Page 100 - Haematologica Vol. 109 - July 2024

ARTICLE - CD47 inhibition is augmented with TLR3 agonism H.E. Ramsey et al.
and magrolimab, representing coverage of CD47 protein (Figure 1A). Complete inhibition of CD47 by ALX90 was confirmed using immunofluorescence in MV-4-11 (Figure 1B) as well as MOLM-13 and THP-1 (Online Supplementary Figure S1A).
The addition of CD47 inhibition to venetoclax/azacitidine treatment regimen leads to decreased tumor burden and lifespan prolongation in multiple in vivo cell line derived models of acute myeloid leukemia
In order to determine the efficacy of CD47 inhibition in vivo, NSGS mice were treated with a VEN/AZA, ALX90 or a combination. On day 28, when vehicle mice became mori- bund, tissues were collected for tricompartmental analysis, and human CD45/CD33+ AML cells were counted relative to the amount of healthy murine CD45+ cells. As shown in Figure 1C the addition of CD47i to a VEN/AZA regimen led to a significant decrease in AML when compared to a VEN/ AZA alone. Furthermore, CD47i amended splenic weight, a result of reduced leukemia within the spleen and de- creased extramedullary hematopoiesis, which is reflective of healthy BM (Online Supplementary Figure S1B), as well as body weight (Online Supplementary Figure S1C). Bone marrow and splenic tissues were stained for human CD45,
and immunohistochemistry revealed a significant decrease of tumor cells in both bone marrow and spleen (Online Supplementary Figure S2A). In an additional MOLM-13 cell line xenograft, the addition of CD47 inhibition led to sig- nificant increases in lifespan compared to ALX or VEN/AZA treatments alone (Figure 1D).
CD47i leads to decreased tumor burden in extramedullary compartments of multiple acute myeloid leukemia patient-derived xenograft models, but not within bone marrow
Given our observation that AML PDX models confluently involved the BM, we began measuring the expression of CD47 in various AML PDX models in an attempt to correlate surface protein expression as a biomarker. As shown in Fig- ure 2A, varying levels of CD47 were noted between patient samples, which were not dependent on any detected mu- tations (Online Supplementary Table S1). In order to further elucidate the role of CD47 as a targetable biomarker, we explored the efficacy of monotherapy in PDX models with both high “++” (18-12-001) and low “+” (18-10-009) CD47 expression. Post-transplantation, peripheral chimerism was tested weekly in these models in search of circulating tumor cells, and leukemic engraftment was established
AB
C
Figure 2. CD47 inhibition monotherapy reveals minimal tumor clearance from bone marrow in a patient-derived xenograft model.
(A) Surface expression of hCD47 was measured by flow cytometry in multiple patient-derived xenograft (PDX) models. (B) CD47 in- hibition leads to decreased leukemia in peripheral blood of PDX models with significant differences shown between 30 mg/mL and vehicle groups. (C) Chimeric analysis of PDX models reveals the effects of ALX in blood, spleen (SPL), and bone marrow (BM). MFI: mean fluorescence intensity; M/W/F: Monday/Wednesday/Friday; NS: not significant. *P<0.05, **P<0.01 and ***P<0.001.
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