Adding CD20, CD52 MoABs to Bortezomib Enhances Killing Power

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[2972] Anti-CD20 and Anti-CD52 Monoclonal Antibodies Enhance

Cytotoxicity of Bortezomib (Velcade) Against Chronic Lymphocytic

Leukemia Cells. Session Type: Poster Session 223-III

Piotr Smolewski, Markus Duechler, Linke, Barbara Cebula, Olga

Grzybowska-Izydorczyk, Medhat Shehata, Tadeusz Robak Department of

Hematology, Medical University of Lodz, Lodz, Poland; Ludwig

Boltzmann Institute for Cytokine Research, Department of Hematology,

Clinic of Internal Medicine I, AKH Vienna, Vienna, Austria

Background: Inhibitor of proteasome, bortezomib (BOR), has high in

vitro cytotoxic activity in chronic lymphocytic leukemia (CLL).

However, first clinical trials showed low efficacy of BOR used alone

in CLL patients. One way to increase the efficacy of BOR in vivo may

be its combinatory use with other agents active in CLL, what can

bring also benefit from lowering their doses. Indeed, there is some

evidence, that efficacy of BOR may be increased by its combination

with other agents active in CLL.

Aim: To assess in vitro cytotoxic effects exerted by BOR in

concomitant treatment with monoclonal antibodies anti-CD20

(rituximab, RIT) or anti-CD52 (alemtuzumab, ALT), agents with

confirmed activity in CLL. Additionally, we investigated mechanisms

responsible for observed interactions.

Methods: The study was performed on cells isolated from 61 untreated

CLL patients. Cell viability was evaluated by propydium iodide and

MTT assays. Proapoptotic effects were measured by detection of

active caspase-3, collapse of mitochondrial transmembrane potential

(Mitotracker Red dye) and annexin V flow cytometry assays.

Additionally, expression of several promoters of apoptosis (Bid,

Bax, Bak, Bcl-w) and anti-apoptotic proteins (Bcl-2, Mcl-1, XIAP and

FLIP) was measured. BOR concentration varied in different

experiments between 1,25nM and 5nM. Based on preliminarily performed

experiments RIT and ALT were used at final doses 10 ìg/ml and 20

ìg/ml, respectively. Cells were cultured for 24 hours in following

sets of samples: 1/ untreated control, 2/ BOR alone, 3/ RIT alone,

4/ ALT alone, 5/ BOR+RIT and 6/ BOR+ALT. Moreover, all those sets of

cultures were either done in RPMI 1640 medium supplemented with 5%

human serum as a source of complement or in the presence of 20 ìg/ml

anti-human IgG crosslinking antibodies. Finally, in additional

series of experiments cells were pretreated for 24 hours with RIT or

ALT, and then BOR was added for the next 24 hours of incubation.

Results: Combinations of BOR+RIT and BOR+ALT showed additive

cytotoxicity, especially when 24-hour incubation with the antibodies

crosslinked with anti-IgG preceded BOR administration (combination

index 1.07 and 0.83, respectively). In these settings both

combinations, BOR+RIT and BOR+ALT, significantly increased caspase-3

activation. Interestingly, addition of complement enhanced

significantly only cell death mediated by ALT. Caspase-3 activation

correlated with collapse of mitochondrial potential in samples

treated with BOR+RIT (R= 0.71; p<0.02). Upregulation of Bax and

downregulation of Bcl-2 or Mcl-1 were found after BOR+RIT treatment

(p<0.005 and p<0.01, respectively). ALT+BOR triggered significant

downregulation of Bcl-2 (p<0.005) and XIAP (p<0.01) proteins in CLL

cells. For both combinations, significant increase in Bax/Bcl-2

ratio was shown (p<0.001).

Conclusions: We found additive cytotoxic effects of both

combinations, BOR+RIT and BOR+ALT. Upregulation of Bax and

downregulation of Bcl-2 and Mcl-1 proteins were main mechanisms of

additive interaction of BOR+RIT. The BOR+ALT combination

significantly increased Bid and p53 expression and downregulated Bcl-

2 and XIAP proteins. These data indicate feasibility of treatment

combining proteasome inhibition with the use of RIT or CAM

monoclonal antibodies in CLL.