Abstract 668: Folate conjugated site-specifically to anti-human-CD3-Fab is efficacious in mouse models of ovarian cancer
Authors: Sandra L. Biroc, Marco Gymnopoulos, Shailaja Srinagesh, Brad Hayes, Nick Knudsen, Anthony Manibusan, Jason Pinkstaff, Tim Buss, Kari Cox, Robin Marsden, Lillian Skidmore, Jinming Xia, Ying Sun, Ning Zou, Tsotne Javahishvili
Journal: Cancer Research (2014)

Abstract Ovarian cancer affects ∼22,000 women in the US each year. Folate receptor (FOLR1) is overexpressed in a high proportion of ovarian tumors and is generally associated with advanced stage disease. We report a first-in-class bi-specific conjugate for ovarian cancer by using RECODE™ technology that site-specifically incorporates novel amino acids into proteins in bacterial cells. The Ambrx bi-specific conjugate (Fol-aCD3) is developed for epithelial ovarian cancer and consists of (i) Folate that can bind to FOLR1 receptor on ovarian cancer cells and (ii) Anti-human CD3ε Fab that can bind to the CD3ε receptor on cytotoxic T cells. The potential engagement of T cells by Fol-aCD3 provides for T cell recruitment especially in an immune evasive, suppressive and tolerogenic tumor microenvironment. Para-acetyl phenylalanine (pAcF) containing anti-CD3ε Fab is conjugated to hydroxyl-amine-folate drug-linker. The resulting compound was analyzed by LC-MS with &amp;gt;95% conjugation of a single folate per Fab and &amp;gt;95% main peak by size-exclusion chromatography. By an in vitro LDH cytotoxicity assay using activated human T-cells, Fol-aCD3 killed SKOV-3 and Ov-90 cells with an EC50 of 7.4 and 4.4 ng/mL, respectively. The pharmacokinetic half-life in CD1 mice is 20 and 60 minutes for IV and IP administration, respectively. Fol-aCD3 delivered IV at 5, 0.5 or 0.05 mg/kg daily for 5 days inhibited tumor growth in a dose-dependent manner for Ov-90 co-implanted subcutaneously with activated T-cells in NOD-SCID mice on a folate deficient diet. There was no body weight loss (BWL). Fol-aCD3 was tested in a peritoneal dissemination mouse model of ovarian cancer where the cancer cells, Fol-aCD3, and activated T-cells were injected IP. At a dose of 1 mg/kg of Fol-aCD3 delivered every 3rd day simultaneously with activated T-cells, the NOD-SCID mice exhibited transient BWL but recovered by the 4th day. The treated mice experienced 30% and 50% increase in life span (ILS) for SKOV-3 and OV-90, respectively. The increased survival was seen for mice treated immediately upon tumor cell inoculation (prevention mode) as well as for mice treated 17 days after inoculation (treatment mode). Thus, Fol-aCD3 is being considered for development as a therapeutic for ovarian cancer.. Citation Format: Sandra L. Biroc, Marco Gymnopoulos, Shailaja Srinagesh, Brad Hayes, Nick Knudsen, Anthony Manibusan, Jason Pinkstaff, Tim Buss, Kari Cox, Robin Marsden, Lillian Skidmore, Jinming Xia, Ying Sun, Ning Zou, Tsotne Javahishvili. Folate conjugated site-specifically to anti-human-CD3-Fab is efficacious in mouse models of ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 668. doi:10.1158/1538-7445.AM2014-668