iPSC-Derived Macrophages Engineered to Express IL-12 Enhance Cancer Cell Killing of a Multi-Cell Type Therapeutic Platform

Ian Hay1, Abigail Blatchford1, Claire Aibel1, Christopher B. Rohde1, Matthew Angel1
1Factor Bioscience Inc., Cambridge, MA

Mol Ther, Vol 32, No 4S1, 2024

 

The immunologically cold microenvironment of solid tumors limits the ability of immune cells to infiltrate and attack cancer cells. The systemic injection of potent cytokines such as interferon gamma (IFNγ) and interleukin 12 (IL-12) have shown tumor shrinkage in mouse models, but have faced challenges in human clinical trials due to dose-limiting toxicities. Myeloid cells, including macrophages, can deliver cargo to the solid tumor microenvironment. However, autologous macrophages are limited by their resistance to genetic engineering, limited expansion potential, cost of characterization and the limited quantity of cells attainable via leukapheresis. Bioreactor-based differentiation of induced pluripotent stem cells (iPSCs) into macrophages can overcome the limitations of autologous macrophages, enabling the generation of large numbers of well characterized, genetically identical, engineered macrophages. Here, we present iPSC-derived macrophages engineered to overexpress IL-12 for delivery to the tumor microenvironment. Bi-allelic knockout of the beta-2-microglobulin gene (B2M-/-) is designed to reduce alloreactivity of transplanted iPSC-derived macrophages. B2M-/- iPSC-macrophages transfected with mRNA encoding both IL-12 subunits joined by a self-cleaving peptide (IL-12 P2A) or a flexible linker (IL-12 fusion) resulted in the production of 20 and 40 ng/mL of the IL-12p70 heterodimer, respectively, as assessed via ELISA 24 hours after transfection. Both IL-12 mRNA constructs resulted in similar levels of IFNγ production when co-cultured with PBMC-derived T cells (17 ng/mL for IL-12 fusion and 15 ng/mL for IL-12 P2A) with no detectable IFNγ in the absence of IL-12 mRNA or T cell co-culture, indicating functional bioactivity. The co-culture of B2M-/- iPSC-derived macrophages with allogeneic PBMC-derived T cells did not upregulate T-cell activation markersCD25 or CD69, as assessed via flow cytometry, suggesting a lack of host-vs-graft-type immunoreactivity of the B2M-/- iPSC-derived macrophages, a feature of importance for durable adoptive cell therapies. When IL-12-mRNA transfected iPSC-derived macrophages were co-cultured with PBMC-derived T-cells and a GFP-expressing ovarian adenocarcinoma cell line SK-OV-3 at a 10:1 effector to target ratio for 72 hours, cancer cell killing was significantly greater when compared to non-transfected iPSC-derived macrophages, as assessed by real-time fluorescence imaging (p=0.03). Engineered iPSC-derived macrophages offer a path to repolarize the tumor microenvironment and translate the clinical success of T cell immunotherapies targeting hematologic cancers to solid tumors, which comprise 90% of cancer cases. Here we show a scalable platform for producing iPSC-derived macrophages engineered to express the potent immunostimulatory protein IL-12. Engineered iPSC-derived macrophages may thus prove useful for the development of therapies designed to deliver biologics previously limited by toxicity or bioavailability directly to the tumor microenvironment.