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Updated: Jul 24, 2025
Production of Human CRISPR-Engineered CAR-T Cells
Published on: March 15, 2021
Rational design of PD-1-CD28 immunostimulatory fusion proteins for CAR T cell therapy
Theo Lorenzini1, Bruno L Cadilha1, Hannah Obeck1
1Division of Clinical Pharmacology, Department of Medicine IV, LMU University Hospital, LMU Munich, Germany, Member of the German Center for Lung Research (DZL), Munich, Germany.
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.
Insights
Immunostimulatory fusion proteins (IFPs) enhance CAR T cell therapy by overcoming immune suppression. Designing IFPs to mimic PD-1/PD-L1 interactions is crucial for tumor-specific CAR T cell efficacy and persistence.
Area of Science:
- Immunology
- Cancer Therapy
- Molecular Engineering
Background:
- CAR T cell therapy efficacy is often limited by tumor immune suppression and poor T cell persistence.
- Immunostimulatory fusion proteins (IFPs) are engineered to convert suppressive signals into stimulatory ones, enhancing T cell persistence.
- A universal IFP design remains elusive, necessitating the identification of key design determinants.
Purpose of the Study:
- To define critical design elements for immunostimulatory fusion protein (IFP) activity using a PD-1-CD28 IFP as a model.
- To evaluate the impact of different IFP design choices on CAR T cell function and therapeutic efficacy.
Main Methods:
- Comparison of various PD-1-CD28 IFP variants in a human leukemia model.
- In vitro assessment of CAR T cell performance against PD-L1+ tumor cells.
- In vivo evaluation of IFP-modified CAR T cells in a xenograft mouse model.
Main Results:
- IFPs exceeding physiological PD-1 length triggered CAR-independent T cell responses, unsuitable for tumor-specific therapy.
- IFP variants with physiological PD-1 length improved CAR T cell effector function, proliferation, and in vivo survival.
- Transmembrane or extracellular CD28 domains could be substituted with PD-1 domains without compromising in vivo efficacy.
Conclusions:
- PD-1-CD28 IFP constructs require mimicry of the natural PD-1/PD-L1 interaction to maintain selectivity.
- CAR-conditional therapeutic activity is achieved when IFPs retain tumor-specific targeting and signaling.
