In vitro and in vivo evaluation of human tumor necrosis factor-α (hTNFα) chemically conjugated to monoclonal antibody

Abstract

Human tumor necrosis-alpha (hTNF-α) was chemically conjugated to the murine anti-Ly-2.1 T cell antibody using heterobifunctional crosslinking agents SAMSA and SPDP. SDS-PAGE analysis of the affinity purified conjugate consisted mainly of 1:1 and 1:2 (Ly-2.1:TNF) complexes. Conjugated hTNF retained 50% of its cytotoxic activity by the L929 cytolytic assay, with an IC50 = 0.12 ng/ml. hTNF-Ly-2.1 was also cytotoxic to E3 cells (Ly-2.1(+ve) with an IC50 = 1.7 μg/ml - 3 times more cytotoxic to these cells than non-conjugated hTNF in vitro. However in vivo hTNF-Ly-2.1 conjugates were more toxic to mice than hTNF. In vivo blood clearance studies in E3 tumor bearing CBF1 mice demonstrated that the h..