A Protease Activatable Interleukin-2 Fusion Protein Engenders Antitumor Immune Responses by Interferon Gamma-Dependent and Interferon Gamma-Independent Mechanisms.

Cytokines are powerful mediators of immune responses and some, such as interleukin-2 (IL-2), have achieved dramatic responses as cancer immunotherapies. Unfortunately, systemic administration often results in deleterious side effects, prompting exploration of strategies to localize cytokine activity to the tumor microenvironment (TME). To this end, we constructed an IL-2/IL2Ra fusion protein (IL-2FP) with an MMP2/9-specific cleavage site, designed to exploit the dysregulated protease activity in the TME to selectively activate IL-2 in the tumor. To determine if TME protease activity is sufficient to cleave the FP and if FP activity is due to specific cleavage, we created Colon 38 tumor cell lines expressing similar levels of IL-2FPs with either a functional cleavage site [H11(cs-1FP)] or a scrambled, noncleavable sequence [H2(scramFP)]. H11(cs-1FP) tumors demonstrated reduced tumor growth, characterized by regressions not observed in H2(scramFP) tumors. Analysis through qRT-PCR, flow cytometry, and immunohistochemistry indicate robust CD8 responses in the H11(cs-1FP) tumors. Interferon gamma (IFNg) knockout mice revealed that the immune effects of the cleavable FP are mediated through both IFNg-dependent and IFNg-independent mechanisms. Collectively, these data suggest that matrix metalloproteinases (MMPs) in the TME can cleave the IL-2FP specifically, thus enhancing an antitumor response, and provide a rationale for further developing this approach.

Superkine IL-2 and IL-33 Armored CAR T Cells Reshape the Tumor Microenvironment and Reduce Growth of Multiple Solid Tumors.

Chimeric-antigen receptor (CAR) T-cell therapy has shown remarkable efficacy against hematologic tumors. Yet, CAR T-cell therapy has had little success against solid tumors due to obstacles presented by the tumor microenvironment (TME) of these cancers. Here, we show that CAR T cells armored with the engineered IL-2 superkine Super2 and IL-33 were able to promote tumor control as a single-agent therapy. IFNγ and perforin were dispensable for the effects of Super2- and IL-33-armored CAR T cells. Super2 and IL-33 synergized to shift leukocyte proportions in the TME and to recruit and activate a broad repertoire of endogenous innate and adaptive immune cells including tumor-specific T cells. However, depletion of CD8+ T cells or NK cells did not disrupt tumor control, suggesting that broad immune activation compensated for loss of individual cell subsets. Thus, we have shown that Super2 and IL-33 CAR T cells can promote antitumor immunity in multiple solid tumor models and can potentially overcome antigen loss, highlighting the potential of this universal CAR T-cell platform for the treatment of solid tumors.