Background Tumors manifest local immune suppression which protects them from antitumor immunity, and effective immunotherapy must overcome that immune suppression. Intratumoral Immunotherapy (ITIT) applies local immune stimulating treatment to reverse local immune suppression and generate effective local and systemic antitumor immunity. There are many options for intratumoral immunotherapy and for combining ITIT with systemic immunotherapy. Among these options is in vivo intratumoral transfection of plasmids encoding immune stimulatory proteins. IL-12 expressed in tumors has local and systemic antitumor stimulatory effects and is being tested in clinical trials. We study expression of other immune stimulatory proteins along with IL-12 to improve tumor treatment. CD40 ligand (CD154) is primarily expressed on activated T cells and stimulates maturation of antigen presenting cells expressing CD40. We evaluated efficacy of co-expressing IL-12 and CD154 in B16F10 and MC38 murine tumors.

Methods IL-12 and CD154 expression is mediated by in vivo electroporation of 50 ug of each LPS-free plasmid into intradermal tumors that are roughly 50 cubic millimeters. Tumors are electroporated once a week for 2 weeks and observed. Efficacy studies monitor tumor growth kinetics and mouse survival. Mechanistic studies employ flow cytometry of tumor and draining lymph nodes, ELISA measurement of cytokines and chemokines, and gene or cell type deletions by antibodies or genetics.

Results Electroporating IL-12 expressing plasmid slows B16F10 tumor growth significantly but does not stop growth or clear tumors. Electroporation of plasmid expressing CD154 only does not significantly slow tumor growth. When CD154 and IL-12 plasmids are electroporated together it stops tumor growth for weeks and roughly 50% of mice clear the treated tumor without relapse. Similar results were demonstrated using MC38 mouse colorectal tumors. Combined treatment efficacy requires T cells, cDC1 cells, and IFNg. B16F10 tumors mediate immune suppression predominantly by recruiting Treg cells. While expression of IL-12 reduces Treg frequency in B16F10 by roughly 50%, expression of IL-12+CD154 reduces intratumoral Treg frequency by >90% and is the basis for the tumor clearance. One strength of the system is the ability to rapidly screen multiple expressed proteins.

Conclusions The studies demonstrate a novel approach to immunotherapy for tumors that recruit Tregs. The studies highlight the potential of intratumoral immunotherapy to safely and inexpensively stimulate antitumor immune responses. Many other expressed proteins could synergize with IL-12 to immunologically clear treated tumors.