Can NK Cells Play a Role in Anti-CD20 Immunotherapy for CLL Patients?.
Authors: Le Garff-Tavernier Magali , De Romeuf Christophe , Teillaud Jean-Luc , Decocq Julie , Dutertre Charles-Antoine , Debre Patrice , Merle-Beral Helene , Vieillard Vincent 
Published: October 2019

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. However, little is known about characteristics of NK cells in CLL patients. For 29 untreated CLL patients and 18 healthy donors, we conducted a complete phenotypic expertise and/or we measured functional capacities of NK cells in presence or not of 2 anti-CD20 mAbs: rituximab or EMAB-6, an optimized chimeric anti-CD20 mAb exhibiting a low fucose content (C. De Romeuf et al, submitted). Four-color FACS analyses were performed on fresh blood cells from 19 CLL patients and 10 healthy donors. NK cells were analyzed on CD3-CD19-CD56+ lymphocyte subset by staining with antibodies against: CD16 (3G8), CD69, activating (NKp30, NKp44, NKp46, NKG2C and NKG2D) and inhibitory (p58a, p58b, p70, NKG2A, ILT2 and LAIR-1) cytotoxicity receptors. We showed that absolute numbers of NK cells were identical or increased in CLL patients (median (m): 410/mm3, range (r): 123–2034) compared to healthy donors (m: 127/mm3, r: 14–314). Immunophenotyping of CLL NK cells revealed a CD16 heterogenous expression. Although no difference in other NK markers expression appeared in patients with normal or high expression of CD16, an heterogeneity and a diminution of activatory cytotoxicity receptors expression were observed on low CD16 NK cells (6/19). In addition, functional tests including NK cell direct cytotoxicity and/or antibody-dependent cellular cytotoxicity (ADCC) were performed simultaneously on NK-enriched PBMC from 10 CLL patients and 8 healthy donors. NK cell direct cytotoxicity was evaluated in a standard 4h 51Cr-release assay against the HLA class-I deficient K562 cell line. NK cells direct cytotoxicity of CLL patients (m: 47%, r: 20–78) was preserved and comparable to that of healthy donors (m: 55%, r: 26–61). Furthermore, preliminary ADCC experiments were performed on 51Cr Raji cells with or without anti-CD20 mAbs at the single dose of 1 μg/ml. Under these conditions, rituximab related ADCC levels obtained with CLL NK cells (m: 45%, r: 25–96) were equal or superior to those obtained with healthy donors NK cells (m: 30%, r: 25–58). EMAB-6 related ADCC levels obtained with CLL NK cells (m: 57%, r: 34–98) were, as for rituximab, also equal or superior to those obtained with healthy donors NK cells (m: 45%, r: 30–61). A moderate enhanced ADCC was observed with EMAB-6 in CLL NK cells or in healthy donors as compared to rituximab, which is in agreement with previous observations (C. De Romeuf et al, submitted). In conclusion, NK cells from CLL patients appeared to be capable of being efficient in anti-CD20 immunotherapy: they are quantitatively and qualitatively similar to those observed in healthy donors, except in a subgroup of patients with low CD16 NK cells, which need to be more investigated. Integrity of ADCC pathway is very encouraging for mAb treatment. Moreover, EMAB-6, an optimized anti-CD20 mAb, induced a higher in vitro ADCC against Raji cells and could be a promising drug candidate for the treatment of CLL.