IN-VITRO AND IN-VIVO EVALUATION OF HUMAN TUMOR-NECROSIS-FACTOR-ALPHA (HTNF-ALPHA) CHEMICALLY CONJUGATED TO MONOCLONAL-ANTIBODY

Human tumor necrosis-alpha (hTNF-alpha) was chemically conjugated to t he murine anti-Ly-2.1 T cell antibody using heterobifunctional crossli nking agents SAMSA and SPDP. SDS-PAGE analysis of the affinity purifie d conjugate consisted mainly of 1:1 and 1:2 (Ly-2.1:TNF) complexes. Co njugated hTNF retained 50% of its cytotoxic activity by the L929 cytol ytic 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 mu g/ml - 3 times more cy totoxic to these cells than non-conjugated hTNF in vitro. However in v ivo hTNF-Ly-2.1 conjugates were more toxic to mice than hTNF. In vivo blood clearance studies in E3 tumor bearing CBF1 mice demonstrated tha t the half life of the conjugate was 2 hr, compared to 20 min for hTNF . In biodistribution studies, tumor accumulation of 3% was seen for hT NF-Ly-2.1 while for unconjugated hTNF no activity in tumor was detecte d 24hr post injection. A single dose of hTNF-Ly-2.1 increased the accu mulation of I-125-anti-Ly-2.1 by 3 fold compared to controls. However, the antitumor effect of hTNF-Ly-2.1 on E3 cells irt vivo was marginal with some tumor growth retardation at day 1-3. The results of these i n vitro and in vivo studies on chemically conjugated h-TNF-MoAb will b e helpful in the design of novel recombinant fusion proteins for targe ting the biologic activity of TNF to tumours.