Background Innate and acquired resistance are major concerns that limit the patient’s response to immune checkpoint therapy.¹ Immune-boosting gene therapies have been shown to improve response in models of checkpoint blockade resistance.^{2, 3} Our group has previously shown that a combined gene therapy with p19Arf and interferon-beta promotes immunogenic cell death in melanoma models.⁴ However, even responsive mice often relapse, which we hypothesize to be partially due to PD-L1 superexpression. Thus, by combining our approach with anti-PD-1/PD-L1 monoclonal antibodies, we aim to overcome limitations of the gene therapy and increase sensibility to checkpoint blockade in resistant models.

Methods B16F10 cells were treated in vitro with non-replicating adenoviral vectors and were analyzed by flow cytometry. Tumor-bearing mice were treated in situ with the gene therapy and PD-L1 expression was analyzed by flow cytometry, RT-qPCR, and immunohistochemistry. Furthermore, tumor-bearing mice received intraperitoneal injections of the monoclonal antibodies and follow-up continued until reaching 1cm³. Blood serum was then collected for cytokine analysis and tumors were excised for immunophenotyping. Alternatively, B16OVA tumor-bearing animals were euthanatized on treatment day 12 and the splenocytes were pulsed with ovalbumin to assess specific tumor antigen presentation via MHC-I by antigen presenting cells by flow cytometry. Lastly, five different melanoma human cell lines were treated with the vectors and PD-L1 surface expression was analyzed by flow cytometry.

Results The analysis showed upregulation of PD-L1 both in vitro and in vivo, which led us to combine the gene therapy with PD-(L)1 inhibitors. In the in vivo model of B16F10, which is a known resistance model for PD-1 blockade, there was an increase of 10% and 20% responders when associating the gene therapy with anti-PD-L1 and anti-PD-1, respectively, in comparison to the gene therapy alone. Tumor immunophenotyping and cytokine profiling showed increased innate immune cell infiltrates in the conditions treated with the vectors and a high increase in NK cells and IFN gamma sera levels in the combination with both gene therapy and anti-PD-1. The gene therapy also increased tumor antigen presentation via MHC-I by antigen presenting cells, compared to mock group. Nevertheless, the gene therapy increased PD-L1 surface expression in all melanoma human cell lines analyzed.

Conclusions These data show that our immunogenic gene therapy approach increases sensibility to PD-1 and PD-L1 blockade in resistant mouse melanoma models and generates tumor antigen-specific immune responses, with further improved effector immune activity when combining it with checkpoint blockade.

Acknowledgements This work has been supported by the São Paulo Research Foundation (FAPESP).

References

1. Barrueto L, Caminero F, Cash L, Makris C, Lamichhane P, Deshmukh RR. Resistance to Checkpoint Inhibition in Cancer Immunotherapy. Transl Oncol. 2020; 13(3): 100738.

2. Wenthe J, Naseri S, Hellström AC, Moreno R, Ullenhag G, Alemany R, Lövgren T, Eriksson E, Loskog A. Immune priming using DC- and T cell-targeting gene therapy sensitizes both treated and distant B16 tumors to checkpoint inhibition. Mol Ther Oncolytics. 2022; 24:429–442.

3. Chada S, Wiederhold D, Menander KB, et al. Tumor suppressor immune gene therapy to reverse immunotherapy resistance. Cancer Gene Ther. 2022; 29:825–834.

4. Hunger A, Medrano R, Zanatta D, et al. Reestablishment of p53/Arf and interferon-ß pathways mediated by a novel adenoviral vector potentiates antiviral response and immunogenic cell death. Cell Death Discov. 2017; 3:17017.

Ethics Approval All procedures and conditions have been approved by the Ethics Committee for Animal Use (CEUA, FMUSP) under the protocol number 1300/2019 and by the National Technical Committee on Biosafety (CTNBio) under the process number 01250.034644/2019-31.