Abstract 5345: Potent ribonuclease-based immunotoxins comprising quadruple ranpirnase (Rap) site-specifically conjugated to B-cell lymphoma-targeting antibodies

Rap, a single-chain ribonuclease originally isolated from the oocytes of Rana pipiens, exhibits cytostatic and cytotoxic effects on a variety of tumor cell lines in vitro, as well as antitumor activity in vivo. Rap enters cells via receptor-mediated endocytosis and selectively degrades tRNA, resulting in inhibition of protein synthesis and induction of apoptosis. Rap was reported to exhibit reversible dose-limiting renal toxicity and no immunogenicity in cancer patients. The Dock-and-Lock (DNL) method was applied to generate a novel class of immunotoxins, each of which contains 4 copies of Rap site-specifically linked to an IgG (MAb-Rap). We combined Rap-DDD2, a dimeric fusion protein comprising Rap and a dimerization and docking domain (DDD), with several IgG-AD2 modules, which comprise two anchor domain (AD) peptides fused to the carboxyl termini of the heavy chains. The DNL method allowed the rapid synthesis of 22-Rap, 20-Rap, C2-Rap and 74-Rap, which target B-cell lymphoma via CD22 (epratuzumab), CD20 (veltuzumab), HLA-DR (MAb hL243) and CD74 (milatuzumab), respectively. The parental MAbs/antigens vary with respect to internalization rate, antigen density and cytotoxicity. A non-targeting control construct, 14-Rap, was derived from a MAb to CEACAM5 (hMN-14), which is not expressed on NHL. The Rap-DDD2 module was expressed in E. coli as inclusion bodies, which were purified by immobilized metal affinity chromatography and efficiently refolded. The IgG-AD2 modules were expressed in myeloma cells and purified from the culture supernatant using Protein A affinity chromatography. MAb-Rap was prepared by mixing the IgG-AD2 modules with the Rap-DDD2 module under mild redox conditions and purified to near homogeneity in a single step using Protein A affinity chromatography. SE-HPLC, RP-HPLC and SDS-PAGE analyses showed that preparations of each MAb-Rap are nearly homogeneous and consistent with a covalent structure of ∼230 kDa, indicative of an IgG and 4 Rap groups. Results of in vitro cytotoxicity assays using several NHL lines demonstrated that each NHL-targeting MAb-Rap was highly cytotoxic, resulting in nearly 100% cell killing at ≥1 nM. In comparison, 14-Rap at 30 nM resulted in only modest (10-40%) inhibition of proliferation for most of the cell lines tested. Interestingly, 14-Rap was considerably more potent than recombinant Rap in some cell lines, suggesting that the multiple copies of Rap may facilitate internalization of MAb-Rap. We are currently investigating the significance of internalization rate and antigen density. The DNL method provides a modular approach to efficiently tether multiple cytotoxins onto a targeting antibody, resulting in novel immunotoxins that are expected to show higher in vivo potency due to improved pharmacokinetics and targeting specificity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5345.