FIGURE 6. Secretion of IgG from encapsulated clone 6B1 cells. (A). Representative image of capsule four in bright field and fluorescence showing homogenous eGFP expression in encapsulated cells. Encapsulation of clone 6B1 cells. Cells were encapsulated in Cell-in-a-Box® and transferred to a 24-well plate with one capsule per well. This was followed by visualization under a fluorescence microscope 2 weeks after encapsulation. Scale bars represent 1 mm at ×4 magnification. (B). Measurement of secreted IgG from capsule 4.20 days after encapsulation, the media in the well with capsule four was replaced with fresh media, which was defined as Day 0. Samples were then collected on Day 0, Day 2 and Day 3. The concentration of secreted IgG was estimated by ELISA in triplicate. Data presented here is the mean with standard deviation.


Therapeutic products that are derived from living organisms are known as biotherapeutics and are the fastest-growing categories of products in the pharmaceutical industry including, but not limited to, monoclonal antibodies, signalling molecules and blood factors that are being produced in mammalian cell lines (Johnson, 2018; Walsh, 2018).

Hence, development and manufacturing of biotherapeutics hinge on genetically stable producer and/or tester cells capable of producing recombinant proteins efficiently. Furthermore, emerging cell encapsulation technologies have enabled possible new applications for mammalian producer cells as mini-bioreactors for in vivo cell-based therapies (Ashimova et al., 2019; Nash et al., 2022b).

Mammalian cells have certain advantages over other expression systems such as those derived from bacterial, yeast or insect origin. They have the desired features to express large and complex proteins with proper folding and post translational modifications (Kim et al., 2012; Kuriakose et al., 2016). Chinese Hamster Ovary (CHO) cells, an immortalized epithelial cell line, are the current workhorse of the biopharmaceutical industry resistant to human pathogen infection (Lalonde and Durocher, 2017). Expi293F cells, derived from human embryonic kidney cells (HEK293) can provide an alternative to CHO cells. These cells can grow in suspension cultures at high density to produce high levels of proteins from episomal or chromosomal transgenes (Fang et al., 2017; Ecker et al., 2020). HEK293 cells have a significant history of use in the development of cell and gene therapy products (Ayuso, 2016; Dumont et al., 2016; Merten et al., 2016), and GMP-qualified HEK293/Expi293F cells are available (Salmons et al., 2007).

Multiple genome-editing tools like zinc finger nucleases (ZFNs), clustered regularly interspaced short palindromic repeats associated protein RNA guided nucleases (e.g. CRISPR-Cas9 system) and transcription-activator like effector nucleases (TALENs), are being used for site-specific transgene insertion (Malphettes et al., 2010; Le et al., 2015; Lee et al., 2015; Sakuma et al., 2015). These programmable endonucleases introduce DNA double strand breaks at a selected locus in the genome, and during the process of repairing this break, the cellular machinery may insert the transgene expression cassette at the break site by employing homologous recombination pathways. Therefore, both the exogenous endonuclease and the cellular repair mechanism are critical to the efficiency of this method (Turan et al., 2011; Kim and Kim, 2014).

Recombinase-Mediated Cassette Exchange (RMCE) using site-specific recombinase systems such as Cre-lox, Flp-FRT, Bxb1-attP/B and ΦC31-attP/B have also been used as genome engineering tools (Kito et al., 2002; Turan et al., 2013; 2014; Kawabe et al., 2015; Inniss et al., 2017; Chi et al., 2019).

These enzymes can perform precise DNA recombination reactions at their respective cognate sites without a need for host factors and can lead to DNA segment insertions, deletions, or inversions (Akopian and Marshall Stark, 2005; Zhou and Droge, 2006).

In RMCE, two different recombinases (e.g. Cre and Flp) are often employed to insert the transgene construct into an artificial genomic landing pad that carries the respective pair of recombination target sequences. The landing pad locus in the host cell chromatin should be accessible for both the recombinases and incoming transgenes. In addition, it must be genetically stable for sustained, high expression of transgenes. A number of these functional hotspots have been identified in CHO and in human cells (Hamaker and Lee, 2018; Aznauryan et al., 2022). Recently, another editing tool based on λ-phage integrase has been engineered for human genome manipulation especially for large transgene insertion reactions. The integrase was genetically modified by directed evolution to generate an enhanced, so-called IntC3 variant for mammalian cells (Siau et al., 2015), that works efficiently in the targeting of a novel
endogenous human target sequence (Vijaya Chandra et al., 2015; Makhija et al., 2018; Chaudhari et al., 2020).

Most genome engineering approaches that aimed to increase production of biotherapeutics have been applied to CHO cells. The relatively new human Expi293F cell line, however, is mostly used for transient transgene expression. Since transient biotherapeutics expression is not always an attractive option from an industrial or cell-therapeutic perspective, an Expi293F platform should entail transgenic master cell lines with modular features as a basis for biopharmaceutical testing/production and innovative therapeutic applications such as transplantable cell-encapsulated mini bioreactors (Zhang et al., 2007; Lathuilière et al., 2015; Bose et al., 2020).

In this study, we presented such a versatile λ-integrase-based platform for site-specific transgenesis in Expi293F cells. A recombination-proficient genomic locus has been selected as a single copy artificial landing pad for λ-integrase-mediated transgenesis. Large transgenic vectors carrying heavy and light chain anti PD-1 monoclonal antibody transgenes in different orientations with respect to each other were inserted, thus permitting direct comparisons of PD-1 antibody expression yields from otherwise isogenic cells. The PD-1 protein is present on the surface of T cells and binds to the PD-1 ligand (PD-L1) expressed on cancer cells resulting in the inhibition of cancer cell killing by the immune cells. Monoclonal anti-PD-1 antibodies impede this interaction by binding to PD-1 as a promising novel anti-cancer strategy (Na et al., 2017; Tan et al., 2022). Our platform-generated PD-1 antibody-expressing cells were encapsulated to create cellulose-based mini bioreactors producing PD-1 antibodies for possible future allogeneic cellbased therapies.

About Austrianova
Austrianova, part of the SG Austria Group, is a biotech company with a global footprint and headquarters in Singapore. Austrianova utilizes a novel and proprietary technology for the encapsulation of living mammalian (Cell-in-a-Box®) and bacterial (Bac-in-a-Box®) cells. Cell-in-a-Box® protects the encapsulated cells from rejection by the immune system, allows cells to be easily transported, stored and implanted at specific sites in patients. The technology, which has been proven safe and efficacious in clinical trials carried out in Europe, allows companies to develop any kind of cells as a one-for-all living pharmaceutical. Bac-in-a-Box® is a similar protective device adapted for encapsulation of probiotic bacteria where it has human food and animal feed applications due to its ability to extend storage under lyophilized conditions and to protect encapsulated bacteria against destruction by stomach acid. Austrianova now also offers GMP4Cells that includes competitively priced Master Cell Bank and Working Cell Bank production as well as “Fill and Finish” services for cell therapy products (such as stem cell therapies, biologics produced from cells e.g. vaccines, antibodies, recombinant proteins etc).

Brian Salmons

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