Exploitable mechanisms of antibody and CAR mediated macrophage cytotoxicity

Introduction

Tumor-associated macrophages (TAMs) are pivotal components of the tumor microenvironment, either promoting or combating cancer progression directly or indirectly through T-cells. T-cells are generally recognized as the main immune mediators of tumor cell death, and tumor mutational burden (TMB) is now a well-recognized predictor of response to many current immune therapies. Direct antitumor effects of macrophages are achieved by phagocytosis or tumor cytotoxicity, and unlike T-cells, do not rely on tumor neoantigens, which makes them particularly well suited to overcome tumors with low TMB. However, the direct killing capacity of a macrophages is low. Numerous approaches attempt to enhance the phagocytic capacity of macrophages, such as by combining antitumor antibodies to engage pro-phagocytic pathways in macrophages with blockade of “don’t eat me” receptors^1,2, however, the effect of these approaches have been surprisingly ineffective at eliminating tumor in patients³.

Recent advances to further improve macrophage tumor-specific phagocytosis leverages a Chimeric Antigen Receptor (CAR), which also utilizes a tumor-binding antibody, directly expressed on the macrophage surface as an extracellular domain with a chimeric transmembrane and intracellular pro-phagocytic signal^4,5. We generated CARs with the same intracellular signaling domain as used by antibodies – the Fc Receptor, which signals through ITAMs. The dominant mechanism by which macrophages directly kill cancer cells, especially when directed to target them with an antibody or a CAR, remains unestablished; better understanding of this mechanism may lead to more effective macrophage directed therapies. Macrophages are often thought to phagocytose and degrade live cancer cells upon recognizing tumors via antibody or CAR binding, a concept influenced by their well-established role in clearing necroptotic dead cells⁶. To test whether direct live tumor phagocytosis is central to tumor killing by macrophages, and to further elucidate the pathways critical to CAR-directed macrophage killing of cancer cells in an unbiased manner, we performed parallel CRISPR screens using two different CARs targeting two different tumor antigens expressed by a human solid cancer cell line.

While many co-culture screens have been conducted to study the interaction between cancer cells and immune cells, such as T cells^7,8 or NK cells^9,10, relatively few have focused on macrophages, and most of these studies target liquid tumors¹¹. Macrophages are particularly abundant...