The vertical growth stage is the most dangerous stage of
melanoma and is often associated with a poor prognosis. The increased invasiveness and
metastasis that is typical for vertically growing
melanoma are mediated by the molecules of cell adhesion (particularly,
integrins).
Integrin αvβ3, which is abundantly expressed on
melanoma cells with high metastatic potentials and is characterized by low expression levels in normal melanocytes, is potentially an attractive target for
melanoma diagnostics and
therapy.
Integrin αvβ3 is known to recognize the
arginine-
glycine-aspartic (RGD) sequence, which has been found in a wide variety of its natural
ligands. Here expression vectors bearing the genes of fusion
proteins have been constructed for producing these
proteins in Escherichia coli. Such fusion
proteins consist of a peptidic 'address,' targeting the
integrins on
melanoma cells, linked to an 'adaptor' for the attachment of a diagnostic or toxic agent. The peptidic 'address' contains the RGD motif, which is stabilized by a
disulfide bond to achieve the optimal receptor binding conformation. The 'adaptor' is a tetrameric
protein, namely,
streptavidin, that is able to achieve high-affinity binding of d-
biotin (K(d) = 10(-15) M) and confer avidity to the address
peptide. This binding ability facilitates the generation of anti-
melanoma diagnostic and therapeutic agents using the appropriate
biotin derivatives. These
recombinant proteins were purified from the periplasm of E.coli using columns with
2-iminobiotin agarose and demonstrated an ability to adhere to the surface of murine and human
melanoma cells.