Abstract 4636: Novel immunoRNases comprising multiple copies of ranpirnase display potent cytotoxicity in human breast cancer cell lines expressing Trop-2
Authors: Liu Donglin , Wang Yang , Rossi Edmund A. , Goldenberg David M. , Chang Chien-Hsing 
Published: August 2012

Abstract
Ranpirnase (Rap) is an amphibian ribonuclease originally isolated from the oocytes of Rana pipiens. Rap shows anti-tumor activity, which can be enhanced by linking Rap to a tumor-targeting antibody, as reported previously in CD22-, CD74-, and Trop-2-expressing human cancer cell lines and xenografts. To further improve the potency of Rap-based ImmunoRNases, we have used the Dock-and-Lock (DNL) method to tether the full IgG of hRS7 (humanized anti-Trop-2) with four copies of Rap and evaluated the resulting conjugate, termed E1-Rap, as a potential therapeutic for breast cancer. The modular nature of DNL also allowed us to substitute hRS7 with other humanized antibodies in-house, such as hLL2 (anti-CD22, epratuzumab), hLL1 (anti-CD74, milatuzumab), hA20 (anti-CD20, veltuzumab), hL243 (anti-HLA-DR), and hR1 (anti-IGF-1R), resulting in 22-Rap, 74-Rap, 20-Rap, C2-Rap, and 1R-Rap, respectively. In the present study, 22-Rap was evaluated along with E1-Rap in a panel of human breast cancer lines, including the basal-like, triple-negative subtype (MDA-MB-468, MDA-MB-231, BT20, HCC1806, and HCC1395), the luminal B, HER2-negative subtype (MCF-7), and the HER2-positive subtype (SKBR3), all except HCC1395 expressing high to moderate levels of Trop-2, but none expressing CD22. As demonstrated by flow cytometry, E1-Rap and hRS7 bound equivalently to MDA-MB-468, indicating the affinity of E1-Rap for Trop-2 is not compromised. Intriguingly, 22-Rap, but not epratuzumab, also bound substantially to MDA-MB-468, albeit to a lesser extent than E1-Rap. Additional experiments revealed 22-Rap was capable of binding to a variety of CD22-negative cell lines, which could be attributed to the enhanced association between the multiple copies of Rap in the DNL conjugate and heparan sulfate proteoglycans on the cell surface. Whereas the individual DNL component (IgG or Rap) alone or in combination showed negligible in vitro cytotoxicity in all the seven breast cancer cell lines examined, E1-Rap exhibited EC50 values of 1 nM or lower in MDA-MB-468 (0.03 nM), MCF-7 (0.1 nM), BT20 (0.18 nM), HCC1806 (0.19 nM), and SKBR3 (1.29 nM). In comparison, the potency of 22-Rap was at least 10-fold lower than E1-Rap in MDA-MB-468, BT20 and HCC1806, with EC50 about 2 nM. Neither E1-Rap nor 22-Rap was very effective in inhibiting the proliferation of the more aggressive MDA-MB231 (EC50 above 50 nM) or the Trop-2-negative HCC1395 (EC50 ∼100 nM). The results of immunofluorescence microscopy showed E1-Rap was effectively internalized in MDA-MB-468 and localized in the cytosol. Thus, these ImmunoRNases, as exemplified by E1-Rap, are potentially new cancer therapeutics for Trop-2-expressing breast and other solid tumors.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4636. doi:1538-7445.AM2012-4636