Cellular Cytotoxicity of Next-Generation CD20 Monoclonal Antibodies

Abstract

CD20 monoclonal antibodies (CD20 mAb) induce cellular cytotoxicity, which is traditionally measured by antibody-dependent cellular cytotoxicity (ADCC) assays. However, data suggest that antibody-dependent cellular phagocytosis (ADCP) is the primary cytotoxic mechanism. We directly compared ADCP versus ADCC using primary human cells. After establishing the primacy of ADCP, we examined next-generation CD20 mAbs, including clinically relevant drug combinations for their effects on ADCP. ADCP and ADCC induction by rituximab, ofatumumab, obinutuzumab, or ocaratuzumab was measured using treatment-naïve chronic lymphocytic leukemia (CLL) target cells and either human monocyte-derived macrophages (for ADCP) or natural killer (NK) cells (for ADCC). Specific effects on ADCP were evaluated for clinically relevant drug combinations using BTK inhibitors (ibrutinib and acalabrutinib), PI3Kδ inhibitors (idelalisib, ACP-319, and umbralisib), and the BCL2 inhibitor venetoclax. ADCP (∼0.5-3 targets/macrophage) was>10-fold more cytotoxic than ADCC (∼0.04-0.1 targets/NK cell). ADCC did not correlate with ADCP. Next-generation mAbs ocaratuzumab and ofatumumab induced ADCP at 10-fold lower concentrations than rituximab. Ofatumumab, selected for enhanced complement activation, significantly increased ADCP in the presence of complement. CD20 mAb-induced ADCP was not inhibited by venetoclax and was less inhibited by acalabrutinib versus ibrutinib and umbralisib versus idelalisib. Overall, ADCP was a better measure of clinically relevant mAb-induced cellular cytotoxicity, and next-generation mAbs could activate ADCP at significantly lower concentrations, suggesting the need to test a wide range of dose sizes and intervals to establish optimal therapeutic regimens. Complement activation by mAbs can contribute to ADCP, and venetoclax, acalabrutinib, and umbralisib are preferred candidates for multidrug therapeutic regimens. .

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

“…Although the combination of a CD20 antibody with ibrutinib does not improve outcomes over ibrutinib monotherapy in randomized studies ( 10 ), this question has not been addressed with BTK inhibitors optimized for selectivity or CD20 antibodies engineered to optimize ADCC. 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.…”

“…In addition, the greater selectivity of acalabrutinib leads to less interference with antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC), as IL2-associated tyrosine kinase (ITK) is not inhibited. This may allow for more effective combination with anti-CD20 antibodies (17,20).…”

Reprogramming the Constant Region of Immunoglobulin G Subclasses for Enhanced Therapeutic Potency against Cancer

“…Their results showed a 24% risk reduction in patients on margetuximab compared to trastuzumab in 536 patients with breast cancer [46]. Another Fc-engineered anti-CD20 antibody, ocaratuzumab, which exhibits 6-fold increased ADCC activity relative to rituximab, is being developed by Mentrik Biotech and is under phase 3 clinical trial for patients with relapsed follicular lymphoma and other oncology indications [47].…”

“…There have been beneficial clinical outcomes of Fc-engineered antibodies with enhanced affinity to FcγRIIIa relative to native IgG1 Fc counterparts, including margetuximab [46], ocaratuzumab [47], obinutuzumab [65], and mogamulizumab [66]. However, it is not clear which leukocytes or FcγRs are responsible for the potentiated therapeutic results because various immune cells exhibit discrete FcγR expression profiles [102].…”

Antibody:CD47 ratio regulates macrophage phagocytosis through competitive receptor phosphorylation

“…Macrophages play a critical role in cancer immunotherapy by recognizing and destroying antibody-opsonized cells. The clinical success of therapeutic antibodies, including anti-CD20 clearance of chronic lymphocytic leukemia cells (Chu et al, 2018;VanDerMeid et al, 2018) and anti-CD38 clearance of multiple myeloma (van de Donk & Usmani, 2018), has been attributed in part to macrophage destruction of tumor cells via antibody-dependent cellular phagocytosis (ADCP). However, potent inhibitory molecules found on tumor cells, such as the ubiquitous "marker of self" CD47, can dampen the pro-phagocytic effect of therapeutic antibodies (Okazawa et al, 2005;Oldenborg et al, 2000).…”