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

1. Siebel C, and Lendahl U (2017). Notch Signaling in Development, Tissue Homeostasis, and Disease. Physiol Rev 97, 1235–1294. 10.1152/physrev.00005.2017. - DOI - PubMed
1. Sprinzak D, and Blacklow SC (2021). Biophysics of Notch Signaling. Annu Rev Biophys 50, 157–189. 10.1146/annurev-biophys-101920-082204. - DOI - PMC - PubMed
1. Luca VC, Kim BC, Ge C, Kakuda S, Wu D, Roein-Peikar M, Haltiwanger RS, Zhu C, Ha T, and Garcia KC (2017). Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity. Science 355, 1320–1324. 10.1126/science.aaf9739. - DOI - PMC - PubMed
1. Biktasova AK, Dudimah DF, Uzhachenko RV, Park K, Akhter A, Arasada RR, Evans JV, Novitskiy SV, Tchekneva EE, Carbone DP, et al. (2015). Multivalent forms of the Notch ligand DLL-1 enhance antitumor T cell immunity in lung cancer and improve efficacy of EGFR targeted therapy. Cancer Res 75, 4728–4741. 10.1158/0008-5472.CAN-14-1154. - DOI - PMC - PubMed
1. Smyrlaki I, Fördős F, Rocamonde-Lago I, Wang Y, Shen B, Lentini A, Luca VC, Reinius B, Teixeira AI, and Högberg B (2024). Soluble and multivalent Jag1 DNA origami nanopatterns activate Notch without pulling force. Nat Commun 15, 465. 10.1038/s41467-023-44059-4. - DOI - PMC - PubMed

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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

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