Background Solid tumors remain a challenging frontier for adoptive cellular therapies (ACT). Armoring T-cells with cytokines, such as interleukin 12 (IL-12), to remodel the tumor microenvironment (TME) has demonstrated preclinical efficacy against solid tumors. However, the clinical utility of IL-12 is limited by systemic toxicities, requiring tight control of expression. Herein, we show that T-cells armored with a small molecule-controlled membrane bound IL-12 (mbIL-12) drives regulation of pharmacodynamic markers and solid tumor efficacy in xenograft and syngeneic solid tumor models.
Methods We regulated mbIL-12 expression using Obsidian’s cytoDRIVE® technology. In this system, a drug responsive domain (DRD) is fused to a protein of interest. In the “off-state” the fusion protein is rapidly degraded by the proteasome. Adding a small molecule ligand stabilizes the complex, enabling expression (the “on-state”). Here, we use a DRD derived from carbonic anhydrase 2 and the FDA-approved inhibitor acetazolamide (ACZ) as a stabilizing ligand. Unlike most other regulation systems, cytoDRiVE® is both fully human, reducing immunogenicity, and induced pharmacologically, allowing on-demand control. Adding oligomerization domains increases the local density of DRDs to form modulation-hubs that further increase the regulation of mbIL-12.
Cytokine levels were determined using flow cytometry and Meso Scale Discovery assays. Human mbIL-12 modulation hubs and CD19-CARs were transduced in peripheral blood T-cells and evaluated in vivo against subcutaneous Raji xenografts that form solid tumors in NSG mice after inoculation in Matrigel. Mouse constructs were evaluated in CD8 gp100 (PMEL) TCR transgenic cells against subcutaneous B16-F10 melanoma in C57BL6 mice.
Results In the xenograft setting, ACZ dosing resulted in 35-fold regulation of IL-12 in the plasma and showed remarkable ACZ-dependent anti-tumor efficacy against large, solid Raji tumors at a 10x lower cell dose than unarmored CAR-Ts. In the immunocompetent PMEL/B16 model, IL-12 modulation-hub PMEL cells slowed tumor growth over unarmored PMEL cells and showed improved tolerability over secreted IL-12. Animals receiving IL-12 modulation-hub cells showed ACZ-dependent regulation of IL-12 and IFNγ in the plasma with levels 100-fold and 20-fold less, respectively, than with constitutive secreted IL-12 cells. This regulation led to functional impacts at the cellular level, including an increase in circulating antigen presenting cells.
Conclusions The cytoDRiVE® platform enables enhanced regulation of IL-12 armored T-cells in multiple preclinical solid tumor models, potentiating an improved therapeutic window for IL12 in ACT.
Ethics Approval All animals studies were IACUC approved.
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