An anti-αv-integrin antibody that blocks integrin function inhibits the development of a human melanoma in nude mice

ABSTRACT A series of murine monoclonal antibodies were raised against purified human αvβ3 integrin and against M21 human melanoma cells. Five notable hybridomas were identified by ELISA on purified integrins, and the isolated antibodies bound the αv-chain. These antibodies, 17E6, 20A9, 23G5, 14D9.F8 and 10G2, recognised the extracellular domains of the integrin, and were shown to be reactive in FACS, immunoprecipitation, ELISA, and ELISA on fixed cells with M21, M21-L4, and UCLA-P3, but not with the αv-deficient M21-L or M21-L-IIb (M21-L transfected with GpIIb integrin). One antibody, 17E6, strongly perturbed cell attachment mediated by αv integrins, reacting at least with αvβ3, αvβ5, and αvβ1, and strongly inhibiting cell attachment to αv-ligands vitronectin and fibronectin with an IC50 of ∼0.1 µg ml-1. Furthermore, 17E6 at this concentration could induce cell retraction from the substrate, while LM609 (anti-αvβ3) and control antibody 14E2 (anti-200 kDa melanoma surface protein) at 1,000-fold higher concentrations had minimal effects on cell morphology. The action of 17E6 was reversible and was not due to toxic effects: in vitro 17E6 at 0.1 mg ml-1 did not affect either cell proliferation or DNA synthesis. In two nude-mouse tumour models, subcutaneous tumour development and a lung colonisation (‘experimental metastasis’) assay, injection of 17E6 strongly inhibited tumour development, while isotype-matched controls had no effect. There was no obvious mechanism of cell or of complement-mediated tumour cytotoxicity; the antibody did not mediate ADCC or AECDC, or complement fixation. The data strongly support previous studies which have indicated the importance of αv-integrins, and especially αvβ3, in the tumour progression of human melanoma.