Supplemental Information. Selective Blockade of the Ubiquitous. Checkpoint Receptor CD47 Is Enabled. by Dual-Targeting Bispecific Antibodies

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1 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

2 κ anti-cd47 arm λ 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.

3 RAJI 100 Binding inhibition (%) 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.

4 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.

5 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) Rajior(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.

6 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 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.

7 Donor #1 + NCI-N87 Donor #2 + NCI-N87 a b Flow cytometry Fluorescent microscopy c d e Donor #3 + HPAC Donor #4 + HPAC f g Flow cytometry Figure S6 ADCP assays with MSLN-positive tumor cells. ThepercentageofphagocytosisofNCI-N87cells(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).

8 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<0.0001

9 A B Figure S8 Binding of the anti-cd47/cd19 bispecific antibody to human and cynomolgus erythrocytes and platelets. Binding to red blood cells (a) and platelets (b) was assessed by flow cytometry following incubation in whole blood. B6H12 bearing a human IgG1 Fc portion (B6H12-hIgG1-Fc) and a control human IgG1 were tested for comparison. Mean fluorescence intensity is shown.

10 mg/kg (n=3) 0.5 mg/kg (n=3) 10 mg/kg (n=3) 0.5 mg/kg (n=3) Time (days) Time (days) Legend 10 mg/kg (n=3) Legend 0.5 mg/kg (n=3) Legend 10 mg/kg (n=3) Legend 0.5 mg/kg (n=3) Time (days) Time (days) Figure S9. Hematotoxicity assessment following a single intravenous injection of the anti-cd47/cd19 bispecific antibody in cynomolgus monkey. Two different doses of antibody were administered as an IV bolus injection, 0.5 mg/kg (dotted lines), and 10 mg/kg (solid lines); n = 3foreachdoselevel. DosingoccurredonstudyDay1andtheverticaldashed line indicates the time of first post-dosing sampling. (a) red blood cell counts, (b) hemoglobin concentration, (c) platelet counts, (d) White blood cells, (e) neutrophilsand(f) lymphocytesareshown. Horizontal dotted lines represent lower and upper limits of normal range for each hematological parameter. No signs of anemia or thrombocytopenia were observed. In general, counts remained within the normal reference range for this species. Occasional deviations observed with individual animals were small, non-dose dependent, and consistent with variability typical for animals of this species. The trough observed at day 3 (e.g., c,d,f) is the result of frequent blood sampling performed at earlier times, necessary for an adequate assessment the alpha phase of the PK profile.

11 parameter value C max (μg/ml) 195 +/ t 1/2 (h) 106 +/ CL (ml/day/kg) /- 2.4 Table S1 anti-cd47/cd19 bispecific antibody pharmacokinetic parameters calculated from data obtained with animals in the 10 mg/kg group. 10

12 Supplementary Materials and Methods CD47-SIRPα competitive binding assay. CD47 + CD19 + Raji or NALM-6 cells were suspended in reaction buffer (PBS, 2% BSA, 0.1% NaN 3 ) containing 5mg/ml of IVIg (CSL Behring, Privigen) at a concentration of 1.5x10 5 cells /ml and incubated for 30 minutes at room temperature (RT). Cells were then dispensed into 384-well clearbottom plates (Corning, Torino, Italy) at 3000 cells per well (20 μl/well), complemented with 50 µl of increasing concentrations of CD47-blocking competitor antibody, and incubated for 50 minutes at RT. In parallel, 4 μg/ml of Alexa Fluor 647-conjugated anti-mouse IgG Fc detection reagent (Jackson Immunoresearch, West Grove, PA, USA) was mixed with 0.5 μg/ml recombinant human SIRPα-mIgG1 Fc fusion protein (produced in house) and incubated for 50 minutes at RT. 30 µl of this mixture per well were then added to the cells and the incubation continued for 3 h. Binding of recombinant SIRPα-Fc to cells was detected using CellInsight CX5 High Content Screening Platform (Thermo Fisher Scientific, Rockford, IL, USA). Assessment of phagocytosis by imaging flow cytometry. Phagocytosis was performed as described in the main text (Materials and Methods), but samples were acquired on an imaging flow cytometer instead (FlowSight, EMD Millipore, Billerica, MA, USA). Direct visualization of phagocytic events allows the determination of the phagocytic index representing the number of engulfed tumor cells per 100 macrophages acquired. Phagocytosis index was calculated following image acquisition using AmnisIDEAS software (EMD Millipore, Billerica, MA, USA). Assessment of phagocytosis by automated high throughput quantitative microscopy: Human peripheral blood mononuclear cells were isolated from buffy coats by Ficoll gradient. Macrophages were generated by culturing PBMC for 4 days in complete medium in presence of 20 ng/ml of human M-CSF (Peprotech, Rocky Hills, NJ, USA). Non adherent cells were subsequently eliminated by washes using PBS and adherent cells representing macrophages were detached with cell dissociation buffer (Sigma Aldrich, Buchs, Switzerland). Harvested macrophages were washed once and plated in complete medium in a clear bottom black wall 96 well plate (Costar, Torino, Italy) at a density of macrophages per well and cultured for 2 additional days at 37 C.

13 For phagocytosis, adherent macrophages were labeled using calcein red-orange (Thermo Fisher Scientific, Rockford, IL, USA) and then co-incubated with calcein AM-labeled target cells (Thermo Fisher Scientific, Rockford, IL, USA) in presence of the test antibody for 2.5 hours at 37 C. This corresponds to an effector/target ratio of 1/1. Phagocytosis data was then acquired using CellInsight CX5 High Content Screening Platform (Thermo Fisher Scientific, Rockford, IL, USA) and analyzed using HCS Studio Cell Analysis Software (Thermo Fisher Scientific, Rockford, IL, USA). Phagocytosis index was calculated as the average number of ingested target cells per 100 macrophages. ADCC assay. Target cells were stained with calcein AM (Sigma, Buchs, Switzerland) and incubated with total human PBMC (E/T ratio 1/50) in the presence of increasing concentrations of test antibody in 96-well plates (Thermo Fisher Scientific, Rockford, IL, USA) for 4 hours at 37 C Target cell killing was determined by dosing the supernatants for calcein release using Synergy Neo (Biotek, Winooski, Vt, USA). Specific killing percentage was calculated according to the formula [(test release release from negative control)/(maximum release spontaneous release)]. Erythrocyte adsorption assay. Antibodies, from a 25 x concentrated stock, were added to 100 μl of undiluted heparinized human blood to a final concentration of 10 μg/ml. After incubation at 37 C for 30 min the mixture was centrifuged in order to retrieve the soluble fraction (plasma). The soluble fraction contains free antibody that has not been adsorbed on blood cells (principally erythrocytes). The concentration of CD47-specific and/or CD19-specific antibody was determined by CD47 or CD19 ELISA using a concentration range of the same antibody spiked in purified plasma from the same donor to calibrate the assay. Data was normalized with a control antibody (anti-cxcl10 IgG1) that does not bind to blood cells. One way Anova analysis of statistical significance was performed using the GraphPad prism5 software (GraphPad Software, San Diego, CA).