Basic Procedures for Detection and Cytotoxicity of Chimeric Antigen Receptors

Methods Mol Biol. 2019:1904:299-306. doi: 10.1007/978-1-4939-8958-4_13.

Abstract

Chimeric antigen receptors against CD19 (anti-CD19-CAR) are widely recognized and used by not only researchers associated with immunology, molecular biology, and cell biology but also physicians to treat B-cell malignancies. Anti-CD19-CAR is currently clinically available as one of the therapeutic modalities for refractory acute B-cell-typed lymphoblastic leukemia (B-ALL) patients. However, to detect CAR on the cell surface and investigate the efficacy of CAR-T cells, there are numerous experimental modalities including flow cytometry, the Cr-releasing assay, immunoblot, and immunostaining. We have chosen several techniques, which are necessary and sufficient as well as reliable and reproducible to detect and assess the killing effect of CAR-T cells. Here, we describe protocols for basic experiments and procedures for the detection of CAR on transduced cells and in in vitro coculture experiments to assess cytotoxicity using CAR-T cells.

Keywords: Antibody capping; Autolysis; CAR; CD19; CD38; Flow cytometry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, CD19 / immunology
  • Antigens, CD19 / metabolism
  • Biomarkers
  • Cells, Cultured
  • Cytotoxicity, Immunologic*
  • Humans
  • Immunophenotyping / methods
  • Protein Binding / immunology
  • Receptors, Antigen, T-Cell / genetics
  • Receptors, Antigen, T-Cell / metabolism*
  • Receptors, Chimeric Antigen / genetics
  • Receptors, Chimeric Antigen / metabolism*
  • Staining and Labeling
  • T-Cell Antigen Receptor Specificity / immunology
  • T-Lymphocytes / immunology*
  • T-Lymphocytes / metabolism*

Substances

  • Antigens, CD19
  • Biomarkers
  • Receptors, Antigen, T-Cell
  • Receptors, Chimeric Antigen