Obinutuzumab is a therapeutic antibody for B cell non-Hodgkin’s Lymphoma (BNHL), which is a glyco-engineered anti-CD20 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) and causes binding-induced direct cell death (DCD) through lysosome membrane permeabilization (LMP). Tumour necrosis factor receptor 1 (TNFR1), a pro-inflammatory death receptor, also evokes cell death, partly through lysosomal rupture. As both obinutuzumab- and TNFR1-induced cell deaths are mediated by LMP and combining TNFR1 and obinutuzumab can amplify LMP-mediated cell death, we made dual-targeting antibody for CD20 and TNFR1 to enhance DCD of obinutuzumab. Obinutuzumab treatment induced CD20 and TNFR1 colocalisation, and TNFR1-overexpressing cells showed increased obinutuzumab-induced DCD. Two targeting modes, anti-CD20/TNFR1 bispecific antibodies (bsAbs), and obinutuzumab-TNFα fusion proteins (OBI-TNFαWT and OBI-TNFαMUT) were designed to cluster CD20 and TNFR1 on the plasma membrane. OBI-TNFαWT and OBI-TNFαMUT showed significantly enhanced LMP, DCD, and ADCC compared with that induced by obinutuzumab. TNFR1 expression is upregulated in many BNHL subtypes compared to that in normal B cells; OBI-TNFαMUT specifically increased DCD and ADCC in a B-cell lymphoma cell line overexpressing TNFR1. Further, OBI-TNFαMUT blocked NF-κB activation in the presence of TNF-α, implying that it can antagonise the proliferative role of TNF-α in cancers. Our study suggests that dual-targeting of CD20 and TNFR1 can be a new therapeutic strategy for improving BNHL treatment. The OBI-TNFαMUT fusion protein enhances DCD and ADCC and prevents the proliferating effect of TNFα signalling; therefore, it may provide precision treatment for patients with BNHL, especially those with upregulated TNFR1 expression.

Obinutuzumab is a therapeutic antibody for B cell non-Hodgkin's Lymphoma (BNHL), which is a glycoengineered anti-CD20 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) and causes binding-induced direct cell death (DCD) through lysosome membrane permeabilization (LMP).Tumour necrosis factor receptor 1 (TNFR1), a pro-in ammatory death receptor, also evokes cell death, partly through lysosomal rupture. As both obinutuzumab-and TNFR1-induced cell deaths are mediated by LMP and combining TNFR1 and obinutuzumab can amplify LMP-mediated cell death, we made dualtargeting antibody for CD20 and TNFR1 to enhance DCD of obinutuzumab.Obinutuzumab treatment induced CD20 and TNFR1 colocalisation, and TNFR1-overexpressing cells showed increased obinutuzumab-induced DCD. Two targeting modes, anti-CD20/TNFR1 bispeci c antibodies (bsAbs), and obinutuzumab-TNFα fusion proteins (OBI-TNFα WT and OBI-TNFα MUT ) were designed to cluster CD20 and TNFR1 on the plasma membrane. OBI-TNFα WT and OBI-TNFα MUT showed signi cantly enhanced LMP, DCD, and ADCC compared with that induced by obinutuzumab. TNFR1 expression is upregulated in many BNHL subtypes compared to that in normal B cells; OBI-TNFα MUT speci cally increased DCD and ADCC in a B-cell lymphoma cell line overexpressing TNFR1. Further, OBI-TNFα MUT blocked NF-κB activation in the presence of TNF-α, implying that it can antagonise the proliferative role of TNF-α in cancers.Our study suggests that dual-targeting of CD20 and TNFR1 can be a new therapeutic strategy for improving BNHL treatment. The OBI-TNFα MUT fusion protein enhances DCD and ADCC and prevents the proliferating effect of TNFα signalling; therefore, it may provide precision treatment for patients with BNHL, especially those with upregulated TNFR1 expression.TNF-α, which successfully inhibits TNFα-mediated activation of TNFR1 as well as inhibited hepatic injury in an experimental hepatitis model ( 35). Based on these results, dual-targeting of TNFR1 and CD20 can be bene cial for BNHL treatment because of the additive effects of LMP induced by simultaneous binding to both antigens. Furthermore, dual-targeting proteins have increased selectivity for cancerous cells with high TNFR1 expression compared to that in normal B cells, thus reducing cross-reactivity.In this study, we generated two types of novel fusion proteins: obinutuzumab-anti-TNFR1 bispeci c antibody and obinutuzumab-cytokine proteins, wild-type TNFα or mutant TNFα, attached to the Fc region of obinutuzumab (OBI-TNFα WT or -TNFα MUT ) and examined their effect on DCD and ADCC in BNHL cells compared to those induced by obinutuzumab alone. Methods Cells, reagents and solutionsRamos, CHO-K1, HEK 293, HEK293T and HeLa cell line were purchased from the Korean Cell Line Bank.Raji cell line was gifted from Prof. Seong Hwan Kim (Chungnam National University, Daejeon). CHO-K1, Ramos and Raji Cells were maintained in RPMI1640 media supplemented with 10% FCS and 1% penicillin/streptomycin at 37°C, 5% CO2. HEK293T and HeLa cells were maintained in DMEM ...