Ranpirnase (Frog RNase) Targeted with a Humanized, Internalizing, Anti–Trop-2 Antibody Has Potent Cytotoxicity against Diverse Epithelial Cancer Cells

Abstract

Ranpirnase (Rap), an amphibian RNase, has been extensively studied both preclinically and clinically as an antitumor agent. Rap can be administered repeatedly to patients without any untoward immune response, with reversible renal toxicity reported to be dose limiting. To enhance its potency and targeted tumor therapy, we describe the generation of a novel IgG-based immunotoxin, designated 2L-Rap(Q)-hRS7, comprising Rap (Q), a mutant Rap with the putative N-glycosylation site removed, and hRS7, an internalizing, humanized antibody against Trop-2, a cell surface glycoprotein overexpressed in variety of epithelial cancers. The immunotoxin was generated recombinantly by fusing Rap(Q) to each of the two hRS7 light (L) chains at the NH 2 terminus, produced in stably transfected myeloma cells, purified by Protein A, and evaluated by a panel of in vitro studies. The results, including size-exclusion high-performance liquid chromatography, SDS-PAGE, flow cytometry, RNase activity, internalization, cell viability, and colony formation, showed its purity, molecular integrity, comparable affinity to hRS7 for binding to several Trop-2-expressing cell lines of different cancer types, and potency to inhibit growth of these cell lines at nanomolar concentrations. In addition, 2L-Rap (Q)-hRS7 suppressed tumor growth in a prophylactic model of nude mice bearing Calu-3 human non-small cell lung cancer xenografts, with an increase in the median survival time from 55 to 96 days (P < 0.01). These results warrant further development of 2L-Rap(Q)-hRS7 as a potential therapeutic for various Trop-2-expressing cancers, such as cervical, breast, colon, pancreatic, ovarian, and prostate cancers. Mol Cancer Ther; 9(8); 2276-86. ©2010 AACR.

Recombinant expression, different downstream processing of the disulfide-rich anti-tumor peptide Ranpirnase and its effect on the growth of human glioma cell line SHG-44

“…1). Ranpirnase is the smallest member of the ribonuclease (RNase A) superfamily, and appears to be a promising drug with broad clinical application in tumor treatment due to its moderate cytotoxicity, unique synergy, low immunogenicity and few side effects (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). Between 1996 and 2004, Tamir Biotechnology, Inc. (formerly Alfacell Corporation) successively conducted clinical investigations regarding the effects of ranpirnase on breast cancer, pancreatic cancer, renal cell carcinoma, non-small cell lung cancer and malignant mesothelioma, for which the therapeutic effect was the most significant with few side effects.…”

Rana catesbeiana ribonuclease induces cell apoptosis via the caspase-9/-3 signaling pathway in human glioblastoma DBTRG, GBM8901 and GBM8401 cell lines

“…Onconase, which is derived from Rana pipiens, and RC-RNase, which is derived from Rana catesbeiana, each belong to the RNase A superfamily (9,10). RC-RNase and Onconase have been revealed to demonstrate antitumor activity (10)(11)(12)(13). Previous studies have reported that RC-RNase is able to exert cytotoxic effects in various tumor cells, including cervical cancer, leukemia, hepatoma and breast cancer cells (11,(14)(15)(16).…”

“…In addition, numerous studies have indicated that RC-RNase is able to trigger distinct cell death mechanisms in various tumor cell types (10,11,14,17). By contrast, previous studies have also revealed that Onconase can exert antitumor activities in numerous tumor cells, including cervical, breast, colon, pancreatic and prostate cancer cells (12,(18)(19)(20)(21)(22).…”

Linker length affects expression and bioactivity of the onconase fusion protein in Pichia pastoris

“…ONC is the smallest member of the ribonuclease A (RNase A) family (Porta et al, 2008) and is considered unique since it has entered clinical trials (Darzynkiewicz et al, 1988;Ardelt et al, 1991). Once ONC is internalized and sorted to the cytosol, it could degrade the target tRNA to inhibit protein synthesis (Chang et al, 2010); however, the mechanism of its cytotoxicity is not completely understood. The company Timair (formerly known as Alfacell) has successively carried out I-III phase clinical trials of ONC on breast cancer, pancreatic cancer, non-small cell lung cancer, and malignant melanoma (Tian et al, 2010).…”