Background Macrophages expressing chimeric antigen receptors (CAR-M) have been shown to reduce tumor burden, remodel the tumor microenvironment (TME), and coordinate a systemic immune response in pre-clinical solid tumor models. Solid tumors overexpress immunosuppressive molecules, such as CD47, which reduces macrophage tumor phagocytosis. We have previously demonstrated that CD47 is a checkpoint that reduces CAR-M function, and have shown that CRISPR-mediated SIRPα knockout (KO) CAR-M are refractory to the anti-phagocytic checkpoint protein CD47.¹ Here, we have enhanced our ability to control gene expression in CAR-M by creating a single vector system that incorporates synthetic shRNA into the CAR intron, enabling simultaneous CAR expression with knockdown of SIRPα. The intronic shRNA platform allows gene-silenced CAR-M and CAR-Monocytes to be manufactured using a single vector in a streamlined single-day process.

Methods To generate gene-silenced CAR-M, we transduced primary human macrophages or monocytes with a novel adenoviral vector comprising a CAR transgene with custom intronic shRNA expressed under a shared promoter. We characterized shRNA-modified CAR-M using a model anti- human epidermal growth factor receptor 2 (HER2) CAR paired with shRNA targeting SIRPα. CAR-M phenotype was characterized using flow cytometry. The anti-tumor efficacy of CAR-M in vitro was monitored by quantifying killing, phagocytosis, and cytokine production in co-culture assays with HER2+ tumor cell lines. The in vivo efficacy of CAR-M was characterized using metastatic solid tumor xenograft models.

Results Intronic shRNA enabled concomitant CAR expression with target SIRPα knockdown. Reduction in SIRPα expression by shRNA was comparable to that achieved by CRISPR/Cas9 ribonucleoprotein. Additionally, the inclusion of an intron significantly augmented expression of the neighboring CAR transgene. Compared to unmodified CAR-M, SIRPα-knockdown CAR-M exhibited enhanced killing, phagocytosis, and cytokine production against HER2+ tumor cells in vitro. Furthermore, SIRPα-knockdown CAR-M significantly delayed tumor growth and prolonged survival in vivo.

Conclusions We show the feasibility of generating gene-silenced primary CAR-M in a single transduction step by integrating CAR delivery with custom intronic shRNA, and we demonstrate that targeted gene knockdown of SIRPα can enhance the anti-tumor activity of CAR-M in vivo. The intronic shRNA design is a generalizable platform that will be valuable for future CAR designs, target tumor antigens, and gene knockout targets.

Reference

1. Sloas C, Gabbasov R, Anderson N, Abramson S, Klichinsky M, Ohtani Y. 144 SIRPα deficient CAR-Macrophages exhibit enhanced anti-tumor function and bypass the CD47 immune checkpoint. J Immunother Cancer. 2021;9:A152-A152.