Leukemia (2011) 25, 101–109
& 2011 Macmillan Publishers Limited All rights reserved 0887-6924/11
ORIGINAL ARTICLE
Analysis of CD16 þ CD56dim NK cells from CLL patients: evidence supporting a
therapeutic strategy with optimized anti-CD20 monoclonal antibodies
M Le Garff-Tavernier1,2,3, J Decocq1,3,4, C de Romeuf4, C Parizot5, CA Dutertre4,6,7,8, E Chapiro2, F Davi1,2,3, P Debré1,3,5,
JF Prost4, JL Teillaud6,7,8, H Merle-Beral2,6,7,9 and V Vieillard1,3,9
1
INSERM, UMR-S 945, Paris, France; 2AP-HP, Hôpital Pitié-Salpêtrière, Service d’Hématologie Biologique, Paris, France;
3
UPMC Université Paris 06, UMR-S 945, Paris, France; 4Laboratoire Français de Fractionnement et des Biotechnologies (LFB),
Les Ulis, France; 5Laboratoire d’Immunologie Cellulaire et Tissulaire, AP-HP, Hôpital Pitié-Salpêtrière, Paris, France;
6
INSERM, UMR-S 872, Paris, France; 7Centre de Recherche des Cordeliers, UPMC Université Paris 06, UMR-S 872,
Paris, France and 8Université Paris Descartes, UMR-S 872, Paris, France
Although anti-CD20 monoclonal antibodies (mAbs) show
promise for the treatment of chronic lymphocytic leukemia
(CLL), the success of the anti-CD20 mAb rituximab in CLL
treatment has been limited. Novel anti-CD20 mAbs with more
potent cytotoxic activity have recently been engineered, but so
far most have only been tested in vitro with natural killer (NK)
cells from healthy donors. Because it is still unclear whether
these optimized cytotoxic mAbs will improve NK-cell killing of
tumor cells in CLL patients, we characterized the relevant
phenotypic and functional features of NK cells from CLL
patients in detail. Expression of inhibitory and activating
NK-cell receptors and of Fc gamma receptor IIIA (FccRIIIA) is
well preserved in CD16 þ CD56dim cytotoxic NK cells from these
patients, independently of disease progression. These cells are
fully functional following cytokine stimulation. In addition,
the FccRIIIA-optimized LFB-R603 anti-CD20 mAb mediates 100
times greater antibody-dependent cell-mediated cytotoxicity by
NK cells from CLL patients and healthy donors than rituximab.
Enhanced degranulation against autologous B-CLL cells is
observed at lower concentrations of LFB-R603 than rituximab,
regardless of CLL prognostic factors. These findings strongly
justify further clinical development of anti-CD20 mAbs
optimized for FccR engagement in CLL patients.
Leukemia (2011) 25, 101–109; doi:10.1038/leu.2010.240;
published online 26 October 2010
Keywords: chronic lymphocytic leukemia (CLL); natural killer (NK)
cells; anti-CD20; monoclonal antibodies
Introduction
Chronic lymphocytic leukemia (CLL) is an accumulation
of CD5 þ /CD19 þ malignant monoclonal B lymphocytes with
a highly variable clinical course. The introduction of therapeutic
monoclonal antibodies (mAbs) has brought a remarkable change
in treatment. The anti-CD52 alemtuzumab and anti-CD20
rituximab mAbs have been widely used in progressive disease.
In contrast to alemtuzumab, rituximab is generally well tole-
rated with infrequent serious adverse effects that are generally
well controlled. Although the combination of fludarabine and
cyclophosphamide with rituximab is currently considered the
Correspondence: Dr M Le Garff-Tavernier, INSERM UMR-S 945,
Bâtiment CERVI, GH Pitié-Salpêtrière, 83 bd de l’Hôpital, 75651 Paris
Cedex 13, France.
E-mail: magali.legarff@psl.aphp.fr
9
These authors are senior co-authors.
Received 19 February 2010; revised 12 August 2010; accepted 1
September 2010; published online 26 October 2010
www.nature.com/leu
‘gold standard’ for the treatment of CLL,1,2 it does not allow
full-disease eradication and the disease can become resistant.
The activity of rituximab is thought to be mediated through
several mechanisms, including complement-dependent cyto-
toxicity, apoptosis, antibody-dependent cellular cytotoxicity
(ADCC) and/or phagocytosis.3 Strategies pursued to improve
the efficacy of mAb therapy have included modifying the
antibody structure to increase its affinity to the Fc gamma
receptor IIIA (FcgRIIIA, CD16), an intermediate-affinity activat-
ing FcgR strongly expressed on natural killer (NK) cells.4
Optimized mAbs, including AME-133, GA-101, veltuzumab
(hA20) and ofatumumab (HuMax-CD20)5–7 are at different
stages of development. We previously described a novel
chimeric anti-CD20 mAb, EMAB-6, characterized by low fucose
content in its Fc region. EMAB-6 shows improved FcgRIIIA
binding, resulting in enhanced ADCC, mediated particularly by
macrophages8 and NK cells.9
The NK cells constitute a unique component of the innate
immune system, able to recognize various targets without
specific sensitization. The NK cells are heterogeneous and
differ in their proliferative potential, homing characteristics,
functional capabilities and in responses to a wide range of
cytokines. Around 10% of NK cells in the peripheral blood are
CD16CD56bright. This immunoregulatory subset of NK cells
produces a wide range of cytokines and chemokines, but its
ability to kill target cells spontaneously is poor. In contrast, the
rest (90%), corresponding to the CD16 þ CD56dim cytotoxic
NK-cell subset, produces relatively lower cytokine levels, but
possesses an abundance of cytolytic granules and can sponta-
neously lyse susceptible target cells.10 The complexity of the
NK-cell compartment is attributable to the vast network
of inhibitory or activating receptors that allow these cells to
recognize target cells. The functional state of NK cells is,
therefore, dictated by the balance between opposing signals.11
Moreover, in the presence of mAbs, strong expression of
FcgRIIIA allows CD16 þ CD56dim NK cells to kill targets through
ADCC, regardless of the presence of any inhibitory receptors.
ADCC has been identified as a critical mechanism underlying
the clinical efficacy of therapeutic antibodies, including
rituximab.12,13 We focused our attention on NK cells in patients
with CLL, because the size of this cell compartment is an
independent factor predictive of the rate of disease progression
in patients with newly diagnosed CLL.14 Several studies have
reported functional alterations in NK cells in these patients,
including defective cytotoxic activity, which are particularly
pronounced in advanced disease and that could be because of
the accumulation of leukemic cells rather than to any intrinsic
 NK cells and optimized anti-CD20 in CLL
M Le Garff-Tavernier et al
102
defect in the former.15,16 This hypothesis was suggested by the
restoration of their functional activity after interferon (IFN)-a
and/or interleukin (IL)-2 treatment.17,18
We conducted the first extensive characterization of the
phenotypic and cytotoxic qualities of NK cells from CLL patients
and compared them with those of healthy subjects. Our data
reveal that NK cells from these patients have the major
phenotypic features of competent NK cells and are able
to function effectively. We also explored the preclinical activity
of LFB-R603, a novel optimized anti-CD20 mAb, and show that
at low doses this antibody promotes a significantly higher
percentage of NK cell-mediated degranulation against auto-
logous CLL cells than rituximab.
Patients and methods
Patients and control donors
Fresh blood samples were collected from 57 CLL patients
followed at Pitié-Salpêtrière Hospital (Paris, France). All patients
met the morphological and phenotypic criteria for typical
CLL (Matutes score X4).19 They had not been treated previously
or had not received any treatment during the 6 months before
the study. None of these patients had received rituximab or
alemtuzumab. Binet stage A patients were considered to have an
unfavorable prognosis if they met any two of the following four
criteria: (1) Presence of unmutated Immunoglobulin heavy
variable chain sequences with a VH gene sequence p2% of
sequence alterations compared with the published germline
sequence; (2) High expression of surface CD38 (420%); (3)
High expression of cytoplasmic ZAP-70 (420%); and (4) Clonal
cytogenetic abnormalities, including deletion of chromosomes
17p or 11q or trisomy of chromosome 12, revealed by
fluorescent-labeled DNA probes used in interphase fluorescence
in situ hybridization (probes 17p13.1, ATM 11q23 and CEP12,
respectively).20 Patients with del (13q) (probe 13q14), have an
excellent prognosis if they do not have additional defects.
Patients with 17p were considered to have an unfavorable
prognosis even if all their other criteria were favorable. All other
Binet stage A patients were considered to have a favorable
prognosis. The control group comprised sex- and age-matched
outpatient volunteers from the rheumatology (n ¼ 29), who
had consulted for noninflammatory arthritic pathology such
as osteoarthritis, or gerontology (n ¼ 38) departments at Pitié-
Salpêtrière or Charles-Foix Hospital, respectively. Our study
applied restrictive health selection criteria, including no
infectious or malignant diseases 6 months before the study
and without acute illness at the time of sampling. None of the
control patients had an autoimmune disease. All case and
control patients provided a written, informed consent before
participation. The local ethics committee approved the study.
Flow cytometry and absolute B cell, T cell, and
NK cell counts
Freshly collected blood cells underwent cell-surface staining for
four-color cytometric analysis. Isotype-matched immunoglobulin
served as the negative control. NK cells were analyzed after
staining with an appropriate antibody cocktail: CD3-FITC
(UCHT1; Beckman-Coulter, Marseille, France), CD19-PerCP
Cy5.5 (SJ25C1; BD Biosciences, Le Pont de Claix, France),
CD56-APC (N901; Beckman-Coulter), and the following PE-
conjugated antibodies: KIR2DL1/KIR2DS1 (EB6B), KIR2DL2/
KIR2DL3/KIR2DS2 (GL183), KIR3DL1/KIR3DS1 (Z27), CD159a/
NKG2A (Z199), NKG2D (ON72), CD336/NKp44 (Z231),
Leukemia
CD335/NKp46 (BAB281), CD85j/ILT2 (HP-F1), 2B4 (C1.7) and
CD69 (TP1.55.3) (Beckman-Coulter), CD16 (3G8) and LAIR1
(DX26) (BD Biosciences), NKG2C (134591) and NKp80/KLFR1
(239127) (R&D Systems, Lille, France) and CD337/NKp30
(AF29-4D12) (Miltenyi Biotec, Paris, France). Erythrocytes were
lysed with a FACS lysing solution kit (BD Biosciences). After
extensive washing, at least 5  103 CD3CD56dim NK cells
were analyzed on a FACSCanto I with FACSDiva 5.03 software
(BD Biosciences). The level of CD16 expression on
CD3CD56dim NK cells was quantified using QuantiBRITE
standard beads (BD Biosciences), as recommended by the
manufacturer. For intracellular IFN-g staining, peripheral blood
mononuclear cells (PBMC) were incubated overnight in the
presence of IL-12 (10 ng/ml) and IL-18 (100 ng/ml) (R&D
Systems). Cells were fixed and permeabilized with a cytofix/
cytoperm kit (BD Biosciences), and stained with IFN-g mAb
(B27; BD Biosciences), as described.21
Direct cytolytic activity of NK cells
NK cell-enriched PBMC (8 CLL patients and 5 controls) were tested
for direct cytotoxicity. NK cell-enriched PBMC fractions were
obtained after negative depletion (anti-CD19-coated beads)
followed by positive selection (anti-CD56-coated beads) (Miltenyi
Biotec, Bergish Gladbach, Germany). Similar proportions of NK
cells were detected in NK cell-enriched PBMC fractions from CLL
patients (38±11%) and controls (42±10%). The B cells accounted
for 30±20% of the residual cells for CLL patients and 1±2% for
controls, whereas the proportions for T cells were 31±17% and
55±11%, respectively. Direct cytotoxicity was assessed after IL-2
stimulation (proleukin-2, 1000 IU/ml) for 72 h, with a standard 4-h
51
Cr-release assay against the human leukocyte antigen (HLA) class
I-deficient human K562 cell line.21
Anti-CD20 LFB-R603 antibody
LFB-R603 is a chimeric mAb directed against CD20 with a
glycosylation profile that allows increased binding to CD16.
This mAb has been granted an orphan-drug status in Europe and
in the USA for CLL treatment (http://www.lfb.fr/en/home.html).
A previous generation of this antibody, named EMAB-6, sharing
the same specificity and a similar glycosylation pattern, was
described in de Romeuf et al.9
Purification of NK cells and ADCC assay
Purified NK cells (498% CD3CD56 þ cells) from 7 CLL
patients and 6 controls were obtained from peripheral blood
after centrifugation on a Ficoll/Hypaque gradient, followed by
negative selection with anti-CD19-coated beads (Miltenyi
Biotec) and cytofluorometric selection of CD3CD56 þ NK
cells on a FACSVantage (BD Biosciences). ADCC assays were
performed with purified NK cells and 51Cr-labeled Raji cells at
an effector-target (E:T) ratio of 10:1. Experiments were
performed in the presence of LFB-R603 (LFB) or rituximab
(Roche Ltd, Basel, Switzerland) at concentrations ranging from
0.1 to 10 000 ng/ml.
Degranulation of NK cells
The degranulation marker CD107a was detected on freshly
isolated PBMC from both CLL patients and controls by
co-staining for NK markers (CD3CD56 þ NK cells). Cells were
incubated with anti-CD107a mAb (H4A3; BD Biosciences),
with 10 or 1000 ng/ml of either anti-CD20 rituximab (Roche) or
LFB-R603 (LFB, Les Ulis, France), as described.21

Statistical analysis
Statistical analyses were performed with Prism 4 software
(GraphPad Software, San Diego, CA, USA). Intergroup
comparisons were assessed with the Mann–Whitney U-test or
Wilcoxon rank-sum test. P-values less than 0.05 were considered
statistically significant; *Po0.05, **Po0.01, ***Po0.001.
Results
CLL patients
Table 1 and Supplementary Table 1 show the main clinical and
biological prognostic characteristics of CLL patients included in
this study. Because the potential benefit of early treatment for
unfavorable prognosis Binet stage A patients is a key therapeutic
question, patients were divided into two groups according to
their biological prognostic factors. Binet stage A CLL patients
with an unfavorable prognosis were classified with Binet stage
B/C patients (group 2, n ¼ 23) and compared with stage A
patients with a favorable prognosis (group 1, n ¼ 34). There was
no significant difference in the sex ratio or median age between
groups 1 and 2.
Distribution of lymphocyte subsets in CLL patients
Flow cytometry was used to measure CD19 þ B, CD3 þ T and
CD3CD56 þ NK subsets in CLL patients and controls. The
B-cell counts were significantly higher in CLL patients (group 1:
median 10 592/mm3 (range: 317–103 402); group 2: 28 084/
mm3 (2918–198 407)) than in controls (median 111/mm3
(22–943)) (groups 1 and 2 vs controls: Po0.0001). The
distribution of lymphocyte subsets showed that CLL patients
had significantly lower T-cell and NK-cell frequencies than
controls (groups 1 and 2 vs controls: Po0.0001 for T cells and
Po0.0001 for NK cells) (Figures 1a and c, respectively).
Interestingly, CLL patients had significantly higher absolute
T-cell and NK-cell counts than controls (groups 1 and 2 vs
controls: Po0.0001 for T cells, group 1 vs controls: P ¼ 0.0066
and group 2 vs controls: P ¼ 0.0003 for NK cells) (Figures 1b and
d, respectively). However, despite higher lymphocyte counts in
group 2 patients, T-cell and NK-cell counts did not differ
significantly between the two groups of CLL patients. Similarly,
the distribution of the CD56dim NK-cell subset did not differ
between the CLL groups and controls (Figure 1e). Finally, there
was no difference in the frequency of CD16 þ /CD56dim NK cells
(Figure 1f) or surface density of CD16, as detected by the
Quantibrite assay (data not shown), between these groups.
Similar results were obtained using the classical stage A vs
stages B/C of the Binet classification (data not shown).
Table 1 Main clinical and biological characteristics of CLL patients
Patients Age median Lymphocytosis
(n) (range) (109/l) median
(range)
Sex Binet
stage
F M A B/C
Group 1 (34) 11 23 62 (34–83) 14.9 (5.1–106.6) 34 0 31/31 0/31 27/34 7/34 27/31 4/31
Group 2 (23) 8 15 66 (40–79) 33.8 (7.3–212.2) 9 14
Abbreviations: CLL, chronic lymphocytic leukemia; F, female; IgHV, immunoglobulin variable heavy chain; M, male.
a
M, mutated; UM, unmutated.
NK cells and optimized anti-CD20 in CLL
M Le Garff-Tavernier et al
103
Phenotypic analysis of NK receptors in CLL patients
The phenotypic properties of the CD3CD56dim cytotoxic NK-
cell subset were investigated further by gating out CD3CD56bright
NK cells. CD56dim NK cells from CLL patients, irrespective of their
prognostic status, were indistinguishable from those of controls in
terms of cell-surface expression of the major NK receptors studied,
including c-lectin receptors (NKG2A, NKG2C and NKG2D),
natural cytotoxicity receptors (NKp30, NKp44 and NKp46) and
other NK-cell markers such as NKp80, ILT-2, LAIR-1 and 2B4
(Figure 2a, upper panels; data not shown). Similar phenotypic
results were observed according to the Binet stages (A vs B/C) (data
not shown). This phenotype was then studied longitudinally, over a
1-year period, comparing the initial samples with samples taken 2,
4, 6 and/or 12 months later. A remarkable phenotypic stability was
observed for each NK receptor studied. Figure 2a (lower panels)
shows representative CLL patients from each of the groups (patients
1 and 2, group 1; patient 3, group 2; patient 4, group 1 (at first
evaluation) and group 2 (at 12 months)). These results were
confirmed with three other patients (data not shown). In addition,
patient 4’s NK receptor repertoire remained stable during disease
progression between stages A (group 1) and C (group 2) after 1
year. The only difference observed in the KIR was a weak, but
significant decrease in expression of KIR2DL1/DS1, which
recognizes group-2 HLA-Cw alleles, in CLL patients with an
unfavorable prognosis compared with controls (P ¼ 0.0370). Binet
B/C patients presented a nonsignificant decrease compared with
controls (data not shown). No differences between groups were
observed for the other KIRs tested, including KIR2DL2-3/DS2,
which binds group-1 HLA Cw ligands, and KIR3DL1/DS1, which
recognizes HLA-Bw4 (Figure 2b).
The expression pattern of the early activation marker CD69 on
CD3CD56 þ NK cells was also investigated. As shown in
Figure 2c, CD69 expression in CD3CD56dim NK cells was
slightly, but significantly lower in CLL patients than in controls
(group 1 vs controls: P ¼ 0.0228, and group 2 vs controls:
P ¼ 0.0044), even if a certain degree of overlap of CD69
expression in CD3CD56dim NK cells was observed between
both groups of CLL patients and controls. Similarly, CD69
expression decreased significantly in CD3CD56bright immuno-
regulatory NK cells from group 1 patients compared with controls
(P ¼ 0.0037). Furthermore, HLA-DR expression, a late cell-activa-
tion marker, remained close to background levels in all control and
CLL subjects, in all NK-cell subsets (data not shown). Similar results
were obtained according to Binet stages (data not shown).
Functional analysis of NK cells in CLL patients
The functional capacities of the NK-cell compartment of CLL
patients were investigated further. As shown in Figure 3a,
intracellular IFN-g in CD3CD56dim NK cells from CLL patients
Prognostic factors
IgHV CD38 ZAP-70 Cytogenetic
mutational abnormalities
status
Ma UMa  +  + 17p  11q  13q  12 +
0/33 0/33 18/33 1/33
7/21 14/21 15/23 8/23 11/22 11/22 4/23 6/23 12/23 2/23
Leukemia
 NK cells and optimized anti-CD20 in CLL
M Le Garff-Tavernier et al
104
*** ***
*** 4 ***
100 10

75

CD3+/mm3
% CD3+
103
50

25
102
0 1
Ctl group 1 group 2 Ctl group 1 group 2

*** ***
*** **
40 4
* 10

CD3-CD56+/mm3
% CD3-CD56+

30
103

20
102
10
101
0 1
Ctl group 1 group 2 Ctl group 1 group 2

100 100
% CD56dim/CD3-CD56+

% CD16+/CD3-CD56dim

75 75

50 50

25 25

0 0
Ctl group 1 group 2 Ctl group 1 group 2
Figure 1 Distribution of lymphocyte subsets in CLL patients. FACS analysis of percentages and absolute cell counts of: (a and b) CD3 þ T cells;
and (c and d) CD3CD56 þ NK cells, gated on lymphocytes from peripheral blood. (e) Expression of CD56dim gated on CD3CD56 þ NK cells.
(f) Expression of CD16 þ gated on CD3CD56dim NK cells. Cells were collected from controls (Ctl, J) and CLL patients in group 1 (}) and group 2
(&). Horizontal bars represent the median values. P-values refer to the comparison between controls and CLL patients, assessed with the
Mann–Whitney U-test. *Po0.05, **Po0.01, ***Po0.001.

(groups 1 and 2) treated with IL-12 and IL-18 was expressed at a
similar level to that in NK cells from controls. Similar results
were observed in the CD3CD56bright NK-cell subset even
if IFN-g expression increased slightly in CLL patients (group 1
and group 2) compared with controls (P ¼ 0.0359 and
P ¼ 0.0176, respectively) (Figure 3b). The direct cytotoxicity of
NK cells was then evaluated after IL-2 activation by their
capacity to lyse HLA class-I negative K562 sensitive-target cells
and Figure 3c shows that the cytotoxicity of NK cell-enriched
PBMC from CLL patients was similar to that in controls.
ADCC potency of NK cells from CLL patients is related
to the concentration of rituximab and LFB-R603
NK-cell cytolysis can also be induced indirectly through Fc
receptors, by antibodies mediating ADCC. ADCC assays with
purified NK cells against Raji target cells, in the presence of
serial concentrations of rituximab or LFB-R603, produced
similar ADCC curves in controls and CLL patients with both
anti-CD20 mAbs, including an exponential phase followed by a
plateau (Figure 4a). The effect in CLL patients was similar
irrespective of their prognostic status. However, the potency
was dose-dependent for both rituximab and LFB-R603, and the
maximum ADCC was detected at 1 ng/ml of LFB-R603, but at
100 ng/ml of rituximab. This indicates that the potency of
LFB-R603 is superior to that of rituximab (Figure 4a). At 1 ng/ml,
the median ADCC value with LFB-R603 was 62.5% for controls
and 65.1% for CLL patients, whereas at a similar dose of
rituximab, the ADCC was significantly lower (P ¼ 0.03) at 7.3
and 8.5%, respectively (Figure 4b, upper panel). In contrast, the
ADCC with both anti-CD20 mAbs was similar at 100 ng/ml in
CLL patients and controls (Figure 4b, lower panel). As shown in
Figure 4c, in CLL patients, EC50 values were 10.5 ng/ml for
rituximab and 0.13 ng/ml for LFB-R603, which indicates that the
ADCC activity of the latter was 81 times higher than that of
rituximab. In the control subjects, EC50 values were 16.2 ng/ml
for rituximab and 0.16 ng/ml for LFB-R603, with LFB-R603
ADCC activity 101 times higher than that of rituximab. Taken
together, these results confirm the strong ADCC capacity of
NK cells from CLL patients and suggest that, at least ex vivo,
LFB-R603 is more potent than rituximab at low concentrations.
The degranulation capacities of NK cells from CLL patients in
the presence of their autologous B-CLL cells provide additional
support for these data because ADCC is influenced not only
by binding of NK cells to their target but also and mainly by their
ability to degranulate. This ex vivo model evaluated the

Leukemia
 NK cells and optimized anti-CD20 in CLL
M Le Garff-Tavernier et al
105
100 100 100

% NKG2A

% NKp30
75 75 75

% ILT-2
50 50 50
25 25 25
0 0 0
Ctl group 1 group 2 Ctl group 1 group 2 Ctl group1 group 2
100 100 100
0
75 2
% NKG2A

75 75

% NKp30

% ILT-2
4
50 50 50 6
12
25 25 25
0 0 0
#1 #2 #3 #4 #1 #2 #3 #4 #1 #2 #3 #4
CLL patients CLL patients CLL patients

* 80
80 % KIR2DL2-3/DS2 80

% KIR3DL1/DS1
% KIR2DL1/DS1

60 60 60
40 40 40
20 20 20
0 0 0
Ctl group1 group 2 Ctl group1 group 2
% CD69+/CD3-CD56bright Ctl group1 group 2

** **
% CD69+/CD3-CD56dim

* 50
75

50
25
25

0 0
Ctl group1 group 2 Ctl group1 group 2

Figure 2 Pattern of receptor expression on NK cells from CLL patients. (a) Expression of NKG2A, NKp30 and ILT-2. Upper panels show
phenotypic expression of each marker gated on CD3CD56dim NK cells. Lower panels show a longitudinal phenotypic study of four representative
CLL patients (patients 1, 2, and 4 in group 1; patient 3 in group 2), every 2 months during a 12-month period after the first phenotypic evaluation.
(b) Expression of killer cell Ig-like receptors, including KIR2DL1/DS1, KIR2DL2-3/DS2, and KIR3DL1/DS1. (c) Expression of the early
cell-activation marker CD69 on CD3CD56dim NK cells and CD3CD56bright NK cells. Cells were collected from controls (Ctl, J) and CLL
patients in group 1 (}) and group 2 (&). Horizontal bars represent the median value. P-values refer to the comparison between controls and
CLL patients, assessed with the Mann-Whitney U-test. *Po0.05, **Po0.01.

proportion of CD107a þ NK cells in whole PBMC from cells, the proportion of degranulating NK cells was considerably
CLL patients and controls. As shown in Figure 5a, analysis of higher with LFB-R603 than with rituximab.
degranulation in the presence of autologous target cells
confirmed the greater potency of LFB-R603 compared with
rituximab: although NK-cell degranulation was not observed in Discussion
samples from any of the 21 CLL patients at low concentrations
of rituximab (10 ng/ml), the median values of degranulation with This report describes the first extensive phenotypic and
the same concentration of LFB-R603 were 16.3% in group 1 and functional study of CD16 þ CD56dim NK cells in CLL patients
15.2% in group 2 (Figure 5a). Similar results were obtained and age-matched controls. CLL patients were found to have
using the Binet stages (data not shown). At a higher concentra- a significantly higher number of NK cells, as previously
tion (1000 ng/ml), NK-cell degranulation remained significantly described,22 but expression of both inhibitory and activating
higher with LFB-R603 than with rituximab (group 1, P ¼ 0.0005 NK-cell receptors was well preserved. In addition, this NK-cell
and group 2, P ¼ 0.0078) (Figure 5b). According to Binet stages, repertoire remained remarkably stable despite disease progres-
this difference remained significant in the A patient group but sion. Intriguingly, only a slight decrease in KIR2DL1/DS1
not in the B/C patient group, probably because of the low expression was noted in patients with an unfavorable prognosis,
number of patients in this latter group (data not shown). Finally, whereas the frequency of HLA-Cw*06, a KIR2DL1 ligand,
we could not exclude that CLL cells inhibited NK-cell function. was significantly higher in CLL cohorts than in controls.23,24 The
To test this hypothesis, we added Raji cells into the CD107 phenotypic features of these CD16 þ CD56dim NK cells cannot
degranulation assay performed with PBMC from CLL patients or explain the lack of antitumor activity or disease control. This
healthy controls. As shown in the Supplementary Figure 1, in the may suggest a failure to express some NK ligands on B-CLL cells,
presence of Raji cells, the CD107 level in NK cells was similar such as MICA and ULBPs, as previously reported.25 In contrast,
in CLL patients and healthy controls, suggesting an absence of the major phenotypic alterations in NK cells observed in acute
effect of CLL cells on NK degranulation. Together, these data leukemia, including the downregulation of natural cytotoxicity
demonstrate that in the presence of autologous B-CLL target receptors,26,27 suggest that chronic and acute leukemia use

Leukemia
 NK cells and optimized anti-CD20 in CLL
M Le Garff-Tavernier et al
106
different strategies to escape from NK-cell immunity. Poor previously reported reduction in NK cytotoxic activity in the
expression of CD69 was also observed on CD16 þ CD56dim absence of exogenous stimulation.15,28 This relative NK-cell
NK cells from CLL patients; this deficit could explain the anergy could be due to the ability of CLL cells and their
*
microenvironment to produce cytokines, such as IL-10, which
* suppress the proliferation of antigen-specific Th1 cells and
100 100
downregulate co-stimulatory molecules on antigen-presenting
% IFN+ cells/
CD3-CD56dim

CD3-CD56bright
cells.29–31 Importantly, NK-cell function was perfectly recovered

% IFNγ+ cells/
75 75
50 50 following activation. This included the ability to produce
25 25 large amounts of IFN-g after IL-12 and IL-18 activation, and
effective cytolysis against major histocompatibility complex
0 0
Ctl group1 group 2 Ctl group1 group 2

100 10 ng/ml 1000 ng/ml

% NK lysis

75 *** ** *** **
75 75
50

+
% CD107a

% CD107a
50 50
25 42.6
35.2
0 25 25
16.3 15.2
12.4
/1

/1

/1

9.5
5/

5/
20

10

25
2.

0.0 1.4
1.

0 0
RTX LFB-R603 RTX LFB-R603 RTX LFB-R603 RTX LFB-R603
E/T ratio
group 1 group 2 group 1 group 2
Figure 3 IFN-g expression and direct cytotoxic activity of NK
cells from CLL patients. Intracellular expression of IFN-g in: Figure 5 Degranulation response determined by CD107a expression
(a) CD3CD56dim and (b) CD3CD56bright NK cells from controls on CD3CD56dim NK cells from CLL patients. PBMC from CLL
(J) and CLL patients in group 1 (}) and group 2 (&), after overnight patients in group 1 (n ¼ 12) and group 2 (n ¼ 9) were incubated
incubation with IL-12 and IL-18. Horizontal bars represent the median with: (a) 10, and (b) 1000 ng/ml of rituximab (RTX) or LFB-R603. The
values. (c) Cytotoxicity of enriched NK cells from controls (J) and median value of the % of CD107a þ NK cells without mAb was 3.5
CLL patients (~) measured by 51Cr release-assays against K562 target (range: 0.8–15.0). Horizontal black bars represent median values.
cells. Mean percentage values±s.d. (vertical bars) are shown. P-values P-values were calculated with the Wilcoxon rank-sum test and refer to
refer to the comparison between controls and CLL patients, assessed the comparison between rituximab and LFB-R603. **Po0.01,
with the Mann–Whitney U-test. *Po0.05. ***Po0.001.

1 ng/ml
* *
100 100
Ctl (n=3) n=6 n=7
% NK ADCC

% NK ADCC

75 75
62.5 65.1
50
50
25
25
0 7.3 8.5
0 0.1 1 10 100 1000 10000 0
100 ng/ml
100 n=5 n=5
100
% NK ADCC

CLL group 1 (n=4)

% NK ADCC

75 71.6
75 66.9
55.6
60.4
50
50
25 25

0 0
0 0.1 1 10 100 1000 10000 RTX LFB-R603 RTX LFB-R603
Ctl CLL
100
CLL group 2 (n=1) 100
% NK ADCC

% NK ADCC

75
75
50
50 RTX CLL
25 LFB-R603 CLL
25 RTX Ctl
0 LFB-R603 Ctl
0
0 0.1 1 10 100 1000 10000 -2 -1 0 1 2 3 4 5
[mAb] ng/mL [mAb] ng/mL (LOG)
Figure 4 NK cell ADCC in the presence of rituximab or LFB-R603. (a) A dose-response study of ADCC against Raji cells was performed with or
without anti-CD20 rituximab (n) or LFB-R603 (~), at six concentrations ranging from 0.1 to 10 000 ng/ml. Assays were performed with purified
NK cells from controls and from CLL patients in group 1 and group 2. Mean percentage values±s.d. (vertical bars) are shown. (b) Comparison of
ADCC activity against Raji cells, after treatment with 1 ng/ml or 100 ng/ml of rituximab (RTX) (n) and LFB-R603 (R603) (~). Horizontal black bars
represent median values. (c) Percentage lysis of Raji cells induced by rituximab or LFB-R603 in the presence of NK cells from control individuals
(n ¼ 3) or CLL patients (n ¼ 5). P-values were calculated with the Wilcoxon rank-sum test and refer to the comparison between rituximab and
LFB-R603, *Po0.05.

Leukemia

class-I-defective K562 target cells after IL-2 activation. Similar
capacities were observed in all patients, regardless of their
prognostic factors in agreement with previous reports.17,18
Altogether, these results contribute to our knowledge on
NK-cell nature and function in CLL.
Our results demonstrate the efficacy of NK cells from CLL
patients, including patients with an unfavorable prognosis, at
killing tumor Raji cells and degranulating against autologous
B-CLL cells. Consequently, these data strongly support the use of
anti-CD20 mAbs optimized for FcgR engagement to induce
efficient ADCC. Surprisingly, very few studies have evaluated
anti-CD20 mAb-mediated ADCC in CLL,9,32 with most
anti-CD20 ADCC studies using non-Hodgkin’s lymphoma cell
lines. Only two studies have investigated the ability of NK cells
to mediate ADCC against primary CLL cells in the presence of
anti-CD20 mAbs.9,32 Weitzman et al.32 showed that rituximab
and veltuzumab, another anti-CD20 mAb optimized for
enhanced complement-dependent cytotoxicity, resulted in
similar levels of ADCC in the presence of primary CLL cells
and NK cell lines. Recently, we have shown that EMAB-6, an
anti-CD20 optimized for FcgRIIIA engagement, induced greater
ADCC against CLL cells than rituximab, in the presence of NK
cells from healthy donors.9 Of note, we focused our attention on
NK cell-mediated ADCC. However, we cannot exclude a
synergistic effect of macrophages and neutrophils with NK cells
in this mechanism.8,33
Other studies have focused on the cytotoxic activity of
anti-CD20 mAbs against non-Hodgkin’s lymphoma cells. With
optimized GA-101 anti-CD20 mAb, the potency of ADCC
against CD20-expressing non-Hodgkin’s lymphoma cells ranges
from 5 to 100 times greater than with rituximab.34 Similarly,
AME-D, another optimized anti-CD20 mAb, produces greater
ADCC at lower mAb concentrations than rituximab against an
non-Hodgkin’s lymphoma cell line.35 In the presence of NK
cells from CLL patients, similar dose-response relationships were
found for the induction of ADCC by the optimized anti-CD20
LFB-R603 mAb and rituximab, except that LFB-R603 could kill
the same number of Raji cells at 1/100 the concentration
of rituximab. ADCC did not increase with concentrations higher
than 100 ng/ml for rituximab or 1 ng/ml for LFB-R603. Of note,
LFB-R603 induces strong ADCC at a low concentration
(1 ng/ml), regardless of the prognostic factors.
Importantly, a robust increase in NK-cell degranulation
against autologous CLL cells was observed in the presence of
LFB-R603, in contrast to the results with rituximab. Together,
these data indicate that the potency of ADCC with these two
anti-CD20 mAbs differs considerably and that the anti-CD20
LFB-R603 mAb triggers better effector functions to clear CLL
cells, which may be therapeutically beneficial. Intriguingly,
considerable interindividual variation in degranulation efficacy
was also observed, regardless of the patients’ prognosis.
Previous studies have shown that the FcgRIIIA polymorphism
is associated with differential affinity and binding to human
IgG1 mAbs. However, in CLL, the FcgR polymorphism did not
predict responses to low-fucosylated mAbs,32,36,37 suggesting
that this polymorphism does not explain the variability in
degranulation responses. Instead, NK-cell degranulation might
be affected by the effector/target ratio, in agreement with Fischer
et al.38 Finally, the individual variability might be also attributed
to intrinsic resistance of CLL cells to degranulation as observed
in 1/21 patients. Moreover, we noted a defect in NK-cell
degranulation in two patients who had received rituximab
before sampling (data not shown). Accordingly, functional
characterization of NK cells against autologous B-CLL targets
from each patient might be useful for predicting the quality of
NK cells and optimized anti-CD20 in CLL
M Le Garff-Tavernier et al
107
the response to anti-CD20 treatment in relationship to clinical
treatment history.
In CLL, the introduction of anti-CD20 mAbs has brought
about a remarkable change in treatment strategy, but limitations
in their therapeutic use have also been noted. Rituximab is not
widely used alone in CLL because of its low-response rate and
the short-response duration. Furthermore, increased doses of
rituximab, from 500 to 2250 mg/m2, are associated with
significant toxicity without any enhancement of clinical
benefits.39 On the basis of these success and limitations,
different types of second-generation mAbs have been engi-
neered. Ofatumumab (HuMax) and veltuzumab are designed to
improve complement-dependent cytotoxicity, whereas GA-101
and LFB-R603, for example, are intended to increase the affinity
of the mAb Fc region for FcgRIIIA, by amino-acid substitutions
or modification of the carbohydrate structure of the mAb,
or both. Lower doses of these antibodies must be sufficient to
produce ADCC levels similar to or higher than those achieved
with rituximab, as demonstrated in this study. Clinical trials with
these antibodies may reveal that they have enhanced activity
compared with rituximab, but without its side effects. Thus, an
interim analysis in an international phase I trial of ofatumumab,
used as single agent at a dose of 2000 mg in refractory
CLL patients, reported a good safety profile and a high overall
response rate in heavily pretreated patients.40 A multicenter
phase I study of GA-101, used as single agent at escalating doses
of 400–2000 mg, showed good responses with two cases of
complete remission at intermediate doses (800/1200 mg), a
tumor-burden decrease and good tolerance in 11/13 evaluable
CLL patients.41 Finally, an ongoing phase I study is testing
LFB-R603 at doses escalating from 75 to 1650 mg/m2.
In summary, our findings clearly demonstrate that peripheral
NK cells from CLL patients are functional in terms
of degranulation and ADCC, although a certain degree of
variability is observed. They support the idea that a functional
characterization of NK cells from each CLL patient could be
useful in predicting the outcome of clinical treatment with
mAbs. Our data also show that LFB-R603 activity is more
promising than that of rituximab. The potency of LFB-R603 at
low doses may have an important impact in patients with severe
disease or in those who are resistant to chemotherapy.
Conflict of interest
J Decocq, C de Romeuf and JF Prost are employed by LFB,
whose potential product was studied in the present report.
CA Dutertre was supported by a CIFRE fellowship from the
ANRT and LFB (#134/2004) between 2004 and 2007. J-L
Teillaud was a LFB consultant until June 2007, which covers
part of the study period. Three of the authors, C de Romeuf, JF
Prost and JL Teillaud, are designated as inventors on patent
application WO2006064121 owned by LFB Biotechnologies,
and claim specific therapeutic use of the antibody studied in this
report. The other authors declare no conflict of interest.
Acknowledgements
We thank Z Azgui, K Maloum, F Nguyen-Khac, JL Binet,
N Marchay, H M’Kada and S Nguyen-Quoc, from the Hematology
Department at Pitié-Salpêtrière Hospital (AP-HP, Paris, France) for
the recruitment of CLL patients. We thank F Gandjbakhch,
C Poulain and the personnel from the Department of Rheuma-
tology at Pitié-Salpêtrière Hospital (AP-HP, Paris, France) and
Leukemia
 NK cells and optimized anti-CD20 in CLL
M Le Garff-Tavernier et al
108
V Siguret, E Pautas and the personnel from the Gerontology
Department at Charles-Foix Hospital (AP-HP, Ivry, France)
for the recruitment of the control group. P Bonnemye, M Brissard,
S Gueguen, M Boudjoghra and A Grelier are acknowledged for
their technical assistance. This study was supported in part
by funds from the Laboratoire Français de Fractionnement et des
Biotechnologies (LFB, Les Ulis, France), the association la Ligue
contre le cancer (RS08/75-4) and INSERM. The anti-CD20
LFB-R603 antibody was purchased by LFB.
Author contributions
MLT, CAD, JLT, HMB and VV conceived and designed the study;
MLT and JD performed the flow cytometric and functional
analyses; CP performed the flow cytometric selection;
EC provided cytogenetic analyses; FD provided IgHV sequences;
HMB cared for the involved patients, provided clinical and
diagnosis/prognosis cytometric data; MLT, JD, CdR, CAD, JLT,
HMB and VV analyzed the flow cytometric and functional data;
MLT, JLT, HMB and VV wrote the manuscript; JD, CdR, CP, CAD,
EC, FD, PD and JFP critically read and approved the manuscript.
References
1 Keating MJ, O’Brien S, Albitar M, Lerner S, Plunkett W, Giles F et al.
Early results of a chemoimmunotherapy regimen of fludarabine,
cyclophosphamide, and rituximab as initial therapy for chronic
lymphocytic leukemia. J Clin Oncol 2005; 23: 4079–4088.
2 Tam CS, O’Brien S, Wierda W, Kantarjian H, Wen S, Do KA et al.
Long-term results of the fludarabine, cyclophosphamide, and
rituximab regimen as initial therapy of chronic lymphocytic
leukemia. Blood 2008; 112: 975–980.
3 Cartron G, Watier H, Golay J, Solal-Celigny P. From the bench to
the bedside: ways to improve rituximab efficacy. Blood 2004; 104:
2635–2642.
4 Lazar GA, Dang W, Karki S, Vafa O, Peng JS, Hyun L et al.
Engineered antibody Fc variants with enhanced effector function.
Proc Natl Acad Sci USA 2006; 103: 4005–4010.
5 Delgado J, Briones J, Sierra J. Emerging therapies for patients with
advanced chronic lymphocytic leukaemia. Blood Rev 2009; 23:
217–224.
6 Pleyer L, Egle A, Hartmann TN, Greil R. Molecular and cellular
mechanisms of CLL: novel therapeutic approaches. Nat Rev Clin
Oncol 2009; 6: 405–418.
7 Cheson BD. Ofatumumab, a novel anti-CD20 monoclonal anti-
body for the treatment of B-cell malignancies. J Clin Oncol 2010;
28: 3525–3530.
8 Leidi M, Gotti E, Bologna L, Miranda E, Rimoldi M, Sica A et al.
M2 macrophages phagocytose rituximab-opsonized leukemic
targets more efficiently than m1 cells in vitro. J Immunol 2009;
182: 4415–4422.
9 de Romeuf C, Dutertre CA, Le Garff-Tavernier M, Fournier N,
Gaucher C, Glacet A et al. Chronic lymphocytic leukaemia
cells are efficiently killed by an anti-CD20 monoclonal antibody
selected for improved engagement of FcgammaRIIIA/CD16.
Br J Haematol 2008; 140: 635–643.
10 Freud AG, Caligiuri MA. Human natural killer cell development.
Immunol Rev 2006; 214: 56–72.
11 Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of
natural killer cells. Nat Immunol 2008; 9: 503–510.
12 Voso MT, Pantel G, Rutella S, Weis M, D’Alo F, Urbano R et al.
Rituximab reduces the number of peripheral blood B-cells in vitro
mainly by effector cell-mediated mechanisms. Haematologica
2002; 87: 918–925.
13 Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc
receptors modulate in vivo cytoxicity against tumor targets.
Nat Med 2000; 6: 443–446.
14 Palmer S, Hanson CA, Zent CS, Porrata LF, Laplant B, Geyer SM
et al. Prognostic importance of T and NK-cells in a consecutive
series of newly diagnosed patients with chronic lymphocytic
leukaemia. Br J Haematol 2008; 141: 607–614.
Leukemia
15 Ziegler HW, Kay NE, Zarling JM. Deficiency of natural killer
cell activity in patients with chronic lymphocytic leukemia.
Int J Cancer 1981; 27: 321–327.
16 Foa R, Lauria F, Lusso P, Giubellino MC, Fierro MT, Ferrando ML
et al. Discrepancy between phenotypic and functional features of
natural killer T-lymphocytes in B-cell chronic lymphocytic
leukaemia. Br J Haematol 1984; 58: 509–516.
17 Kay NE, Zarling J. Restoration of impaired natural killer cell
activity of B-chronic lymphocytic leukemia patients by recombi-
nant interleukin-2. Am J Hematol 1987; 24: 161–167.
18 Guven H, Gilljam M, Chambers BJ, Ljunggren HG, Christensson B,
Kimby E et al. Expansion of natural killer (NK) and natural
killer-like T (NKT)-cell populations derived from patients with
B-chronic lymphocytic leukemia (B-CLL): a potential source for
cellular immunotherapy. Leukemia 2003; 17: 1973–1980.
19 Matutes E, Owusu-Ankomah K, Morilla R, Garcia Marco J,
Houlihan A, Que TH et al. The immunological profile of B-cell
disorders and proposal of a scoring system for the diagnosis of CLL.
Leukemia 1994; 8: 1640–1645.
20 Van Bockstaele F, Verhasselt B, Philippe J. Prognostic markers
in chronic lymphocytic leukemia: a comprehensive review.
Blood Rev 2009; 23: 25–47.
21 Beziat V, Nguyen S, Lapusan S, Hervier B, Dhedin N, Bories D
et al. Fully functional NK cells after unrelated cord blood
transplantation. Leukemia 2009; 23: 721–728.
22 Vuillier F, Tortevoye P, Binet JL, Dighiero G. CD4, CD8 and NK
subsets in B-CLL. Nouv Rev Fr Hematol 1988; 30: 331–334.
23 Linet MS, Bias WB, Dorgan JF, McCaffrey LD, Humphrey RL. HLA
antigens in chronic lymphocytic leukemia. Tissue Antigens 1988;
31: 71–78.
24 Mueller LP, Machulla HK. Increased frequency of homozygosity
for HLA class II loci in female patients with chronic lymphocytic
leukemia. Leuk Lymphoma 2002; 43: 1013–1019.
25 Poggi A, Venturino C, Catellani S, Clavio M, Miglino M, Gobbi M
et al. Vdelta1 T lymphocytes from B-CLL patients recognize ULBP3
expressed on leukemic B cells and up-regulated by trans-retinoic
acid. Cancer Res 2004; 64: 9172–9179.
26 Verheyden S, Demanet C. NK cell receptors and their ligands in
leukemia. Leukemia 2008; 22: 249–257.
27 Costello RT, Sivori S, Marcenaro E, Lafage-Pochitaloff M,
Mozziconacci MJ, Reviron D et al. Defective expression and
function of natural killer cell-triggering receptors in patients with
acute myeloid leukemia. Blood 2002; 99: 3661–3667.
28 Jewell AP, Worman CP, Giles FJ, Goldstone AH, Lydyard PM.
Resistance of chronic lymphocytic leukaemia cells to inter-
feron-alpha generated lymphokine activated killer cells.
Leuk Lymphoma 1992; 7: 473–480.
29 Stevenson FK, Caligaris-Cappio F. Chronic lymphocytic leukemia:
revelations from the B-cell receptor. Blood 2004; 103: 4389–4395.
30 Jak M, Mous R, Remmerswaal EB, Spijker R, Jaspers A, Yague A
et al. Enhanced formation and survival of CD4+ CD25hi Foxp3+
T-cells in chronic lymphocytic leukemia. Leuk Lymphoma 2009;
50: 788–801.
31 Pallasch CP, Ulbrich S, Brinker R, Hallek M, Uger RA,
Wendtner CM. Disruption of T cell suppression in chronic
lymphocytic leukemia by CD200 blockade. Leuk Res 2009; 33:
460–464.
32 Weitzman J, Betancur M, Boissel L, Rabinowitz AP, Klein A,
Klingemann H. Variable contribution of monoclonal antibodies
to ADCC in patients with chronic lymphocytic leukemia.
Leuk Lymphoma 2009; 50: 1361–1368.
33 Lefebvre ML, Krause SW, Salcedo M, Nardin A. Ex vivo-activated
human macrophages kill chronic lymphocytic leukemia cells in
the presence of rituximab: mechanism of antibody-dependent
cellular cytotoxicity and impact of human serum. J Immunother
2006; 29: 388–397.
34 Umana P, Moessner E, Bruenker P, Unsin G, Puentener U, Suter T
et al. Novel 3rd generation humanized type II CD20 antibody with
glycoengineered Fc and modified elbow hinge for enhanced
ADCC and superior apoptosis induction. Blood Rev 2006; 108:
Abs 229.
35 Bowles JA, Wang SY, Link BK, Allan B, Beuerlein G, Campbell MA
et al. Anti-CD20 monoclonal antibody with enhanced affinity for
CD16 activates NK cells at lower concentrations and more
effectively than rituximab. Blood 2006; 108: 2648–2654.

36 Farag SS, Flinn IW, Modali R, Lehman TA, Young D, Byrd JC.
Fc gamma RIIIa and Fc gamma RIIa polymorphisms do not predict
response to rituximab in B-cell chronic lymphocytic leukemia.
Blood 2004; 103: 1472–1474.
37 Niwa R, Hatanaka S, Shoji-Hosaka E, Sakurada M, Kobayashi Y,
Uehara A et al. Enhancement of the antibody-dependent
cellular cytotoxicity of low-fucose IgG1 Is independent of
FcgammaRIIIa functional polymorphism. Clin Cancer Res 2004;
10: 6248–6255.
38 Fischer L, Penack O, Gentilini C, Nogai A, Muessig A, Thiel E et al.
The anti-lymphoma effect of antibody-mediated immuno-
therapy is based on an increased degranulation of peripheral
Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)
NK cells and optimized anti-CD20 in CLL
M Le Garff-Tavernier et al
109
blood natural killer (NK) cells. Exp Hematol 2006; 34:
753–759.
39 O’Brien SM, Kantarjian H, Thomas DA, Giles FJ, Freireich EJ,
Cortes J et al. Rituximab dose-escalation trial in chronic
lymphocytic leukemia. J Clin Oncol 2001; 19: 2165–2170.
40 Wierda WG, Kipps TJ, Mayer J, Stilgenbauer S, Williams CD,
Hellmann A et al. Ofatumumab as single-agent CD20 immuno-
therapy in fludarabine-refractory chronic lymphocytic leukemia.
J Clin Oncol 2010; 28: 1749–1755.
41 Morschhauser F, Cartron G, Lamy T, Milpied NJ, Thieblemont C,
Tilly H et al. Phase I study of RO5072759 (GA101) in relapsed/
refractory chronic lymphocytic leukemia. Blood 2009: Abs 884.
Leukemia