Intratumoral injection of IL-12-encoding mRNA targeted to CSFR1 and PD-L1 exerts potent anti-tumor effects without substantial systemic exposure

Claudia Augusta Di Trani; Assunta Cirella; Leire Arrizabalaga; Maite Alvarez; Ángela Bella; Myriam Fernandez-Sendin; Joan Salvador Russo-Cabrera; Celia Gomar; Nuria Ardaiz; Alvaro Teijeira; Elixabet Bolaños; José González-Gomariz; Itziar Otano; Fernando Aranda; Belén Palencia; Aina Segués; Shuyu Huang; Sander M J van Duijnhoven; Andrea van Elsas; Ignacio Melero; Pedro Berraondo

doi:10.1016/j.omtn.2023.07.020

Intratumoral injection of IL-12-encoding mRNA targeted to CSFR1 and PD-L1 exerts potent anti-tumor effects without substantial systemic exposure

Mol Ther Nucleic Acids. 2023 Jul 19:33:599-616. doi: 10.1016/j.omtn.2023.07.020. eCollection 2023 Sep 12.

Authors

Claudia Augusta Di Trani^{1

2}, Assunta Cirella^{1

2}, Leire Arrizabalaga^{1

2}, Maite Alvarez^{1

2

3}, Ángela Bella^{1

2}, Myriam Fernandez-Sendin^{1

2}, Joan Salvador Russo-Cabrera^{1

2}, Celia Gomar^{1

2}, Nuria Ardaiz^{1

2}, Alvaro Teijeira^{1

2

3}, Elixabet Bolaños^{1

2}, José González-Gomariz^{1

2}, Itziar Otano^{1

2}, Fernando Aranda^{1

2}, Belén Palencia¹, Aina Segués^{4

5}, Shuyu Huang^{4

5}, Sander M J van Duijnhoven⁶, Andrea van Elsas⁷, Ignacio Melero^{1

2

3

8

9}, Pedro Berraondo^{1

2

3}

Affiliations

¹ Program of Immunology and Immunotherapy, Cima Universidad de Navarra, Pamplona, Spain.
² Navarra Institute for Health Research (IDISNA), Pamplona, Spain.
³ Spanish Center for Biomedical Research Network in Oncology (CIBERONC), Madrid, Spain.
⁴ Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, EH9 3FL Edinburgh, UK.
⁵ Faculty of Veterinary Medicine, Department of Infectious Diseases and Immunology, Utrecht University, 3584 CS Utrecht, The Netherlands.
⁶ ImmunoPrecise Antibodies Ltd., 5349 AB Oss, The Netherlands.
⁷ Third Rock Ventures, San Francisco, CA 94158, USA.
⁸ Department of Immunology and Immunotherapy, Clínica Universidad de Navarra, Pamplona, Spain.
⁹ Department of Oncology, Clínica Universidad de Navarra, Pamplona, Spain.

Abstract

IL-12 is a potent cytokine for cancer immunotherapy. However, its systemic delivery as a recombinant protein has shown unacceptable toxicity in the clinic. Currently, the intratumoral injection of IL-12-encoding mRNA or DNA to avoid such side effects is being evaluated in clinical trials. In this study, we aimed to improve this strategy by further favoring IL-12 tethering to the tumor. We generated in vitro transcribed mRNAs encoding murine single-chain IL-12 fused to diabodies binding to CSF1R and/or PD-L1. These targeted molecules are expressed in the tumor microenvironment, especially on myeloid cells. The binding capacity of chimeric constructs and the bioactivity of IL-12 were demonstrated in vitro and in vivo. Doses as low as 0.5 μg IL-12-encoding mRNA achieved potent antitumor effects in subcutaneously injected B16-OVA and MC38 tumors. Treatment delivery was associated with increases in IL-12p70 and IFN-γ levels in circulation. Fusion of IL-12 to the diabodies exerted comparable efficacy against bilateral tumor models. However, it achieved tethering to myeloid cells infiltrating the tumor, resulting in nearly undetectable systemic levels of IL-12 and IFN-γ. Overall, tethering IL-12 to intratumoral myeloid cells in the mRNA-transferred tumors achieves similar efficacy while reducing the dangerous systemic bioavailability of IL-12.

Keywords: CSF1R; MT: Delivery Strategies; PD-L1; cancer immunotherapy; fusion molecule; immunocytokines; interleukin 12; locoregional treatment; mRNA; myeloid cells.