CD40 stimulation sensitizes CLL cells to rituximab-induced cell death

Jak, Margot; Bochove, Gregor G. W. van; Lier, R. A. W. Van; Eldering, Eric; Oers, Marinus H. J. van

doi:10.1038/leu.2011.39

Cited by 31 publications

(33 citation statements)

References 57 publications

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“…Indeed, it would appear that increased expression of these molecules on the surface of CLL cells may even augment blinatumomab-directed T-cell activation and, ultimately, CLL cell killing (Online Supplementary Figure S2). These findings are in agreement with recent studies demonstrating the enhancing effect of CD40 stimulation on cytotoxicity mediated by rituximab 46 and GA101 47 antibodies. Five of the samples analyzed in this study were derived from patients with relapsed or refractory CLL with one patient also exhibiting a p53 deletion.…”

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confidence: 83%

Blinatumomab induces autologous T-cell killing of chronic lymphocytic leukemia cells

et al. 2013

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© F e r r a t a S t o r t i F o u n d a t i o n 2 0 1 3of CLL activation and pro-survival signals. Given these data, this approach represents a novel and promising therapeutic strategy for the treatment of CLL and, potentially, other lymphoid malignancies. Methods Blood samples from patients with chronic lymphocytic leukemiaTwenty-eight CLL patients (aged 53-83 years) were studied with full ethical approval. All blood samples from patients were obtained with informed consent (see Online Supplementary Methods). The majority of the assays were performed on fresh samples from treatment-naïve patients, except those for which the results are shown in Figure 1: in these assays, samples from patients who had received standard chemotherapy treatment were also tested. Antibodies and flow cytometryA full list of the antibodies used can be found in the Online Supplementary Methods. Blinatumomab (MT103, AMG103) was provided by Amgen Inc. (Munich, Germany). T cells were identified using CD4 and CD8 markers, while CLL cells were CD5 + CD4-CD8 -(>99% CD19 + ). Cell culturesPeripheral blood mononuclear cells (PBMC) were incubated in RPMI 1640 supplemented with 5% AB serum (AB media) for 3-7 days. Blinatumomab was added to PBMC cultures at a concentration of 10 or 100 ng/mL. Human T-cell activator CD3/CD28 dynabeads (Invitrogen) were added at 1x10 5 beads/1x10 6 PBMC as a positive control for T-cell activation. For absolute counts of T cells and CLL cells, an anti-CD3 antibody (Invitrogen) at a dose of 27.7 or 277 ng/mL was used as a positive control (same molar concentration as 10 or 100 ng/mL blinatumomab). Intracellular Ki67 stainingPBMC were labeled with antibodies against CD4, CD8, CCR7 and CD45RA, fixed and permeabilized (Fix and Perm with 1% NP40, Caltag-Medsystems, Buckingham, UK) before staining with a Ki67-FITC antibody for flow cytometric analysis. Cytokine secretion assayCytokines in tissue culture supernatants were measured using a Human Th1/Th2 11plex RTU Flowcytomix kit (eBioscience) for simultaneous detection of 11 cytokines. Intracellular cytokine staining and the determination of CD107 and granzyme B expressionPBMC were cultured (3 days) with 10 ng/mL blinatumomab, before treatment with Golgi plug and Golgi stop (BD biosciences), and incubation with an anti-CD107 antibody (5 h at 37°C). Intracellular expression of granzyme B or interferon (IFN)-g and tumor necrosis factor (TNF)-α in CD4 or CD8 T cells was measured by flow cytometry. Absolute counts of T cells and chronic lymphocytic leukemia cellsPBMC samples that had or had not been treated with blinatumomab (7 days) were surface-stained with antibodies against annexin V, CD5, CD8 and CD4. Absolute counts were determined by flow cytometry using Cytocount beads (Dako, Stockport, UK). Flow cytometry-based cytotoxicity assayMouse fibroblast cells transfected with CD40L [TL(CD40L)] and non-transfected cells (NTL) were cultured as previously described. 21 For co-cultures, irradiated (80 Gy) NTL or TL(CD40L) cells were seeded into a 48-well plate, and allow...

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Blinatumomab induces autologous T-cell killing of chronic lymphocytic leukemia cells

et al. 2013

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“…23 Of note, whereas the ROS scavenger NAC significantly inhibited RXL-induced cell death ( Figure 5E), no inhibition was observed in GA101-induced cell death in the presence of NAC (Figure 5E), making a role for ROS in GA101-induced cell death less likely. An important question is whether lysosomes are involved in RXLinduced cell death of CD40 stimulated CLL cells.…”

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confidence: 98%

“…21,22 Rather unexpectedly, we recently found that, in contrast to its induction of chemoresistance, CD40 stimulation enhanced sensitivity of CLL cells to rituximab via a reactive oxygen species (ROS)-dependent mechanism. 23 Because of the largely different mechanism of action of type I compared with type II anti-CD20 mAbs, we investigated in the present study whether CD40 stimulation also affects the sensitivity of CLL cells to GA101 and, if so, by what mechanism. …”

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“…3,44 We and others have shown that Bcl-2 members and classic apoptosis involving activated caspase 3 do not play a role in anti-CD20-induced cell death. 21,23 To translate the observation that CLL cells cultured on CD40L-expressing fibroblasts were more sensitive to anti-CD20 mAbs to a more physiologic setting, we also tested CLL cells cocultured with autologous CD3/CD28 activated T cells. The fraction of T cells in this setup is far less than that of the CLL cells, and the CD40 stimulation is likely to be less potent than in the coculture system, but enhanced sensitivity to anti-CD20 mAbs was still detectable.…”

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confidence: 99%

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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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“…[27][28][29][30][31] However, this cross-linking can hardly be realized in vivo. It seems, although we acknowledge that the activation of more effector mechanisms may lead to improved curative effects, 7,22,25,32,33 the abovementioned immunotherapeutic mechanisms could hardly be synchronously activated by any reported anti-CD20 mAb or mAb derivative until now.…”

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Construction and characterization of an anti-CD20 mAb nanocomb with exceptionally excellent lymphoma-suppressing activity

Li¹,

Wu²,

Chen³

et al. 2015

IJN

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The CD20-directed monoclonal antibody rituximab (RTX) established a new era in the treatment of non-Hodgkin lymphoma (NHL); however, suboptimal response and/or resistance to RTX still limit its clinical merits. Although four effector mechanisms are validated to participate in CD20-based immunotherapy, including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, caspase-dependent apoptosis, and lysosome-mediated programmed cell death (PCD), they could hardly be synchronously activated by any anti-CD20 mAb or mAb derivative until now. Herein, a novel mAb nanocomb (polyethylenimine polymer–RTX–tositumomab [PPRT nanocomb]) was firstly constructed through mass arming two different anti-CD20 mAbs (RTX and tositumomab) to one polymer by nanotechnology. Comparing with free mAbs, PPRT nanocomb possesses a comparable binding ability and reduced “off-rate” to surface CD20 of NHL cells. When treated by PPRT nanocomb, the caspase-dependent apoptosis was remarkably enhanced except for concurrently eliciting complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and lysosome-mediated PCD. Besides, “cross-cell link”-assisted homotypic adhesion by PPRT nanocomb further enhanced the susceptibility to PCD of lymphoma cells. Pharmacokinetic assays revealed that PPRT nanocomb experienced a relatively reduced clearance from peripheral blood compared with free antibodies. With the cooperation of all the abovementioned superiorities, PPRT nanocomb exhibits exceptionally excellent in vivo antitumor activities in both disseminated and localized human NHL xenotransplant models.

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CD40 stimulation sensitizes CLL cells to rituximab-induced cell death

Cited by 31 publications

References 57 publications

Blinatumomab induces autologous T-cell killing of chronic lymphocytic leukemia cells

Blinatumomab induces autologous T-cell killing of chronic lymphocytic leukemia cells

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

Construction and characterization of an anti-CD20 mAb nanocomb with exceptionally excellent lymphoma-suppressing activity

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