Background Interleukin-12 (IL-12) is a 70-kDa heterodimeric cytokine composed of a 35 kDa(p35) and a 40 kDa subunit(p40). IL-12 has been proven to be a potent anticancer agent as it promotes the development of anti-tumor CD8+ T cells and NK cells.

Although IL-12 has shown robust antitumor activity in preclinical studies and potent immune-stimulating potential in humans, there are no approved IL-12 therapeutic products due to its systemic toxicities.

The IL-12 vectors described here are non-replicating MLV retrovectors which transduce rapidly dividing cells. Two types of IL-12 vectors were engineered. In the first type, both p35 and p40 are encoded for and linked by self-cleaving T2A peptide whereas in the second, only the p35 is encoded. The latter vector may be suitable for transducing tumor types that overexpress p40 such as squamous cell tumors.¹ IL-12 tumor specificity can be achieved through local delivery of the vector, targeting via PiT2 receptors, the transduction and integration of rapidly dividing tumor cells, and/or pseudotyping the vector envelope to target tumor specific surface receptors.

Methods All IL-12 genes were synthesized at Genscript and the final plasmids were confirmed by whole plasmid sequencing at Primordium Labs. Human A375 melanoma cells were transduced by the first IL-12 vector and subjected to Western blotting to confirm the protein expression of the two subunits. Both human and murine IL-12 as well as human p35 vectors were produced and tested. An mIL-12 expressing mouse colorectal CT26 stable cell line was established. P40 subunit vector was also separately engineered and used to generate A375-P40 overexpressing cells. Functional IL-12(p70) was tested by mIL-12 or hIL-12 ELISAs and in vitro assays.

Results The IL-12 vector encoding for the two subunits can successfully transduce mouse and human cancer cells which then express and secrete biologically functional IL-12. The other vector encoding for p35 only, when transducing a p40 expressing cell line, produces and secretes a fully functional p70(IL-12) as well.

Conclusions The data indicate the potential use of our IL-12 vectors as an efficient delivery system in IL-12 cancer gene therapy.

Pre-clinical studies in a mouse tumor model of colorectal cancer demonstrate that IL-12 vector transduced tumors generate a significant immune response, inhibit tumor growth, and increase survival rate.

Our current focus is the investigation of pseudotyped IL-12 vectors to target tumor specific surface receptors in in vivo tumor models.

Reference

1. Karlin, et al. J Cancer Metastasis Treat, 2022;8:15, Review: Thymic carcinoma: review and update.