Page 104 - Haematologica Vol. 109 - July 2024

ARTICLE - CD47 inhibition is augmented with TLR3 agonism H.E. Ramsey et al.
and began treatment with VEN/AZA + ALX + poly(I:C). The “quadruplet therapy” of poly(I:C) together with VEN/AZA + ALX led to a drop in peripheral chimerism (Figure 6A). Again, at 8 weeks, mice were sacrificed and a significant decrease in leukemia was seen in the blood, spleen and, notably, BM in instances in which poly(I:C) was added to a Ven/ AZA + ALX regimen (Figure 6B, C). We analyzed the quality of the remaining murine BM in this model for functional hematopoiesis. Erythropoiesis was noted through higher counts of TER119+ cells with the BM, along with higher levels of myeloid (CD11b+) and neutrophil (CD11b+/Ly6g+) levels (Figure 7A). Furthermore, the macrophage popula- tion within the BM of VEN/AZA+ALX-treated mice versus VEN/AZA + ALK + poly(I:C)-treated mice was significantly diminished. The resident macrophages within the BM of these VEN/AZA + ALX + poly(I:C)-treated mice included a greater portion with a M1 phenotype (Figure 4B). In cyto- spins, the the return to more normal hematopoiesis was evident (Figure 7B). Furthermore, the addition of poly(I:C) to a VEN/AZA + ALX regimen significantly extended survival in treated mice, even after ceasing treatment (Figure 7C). Finally, to confirm active phagocytosis we conducted an ex vivo phagocytosis assay with two additional AML primary patient samples. For this, naïve NSGS mice were again
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treated with poly(I:C) or control vehicle and after 2 weeks of treatment, the two BM groups were harvested and incu- bated with pHrodo-labeled patient samples pre-incubated with or without ALX90. Target cells labeled with pHrodo will only fluoresce when under the highly acidic condition of the phagolysosome. This fluorescence, when emitted from a macrophage, ensures that the engulfed cell was successfully phagocytosed and further incorporated into a functional phagolysosome. As shown in Figure 8A, F4/80+ bone marrow macrophages displayed increased phago-ly- sosomal acidification following phagocytosis of patient samples when ALX is employed in combination with poly(I:C) treated BM versus BM from vehicle-treated mice. In order to similarly validate the cooperativity of ALX and poly(I:C) in human cells, peripheral blood monocytes were isolated from healthy donors, differentiated into macrophages and further subjected to macrophage stimulation with poly(I:C). These macrophages were incubated with pHrodo-treated AML cells which were then subsequently treated with CD47i (ALX90 or magrolimab). Baseline phagocytosis in these assays was measured in cultures where AML target cells were incubated with drug corresponding IgG antibodies. While there was expected variation between patients, con- sistently the greatest fold changes in phagocytosis were
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Figure 7. Murine hematopoiesis and lifespan after a venetoclax/azacitidin plus ALX plus polyinosinic:polycytidylic acid regimen.
(A) Flow cytometry of remaining murine bone marrow (BM) in patient-derived xenograft (PDX) models reveals significant differ- ences in erythroid, myeloid, and neutrophil populations. (B) Wright Giemsa staining of BM cytospins reveal decreased acute my- eloid leukemia (AML) blast populations in venetoclax/azacitidin plus ALX plus polyinosinic:polycytidylic acid (VEN/AZA + ALX + poly(I:C))-treated PDX mice. (C) Kaplan Mayer analysis affirms increased lifespan when poly(I:C) is added to a VEN/AZA + ALX regimen.
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