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

Chien-Hsing Chang,Rosana Michel,David M Goldenberg,Meiyu Loo,Yang Wang,Pankaj Gupta,Thomas M Cardillo

doi:10.1158/1535-7163.mct-10-0338

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.

Highlights

RNases, in particular, ranpirnase (Rap; ref. 1) and its more basic variant, amphinase [2], are potential antitumor agents [3]
The method for generating 2L-Rap-hLL1-γ4P allowed us to develop a series of structurally similar immunotoxins, referred to in general as 2L-Rap-X, all of which consist of two Rap molecules, each connected through a flexible linker to the NH2 terminus of one light chain (L chain) of an antibody of interest (X)
We provide a further example to illustrate that the NH2-terminal fusion of Rap(Q) to a tumortargeting monoclonal antibody (mAb) is a valid and versatile approach to generate novel immunotoxins by showing that (Q)-humanized RS7 (hRS7) (a) can be produced in a mammalian host and purified to homogeneity, (b) retains the binding specificity and affinity of hRS7, as well as the RNase activity of Rap, (c) suppresses the proliferation of various Trop-2–expressing cancer cell lines at nanomolar concentrations in vitro, and (d) inhibits the growth of a human lung tumor xenograft in vivo

Summary

Introduction

RNases, in particular, ranpirnase (Rap; ref. 1) and its more basic variant, amphinase [2], are potential antitumor agents [3]. We have generated another series of immunotoxins of the same design, called 2L-Rap(Q)-X, by substituting Rap with its nonglycosylation form, designated as Rap(Q) to denote that the potential glycosylation site at Asn is changed to Gln (or Q, single letter code). For both series, we made the IgG as either IgG1(γ1) or IgG4(γ4), and to prevent the formation of IgG4 half molecules [11], we converted the serine residue in the hinge region (S228) of IgG4 to proline (γ4P). This design is dictated by the requirement of a pyroglutamate residue at the NH2 terminus of Rap for the RNase to be fully functional [12]

Methods

Results

Conclusion