R603: A New Low Dose Efficient Anti-CD20 Immunotherapy for CLL Patients ?
Authors: Le Garff-Tavernier Magali , Decocq Julie , de Romeuf Christophe , Teillaud Jean-Luc , Dutertre Charles-Antoine , Debre Patrice , Merle-Beral Helene , Vieillard Vincent 
Published: April 2020

Abstract
Immunotherapy with monoclonal antibodies (mAbs), such as anti-CD20, is used in CLL treatment and represents a promising approach for achieving MRD eradication. Given their FcγRIIIa expression, NK cells are known to be involved in mAb therapy. We previously conducted a complete NK cells phenotypic expertise and functional assays including cytotoxicity against K562 cell line and antibody-dependent cellular cytotoxicity (ADCC) with rituximab, showing no major differences between NK cells from CLL patients and NK cells from healthy donors. We are now interested in functional capacities of NK cells in presence or not of a new anti-CD20 mAb: R603, a chimeric anti-CD20 mAb exhibiting a low fucose content as described for EMAB-6 mAb (C. de Romeuf et al, BJH 2008) in comparison to rituximab. To assess the degranulation of NK cells from CLL patients in response to anti-CD20 mAbs, we examined the surface expression of CD107a (percentage of CD107a+ NK cells) after co-incubation of PBMC from untreated CLL patients (n=8) with Raji cells (E/T ratio: 1/1) at 2 concentrations of each anti-CD20 mAb (10 and 1,000 ng/ml). At the higher mAb dose (1,000 ng/ml), R603 related degranulation of CLL NK cells (median (m): 43.6%; range (r): 27.0–79.8) was similar to the one obtained with rituximab (m: 38.9%; r: 22.4–75.2). At the lower dose (10 ng/ml), R603 related degranulation of CLL NK cells (m: 45.7%; r: 28.7–79.2) was similar to the one obtained with the high mAb concentration, contrary to rituximab related degranulation which was significantly decreased (m: 14.1%; r: 1.4–45.4) (p&lt;0.0001). These results are emphasized by ADCC chromium assay performed with purified CLL NK cells (E/T ratio: 5/1 and 10/1) against Raji cells and with or without anti-CD20 mAbs (at 3 doses: 1, 10 and 1,000 ng/ml). R603 related ADCC levels were high whatever the mAb concentration, contrary to rituximab related ADCC levels which were very low at 1 ng/ml and only reached R603 ADCC levels at 1,000 ng/ml. Similar results were obtained with healthy donors. Without addition of Raji cells (CLL PBMC + mAb), at the lower dose (10 ng/ml), none of the NK cells from CLL patients exhibited degranulation with rituximab, contrary to R603 where 5/8 CLL patients exhibited degranulation (cut-off: more than 10% of CD107a+ NK cells). At the higher dose (1,000 ng/ml), NK cells from 6/8 CLL patients with rituximab and from 7/8 CLL patients with R603 showed degranulation and R603 related degranulation levels (m: 32.3%, r: 0.8–51.0) were significantly superior to rituximab related degranulation levels (m: 12.1%, r: 0.1–30.6) (p=0.0005). These results showed that R603 in the presence of CLL B cells might induce CLL NK degranulation. In conclusion, NK cells from CLL patients appeared to be capable of being efficient in anti-CD20 immunotherapy by the ADCC pathway. Moreover, R603 a new anti-CD20 mAb, induced at low dose a significantly higher in vitro ADCC against Raji cells and autologous CLL B cells, compared to rituximab. This R603 mAb feature may be useful in therapeutic strategy for CLL patients.