CD19 alterations emerging after CD19-directed immunotherapy cause retention of the misfolded protein in the endoplasmic reticulum

Asen Bagashev, Elena Sotillo, Chih Hang Anthony Tang, Kathryn L. Black, Jessica Perazzelli, Steven H. Seeholzer, Yair Argon, David M. Barrett, Stephan A. Grupp, Chih Chi Andrew Hu, Andrei Thomas-Tikhonenkoa

Research output: Contribution to journalArticle

24 Scopus citations

Abstract

We previously described a mechanism of acquired resistance of B-cell acute lymphoblastic leukemia to CD19-directed chimeric antigen receptor T-cell (CART) immunotherapy. It was based on in-frame insertions in or skipping of CD19 exon 2. To distinguish between epitope loss and defects in surface localization, we used retroviral transduction and genome editing to generate cell lines expressing CD19 exon 2 variants (CD19ex2vs) bearing vesicular stomatitis virus G protein (VSVg) tags. These lines were negative by live-cell flow cytometry with an anti-VSVg antibody and resistant to killing by VSVg-directed antibody-drug conjugates (ADCs), suggestive of a defect in surface localization. Indeed, pulse-chase and -mannosidase inhibitor assays showed that all CD19ex2vs acquired endoplasmic reticulum (ER)-specific high-mannose-type sugars but not complex-type glycans synthesized in the Golgi apparatus. When fused with green fluorescent protein (GFP), CD19ex2vs (including a mutant lacking the relevant disulfide bond) showed colocalization with ER markers, implying protein misfolding. Mass spectrometric profiling of CD19-interacting proteins demonstrated that CD19ex2vs fail to bind to the key tetraspanin CD81 and instead interact with ER-resident chaperones, such as calnexin, and ER transporters involved in antigen presentation. Thus, even the intact domains of CD19ex2vs cannot be easily targeted with ADCs or current CD19 CARTs but could serve as sources of peptides for major histocompatibility complex (MHC)-restricted presentation and T-cell receptor (TCR)-mediated killing.

Original languageEnglish (US)
Article numbere0038318
JournalMolecular and Cellular Biology
Volume38
Issue number21
DOIs
StatePublished - Nov 1 2018

Keywords

  • Immunotherapy
  • Membrane proteins
  • Membrane transport
  • Protein folding
  • RNA splicing

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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