Cytokine driven effector functions of CD8 cells: application to protease-activated cytokine therapy for cancer

Abstract

Abstract Cytokines are potent immune mediators and play a crucial role in the differentiation and activation of effector CD8 T cells which are critical in anti-tumor responses. Here we tested the ability of IL-2, IL-12, and IL-18 to drive expression of gamma interferon (IFNg) in the absence of intentional antigen stimulation. We show in vitro that spleen cells from both BALB/c and C57BL/6 mice produced large amounts of IFNg and downstream mediators in response to these cytokines individually or synergistically in combination. Moreover, CD8 cells purified from Colon38 tumors can be similarly activated, demonstrating that they are not permanently inactivated or exhausted. These data illustrate innate-like function of CD8 cells, including those in the tumor microenvironment (TME). To address concerns of severe toxicities that can accompany systemic delivery of cytokines, we designed protease-activated cytokine fusion proteins (FPs) which exploit the overexpression of proteases in tumors to release active cytokine in the TME. Using gene transfection techniques, we show that an IL-2FP with a matrix metalloproteinase 2, 9 cleavage sequence can be activated in the TME and reduce tumor growth. In contrast, a FP with a scrambled non-cleavable sequence does not reduce tumor growth, illustrating the specificity of cleavage of the FP in vivo. Additionally, we demonstrate that systemic expression of the IL-2FP elicits little systemic toxicity in striking contrast to the delivery of free IL-2. Collectively, these data support the development of cytokine fusions proteins as a new class of therapeutics to promote anti-tumor effectors with limited detrimental cytokine side effects.