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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 (NCT01502293)., 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., Competing Interests: Disclosure AA is a paid advisor to OncoSec Medical, Inc. and he holds stock options in the company. He is also a paid advisor to Array, Regeneron, and Valitor. He also receives research funding from Acerta, Amgen, AstraZeneca, BMS, Dynavax, Genentech, Idera, Incyte, Idera, ISA, LOXO, Merck, Novartis, Regeneron, Sensei, and Tessa. He previously received research funding from Amgen, Celldex, GlaxoSmithKline, Lilly, Medimmune, Plexxicon, Roche, OncoSec Medical, Inc. SB, research funding from OncoSec Inc and Merck Inc. SA, research funding from OncoSec Inc. and Merck Inc. KL receives research funding from OncoSec Inc. and Merck Inc. MF receives research funding from OncoSec Inc; Advisor Boards of Novartis, Pulse Bioscience, Array Bioscience, Bristol Myers Squibb, Sanofi. LF receives research funding from OncoSec, Merck, AbbVie, Bavarian Nordic, BMS, Dendreon, Janssen, Roche/Genentech. CBB is advisor to PrimeVax, BMS. Stock ownership in BMS, Patent US20180322632A1: image processing systems and methods for displaying multiple images of a biological specimen. DB is an employee of OncoSec Medical, Inc. RT is an employee of OncoSec Medical, Inc. EB is an employee of OncoSec Medical, Inc. MHL is a former employee of OncoSec Medical, Inc. Consulting: Immunomic Therapeutics, Inc., Pulse Biosciences, Inc., Juno Therapeutics, Inc., Genexine, Inc., NKarta Therapeutics, Inc., Seattle Genetics, Inc., Ideaya Biosciences, Inc., Apros Therapeutics, Inc., IgM Biosciences, Inc., Immune Design Corporation, Plexxikon, Inc., Curis, Inc. She is married to RHP. with conflict listed next. RHP is a former employee of OncoSec Medical, Inc. with equity. He also has consulting income from Immunomic Therapeutics, Inc., Pulse Biosciences, Sensei Biotherapeutics, AbbVie, Calithera Biosciences, Minerva Biosciences, AstraZeneca, Curis, Inc. He has received research funding from Exicure Therapeutics, X4-Pharma, Incyte Pharmaceuticals. He is married to MHL with conflicts listed earlier. SG is a former employee of OncoSec Medical, Inc., currently employed by Alloplex Biotherapeutics, Inc. KKT receives research funding from OncoSec, Merck Inc., Takeda, and Regeneron; consulting for Compugen, Pulse Biosciences. CT is an employee of OncoSec Medical, Inc. with equity. AD receives research funding from OncoSec Inc., Merck, BMS, Pfizer, Novartis, Roche/Genentech, Xencor, Consultant for Incyte, Novartis, Amgen, Caris Inc. All other authors have declared no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Myeloid-origin dendritic cells (DCs) can develop into IL-12-secreting DC1 or non-IL-12-secreting DC2 depending on signals received during maturation. Through rapid culture techniques that prepared either mature, CD83+ DC1 or DC2 from CD14+ monocytes in only 2 days followed by a single 6-7 day DC-T cell coculture, we sensitized normal donor CD8+ T cells to tumor Ags (HER-2/neu, MART-1, and gp100) such that peptide Ag-specific lymphocytes constituted up to 16% of the total CD8+ population. Both DC1 and DC2 could sensitize CD8+ T cells that recognized peptide-pulsed target cells. However, with DC2, a general decoupling was observed between recognition of peptide-pulsed T2 target cells and recognition of Ag-expressing tumor cells, with peptide-sensitized T cells responding to tumor only about 15% of the time. In contrast, direct recognition of tumor by T cells was dramatically increased (to 85%) when DC1 were used for sensitization. Enhanced tumor recognition was accompanied by 10- to 100-fold increases in peptide sensitivity and elevated expression of CD8beta, characteristic of high functional avidity T cells. Both of these properties were IL-12-dependent. These results demonstrate the utility of rapid DC culture methods for high efficiency in vitro T cell sensitization that achieves robust priming and expansion of Ag-specific populations in 6 days. They also demonstrate a novel function of IL-12, which is enhancement of CD8+ T cell functional avidity. A new approach to DC-based vaccines that emphasizes IL-12 secretion to enhance functional avidity and concomitant tumor recognition by CD8+ T cells is indicated.

Mature dendritic cells (DCs), in addition to providing costimulation, can define the Th1, in contrast to the Th2, nature of a T-cell response through the production of cytokines and chemokines. Because calcium signaling alone causes rapid DC maturation of both normal and transformed myeloid cells, it was evaluated whether calcium-mobilized DCs polarize T cells toward a Th1 or a Th2 phenotype. After human monocytes were cultured for 24 hours in serum-free medium and granulocyte-macrophage colony-stimulating factor to produce immature DCs, additional overnight culture with either calcium ionophore (CI) or interferon gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and soluble CD40L resulted in phenotypically mature DCs that produced interleukin-8 (IL-8) and displayed marked expression of CD80, CD86, CD40, CD54, CD83, DC-LAMP, and RelB. DCs matured by IFN-gamma, TNF-alpha, and soluble CD40L were additionally distinguished by undetectable CD4 expression, marked secretion of IL-12, IL-6, and MIP-1beta, and preferential ability to promote Th1/Tc1 characteristics during T-cell sensitization. In contrast, DCs matured by CI treatment were distinguished by CD4 expression, modest or absent levels of IL-12, IL-6, and MIP-1beta, and preferential ability to promote Th2/Tc2 characteristics. Calcium signaling selectively antagonized IL-12 production by mature DCs activated with IFN-gamma, TNF-alpha, and soluble CD40L. Although the activation of DCs by calcium signals is largely mediated through calcineurin phosphatase, the inhibition of IL-12 production by calcium signaling was independent of this enzyme. Naturally occurring calcium fluxes in immature DCs, therefore, negatively regulate Dc1 differentiation while promoting Dc2 characteristics and Th2/Tc2 polarization. Calcium-mobilized DCs may have clinical usefulness in treating disease states with excessive Th1/Tc1 activity, such as graft-versus-host disease or autoimmunity.

The authors previously showed that monocytes treated with calcium ionophore (CI) acquire characteristics of mature dendritic cells (DC) in part through a calcineurin-dependent pathway. In this study, the authors evaluated the ability of granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), and interleukin-12 (IL-12) alone or in combination with CI to induce DC characteristics in peripheral blood monocytes. Monocytes obtained by leukapheresis and countercurrent centrifugal elutriation were cultured with calcium, cytokines, or both, profiled by flow cytometry, and assessed for antigen uptake and sensitization of autologous CD8+ T cells to antigen. Monocytes treated with the combination of GM-CSF, IL-2, and IL-12 resulted in immunophenotypic and antigen uptake profiles typical of immature DC, including loss of surface CD14 expression, de novo low-level expression of B7.1, negligible CD83 expression, marked enhancement of CD40 and ICAM-1, and high major histocompatibility complex class I and II levels. A high level of antigen uptake by macro-pinocytosis was observed. In contrast, CI treatment significantly up-regulates B7.1, B7.2, CD40, CD54, and CD83 and substantially down-regulates CD14 and macro-pinocytosis, a profile consistent with mature DC. Many CI-induced modulations, but none resulting from cytokine treatment alone, were inhibited by the calcineurin phosphatase inhibitor cyclosporin A. Compared with monocytes treated with CI alone, combined treatment of monocytes with GM-CSF, IL-2, IL-12, and CI augmented B7.1 and CD83 expression and enhanced sensitization of autologous CD8+ T cells to melanoma-antigen-derived peptides. These results suggest that several independent pathways of DC activation can cooperatively enhance the function of monocyte-derived DC.