Generation of cell lines with high productivity and stability for the production of humanized monoclonal antibodies

Abstract

This project, conducted between Instituto Butantan and PR&D/Recepta biopharma under Fapesp's Partnership for Technological Innovation program (PITE) , aims to generate cell lines for the production of humanized monoclonal antibodies (Hu-Mabs). Those recombinant cell lines will be the basis for the future GMP production and use of the Hu-Mabs by Recepta to test their efficacy on cancer patients in Phase I/II clinical trials. A few monoclonal antibodies are established worldwide in the clinic as potent immunotherapeutics for the treatment of a variety of cancer types. The success of Hu-Mabs against cancer is due to their targeting at antigens highly expressed on the surface of cancer cells, and the effector function they are able to exert. Cell line development and engineering are results of complex technologies that are not widely accessible. We started the project with the Hu-Mab Rebmab 200 that recognizes the sodium-dependent phosphate transport protein 2b (NaPi2b), a transmembrane protein over-expressed in 70-90 % of human ovarian epithelial cancers, but displaying restricted expression in normal tissues. To generate stable cell lines, PER.C6® human cells (Crucell, Netherlands) were transfected with a vector containing the genes coding for the heavy and light chains of Rebmab 200 antibody. The stable pool was generated by selective pressure, and the pool was cloned by limiting dilution. After 3 to 4 weeks, 96-well plates were screened for cell growth and a total of 210 clones were transferred to 24-well plates. At that point, a titer ranking was evaluated by surface plasma resonance technology to eliminate non- or low-producing clones. During the expansion of the clones, some of them stopped growing and died. A total of 63 clones were transferred to T-flasks, and a 3-day pcd (picograms/cell/day) was calculated to select 30 clones. These clones were transferred to shaker flasks and after a growth adaptation period of 3 weeks they were assessed for productivity in batch and fed-batch processes. Based on the performance in those situations, 10 clones were selected for stability study during 50 generations, and it was then determined whether their growth and productivity remained stable. During this study, each clone was subjected to batches tests at regular time points. As a result of that, 8 out of the 10 tested clones showed stability for antibody production in the absence of selective pressure. Concomitantly during the stability study, the Rebmab 200 was purified by protein A chromatography from the cell supernatants collected at the end of the fed-batch experiment. The purified antibodies were used in FACS and ADCC assays to evaluate, respectively, the binding to the target and the immune-effector function of Rebmab 200 on NaPi2b positive cancer cells. The FACS analysis showed no significant differences among the clones. On the other hand, the ADCC analysis showed a higher cytotoxic activity for the monoclonal antibody purified from some clones, which were ranked accordingly. All the accumulated data on cellular growth (pdt), overall production, productivity per cell (Qp), performance in batch and fed-batch conditions, binding properties to cancer cell lines (FACS) and immune-effector function in cancer cell lines (ADCC) were compared so as to choose the three top clones. Currently we are evaluating attributes related to the scale-up and GMP production, soon to be ordered by Recepta to a CMO.A second Hu-Mab (Rebmab 100) is underway for the generation of cell lines. This HuMab targets the LewisY epitope highly present on the surface of a variety of epithelial carcinomas. A cell line for this antibody was generated previously by a contracted biotech company in the U.S. (Gala Biotech), and the clone belongs to the company that generated the cell lines. The production of the mAb expressed by this cell line has been ordered to Gala and is under clinical evaluation in a Phase II trial for some types of ovarian cancer. One clinical trial has finished recruiting (the patients are in the final steps of clinical evaluation), and a second trial has started with patients sensitive to platinum chemotherapy. While Recepta is conducting these clinical trials, we have started at Butantan the generation of a cell line that will belong to Recepta. Another reason for developing a new cell line is to obtain higher productivity. We have already noticed that transfection and establishing of conditions for cell line generation are different than those attained with the first antibody, Rebmab 200. This observation is largely reported for the generation of recombinant cell lines. The gene sequence and other protein characteristics seem to be determinant for obtaining the clones, which makes it a process that is not merely a repetition of the same protocol. At Butantan the work is conducted by four dedicated post-docs and one technician paid by Recepta besides members from “Biopharmaceuticals in Animal Cells” laboratory’s team. This work depends heavily on the team integration, with frequent meetings for discussing the results and planning further steps. The partnership between Butantan and Receptahas been an interactive one, with progress reports and workshops regularly maintained. (AU)