3913 Anti-CD20 monoclonal antibodies (mAb) eliminate CD20-positive cells mainly through three different molecular mechanisms including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and apoptosis. They are indicated as monotherapy or in combination with other compounds to treat B-cell malignancies such as Chronic Lymphocytic Leukemia (CLL). In contrast to normal B-lymphocytes, B malignant cells in CLL patients usually express low levels of CD20 molecules at their cell surface. Rituximab (Rituxan®/MabThera®), the first-in-class anti-CD20 mAb, has been described to be poorly active in CLL patients as a single agent. Ofatumumab (Arzerra®), the second generation approved anti-CD20 mAb, displays a higher CDC-activity when compared to rituximab. However, the contribution of this mechanism of action to control this disease is not fully characterized and may not be sustained when low CD20-density malignant cells are targeted. We have developed a third generation anti-CD20 mAb (LFB-R603) on our EMABling® platform, known to select antibodies with improved ADCC, based on its glycosylation pattern. We have previously demonstrated that LFB-R603 is able to mediate a more potent ADCC against several cell lines as compared to rituximab. In this study, we further investigated lysis activity of LFB-R603 as compared to ofatumumab and rituximab. In terms of CDC-mediated lysis, we observed a higher CDC activity of ofatumumab compared to LFB-R603 on several cell lines (Raji, MEC-1, Wil-2S) expressing high CD20 levels. Using a FACS-based methodology and MEC-1 cells as targets, C1q-dependent-CDC triggered by ofatumumab was shown to induce approximately 90% of specific cell-lysis as compared to 60% cell-lysis triggered by LFB-R603. Rituximab displayed a CDC activity slightly superior to that of LFB-R603. In contrast, CDC activity against CD20-low expressing cells, illustrated by SUDHL-8, was very low and almost identical with all three mAbs, rituximab, ofatumumab and LFB-R603. Indeed, ofatumumab was only able to mediate 5% CDC lysis, comparable to the 1% observed with LFB-R603. Regarding the ADCC efficacy, and consistently to previous results, LFB-R603 was shown to mediate ADCC at a very high rate against all cell lines tested. Around 55% lysis of LFB-R603-opsonized MEC-1 was obtained using PBMC as effectors from 5 independent healthy donors, while less than 30% lysis of rituximab- or ofatumumab-opsonized MEC-1 was observed using the same PBMC samples. In addition, LFB-R603 still mediated a potent ADCC against CD20-low expressing cells (SUDHL-8 and JYE5.1), whereas both ofatumumab and rituximab mediated ADCC at low levels. Indeed, only 5% of rituximab-opsonized SUDHL-8 cells were killed by PBMC from healthy donor, whereas around 30% of the SUDHL-8 opsonized by LFB-R603 were lysed by the same effectors. Altogether, these results support the fact that the therapeutic use of LFB-R603 may be advantageous over currently approved anti-CD20 mAbs to target malignant cells where surface CD20 molecules are known to be expressed at low levels such as CLL and potentially other lymphoma subtypes. Disclosures: Bellon: LFB Biotechnologies: Employment. Sadoun:LFB Biotechnologies: Employment. Grivel:LFB Biotechnologies: Consultancy. Moulard:LFB Biotechnologies: Consultancy. Brune:LFB Biotechnologies: Employment. Prost:LFB Biotechnologies: Employment. Salcedo:LFB Biotechnologies: Employment.
4728 Introduction. Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates survival, proliferation, and differentiation of monocytes/macrophages. The GM-CSF receptor (CD116) is expressed on primary monocytes and mediates cell signaling through STAT5 phosphorylation. Therefore STAT5 is central to GM-CSF action. BioCytex has developed a new semi-quantitative flow cytometry (FC) assay to measure the phosphorylation status of STAT5 in human primary monocytes and lymphocytes in whole blood samples. The assay was used to investigate the potency of recombinant monoclonal antibodies (MAbs) for the neutralization of human GM-CSF. Methods. Engineered anti-GM-CSF MAbs were generated and characterized by Merck Serono. Several clones and preparation were tested, including MAbs 343 (low and high LPS), 467; 340; 339; 366, and MAb 285 as isotype control. Their different in vitro potencies for blocking recombinant GM-CSF activity in stable cell lines was determined using the TF-1 proliferation and IL-8 secretion in U937 cell assays. Whole blood FC assay was designed and validated at BioCytex using either a rabbit MAb or a rabbit polyclonal antibody specific for phospho-STAT5 (Tyr694). Monocytes were gated using a PE-labeled anti-CD14 probe. Inhibition of STAT5 phosphorylation by anti-GM-CSF MAbs was performed using recombinant GM-CSF at EC90. Results. EC90 of GM-CSF was determined at 2.3 pM from n=5 healthy volunteers. All GM-CSF specific MAbs inhibited STAT5 phosphorylation in monocytes, whereas the control (MAb 285) did not. More interestingly, there was a marked effect of LPS content since inhibition of STAT5 phosphorylation was not completed in presence of high LPS load (MAb 343). IC50 and IC90 for MAbs 339 and 366 were 10-fold increased as compared to MAbs 467 and 340. IC50 were ranging from 0.12 to 0.19 nM for clones 343 (low LPS), 467 and 340. IC50 and IC90 for clone 339 were 0.016 and 0.034 nM, respectively. Conclusion. Inhibition of GM-CSF mediated monocyte activation by neutralizing MAbs can be monitored in a whole blood assay. STAT5 phophorylation could be viewed as a potential biomarker for GM-CSF receptor activation. The cell-signaling inhibition potency in primary cells correlated with in vitro potency using stable cell lines. In summary, the new validated FC could be used for research and clinical development purposes. Disclosures: Grivel: LFB Biotechnologies: Consultancy. Magnenat:Merck Serono S.A.: Employment. Moulard:BioCytex: Consultancy, Employment.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.