TLR3 agonism augments CD47 inhibition in acute myeloid leukemia

Abstract

CD47-SIRPa is a myeloid check point pathway that promotes phagocytosis of cells lacking markers for self-recognition. Tumor cells can overexpress CD47 and bind to SIRPa on macrophages, preventing phagocytosis. CD47 expression is enhanced and correlated with a negative prognosis in acute myeloid leukemia (AML), with its blockade leading to cell clearance. ALX90 is an engineered fusion protein with high affinity for CD47. Composed of the N-terminal D1 domain of SIRPα genetically linked to an inactive Fc domain from human immunoglobulin (Ig) G, ALX90 is designed to avoid potential toxicity of CD47-expressing red blood cells. Venetoclax (VEN) is a specific B-cell lymphoma-2 (BCL-2) inhibitor that can restore apoptosis in malignant cells. In AML, VEN is combined with azanucleosides to induce superior remission rates, however treatment for refractory/relapse is an unmet need. We questioned whether the anti-tumor activity of a VENbased regimen can be augmented through CD47 inhibition (CD47i) in AML and how this triplet may be enhanced. Human AML cell lines were sensitive to ALX90 and its addition increased efficacy of a VEN plus azacitidin (VEN+AZA) regimen in vivo. However, CD47i failed to clear bone marrow tumor burden in PDX models. We hypothesized that the loss of resident macrophages in the bone marrow in AML reduced efficiency of CD47i. Therefore, we attempted to enhance this medullary macrophage population with agonism of TLR3 via polyinosinic:polycytidylic acid (poly(I:C)), which led to expansion and activation of medullary macrophages in in vivo AML PDX models and potentiated CD47i. In summary, the addition of poly(I:C) can enhance medullary macrophage populations to potentiate the phagocytosis merited by therapeutic inhibition of CD47.

Figure 1.

ALX90 conceals CD47 both in vivo and in vitro. In vitro incubation of MV-4-11, MOLM-13 and THP-1 with CD47 inhibitors ALX90 (100 nM) or magrolimab (10 ug/mL) conceals surface expression of CD47 in acute myeloid leukemia (AML) cell lines when analyzed via (A) flow cytometry and (B) in the MV-4-11 cell line by confocal microscopy (green: hCD47, blue: DAPI). (C) The addition of ALX90 to a venetoclax/azacitidine (VEN/AZA) regimen leads to decreased tumor burden in an MV-4-11 as shown through flow cytometric analysis. (D) Survival was increased in a MOLM13 cell line-derived xenograft (CDX) when ALX90 was added to VEN/AZA (P=0.01). (D). MFI: mean fluorescence intensity; Mag: magrolimab; BM: bone marrow; SPL: spleen. *P<0.05, **P<0.01 and ***P<0.001.

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 inhibition 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.

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.

Figure 4.

Analysis of acute myeloid leukemia patient-derived xenograft resident murine macrophage populations. (A-C) Murine macrophage (F4/80) levels were measured by flow cytometry in both patient-derived xenograft (PDX) models under ALX treatment, and within a naïve untreated NSGS mouse in peripheral blood, splenic (SPL) and bone marrow (BM) compartments. *P<0.05, **P<0.01 and ***P<0.001.

Figure 5.

The activity of polyinosinic:polycytidylic acid on murine macrophages. (A) Polyinosinic:polycytidylic acid (poly(I:C)) leads to overall shifts in macrophage populations in the bone marrow (BM), spleen (SPL) and peripheral blood of treated mice. Shifts in M1/M2 ratio develop across compartments (B) while macrophage numbers increase (C) upon poly(I:C) administration. *P<0.05, **P<0.01 and ***P<0.001.

Figure 6.

The addition of polyinosinic:polycytidylic acid to a venetoclax/azacitidin regimen augments CD47 inhibition in a patient-derived xenograft model. (A) Peripheral chimerism and (B) endpoint analysis of a venetoclax/azacitidin plus ALX (VEN/AZA + ALX) regimen with the addition of polyinosinic:polycytidylic acid (poly(I:C)). (C) Immunohistochemistry for hCD45 reveals visible decreases in human cells with the bone marrow (BM) and spleen (SPL) of patient-derived xenograft (PDX) mice treated with VEN/AZA + ALX + poly(I:C). **P<0.01 and ***P<0.001.

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 differences in erythroid, myeloid, and neutrophil populations. (B) Wright Giemsa staining of BM cytospins reveal decreased acute myeloid 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.

Figure 8.

Polyinosinic:polycytidylic acid enhances CD47 inhibitor-induced phagocytosis. (A) Phagocytosis assays using macrophages from vehicle- or polyinosinic:polycytidylic acid (poly(I:C))-treated mouse bone marrow (BM) incubated with pHrodo-labeled cells from patient acute myeloid leukemia (AML) 18-12-001 and 19-03-007 reveal increases in phagocytosis in poly(I:C)-treated BM. (B) Healthy donor macrophages, when pretreated with poly(I:C) reveal increases in phagocytosis of CD47-treated AML (18-12-001) cells. Statistical comparisons in 3 individual donors shown here as ALX monotherapy, poly(I:C) monotherapy and combination against IgG1 control. Ig: immunoglobulin.