Selective Blockade of the Ubiquitous Checkpoint ...

YMTHE, Volume 25

Supplemental Information

Selective Blockade of the Ubiquitous Checkpoint Receptor CD47 Is Enabled by Dual-Targeting Bispecific Antibodies Elie Dheilly, Valéry Moine, Lucile Broyer, Susana Salgado-Pires, Zoë Johnson, Anne Papaioannou, Laura Cons, Sébastien Calloud, Stefano Majocchi, Robert Nelson, François Rousseau, Walter Ferlin, Marie Kosco-Vilbois, Nicolas Fischer, and Krzysztof Masternak

λ

κ anti-CD47 arm

anti-TAA arm human IgG1 Fc

Figure S1 Schematic representation anti-CD47/TAA bispecific κλ body. Anti-CD47 antibody arm (κ light chain, white) and a high-affinity anti-TAA arm (λ light chain, black) are associated through a native human IgG1 heavy chain constant region.

RAJI

Binding inhibition (%)

100 80 60 40 20 0 0.00010.001 0.01 0.1 1 10 100 Antibody concentration ( g/ml) anti-CD47/CD19 biAb anti-CD47 monovalent antibody mAb B6H12 control IgG

Figure S2 Blockage of CD47-SIRPα interaction on target cells. Binding of fluorescently-labeled high-affinity human SIRPα-Fc to CD47 expressed on the surface of (a) Raji and (b) NALM-6 cells in the presence of increasing concentrations of the indicated competitor CD47-blocking antibodies . The high-affinity anti human CD47 mAb B6H12 was tested for comparison. Data points represent mean percentage of inhibition ± S.E.M of four replicates.

a

b

Figure S3 Phagocytosis induced by anti-CD47/CD19 bispecific antibody depends on CD19 expression: (a) CD19-positive Raji cells and (b) CD19-negative mutant cells (Raji CD19KO) were incubated with donor-derived macrophages (E:T ratio 1:5) and increasing concentrations of anti-CD47/CD19 biAb or the indicated control antibodies. Phagocytosis was assessed by flow cytometry and is expressed as the percentage of macrophages that ingested at least one target cell.

a

b

Figure S4 High phagocytic activity of macrophages in the presence of anti-CD47/anti-CD19 bispecific antibody. Donor-derived macrophages were incubated with (a) Raji or (b) NALM-6 CFSE-labeled B cell lines at effector to target ratio of 1 : 5 in the presence of indicated antibodies at 0.1 mg/ml (a) or 10 mg/ml (b). Samples were acquired on an image-based flow cytometer (FlowSight). Phagocytosis index corresponds to the average number of target cells ingested by 100 macrophages. Representative images of macrophages are shown under the bar graphs.

Specific killing (%)

Figure S5 Antibody dependent cell-mediated cytotoxicity (ADCC). Comparison of ADCC activity induced by the anti-CD47/CD19 biAb, individual monovalent antibodies , and a 1 + 1 mixture thereof. Peripheral blood mononuclear cells (PBMC) and calcein AM labeled Raji cells (E:T ratio 50:1) were incubated with the indicated antibodies for 4 hours. Shown is the percentage of specific killing ± SD of triplicate samples .

Donor #1 + NCI-N87

Donor #2 + NCI-N87

b

c

d

Fluorescent microscopy

Flow cytometry

a

Donor #3 + HPAC

Donor #4 + HPAC

g

Flow cytometry

f

e

Figure S6 ADCP assays with MSLN-positive tumor cells. The percentage of phagocytosis of NCI-N87 cells (a,b) or HPAC cells (f,g) was determined by flow cytometry as described in Figure 4. Macrophages used in these experiments were derived from 4 different blood donors. (c,d) ADCP of NCI-N87 cells by donor #1 and donor #2 macrophages was assayed in parallel using automated cellular quantitative fluorescent microscopy (CellInsight™ CX5 HCS Platform). Contrary to the FACS-based method, phagocytosis is performed here by macrophages adhering to the assay plate (see Supplementary Materials and Methods for experimental details). Highresolution cell imaging allows to calculate the phagocytosis index. (e) Example image acquired with the CellInsight™ CX5 instrument showing ADCP of calcein AM-labeled NCI-N87 target cells (green) by calcein red-orange labeled macrophages (magenta).

Figure S7 Blood cell adsorbtion assay. The indicated antibodies (10 μg/ml) were incubated with whole human blood for 30 minutes at 37°C. The proportion of antibody remaining in the plasma fraction was determined by ELISA and is represented as % recovery (normalized to non-binding control mAbs). Mean % recovery obtained with blood of 3 different donors ± SD is shown. Statistical significance was determined using one way ANOVA; p-values: n.s. = not significant; **** for p