The arrival of immune checkpoint inhibitors has improved the prognosis of various types of cancer. However, many patients still do not respond to these antibodies, or suffer from severe adverse events. Thousands of clinical trials are currently evaluating combination strategies to tackle the resistance to checkpoint inhibitors, yet these often observe higher toxicity rates than with the single treatments. Overall, this indicates that there is ample room for innovation in the delivery of and treatment with checkpoint-inhibiting antibodies. What if we can force the cancer cells into producing these immunotherapeutics themselves, in combination with other drugs? That was one of the questions we wanted to investigate within our Antibody Gene Transfer Program.

Our laboratory started this research program in 2013, envisioning the development of a broadly applicable and effective platform for the evaluation of various forms of DNA-encoded therapies. In antibody gene transfer, the genetic blueprint of the antibody instead of the antibody protein is administered, enabling the body to produce the drug for a prolonged period of time. Compared to conventional antibody therapy, this approach avoids the frequent high-dose protein administrations, and simplifies the complex and costly in vitro manufacturing of the drugs. Amongst the various platforms available for packaging the antibody blueprint, such as viral vectors and mRNA, our laboratory focuses on plasmid DNA (pDNA). As these small circular DNA molecules do not readily enter cells, we deliver them by means of electroporation, a technique that entails the application of short electrical pulses to the pDNA injection site. The pulses transiently enhance the permeability of the cell membranes and drive the negatively charged pDNA into the cells.  

We initially focused on the muscle as pDNA administration site, and demonstrated in mice and sheep how intramuscular DNA-based delivery of tumor-targeting antibodies and nanobodies allowed for prolonged and therapeutically effective antibody production.^1,2,3 Intramuscular gene transfer can therefore present an attractive alternative to conventional antibody treatment, especially for indications in which sustained systemic antibody exposure is preferred and does not lead to significant side effects. However, we realized that this approach is not ideal in case the expressed antibodies are associated with systemic toxicity, such as with immune checkpoint inhibitors. We therefore shifted to the tumor as site of delivery for DNA-based checkpoint inhibitors, presuming that this enables localized antibody production with only limited systemic exposure. Indeed, we demonstrated that intratumoral gene transfer of an anti-PD-1 and anti-CTLA-4 antibody was equally effective as intramuscular gene transfer, albeit antibody plasma levels were up to 70-fold lower and less persistent.⁴ This intratumoral approach could consequently reduce or avoid the toxicity typically observed with intravenous delivery of these immunomodulatory antibodies.

Since intratumoral gene transfer limits the systemic exposure to the expressed drugs, it might also allow for gene-based medicine cocktails that have the potential to increase the anti-tumor response but would be highly toxic when delivered via conventional systemic routes. With that hypothesis in mind, we tested in our latest study combined DNA-based delivery of checkpoint-inhibiting antibodies and the cytokine IL-12. We considered IL-12 as a good candidate to combine with the DNA-based checkpoint inhibitors, as intratumoral gene transfer of this cytokine is already under clinical evaluation, alone as well as with anti-PD-1 antibody protein therapy (ClinicalTrials.gov: e.g. NCT01502293, NCT03132675).

Combined administration of plasmids encoding IL-12 and an anti-PD-1 antibody, however, did not outperform the DNA-based monotherapies in a mouse tumor model. On the other hand, when we added a DNA-based anti-CTLA-4 antibody to the treatment, this triple combination did improve responses in treated tumors compared to DNA-based IL-12 alone and to the combination of the DNA-based anti-PD-1 and anti-CTLA-4 antibodies. The triple combination also induced regressions of distant tumors, eliminating the need to inject every single tumor lesion in a patient. On immune-cell level, the various DNA-based immunotherapies increased the infiltration of T cells and especially cytotoxic T cells in treated tumors, confirming the immune-mediated nature of the observed local anti-tumor responses. They did not trigger a major immune activation in the spleen that could potentially lead to adverse events, nor did they lead to substantial leakage of the expressed drugs into the circulation, which again highlights the favorable safety profile of the intratumoral gene transfer approach.

In summary, our latest study illustrates how we can turn the cancer against itself, by forcing it into producing an effective cocktail of therapeutics. It suggests that intratumoral DNA-based gene transfer can present an effective approach to facilitate the evaluation of various other combinations of biologicals. In particular, drugs that are associated with severe toxicity or reach insufficient intratumoral exposure with conventional delivery routes are suitable candidates. The DNA platform can also allow for innovative, tailored treatments, in which expression is, for example, targeted to particular cell types or cellular structures at the tumor site.

¹ Hollevoet K, De Smidt E, Geukens N, Declerck P. Prolonged in vivo expression and anti-tumor response of DNA-based anti-HER2 antibodies. Oncotarget 2018; 9(17): 13623-13636.

² Hollevoet K, De Vleeschauwer S, De Smidt E, Vermeire G, Geukens N, Declerck P. Bridging the Clinical Gap for DNA-based Antibody Therapy through Translational Studies in Sheep. Hum Gene Ther 2019; 30(11): 1431-1443.

³ Vermeire G, De Smidt E, Casteels P, Geukens N, Declerck P, Hollevoet K. DNA-based delivery of anti-DR5 nanobodies improves exposure and anti-tumor efficacy over protein-based administration. Cancer Gene Ther 2021; 28(7-8): 828-838.

⁴ Jacobs L, De Smidt E, Geukens N, Declerck P, Hollevoet K. DNA-Based Delivery of Checkpoint Inhibitors in Muscle and Tumor Enables Long-Term Responses with Distinct Exposure. Mol Ther 2020; 28(4): 1068-1077.