Complementarity-determining region clustering may cause CAR-T cell dysfunction

Nat Commun. 2023 Aug 10;14(1):4732. doi: 10.1038/s41467-023-40303-z.


Chimeric antigen receptor (CAR)-T cell therapy is rapidly advancing as cancer treatment, however, designing an optimal CAR remains challenging. A single-chain variable fragment (scFv) is generally used as CAR targeting moiety, wherein the complementarity-determining regions (CDRs) define its specificity. We report here that the CDR loops can cause CAR clustering, leading to antigen-independent tonic signalling and subsequent CAR-T cell dysfunction. We show via CARs incorporating scFvs with identical framework and varying CDR sequences that CARs may cluster on the T cell surface, which leads to antigen-independent CAR-T cell activation, characterized by increased cell size and interferon (IFN)-γ secretion. This results in CAR-T cell exhaustion, activation-induced cell death and reduced responsiveness to target-antigen-expressing tumour cells. CDR mutagenesis confirms that the CAR-clustering is mediated by CDR-loops. In summary, antigen-independent tonic signalling can be induced by CDR-mediated CAR clustering, which could not be predicted from the scFv sequences, but could be tested for by evaluating the activity of unstimulated CAR-T cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Complementarity Determining Regions* / genetics
  • Complementarity Determining Regions* / metabolism
  • Immunotherapy, Adoptive / methods
  • Receptors, Antigen, T-Cell / metabolism
  • Signal Transduction
  • Single-Chain Antibodies*
  • T-Lymphocytes


  • Complementarity Determining Regions
  • Single-Chain Antibodies
  • Receptors, Antigen, T-Cell