Antibody-Dependent Phagocytosis by SIRPα-Knockout Macrophages for Cancer Immunotherapy

Introduction:: Macrophage immunotherapy is emerging as a possible therapy for solid tumors based on an ability to phagocytose or ‘eat’ other cells. Macrophage phagocytosis is impeded by SIRPα which binds CD47 on healthy and cancer cells to convey a ‘don’t eat me’ signal (Figure 1). Most recent clinical efforts aimed at blocking the CD47-SIRPα interaction focus on anti-CD47 monoclonal antibodies which show significant side effects including depletion of healthy cells. While macrophage targeted immunotherapy is promising for liquid tumors, there are still substantial limitations against solid tumors that motivates a deeper understanding of the potency of CD47-SIRPα against phagocytosis. Here, we deleted SIRPα in conditionally immortalized macrophages (CIM) progenitors and showed that these engineered macrophages readily engulf opsonized targets, including B16 melanoma cells.

Materials and Methods:: SIRPα knockout CIM progenitors (SIRPα-KO CIMs) were made by transducing Cas9⁺ ER (estrogen receptor)-Hoxb8 CIMs with lentivirus encoding a guide RNA targeting the Sirpa gene [1]. CIMs were passaged in suspension culture in media supplemented with β-estrogen and recombinant mouse GM-CSF. To differentiate SIRPα-KO into macrophages, progenitors were washed with PBS and plated in media supplemented with recombinant mouse M-CSF for 7 days. For phagocytosis assays, opsonized mouse red blood cells (RBCs) or streptavidin-coated polystyrene beads were added to macrophages and incubated for 90 min. Similarly, B16 cells opsonized with a melanoma-specific monoclonal antibody were added to macrophages and incubated for 2 hours. Non-phagocytosed targets were washed with PBS and the remaining cells were fixed and imaged by fluorescence microscopy. The number of phagocytic events (complete engulfment of targets by macrophages) was recorded, and the phagocytic index was calculated as the percentage of phagocytic events multiplied by the number of engulfed cells per macrophage [1].

Results, Conclusions, and Discussions:: Results and Discussion: We confirmed successful knockout of SIRPα in CIMs by flow cytometry and similarly demonstrated expression of all four murine Fc receptors to engage the melanoma-specific monoclonal antibody. Gene-edited CIMs differentiated into adherent cells resemble bone-marrow derived macrophages based on their morphology, surface marker staining, and engulfment of polystyrene microbeads opsonized with IgG antibody. Importantly, SIRPα-KO CIMs exhibited greater phagocytosis of mouse RBCs than WT CIMs, demonstrating the lack of inhibition by CD47 by targets on macrophages lacking SIRPα. To determine the potential of SIRPα-KO CIMs as a cell-based immunotherapy against solid tumors, we investigated their ability to phagocytose B16 melanoma cells that also express CD47. SIRPα-KO CIMs were again highly phagocytic towards the opsonized B16 cells in vitro (Figure 2).



Conclusion: Our results are the first to use engineered CIMs in studies of antibody-dependent cellular phagocytosis, an important immune effector function in host defense as well as cancer immunotherapy. We further demonstrate that genetic deletion of SIRPα in CIMs enhances phagocytosis of cellular targets expressing CD47 including mRBCs and mouse melanoma cells. Key advantages of CIMs include their close resemblance to primary mouse macrophages together with the capacity for further engineering and to expand in large numbers.




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References (Optional): : [1] Dooling, L.J., Andrechak, J.C. et al. Cooperative phagocytosis of solid tumours by macrophages triggers durable anti-tumour responses. Nat. Biomed. Eng (2023). https://doi.org/10.1038/s41551-023-01031-3