The Notch signaling pathway plays a pivotal role in determining cell fate, especially in the development and function of T cells. But mimicking this highly mechanical, contact-dependent pathway in the lab has been a formidable challenge—until now.
A team of researchers at Boston Children’s Hospital and Harvard Medical School has developed a solution by designing a synthetic protein using AI-powered tools to activate Notch signaling. These soluble protein agonists can replicate Notch activation in suspension culture, opening the door to scalable, precision-controlled T-cell therapies.
The study published in Cell, “Design of Soluble Notch Agonists that Drive T Cell Development and Boost Immunity” was led by George Daley, MD, PhD, Dean of Harvard Medical School and co-founder of the Stem Cell and Regenerative Biology Program at Boston Children’s Hospital. The researchers used AI-based computational design tools to build synthetic molecules with similar geometry and multivalency required for Notch activation.
The Notch pathway regulates cell differentiation, particularly in the immune system. In hematopoietic stem cells, Notch activation initiates their differentiation into T cells.
Daley and his team, including lead author Rubul Mout, PhD, research fellow at Boston Children’s Hospital who previously worked with Nobel laureate David Baker, PhD, utilized the Rosetta software platform developed in the Baker lab.
Using the AI program, they constructed and screened a panel of multivalent Notch ligands with distinct structures to test how different spatial configurations impacted signaling potency.
“We exploit computationally designed protein complexes with precise valencies and geometries to generate soluble cytokine-like Notch agonists,” wrote the authors. The resulting proteins enhanced cell-to-cell contact, clustering Notch receptors to amplify the signal cascade.
Each design was screened for its ability to bind Notch receptors and trigger downstream signaling. In cultured human cord blood progenitors or induced pluripotent stem cells (iPSCs), the designed Notch agonists drove differentiation of T cells in liquid suspension, a format highly compatible with industrial-scale bioreactor systems.
The engineered proteins mimic the function of membrane-bound ligands by applying mechanical force during cell-to-cell contact. The ligands undergo trans-binding, forming bridges with receptors on adjacent cells, which effectively clustered the receptors at cell surfaces to mimic the spatial dynamics of natural signaling synapses. This receptor clustering amplified Notch activation, mimicking the physiological synapse formation observed in vivo and initiating robust T-cell development in suspension cultures.
“Being able to activate Notch signaling opens up lots of opportunities in immunotherapy, vaccine development, and immune cell regeneration,” said Mout.
Beyond boosting T-cell output in vitro, the synthetic Notch agonists also showed promise in animal models, where they enhanced T-cell function and antitumor responses. When injected intravenously into mice, the agonists enhanced immune activity in vivo, stimulating the expansion of antigen-specific CD4+ T cells, increasing cytokine production, and cytotoxic function.
“AI-driven protein design is a broadly enabling platform technology that we’ve exploited to develop a synthetic molecule that facilitates T-cell manufacture for clinical use and enhances immune responses when delivered in vivo,” says Daley. “We’re excited that this approach can target T cells to tumors while also stimulating their cytotoxic functions.”
Mout added: “I’m using this technology to engineer a variety of synthetic protein molecules that bridge T cells and cancer cells while boosting T-cell killing and neutralizing the immunosuppressive tumor microenvironment. My goal is to make better immunotherapies.”
Because the molecules are fully synthetic and soluble, they offer advantages in terms of manufacturing, storage, and clinical delivery compared to traditional ligand presentation systems. This platform approach could improve CAR-T cell manufacturing, vaccine development, and regenerative therapies where T-cell generation or function is limiting. It also represents one of the first demonstrations of using de novo designed proteins to modulate a complex signaling pathway with clinical relevance.