Two independent runs demonstrated that MUT-FRL retained comparable binding kinetics as the WT-FRL (Table S1). We also performed FACS analysis to rule out that this mutations in MUT-FRL affected its ability to recognize native FOLR1 on target cells. and therefore has potential as a target for passive immunotherapy. The anti-FOLR1 monoclonal antibody MORAb-003 (farletuzumab) was previously shown to elicit antibody dependent cellular cytotoxicity (ADCC) and inhibit tumor growth of human tumor xenografts in nude mice. Because of its promising preclinical profile, farletuzumab has been evaluated in clinical trials as a potential therapeutic agent for ovarian cancer. In this report, we exhibited that farletuzumabs antitumor effect Golgicide A against an experimental model of ovarian cancer is usually mediated by its ADCC activity. Keywords: FOLR1, ovarian cancer, farletuzumab, antibody-dependent cellular cytotoxicity, CD16a Golgicide A Introduction Ovarian cancer is the leading cause of death among women with gynecologic malignancies and the American Cancer Society estimates that about 14?000 women will die from the disease in 2013. Because its initial presentation is usually often at an advanced stage and despite advances in chemotherapy, the majority of ovarian cancer patients eventually die of the disease. Therefore, new therapeutic agents that are effective and well tolerated are needed. Cancer antigen-specific monoclonal antibodies that have direct pharmacologic effects or can stimulate immunological responses represent a promising class of brokers for acute therapy or chronic maintenance of remission. The human folate receptor (FOLR1) is usually overexpressed in ovarian cancer but largely absent in normal tissues.1-4 FOLR1 is also frequently expressed in other gynecologic malignancies5 as well as lung adenocarcinoma.6 It is hypothesized that the presence of elevated levels of FOLR1 can be correlated with the transformed phenotype in ovarian cancer, cisplatin sensitivity, and growth in Golgicide A depleted folate conditions.2,7-11 The highly restricted distribution of FOLR1 in normal tissues and its expression in tumors suggest that this antigen might have potential as a diagnostic marker6 as well as a target for passive immunotherapy. In a previous report,7 we described the in vitro and in vivo activity exerted by the anti-FOLR1 monoclonal antibody MORAb-003, which recently acquired the generic name of farletuzumab. MORAb-003 (farletuzumab) was shown to elicit antibody-dependent cellular cytotoxicity (ADCC) and complement mediated cytotoxicity (CDC) in vitro, to inhibit tumor growth of human tumor xenografts in nude mice, and to cause no observable toxicity in cynomolgus monkeys.7 Because of its promising preclinical profile, farletuzumab has been evaluated in Phase I and Phase II clinical trials12,13 and is currently being Rabbit Polyclonal to AGR3 tested in a Phase III as a potential therapeutic agent for ovarian cancer. In this report, we exhibited that farletuzumabs antitumor effect against an experimental model of ovarian cancer is usually mediated by its ADCC activity. Results MUT-FRL binds to recombinant as well as cell surface FOLR1 with comparable affinity as WT-FRL To investigate the contribution of ADCC activity to the in vivo antitumor activity mediated by wild type farletuzumab (WT-FRL), we mutated key residues involved with effector cells-antibody conversation and generated the antibody mutant, MUT-FRL (see Materials and Methods). We initially compared MUT-FRL and WT-FRL binding affinity to recombinant human FOLR1 by using surface plasmon resonance analysis. Two independent runs exhibited that MUT-FRL retained comparable binding kinetics as the WT-FRL (Table S1). We also performed FACS analysis to rule out that this mutations in MUT-FRL affected its ability to recognize native FOLR1 on target cells. Physique?1 shows that WT-FRL produced in 293F cells for use in these studies could bind target cells (human ovarian cancer IGROV-1) with comparable affinity as GMP-grade WT-FRL (GMP). Importantly, MUT-FRL also bound target cells with comparable affinity as WT-FRL. All three antibodies reached maximum steady-state binding at 1000 ng/mL. Expectedly, irrelevant human IgG did not bind target cells. Open in a separate window Physique?1. MUT-FRL binds to cell surface FOLR1 with comparable affinity as WT-FRL. Human ovarian cancer IGROV-1 cells were stained with MUT-FRL or WT-FRL at the indicated concentrations. Overall, these analyses exhibited that MUT-FRL and WT-FRL bind FOLR1 with comparable affinity. MUT-FRL exhibits decreased binding to CD16a CD16a (Fc gamma receptor IIIa) is usually a receptor that allows natural killer cells to recognize and bind target cell-bound antibodies and mediate ADCC. We generated CHO cells expressing human CD16a (CHO-CD16a) made up of valine at position 158 (CD16a-158V). This allele encodes for a high-affinity antibody receptor while the CD16a-158F allele encodes for a low-affinity receptor.14 CHO-CD16a cells also co-expressed human FcRI, as this subunit is required for antibody binding mediated by the CD16a receptor. CHO-CD16a cells were then used to determine the relative affinities of WT-FLR and MUT-FRL to CD16a/FcRI. As shown in Physique?2 (upper panels), neither WT-FRL nor MUT-FRL bound to parental CHO-K1 cells. Open in a separate window Physique?2. MUT-FRL shows a significantly decreased binding to CD16A. CHO-CD16A cells co-expressing human FcRI were reacted with MUT-FRL or WT-FRL. The test was used and labeled as: *< 0.05; **< 0.01. Discussion The.