In Vitro and in Vivo Cytotoxic Activities of Recombinant Immunotoxin 8H9(Fv)-PE38 against Breast Cancer, Osteosarcoma, and Neuroblastoma
Recombinant Fusion Proteins
The 8H9 monoclonal antibody (MAb) is highly reactive with a cell surface glycoprotein expressed on human breast cancers, childhood sarcomas, and neuroblastomas but is not reactive with the cell surface of normal human tissues. This specific reactivity suggests that MAb 8H9 may be useful for targeted cancer therapy. To explore this possibility, we generated two recombinant immunotoxins (ITs) using the single-chain Fv (scFv) of MAb 8H9. Initially the 8H9(scFv) cDNA was fused to a DNA encoding a 38-kDa truncated form of Pseudomonas exotoxin (PE38) to generate the IT 8H9(scFv)-PE38. The fusion gene was expressed in Escherichia coli, and the IT was purified to near homogeneity from inclusion bodies. The purified IT showed specific cytotoxicity on nine different cancer cell lines derived from breast cancer, osteosarcoma, and neuroblastomas, known to react with MAb 8H9. The cytotoxic activity was inhibited by MAb 8H9, showing the cytotoxic activity is specific. The antitumor activity of 8H9(scFv)-PE38 was evaluated in severe combined immunodeficient mice bearing MCF-7 breast cancers or OHS-M1 osteosarcomas. The IT showed a specific dose-dependent antitumor activity at 0.075 and 0.15 mg/kg. Next, a more stable disulfide-linked IT, 8H9(dsFv)-PE38, was constructed. It was produced in high yield (16%) and showed cytotoxic and antitumor activities similar to those of 8H9(scFv)-PE38. 8H9(dsFv)-PE38 was given to two cynomolgus monkeys at doses of 0.1 and 0.2 mg/kg i.v. QOD x 3 and was well tolerated. This shows that a dose that causes significant tumor regressions in mice is well tolerated by monkeys. These results make 8H9(dsFv)-PE38 a candidate for further development as a therapeutic agent for breast cancers, osteosarcomas, and neuroblastomas.