In vitro and in vivo evaluation of human tumor necrosis factor-alpha (hTNFalpha) chemically conjugated to monoclonal antibody.
Human tumor necrosis-alpha (hTNF-alpha) 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 microg/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 half life of the conjugate was 2 hr, compared to 20 min for hTNF. In biodistribution studies, tumor accumulation of 3% was seen for hTNF-Ly-2.1 while for unconjugated hTNF no activity in tumor was detected 24hr post injection. A single dose of hTNF-Ly-2.1 increased the accumulation of 125I-anti-Ly-2.1 by 3 fold compared to controls. However, the antitumor effect of hTNF-Ly-2.1 on E3 cells in vivo was marginal with some tumor growth retardation at day 1-3. The results of these in vitro and in vivo studies on chemically conjugated h-TNF-MoAb will be helpful in the design of novel recombinant fusion proteins for targeting the biologic activity of TNF to tumours.
Pietersz
G A
GA
The Austin Research Institute, Austin Hospital, Heidelberg Vic, Australia.
Toohey
B
B
McKenzie
I F
IF
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002460:Cell Line; D004341:Drug Evaluation; D006207:Half-Life; D006801:Humans; D018796:Immunoconjugates / Q000008:administration & dosage / Q000302:isolation & purification / Q000493:pharmacokinetics*; D051379:Mice; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000493:pharmacokinetics; D013945:Thymoma / Q000378:metabolism; D013953:Thymus Neoplasms / Q000378:metabolism; D014018:Tissue Distribution; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000493:pharmacokinetics*
D000911:Antibodies, Monoclonal; D018796:Immunoconjugates; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha