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.

Acknowledgements

We thank Dr. Mark J. Shulman of Immunology Department, University of Toronto for giving us GP (J chain negative) and XP (J chain positive) hybridomas which were used as a control for western blot analysis.

Figures and Tables

Figure 1 Expression vectors expressing the SM-6 (J+) and SM-6 (J−) IgM. The heavy chain (HC), light chain (LC) and J chain are all expressed from the hCMV promoter.

Figure 2 Summary of PER.C6® cell line generation. The expression construct is introduced into serum-free and suspension adapted PER.C6® cells by electroporation. Following a short recovery period, cells are seeded into 96-well plates by limiting dilution, whereupon stable transfectants are selected by survival in the presence of the antibiotic Geneticin®. Following productivity screens in the multi-well plates, candidate cell lines are expanded and cultured in shake flasks. Batch and fed-batch assays were performed on the candidate cell lines to determine Qp_max and volumetric productivity.

Figure 3 Productivity of IgG and IgM expressing cell lines in PER.C6® cells, in a 7-day batch process. The average Qp_max values for the top 4 cell lines, for 6 independent IgG stable cell line generation programs, are shown in the red bar (n = 24). For the SM-6 (J−) and SM-6 (J+) IgMs, the average of the top 4 cell lines for each variant is shown in the blue bars. For each variant, the Qp_max for the leading cell line is indicated by the yellow circle.

Figure 4 Cell growth profiles from a small scale fed-batch screen of 21 candidate LM-1 IgM expressing cell lines (A) and 22 candidate CM-1 IgM expressing cell lines (B). Colored traces represent results from individual clones.

Figure 5 Production profiles from a small scale fed-batch screen of 21 candidate LM-1 IgM expressing cell lines (A) and 22 candidate CM-1 IgM expressing cell lines (B). Colored traces represent results from individual clones.

Figure 6 Evaluation of pentameric/hexameric expression of IgM in PER.C6® cells. Samples were run on 3.7% polyacrylamide-agarose composite SDS-PAGE gel and subjected to western blotting analysis. Ten micrograms of purified IgM was loaded for each recombinant IgM. Lane 1: CM-1 clone 064 (J+); Lane 2: LM-1 clone 041 (J+); Lane 3: XP control (J chain positive); Lane 4: GP control (J chain deficient); Lane 5: XP control (J chain positive); Lane 6: GP control (J chain deficient); Lane 7: SM-6 clone 089 (J−); Lane 8: SM-6 clone 130 (J−); Lane 9: SM-6 clone 450 (J+); Lane 10: SM-6 clone 528 (J+). Control IgMs were described by Collins et al.3

Figure 7 FACS analysis of hybridoma and PER.C6® derived IgM antibodies on tumor cells. Top: FACS analysis of SM-6 antibody binding to tumor cell line BXPC-3. Positive control SM-6 antibody (black line, 100 µg/mL) is hybridoma produced IgM. CP IgM (Chrompure; red line 100 µg/mL) was used as a negative control. PER.C6® SM-6 (J−) clones 089 and 130, and SM-6 (J+) clones 450 and 528 were tested (black line, 100 µg/mL). Middle: FACS analysis of LM-1 antibody binding to the BXPC-3 cell line. Positive control LM-1 (black line, 100 µg/mL) is hybridoma produced IgM. Negative control is CP IgM (Chrompure; red line 100 µg/mL). PER.C6® LM-1 clones 041 and 170 were tested (black line, 100 µg/mL). Bottom: FACS analysis of CM-1 antibody to the tumor cell line A549. Positive control CM-1 (black line, 100 µg/mL) is hybridoma produced IgM. Negative control is CP IgM (Chrompure; red line 100 µg/mL). PER.C6® CM-1 clones 027 and 064 were tested (100 µg/mL).

Figure 8 Calcein-AM assay with purified PER.C6® derived IgM antibodies. HeLa cells were seeded into 96-well plates and incubated with increasing amounts of antibodies or formulation buffer alone. Cell viability was measured after incubation for 2 hours with Calcein-AM. The percentage cytotoxicity was calculated using the following formula: Cytotoxicity [%] = [100/(cells^only × cells+^{formulation buffer})] − [100/(cells^only × cells^+antibody)], where cells^only are cells in RPMI medium without formulation buffer or antibody. Dose-dependent increase in cytotoxicity was observed with LM-1 IgM (A) and SM-6 IgM (B) antibodies incubated with HeLa tumor cells.

Table 1 Productivities of lead clones for each of the four IgM variants in a fed-batch process