Antigen-associated determinants of response to CD20xCD3 bispecific antibodies in diffuse large B cell lymphoma

CD20xCD3 bispecific antibodies (BsAbs), which engage CD20 on B cells and CD3 on T cells, have shown unprecedented activity in relapsed/refractory diffuse large B cell lymphoma (DLBCL). However, molecular determinants of BsAb response – including construct-specific and tumour-intrinsic factors – remain poorly understood. Importantly, CD20 epitopes vary between BsAb constructs and glofitamab retains bivalent CD20 binding. Furthermore, CD20 expression levels poorly predict BsAb response and mutations represent an uncommon mechanism of resistance. Using a flow cytometry-based cytotoxicity assay, I compared the in vitro activity of glofitamab, epcoritamab, odronextamab, and mosunetuzumab biosimilars against DLBCL cell lines with varying CD20 expression. Cells were treated for 24 hours with healthy volunteer peripheral blood mononuclear cells as effectors. All BsAbs demonstrated dose-dependent killing of sensitive cell lines with concurrent T cell activation and degranulation. Glofitamab showed the highest maximal efficacy and potency, likely due to bivalent CD20 binding. BsAb activity occurred at CD20 levels as low as 2,000 molecules per cell. Two cell lines exhibited resistance despite substantial CD20 expression and high T cell activation, indicating tumour-intrinsic resistance mechanisms. Target antigen-associated determinants of BsAb response – i.e. relating to CD20 and its interactome – were investigated using co-immunoprecipitation followed by mass spectrometry in a panel of B cell lymphoma models. The relative abundance of co-precipitated proteins was highly heterogeneous and implicated CD20 in B cell signalling, lipid raft clustering, immune synapse formation and metabolism. A preliminary analysis also identified CD20-associated proteins that may contribute to BsAb resistance – such as NEDD4L and CD44 – or to sensitivity, including MHC II, CD38, CD48, CD74, CD147, MCT-1, and GLUT1. Finally, a cell surface proteomics workflow was optimised and applied to one representative cell line.Collectively, this work highlights the importance of BsAb construct selection in DLBCL and proposes that CD20-associated proteins modulate therapeutic response, providing a foundation for predictive biomarker development.