Mechanism of Action of Type II, Glycoengineered, Anti-CD20 Monoclonal Antibody GA101 in B-Chronic Lymphocytic Leukemia Whole Blood Assays in Comparison with Rituximab and Alemtuzumab

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BlankMechanism of Action of Type II, Glycoengineered, Anti-CD20 Monoclonal

Antibody GA101 in B-Chronic Lymphocytic Leukemia Whole Blood Assays in

Comparison with Rituximab and Alemtuzumab

1.. Luca Bologna,

2.. Gotti,

3.. Massimiliano Manganini,

4.. Alessandro Rambaldi,

5.. Tamara Intermesoli,

6.. o Introna and

7.. Josée Golay

+ Author Affiliations

1.. Laboratory of Cellular Therapy “G. Lanzani,” Division of Hematology,

Ospedali Riuniti, 24128 Bergamo, Italy

1.. Address correspondence and reprint requests to Dr. o Introna,

Laboratory of Cellular Therapy “G. Lanzani,” Division of Hematology, Ospedali

Riuniti, c/o Presidio Matteo Rota, Via Garibaldi 11-13, 24128 Bergamo, Italy.

E-mail address: mintrona@...

Abstract

We analyzed in B-chronic lymphocytic leukemia (B-CLL) whole blood assays the

activity of therapeutic mAbs alemtuzumab, rituximab, and type II glycoengineered

anti-CD20 mAb GA101. Whole blood samples were treated with Abs, and death of

CD19+ B-CLL was measured by flow cytometry. Alemtuzumab efficiently lysed B-CLL

targets with maximal lysis at 1–4 h (62%). In contrast, rituximab induced a more

limited cell death (21%) that was maximal only at 24 h. GA101 killed B-CLL

targets to a similar extent but more rapidly than rituximab, with 19.2 and 23.5%

cell death at 4 and 24 h, respectively, compared with 7.9 and 21.4% for

rituximab. Lysis by both rituximab and GA101 correlated directly with CD20

expression levels (r2 = 0.88 and 0.85, respectively). Interestingly, lysis by

all three Abs at high concentrations was mostly complement dependent, because it

was blocked by the anti-C5 Ab eculizumab by 90% in the case of alemtuzumab and

rituximab and by 64% in the case of GA101. Although GA101 caused homotypic

adhesion, it induced only limited (3%) direct cell death of purified B-CLL

cells. Both rituximab and GA101 showed the same efficiency in phagocytosis

assays, but phagocytosis was not significant in whole blood due to excess Igs.

Finally, GA101 at 1–100 µg/ml induced 2- to 3-fold more efficient NK cell

degranulation than rituximab in isolated B-CLL or normal PBMCs. GA101, but not

rituximab, also mediated significant NK cell degranulation in whole blood

samples. Thus, complement and Ab-dependent cellular cytotoxicity are believed to

be the major effector mechanisms of GA101 in whole blood assays.