Comparison of Cell Lysis Mediated by LFB-R603 with That Mediated by Ofatumumab Against Cells Expressing Low Levels of CD20,

Abstract

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.

A phase 1/2 trial of ublituximab, a novel anti‐CD20 monoclonal antibody, in patients with B‐cell non‐Hodgkin lymphoma or chronic lymphocytic leukaemia previously exposed to rituximab

“…Similarly, rituximab monotherapy has shown modest activity in CLL (Huhn et al , ; Furman et al , ), which has been attributed to low CD20 antigen expression by CLL cells (Prevodnik et al , ). The collective experience with ublituximab supports improved ADCC and improved activity in low CD20‐expressing malignancies (De Romeuf et al , ; Bellon et al , ).…”

“…The low fucose content in ublituximab's Fc region results in increased affinity for the FcγRIIIa (or CD16) (Le Garff‐Tavernier et al , , ). This increased affinity is associated with potent in vitro ADCC against B cells compared with both rituximab and ofatumumab, particularly against tumour cells that express low levels of CD20, such as CLL (De Romeuf et al , ; Bellon et al , ; Le Garff‐Tavernier et al , ). Ublituximab differs from obinutuzumab in at least 3 distinct ways: (i) ublituximab is an IgG1 class MAb, which exhibits improved ADCC, in contrast to obinutuzumab, which is an IgG2 class MAb; (ii) ublituximab and obinutuzumab bind to distinctly different epitopes on the CD20 domain (Figure S1); and (iii) obinutuzumab is humanised, while ublituximab is chimeric.…”

Ublituximab: A Novel Anti-CD20 Therapy for Multiple Sclerosis

“…While current anti-CD20 monoclonal antibodies are among the most effective DMTs, they differ structurally (Figure 1), leading to variations in the relative contributions of each of these mechanisms to B-cell depletion, epitopes on CD20, immunogenicity and administration profiles (Table 1). 7,8,9 Rituximab, ocrelizumab, ofatumumab and ublituximab are all type I antibodies, which localize CD20…”