“Don't Eat me”: CD47 and LFA-1 Modulate CLL Response to Nurselike Cells-Mediated Antibody-Dependent Phagocytosis Induced by Rituximab and Obinutuzumab (GA101)

Antibody-mediated B cell depletion was determined by flow cytometry after antibody treatment. PBMCs from CLL patients were cultured for 14 days to allow outgrowth of NLC [Tsukada N, 2002]. Cells were collected and either (i) put in 6-well plates in the same medium without NLC (w/o NLC), or (ii) seeded back onto NLC (CLL+NLC), before anti-CD20 antibodies were added at 10μg/ml for 7d. For CD47 blocking experiments, cells were incubated for 15 min with blocking antibodies before being seeded back onto NLC. An Affymetrix Uplus2.0 chip was used for gene expression profiling (GEP) of 19 NLC and 5 normal CD14+ monocytes samples. For phenotype studies, CLL cells in suspension were thoroughly harvested with medium, and remaining adherent CLL cells were discarded from NLC by vigorous pipetting, before staining with CD47-FITC/LFA-1-PE antibodies.

1. NLC phagocytic activity: GEP indicated NLCs are enriched in genes associated with phagocytic machinery compared to normal monocytes. Confocal microscopy experiments showed that NLCs are able to phagocytose CLL cells (though at a low baseline level). This phagocytic activity was significantly increased with the addition of anti-CD47 blocking antibody.

2. NLC-ADCP is induced by anti-CD20 antibodies: When comparing B-cell depletion induced by RTX and GA101 against CLL cells with or without NLC, a significant ADCP activity was measured with RTX (15%-NLC vs 36%+NLC, two-sided p=0.028), while GA101 already had significant levels of ADCP at baseline without NLCs that was only marginally increased with the addition of NLC (45%-NLC vs 53%+NLC).

3. Phenotype of NLC-adherent versus suspension CLL cells (n=50) in vitro: 44% and 61% of patients had an increase of CD47 and LFA-1 levels respectively (defined by an MFI increase>20% between adherent and suspension CLL cells in the same well). Mean RTX-induced B-cell depletion mediated by NLC was significantly decreased in LFA-1hi versus LFA-1lo pts (25.05% vs 45.46%, n=32, p=0.048), but GA101-induced ADCP was not inhibited (45.5% vs 53%). On the other hand, RTX (20.8% vs 48.3%, p=0.005) and GA101 (33% vs 66%, n=37, p=0.002) induced B-cell depletion was significantly reduced in CD47hi versus CD47lo patients. Anti-CD47 blocking antibody could restore both RTX and GA101-induced mean B-cell depletion (n=13, p=0.003 for RTX, p=0.025 for GA101). The combination of anti-CD47 with anti-CD20 antibodies suggest both FcR-dependent (anti-CD20) and FcR-independent (anti-CD47) mechanisms promote the observed depletion effect.

4. NLC gene expression profiles were compared between CD47hi (n=3) versus CD47lo (n=8). Among the 15 most down-regulated genes (fold-change>2) in CD47hi samples were FCGR3A/B (fc −4.6, p=0.048) and FCGR1 (fc −2.15, p=0.01), encoding for the ADCC/ADCP receptors CD16 and CD64, respectively. This phenomenon may explain the decreased ADCP activity with RTX and GA101.

Our results suggest that NLC may be therapeutically exploited through mediators of ADCP, especially in lymphoid organs. For that purpose, the combination of CD47 and CD20 antibodies may be considered. Due to its efficacy even in LFA-1hi patients, GA101 is considered a better mediator of NLC-ADCP. Exploring the regulation of CD47 in CLL cells is important because it may interfere with strategies targeting surface antigens through FcgammaR-dependent mechanisms.

Ysebaert:Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.