FORMULATION AND ANTITUMOR EFFICACY OF LIPOSOMAL-CAPRYLATED-TNF-SAM(2)

The tumor necrosis factor (TNF) mutant TNF-SAM(2) has previously been shown to have a therapeutic profile superior to parental TNF. To initi ally evaluate the characteristics of liposomal formulations of TNF-SAM (2), it was modified with the N-hydroxysuccinimide ester of caprylic a cid to increase its hydrophobic binding to multilamellar and small uni lamellar vesicles (MLVs and SUVs). Native PAGE and fluorescamine analy sis of acetylated parental TNF and TNF-SAM(2), indicated that these pr oteins both displayed trimeric structures based on crosslinking/SDS-PA GE analysis and behaved similarly with respect to reactivity of their amino functions. Limited N-terminal sequencing analysis of partially a cetylated (approx 3 acetyl groups per trimer) TNF-SAM(2) indicated tha t the N-terminal Val was not modified; this was also concluded based o n HPLC/mass spectrometric (LC-MS) analysis of Glu C digests. LC-MS ana lysis of tryptic digests of the acetylated TNFSAM(2) indicated that Ly s-98 was unreactive. Molecular ions corresponding to acetylated Lys-co ntaining peptides for all five other Lys residues could be detected; n one appeared hyperreactive, but Lys-11 appeared hyporeactive. MLVs com posed of DMPC/DMPG (7 : 3) and SUVs composed of DPPC/DSPC (1 : 1) disp layed high capacity for binding to caprylated TNF-SAM(2). These formul ations of caprylated TNF-SAM(2) displayed tumor necrotizing and growth -inhibitory activity in a syngeneic tumor model, and may be candidates for clinical development.