Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-06-08
2004-08-03
Nickol, Gary (Department: 1642)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600, C530S350000
Reexamination Certificate
active
06770622
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates to methods and compositions for treating cancer.
2. Background Art
There is evidence that tumor cell metastasis is, in part, due to complex intercellular interactions involving adhesion and aggregation. A specific class of tumor proteins, lectins, that play a role in cell adhesion are now known to be important in tumor formation and metastasis.
Lectins are, by definition, proteins with at least one carbohydrate-binding domain. By immobilizing monosaccharides, oligosaccharides, or glycoproteins in affinity columns, lectins have been isolated from tumor tissue extracts. Generally, a tissue extract in acetone or the like is prepared to isolate the protein component from the lipid component. The acetone is then evaporated, whereupon the residue is solublized in a buffered aqueous solution. This solution is then passed through an affinity column containing the immobilized carbohydrates or glycoproteins. A number of lectins, which selectively bind to galactosides, have been isolated in this manner.
Galectin-3 is one member of the family of lectins termed galectins, formerly known as S-type or S-Lac lectins. Galectins-are classified as such due to structural similarity and characteristic affinity for &bgr;-galactoside sugars (1, 2). The highest levels of galectin-3 are found in activated macrophages, basophils, mast cells, some epithelial cells, and sensory neurons. An early observation was that many tumor cells express galectins on their surface and that their expression could be involved in adhesion and invasion processes. Experimental evidence also suggested that these galectins could be cross-linked by an exogenous glycoprotein resulting in the aggregation of tumor cells. Based on these results, Raz and Lotan proposed that galectin-1 and galectin-3 could promote tumor metastasis (3). Since that time, the evidence for the role of galectin-3 in tumor adhesion, invasion and metastasis has mounted.
Galectin-3 is composed of three distinct structural motifs: a short amino terminal region of 12 amino acids, a sequence rich in G-X-Y tandem repeats characteristic of the collagen supergene family, and a carboxy-terminal half containing the globular carbohydrate recognition domain (2, 4-6). There is close homology between the galectin-3 proteins of different species, but the number of N-terminal tandem repeats differs, hence, the sizes of the proteins vary (7). The human protein is composed of 250 amino acid residues with a M
r
of ~31,000 (5) and a carbohydrate recognition domain extending from 117 to 250. The X-ray crystal structure of the human galectin-3 carbohydrate recognition domain complexed with lactose and N-acetyllactosamine has been published (8).
Although all galectins bind lactose with similar affinity, each galectin is more specific and has higher affinity for certain more complex saccharides (9, 10). Galectins, in general, are unusual among extracellular proteins in that they are initially mainly cytosolic but can be secreted by non-classical pathways, translocated to the cell nucleus, and endocytosed and trancytosed by cells. Galectins are thought to interact with various cell-surface and extracellular glycoproteins and glycolipids, thereby playing a role in cell adhesion, migration, and signaling. The relationship between the intra- and extracellular functions of galectins may be of great biological importance. A number of reviews of the biology of the galectins have been published (11-15).
Galectin-3 can be found on the plasma membrane, and depending on the cell type can be both the nuclear and cytoplasmic or limited to the cytoplasm (6, 16-18). Galectin-3 can be secreted and reuptaken into cells by a nonclassical mechanism (19-21). Studies of mutants of hamster galectin-3 with various deletions in the N-terminal domain have shown that the even if lacking the first 103 amino acid residues the protein is localized in the nucleus. Deletion of the first 110 amino acid residues, however, prevented nuclear localization, although the exact sequence of amino acid residues 104-110, APTGALT, was not obligatory and substitution of other unrelated sequences permitted nuclear sequestration (18). The amino acid residues 104-110 of the hamster galectin-3 protein according to the consensus sequence correspond to the amino acid residues 109-115 of the homologous human galectin-3 protein (7).
Galectin-3 shares the ability to be secreted despite the absence of a signal peptide with a number of other proteins that have unconventional intercellular transfer. These proteins are internalized by cells and are able to directly access the cytoplasm and the nucleus by a process that does not involve classical endocytosis (22). This is in contrast with the modulation of intercellular events by second messengers that bind to extracellular receptors and initiate a cascade of intracellular events that often involve transciptional regulation. Although the mechanisms for the ability of some proteins to cross biological membranes in the absence of a signal sequence are poorly understood, a number of common features have been identified. Many of the proteins can directly access the nucleus, their mechanisms for secretion often vary from their mechanisms for entry, and apolipoproteins and cholesterol can play a role (22).
Galectin-3 is isolated as a monomer but undergoes multimerization on binding to surfaces that contain glycoconjugate ligands, and the N-terminal half of the protein is required for this property (23, 24). The N-terminal domain of the protein is required for galectin-3 to have affinity for multivalent carbohydrate ligands (23, 25) and to transmit intracellular signals (6, 26). Galectin-3 promotes binding of cells to laminin and fibronectin, but the N-terminally truncated protein does not (27). Thus, the N-terminal domain appears to be necessary for the self-association of galectin-3 that are required for some of its biological functions. Galectin-3 null cells were transfected to express recombinant galectin-3 and induced tumors within 4 weeks when injected into mice. When the same galectin-3 null cells were transfected to express a mutant galectin-3 that was lacking the 11 amino terminal amino acids no tumors developed within 4 weeks (6).
A number of laboratories have studied the biology of galectin-3 that apparently is significant in cell growth, differentiation, adhesion, RNA processing, apoptosis, and malignant transformation (28). Laminin is the major non-collagenous polypeptide of basement membranes, and galectin-3 binds preferentially to mouse tumor laminin compared to human placental laminin (29). Galectin-3 has been shown to increase the binding of breast cancer cells to other extracellular matrix proteins (29, 30). In addition to increasing the binding of tumor cells to basement membranes, the interaction of cell surface galectin-3 with complementary serum glycoproteins appears to promote aggregation of tumor cells in circulation, thereby playing another important role in the pathogenesis of metastasis (31).
Expression of recombinant galectin-3 in weakly metastatic fibrosarcoma cells resulted in an increased incidence of experimental lung metastases in syngeneic and nude mice (32). In human umbilical vein endothelial cells (HUVEC) galectin-3 induces angiogenesis (33). Increased expression of galectin-3 in human colon cancer cells resulted in increased metastases, and reduction in galectin-3 expression from antisense DNA was associated with decreased liver colonization and spontaneous metastasis in athymic nude mice (34). Exogenous galectin-3 has been shown to increase invasiveness of human breast cancer cells (35), and to be a chemotactic factor for human umbilical vein endothelial cells (34). However, the endogenous expression of galectin-3 by the cells was not correlated with their invasivness (35). Introduction of human galectin-3 cDNA into the human breast cancer cells BT-549 which are galectin-3 null and non-tumorigenic in nude mice resulted in the establishment of four galectin-3 expressing clones
Jarvis Gary A.
John Constance M.
Leffler Hakon
Kohn Kenneth I.
Kohn & Associates PLLC
Nickol Gary
Rinaldo Amy E.
Yaen Christopher
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