## 2 Results
### Tumor Clearance Induced by Opsonization and CD47 Deletion Prolongs Survival, but Some Tumors Escape
Antibody-Engineered Marrow Macrophages/Monocytes Eliminate Lung Tumor Modules in WT Metastases, While Anti-Tyrp1 Treatment Alone Does Not  
WT metastases that express CD47 are more clinically relevant than CD47 KO tumors, and so we sought a translatable approach to the treatment of WT B16F10 lung metastases. To do this, we extended an adoptive cell therapy we had developed [29], in which an anti-SIRP\(\alpha\) blocking antibody is added to fresh marrow macrophages and monocytes together with anti-Tyrp1 loaded onto Fc receptors (Figure 5A). Tail-vein injections of these Antibody-primed Plus SIRP\(\alpha\)-Blocked (A'PB) engineered macrophages previously proved to be safe and effective against subcutaneous human tumor lines in immunodeficient mice but had not been tested on metastases in syngeneic, immunocompetent models. Our earlier studies also showed that sorting of macrophages/monocytes from marrow gave the same results in terms of tumor suppression as unsorted marrow, likely because those cell fractions are the main marrow cell types that express SIRP\(\alpha\). Pre-incubation of freshly isolated marrow maximizes the saturable antibody binding to the effector cells and distinguishes our antibody-engineered macrophage approach from systemic injections of antibodies. In particular, systemic anti-SIRP\(\alpha\) will preferentially bind circulation-accessible SIRP\(\alpha\), such as the SIRP\(\alpha\) displayed on splenic macrophages and on liver Kupffer macrophages [30]. A'PB cells were tail-vein injected into mice with established lung metastases (\(2\times 10^{7}\) A'PB at day 4 post-inoculation). To continue to drive phagocytosis via tumor opsonization, anti-Tyrp1 was administered per above (Figure 2B). WT metastases are clearly suppressed by A'PB (Figure 5B and Figure S8), whereas anti-Tyrp1 does not show significant effects relative to controls including isotype IgG2a (in agreement with Figure 2 Aii and Figure S2A,B). Survival estimations for these day 14 timepoints show prolonged survival with A'PB, giving a projected median survival that is almost 2-fold longer than the control cohort (again showing \(\sim\)23 day median survival) (Figure 5C). To further enhance the anti-tumor efficacy, a second dose of APB at day 6 (denoted as 2\(\times\) A\({}^{\prime}\)PB) was administered, which confirmed significant suppression of tumor growth (Figure 5B) and prolonged the projected survival. Compared to the median survival of the control cohort in this separate study (-25 days), all of the treated cohort survived longer (Figure 5C), and 20% were projected to survive 3-4-fold longer (82 days), which is the longest survival time in any of our studies. Multiple doses of our antibody-engineered marrow macrophage/monocyte therapy can thus offer further opportunity to enhance suppression of WT metastases. Figure 5: SIRP\(\alpha\)-blocked, Tyrp1-targeted marrow cells suppress wild-type metastases, while anti-Tyrp1 treatment alone does not. (**A**) Schematic of SIRP\(\alpha\)-blocked, Tyrp1-targeted (A\({}^{\prime}\)PB) fresh marrow cell production for tail-vein infusion into mice with WT B16F10 lung metastases. Monocytes and macrophage pre-cursors are the main marrow cells with SIRP\(\alpha\) and Fc receptors, and the latter are pre-loaded with anti-Tyrp1 prior to infusion. Mice receive 2 \(\times\) 10\({}^{\prime}\) antibody-engineered marrow cells intravenously on day 4 (1\(\times\) A\({}^{\prime}\)PB) and 6 (2\(\times\) A\({}^{\prime}\)PB). Additional anti-Tyrp1 was administered i.v. on days 4, 5, 7, 9, 11, and 13 (250 \(\upmu\)g doses). (**B**) WT B16F10 tumor burden in mice treated with antibody-engineered marrow. Left: Control mice were treated with either isotype IgG2a antibody (gray, 250 \(\upmu\)g dosing regimen) or received no treatment (black), while treated mice received either anti-Tyrp1 (250 \(\upmu\)g dosing regimen) or A\({}^{\prime}\)PB marrow with anti-Tyrp1 (250 \(\upmu\)g dosing regimen). Mean and s.e.m. (* \(p<\) 0.05, n.s.