Dendritic cells engineered to secrete interleukin-12 p70 when delivered into tumors results in the crosspriming of an expanded anti-tumor CD8+ T cell repertoire and therapeutic benefit.

2722 Bone marrow-derived dendritic cells (DCs) were engineered using recombinant adenovirus to secrete high levels of IL-12p70 and subsequently evaluated for their ability to promote therapeutic anti-tumor immunity in a subcutaneous CMS4 (H-2 d ) murine sarcoma model. Material and methods: Day 5 bone marrow DCs developed in cultures containing rIL-4 and rGM-CSF were infected with control (Ad.ψ5) or IL-12 encoding (Ad.IL12) adenoviruses for 48h at 37 o C. Fifty microliters of PBS (as a control) or DC cell suspensions (10 6 DC total) were injected into established day 7 CMS4 tumors, with repeat injections provided on day 14. Tumor growth was then monitored through day 28. To evaluate the repertoire of tumor peptide epitopes recognized by CD8 + T cells in treated mice, splenocytes were harvested on d28 and analyzed for reactivity against reverse-phase HPLC fractionated tumor peptides (that had been acid extracted from MHC complexes isolated from resected CMS4 tumors) presented by syngenic DC using an IFN-γ ELISA. RP-HPLC fractions containing peptides recognized by therapeutic CD8 + T cells were also submitted to mass spectrometric analysis for determination of their identity. Results: Ad.IL-12 infection (MOI = 20-50) of DC resulted in significant production of IL-12p70 (7.3-17 ng/ml/10 6 DC) and increased expression of MHC class I molecules vs. DC controls. When compared to the PBS or DC.ψ5 treated group, tumors in mice treated with DC.IL12 (at either MOI of Ad.IL12 infection) were suppressed in their growth. CD8 + T cells isolated from DC.IL-12 (vs. PBS or DC-ψ5) treated animals also displayed a stronger and more diverse pattern of reactivity against CMS4-derived (naturally-processed and -presented) peptides, with several immunodominant HPLC fractions identified. Conclusion: Intratumoral administration of DC.IL12 promoted the crosspriming of a stronger and more diverse anti-tumor CD8 + T cell response in association with improved therapeutic benefit than similar therapies using control DC.