High level expression of functional human IgMs in human PER.C6® cells

Abstract

Natural IgM antibodies play an important role in the body's defense mechanisms against transformed cells in the human body and are currently being exploited both in prognoses of malignant lesions and in the therapy of cancer patients. However, despite growing interest and clinical promise, thus far the IgM class of antibodies has failed to gain widespread commercial interest as these are considered to be difficult to produce recombinantly. IgMs are polymeric and have a relatively large mass. In addition, IgM molecules are heavily glycosylated and, when produced in non-human cell lines, they may contain non-human glycan structures which may be potentially immunogenic. Clearly, production systems capable of expressing human recombinant IgM antibodies are needed. We have successfully used PER.C6 cells-a human cell line-to generate three separate human recombinant monoclonal IgMs in suspension cultures in protein-free medium. All three of the IgMs were constructed with joining (J) chain and were expressed in the pentameric form. One of the IgMs was also expressed as a hexamer without J chain. Clones with cell specific productivities greater than 20 pg/cell/day were generated, which led to yields of 0.5 g/L to 2g/L in fed-batch production. All the IgMs expressed were biologically active as shown in binding and cytotoxicity assays. These studies demonstrate the potential of PER.C6 cells for the production of high levels of functional recombinant IgM and other polymeric molecules, using a straightforward and rapid stable cell line generation method.

Expression and glycoengineering of functionally active heteromultimeric IgM in plants

“…Binding of SM6 to tumor cell lines was assayed on an FACS (FACSCalibur, Becton & Dickinson) as described previously (5). In brief, the human-lung carcinoma cell line A549 was detached with enzyme-free detaching solution (Millipore), washed and incubated with 0, 5, or 25 μg/mL of SM6 or unrelated IgM (ChromPure IgM, Dianova).…”

“…As a next step the glycosylation profile of SM6 produced in the human PER.C6 cell line (SM6 PER ) was determined (5). In general, the glycosylation pattern largely resembled that of human-serumderived IgM: the three N-terminal glycosylation sites are occupied by complex-type N-glycans and the two C-terminal sites carried oligomannosidic structures.…”

“…Thus, monoclonal IgM antibodies have increasingly gained interest for the treatment of various diseases (2)(3)(4). However, despite the recognized relevance for pharmaceutical applications the (recombinant) production has long been considered a challenge (5,6). This is mainly due to the large size and extensive coand posttranslational modifications, which rank IgMs among the most complex human proteins known.…”

Site-SpecificN-Glycosylation of Recombinant Pentameric and Hexameric Human IgM

“…Recently, in-depth characterization of the glycosylation of therapeutic proteins has become of interest to the pharmaceutical industry and the regulatory bodies because of the implications of glycosylation on host immune response and protein function [43]. In this study, a recombinant pentameric and hexameric human IgM, PAT-SM6 produced in the same human cell expression system as PER.C6® cells [44] under identical culture conditions, were used to investigate whether there are differences between the glycosylation of the two oligomeric variants of IgM. The original PAT-SM6 antibody was a natural pentameric IgM isolated from a patient with stomach cancer, and the recombinant form was previously under Phase I/II clinical trials for the treatment of multiple myeloma [37].…”

Designed IgM from glycoengineering

“…This has the potential to jump start research into the structures of IgM that are most appropriate for high level production and ultimately for clinical development. A number of anticancer IgMs have been isolated in the last two decades, some of which are specific for malignant tissues and mediate tumor-specific apoptosis (16,17). It is a timely development that we now have the tools to explore glycoengineered IgM variants that may have enhanced clinical efficacy.…”