From hybridomas to a robust microalgal-based production platform: molecular design of a diatom secreting monoclonal antibodies directed against the Marburg virus nucleoprotein

Abstract

Background: The ideal protein expression system should provide recombinant proteins in high quality and quantity involving low production costs only. However, especially for complex therapeutic proteins like monoclonal antibodies many challenges remain to meet this goal and up to now production of monoclonal antibodies is very costly and delicate. Particularly, emerging disease outbreaks like Ebola virus in Western Africa in 2014-2016 make it necessary to reevaluate existing production platforms and develop robust and cheap alternatives that are easy to handle.

Results: In this study, we engineered the microalga Phaeodactylum tricornutum to produce monoclonal IgG antibodies against the nucleoprotein of Marburg virus, a close relative of Ebola virus causing severe hemorrhagic fever with high fatality rates in humans. Sequences for both chains of a mouse IgG antibody were retrieved from a murine hybridoma cell line and implemented in the microalgal system. Fully assembled antibodies were shown to be secreted by the alga and antibodies were proven to be functional in western blot, ELISA as well as IFA studies just like the original hybridoma produced IgG. Furthermore, synthetic variants with constant regions of a rabbit IgG and human IgG with optimized codon usage were produced and characterized.

Conclusions: This study highlights the potential of microalgae as robust and low cost expression platform for monoclonal antibodies secreting IgG antibodies directly into the culture medium. Microalgae possess rapid growth rates, need basically only water, air and sunlight for cultivation and are very easy to handle.

Keywords: Antibody production; Biotechnology; Expression systems; Microalgae.

Fig. 1

Phaeodactylum tricornutum secretes fully assembled α MARV NP IgG antibodies into the medium. a In vivo localization studies on the expression of heavy and light α MARV NP antibody chains in P. tricornutum. Sequences for the light and heavy chain of the murine IgG2a antibody against the Marburg virus nucleoprotein were retrieved from a hybridoma cell line and expressed as GFP fusion proteins in the diatom P. tricornutum. Both fusion proteins are expressed in the heterologous system without codon optimization and accumulate within the ER of the cells demonstrating that the murine signal peptide is sufficient for ER targeting. Scale bar represents 10 µm. b Purity analysis of secreted α MARV NP IgG antibodies. Proteins of 30 ml culture medium were concentrated with centrifugal filter units (cut off of 10 kDa), separated by SDS-PAGE and stained with Coomassie. Besides the antibody heavy and light chain (marked with asterisks) two dominant signals were detected at ~130 kDa representing a known phosphatase (Phatr2 ID: 47612, verified by mass spectrometry). After protein A purification no background remained, but a weak signal for a HC variant with reduced molecular weight is detected (verified by mass spectrometry). c Biochemical analyses of secreted α MARV NP IgG antibodies. 100 ng of purified hybridoma produced antibody and antibody from the algal medium were prepared under reduced and non reduced conditions, separated by SDS-PAGE and analysed in western blot studies using an α mouse secondary antibody for detection. Under reduced conditions heavy and light chain of the antibody are clearly separated and appear as distinct signals of 55 and 30 kDa. In case of the algal produced antibody the light chain seems to accumulate to slightly higher amounts. Under non reduced conditions disulfide bridges stay intact and the assembled antibody consisting of two heavy and two light chains is detected with a size of about 170 kDa. Lower molecular weight signals are observed in case of both antibodies with no significant difference. Ab antibody, HC heavy chain, LC light chain, MARV Marburg virus, NP nucleoprotein, PAF plastid autofluorescence

Fig. 2

Comparing functionality of algae and hybridoma produced α MARV NP antibodies in different in vitro assays. a Western blot analyses demonstrate that algae and hybridoma produced antibodies recognize the MARV NP protein equally good. Cell extract of MARV infected HuH7 cells was separated by SDS-PAGE, followed by western blot analyses using 500 ng/ml antibody for detection. In case of both antibodies the nucleoprotein is detected with a clear and distinct signal at about 120 kDa. b ELISA studies measuring the binding affinity for MARV NP protein reveal that the algal produced antibody, which was retrieved directly from the culture medium is able to bind the native protein, but not as good as the hybridoma produced antibody. Medium of wild type cells and samples without NP protein served as negative controls. c Immunofluorescence assays with MARV infected HuH7 cells (20 h post infection) show for both antibodies a clear labeling for MARV NP, which accumulates in dot like structures (green) around the nucleus (blue DAPI stained). No labeling is observed for mock HuH7 cells. Error bars indicate standard deviation (n = 3)

Fig. 3

Production of chimeric α MARV NP IgG antibodies in P. tricornutum. a Chimeric antibody variants with rabbit and human Fc-regions are like the original murine IgG complex secreted by P. tricornutum. The secreted antibodies are assembled as demonstrated by gelelectrophoretic separation of reduced and non-reduced samples followed by western blot assays with α human and α rabbit secondary antibodies respectively (100 ng of algal produced antibody were loaded). Beside the ~170 kDa signal of the completely assembled IgG complex a second signal of about 130 kDa was detected for the humanized antibody version, however this is also true for a sample of human IgG standard. b Western blot assays using both chimeric variants as detection antibody for MARV NP demonstrate that both antibodies are functional and recognize the target antigen. Cell extract of MARV infected HuH7 cells was separated by SDS-PAGE, followed by western blot analyses using 500 ng/ml antibody for detection