Depletion of Chronic Lymphocytic Leukemia Cells From Whole Blood Samples Mediated by the Anti-CD20 Antibodies Rituximab and GA101.
Authors: Patz Michaela , Forcob Nche , Müller Bianka , Klein Christian , Umana Pablo , Hallek Michael , Krause Günter 
Published: October 2019

Abstract
Abstract 2365
Poster Board II-342

Introduction:
Monoclonal antibodies against CD20 have great potential as therapeutics for the treatment of B-cell lymphomas and chronic lymphocytic leukemia (CLL), since CD20 is expressed on all mature B-cells except stem or plasma cells. Therefore treatment with anti-CD20 antibodies leads to both, direct cell death induction (DCD) by receptor engagement and to cell killing by Fc-mediated mechanisms, namely antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In a pre-clinical in vitro assessment we compared the effects of rituximab, which was approved for treatment of non-Hodgkin lymphoma more than a decade ago, with GA101, a third-generation glyco-engineered anti-CD20-antibody recognizing a type II epitope.

Methods:
CLL cells were isolated from blood samples by Ficoll gradient centrifugation after treatment with a RosetteSep antibody cocktail. The effects of antibody treatment on membrane integrity were assessed by annexin V binding. Antibody-mediated B cell depletion in whole blood samples was determined flow cytometrically by enumerating CD19- and CD3-positive B and T lymphocytes and by relating the B/T cell ratios after antibody treatment with those of the corresponding untreated samples. Whole blood samples from 25 patients were investigated. Significance thresholds were determined by unpaired Student's T-test and linear correlation analysis.

Results:
The average decreases in viability of freshly isolated CLL cells due to treatment with 10 μg/ml of anti-CD20 antibodies were 21 % for GA101 (n=8) and 6 % for rituximab (n=6), while the mean B cell depletion in whole blood samples from CLL patients was 32 % for GA101 (n=18) and 11 % for rituximab (n=17). Thus the whole blood assay indicated the antibody effects with greater sensitivity than analysis of annexin V binding in isolated cells. In both assays GA101 performed better than rituximab as evidenced by superior DCD (p=0.021) and antibody-dependent B cell depletion (p=0.001). In about two thirds of whole blood samples from CLL patients treatment with 1 μg/ml of GA101 led to more than 20 % antibody-mediated B-cell depletion. The maximum B-cell depletion observed among the investigated CLL samples after treatment with 10 μg/ml of antibody was 85 % for GA101 and 25 % for rituximab. The B cell depletion from whole blood samples mediated by GA101 and rituximab correlated with the CD20 surface expression on CLL cells from the same patients (r=0.643, n=15, p<0.01 and r=0.610, n=14, p<0.05). In contrast, CLL cell depletion in six whole blood samples did not correlate with the increases in annexin V binding of the corresponding isolated CLL cells.

Conclusions:
The size of GA101 effects observed in the present B cell depletion assay in a majority of whole blood samples encourages attempts to dissect antibody-mediated killing mechanisms in individual patient samples by specifically inhibiting ADCC or CDC to determine their contribution to B cell depletion. As compared to rituximab, GA101 had stronger effects on isolated CLL cells and, to an even higher degree, led to superior B cell depletion in whole blood samples. These pre-clinical in vitro data obtained with CLL cells indicate that many patients might benefit from this promising therapeutic agent.

Disclosures:
Klein: Roche: Employment, Equity Ownership, Patents & Royalties. Umana:Roche: Employment, Equity Ownership, Patents & Royalties. Hallek:Roche: Honoraria, Research Funding. Krause:Roche: Research Funding.