Lab-Led

• Design of a potent interleukin-21 mimic for cancer immunotherapy
  Agonists Cytokines Cancer Therapeutics
  Chun JH, Lim BS, Roy S, Walsh MJ, Abhiraman GC, Zhangxu K, Atajanova T, Revach OY, Clark EC, Li P, Palin CA, Khanna A, Tower S, Kureshi R, Hoffman MT, Sharova T, Lawless A, Cohen S, Boland GM, Nguyen T, Peprah F, Tello JG, Liu SY, Kim CJ, Shin H, Quijano-Rubio A, Jude KM, Gerben S, Murray A, Heine P, DeWitt M, Ulge UY, Carter L, King NP, Silva DA, Kueh HY, Kalia V, Sarkar S, Jenkins RW, Garcia KC, Leonard WJ, Dougan M, Dougan SK, Baker D.
  Sci Immunol, 2025 | doi:10.1126/sciimmunol.adx1582
  
  Abstract

  Long-standing goals of cancer immunotherapy are to activate cytotoxic antitumor T cells across a range of affinities for tumor antigens while suppressing regulatory T cells. Computational protein design has enabled the precise tailoring of proteins to meet specific needs. Here, we report a de novo designed IL-21 mimic, 21h10, with high stability and signaling potency in humans and mice. In murine and ex vivo human organotypic tumor models, 21h10 showed robust antitumor activity, with more prolonged signaling and stronger antitumor activity than native IL-21. 21h10 induced pancreatitis that could be mitigated by TNF blockade without compromising antitumor efficacy. Although antidrug antibodies to 21h10 formed, they were not neutralizing. 21h10 induced highly cytotoxic T cells with a range of affinities, robustly expanding intratumoral low-affinity cytotoxic T cells and driving high expression of IFN-γ and granzyme B compared with native IL-21, while increasing the frequency of IFN-γ T helper 1 cells and reducing regulatory T cells. The full human-mouse cross-reactivity, high stability and potency, and low-affinity antitumor responses support the translational potential of 21h10.

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• De novo designed proteins neutralize lethal snake venom toxins
  Agonists
  Vázquez Torres S, Benard Valle M, Mackessy SP, Menzies SK, Casewell NR, Ahmadi S, Burlet NJ, Muratspahić E, Sappington I, Overath MD, Rivera-de-Torre E, Ledergerber J, Laustsen AH, Boddum K, Bera AK, Kang A, Brackenbrough E, Cardoso IA, Crittenden EP, Edge RJ, Decarreau J, Ragotte RJ, Pillai AS, Abedi M, Han HL, Gerben SR, Murray A, Skotheim R, Stuart L, Stewart L, Fryer TJA, Jenkins TP, Baker D.
  Nature, 2025 | doi:10.1038/s41586-024-08393-x
  
  Abstract

  Snakebite envenoming remains a devastating and neglected tropical disease, claiming over 100,000 lives annually and causing severe complications and long-lasting disabilities for many more. Three-finger toxins (3FTx) are highly toxic components of elapid snake venoms that can cause diverse pathologies, including severe tissue damage and inhibition of nicotinic acetylcholine receptors, resulting in life-threatening neurotoxicity. At present, the only available treatments for snakebites consist of polyclonal antibodies derived from the plasma of immunized animals, which have high cost and limited efficacy against 3FTxs. Here we used deep learning methods to de novo design proteins to bind short-chain and long-chain α-neurotoxins and cytotoxins from the 3FTx family. With limited experimental screening, we obtained protein designs with remarkable thermal stability, high binding affinity and near-atomic-level agreement with the computational models. The designed proteins effectively neutralized all three 3FTx subfamilies in vitro and protected mice from a lethal neurotoxin challenge. Such potent, stable and readily manufacturable toxin-neutralizing proteins could provide the basis for safer, cost-effective and widely accessible next-generation antivenom therapeutics. Beyond snakebite, our results highlight how computational design could help democratize therapeutic discovery, particularly in resource-limited settings, by substantially reducing costs and resource requirements for the development of therapies for neglected tropical diseases.

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Collaborator-Led

• Design of soluble Notch agonists that drive T cell development and boost immunity
  Agonists
  Mout R, Jing R, Tanaka-Yano M, Egan ED, Eisenach H, Kononov MA, Windisch R, Najia MAT, Tompkins A, Hensch L, Bingham T, Gunage R, Zhao Y, Edman NI, Li C, Wang D, Schlaeger TM, Zon LI, North TE, Lendahl U, Rowe RG, Baker D, Blacklow SC, Daley GQ.
  Cell, 2025 | 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.

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• Target-conditioned diffusion generates potent TNFR superfamily antagonists and agonists
  Agonists
  Glögl M, Krishnakumar A, Ragotte RJ, Goreshnik I, Coventry B, Bera AK, Kang A, Joyce E, Ahn G, Huang B, Yang W, Chen W, Sanchez MG, Koepnick B, Baker D.
  Science, 2024 | doi:10.1126/science.adp1779
  
  Abstract

  Despite progress in designing protein-binding proteins, the shape matching of designs to targets is lower than in many native protein complexes, and design efforts have failed for the tumor necrosis factor receptor 1 (TNFR1) and other protein targets with relatively flat and polar surfaces. We hypothesized that free diffusion from random noise could generate shape-matched binders for challenging targets and tested this approach on TNFR1. We obtain designs with low picomolar affinity whose specificity can be completely switched to other family members using partial diffusion. Designs function as antagonists or as superagonists when presented at higher valency for OX40 and 4-1BB. The ability to design high-affinity and high-specificity antagonists and agonists for pharmacologically important targets in silico presages a coming era in protein design in which binders are made by computation rather than immunization or random screening approaches.

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• Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies
  Agonists
  Edman NI, Phal A, Redler RL, Schlichthaerle T, Srivatsan SR, Ehnes DD, Etemadi A, An SJ, Favor A, Li Z, Praetorius F, Gordon M, Vincent T, Marchiano S, Blakely L, Lin C, Yang W, Coventry B, Hicks DR, Cao L, Bethel N, Heine P, Murray A, Gerben S, Carter L, Miranda M, Negahdari B, Lee S, Trapnell C, Zheng Y, Murry CE, Schweppe DK, Freedman BS, Stewart L, Ekiert DC, Schlessinger J, Shendure J, Bhabha G, Ruohola-Baker H, Baker D.
  Cell, 2024 | doi:10.1016/j.cell.2024.05.025
  
  Abstract

  Many growth factors and cytokines signal by binding to the extracellular domains of their receptors and driving association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affect signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo-designed fibroblast growth factor receptor (FGFR)-binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca release and mitogen-activated protein kinase (MAPK) pathway activation. The high specificity of the designed agonists reveals distinct roles for two FGFR splice variants in driving arterial endothelium and perivascular cell fates during early vascular development. Our designed modular assemblies should be broadly useful for unraveling the complexities of signaling in key developmental transitions and for developing future therapeutic applications.

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• designed Hsp70 activator dissolves intracellular condensates
  Agonists
  Zhang JZ, Greenwood N, Hernandez J, Cuperus JT, Huang B, Ryder BD, Queitsch C, Gestwicki JE, Baker D.
  bioRxiv, 2023 | doi:10.1101/2023.09.18.558356
  
  Abstract

  Protein quality control (PQC) is carried out in part by the chaperone Hsp70, in concert with adapters of the J-domain protein (JDP) family. The JDPs, also called Hsp40s, are thought to recruit Hsp70 into complexes with specific client proteins. However, the molecular principles regulating this process are not well understood. We describe the design of a set of Hsp70 binding proteins that either inhibited or stimulated Hsp70’s ATPase activity; a stimulating design promoted the refolding of denatured luciferase , similar to native JDPs. Targeting of this design to intracellular condensates resulted in their nearly complete dissolution. The designs inform our understanding of chaperone structure-function relationships and provide a general and modular way to target PQC systems to condensates and other cellular targets.

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