On 2017 Feb 09, K Hollevoet commented:

Intramuscular antibody gene transfer as a means for prolonged in vivo antibody expression continues to gain traction as an alternative to conventional antibody production and delivery. The study by Kim et al. presents yet another elegant example of said approach, specifically expressing the anti-HER2 4D5 monoclonal antibody (mAb) in mice via plasmid-based gene electrotransfer. The authors report an average 4D5 peak concentration of up to 152 μg ml−1 in BALB/c mice, two weeks after intramuscular electrotransfer of the 4D5-encoding plasmid DNA (pDNA). mAb levels remained above 120 μg ml−1 for at least a month, as depicted in Figure 3d of the manuscript.¹ These results raise some questions, which, in our opinion, are not sufficiently addressed in the manuscript discussion.

Firstly, plasmid-based antibody gene electrotransfer in mice typically results in mere single-digit μg ml−1 mAb serum levels.² After careful consideration, we found no novelties in pDNA design, optimization or delivery in Kim et al.¹ that could explain their quantum leap in attained mAb titers – up to two log higher than the current available literature.

Secondly, the presented data by Kim et al.¹ surpass the expression levels of viral-based anti-HER2 antibody gene transfer studies in mice, with reported peak 4D5 and trastuzumab concentrations of 30 to 40 μg ml–1.^3,4 This further adds to the surprise, given viral vectors consistently outperform plasmid electrotransfer in terms of transgene expression.²

Thirdly, Figure 4f shows an average 4D5 serum concentration of 3.8 μg ml–1 in athymic nude mice, 22 days after tumor cell injection and, so we assume, approximately two weeks after pDNA delivery.¹ mAb titers in these tumor-bearing mice are thus about 40-fold lower than those in the BALB/c mice. Given identical dosing and delivery conditions were applied, the reason for this discrepancy is unclear. The difference in mAb titers appears too high to e.g. attribute it to inter-experiment or mice strain variability. Target-mediated drug disposition in the tumor-bearing mice, i.e. the binding of 4D5 to HER2, is also unlikely to have such impact, given the continuous and robust in vivo mAb production the authors found.

In conclusion, prolonged in vivo mAb expression above 100 μg ml–1 is unprecedented in non-viral antibody gene transfer. The impact of these findings, however, is mortgaged by the lack of explanation Kim et al. provide on the obvious differences with the available literature and with their own subsequent results – as outlined earlier. To allow for these remarkable findings to advance the field, we respectfully invite the authors to address the above concerns, and provide additional support for their data.

References: 1. Kim H, Danishmalik SN, Hwang H, Sin JI, Oh J, Cho Y, et al. Gene therapy using plasmid DNA-encoded anti-HER2 antibody for cancers that overexpress HER2. Cancer Gene Ther 2016 doi: 10.1038/cgt.2016.37. 2. Suscovich TJ, Alter G. In situ production of therapeutic monoclonal antibodies. Expert Rev Vaccines 2015; 14: 205-19. 3. Jiang M, Shi W, Zhang Q, Wang X, Guo M, Cui Z, et al. Gene therapy using adenovirus-mediated full-length anti-HER-2 antibody for HER-2 overexpression cancers. Clin Cancer Res 2006; 12: 6179-6185. 4. Wang G, Qiu J, Wang R, Krause A, Boyer JL, Hackett NR, et al. Persistent expression of biologically active anti-HER2 antibody by AAVrh.10-mediated gene transfer. Cancer Gene Ther 2010; 17: 559-570.

EDIT: A correction of the manuscript by the authors is ongoing based on the above comments. While awaiting the revision, our comments remain posted.

On 2017 Feb 09, K Hollevoet commented:

Intramuscular antibody gene transfer as a means for prolonged in vivo antibody expression continues to gain traction as an alternative to conventional antibody production and delivery. The study by Kim et al. presents yet another elegant example of said approach, specifically expressing the anti-HER2 4D5 monoclonal antibody (mAb) in mice via plasmid-based gene electrotransfer. The authors report an average 4D5 peak concentration of up to 152 μg ml−1 in BALB/c mice, two weeks after intramuscular electrotransfer of the 4D5-encoding plasmid DNA (pDNA). mAb levels remained above 120 μg ml−1 for at least a month, as depicted in Figure 3d of the manuscript.¹ These results raise some questions, which, in our opinion, are not sufficiently addressed in the manuscript discussion.

Firstly, plasmid-based antibody gene electrotransfer in mice typically results in mere single-digit μg ml−1 mAb serum levels.² After careful consideration, we found no novelties in pDNA design, optimization or delivery in Kim et al.¹ that could explain their quantum leap in attained mAb titers – up to two log higher than the current available literature.

Secondly, the presented data by Kim et al.¹ surpass the expression levels of viral-based anti-HER2 antibody gene transfer studies in mice, with reported peak 4D5 and trastuzumab concentrations of 30 to 40 μg ml–1.^3,4 This further adds to the surprise, given viral vectors consistently outperform plasmid electrotransfer in terms of transgene expression.²

Thirdly, Figure 4f shows an average 4D5 serum concentration of 3.8 μg ml–1 in athymic nude mice, 22 days after tumor cell injection and, so we assume, approximately two weeks after pDNA delivery.¹ mAb titers in these tumor-bearing mice are thus about 40-fold lower than those in the BALB/c mice. Given identical dosing and delivery conditions were applied, the reason for this discrepancy is unclear. The difference in mAb titers appears too high to e.g. attribute it to inter-experiment or mice strain variability. Target-mediated drug disposition in the tumor-bearing mice, i.e. the binding of 4D5 to HER2, is also unlikely to have such impact, given the continuous and robust in vivo mAb production the authors found.

In conclusion, prolonged in vivo mAb expression above 100 μg ml–1 is unprecedented in non-viral antibody gene transfer. The impact of these findings, however, is mortgaged by the lack of explanation Kim et al. provide on the obvious differences with the available literature and with their own subsequent results – as outlined earlier. To allow for these remarkable findings to advance the field, we respectfully invite the authors to address the above concerns, and provide additional support for their data.

References: 1. Kim H, Danishmalik SN, Hwang H, Sin JI, Oh J, Cho Y, et al. Gene therapy using plasmid DNA-encoded anti-HER2 antibody for cancers that overexpress HER2. Cancer Gene Ther 2016 doi: 10.1038/cgt.2016.37. 2. Suscovich TJ, Alter G. In situ production of therapeutic monoclonal antibodies. Expert Rev Vaccines 2015; 14: 205-19. 3. Jiang M, Shi W, Zhang Q, Wang X, Guo M, Cui Z, et al. Gene therapy using adenovirus-mediated full-length anti-HER-2 antibody for HER-2 overexpression cancers. Clin Cancer Res 2006; 12: 6179-6185. 4. Wang G, Qiu J, Wang R, Krause A, Boyer JL, Hackett NR, et al. Persistent expression of biologically active anti-HER2 antibody by AAVrh.10-mediated gene transfer. Cancer Gene Ther 2010; 17: 559-570.

EDIT: A correction of the manuscript by the authors is ongoing based on the above comments. While awaiting the revision, our comments remain posted.