Comparative in-Vitro Efficacy of CD20xCD3 IgG Bispecific Biosimilar Constructs Against Diffuse Large B Cell Lymphoma (DLBCL) Cell Lines with Different Levels of Expression of CD20
Authors: Bray Joshua S. , Thomas Gethin R. , Smith Victoria M. , Jayne Sandrine , Dyer Martin J.S. , Walter Harriet S. 
Published: December 2024

Introduction
Multiple IgG anti-CD20 bispecific antibodies (BsAbs), that bind CD20 on B cells and CD3 on T cells show unprecedented activity in relapsed/refractory (R/R) B cell malignancies, including CAR T-cell failures. Multiple studies are ongoing to assess activity in combination and in earlier treatment settings. With several BsAbs now undergoing clinical evaluation with apparently similar toxicity profiles but different routes of administration and treatment duration, selection of construct may be key to optimize outcomes. Importantly, whilst CD3 binding in all the various constructs is similar, the CD20 epitopes vary, and moreover, glofitamab, retains bivalent and Type 2 CD20 binding. Using biosimilar versions of glofitamab, epcoritamab, odronextamab and mosunetuzumab we compared head-to-head the in vitro activity of these constructs against a panel of DLBCL cell lines expressing variable levels of cell surface CD20.
Methods
BsAb activity was assessed using a flow cytometry-based cytotoxicity assay. DLBCL not otherwise specified cell lines with varying levels of CD20 expression; SU-DHL-10 (588400 mol/cell), UoL-RAD (148600 mol/cell), Karpas-1718 (52820 mol/cell) and UoL-AME (2300 mol/cell) were treated with 0.01-10,000pM BsAb biosimilar (Proteogenix, https://www.proteogenix.science/therapeutic-antibody/) for 24 hours, using healthy volunteer peripheral blood mononuclear cells as effector cells. B cell depletion (BCD) was calculated by comparing live target cell counts with a no drug control. Effector T and natural killer (NK) cell activity was determined by the expression of CD69 (early activation), CD25 (late activation) and CD107a (degranulation). Comparative data with biosimilar anti-CD20 monospecific antibodies, rituximab and obinituzumab, were obtained. Absolute EC50 values were estimated using a four-parameter log-logistic regression model. BsAb binding to DLBCL cells was confirmed by flow cytometry.
Results
All CD20xCD3 biosimilar constructs demonstrated dose-dependent killing against UoL-RAD and SU-DHL-10, with simultaneous activation and degranulation of T cells, but not NK cells. Glofitamab biosimilar demonstrated the highest activity against UoL-RAD and SU-DHL-10 (EC50 2pM and 59pM respectively). Comparative EC50 values for epcoritamab were 71pM and 537pM, for odronextamab 65pM and 129pM and mosunetuzumab 156pM and &gt;10,000pM respectively. Despite low expression of CD20, UoL-AME was sensitive to glofitamab and odronextamab (EC50 802pM and 3248pM), but resistant to epcoritamab and mosunetuzumab (EC50 &gt;10,000pM). Karpas-1718 was resistant to all biosimilar BsAbs tested (EC50 &gt;10,000pM) despite BsAb binding and high levels of T cell activation and degranulation, suggesting an intrinsic mechanism of resistance. UoL-RAD and SU-DHL-10 were sensitive to obinituzumab (EC50 95pM and 99pM respectively), and UoL-RAD alone sensitive to rituximab (EC50 1437pM). Rituximab and obinutuzumab did not engage T cells, but induced activation and degranulation of NK cells.
Conclusions
These data indicate that CD20 monospecific and BsAbs may have variable effects on CD20+ B cell malignancies, independent of levels of cell surface expression of CD20. Glofitamab biosimilar exhibited superior efficacy against DLBCL cell lines compared to other CD20xCD3 biosimilar BsAbs, which may be due its bivalent CD20 binding. Interestingly, significant responses to glofitamab and odronextamab were observed in vitro for UoL-AME, despite low CD20 expression. These data may have important clinical sequencing considerations as CD19 and CD20 antigen loss is reported as a result of selective pressure after CD19 and CD20 targeted T cell redirecting therapy. The biological basis of inherent resistance to all CD20xCD3 BsAbs in the Karpas-1718 cell line requires further investigation.