HeLa cells were grown and maintained with Dulbecco’s Modified Eagle Medium (DMEM) with GlutaMAX (Gibco, Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific) and 1% penicillin-streptomycin solution (Gibco, Thermo Fisher Scientific). Cells were maintained in an incubator at 37°C, with 5% CO₂, and were subcultured every 3–4 days.

ExpiCHO-S cell line (Thermo Fisher Scientific) was maintained in 30 mL of ExpiCHO Expression Medium (Gibco, Thermo Fisher Scientific), in 125 mL Nalgene Single-Use PETG Erlenmeyer Flasks with Plain Bottom (Thermo Fisher Scientific). Cells were kept in an incubator at 37°C, with 8% CO₂ and 80% of relative humidity, with agitation at 125 rpm. When cells reached a concentration between 4 × 10⁶ and 6 × 10⁶ viable cells/mL, routine splitting to the desired dilution was performed, following the manufactures procedure.

HeLa cells were transfected with the different vectors using Lipofectamine 2000 reagent (Thermo Fisher Scientific). For that, HeLa cells were seeded in 24-well plates, in a concentration that allowed them to reach 70%–90% confluency in the following day. For transfections, 1 µL of Lipofectamine 2000 and 500 ng of each plasmid was diluted in Opti-MEM (Thermo Fisher Scientific), following the manufacturer’s protocol. Cells were allowed to grow at 37°C for 48 h, after which they were collected and processed, either for total RNA or protein extraction. ExpiCHO-S cells were transfected using ExpiFectamine CHO reagent (Thermo Fisher Scientific), following the manufacturer’s protocol. In the day prior to transfection (day −1), cells were diluted to a final density of 4 × 10⁶ viable cells/mL, in order to reach a density of 7 × 10⁶–10 × 10⁶ viable cells/mL in the following day (day 0). On the day of transfection, cell viability was assessed, and cells were split to a concentration of 6 × 10⁶ viable cells/mL in a 25 mL final volume. Cells were transfected with 20 μg of plasmid DNA in a final volume of 20 μL and, for Transtuzumab constructs, a 2:1 LC:HC ratio was followed. Following the Standard Protocol for transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed (Thermo Fisher Scientific) were added the day after transfection (day 1) and cells were kept at 37°C with 8% CO₂ for the remaining 9 days. At the time of cell harvest for downstream analysis, cell viability was at least 75%.

Total RNA extraction was performed using TRIzol Reagent (Ambion by Life Technologies) following the manufacturer’s standard protocol, with the exception that all RNA precipitations were carried out at −80°C, during at least 2 h, and using 1 μL of glycogen (15 mg/mL; Roche). Quantification and quality assessment of RNA was performed using the Nanodrop 1000 Spectrophotometer (Thermo Fisher Scientific). RNA samples were stored at −80°C until further use.

To eliminate any potential contamination from genomic and/or plasmid DNA in the RNA samples, these were incubated with DNase I (Roche) for 2 h at 37°C following the manufacturer protocol.

Following DNase I treatment, 1 µL of Random Hexamers (50 µM) and 1 µL of dNTPs mix (10 nM each) (Thermo Fisher Scientific) were added and samples were incubated at 65°C for 5 min followed by 5 min at 4°C. After that, 4 µL of 5x SSIV Buffer (Invitrogen), 1 µL of dithiothreitol (DTT; 100 mM; Invitrogen), 0.5 µL of Ribolock Recombinant RNase Inhibitor (40 U/µL; Thermo Fisher Scientific), and 0.5 µL of Superscript IV (SSIV) Reverse Transcriptase (200 U/µL; Invitrogen) were added to each sample. As a negative control to detect genomic DNA, the same mixtures were prepared for each sample, but without the addition of SSIV RT (-RT). Samples were incubated for 10 min at 23°C, 10 min at 55°C, and finally, 10 min at 80°C to inactivate the enzyme. Samples were stored at −20°C until further use.

RT-qPCRs were prepared using 1 μL of cDNA and 9 μL of a mixture containing 5 μL of 2x SYBR Select Master Mix (Applied Biosystems), 0.125 μL (0.125 μM) of each primer (listed in Supplementary Table S1) and 3.75 μL of nuclease-free water. Samples were run in triplicate on a 7500 Fast Real-Time PCR System (Applied Biosystems), using the program: 50°C for 2 min, 95°C for 2 min; 40 cycles at 95°C for 15 s, 60°C for 1 min followed by a melt curve stage. Non-template controls and -RT samples were also used in each experiment. Results were analyzed using the ΔΔCt method (Normalized fold expression) using ACTB (for ExpiCHO-S cells) or 18S (for HeLa cells) reference genes to normalize gene expression (Pfaffl, 2001).

For recombinant trastuzumab protein quantification, the human IgG (total) uncoated ELISA kit with plates (Invitrogen) was used. For sample preparation, ExpiCHO-S cells were collected and centrifuged at 4,000 g for 30 min. The supernatant containing the recombinant protein was recovered and used for the ELISA protocol using 1:250 or 1:500 dilutions. For each condition, duplicates were made and non-transfected cells were used as negative controls. The ELISA manufacturer’s protocol was followed and the 96-well plate was read at 450 nm and 630 nm in a Synergy2 plate reader (BioTek).

Whole-cell protein extracts were obtained by cell lysis with NP-40 cell lysis buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% NP-40), supplemented with 1x protease inhibitor cocktail (Sigma, P8340). Protein extracts were quantified using the Quick Bradford reagent (Bio-Rad) and 20–40 µg were added to Laemmli Buffer 2x (Bio-Rad) with 5% of β-mercaptoethanol in a 1:1 ratio, and boiled for 5 min at 95°C. Samples were run in a 3% stacking and 10% resolving Tris-glycine SDS-polyacrylamide gel and transferred into a nitrocellulose membrane, using a dry transfer method with the iBlot Gel Transfer Device (Thermo Fisher Scientific). The membrane was incubated with the respective primary antibody diluted in TBS-0.1% Tween 20 containing 5% non-fat dried milk, followed by three washes with TBS-0.1% Tween 20 and incubation with the appropriate secondary antibody. The antibodies used in this study were: anti-SARS-CoV-2 Spike mouse monoclonal antibody (1A9; GeneTex; 1:2,000 dilution), anti-α-tubulin mouse monoclonal antibody (B-5-1-2; Sigma; 1:150,000 dilution), anti-GAPDH mouse monoclonal antibody (ab8245; Abcam; 1:1,500,000 dilution), recombinant anti-MAGEC2 antibody (ab209667; Abcam; 1:2,500 dilution), goat anti-mouse IgG-HRP secondary antibody (Santa Cruz Biotechnologies; 1:10,000 dilution) and goat anti-rabbit IgG-HRP secondary antibody (Santa Cruz Biotechnologies; 1:10,000 dilution). Detection was achieved using enhanced chemiluminescence (ECL Prime, GE Healthcare) and the immunoblots were visualized on the ChemiDoc XRS + System (Bio-Rad). Quantification of the protein intensity was performed using the Image lab Software (Bio-Rad).

These experiments were done as a subcontracted service by Trenzyme GmbH (Konstanz, Germany). After transformation of the expression vectors pTZ_P04_GFP (control plasmid without iPLUS sequences), pTZ_P04_GFP_iPLUS 3X and pTZ_P04_GFP_iPLUSv2 into Pichia pastoris strain KM71H, positive integrants (stable clones) were selected by their ability to grow in presence of Zeocin. After confirmation of proper integration at the AOX locus by PCR based methods, five stable clones per construct were analyzed in duplicates for their ability to synthesize GFP after induction with methanol (0.5% methanol every 24 h) in a 72 h experiment. Screening process was done in a microbioreactor system (BioLector, equipped with 48-well “flower plates”) in 48 -well format, sample volume up to 1,000 µL per well in all clones in parallel.

Data are presented in the graphs as mean ± SD (standard deviation) and are from three independent experiments. Statistical differences were tested using two-tailed unpaired t-test. Only p-values <0.05 were considered statistically significant. All graphs were done using GraphPad Prism version 8.01 (GraphPad Software).

The value of the biopharmaceutical industry has been pushed by the recombinant monoclonal antibodies (mAbs) market (Walsh, 2018). Therefore, we applied iPLUS to the production of trastuzumab, a human recombinant monoclonal antibody used in cancer immunotherapy (Albanell and Baselga, 1999; Krasniqi et al., 2019). As other mAbs, trastuzumab is composed of four polypeptide chains: two identical light chains (LC) and two identical heavy chains (HC), bound by covalent bonds, mainly disulfide bonds, forming a heterodimeric Y-shaped structure (Castelli et al., 2019). We started by subcloning trastuzumab LC and HC chains separately in the pcDNA3.4 expression vector (Figure 1A). We chose pcDNA3.4 as it is widely used in industrial settings for monoclonal antibodies production (Kosuge et al., 2020). This vector contains a full-length CMV promoter that drives high levels of expression, and was modified by the vector’s industry (Thermo Fisher Scientific) to include the WPRE downstream of the multiple cloning site, which increases the expression of several proteins, such as GFP and luciferase (Zufferey et al., 1999).

Our hypothesis was that the inclusion of the iPLUS in the pcDNA3.4 expression vector could further improve the yield of production. We thus incorporated in the LC and HC containing vectors the 28-nucleotide long iPLUS non-coding sequence downstream of WPRE and upstream of the herpes simplex virus (HSV) type 1 thymidine kinase (TK) PAS (Figure 1A). Co-transfection of LC:HC antibody chains containing vectors with and without iPLUS was performed in ExpiCHO-S cells and antibody protein levels were quantified by an IgG ELISA assay and mRNA levels evaluated by RT-qPCR. ExpiCHO-S cells were selected as they are commonly used in the biopharmaceutical industries for therapeutic recombinant protein production due to their ability to survive in animal-component-free medium and grow in suspension at high densities. As can be observed in Figure 1B, vectors containing iPLUS result in an increase in LC and HC mRNA levels, as well as in a 2.2-fold increase in trastuzumab protein levels (Figure 1C). These results show that iPLUS doubles the production of this important therapeutic monoclonal antibody at the laboratory scale.

The next step was to evaluate the use of iPLUS in yeast. Pichia pastoris may use methanol as a carbon source, allowing the induction of protein expression in a highly controlled manner through the AOX1 promoter, making it especially suitable for large-scale and industrial applications. In fact, Pichia is widely used by the biopharmaceutical industry to produce recombinant proteins (Cregg et al., 2000).

We used the GFP reporter protein as a read-out for the use of iPLUS in Pichia pastoris, and started by making GFP constructs with and without iPLUS (GFP-control) sequences by gene synthesis. The iPLUS constructs contained the iPLUS sequence in tandem 3X (iPLUS 3X), to test if the inclusion of several iPLUS repeats would increase protein levels, and a variant sequence of iPLUS (iPLUSv2), which corresponds to the most conserved region of the iPLUS contained in Drosophila’s polo gene (Oliveira et al., 2019), also repeated 3 times (Figure 2A). These sequences were then cloned into a Pichia expression vector having GFP under the control of methanol inducible AOX promoter (Figure 2A). After Pichia transformation with the different constructs, five stable clones (positive integrants) of each construct were selected and analyzed for their ability to synthesize GFP after induction with methanol. The parallel screening of all clones in a microbioreactor system demonstrates that both iPLUS 3X (Figure 2B) and iPLUSv2 (Figure 2C) can significantly increase GFP expression in Pichia. In comparison to the control GFP without any iPLUS sequence (Supplementary Figure S1), the best performing clones showed an increase in GFP expression of 114-fold for iPLUS 3X and 303-fold for iPLUSv2.

This proof-of-concept experiment underscore the versatility and efficacy of iPLUS and reveals that different iPLUS variants, such as the new iPLUSv2, enhance protein production in Pichia pastoris expression systems. Furthermore, it clearly shows that iPLUS may be a valuable asset for the improvement of Pichia expression vectors with biopharmaceutical applications.

In the post COVID-19 pandemic landscape, mRNA vaccines are positioned as a dynamic and groundbreaking technology platform for addressing global health threats (Lorentzen et al., 2022). However, moving from a novel technology to a cornerstone of vaccination strategies in a short time opens space for improvements, such as in the capacity of increasing antigen production levels.

Given our results in improving trastuzumab expression in mammalian cells, as well as with the new variant iPLUSv2 in Pichia, we hypothesized that the inclusion of the new activator sequence, iPLUSv2, in the 3′end of Spike’s coding sequence could be a valuable asset for the mRNA vaccine technology. In fact, the inclusion of cis-regulatory sequences in the 3′ end of Spike was also applied by Moderna and Pfizer in their COVID-19 mRNA vaccines (Xia, 2021).

As in the trastuzumab experiments, the pcDNA3.4 constructs were used (Figure 3A), with iPLUSv2 downstream of Spike. We tested two different cell lines, HeLa and ExpiCHO-S. Human HeLa cells are excellent models for proof-of-concept experiments, due to their easiness in manipulation and analyses. ExpiCHO-S cells were selected not only because they are commonly used by the industry but also as iPLUS increases trastuzumab expression in these cells (Figure 1).

As observed previously for Pichia (Figure 2), in HeLa cells the inclusion of iPLUSv2 leads to an increase in both mRNA and protein levels of Spike (Figures 3B–D), in comparison to the control vector without iPLUSv2. For the protein levels, this increase is approximately 2.1-fold, while for the mRNA levels the increase is approximately 1.4-fold. In ExpiCHO-S cells, iPLUSv2 promoted, once again, an increase in both Spike’s mRNA and protein levels, as illustrated in Figures 3E–G. Here, the increase in protein production is approximately 2.5-fold, while for the mRNA levels the increase is approximately 1.6-fold.

These results clearly show that iPLUSv2 can increase Spike’s mRNA levels and double protein production, in two different cell lines from different species (human and hamster).

The mRNA vaccines are a potential immunotherapy solution to combat various types of cancer, prompting the immune system to destroy cancer cells by triggering a specific and enduring immune response against tumor antigens (TAs), inhibiting tumor growth (Miao et al., 2021). Nevertheless, in clinical trials it has been observed that although there is a certain level of positive response from patients (Papachristofilou et al., 2019), it does not seem to be a sufficiently robust response to cause tumor recession or an increase in overall survival for all the patients (Chehelgerdi and Chehelgerdi, 2023), leading to termination of their production. For that reason, pharmaceutical companies ought to incorporate novel molecular tools to improve cancer mRNA vaccines, to promote good immunological responses.

Therefore, our next step was to assess whether subcloning iPLUSv2 downstream of the melanoma antigen family C2 (MAGEC2) would result in increased mRNA levels and consequent protein production. For these proof-of-concept experiments, MAGEC2, a tumor-specific antigen for non-small cell lung cancer (NSCLC), was selected since it is included in CureVac’s CV9202 NSCLC mRNA vaccine and whose information was made public (Papachristofilou et al., 2019). CV9202 is a vaccine encoding 6 tumor associated antigens, being MAGE-C2 one of them. Antigen-specific immune responses were detected in a phase Ib study enrolling 26 patients with stage IV NSCL (Papachristofilou et al., 2019; Sebastian et al., 2019).

As before, the initial step was the construction of vectors containing the MAGEC2 with and without iPLUSv2 downstream of this antigen (Figure 4A) and these vectors were used to transfect HeLa cells. As it can be observed in Figures 4B, C, the inclusion of iPLUSv2 sequence in MAGEC2 vectors resulted in an increase in approximately 1.8-fold in MAGEC2 mRNA levels (Figure 4B) and a 2.8-fold increase in MAGEC2 protein levels, in comparison to vectors without this sequence (Figures 4C, D).

As observed with iPLUSv2 in Spike or iPLUS in trastuzumab, these results show that the presence of iPLUS non-coding sequences in vectors used for recombinant protein expression can at least double protein production. Moreover, the impact of iPLUSv2 in MAGEC2 bears substantial implications for its application in cancer mRNA vaccines, as the increase in antigen production certainly influences the efficiency of the process.