Antibody-mediated delivery of tumor necrosis factor (TNF-alpha): improvement of cytotoxicity and reduction of cellular resistance.

Recombinant human tumor necrosis factor-alpha (TNF-alpha) is a macrophage-derived, non-glycosylated (17 kDa) peptide that has a remarkably broad range of biological and immunological effects including antiviral action and cytotoxic and cytostatic effects. TNF-alpha was coupled to murine antibody ZME-018, which recognizes a 240 kDa glycoprotein present on over 80% of melanoma cells. The crosslinking was accomplished using the heterobifunctional crosslinking reagent, N-succimindyl 3-(2-pyridyldithio)proprionate (SPDP). After purification on gel-permeation and affinity columns, the resulting eluate was analyzed by non-reducing SDS-PAGE, which confirmed that the product was a mixture of ZME-018 coupled to one or two TNF-alpha molecules. The ZME-TNF conjugate was titered against murine L-929 cells to demonstrate the presence of active TNF. ELISA of the conjugate against target BRO human melanoma cells or non-target T-24 cells demonstrated specific binding only to target cells. Melanoma BRO cells were killed by the immunoconjugate (IC50 of 10 units/mL), whereas native TNF-alpha had no effect at concentrations greater than 50,000 units/mL. The immunoconjugate and TNF-alpha were inactive against T-24 non-target cells. These studies suggest that the sensitivity of cells to TNF was dramatically augmented by specific antibody mediated delivery to tumor cells.