Review article
The clinical application of monoclonal antibodies in chronic
lymphocytic leukemia
Samantha M. Jaglowski,1 Lapo Alinari,1 Rosa Lapalombella,1 Natarajan Muthusamy,1 and John C. Byrd1,2
1Division
of Hematology-Oncology, Department of Internal Medicine, College of Medicine, and 2Division of Medicinal Chemistry, College of Pharmacy, The Ohio
State University, Columbus, OH
CLL and as salvage therapy. Recent data
suggest that the addition of rituximab to
fludarabine with or without cyclophosphamide prolongs survival in younger patients with CLL. Other improved CD20
antibodies with promising clinical activity, including ofatumumab and GA-101,
are coming forward. Alemtuzumab, a
CD52 antibody, likewise has demonstrated
benefit in both symptomatic, previously
untreated CLL and in patients with relapsed disease but has less selectivity.
Development of other therapeutic antibod-
ies targeting alternative B-cell–specific
antigens in CLL has been less successful, although many promising candidate
antibodies and/or small modular immune
pharmaceuticals (SMIPs) are coming forward. In addition, recent efforts to combine currently applied therapeutic antibodies with other biologic and targeted
therapies with efficacy in CLL offers the
potential to move toward alternative non–
chemotherapy-based treatment approaches.
(Blood. 2010;116(19):3705-3714)
Introduction
Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/
SLL) is the most common leukemia, with an incidence rate of 2 to
6 cases per 100 000 people per year.1 The median survival is highly
variable with some patients exhibiting an indolent natural history,
whereas others develop aggressive disease with a survival of less
than 2 to 3 years. Genomic features such as immunoglobulin
herpesvirus (IGHV) mutational status, ␤2-microglobulin, ZAP70
expression, interphase cytogenetics, and complex karyotype on
metaphase cytogenetics, recently reviewed by Zenz et al,2 provide
further differentiation of disease prognosis. Because there is no
proven survival benefit with early treatment, CLL patients are
observed until symptoms appear.
Therapy for CLL has evolved significantly from 1970 when
alkylator-based therapy such as chlorambucil or cyclophosphamide
was used. Late 1990 saw multiple randomized phase 3 studies
comparing fludarabine to alkylator-based therapy and demonstrating improved response and progression-free survival (PFS), as
recently reviewed in Ricci et al.3 Success prompted phase 3 studies
combining fludarabine with cyclophosphamide, in which further
improved response and PFS were noted.3 Treatment intensification
in CLL from alkylator to fludarabine and cyclophosphamide
combinations resulted in increased cellular immune suppression
and myelosuppression. In addition, chemotherapy intensification
did not greatly improve treatment outcomes in patients with
high-risk genomic features, such as those with IGHV unmutated
disease, del(17p13.1), and p53 mutations.2 These patients all
display poorer outcomes, with markedly reduced survivals compared with patients with normal genomic features or good-risk
features, as recently reviewed by Zenz et al.4 Of all prognostic
factors examined in CLL, patients with mutated or deleted
Submitted April 24, 2010; accepted June 23, 2010. Prepublished online as
Blood First Edition paper, July 7, 2010; DOI 10.1182/blood-2010-04-001230.
BLOOD, 11 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 19
p53 respond very poorly to standard therapies that mainly act
through mechanisms relying on an intact p53 pathway. Identifying
therapies that circumvent p53 is therefore a priority for the
treatment of this high-risk population. Attempts to intensify
chemotherapy beyond fludarabine/alkylator-based combinations
have been pursued with enhanced toxicity but little evidence of
clinical benefit. As with many other types of cancers, treatment
outcomes of CLL patients with chemotherapy-based approaches
reached a plateau with no improvements in survival or hints of cure
in even a subset of patients. This review will focus on how the
clinical application of therapeutic monoclonal antibodies more than
the past decade has impacted the therapeutic approach to CLL and
point to potential opportunities in the future with other targeted
therapies currently being explored.
History of monoclonal antibodies in B-cell malignancies
Monoclonal antibodies have a fixed effector cell binding region
(Fc) and a variable region with affinity toward a specific antigen.
Antibodies can mediate cytotoxicity toward tumor cells via both
direct and indirect mechanisms based upon the target. Direct
cytotoxicity of tumor cells can occur though transmembrane
signaling, and recruitment of effector cells (natural killer [NK]
cells, macrophages, neutrophils) that mediate antibody-dependent
cell cytotoxicity (ADCC) and complement that mediates complement-dependent cytotoxicity (CDC). Indirect cytotoxicity can
occur by interfering with both the interaction of a tumor cell with
the microenvironment-generated survival signal and with its binding to soluble factors that enhance tumor cell survival. Given the
specificity of antibodies for a single antigen and the multiple
© 2010 by The American Society of Hematology
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Chronic lymphocytic leukemia (CLL) represents the most prevalent adult leukemia. Treatment with chemotherapy over
the past 3 decades has been palliative.
The introduction of therapeutic antibodies has increased the number of treatment options for this disease. Despite
this increase, our true understanding of
the mechanism of action of antibody
therapy in CLL remains limited. Rituximab, a CD20 antibody, is currently widely
used in combination-based strategies for
both previously untreated symptomatic
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BLOOD, 11 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 19
JAGLOWSKI et al
Table 1. Newer monoclonal antibodies in clinical development
Agent
Target
Humanized/chimeric
Direct cell death
ADCC
CDC
Development status
MDX-1342
CD19
Humanized
Yes
Yes
No
Phase 1
XmAb5574
CD19
Humanized
Modest
Yes
No
Phase 1
Ofatumumab
CD20
Humanized
Yes
Yes
Yes
Phase 3
GA-101
CD20
Humanized
Yes
Yes
Modest
Phase 2
PRO131921
CD20
Humanized
Yes
Yes
Yes
Phase1/2
Veltuzumab
CD20
Humanized
Yes
Yes
Yes
Phase1/2
LFB-R603
CD20
Chimeric
Yes
Yes
Yes
Phase 1
Lumiliximab
CD23
Primatized
Yes
Yes
Yes
Phase 3
TRU-016
CD37
Humanized
Yes
Yes
No
Phase 1
SGN40
CD40
Humanized
Yes
Yes
No
Phase1/2
HCD122
CD40
Humanized
No
Yes
No
Phase 1
CD70
Humanized
No
Yes
No
Phase 1
Milatuzumab
CD74
Humanized
Yes
No
No
Phase1/2
Ipilimumab
CTLA-4
Humanized
Yes
No
No
Phase 2
Bevacizumab
VEGF
Humanized
No
No
No
Phase 2
ADCC indicates antibody-dependent cell-mediated cytotoxicity; and CDC, complement-dependent cytotoxicity.
mechanisms by which they can mediate cytotoxicity, antibodybased cancer therapy was seen as a potential “silver bullet” therapy
for patients with CLL, particularly if the antigen is selectively
expressed on B cells. Numerous target antigens offered the opportunity to selectively target B cells, including CD19, CD37, CD20,
and idiotype. Murine antibodies derived from mouse plasma cell
hybridoma cells directed toward these targets were the firstgeneration agents evaluated in multiple clinical studies from 1980.
These studies were impaired by production issues that limited
antibody supply, diminished antibody activity toward the tumor
cell, and development of human antibody–mouse antibody reactions with repeated administration. As a consequence, very modest
activity with essentially all murine antibody-based treatments was
observed, limiting the development of this modality. Technologic
advances allowing engineering of mouse-derived antibodies including a minimal mouse component of the variable complementaritydetermining region in the final product (chimeric or humanized)
represented a major advance for this modality. In general, chimeric
and humanized therapeutic antibodies directed toward human
B-cell antigens mediate improved ADCC and CDC compared with
their murine counterparts. In addition, chimeric and humanized
antibodies generally lack human anti–mouse antibody even on
repeated administration. Concurrent with advances in chimeric and
humanization technologies were improvements in the ability to
produce larger amounts of antibodies. These advances fostered the
rebirth of antibody-based therapeutics, impacting treatment of
many diseases including CLL. This review will summarize evaluations of antibody and peptide therapies that directly target CLL
cells that are either approved or under clinical investigation at this
time (Table 1).
B cells.6 These data support CD20 as an ideal target for antibodybased therapy in mature B-cell malignancies.
Rituximab was the first approved therapeutic antibody for the
treatment of cancer. Not surprisingly, the majority of mechanism of
action studies of therapeutically used antibodies come from
preclinical studies with rituximab. As with most immunoglobulin
G1 (IgG1) therapeutic antibodies, rituximab can mediate CDC,
ADCC, and direct apoptosis with a cross-linking antibody (Figure
1).7 Extensive investigation of each mechanism has been pursued
in lymphoma and CLL. Although CDC is relevant to rituximabmediated cytotoxicity in some B-cell lines, CLL cells express dim
CD20 and only a small subset of cells are susceptible to CDC by
Rituximab
Rituximab target and mechanism of action
Rituximab is a chimeric murine/human antibody directed against
CD20. CD20 is expressed relatively selectively on B cells from the
pre–B-cell stage until postgerminal cells differentiate to become
plasma cells. CD20 knockout mice demonstrate normal B-cell
development and function, but CD19-induced calcium responses
and B-cell receptor signaling are significantly altered.5 Unlike other
antigens, CD20 is neither shed nor internalized in resting normal
Figure 1. Mechanisms of rituximab-mediated cell death. Rituximab coated B cells
are killed by at least 4 different mechanisms. (A) Binding of rituximab to CD20 on
B cell surface causes activation of the complement cascade, which generates the
membrane attack complex (MAC) that can directly induce B-cell lysis by complementmediated cytotoxicity (CDC). (B) Binding of rituximab allows interaction with NK cells
via Fc receptors III (FcRIII), which leads to antibody-dependent cell-mediated
cytotoxicity (ADCC). (C) The Fc portion of rituximab and the deposited complement
fragments allow for recognition by both FcR and complement receptors on macrophages, which lead to phagocytosis and ADCC. (D) The crosslinking of several
molecules of rituximab and CD20 in the lipid raft determine the interaction of these
complexes with elements of a signaling pathway involving Src kinases that mediate
direct apoptosis.
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MDX-1411
BLOOD, 11 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 19
Early rituximab studies and single-agent CLL trials
Although the phase 3 pivotal approval study of rituximab in
non-Hodgkin lymphomas (NHL) demonstrated promising clinical
activity, the response among the 33 patients with SLL was modest,
with only 12% of patients achieving a partial response (PR).37
Similarly disappointing results were obtained in several other small
studies (reviewed in Jaglowski and Byrd27). The very modest
response to rituximab compared with follicular lymphoma provided some pause for developing this agent in CLL/SLL. Potential
3707
reasons for lower response in CLL included diminished CD20
expression,38 altered innate immune function,17,18 and different
pharmacokinetic features37 compared with lymphoma. Two trials
performed by our group39 and the M. D. Anderson Cancer Center
(MDACC)40 administered either thrice weekly doses or higher
doses of rituximab weekly (up to 2250 mg/m2 per dose) to relapsed
CLL patients with improved response. Benefit was predominately
in the blood and nodal compartment with response duration
approaching that achieved in follicular B-NHL trials. These
2 studies established a role for single-agent rituximab in relapsed
CLL and encouraged subsequent trials of rituximab as a single
agent in previously untreated patients where improved efficacy was
observed41 and in combination strategies with chemotherapy.
Rituximab chemoimmunotherapy studies in CLL
Numerous phase 2 studies combining rituximab with other therapies used in CLL have been pursued. Due to reference limitations,
those impacting current CLL therapy approaches are outlined
below.
A phase 2 study performed by the German CLL Study Group
(GCLLSG) of fludarabine and rituximab (FR) in both refractory
and previously untreated patients resulted in an overall response
rate (ORR) of 87% with a subset achieving complete response
(CR).42 CALGB 9712 evaluated fludarabine in combination with
rituximab given either concurrently or sequentially. Patients in the
concurrent arm experienced more severe hematologic and infusionrelated toxicity, but the ORR was 90% with a CR of 47% compared
with an ORR of 78% and CR of 28% in the sequential arm.43 An
evaluation of outcome based on genetic features demonstrated
worse 3 year survival among those patients with del(17p13.1) or
del(11q22.3) compared with those with normal cytogenetics or
other abnormalities, with 33% and 53% of patients with del(17p13.1)
and del(11q22.3) respectively surviving at 3 years compared with
86% of patients with normal cytogenetics.44 A retrospective
comparison of outcome of patients on this trial to a similarly
designed CALGB study evaluating, in part, fludarabine alone
demonstrated chemoimmunotherapy improved PFS and overall
survival (OS).45 Long-term follow-up data recently presented
shows no increased risk of treatment-related acute myeloid leukemia (AML).24 An Italian phase 2 study of sequential FR confirmed
good response rates, with 78% of patients achieving a CR, but only
patients who had stable disease (SD) or better with fludarabine
remained on the study to receive rituximab.46
The combination of fludarabine, cyclophosphamide, and rituximab (FCR) has been extensively explored. A single-arm study of
300 previously untreated patients with progressive CLL from the
MDACC reported an ORR of 95% with 72% of patients attaining a
CR, 10% nodular partial remission (nPR), and 13% a PR.47 The
6-year OS and PFS were 77% and 51%, respectively.47 Toxicity in
this study included predominately cytopenias and associated infection. Eight patients developed treatment-related myelodysplasia. A
second study of 177 previously treated patients at this same
institution was pursued using the same schedule of FCR.48 The
results of this study demonstrated a CRR in 25% of patients with an
ORR of 73%.48 The median time to progression was 28 months,
which varied significantly based upon response. The median time
to progression for patients achieving CR, nPR, and PR was 39,
33, and 15 months, respectively. Toxicities observed were predominately grade 3 or 4 neutropenia (81%) and grade 3 or greater
infection (16%). Whereas patients with del(11q22.3) appeared to
benefit from FCR with loss of the adverse PFS observed in
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rituximab.8,9 Very elegant preclinical in vitro and in vivo studies
have demonstrated that CLL cells are prone to CD20-shaving after
treatment, and this diminishes the ability of CDC to occur.10-12
Attempts to abrogate the phenomenon of shaving have been
undertaken by administering very low doses of rituximab on a
thrice weekly schedule with very modest clinical activity.11,13 Thus,
although a very strong hypothesis with supportive preclinical data
support shaving as a reason for modest rituximab clinical activity
when administered at higher doses, it is not clear what CDC
contributes to tumor elimination. Monocytes mediate antibodydependent cellular phagocytosis (ADCP),14 and NK cells mediate
ADCC14 against rituximab-labeled CLL cells in vitro. Despite this,
the function of both monocytes15,16 and NK cells17,18 to mediate
ADCP and ADCC, respectively, are certainly compromised in vitro
and are likely compromised in vivo in CLL patients. The mechanism of innate immune ADCP and ADCC is not known but may be
from the increased T regulatory cells documented in CLL patients
with active disease.19 A recent study demonstrated that T regulatory
cells can dramatically dampen ADCC mediated toward rituximablabeled tumor cells.20 With the increased T regulatory cells in CLL
patients,19 diminished monocyte15,16 and NK cell17,18 function, the
contribution of these cells to tumor elimination is not clear. In
addition, single nucleotide polymorphisms of Fc␥RIIIa and Fc␥RIIa
that enhance ADCC and are associated with improved response to
rituximab,21,22 in lymphoma have no impact on rituximab treatment
response in CLL.23,24 Strategies directed at either reversing the
innate immune dysfunction with immune modulating agents such
as interleukin-21,14 TLR agonists,25 or agents that deplete T
regulatory cells26 offer the best opportunity to optimize immune
cell participation in tumor clearance by rituximab. Finally, several
groups have demonstrated that rituximab can mediate both caspasedependent and -independent apoptosis in vitro (reviewed in Jaglowski and Byrd27) and in vivo.28 Apoptosis appears to be the most
important mechanism of action in CLL and involves the activation
of the p38 mitogen-activated protein kinase, a pathway that
requires an intact p53 gene, and caspase 9 cleavage.7-9 Ofatumumab, a second-generation fully humanized anti-CD20 that
recognizes a different CD20 epitope than rituximab, has similar
ADCC, stronger CDC, and requires cross-linking to induce direct
apoptosis similar to riutximab.29,30 GA101 binds with high affinity
to the CD20 epitope and, as a result, induction of ADCC is 5 to
100 times greater than with rituximab.31-34 Type II anti-CD20
antibodies such as B1 and GA101 promote direct apoptosis without
a cross-linking antibody.35 Other differences between type I
(rituximab, ofatumumab) and type II (GA101, B1) anti-CD20
antibodies lie predominately in their ability to redistribute CD20
into plasma membrane lipid rafts.36 Type II anti-CD20 antibodies
do not segregate CD20 into lipid rafts and are very effective at
activating a caspase-independent, lysosomal-dependent mechanism of death that is dependent upon homotypic adhesion.35 The
relevance of this observation in vivo among CLL patients receiving
type II CD20 antibody therapy remains unexplored.
ANTIBODY THERAPY FOR CLL
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JAGLOWSKI et al
Phase 3 studies with rituximab in CLL
Two phase 3 studies, CLL8 from the GCLLSG and the REACH
trial, have been presented and confirm improved response rates
and overall survivals with the addition of rituximab. In CLL8, at
a median observation time of 37.7 months, the ORR for FCR
among the 761 previously untreated patients evaluable for
response was 95.1% versus 88.4% for FC, and the CRR was
44.1% compared with 21.8% for FC.55,56 The median PFS was
32.8 months for FC and 51.8 months for FCR (P ⬍ .001, hazard
ratio [HR] .56), and the OS was longer for those patients who
received FCR at 84.1% versus 79.0% in the FC arm (P ⫽ .01),
with statistically significant differences seen in patients with
Binet stages A (P ⫽ .09, HR .19) and B (P ⬍ .001, HR .45) but
not in patients with Binet stage C disease (P ⫽ .168, HR 1.4).
Patients with del(17p13.1) had particularly poor outcome, and
shorter overall survival was seen in the FC arm; likewise, there
was no significant improvement in CR, PFS, and OS among the
subgroup of patients with del(17p13.1) treated with FCR. A
trend toward shorter overall survival in the FCR arm in patients
with unmutated IGHV status was observed. Patients with
del(11q22.3) again appeared to benefit from the addition of
cyclophosphamide, with response rates approaching that of
patients without this mutation.57 There was not an increased
infection rate observed with the addition of rituximab, and more
deaths occurred in the FC arm.55 The REACH trial compared FC
to FCR in 552 patients with relapsed/refractory CLL. These
patients had received a median one line of previous treatment
with the majority consisting of single-agent alkylator therapy;
patients who had received combination FC or rituximab were
not eligible. Treatment with FCR resulted in an ORR of 70%,
versus 58% for FC alone, and the CRRs were 24% versus 13%
for FCR and FC, respectively. Observed PFS in the FCR arm
was 30.6 months compared with 20.6 months in the FC arm.58
Hematologic toxicities remained the most significant adverse
events. Collectively, these 2 phase 3 studies provide justification
for the use of rituximab as part of combination chemoimmunotherapy in both newly diagnosed and relapsed CLL. The use of
cyclophosphamide in this regimen seems to be important for
patients with del(11q22.3) as both the MDA and German CLL
study group showed no adverse outcome in this group compared
with low risk karyotype whereas CALGB 9712 did. For other
genetic groups the benefit of cyclophosphamide is uncertain as
there has not been a comparison of FCR to FR. A randomized
study (CALGB 10404) is ongoing and seeks to answer whether
the combination of FCR has an advantage over FR and, if so,
whether the increased risk of secondary AML associated with
cyclophosphamide justifies that advantage.
Maintenance rituximab in CLL
The use of maintenance rituximab is common in NHL. Contrasting
with this approach, no randomized trials have been performed to
determine whether benefit is derived in CLL/SLL from extended
maintenance therapy using rituximab. A phase 2 study of 75 previously untreated patients with CLL evaluated the efficacy of
rituximab maintenance after treatment with fludarabine for
6 cycles.59 All patients received 4 weekly doses of 375 mg/m2
rituximab after therapy, and then those who were minimal residual
disease (MRD)–positive went on to consolidation with 4 monthly
cycles of 375 mg/m2 rituximab followed by 12 monthly cycles of
150 mg/m2. MRD-positive patients in CR or PR receiving consolidation had a longer PFS than the patients not receiving consolidation (87% vs 32% at 5 years). A randomized study comparing
maintenance rituximab is now underway by the Polish CLL group
and until results from this trial are available, this approach should
only be applied as part of clinical trials.
Rituximab in the treatment of autoimmune complications
Autoimmune complications of CLL occur in 10% to 25% of
patients during their disease course. Autoimmune hemolytic anemia (AIHA) is the most common, followed by immune thrombocytopenia (ITP). These occur both as an intrinsic process associated
with the pathogenesis of CLL and as a result of treatment with
purine analogues such as fludarabine.60 Rituximab was initially
described for the treatment of steroid-refractory pure red cell
aplasia or AIHA61; subsequently, the successful treatment of
2 patients with CLL who developed red cell aplasia with 375 mg/
m2 rituximab weekly for 2 weeks was described.62 A series of 8
patients with CLL and steroid-refractory AIHA was treated with a
combination of rituximab and dexamethasone, and all patients
achieved a remission of their AIHA, with 5 patients achieving a
Coombs-negative status. Retreatment was also found to be successful.63 In a series of 14 patients with CLL and AIHA treated with
rituximab monotherapy, all but 2 patients had an increase in their
hemoglobin levels after treatment.64 Rituximab is effective in
patients with chronic refractory ITP as well and is of interest in
CLL specifically as fludarabine-related ITP is not responsive to
steroids. Three patients who developed ITP while receiving
fludarabine and who did not respond to treatment with steroids or
IVIG were treated with weekly rituximab for 4 doses. All patients
had rapid and dramatic improvements in their platelet counts, and
the response durations were 6 months or greater for all 3 patients.65
While randomized data demonstrating the exact benefit of rituximab in autoimmune complications is lacking, these data provide
evidence for its effectiveness. In the author’s opinion, rituximab
represents one of the more active therapies for the treatment of
autoimmune complications of CLL not responding to initial steroid
treatment.
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fludarabine-monotherapy studies, based on a retrospective evaluation of patients treated at the MDACC,49 those with IGHV
unmutated disease and del(17p13.1) continued to have an inferior
outcome with FCR.4750
Pentostatin is a nucleoside analog that has been suggested to be
less myelotoxic than fludarabine while still active in CLL. This
prompted a study in patients with previously treated CLL51
substituting pentostatin for fludarabine. This pentostatin, cyclophosphamide, and rituximab (PCR) regimen had an ORR of 75% with a
CR rate of 25%. The major toxicities were infections and myelosuppression. When evaluated in previously untreated patients, 91%
had responses with a 41% CR rate. Similar to the study with FCR,
patients with del(11q22.3) had similar PFS as those without this
aberration,52 again suggesting that cyclophosphamide may be an
important addition for del(11q22.3) patients. Infections and myelosuppression were again the predominant toxicities observed.
With the approval of bendamustine for clinical use in newly
diagnosed CLL, pilot studies combining this agent with rituximab
have recently been reported in previously untreated patients where
a 90% ORR and 33% CRR was observed.53 A parallel study in
relapsed CLL demonstrated a 76% ORR and 13% CRR.54 Toxicity
in both of these studies included myelosuppression and infection. A
randomized phase 3 study comparing bendamustine and rituximab
to FCR is currently ongoing.
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Newer CD20 antibodies for CLL
Ofatumumab
3709
rates were 77% and 73% respectively. The most common grade
3-4 toxicities were infections, reported in 11 patients, and hematologic adverse events, including neutropenia in 29 patients, anemia
in 8 patients, and thrombocytopenia in 9 patients. Grade 3-4 hemolytic anemia occurred in 3 patients.70
Currently, NCCN guidelines provide the recommendation for
use of ofatumumab in previously treated CLL patients.71 Clinical
trials to define the activity of ofatumuamb in previously untreated
CLL and in combination with most forms of chemoimmunotherapy
explored with rituximab are ongoing. Determining the actual
scientific advance of ofatumumab over rituximab will require
randomized phase 3 trials.
GA101
GA101 is a type II glycoengineered humanized CD20 monoclonal
antibody that binds CD20 in a completely different orientation than
rituximab and over a larger surface area.31 It initiates nonapoptotic
cell death via an actin-dependent lysosome-mediated mechanism
that is reliant on cell-to-cell contact.32 Depletion of CLL cells in
whole blood samples has been demonstrated, and it may be more
potent than rituximab at similar concentrations.33,34 GA-101 is
internalized less by CLL cells compared with type I CD20
antibodies, making it an ideal agent for investigation. A recently
reported phase 1 study in 13 relapsed/refractory CLL patients
demonstrated that GA101 is relatively well tolerated, with the most
common grade 3-4 toxicity being transient neutropenia in 9
patients. One CRi, 7 PRs, and 3 patients with SD were observed.
No clear dose-effect relationship was established.72 GA101 is being
evaluated in a phase 2 study as a single agent in relapsed/refractory
CLL and in combination with chlorambucil in previously untreated
elderly patients. A phase 3 study is currently underway in patients
with comorbidity comparing monotherapy with chlorambucil,
chlorambucil plus rituximab, and chlorambucil plus GA-101.
Alemtuzumab
Target and mechanism of action
Alemtuzumab (Campath-1H, Genzyme) is a recombinant DNAderived humanized IgG1 kappa monoclonal antibody that recognizes the cell-surface antigen CD52. CD52 is a 21- to 28-kDa,
heavily glycosylated, membrane-anchored glycoprotein, highly
expressed on all B and T lymphocytes at most stages of differentiation (except plasma cells), as well as on granulocytes, monocytes,
macrophages, eosinophils, NK cells, and dendritic cells.73 The
CD52 antigen is also expressed on tumor cells, particularly T-cell
prolymphocytic leukemia (T-PLL) as well as CLL, hairy cell
leukemia, NHL, and acute lymphoblastic leukemia.74 Despite the
frequent use of alemtuzumab in clinical trials, detailed mechanistic
studies to elucidate specific pathways of cell killing have been
hampered by the lack of cell lines expressing CD52. Thus, the
mechanism of action of alemtuzumab remains to be completely
clarified. Alemtuzumab can act through immunologic mechanisms,
such as CDC75,76 and/or ADCC by virtue of its IgG Fc region.77,78 It
has also been shown that alemtuzumab can induce direct CLL cell
death through a membrane raft-dependent mechanism.79 Unlike
rituximab, alemtuzumab has been shown to induce cell death in
vitro in CLL cells through a mechanism that is independent of p53
status and caspase activation.79 Moreover several in vivo clinical
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Ofatumumab is a newly approved, human type I CD20 monoclonal
antibody. In vitro, it has been demonstrated to mediate CDC against
rituximab-resistant Raji cells and CLL cells with low expression of
CD20. It appears to have greater potency in CDC than rituximab, as
well as a slower off-rate and more stable CD20 binding.29 In
addition, it appears to bind a different epitope of CD20 than
rituximab.30 A phase 1/2 study of ofatumumab in relapsed/
refractory patients demonstrated that it is generally well tolerated,
even at high doses and is active, with an ORR of 50%. Infusionrelated adverse events are similar to those reported with rituximab
and decrease after the first infusion. Infections were fairly common,
occurring in 51% of patients, including one fatal infection.66 A
planned interim analysis of the seminal study of 138 patients
treated with 8 weekly infusions of ofatumumab followed by 4
monthly infusions over a 24-week period has been reported. The
patients included in this study were required to be refractory to at
least one fludarabine-containing regimen and either refractory to at
least one alemtuzumab-containing regimen (FA-refractory group)
or to have bulky lymphadenopathy (⬎ 5 cm) rendering them less
suitable for alemtuzumab treatment (BF-refractory group). Response criteria were based on physical examination or hematologic
criteria alone, limiting assessment of internal lymphadenopathy.
The ORR was 58% for the FA-refractory group and 47% for the
BR-refractory group with one CR observed in the BF-refractory
group. All other responses were partial. The median duration of
response was 7.1 months in the FA-refractory group and 5.6 months
in the BF-refractory group with most patients progressing during
treatment. The presence of del(17p13.1) in the BF-refractory group
was associated with lower response, which was most apparent
among patients with bulky lymph nodes. The median PFS was
5.7 months in the FA-refractory group and 5.9 months in the
BF-refractory group with median OS of 13.7 months and
15.4 months, respectively. A landmark analysis done at week
12 demonstrated that median OS was significantly longer among
responding patients compared with those who did not respond
although the meaning of this is uncertain. Infusion-related reactions
were seen in 64% of patients in the FA-refractory group and 61% in
the BF-refractory group, almost all of which were grade 1 or 2. One
hundred eighty-nine infectious events were reported, 74% of which
were grade 1 or 2. Thirteen infections with onset during treatment
resulted in death, including one reported case of progressive
multifocal leukoencephalopathy (PML).67 Although the lack of CT
scan monitoring of nodal disease in this study has been criticized, it
is unclear that such monitoring adds benefit in terms of predicting
PFS to therapy in CLL.68,69 From this study and other data
presented, it is unlear that ofatumumab offers relative benefit over
the use of high dose rituximab previously reported.39,40 Only a
randomized comparative study will be able to discern such a
difference.
Combination studies are being done to enhance the therapeutic
efficacy of ofatumumab. A study evaluating the combination of
ofatumumab with fludarabine and cyclophosphamide (O-FC) was
recently presented. Sixty-one previously untreated patients received either 500 mg or 1000 mg ofatumumab combined with
fludarabine and cyclophosphamide every 4 weeks for a total of
6 courses. The CRR was 32% for patients who received 500 mg
ofatumumab and 50% for those who received 1000 mg. The OR
ANTIBODY THERAPY FOR CLL
3710
JAGLOWSKI et al
studies80-82 report that alemtuzumab therapy is effective in subgroup of patients with high-risk cytogenetic markers (such as
del(17p13.1), which points to its unique mechanism of action.
Alemtuzumab single-agent activity
neutropenia or major bacterial infections. CMV reactivation occurred in 10% of patients. These promising results promoted a
recent phase 3 trial of 297 CLL patients who were prospectively
randomized to receive either IV alemtuzumab 30 mg 3 times
weekly for up to 12 weeks or oral chlorambucil 40 mg/m2 every
4 weeks for up to 12 cycles.88 Alemtuzumab-treated patients
obtained a significantly superior response rate with significantly
improved PFS compared with chlorambucil (ORR 83% vs 56%
and CRR 24% vs 2%). In addition, this trial prospectively
demonstrated the superiority of alemtuzumab versus chlorambucil
in patients with del(17p13). No differences in terms of grade
3-4 hematologic toxicities were noticed between the 2 arms;
however, 52% of alemtuzumab-treated patients developed CMV
reactivation, in contrast to only 2% of the patients treated with
chlorambucil. Despite approval for this indication, the use of
alemtuzumab as monotherapy for primary therapy of CLL is not
generally used, given both the improved results with chemoimmunotherapy and the immune suppression associated with this
treatment.
Alemtuzumab as consolidation therapy for CLL
Given that alemtuzumab works best against blood and bone
marrow disease, efforts to apply alemtuzumab as a consolidation
approach occurred early in its development (reviewed in Alinari et
al83). O’Brien and colleagues89 administered alemtuzumab 10 or
30 mg IV 3 times weekly to 41 CLL patients with residual disease
after their most recent therapy. The ORR was 46% including 56%
among the 29 patients treated with 30 mg. Eleven of these 29
patients (38%) achieved a MRD-negative marrow, assessed by
polymerase chain reaction. Infectious occurred in 15 patients
(37%) with 9 being CMV reactivation. The authors reported that 3
patients developed Epstein-Barr virus (EBV)–positive large B-cell
lymphoma (LCL) within 6 weeks after finishing therapy. It is
known that T and B lymphocytes subpopulations are heavily
depleted by alemtuzumab and remain subnormal for more than a
year.83 Therefore the development of these LCLs is probably due to
proliferation of EBV-positive B cells in severely immunocompromised patients. Development of LCL represents a major concern in
alemtuzumab treated patients, although fortunately it is not that
common. The GCLLSG90 reported the results of a phase 3 trial
where patients responding to fludarabine-based induction therapy
were randomized to receive IV alemtuzumab 30 mg 3 times weekly
for a maximum of 12 weeks or observation. Of 21 evaluable
patients, 11 were randomized to receive alemtuzumab. This study
was prematurely closed because of severe infections in 7 of 11
patients in the alemtuzumab arm. The PFS was significantly
improved for patients receiving alemtuzumab at a median follow-up of 21.4 months. The CALGB performed 2 studies administering alemtuzumab after fludarabine91 or fludarabine and rituximab.92 Both of these studies demonstrated the ability of
alemtuzumab to improve response to treatment.92 However, reactivation of CMV was observed in both studies and unacceptable
infectious toxicity was observed in patients when alemtuzumab
was administered after fludarabine and rituximab. A communitybased clinical trial administering alemtuzumab after fludarabine
and rituximab also noted problematic toxicity with combined
chemoimmunotherapy.93 Future attempts to explore this consolidation approach after chemoimmunotherapy regimens should allow
an extended recovery time before administration of alemtuzumab.
Consolidation with alemtuzumab should only be considered in the
context of a clinical trial.
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The dosing schedule of alemtuzumab was developed empirically
using primarily clinical response as a surrogate end point in initial
phase 1 studies. The intravenous (IV) dosing schedule currently
used as the standard regimen for alemtuzumab therapy comprises a
2-hour IV infusion at a starting dose of 3 mg on day 1, 10 mg on
day 2, and 30 mg 3 times weekly for a total of 8 to 12 weeks.
The effectiveness of single-agent alemtuzumab in refractory/
relapsed CLL patients has been demonstrated (reviewed in Alinari
et al83). In this setting, alemtuzumab produced an ORR of 33% to
54%. In the majority of these studies, antitumor effects of
alemtuzumab were more significant in blood and bone marrow than
in lymph nodes (especially if larger than 5 cm). The reason for this
differential response is not clear, although it has been postulated to
be related to poor bioavailability of the drug in bulky sites causing a
low saturation of the binding sites on the neoplastic cells’ surface.
Moreover, there may be variability in the immune effector mechanisms in lymph nodes compared with other sites.
Alemtuzumab was initially approved in 2001 as a consequence
of the pivotal CAM 211 phase 3 study, in which 93 patients with
relapsed or refractory CLL who had failed prior therapy with
fludarabine and an alkylating agent were treated with stepped-up
dosing followed by 30 mg 3 times weekly for a total of 12 weeks.84
The ORR was 33% (2% CR, 31% PR) and the median duration of
response was 8.7 months. Given that the majority of patients
treated with IV alemtuzumab experience infusional toxicity combined with the observation that subcutaneous (SC) alemtuzumab
had comparable biologic activity with diminished infusion-related
events, interest in SC administration has progressively increased.
In a phase 2 study by the GCLLSG,80 103 patients with fludarabinerefractory CLL received at least one dose of alemtuzumab,
administered subcutaneously at 30 mg 3 times weekly for up to
12 weeks. The ORR was 34% (4% CR, 30% PR). The median PFS
was 7.7 months and the median OS was 19.1 months. This trial
confirmed earlier studies that alemtuzumab was effective for
del(17p13.1) CLL.81,82
In studies involving patients with relapsed/refractory CLL
treated with alemtuzumab, the most common adverse events were
cytopenia and infection as a consequence of profound cellular
immune suppression. Reactivation of herpesvirus including cytomegalovirus (CMV) were the most common opportunistic infections observed. Prophylaxis against opportunistic infections together with monitoring for CMV reactivation is highly
recommended in these patients. Recently, O’Brien et al85 demonstrated that the addition of valganciclovir 450 mg orally twice daily
was highly effective for prophylaxis of CMV reactivation in
patients receiving alemtuzumab. Our own approach is to administer
bactrim (or equivalent) and anti-herpes antiviral (acyclovir, valacylovir) and to monitor for reactivation of CMV with subsequent
preemptive early treatment due to the prohibitive cost of
valgancyclovir.
Several pilot studies in previously untreated CLL patients86,87
have shown benefit with alemtuzumab. Lundin and colleagues87
treated 41 CLL patients with SC alemtuzumab as first-line therapy
for up to 18 weeks, with an observed ORR of 87%, including a 19%
CR rate with a median TTF of more than 18 months. The treatment
was generally well tolerated, with adverse events mainly comprising local injection site reactions, and with no episodes of febrile
BLOOD, 11 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 19
BLOOD, 11 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 19
Alemtuzumab combination strategies in CLL
3711
1/2 combination study with FCR showing potential benefit,105
results from a phase 3 study comparing FCR plus lumiliximab
versus FCR did not confirm benefit in terms of improved response
or PFS. Therapeutic antibodies directed at CD40 with partial
agonist (SGN40106) and blocking properties (HCD122; personal
communication, J.C.B., September 5, 2010) have completed phase
1 testing with minimal clinical activity in CLL and likely will be
applied as combination therapies. Other new antibodies targeting
CD19 (MDX-1342 and XmAb5574) and CD74 (milatuzamab) are
either entering into or currently being tested in early phase 1
clinical trials for CLL. Extending beyond new targets for therapeutic antibodies and SMIPs is the opportunity to combine these
therapies with new immune-modulating agents (interleukin-21,
lenalidomide, CpG oligonucleotides) and targeted therapies (CAL101, fostamatinib, PCI-32 765, SCH727965, ABT-263, and flavopiridol) with the potential to avoid the immunosuppressive
effects of chemotherapy treatments.
Conclusions
Monoclonal antibodies represent an exciting addition to the
growing armamentarium of agents used to treat CLL. To date, those
agents targeting CD20 and CD52 have shown greatest promise.
While both CD20- and CD52-directed antibodies have demonstrated activity as single agents, their greatest contribution (as has
been demonstrated with rituximab) may lie in their combination
with more traditional chemotherapies. When added to chemotherapy, the use of rituximab has resulted in improved ORR and CR
rate and longer PFS and OS compared with chemotherapy alone,
and this has been borne out in phase 3 studies. Monoclonal
antibodies promise to be a fertile source of preclinical and clinical
investigation and will likely continue to revolutionize the way we
care for patients with CLL.
Acknowledgments
Other targets
Efforts to target other B cell–specific antigens are underway as part
of clinical trials at this time. Of antibody and peptide therapeutics
currently in clinical development, those targeting CD37 appear the
most promising. CD37 is expressed on B cells and transformed
mature B cell leukemias and lymphomas but not on T cells.
TRU-016 is a CD37 small modular immunopharmaceutical (SMIP)
that represents a structural modification of a CD37 antibody that
lacks the CH1 domain. In vitro studies with the chimeric version of
TRU-016 demonstrate that this is a potent inducer of apoptosis and
ADCC-dependent cytotoxicity against CLL cells.102 Interim results
of a phase 1 study of TRU-016 reported a favorable toxicity profile
and partial responses at higher doses.103 Whereas the CD23 antibody lumiliximab is most mature with respect to clinical investigation in CLL, with a completed phase 1 single-agent study showing
minimal activity but favorable toxicity profile104 and a phase
This work was supported by grants from Leukemia & Lymphoma
Society (P50-CA140158, PO1-CA95426, PO1 CA81534) and by
the D. Warren Brown Foundation.
Authorship
Contribution: S.J. and J.C.B. wrote the entire manuscript; and L.A.,
R.L., and R.M. wrote components of the manuscript and reviewed
and approved the final version.
Conflict-of-interest disclosure: J.C.B. is a consultant for Calistoga Pharmaceuticals and has a financial interest in the development of this compound. The remaining authors declare no competing financial interests.
Correspondence: John C. Byrd, MD, 455B, OSUCCC, 410 W.
12th Ave, Columbus, OH 43210; e-mail: John.byrd@osumc.edu.
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BLOOD, 11 NOVEMBER 2010 䡠 VOLUME 116, NUMBER 19