Osteopontin derived chemotactic peptides and methods of use

#The formation of metastases from a primary tumor is a complex temporal process that involves angiogenesis, invasion of the circulatory system by tumor cells, intravasation of the endothelium, arrest in the venous or capillary bed of the target organ, extravasation, entry into the target parenchyme and proliferation of the secondary tumor in tissue different from its tissue of origin. Throughout this process metastatic tumor cells are constantly interacting with their host tissue modulating their adhesiveness to cells and extracellular matrices, degrading matrices and migrating into interstitial stroma. These events are not unique to tumor metastasis and occur in other processes such as angiogenesis, tissue remodeling, bone remodeling and embryogenesis. Experimental studies have demonstrated that invasion involves the active locomotion of tumor cells through and into tissue barriers. The molecular mechanisms regulating such invasive tumor cell migration and subsequent implantation are poorly understood but it appears that there is organ specificity of colonization.

#Several substances that stimulate tumor cell locomotion in vitro have been described. These include factors derived from resorbing bone, liver and smooth muscle. Locomotion of tumor cells can also be induced by endogenous substances in an autocrine fashion. These factors can induce two types of directed cell locomotion: (1) chemotaxis, i.e., directed locomotion of cells up a soluble gradient; or (2) haptotaxis, i.e., migration of cells up a gradient of attached molecules.

#Bone metastases are frequently one of the first signs of disseminated disease in certain carcinomas of lung, breast, prostate, kidney or thyroid. Osteoblasts, the bone forming cells, have been shown to secrete substances that induce the chemotaxis and invasion of melanoma and breast cancer cells. One product of osteoblasts that induces the migration of several cell types and is secreted by several tumors is osteopontin.

#The present invention pertains to novel osteopontin derived peptides that induce the chemotaxis of several cell types including endothelial cells, tumor cells, macrophages and osteoprogenitor cells. Antibodies against these peptides neutralizes the migration of various cell types in response to osteopontin both in vitro and in vivo.

#In accordance with this invention, an “osteopontin derived chemotactic peptide” is a peptide derived from osteopontin having chemotactic activity. Peptides of this invention include peptides comprising no more than about 60 amino acid residues and comprising at least approximately five amino acid residues in length, and preferably at least about 6-45 amino acid residues in length, and more preferably at least about 10-35 amino acid residues in length, from the C-terminal region of the osteopontin polypeptide. The term “peptides of this invention” as used herein is intended to include both peptides and analogs, e.g., agonists or antagonists, thereof. Furthermore, the peptides of the invention are not intended to include the full length osteopontin polypeptide. The peptides of the invention have chemotactic activity. Examples of the peptides of the invention include: a purified osteopontin derived chemotactic peptide comprising a sequence of the formula n-R1−R2-R3-R4-R5-R6-R7-R8−c, wherein R1 is 0, 1, 3, 5, 10, 15, 20, 25, or 27 amino acids long; R2 is Leu, Val, Met or absent; R3 is Val, Leu, Ile or Met; R4 is Leu, Val, Pro or Ile; R5 is aspartic acid or any acidic amino acid; R6 is Pro or Ser; R7 is Lys, Arg, Met or Ile; R8 is 0, 1, 3, 5, 10, 15, 20, 25 or 27 amino acids long, and wherein c indicates the carboxy terminal direction of the peptide and n indicates the amino terminal direction of the peptide; LVLDPK (SEQ ID NO:1); KFHSHKDKLVLDPKSK (SEQ ID NO:2); LVVDPK (SEQ ID NO:3); LVPDPK (SEQ ID NO:4); LVPDSK (SEQ ID NO:5); LVIDPK (SEQ ID NO:6); and VLDPK (SEQ ID NO:7).

#The term “isolated” as used herein refers to nucleic acids of the invention which are in a non-naturally occurring form. For example, isolated DNA is a nucleic acid which is one or both of: not immediately contiguous with both of the coding sequences with which it is immediately contiguous (i.e., one at the 5′ end and one at the 3′ end) in the naturally-occurring genome of the organism from which the nucleic acid is derived; or which is substantially free or free of a nucleic acid sequence with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences.