Antigen density quantification of cell-surface immunotherapy targets by flow cytometry: Multi-antigen assay of neuroblastoma bone marrow metastasis.

The central role of target antigen density on chimeric antigen receptor T cell potency highlights the need for accurate measurement of antigen levels on clinical tumor samples. Here, we present a protocol for quantifying antigen density for six cell-surface antigens on neuroblastoma cells metastatic to bone marrow. We describe steps for patient sample acquisition, flow cytometry panel development, instrument setup, and compensation and detail procedures for running clinical samples and data analysis. For complete details on the use and execution of this protocol, please refer to Heitzeneder et al. (2022). ¹ .
Competing Interests: Declaration of interests S.H. and C.L.M. are co-inventors on patents relevant to CAR T cells targeting GPC2. C.L.M. has multiple patents pertinent to CAR T cells and is a co-founder of Lyell Immunopharma and CARGO Therapeutics, formerly Syncopation Life Sciences, which develop CAR-based therapies, and consults for Lyell, NeoImmune Tech, Apricity, Nektar, and Immatics. S.J.B. and M.M. are employees of BD Biosciences.
(Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)