IDENTIFICATION & CHARACTERIZATION OF A MELANOMA SPECIFIC LIGAND FOR NK CELL RECEPTOR 2B4

Purpose (a): Natural Killer (NK) cells are lymphocytes that play a vital role in defense against cancer and infections. NK cell function is regulated by a balance between activating & inhibitory signals transmitted through NK cell surface receptors upon interaction with their ligands. Identification of NK cell receptors and their ligands on tumor cells allow targeted therapy for the specific tumor. CD48 is the only known natural ligand for NK receptor 2B4. Previous studies indicate an in vivo role of 2B4 in B16 melanoma tumor rejection. B16 melanoma cells do not express CD48. We hypothesize that 2B4 interacts with a melanoma specific ligand and that this interaction regulates NK cell function. Methods (b): We generated m2B4-Fc fusion protein, consisting of the extracellular domain of mouse 2B4 and human IgG1 Fc portion. To produce the soluble m2B4-Fc, the p2B4-Fc plasmid was transiently transfected into HEK cells. The purified protein was tested for binding by flow cytometry to mouse CD48 of P815 cells, which express CD48, and for binding to mouse 2B4 melanoma cells, which do not express CD48. The melanoma specific ligand was isolated by immunoprecipitation. Results (c): We show that NK cell receptor 2B4 binds to a melanoma specific ligand on B16 melanoma cells. Conclusions (d): Identification and molecular characterization of the melanoma specific ligand for 2B4 will help in developing new strategies to target melanoma with NK cells.