Abstract 2182: Optimizing immunolabeling strategies in preparation for magnetic activated cell sorting for chimeric antigen receptor-natural killer and multiple myeloma cells

Chimeric antigen receptor (CAR) natural killer (NK) cells are of increased interest compared to CAR-T cell therapy because CAR-NK cells can circumvent the challenges of graft-versus-host disease, serve as an off-the-shelf therapy, and avoid the neurotoxic side effects and cytokine release syndrome that accompany CAR-T cell immunotherapy. However, CARs are currently optimized for use in T cells and are not optimized for NK cell mechanisms. There are currently no comprehensive studies that characterize CAR-NK cellular pathways for CAR-NK optimization. In preparation for a comprehensive CAR-NK phospho-proteomic pathway analysis, we evaluated strategies for magnetic-activated cell sorting (MACS) to isolate CAR-NK cells prior to phospho-proteomics including alternating formalin fixation and immunolabeling. To identify the most optimal immunolabeling strategy, NK cells transduced with a viral construct that included a truncated CD34 component (tCD34) were tested for an ability to optimally bind Human CD34 Biotinylated Antibody followed by Streptavidin-Allophycocyanin (SAv-APC) with varying formalin fixation sequences. Cells were either formalin fixed before antibody and SAv-APC labeling, after antibody and SAv-APC labeling, or after antibody and before SAv-APC labeling. Human CD34 APC-conjugated Antibodies, NK cells without tCD34, and OPM-2 multiple myeloma cells were used as controls for understanding SAv-APC background binding. Lastly, CAR-NK and control NK cell labeling effectiveness was also tested against the cells’ incubation periods. There were no consistent notable differences in CAR-NK labeling efficiency with alternating fixation and immunolabeling patterns. However, increased labeling was consistently found in unfixed samples. Formalin fixation also leads to unintentional background binding as demonstrated by labeled control NK and OPM-2 cell numbers. Furthermore, incubation time for CAR-NK cell samples proves advantageous in labeling CAR-NK cells irrespective of fixation and staining patterns. By analyzing control NK and CAR-NK samples over four weeks, we found that control NK cells were dying at a faster rate while CAR-NK cells remained at a consistently high viability. This increased death rate of the control NKs in the CAR-NK samples allowed for a greater proportion of CAR-NK detection with reduced noise from background control NK binding. Formalin fixation poses a challenge for optimal CAR-NK labeling due to background binding and a decrease in labeling levels when compared to non-fixed samples. However, incubation time of CAR-NK cells can potentially circumvent these difficulties and serve as a tool for cell sample preparation due to biological differences in CAR-NK and control NK proliferation. This can be a novel strategy for optimizing MACS for phosphoproteomic analysis of CAR-NK cells. Citation Format: Kaitlyn Bali, Yukiko Higa, Long Huynh, Lei Tian, Jianhua Yu, Nicholas Graham. Optimizing immunolabeling strategies in preparation for magnetic activated cell sorting for chimeric antigen receptor-natural killer and multiple myeloma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2182.