Background: Interleukin 12 (IL-12) is a pivotal regulator of innate and adaptive immunity. We conducted a prospective open-label, phase II clinical trial of electroporated plasmid IL-12 in advanced melanoma patients (NCT 01502293). Patients and methods: Patients with stage III/IV melanoma were treated intratumorally with plasmid encoding IL-12 (tavokinogene telseplasmid; tavo), 0.5 mg/ml followed by electroporation (six pulses, 1500 V/cm) on days 1, 5, and 8 every 90 days in the main study and additional patients were treated in two alternative schedule exploration cohorts. Correlative analyses for programmed death-ligand 1 (PD-L1), flow cytometry to assess changes in immune cell subsets, and analysis of immune-related gene expression were carried out on pre-and post-treatment samples from study patients, as well as from additional patients treated during exploration of additional dosing schedules beyond the pre-specified protocol dosing schedule. Response was measured by study-specific criteria to maximize detection of latent and potentially transient immune responses in patients with multiple skin lesions and toxicities were graded by the Common Terminology Criteria for Adverse Events version 4.0 (CTCAE v4.0). Results: The objective overall response rate was 35.7% in the main study (29.8% in all cohorts), with a complete response rate of 17.9% (10.6% in all cohorts). The median progression-free survival in the main study was 3.7 months while the median overall survival was not reached at a median follow up of 29.7 months. A total of 46% of patients in all cohorts with uninjected lesions experienced regression of at least one of these lesions and 25% had a net regression of all untreated lesions. Transcriptomic and immunohistochemistry analysis showed that immune activation and co-stimulatory transcripts were up-regulated but there was also increased adaptive immune resistance. Conclusions: Intratumoral Tavo was well tolerated and led to systemic immune responses in advanced melanoma patients. While tumor regression and increased immune infiltration were observed in treated as well as untreated/ distal lesions, adaptive immune resistance limited the response.

No abstract

Background: Interleukin 12 (IL-12) is a pivotal regulator of innate and adaptive immunity. We conducted a prospective open-label, phase II clinical trial of electroporated plasmid IL-12 in advanced melanoma patients (NCT 01502293). Patients and methods: Patients with stage III/IV melanoma were treated intratumorally with plasmid encoding IL-12 (tavokinogene telseplasmid; tavo), 0.5 mg/ml followed by electroporation (six pulses, 1500 V/cm) on days 1, 5, and 8 every 90 days in the main study and additional patients were treated in two alternative schedule exploration cohorts. Correlative analyses for programmed death-ligand 1 (PD-L1), flow cytometry to assess changes in immune cell subsets, and analysis of immune-related gene expression were carried out on pre-and post-treatment samples from study patients, as well as from additional patients treated during exploration of additional dosing schedules beyond the pre-specified protocol dosing schedule. Response was measured by study-specific criteria to maximize detection of latent and potentially transient immune responses in patients with multiple skin lesions and toxicities were graded by the Common Terminology Criteria for Adverse Events version 4.0 (CTCAE v4.0). Results: The objective overall response rate was 35.7% in the main study (29.8% in all cohorts), with a complete response rate of 17.9% (10.6% in all cohorts). The median progression-free survival in the main study was 3.7 months while the median overall survival was not reached at a median follow up of 29.7 months. A total of 46% of patients in all cohorts with uninjected lesions experienced regression of at least one of these lesions and 25% had a net regression of all untreated lesions. Transcriptomic and immunohistochemistry analysis showed that immune activation and co-stimulatory transcripts were up-regulated but there was also increased adaptive immune resistance. Conclusions: Intratumoral Tavo was well tolerated and led to systemic immune responses in advanced melanoma patients. While tumor regression and increased immune infiltration were observed in treated as well as untreated/ distal lesions, adaptive immune resistance limited the response.

No abstract

No abstract

Mass cytometry permits high-dimensional analysis of diverse aspects of cellular behavior. Here, we adapted this platform to simultaneously profile metabolism, signaling, cell cycle, and effector function with single-cell resolution. Using this approach, we measured enzymes characterizing glycolysis, the TCA cycle, fatty acid oxidation, oxidative phosphorylation, and nutrient transport.In conjunction, we measured downstream targets of TCR signaling regulating translation, proliferation and cytotoxicity as well as surface markers and transcription factors delineating CD8 T cell differentiation. High-dimensional single-cell metabolic analysis by mass cytometry permits identification of unique metabolic and differentiation states of extremely rare cell populations, such as antigen-specific T cells. We interrogated antigen-specific CD8 T cell activation in vitro as well as the trajectory of CD8 T cells responding to Listeria monocytogenes infection, a well-characterized model for studies of T cell differentiation. This integrated, highdimensional approach revealed a novel, activated, and highly metabolically active transitional T cell subset emerging at four days post-infection. These cells simultaneously exhibit glycolytic and oxidative functional programs, which we propose represents a key metabolic inflection point in CD8+ T cell differentiation. This approach should be useful for mechanistic investigations of metabolic regulation of immune responses in vivo.Recombinant human IL-2 (IL-2; TECIN (Teceleukin)) was provided by the National Cancer Institute.

No abstract

No abstract