Sensitive Detection of the Natural Killer Cell-Mediated Cytotoxicity of Anti-CD20 Antibodies and Its Impairment by B-Cell Receptor Pathway Inhibitors

Abstract

The antibody-dependent cell-mediated cytotoxicity (ADCC) of the anti-CD20 monoclonal antibodies (mAbs) rituximab and obinutuzumab against the cell line Raji and isolated CLL cells and its potential impairment by kinase inhibitors (KI) was determined via lactate dehydrogenase release or calcein retention, respectively, using genetically modified NK92 cells expressing CD16-176V as effector cells. Compared to peripheral blood mononuclear cells, recombinant effector cell lines showed substantial alloreactivity-related cytotoxicity without addition of mAbs but afforded determination of ADCC with reduced interassay variability. The cytotoxicity owing to alloreactivity was less susceptible to interference by KI than the ADCC of anti-CD20 mAbs, which was markedly diminished by ibrutinib, but not by idelalisib. Compared to rituximab, the ADCC of obinutuzumab against primary CLL cells showed approximately 30% higher efficacy and less interference with KI. Irreversible BTK inhibitors at a clinically relevant concentration of 1 μM only weakly impaired the ADCC of anti-CD20 mAbs, with less influence in combinations with obinutuzumab than with rituximab and by acalabrutinib than by ibrutinib or tirabrutinib. In summary, NK cell line-based assays permitted the sensitive detection of ADCC of therapeutic anti-CD20 mAbs against CLL cells and of the interference of KI with this important killing mechanism.

Acalabrutinib plus Obinutuzumab in Treatment-Naïve and Relapsed/Refractory Chronic Lymphocytic Leukemia

“…Ibrutinib, through inhibition of ITK and other non-BTK kinases, abrogates NK-cell ADCC and ADCP in vitro (20)(21)(22) and may reduce the effectiveness of the CD20 antibody in patients. Acalabrutinib is highly selective for BTK and is not a potent inhibitor of most other kinases ( 15,23 ), suggesting that this BTK inhibitor might be more appropriate for combination therapy with antibodies. Similarly, several groups have demonstrated that the engineered CD20 antibody obinutuzumab mediates improved ADCC over rituximab ( 24,25 ).…”

Macrophage-mediated antibody dependent effector function in aggressive B-cell lymphoma treatment is enhanced by Ibrutinib via inhibition of JAK2

“…While monoclonal antibodies exert various mechanisms of targeting malignant cells in B-cell lymphoma, macrophage ADCP has been identified as a pre-dominant mechanism (18,28). In this light, previous reports examining ibrutinib in combination with rituximab that did not reveal synergy were mostly focused on NK-cell ADCC or relied on monocyte derived macrophages (20)(21)(22)(23)35). Exploring effector cell mechanisms is technically challenging since co-culture systems demand high levels of standardization.…”

“…Pre-clinical studies assessing the synergistic interaction between ibrutinib and monoclonal antibodies have provided conflicting results, with a number of studies reporting that ibrutinib not only has no impact on antibody-mediated effects, rather a negative effect specifically on NK-cell mediated effects of the antibody (20)(21)(22)(23).…”

Enhancing antibody-dependent cell-mediated cytotoxicity: a strategy for improving antibody-based immunotherapy

“…There are several newer techniques that rely on similar principles as the 51 Cr release assay but replace radioisotopes with natural cell products. As cells are killed they release lactate dehydrogenase and other proteases that can be quantified by supplying fluorogenic substrates in order to more accurately assess cytotoxicity without the need to perform any labeling or manipulation of target cells [38,39]. A potential drawback of relying on reporter molecules is that they may spontaneously release even from live cells.…”