A Novel Ribonuclease-Based Immunotoxin Comprising Quadruple Ranpirnase (Rap) Site-Specifically Conjugated to An Anti-CD22 IgG Showing Potent Anti-Lymphoma Activity. (2009)

Abstract Abstract 3721 Poster Board III-657 Background Rap is a single-chain ribonuclease of 104 amino acids originally isolated from the oocytes of Rana pipiens. Rap exhibits cytostatic and cytotoxic effects on a variety of tumor cell lines in vitro, as well as antitumor activity in vivo. The amphibian ribonuclease enters cells via receptor-mediated endocytosis and once internalized into the cytosol, selectively degrades tRNA, resulting in inhibition of protein synthesis and induction of apoptosis. Rap is in advanced Phase IIIb clinical trials against malignant mesothelioma, with reversible dose-limiting renal toxicity and no reported immunogenicity. We have previously shown that effective therapy of human lymphoma xenografts could be achieved with a recombinant fusion protein comprising Rap and a rapidly internalizing anti-CD74 humanized IgG4 antibody (Chang et al., Blood, 2005; 106: 4308-14). In this study, we applied the Dock-and-Lock (DNL) method to generate another novel class of immunotoxins, each of which contains four copies of Rap site-specifically linked to a bivalent IgG. Methods The DNL platform technology exploits a pair of distinct protein domains involved in the natural binding of cAMP-dependent protein kinase (PKA) and A-kinase anchoring proteins (AKAPs). These domains serve as linkers for site-specific conjugation of 2 types of modules, one containing the dimerization and docking domain (DDD) of PKA and the other containing the anchoring domain (AD) of an interactive AKAP. We have combined a recombinant Rap-DDD module with a recombinant IgG-AD module derived from the internalizing anti-CD22 humanized antibody, epratuzumab, to generate 22-Rap, in which a dimer of Rap is covalently tethered to the c-terminus of each heavy chain of epratuzumab. The Rap-DDD module was expressed in E. coli as inclusion bodies, which were purified by immobilized metal affinity chromatography and refolded. The epratuzumab-AD module was expressed in myeloma cells and purified from the culture supernatant using Protein A affinity chromatography. Results 22-Rap was prepared by mixing the epratuzumab-AD module with the Rap-DDD module under mild redox conditions and purified to near homogeneity in a single step using Protein A affinity chromatography. Size exclusion HPLC revealed the presence of a single peak of a retention time consistent with the molecular size of ∼230 kDa, indicative of an IgG and four Rap groups. In vitro transcription/translation assays showed that both the Rap-DDD2 module and 22-Rap retained the activity of Rap. Results of in vitro cytotoxicity assays using Daudi Burkitt lymphoma cells demonstrated that 22-Rap was highly cytotoxic, resulting in nearly 100% cell killing at 1 nM, with an IC50 <50 pM. In comparison, recombinant Rap at 100 nM, either alone or in combination with epratuzumab, resulted in only 50% inhibition of Daudi proliferation. Thus, 22-Rap is about 3 logs more potent than free Rap against Daudi cells. Conclusion 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. Disclosures: Rossi: Immunomedics, Inc: Employment. Chan:Immunomedics, Inc: Employment. Gupta:Immunomedics Inc.: Employment. Goldenberg:Immunomedics Inc.: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Chang:Immunomedics Inc.: Employment.