Preclinical study of CD19 detection methods using monoclonal antibodies post tafasitamab treatment

Abstract Introduction Tafasitamab (TAFA), an Fc-enhanced antibody immunotherapy against CD19 received accelerated/conditional approval in relapsed or refractory diffuse large B cell lymphoma (R/R DLBCL) in combination with lenalidomide. Several other CD19 targeting therapies including CAR Ts are now available for R/R DLBCL treatment warranting evaluation of potential therapeutic sequencing strategies. In this context, CD19 expression status may be assessed on patient samples post TAFA treatment. However, methods for detecting CD19 using monoclonal antibodies (mAbs) following TAFA treatment are not standardized which may lead to data misinterpretations including confusion of CD19 epitope masking with CD19 loss or downregulation. Aim This study evaluated various CD19 detection methods following TAFA treatment using comprehensive panels of available flow cytometry (FC) and immunohistochemistry (IHC) anti-CD19 mAbs, routinely used in laboratory practice. Methods FC and IHC competition assays were used on TAFA pre-treated CD19+ cell lines to differentiate non-competing from competing mAbs, the latter resulting in epitope masking by TAFA. Bound TAFA was removed from CD19+ cells using an acidic dissociation protocol. Antibody affinities for CD19 were determined using Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI). Results All but one of the 8 mAbs tested in FC competed with TAFA and their affinities for CD19 were shown to be lower than that of TAFA. The non-competing clone OTI3B10, reported to recognize a linear epitope of CD19 (1), bound to only one of three tested CD19+ cell lines, which rendered it unsuitable for reliable FC detection of CD19. In contrast, several clones could detect CD19 on TAFA pre-treated samples using IHC. To circumvent TAFA masking in FC, we successfully unmasked CD19 via acidic dissociation and similar CD19 levels on TAFA pre-treated as on untreated cells could be subsequently detected by a competing CD19 antibody (HIB19). Conclusion Our study provides an overview of commonly used mAb tools for CD19 detection post TAFA treatment, some of which may be used in routine clinical practice. Our findings reveal that CD19 expression post TAFA could be detected by IHC but lack of TAFA non-competing anti-CD19 antibodies for FC poses a risk of mistaking CD19 masking by TAFA with CD19 loss or downregulation. Prior acidic dissociation is critical in the staining protocol to avoid data misinterpretations. 1. Klesmith et al, 2019, Biochem. Citation Format: Kristina M. Ilieva, Markus Eberl, Jan Jaehrling, Carmen Ginzel, Britta Voland, Michaela Zankl, Andrea Polzer, Derek Blair, Rainer Boxhammer, Liang-Chuan Wang, Diana Alvarez Arias, Christina Heitmüller. Preclinical study of CD19 detection methods using monoclonal antibodies post tafasitamab treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6329.