A versatile genomic transgenesis platform with enhanced λ integrase for human Expi293F cells

Abstract

Reliable cell-based platforms to test and/or produce biologics in a sustainable manner are important for the biotech industry. Utilizing enhanced λ integrase, a sequence-specific DNA recombinase, we developed a novel transgenesis platform involving a fully characterized single genomic locus as an artificial landing pad for transgene insertion in human Expi293F cells. Importantly, transgene instability and variation in expression were not observed in the absence of selection pressure, thus enabling reliable long-term biotherapeutics testing or production. The artificial landing pad for λ integrase can be targeted with multi-transgene constructs and offers future modularity involving additional genome manipulation tools to generate sequential or nearly seamless insertions. We demonstrated broad utility with expression constructs for anti PD-1 monoclonal antibodies and showed that the orientation of heavy and light chain transcription units profoundly affected antibody expression levels. In addition, we demonstrated encapsulation of our PD-1 platform cells into bio-compatible mini-bioreactors and the continued secretion of antibodies, thus providing a basis for future cell-based applications for more effective and affordable therapies.

Keywords: Expi293F cells; biotherapeutics; human cell line engineering; microencapsulation; site-specific transgenesis; λ integrase.

FIGURE 1

Generation of landing-pad inserted clones. (A). An illustration of pEF_attP_mCherry and of the experimental strategy for creating EF_attP_mCherry or landing pad inserted clonal Expi293F cells. (B). Screening of mCherry positive clones. Expi293F cells were sorted for mCherry positive fluorescence after transfection with pEF_attP_mCherry. Once a stable population had been obtained, single cell clones were obtained by dilution. Single cell clones were next analyzed by flow cytometry. Clone 17 (black border) was selected for further experiments.

FIGURE 2

Targeted integration of pattB_HygroR_eGFP landing pad. (A). Schematic figure showing intC3 facilitated attP X attB recombination and targeted integration of pattB_HygroR_eGFP at the landing pad and predicted recombination construct and positions of PCR primers: 39_EF_fwd and 238_Hygro_rev (Left junction) and 201_ori_fwd and 66_mCherry_rev (Right junction). (B). PCR confirmation of attP X attB recombination resulting in attR and attL sites at the left (0.577 kb) and right (1.366 kb) junctions, respectively. PCR was performed with genomic DNA as a template from a green negative, a green positive colony and clone 17 with the mentioned primers. Genomic DNA from bulk targeted and antibiotic selected cells was used as positive control and no template DNA as water control. Ladder denotes 1 kb DNA ladder. (C). Flow cytometric analysis of the selected colony. Dot plots representing mCherry negative and eGFP negative Expi293F cells in the lower left quadrant in the first panel, mCherry positive and eGFP negative clone 17 cells in the upper left quadrant in the second panel and mCherry negative and eGFP positive cells from green positive colony in the lower right quadrant in the third panel.

FIGURE 3

Single copy of landing pad in chromosome two of clone 17. (A). Schematic representation of pattB_HygroR_eGFP integrated construct with positions of BsrGI restriction sites and of the ∼5.3 kb predicted product after digestion. (B). Southern blot confirmation of single landing pad site. Southern blot was performed with BsrGI digested genomic DNA from Expi293F, clone 17 and green positive colony cells and incubated with a mCherry gene probe followed by an eGFP gene probe after stripping the same blot. As a positive control 0.5 million copies of pEF_attP_mcherry (5.217 kb) and pattB_HygroR_eGFP (7.550 kb) were used after linearization by BsrGI. (C). Schematic drawing of the landing pad site in the SH3RF3 intron of chromosome two of clone 17 depicting NheI and AgeI restriction sites with the primers used for nested PCR after inverse PCR and junction PCR. (D). Detection of the specific site of landing pad insertion by inverse PCR. Inverse PCR and nested PCR were performed, after NheI (for left junction or EF promoter side) or AgeI (for right junction or mCherry side) digestion of clone 17 genomic DNA, with EF_rev_104 and mCherry_fwd_597 primers and nested PCR products were resolved on an agarose gel. DNA bands marked with an arrow were excised and extracted DNA was sequenced to identify the site of the landing pad insertion in the genome. (E). Genomic location confirmation by junction PCR. Junction PCR was performed with clone 17 genomic DNA using C17_gnmc_fwd and 255_pUC_ori_rev primers for the left junction or mCherry_fwd_597 and C17_gnmc_rev primers for the right junction. Amplified products were sequenced to confirm the site of insertion. Ladder denotes 1 kb DNA ladder.

FIGURE 4

Targeted integration of IgG genes containing plasmids at the landing pad in clone 17. (A)n il. Alustration of attP X attL recombination between the landing pad and anti-PD1 IgG heavy and light chain genes containing plasmid with either CW or CCW orientations. Predicted integrated constructs are depicted with the primers used to confirm integration by junction PCR analysis. (B). PCR confirmation of the left and right junctions. PCR was performed with genomic DNA from different subclones of clones 6, 8, 12, 19 and 23 using either 39_EF_fwd and Amp_rev_498 primers for the left junction with an expected 1.289 kb product or 231_Puro_rev and 66_mCherry_rev primers for the right junction with an expected 1.101 kb product. Bands obtained after resolution on an agarose gel were later confirmed by sequencing. Clones 6B1 and 23A4, marked by black border, were further used for protein expression. Ladder denotes 1 kb DNA ladder.

FIGURE 5

IgG expression and purification from landing pad targeted clones. (A). Schematic presentation of IgG genes carrying plasmids, with either CW or CCW arrangement, integrated at the landing pad in clone 17. SphI restriction sites are shown in the figure that would yield a 5.995 kb product after digestion of the genomic DNA clones with both CW or CCW arrangement of the transgenes. (B). Southern blot confirmation of the single copy integration of IgG transgenes for both orientations. Southern blot was performed with SphI digested genomic DNA from clone 17, 6B1 (CW) and 23A4 (CCW) cells and analyzed with an IgG light chain gene probe. As a positive control, 0.5 million copies of targeting plasmid with CW arrangement (10.793 kb) were used after digestion with SphI, yielding an 8.468 kb fragment when hydridized to the light chain gene probe. (C). Purification of secreted IgG. Secreted IgG was purified from both clone 6B1 and 23A4 media by protein G agarose resin and an equal volume (30 μL) from each eluant was resolved on SDS-PAGE. (D). PD-1 antibody binding assay. The functionality of the combined monoclonal IgG fractions was analysed by ELISA using immobilized human PD-1 protein. Purified monoclonal anti-PD1 IgG produced by transiently transfected CHO cells was used as control. Data presented are mean of two technical repeats.

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.