Strategies for clone detection, selection and isolation in Per.C6 cells - case for Rebmab100

Abstract

Background A successful monoclonal antibody (mAb) cell line development requires efficient clone detection and screening. Cloning by limiting dilution (LDC) is the traditional method to isolate mAbs expressing clones [1]. Although effective, LDC is time-consuming, with limited workflow and therefore a critical step of cell line development. To compare to LDC in terms of timelines and productivities for Rebmab100 mAb cell line development we have implemented ClonePix FL (CP-FL), an automated system for high throughput clone detection. The robotic colony picker has the advantages of reducing the process time and increasing the probability to isolate highproducing clones. Moreover, we have combined these two approaches with high throughput screening assays for early detection of high productive clones. Rebmab100 mAb targets Lewis-Y, a blood group-related antigen expressed in over 70% of epithelial cancers, including breast, colon, ovary and lung carcinomas. The murine monoclonal 3S193 was generated in BALB/c mice by immunization with Le-expressing cells from the MCF-7 breast carcinoma cell line [2]. The humanized version of antiLe 3S193 mAb was obtained by CDR-grafting method [3]. The hu3S193 (Rebmab 100) mAb has potent immune effector function (ADCC and CDC), is rapidly internalized into Le expressing cancer cells, and has been shown to cause significant regressions in xenograft models in preclinical studies, alone or in conjunction with isotope and toxins [3,4]. Safety and desirable pharmacokinetic profiles of Rebmab100 were demonstrated in a Phase I clinical trial in patients with epithelial carcinomas [5] and promising results have been obtained in a Phase II clinical trial conducted in Brazil [6]. Very importantly, Rebmab100 was granted orphan-drug status by the FDA for ovary cancer. Aiming the next step of Rebmab100 mAb development we generated a new Rebmab100 cell line that shows stability and high productivity allowing its scale-up to later clinical trials.