Permanent Efferocytosis Prevention by Terminating MerTK Recycle on Tumor-Associated Macrophages for Cancer Immunotherapy

Efferocytosis of apoptotic tumor cells by tumor-associated macrophages mediated through the phosphatidylserine (PtdSer)/MER proto-oncogene tyrosine kinase (MerTK) axis can exacerbate tumor immunosuppression, and conversely, prevention of efferocytosis via blocking PtdSer-MerTK association using prevalent antibodies represents a promising strategy for reversing tumor immunosuppression and boosting antitumor immunity. However, it remains unclear whether the antibody blockade can induce durable efferocytosis prevention and achieve sustained tumor growth inhibition. Here, we have shown that utilizing PtdSer and MerTK antibodies induced only a transient rather than a persistent efferocytosis prevention effect, and little enhancement was observed even after improving antibody enrichment in tumor sites. Further mechanistic studies suggested that degradation of <i>anti</i>-MerTK antibody and recycling of the MerTK receptor to the cell membrane would compromise the therapeutic benefits of antibody blockade. Based on these findings, we developed a CRISPR/Cas9 gene editing system deployed using Cas9 mRNA and MerTK sgRNA to permanently knock out MerTK, which achieved durable efferocytosis prevention, elicited persistent in situ vaccination immune responses via enhancing X-ray irradiation-induced immunogenic cell death, and led to sustained tumor suppression effects together with <i>anti</i>-PtdSer antibody and X-ray irradiation treatment in multiple B16 melanoma tumor models. Our findings provide a reliable gene-editing-mediated strategy for long-term modulating MerTK homeostasis and overcoming MerTK-dependent cancer immune evasion, generating adaptive antitumor immune responses for sustained cancer immunotherapy.