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CD20/TNFR1 dual-targeting antibody enhances lysosome rupture-mediated cell death in B cell lymphoma

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Abstract

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.

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Abbreviations

ADCC:

Antibody-dependent cellular cytotoxicity

BNHL:

B cell non-Hodgkin lymphoma

bsAb:

Bispecific antibody

CLL:

Chronic lymphocytic leukaemia

DCD:

Direct cell death

DLBCL:

Diffuse large B cell lymphoma

EGFR:

Epidermal growth factor receptor

EV:

Empty vector

FL:

Follicular lymphoma

LMP:

Lysosomal membrane permeabilization

MMML:

Molecular mechanism in malignant lymphoma

NHL:

Non-Hodgkin lymphoma

OBI-TNFαWT :

Obinutuzumab-tumour necrosis factor alpha wild-type fusion protein

OBI-TNFαMUT :

Obinutuzumab-tumour necrosis factor alpha mutant fusion protein

TNFR1:

Tumour necrosis factor receptor 1

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Acknowledgements

The authors are very grateful to Dr. Henry Löffler-Wirth and Dr. Hans of the Interdisciplinary Center for Bioinformatics at the University of Leipzig for making the whole expression data set of mature B cell lymphomas available to the public in the repository ‘The Leipzig Health Atlas’ (https://www.health-atlas.de/index.php/en/lha/7VT47TM4GV-1). We would also thank Professor Joon Yong Ahn of the Department of Biosystem and Biomedical Science, Korea University, for his advise on big data analysis.

Funding

This work was supported by grants from the National Research Foundation of Korea, Project Nos. NRF-2021R1I1A1A01059867 to D.H.L and Project Nos. NRF-2019R1A2C1086348 to J.Y.K.

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JRK performed and analyzed most of the experiments. JYK organized and supervised the whole project. YK helped and reproduced Lysotracker assays, DL produced obinutuzumab-TNFα fusion protein and provided technical advice. JRK and JYK wrote the manuscript and YK and DL edited the English.

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Correspondence to Joo Young Kim.

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Patient-derived PBMC samples were isolated with approval of Yonsei University Institutional Review Committee after obtaining informed consent, under IRP procedure (#4-2016-0600).

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Kim, J.R., Lee, D., Kim, Y. et al. CD20/TNFR1 dual-targeting antibody enhances lysosome rupture-mediated cell death in B cell lymphoma. Cancer Immunol Immunother 72, 1567–1580 (2023). https://doi.org/10.1007/s00262-022-03344-9

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