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.

A phase I/II study of the intralesional injection of ricin-monoclonal antibody conjugates in patients with hepatic metastases.

A phase I/II study of the intralesional administration of ricin-labelled monoclonal antibodies was conducted in patients with hepatic metastases of gastrointestinal origin. The anti-carcinoembryonic antigen (CEA) antibody I-1 was conjugated to blocked ricin via a disulphide bridge. After a test dose of antibody, patients were injected with ricin-antibody conjugates under computed tomography (CT) guidance on two occasions 1 week apart. Patients with stable or responding disease would receive a third course. The dose of ricin relative to surface area was increased in a predefined manner in cohorts of 3 patients. A total of 27 patients with hepatic metastases were entered into this study. All patients had metastatic colorectal cancer (26 patients) or adenocarcinoma of unknown primary with elevated CEA levels (1 patient). The presence of malignancy was documented cytologically in 9 of 11 patients tested. Minor responses were seen in 7 patients. However, no major objective responses or changes in the growth rate of injected lesions were observed. Toxicity was generally mild, the most common being hepatic capsular pain 24-48 h after each injection. 6 patients experienced rigors. One patient had anaphylaxis. Human anti-mouse and anti-ricin antibody responses were observed. Although substantial amounts of ricin conjugated to monoclonal antibodies were delivered into single lesions, this therapeutic approach was unsuccessful. Future studies of ricin-labelled antibodies should incorporate the systemic administration of immunoconjugates.