Abstracts / Molecular Immunology 89 (2017) 200–206
173
Monitoring of hemolytic activity of serum
during alemtuzumab/ofatumumab therapy of
chronic lymphocytic leukemia
Grzegorz Stasiłojć 1,∗ , Maria Winqvist 2 , Anna
Blom 3 , Anders Österborg 2 , Marcin Okrój 1
1 Intercollegiate Faculty of Biotechnology, University
of Gdańsk and Medical University of Gdańsk, Gdańsk,
Poland
2 Department of Oncology & Pathology, Cancer
Centre Karolinska, Karolinska Institutet, Stockholm,
Sweden
3 Department of Translational Medicine, Lund
University, Malmö, Sweden
Background: Available therapies to combat chronic lym-
phocytic leukemia (CLL) employ specific antitumor monoclonal
antibodies (mAb) like anti-CD20 or anti-CD52 specimens. Injection
of mAb may initiate classical complement pathway and subsequent
complement dependent cytotoxicity is supposed to play a role in a
therapeutic effect. However, the complement system is exhaustible
and therefore optimal dosing is critical to preserve cytotoxic poten-
tial for consecutive drug infusions, as shown in model experiments
(J Immunol. 2012; 188: 3532–41, J Immunol. 2014;192: 1620–9).
Overdosing may not only deplete immune effector but also induce
cancer resistance mechanisms like shedding or trogocytosis of tar-
get molecules.
Materials and methods: The materials for our study were serum
samples from patients with previously untreated CLL, who received
alemtuzumab (anti-CD52) three times per week for 18 weeks and
ofatumumab (anti-CD20) once a week starting from week 3 (clin-
ical trial NCT01361711). The activity of the classical complement
pathway was measured by haemolytic assay on sensitized sheep
erythrocytes. We analysed samples from 22 patients collected at
baseline and before ofatumumab infusions at weeks 3, 5, 7, 9, 13,
17 as well as follow-up samples.
Results and conclusions: Two patients showed low hemolytic
activity through the whole period of the study, which suggests
limited ability to use complement as effector mechanism. One
patient showed substantial loss of haemolytic activity after week 5
and further rebound, which may suggest depletion of complement
components and subsequent selection of resistant tumor cells. The
majority of the patients did not show a particular trend in hemolytic
activity through the study indicating that their pool of the classical
complement pathway components was not markedly depleted.
http://dx.doi.org/10.1016/j.molimm.2017.06.231
203
174
The effect of complement inhibition on
erythrocyte destruction in AIHA
Inge Baas 1,∗ , Edimara S. Reis 2 , Daniel Ricklin 2 ,
John D. Lambris 2 , Masja de Haas 3 , Diana
Wouters 1 , Sacha S. Zeerleder 4
1 Sanquin Research, Dept of Immunopathology,
Amsterdam and Landsteiner Laboratory, Academic
Medical Center, University of Amsterdam,
Amsterdam, The Netherlands
2 Department of Pathology and Laboratory Medicine,
Perelman School of Medicine, University of
Pennsylvania, Philadelphia, PA 19104, USA
3 Sanquin Diagnostic Services, Amsterdam, The
Netherlands
4 Department of Hematology, Academic Medical
Center, University of Amsterdam, Amsterdam, The
Netherlands
Background: Autoimmune hemolytic anemia (AIHA) is a rare
disease characterized by autoantibodies against erythrocytes.
These autoantibodies may activate the classical complement path-
way leading to opsonization by complement proteins C3b and C4b,
resulting in increased clearance of erythrocytes by phagocytes,
called extravascular hemolysis. Occasionally, complement activa-
tion results in formation of the membrane attack complex (MAC)
resulting in intravascular hemolysis. C3-inhibitor compstatin pre-
vents C3b deposition and thus it is expected that C3 inhibition will
inhibit formation of the MAC. However, no effect on C4b deposition
is expected, thus this inhibitor can be used as a tool to distinguish
the roles of C3b and C4b on clearance of erythrocytes by phagocytes.
The current aim of this research is to investigate in vitro whether
compstatin would be a suitable drug for AIHA treatment.
Materials and methods: Healthy donor erythrocytes were incu-
bated with AIHA patient serum and opsonization was analyzed
by FACS using anti-C3-FITC and anti-C4-APC antibodies. To assess
the effect of compstatin Cp40 on uptake of erythrocytes by phago-
cytes, erythrocytes were fluorescently labeled before opsonization.
Opsonized erythrocytes were then incubated with healthy mono-
cyte derived macrophages and phagocytosis of erythrocytes was
measured with ImageStream after lysing the non-phagocytosed
erythrocytes.
Results and conclusions: Compstatin completely inhibited C3
deposition on erythrocytes, while unexpectedly C4 deposition
appeared to be increased. As expected, compstatin prevented for-
mation of the MAC and also phagocytic uptake of erythrocytes by
macrophages was decreased by compstatin. However, when eryth-
rocytes were opsonized with both complement and IgG there was
less inhibition of phagocytosis compared to erythrocytes opsonized
with only complement.
Since compstatin inhibits both intravascular and extravascular
hemolysis it is an interesting candidate to consider for treatment
of AIHA. However, it is important not to overlook the role of IgG
mediated clearance.
http://dx.doi.org/10.1016/j.molimm.2017.06.232