Intratumoral gene transfer of immunomodulatory antibody combinations: a preclinical proof-of-concept study

Immune checkpoint inhibitors have emerged as a new promising treatment modality for cancer, as these monoclonal antibodies (mAbs) have been shown to enable durable responses in patients with various advanced and metastatic tumors. Still, a large proportion of cancer patients are refractory to this type of immunotherapy, which has led to a surging interest in combination treatments to improve response rates. These combination treatments, however, further increase the risk of severe immune-related toxicity associated with checkpoint inhibitors. We hypothesized that intratumoral antibody gene transfer can address these specific challenges. In this approach, one administers the mAb-encoding nucleotides into the tumor, enabling the body to locally produce and secrete the mAbs in vivo for a prolonged period of time. Intratumoral antibody gene transfer has repeatedly been reported with viral vectors, yet data with other in vivo gene delivery platforms, such as mRNA and plasmid DNA (pDNA), were lacking at the start of this PhD project. pDNA offers several advantages compared to the other platforms, including a lower immunogenicity, a higher stability and a less complex manufacturing process. The resulting expression can be more moderate, but can still be effective, as e.g. shown in multiple clinical trials of intratumoral DNA-based delivery of the cytokine interleukin 12 (IL-12). In this PhD project, we aimed to demonstrate preclinical proof of concept for intratumoral DNA-based antibody gene transfer. Two checkpoint-inhibiting mAbs targeting cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1), respectively, were used as model therapeutics. To improve the low transfection efficiency of naked pDNA, we first compared two transfection methods that are already applied in the clinic for intratumoral pDNA delivery, i.e. electroporation and complexation with the polycation in vivo-jetPEI (Chapter 3). In the syngeneic MC38 mouse tumor model, intratumoral electroporation resulted in more robust, prolonged and tumor-specific reporter expression, and was therefore selected as transfection method for subsequent experiments. Next, a DNA-based anti-CTLA-4 mAb and DNA-based anti-PD-1 mAb were designed and validated by means of intramuscular gene electrotransfer (Chapter 4). Engineering of the mAb sequences and expression cassette improved mAb expression, resulting in plasma concentrations up to 4 and 14 µg/mL for the anti-CTLA-4 mAb and anti-PD-1 mAb, respectively. mAb levels remained detectable for at least six months, and significant tumor regressions were obtained in the MC38 tumor model. Intratumoral electrotransfer of the DNA-based anti-CTLA-4 and anti-PD-1 mAbs, either alone or combined, resulted in similar anti-tumor responses as the intramuscular route (Chapter 4). mAb plasma levels were however up to 70-fold lower and substantially more transient, suggesting that intratumoral gene transfer can reduce the toxicity risk of the expressed checkpoint inhibitors. Importantly, despite the low systemic mAb exposure, the local treatment did trigger a systemic anti-tumor response, illustrated by moderate abscopal effects in contralateral tumors and protection of cured mice against a tumor rechallenge. Finally, we evaluated intratumoral gene electrotransfer for the simultaneous delivery of checkpoint-inhibiting mAbs and the cytokine IL-12 (Chapter 5). In MC38-bearing mice, the DNA-based triple combination of the anti-PD-1 mAb, anti-CTLA-4 mAb and IL-12 improved local and systemic anti-tumor responses compared to DNA-based IL-12 alone and to the combination of both DNA-based checkpoint inhibitors. The immune-mediated nature of the local effects was confirmed by an increase in tumor-infiltrating T cells, especially CD8+ T cells, and upregulated expression of CD8+ effector markers in electroporated tumors. No general immune activation, which could lead to adverse events, was detected in the spleen following either intratumoral treatment. In the syngeneic B16F10 tumor model, high complete response rates were observed following treatment with control plasmids, the DNA-based checkpoint inhibitors and DNA-based IL-12 alone, hampering the evaluation of the respective triple combination. In conclusion, we demonstrated effective, localized delivery of DNA-based checkpoint-inhibiting mAbs, alone as well as in combination with other biologicals, by means of intratumoral gene electrotransfer. This gene therapy approach can present a suitable platform to facilitate the development of other promising (combination) therapies that might be too toxic or reach insufficient intratumoral levels when delivered systemically.

Publication year:2021

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