The invention relates to functionalized saccharides and methods of making and using such saccharides.
The early diagnosis and treatment of cancer, a family of diseases that affects one in three Americans, continues to present a major challenge (1). While primary tumors can be removed surgically, by the time they are identified, metastatic cells may have spread throughout the body resulting in the lethal growth of tumors in secondary sites. As a result, most treatment protocols involve the additional use of chemotherapeutics, many of which are characterized by severe toxic side effects. These problems have motivated the search for cancer-cell specific agents capable of facilitating the early diagnosis of tumors (i.e., before metastases have developed) and targeting the toxic effects of drugs to cancer cells and away from normal cells. A central feature of these efforts is the identification of cell-surface antigens that are specific to cancer cells. Several such antigens have been identified and, likewise, some progress has been achieved using monoclonal antibodies (mAbs) as delivery agents for drugs (xe2x80x9cimmunotoxinsxe2x80x9d) (2) and diagnostic probes (xe2x80x9cimmunodiagnosticsxe2x80x9d) (3). Unfortunately, the heterogeneity of cancer-associated epitopes has necessitated laborious and cumbersome mAb preparation for each of the myriad of different cancer-associated antigens (4,5). Furthermore, the murine-based mAbs in common usage have proven to be immunogenic in human patients (6) and attempts to xe2x80x9chumanizexe2x80x9d such mAbs have not yet reached maturity (7). A particularly refractory problem is the development of resistant cells that are able to mask or downregulate the targeted antigens (4,8). Thus, alternative approaches are urgently needed for selective cancer cell targeting.
The composition of cell surface carbohydrates is dramatically altered in many epithelial and blood-derived cancers. Alterations in cancer-associated cell surface carbohydrate structures can be divided into two broad categories: a) increased levels and inappropriate expression of normal oligosaccharide epitopes and b) expression of novel carbohydrate moieties that do not normally occur on healthy cells. The major antigen families associated with transformed cells are the sialyl Lewis a, sialyl Lewis x, Lewis y, A and Tn-related oligosaccharides (4). A common feature of these antigens is that each contains one or more terminal sialic acid or fucose residues, and the Lewis antigens contain both. The overexpression of sialic acid and fucose residues is highly correlated with increased metastatic potential in several cancers with the greatest impact on human health including gastric, uterine endometrial, colonic, epithelial, pancreatic, bladder, liver, lung, prostate and breast cancers as well as several types of leukemia (9). Consequently, therapeutic and diagnostic strategies that target sialic acid and/or fucose residues may have broad applicability to a variety of cancers.
The invention provides methods and compositions for making and using functionalized glycoconjugates. For example, novel and/or unnatural sugars are incorporated into cell associated oligosaccharides using resident pathways of oligosaccharide biosynthesis to expand the informational repertoire of the plasma membrane.
Disclosed methods for making glycoconjugates include (a) contacting a cell with a first monosaccharide, and (b) incubating the cell under conditions whereby the cell (i) internalizes the first monosaccharide, (ii) biochemically processes the first monosaccharide into a second monosaccharide, (iii) conjugates the second monosaccharide to a carrier to form a glycoconjugate, and (iv) extracellularly expresses the glycoconjugate to form an extracellular glycoconjugate comprising a selectively reactive functional group. In a particular embodiments, the first monosaccharide comprises a chemically reactive functional group such as a ketone, which is incorporated into the second monosaccharide, the glycoconjugate and the extracellular glycoconjugate, the first functional group is N-linked in the first monosaccharide, and the first monosaccharide comprises ManLev.
The invention also includes methods for forming a wide variety of products at a cell. The products may provide a label, a binding site, a modulator of cell function such as a drug or toxin, a radiative emission, etc. These methods comprise the steps of making a glycoconjugate according to the invention and then contacting the functional group of the extracellularly expressed glycoconjugate with an agent which selectively reacts with the functional group to form a product. In a particular embodiment, the agent comprises a functional group moiety, such as a hydrazide, which selectively reacts with the functional group of the extracellular glycoconjugate to form a covalent bond, and an effector moiety, such as a drug, which modulates a function of a cell.
The subject compositions include cyto-compatible monosaccharides comprising a nitrogen or ether linked functional group, such as a ketone, selectively reactive at a cell surface and compositions and cells comprising such saccharides.