Possible misinterpretation of the mode of action of therapeutic antibodies in vitro: homotypic adhesion and flow cytometry result in artefactual direct cell death

Golay, Joseé; Bologna, Luca; André, Pierre-Alain; Buchegger, Franz; Mach, J. P.; Boumsell, Laurence; Introna, Martino

doi:10.1182/blood-2010-06-289736

Cited by 23 publications

(21 citation statements)

References 4 publications

(6 reference statements)

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“…by guest www.bloodjournal.org From chose glycoengineered rituximab as a positive control because obinutuzumab induces stronger homotypic adhesion, a phenomenon that may cause artifacts by flow cytometry. 19,44 We observed that phagocytosis of glycoengineered rituximab-opsonized targets by purified PMNs in whole blood was partially blocked by either Fab fragment, but fully inhibited when both were present ( Figure 6A). These data suggest that both CD16B and CD32A are involved in phagocytosis by glycoengineered CD20 antibodies.…”

Section: Phagocytosis Of Cll Targets By Pmns Requires Cd16b and Cd32amentioning

confidence: 87%

Glycoengineered CD20 antibody obinutuzumab activates neutrophils and mediates phagocytosis through CD16B more efficiently than rituximab

Golay

Roit

Bologna

et al. 2013

Blood

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203

143

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Key Points• Phagocytosis of CLL targets by neutrophils is a novel mechanism of action of the glycoengineered anti-CD20 antibody obinutuzumab.• This mechanism takes place in physiological conditions and requires CD16B and CD32A.Obinutuzumab (GA101) is a glycoengineered type 2 CD20 antibody with enhanced CD16A-binding and natural killer-mediated cytotoxicity. CD16B is highly homologous to CD16A and a major FcgR on human polymorphonuclear neutrophils (PMNs). We show here that glycoengineered obinutuzumab or rituximab bound CD16B with approximately sevenfold higher affinity, compared with nonglycoengineered wild-type parental antibodies. Furthermore, glycoengineered obinutuzumab activated PMNs, either purified or in chronic lymphoblastic leukemia whole blood, more efficiently than wild-type rituximab. Activation resulted in a 50% increase in CD11b expression and 70% down-modulation of CD62L on neutrophils and in release of tumor necrosis factor alpha, IL-6, and IL-8. Activation was not accompanied by generation of reactive oxygen species or antibody-dependent cellular cytotoxicity activity, but led to up to 47% phagocytosis of glycoengineered anti-CD20 opsonized chronic lymphoblastic leukemia targets by purified PMNs. Significant phagocytosis was observed in whole blood, but only in the presence of glycoengineered antibodies, and was followed by up to 50% PMN death. Finally we show, using anti-CD16B and anti-CD32A Fab and F(ab') 2 fragments, that both of these receptors are involved in PMN activation, phagocytosis, and cell death induced by glycoengineered antibodies. We conclude that phagocytosis by PMNs is an additional mechanism of action of obinutuzumab mediated through its higher binding affinity for CD16B. (Blood. 2013;122(20):3482-3491) IntroductionThe chimeric unmodified, wild-type CD20 IgG1 monoclonal antibody rituximab (MabThera and Rituxan) has shown significant therapeutic activity in B-non-Hodgkin lymphoma (B-NHL) and chronic lymphocytic leukemia (CLL).1,2 Rituximab is thought to act largely through immune-mediated mechanisms: complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and/or antibody-dependent phagocytosis (ADCP) by macrophages/monocytes.3,4 CD20 IgG1 antibodies can differ in their functional properties according to their binding mode to CD20. They are classified as type 1 when they show high CDC such as rituximab or as type 2 when they show high homotypic adhesion and direct cell death, respectively. 5,6 Improved versions of rituximab have been developed with the scope of enhancing response rates and reducing relapse or resistance. 7,8 One modification applied for CD20 and other therapeutic antibodies is termed "glycoengineering" and results in the decreased fucosylation of the carbohydrate attached to the Asn-297 glycosylation site of the Fc portion of the antibody. [9][10][11][12][13][14][15] Obinutuzumab (GA101) is a glycoengineered CD20 antibody derived from the murine Bly-1 antibody and is currently in pivotal clinical trials for the treatment of...

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Section: Phagocytosis Of Cll Targets By Pmns Requires Cd16b and Cd32amentioning

confidence: 87%

Glycoengineered CD20 antibody obinutuzumab activates neutrophils and mediates phagocytosis through CD16B more efficiently than rituximab

Golay

Roit

Bologna

et al. 2013

Blood

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203

143

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“…Golay et al recently proposed that type II anti-CD20-induced cell death detected by flow cytometry is an artifact of HA. 46 An argument against this hypothesis is the prominent cell death inhibition by concA in the presence of HA, as has also been observed with cathepsin inhibitors in some circumstances. 22,46 An interesting observation was the increased LysoTracker signal in the absence of HA in CD40-stimulated CLL cells incubated with GA101 plus cytoD.…”

Section: Discussionmentioning

confidence: 99%

“…46 An argument against this hypothesis is the prominent cell death inhibition by concA in the presence of HA, as has also been observed with cathepsin inhibitors in some circumstances. 22,46 An interesting observation was the increased LysoTracker signal in the absence of HA in CD40-stimulated CLL cells incubated with GA101 plus cytoD. This suggests that the process of actin polymerization, HA, and increased lysosomal number and activity are not functionally linked.…”

Section: Discussionmentioning

confidence: 99%

CD40 stimulation sensitizes CLL cells to lysosomal cell death induction by type II anti-CD20 mAb GA101

Jak¹,

Bochove²,

Reits

et al. 2011

Blood

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Sensitivity of chronic lymphocytic leukemia (CLL) cells to anti-CD20 mAbs is low and, therefore, the efficacy of monotherapy with current anti-CD20 mAbs is limited. At present, it is not known whether sensitivity of CLL cells to CD20 mAbs is modulated by microenvironmental stimuli. We have shown previously that in vitro CD40 stimulation of peripheral bloodderived CLL cells results in resistance to cytotoxic drugs. In the present study, we show that, in contrast, CD40 stimulation sensitizes CLL cells to the recently described novel type II anti-CD20 mAb GA101. Cell death occurred without crosslinking of GA101 and involved a lysosomedependent mechanism. Combining GA101 with various cytotoxic drugs resulted in additive cell death, not only in CD40-stimulated CLL cells, but also in p53-dysfunctional CLL cells. Our findings indicate that GA101 has efficacy against chemoresistant CLL, and provide a rationale for combining cytotoxic drugs with anti-CD20 mAbs. (Blood. 2011;118(19): 5178-5188) IntroductionAlthough treatment results for chronic lymphocytic leukemia (CLL) have improved considerably over the last decade, a curative drug regimen is still lacking. Similar to other B-cell malignancies such as follicular lymphoma and multiple myeloma, in CLL, the interaction of the malignant cells with their microenvironment in the lymph nodes, spleen, and possibly BM has been shown to play an important role in the biology of the disease. 1 We and others have previously shown that in vitro CD40 stimulation of CLL cells can to a certain extent mimic the lymph node environment and results in the induction of resistance of the CLL cells to cytotoxic drugs such as fludarabine, chlorambucil, bortezomib, and roscovitine. [2][3][4][5][6] These microenvironmental niches might be an important localization of minimal residual disease and form the basis for the relapses characterizing this disease. 1,[3][4][5]7,8 Moreover, after sequential treatments, selection of p53 dysfunctional clones occurs in up to 50% of patients, 9,10 which also results in chemoresistance. Therefore, there is a need for new treatments that circumvent microenvironmental chemoresistance and act independently of p53, possibly including anti-CD20 mAb-containing regimens. 11,12 However, sensitivity of CLL cells to anti-CD20 mAbs in vitro is low and monotherapy with conventional doses of the type I anti-CD20 mAb rituximab has only limited efficacy in CLL. Because of rituximab resistance or unresponsiveness, more potent anti-CD20 mAbs are currently being sought. Two types of anti-CD20 mAbs have been described. A prime difference is that, in contrast to type I anti-CD20 mAbs, type II mAbs are unable to translocate CD20 into lipid rafts or to evoke Ca 2ϩ flux. [13][14][15][16] Ofatumumab, a second-generation type I anti-CD20 mAb seems promising for the treatment of CLL, 17 although large amounts seem to be required. GA101 is a novel glycoengineered type II anti-CD20 mAb. Compared with rituximab, GA101 has enhanced direct cell death-inducing capacity and improved Ab-depende...

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“…3A). To confirm that cell-death induction by GA101 is unrelated to mechanical disruption, as recently hypothesized (13,14), and to gain further insights into the kinetics and mechanisms of cell death, direct cell death was assessed using time-lapse confocal microscopy and Annexin V/PI labeling of Z138 tumor cells. Figure 3B shows representative images taken at the indicated time points (cf.…”

Section: Induction Of Direct Cell Deathmentioning

confidence: 99%

Preclinical Activity of the Type II CD20 Antibody GA101 (Obinutuzumab) Compared with Rituximab and Ofatumumab In Vitro and in Xenograft Models

Herter

Herting

Mundigl

et al. 2013

Molecular Cancer Therapeutics

302

252

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We report the first preclinical in vitro and in vivo comparison of GA101 (obinutuzumab), a novel glycoengineered type II CD20 monoclonal antibody, with rituximab and ofatumumab, the two currently approved type I CD20 antibodies. The three antibodies were compared in assays measuring direct cell death (AnnexinV/PI staining and time-lapse microscopy), complement-dependent cytotoxicity (CDC), antibody-dependent cellmediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and internalization. The models used for the comparison of their activity in vivo were SU-DHL4 and RL xenografts. GA101 was found to be superior to rituximab and ofatumumab in the induction of direct cell death (independent of mechanical manipulation required for cell aggregate disruption formed by antibody treatment), whereas it was 10 to 1,000 times less potent in mediating CDC. GA101 showed superior activity to rituximab and ofatumumab in ADCC and whole-blood B-cell depletion assays, and was comparable with these two in ADCP. GA101 also showed slower internalization rate upon binding to CD20 than rituximab and ofatumumab. In vivo, GA101 induced a strong antitumor effect, including complete tumor remission in the SU-DHL4 model and overall superior efficacy compared with both rituximab and ofatumumab. When rituximab-pretreated animals were used, second-line treatment with GA101 was still able to control tumor progression, whereas tumors escaped rituximab treatment. Taken together, the preclinical data show that the glyoengineered type II CD20 antibody GA101 is differentiated from the two approved type I CD20 antibodies rituximab and ofatumumab by its overall preclinical activity, further supporting its clinical investigation. Mol Cancer Ther; 12(10); 2031-42. Ó2013 AACR.

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Possible misinterpretation of the mode of action of therapeutic antibodies in vitro: homotypic adhesion and flow cytometry result in artefactual direct cell death

Cited by 23 publications

References 4 publications

Glycoengineered CD20 antibody obinutuzumab activates neutrophils and mediates phagocytosis through CD16B more efficiently than rituximab

Glycoengineered CD20 antibody obinutuzumab activates neutrophils and mediates phagocytosis through CD16B more efficiently than rituximab

CD40 stimulation sensitizes CLL cells to lysosomal cell death induction by type II anti-CD20 mAb GA101

Preclinical Activity of the Type II CD20 Antibody GA101 (Obinutuzumab) Compared with Rituximab and Ofatumumab In Vitro and in Xenograft Models

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