Molecular Downsizing Of Monoclonal Antibody-Drug Immunoconjugates And Their Antitumor Effects

Monoclonal antibody (mAb)-targeted cancer therapy is one of the key projects in the field of cancer therapy and medicinal biotechnology. On account of the large molecular size, poor penetration into solid tumors, relatively non-specific distribution in vivo, and human anti-mouse antibody response (HAMA), the conventional antibody-drug conjugates failed to show satisfactory antitumor effects in humans. Finding of novel molecular targets, reconstruction of mAb and discovery of novel "effector" agents are critical for overcoming the difficiency of conventional mAb conjugates. The studies focus on these fields.1. Antitumor effects of pingyangmycin conjugated with Fab' fragment of mAb 3A5 directed against hepatomaFc fragment of mAb is one of the critical factors related to non-specific distribution of mAb conjugate and HAMA reaction; therefore, it is beneficial to remove Fc fragment. In the study, 3A5, an IgG1 rat mAb directed against human hepatoma BEL-7402 cells, was digested with pepsin and then reduced by dithioerytol (DTT) to produce its Fab' fragment. The Fab' fragment was then conjugated to pingyangmycin (PYM), an antitumor antibiotic, through dextran T-40 to produce Fab'-PYM conjugate. Fab'-PYM conjugate retained 15% activity of PYM and showed immunoreactivity with antigen-related cancer cells including BEL-7402, hepatoma 22 (H22), colon carcinoma HT-29 and colon carcinoma 26 (C26) cells. Determined by clonogenic assay, the Fab'-PYM conjugate showed obvious cytotoxicity to BEL-7402 and HT-29 cells with IC₅₀ values of 0.02 μM and 0.08 μM, respectively, whereas the IC₅₀ values of PYM to these cells were 0.51 μM and 1.15μM, respectively. As for KB cells, the antigen irrelatvant cells, IC₅₀ values of Fab'-PYM and PYM were 0.51 μM and 0.42 μM, respectively. Determined by MTT assay with C26 and KB cells, IC₅₀ values of Fab'-PYM were 1.61 μM and 3.45 μM, respectively, while IC₅₀ values of PYM were 21.46 μM and 2.63 μM, respectively. By ip route of 6 injections at dose of 5 mg/kg, Fab'-PYM conjugate and PYM suppressed the growth of C26 in BALB/c...