Design of soluble Notch agonists that drive T cell development and boost immunity

Rubul Mout¹, Ran Jing¹, Mayuri Tanaka-Yano², Emily D Egan³, Helen Eisenach⁴, Martin A Kononov², Roland Windisch¹, Mohamad Ali Toufic Najia⁵, Allison Tompkins², Luca Hensch², Trevor Bingham², Rajesh Gunage⁶, Yunliang Zhao², Natasha I Edman⁷, Christopher Li⁸, Dahai Wang², Thorsten M Schlaeger², Leonard I Zon⁹, Trista E North², Urban Lendahl¹⁰, R Grant Rowe¹¹, David Baker¹², Stephen C Blacklow¹³, George Q Daley¹⁴

Affiliations

¹ Stem Cell & Regenerative Biology Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA.
² Stem Cell & Regenerative Biology Program, Boston Children's Hospital, Boston, MA 02115, USA.
³ Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA.
⁴ Department of Biochemistry, University of Washington, Seattle, WA 98195, USA; Institute for Protein Design, University of Washington, Seattle, WA 98195, USA.
⁵ Stem Cell & Regenerative Biology Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁶ Stem Cell & Regenerative Biology Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.
⁷ Department of Biochemistry, University of Washington, Seattle, WA 98195, USA; Institute for Protein Design, University of Washington, Seattle, WA 98195, USA; Medical Scientist Training Program, University of Washington, Seattle, WA 98195, USA.
⁸ Stem Cell & Regenerative Biology Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA.
⁹ Stem Cell & Regenerative Biology Program, Boston Children's Hospital, Boston, MA 02115, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Harvard Stem Cell Institute, Boston, MA 02115, USA.
¹⁰ Department of Cell and Molecular Biology, Karolinska Institutet, 17177 Stockholm, Sweden.
¹¹ Stem Cell & Regenerative Biology Program, Boston Children's Hospital, Boston, MA 02115, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.
¹² Department of Biochemistry, University of Washington, Seattle, WA 98195, USA; Institute for Protein Design, University of Washington, Seattle, WA 98195, USA; Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.
¹³ Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA; Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.
¹⁴ Stem Cell & Regenerative Biology Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA. Electronic address: george_daley@hms.harvard.edu.

PMID: 40752493
PMCID: PMC12327808 (available on 2026-07-29)
DOI: 10.1016/j.cell.2025.07.009

Design of soluble Notch agonists that drive T cell development and boost immunity

Rubul Mout et al. Cell. 2025.

. 2025 Jul 29:S0092-8674(25)00798-6.

doi: 10.1016/j.cell.2025.07.009. Online ahead of print.

Authors

Affiliations

¹ Stem Cell & Regenerative Biology Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA.
² Stem Cell & Regenerative Biology Program, Boston Children's Hospital, Boston, MA 02115, USA.
³ Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA.
⁴ Department of Biochemistry, University of Washington, Seattle, WA 98195, USA; Institute for Protein Design, University of Washington, Seattle, WA 98195, USA.
⁵ Stem Cell & Regenerative Biology Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁶ Stem Cell & Regenerative Biology Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.
⁷ Department of Biochemistry, University of Washington, Seattle, WA 98195, USA; Institute for Protein Design, University of Washington, Seattle, WA 98195, USA; Medical Scientist Training Program, University of Washington, Seattle, WA 98195, USA.
⁸ Stem Cell & Regenerative Biology Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA.
⁹ Stem Cell & Regenerative Biology Program, Boston Children's Hospital, Boston, MA 02115, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Harvard Stem Cell Institute, Boston, MA 02115, USA.
¹⁰ Department of Cell and Molecular Biology, Karolinska Institutet, 17177 Stockholm, Sweden.
¹¹ Stem Cell & Regenerative Biology Program, Boston Children's Hospital, Boston, MA 02115, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.
¹² Department of Biochemistry, University of Washington, Seattle, WA 98195, USA; Institute for Protein Design, University of Washington, Seattle, WA 98195, USA; Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.
¹³ Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA; Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.
¹⁴ Stem Cell & Regenerative Biology Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA. Electronic address: george_daley@hms.harvard.edu.

PMID: 40752493
PMCID: PMC12327808 (available on 2026-07-29)
DOI: 10.1016/j.cell.2025.07.009

Abstract

The rational design of receptor agonists to control cell signaling is an emerging strategy for developing disease therapeutics. Creating a soluble cytokine-like agonist for the Notch receptor, which regulates cell fate in embryonic and adult development, is challenging, as receptor activation requires a mechanical force that is usually mediated by cell-associated transmembrane ligands. Here, we exploit computationally designed protein complexes with precise valencies and geometries to generate soluble cytokine-like Notch agonists. These molecules promote cell-cell bridging, cluster Notch receptors at cell synapses, and activate receptor signaling. We show that these agonists drive T cell differentiation from cord blood progenitors and human induced pluripotent stem cells (iPSCs) and in bioreactor production of T cells in liquid suspension. When delivered intravenously in mice, they stimulate cytokine production, expansion of antigen-specific CD4⁺ T cells, and antibody class switching. These de-novo-designed ligands can be broadly applied to optimize in vitro cell differentiation and advance immunotherapy development.

Keywords: Notch signaling; T cell development; T cell immunity; computational protein design; iPSC-derived T cells; immunotherapy; protein agonist; soluble Notch activation; vaccine.

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Conflict of interest statement

Declaration of interests R.M., R.J., E.D.E., D.B., S.C.B., and G.Q.D. filed patents related to C3-DLL4 soluble Notch agonist. R.M. and G.Q.D. filed patents related to C5(15H)-DLL4 soluble Notch agonist. G.Q.D. is an advisory board member at Cell.

References

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Design of soluble Notch agonists that drive T cell development and boost immunity

Affiliations

Design of soluble Notch agonists that drive T cell development and boost immunity

Authors

Affiliations

Abstract

Conflict of interest statement

References

Grants and funding

LinkOut - more resources

Full Text Sources

Research Materials