Rational design of PD-1-CD28 immunostimulatory fusion proteins for CAR T cell therapy

Br J Cancer. 2023 Sep;129(4):696-705. doi: 10.1038/s41416-023-02332-9. Epub 2023 Jul 4.


Background: In many situations, the therapeutic efficacy of CAR T cells is limited due to immune suppression and poor persistence. Immunostimulatory fusion protein (IFP) constructs have been advanced as a tool to convert suppressive signals into stimulation and thus promote the persistence of T cells, but no universal IFP design has been established so far. We now took advantage of a PD-1-CD28 IFP as a clinically relevant structure to define key determinants of IFP activity.

Methods: We compared different PD-1-CD28 IFP variants in a human leukemia model to assess the impact of distinctive design choices on CAR T cell performance in vitro and a xenograft mouse model.

Results: We observed that IFP constructs that putatively exceed the extracellular length of PD-1 induce T-cell response without CAR target recognition, rendering them unsuitable for tumour-specific therapy. IFP variants with physiological PD-1 length ameliorated CAR T cell effector function and proliferation in response to PD-L1+ tumour cells in vitro and prolonged survival in vivo. Transmembrane or extracellular CD28 domains were found to be replaceable by corresponding PD-1 domains for in vivo efficacy.

Conclusion: PD-1-CD28 IFP constructs must mimic the physiological interaction of PD-1 with PD-L1 to retain selectivity and mediate CAR-conditional therapeutic activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • B7-H1 Antigen
  • CD28 Antigens
  • Cell Line, Tumor
  • Humans
  • Immunotherapy, Adoptive*
  • Leukemia*
  • Mice
  • Programmed Cell Death 1 Receptor


  • CD28 Antigens
  • Programmed Cell Death 1 Receptor
  • B7-H1 Antigen