in many developmental and oncogenic processes (J. L. Magnani et al., J. Biol. Chem. (1982) 257:14365). Smaller quantities of most of these carbohydrates can be found in normal human tissue (see Fukushi, Y., et al., J. Exp. Med. (1984) 160:506), but until now no function for these structures has been reported.
Adhesion of circulating neutrophils to stimulated vascular endothelium is a primary event of the inflammatory response. Several receptors have been implicated in this interaction, including a family of putative lectins that includes gp90.sup.MEL (Leu8), GMP-140 (PADGEM) and ELAM-1 (Gong, J.-G., et al., Nature (1990) 343:757; Johnston, G. I., et al., Cell (1989) 56:1033; Geoffrey, J. S., and Rosen, S. D., J. Cell Biol. (1989) 109:2463; Lasky, L. A., et al., Cell (1989) 56:1045). These receptors each contain a domain with sequence homology to calcium dependent lectins, and gp90.sup.MEL has been demonstrated to recognize a carbohydrate (see J. S. Geoffrey et al., J Cell Biol. (1989) 109:2463). Endogenous ligands for these receptors are beginning to be characterized (see U.S. Pat. No. 5,143,712 issued Sep. 1, 1992, incorporated herein by reference).
One receptor referred to as E- selectin or ELAM-1 provides transient expression on endothelial cells in response to IL-1 or TNF (Bevilacqua, M. P., et al., Science (1989) 243:1160). The time course of this induced expression (2-8 h) suggests a role for this receptor in initial neutrophil extravasation in response to infection and injury. Furthermore, Bevilacqua et al. (see Bevilacqua, M. P., et al., Proc. Natl. Acad. Sci. USA (1987) 84:9238) have demonstrated that human neutrophils or HL-60 cells will adhere to COS cells transfected with a plasmid containing a cDNA encoding for the ELAM-1 receptor.
Recently, several different groups have published papers regarding ELAM-1 ligands which ligands are also referred to as LECAM-2 ligands. Lowe et al. (1990) demonstrated a positive correlation between the LECAM-2 dependent adhesion of HL-60 cell variants and transfected cell lines, with their expression of the sialyl Lewis x (sLex) oligosaccharide, Neu NAc .alpha.2-3Gal-.beta.1-4(Fuc .alpha.1-3)-GlcNAc. By transfecting cells with plasmids containing an .alpha.(1,3/1,4) fucosyltransferase, they were able to convert non-myeloid COS or CHO lines into sLex-positive cells that bind in an LECAM-2 dependent manner. Attempts to block LECAM-2 dependent adhesion using anti-sLex antibodies were uninterpretable due to the agglutination of the test cells by the antibody. They conclude that one or more members of a family of oligosaccharides consisting of sialylated, fucosylated, lactosaminoglycans are the ligands for the lectin domain of LECAM-2. Phillips et al. (1990) used antibodies with reported specificity for sLex to inhibit the LECAM-2 dependent adhesion of HL-60 or LEC11 CHO cells to activated endothelial cells. Liposomes containing difucosylated glycolipids with terminal sLex structures inhibited adhesion, while those containing nonsialylated Lex structures were partially inhibitory. Walz et al. (1990) were able to inhibit the binding of a LECAM-2-1gG chimera to HL-60 cells with a monoclonal antibody directed against sLex or by glycoproteins with the sLex structure, but could not demonstrate inhibition with CD65 or CD15 antibodies. Both groups concluded that the sLex structure is the ligand for LECAM-2.
LECAM-1 is particularly interesting because of its ability to block neutrophil influx (Watson et al., Nature (1991) 349:164-167). It was expressed in chronic lymphocytic leukemia cells which bind to HEV (see Spertini et al., Nature (1991) 349:691-694). It is believed that HEV structures at sites of chronic inflammation are associated with the symptoms of disease such as rheumatoid arthritis, psoriasis, and multiple sclerosis.
Information regarding the DNA sequences encoding endothelial cell-leukocyte adhesion molecules are disclosed in PCT published application WO90/13300 published November 15, 1990 incorporated herein by reference. The PCT publication cites numerous articles which may be related to endothelial cell-leukocyte adhesion molecules. The PCT publication claims methods of identifying ELAM-ligands, as well as methods of inhibiting adhesion between leukocytes and endothelial cells using such ligands and specifically refers to MILAs which are described as molecules involved in leukocyte adhesion to endothelial cells.
In general, the above publications are directed toward identifying and characterizing endogenous ligands which are believed to be carbohydrates. The present invention is directed toward synthetic compounds which act as agonists or antagonists of naturally occurring ligands.
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