Page 101 - Haematologica Vol. 109 - July 2024

ARTICLE - CD47 inhibition is augmented with TLR3 agonism H.E. Ramsey et al.
levels remained unchanged in that compartment (Figure 3B). In order to understand this, we examined the resident macrophages responsible for phagocytosis of cells treated with CD47i. Using the remaining cells from our PDX models from the experiment in Figure 3, we calculated the number of macrophages residing within the compartments and compared with the number of macrophages found with- in a healthy, naïve NSGS mouse. In all treatment groups, macrophages were increased in the blood and significantly decreased in the other tissues, with the largest diminish- ment observed in the BM (Figure 4A-C). Leukemia-associated macrophages (LAM) are a type of tissue-resident macrophages noted in the presence of leukemia. LAM are largely noted to be CD206+ ‘pro-tu- moral’, M2 macrophages. M2 macrophages are considered pro-tumoral due to their decreased phagocytotic ability and production of immunosuppressive cytokines.21,22 Inter- estingly, CD206 expression has been noted as a negative prognostic factor in AML.21 By contrast, M1 (CD11b+) macro- phages are associated with pro-inflammatory/anti-tumoral activity with increased phagocytosis and the production of inflammatory cytokines.23 Treatments such as lipopoly- saccharide and interferon-γ (IFN-γ) have been shown to promote proliferation and polarization of macrophages to an M1 status, supporting TH1 activation and tumoricidal activity.24,25 While little is known about the effect that AML blasts have on the tissue-resident macrophages in mu- rine PDX models, we began to look at these populations. To this end, we began characterizing the F4/80+ resident macrophage populations in two additional PDX models 8 weeks post-transplantation (20-09-002 and 19-03-007),
Figure 3. The activity of CD47i in the bone marrow. (A) Flow cytometric analysis denoting coverage of hCD47 epitope on human acute myeloid leukemia (AML) cells of 2 AML patient-derived xenograft (PDX) models. The addition of ALX90 to a venetoclax/ azacitidin (VEN/AZA)-based regimen in AML PDX (18-10-009), leads to decreases in intercompartmental tumor burden (B) with Kaplan-Maier analysis of an VEN/AZA and VEN/AZA + ALX regimen showing failure to increase lifespan (C). MFI: mean fluorescence intensity; BM: bone marrow; SPL: spleen. *P<0.05, **P<0.01 and ***P<0.001.
Haematologica | 109 July 2024
at 4 weeks post-transplant. At this juncture, mice were treated with ALX90 monotherapy at a low 5 mg/kg and high 30 mg/kg dose (Figure 2B). Four weeks after treat- ment, mice were euthanized, and tissues were harvested for tricompartmental analysis. CD47i monotherapy led to a significant decrease in tumor burden in both the blood and spleen (Figure 2C). Contrary to CDX models, much of the BM remained unaffected in these PDX (Figure 2C). In order to confirm that ALX90 bound to CD47 antigen on PDX cells, we harvested human cells from the blood, BM and spleen from these mice, stained them for CD47, and quantified ALX90 bound human hematopoietic CD45+/CD33+ cells in a dose-dependent fashion (Figure 3A).
In order to determine if VEN/AZA increased efficacy of CD47i monotherapy, we treated the CD47-low AML PDX (18-10- 009), with VEN/AZA and ALX90. Tumor kinetics were mon- itored peripherally through weekly measurements in blood chimerism, showing a significant decrease in chimerism in a regimen of all three drugs (Online Supplementary Figure S2B). Mice were sacrificed at 8 weeks, and VEN/AZA+ALX was found to lead to significant decreases of AML in both the blood and spleen of mice, with a more modest effect is more modest in the bone marrow (Figure 3B); however, the addition of CD47i failed to prolong the lifespan of VEN/ AZA treated mice in this experiment (Figure 3C).
CD47 inhibition in the bone marrow may be dependent upon the number and type of macrophages residing within the bone marrow during treatment
CD47 inhibition was detected on BM resident tumor cells in a PDX model of AML (Figure 3A), yet tumor chimerism
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