Page 99 - Haematologica Vol. 109 - July 2024

ARTICLE - CD47 inhibition is augmented with TLR3 agonism H.E. Ramsey et al.
ing cells washed and resuspended in 1x phosphate-buffered saline (PBS) with 1% bovine serum albumin and stained for 15 minutes with the following antibodies: human CD45- APC, human CD33-PE-Cy7, murine CD45-PE and DAPI (Bi- olegend). For CD47 measurement in cell line experiments, human CD47-FITC (Biolegend) was used. As a background control, positive mean fluorescence intensity (MFI) from either control human IgG4-FITC or human IgG1-FITC was subtracted from CD47 MFI to estimate final value. Cells were washed and submitted for flow cytometric analysis using a 3-laser LSRII (Becton Dickinson).
Immunohistochemistry
Statistics
Unless otherwise noted, data were summarized using the mean (+/- standard deviation). Per group sample sizes are presented in figures and results reported from two separate experiments, unless stated otherwise. In order to avoid normality assumptions, pairwise group comparisons were made using the non-parametric Mann-Whitney U test. The non-parametric Spearman correlation was used to assess pairwise variable associations. Data were analyzed using Graph Pad Prism 6.0 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com). *P<0.05, **P<0.01 and ***P<0.001.
Results
ALX90 significantly conceals surface expression of CD47 on multiple acute myeloid leukemia cell lines
In order to ensure CD47 specificity of the CD47i ALX90, we incubated AML cell lines MV-4-11, MOLM-13, and THP- 1 with ALX90 or magrolimab and stained for CD47. Flow cytometry revealed a significant decrease in detectable surface expression of CD47 MFI in the presence of ALX 90
Tissues were fixed in 4% paraformaldehyde for 48 hours and stored in 70% ethanol before being embedding in par- affin and sectioned at 5 μm. The bone tissue was decal- cified prior to being embedded in paraffin. Sections were dewaxed in Xylene and rehydrated in ethanol. Standard Mayer’s Hematoxylin and Eosin staining was performed. Antigen retrieval using a standard pH 6 sodium citrate buf- fer (BioGenex) was performed and sections were stained with monoclonal mouse anti-human CD45 (Dako, M0701, dilution 1:200) using M.O.M. Kit (Vector).
AB
CD
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 ad- dition 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.
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