1214 IL-12 vectors engineered to encode a functional p35-p40 heterodimer or a single p35 subunit for cancer immunotherapy

BackgroundInterleukin-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.MethodsAll 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.ResultsThe 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.ConclusionsThe 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 surfa