CD47-SIRP\(\alpha\) Checkpoint Disruption in Metastases Requires Tumor-Targeting Antibody for Molecular and Engineered Macrophage Therapies  
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macrophages by passivating phagocytic activation of the cytoskeleton [2]. Recent interest in CD47 as a cancer therapeutic target has led to rapid expansion of preclinical and clinical efforts to physically block its inhibitory signaling in liquid and solid tumors that express CD47 at normal or elevated levels [3; 4]. CD47-blocking antibodies in human trials have demonstrated promising early results in lymphoma that indicate a requirement for combination with a tumor-opsonizing IgG (e.g., anti-CD20, rituximab) [5]. However, efficacy with anti-CD47 antibodies in solid tumors remains unclear in the clinic [4], and a pre-clinical replication study of a syngeneic immunocompetent mouse model also cast doubt on early reports of success in mouse models with anti-CD47 monotherapy [6]. More generally, some immunotherapies (e.g., engineered T cells) show impressive success against liquid tumors with durable long-term responses [7; 8; 9], but solid tumors remain a general challenge--especially with poorly immunogenic tumors and with metastatic tumors that are most relevant to patients. Infusions of species-relevant anti-CD47 IgG (e.g., mouse IgG into mouse, or humanized IgG into human) cause loss of blood cells, which confirms biological activity but raises concerns over safety [5; 6]. Such on-target, off-tumor effects underscore the limited efficacy of monotherapy and motivate an increasing focus on combination therapies (Figures 1 and 2 Ai) [3; 4; 5; 10; 11; 12; 13; 14]. Trials are now beginning with anti-SIRP\(\alpha\), but SIRP\(\alpha\) is expressed on many cell types and clinical advantages remain unclear [15; 16; 17]. Transport of antibodies and effector cells into solid tumors including metastatic lesions can also be problematic. Figure 1: CD47-SIRP\(\alpha\) signaling and pro-phagocytic cues between macrophages and solid tumor cells. (**A**) Fc regions of tumor-opsonizing antibodies bind to Fc receptors on macrophages, and Fab regions bind to tumor cell antigens. These signals act as a pro-phagocytic cues, but phagocytosis is ineffective when CD47 is only partially blocked by anti-CD47. (**B**) Complete knockout (KO) of tumor cell CD47 to ablate CD47-SIRP\(\alpha\) signaling without a pro-phagocytic signal results in ineffective phagocytosis. (**C**) Both tumor cell CD47 KO (or anti-SIRP\(\alpha\) block on macrophages) and tumor-opsonizing antibodies results in effective phagocytosis. Here, we focus first on poorly immunogenic, syngeneic B16F10 melanoma cells in lung [18] to model solid tumor metastasis within immunocompetent C57 BL/6 mice. Deletion of CD47 on opsonized B16F10s increases the engulfment of cell suspensions in vitro by bone marrow-derived macrophages in our recent studies [19]. We now find that in vivo efficacy against established B16F10 metastatic nodules requires both CD47 deletion and a pro-phagocytic, melanocyte-specific IgG. Our quantitative analyses of tumor growth with CD47 deletion together with high-resolution imaging indicates on-target phagocytosis with IgG opsonization in B16F10 tumor suppression. In a second approach, we modify marrow macrophages for an adoptive cell therapy by pre-blocking SIRP\(\alpha\) and pre-loading Fc-receptors with tumor-opsonizing IgG prior to infusion. Human lung carcinoma A549 liver metastases in immunodeficient mice confirm the effects of this cell therapy and thus confirm the benefits of the combination approach. Figure 2: Tumor-opsonizing antibodies can drive regression of metastases only when CD47 is disrupted. (**A**) (i) Human clinical CD47 blockade in solid tumors compared to our main design of a suitable preclinical model (mouse). Anti-CD47 monotherapies show no efficacy, motivating a combination with tumor-opsonizing antibodies (e.g., Cetuximab in colorectal cancer). Anti-Tyrp1 monotherapy also showed no efficacy against human melanoma. Two distinct approaches are taken in our model.