Publication

Ma Z, Hellweg L, Elledge S, Lee J, Rotiroti M, Wucherpfennig K, Majzner R, Zhou, X., “Synthetic signaling platform uncovers and rewires cellular responses to PD-1 perturbation”, bioRxiv, 2025.

Tyrosine phosphorylation motifs are central regulators of cell signaling, yet methods to selectively detect and reprogram these events have been lacking. Here we introduce Sphyder (Selective PHosphotYrosine DEtection and Rewiring), which enables precise detection of signaling at the resolution of individual phosphorylation motifs. Using Sphyder biosensors, we resolved phosphorylation dynamics and uncovered regulatory mechanisms of the checkpoint receptor PD-1 in living cells. Sphyder also provided a framework for reconstructing phosphosignaling pathways. With this approach, we redirected PD-1 signaling from immunosuppressive to immunoactivating outputs and engineered synthetic receptors that linked extracellular sensing to customized transcriptional programs. In addition, Sphyder biosensors revealed previously unrecognized mechanisms of the PD-1/VEGF bispecific antibody Ivonescimab, showing that it induces VEGF-dependent clustering, phosphorylation, and degradation of PD-1. These findings may underlie its promising clinical activity relative to conventional PD-1 blockade. Together, our study establishes a broadly applicable strategy for sensing and reprogramming cell signaling, while also providing mechanistic insights into a new class of immune checkpoint inhibitors of major clinical interest.

Algov, I.*, Heest, A.*, Hopton, M., Liang, F., Holmes, A., Hao, L.†, Zhou, X.†, A tissue proteolysis mapping and substrate discovery platform for identifying novel tumor-activated biosensors, bioRxiv, 2025.

Dysregulated extracellular proteolytic activity is a prominent hallmark of cancer and can thus be exploited for tumor detection and therapeutic development. However, the discovery of tumor-responsive probes has been hindered by the lack of methods capable of capturing proteolytic events directly in tissue samples. Here, we report PSurf, a platform that enables the identification of tissue-specific protease sensors with tissue specimens. Through differential selection of tumor-specific sequences over healthy tissue, PSurf identified context-specific tumor-activated probes that precisely distinguish metastatic lesions in lung tissue slices. Using these substrates, we engineered nanobody-targeted biosensors that release urinary reporters upon tumor-specific cleavage in vivo, enabling precise noninvasive tumor detection in a murine lung metastasis model. PSurf provides a foundation for developing conditionally activated agents through tissue-specific activity mapping and probe discovery.

Rhee, K., Shue, L., Adachi, A., Osei-Owusu, J., Zhou, D., Cheng, A., Baluapuri, A., Harvey, E., Adelman, K., Skiba, M., Jun, H., Kruse, A.†, Zhou, X.†, GPCR antagonism via antibody-mediated rewiring of receptor trafficking and degradation, bioRxiv, 2025.

G-protein coupled receptors (GPCRs) represent one of the most important yet incompletely targeted classes of therapeutic proteins. Here, we report a novel strategy for functional GPCR antagonism through antibody-mediated endocytosis and lysosomal degradation. Our engineered bispecific antibodies, termed GPCR-TfR1 Targeting Chimeras (GTACs), achieve potent and selective downregulation of various GPCRs, including RXFP1 and CCR6, critical cancer and immune drug targets that are difficult to antagonize with conventional approaches. GTACs led to complete inhibition of receptor activity with over 100-fold greater potency than conventional antibody antagonists. Using four-color imaging, we elucidated the trafficking mechanism of both the target protein and TfR1 in living cells. The GTAC platform enables robust antagonism of signaling across diverse GPCR families and establishes induced endocytosis and lysosomal trafficking as a fundamentally new paradigm for therapeutic GPCR modulation.

Ali, R.†, Jenkins, B.†, Karthaus W.†, Lipkova, J.†, Misale, S.†, Ruschhof, J.†, Smith, M.†, Vardhana, S.†, Zhou, X.†, Tackling the next decade of cancer research, Trends in Cancer, 4, 267-273, 2025.

Zhang, D., Duque-Jimenez, J., Facchinetti, F., Brixi, G., Rhee, K., Jänne, P., Zhou, X., Transferrin receptor targeting chimeras for membrane protein degradation, Nature, 638: 787–795, 2025.

Cancer cells require high levels of iron for rapid proliferation, leading to significant upregulation of cell-surface transferrin receptor 1 (TfR1), which mediates iron uptake by binding to the iron-carrying protein transferrin. Leveraging this phenomenon and the fast endocytosis rate of TfR1, we developed transferrin receptor targeting chimeras (TransTACs), a heterobispecific antibody modality for membrane protein degradation. TransTACs are engineered to drive rapid co-internalization of a target protein of interest and TfR1 from the cell surface, and to enable target protein entry into the lysosomal degradation pathway. We show that TransTACs can efficiently degrade a diverse range of single-pass, multi-pass, native or synthetic membrane proteins, including epidermal growth factor receptor, programmed cell death 1 ligand 1, cluster of differentiation 20 and chimeric antigen receptor. In example applications, TransTACs enabled the reversible control of human primary chimeric antigen receptor T cells and the targeting of drug-resistant epidermal growth factor receptor-driven lung cancer with the exon 19 deletion/T790M/C797S mutations in a mouse xenograft model. TransTACs represent a promising new family of bifunctional antibodies for precise manipulation of membrane proteins and targeted cancer therapy.

Rhee, K., Zhou X.‡, Two in one, the emerging concept of bifunctional antibodies, Curr. Opin. Biotechnol., 85:103050, 2024.

Zhou, X.*, Kong, S.*, Maker, A., Remesh, S., Leung, K., Verba, K., Wells, J.‡, Antibody discovery identifies regulatory mechanisms of protein arginine deiminase 4”, Nat. Chem. Biol., 20, 742–750, 2024.

Prior to 2024:

Antibody Engineering

Jiang, Z., Kuo, Y., Zhong, M., Zhang, J., Zhou, X., Xing, L., Wells, J., Wang, Y., Arkin, M.‡, Adaptor-Specific Antibody Fragment Inhibitors for the Intracellular Modulation of p97 (VCP) Protein-Protein Interactions. J. Am. Chem. Soc. 144(29):13218-13225, 2022.

Zhou, X., Bracken, C., Zhang, K., Zhou, J., Mou, Y., Wang, L., Cheng, Y., Leung, K., Wells, J.‡, Targeting phosphotyrosine in native proteins with conditional, bi-specific antibody traps. J. Am. Chem. Soc. 142(41), 17703-17713, 2020.

Engineering sequence-specific antibodies (Abs) against phosphotyrosine (pY) motifs embedded in folded polypeptides remains highly challenging because of the stringent requirement for simultaneous recognition of the pY motif and the surrounding folded protein epitope. Here, we present a method named phosphotyrosine Targeting by Recombinant Ab Pair, or pY-TRAP, for in vitro engineering of binders for native pY proteins. Specifically, we create the pY protein by unnatural amino acid misincorporation, mutagenize a universal pY-binding Ab to create a first binder B1 for the pY motif on the pY protein, and then select against the B1–pY protein complex for a second binder B2 that recognizes the composite epitope of B1 and the pY-containing protein complex. We applied pY-TRAP to create highly specific binders to folded Ub-pY59, a rarely studied Ub phosphoform exclusively observed in cancerous tissues, and ZAP70-pY248, a kinase phosphoform regulated in feedback signaling pathways in T cells. The pY-TRAPs do not have detectable binding to wild-type proteins or to other pY peptides or proteins tested. This pY-TRAP approach serves as a generalizable method for engineering sequence-specific Ab binders to native pY proteins.

Mou, Y., Zhou, X., Leung, K., Martinko, A., Yu, J., Chen, W., Mayo, S., Wang, L., Wells, J.‡, Engineering Improved Antiphosphotyrosine Antibodies Based on an Immunoconvergent Binding Motif. J. Am. Chem. Soc. 140(48):16615–16624, 2018.

Bracken, C., Lim, S., Solomon, P., Rettko, N., Nguyen, D., Zha, B., Schaefer, K., Byrnes, J., Zhou, J., Lui, I., Liu, J., Pance, K., QCRG Structural Biology Consortium, Zhou, X., Leung, K., Wells, J.‡, Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2. Nat. Chem. Biol., 17: 113-121, 2021.

Lim, S., Gramespacher, J., Pance, K.*, Solomon, P., Jin, J., Rettko, N., Lui, I., Elledge, S., Liu, J., Bracken, C., Simmons, G., Zhou, X., Leung, K., Wells, J.‡, Bispecific VH/Fab antibodies targeting neutralizing and non-neutralizing Spike epitopes demonstrate enhanced potency against SARS-CoV-2. mAbs, 13(1): 1893426, 2021.

Glasgow, A., Glasgow, J., Limonta, D., Solomon, P., Lui, I., Zhang, Y., Nix, M., Rettko, N., Zha, B., Yamin, R., Kao, K., Rosenberg, O., Ravetch, J., Wiita, A., Leung, K., Lim, S., Zhou, X., Hobman, T., Kortemme, T., Wells, J.‡, Engineered ACE2 receptor traps potently neutralize SARS-CoV-2. PNAS, 117 (45): 28046-28055, 2020.

Decoding and Reprogramming of Cell Signaling

Ju, J., Lee, H., Ning, L., Ryu, H., Zhou, X., Chun, H., Lee, Y., Lee-Richerson, A., Jeong, C., Lin, M., Seong, J.‡, Optical regulation of endogenous RhoA reveals selection of cellular responses by signal amplitude. Cell Rep. 40(2):111080, 2022.

Zhou, X., Zou, X., Chung, H., Gao, Y., Liu, Y., Qi, L., Lin, M.‡, A single-chain photoswitchable Cas9 architecture for inducible gene editing and transcription. ACS Chemical Biology, Special Issue on the Chemical Biology of CRISPR, 13(2): 443–448, 2018.

Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.

Zhou, X., Fan, L., Li, P., Shen, K., Lin, M.‡, Optical Control of Cell Signaling by Single-chain Photoswitchable Kinases. Science, 355: 836 – 842, 2017.

Protein kinases transduce signals to regulate a wide array of cellular functions in eukaryotes. A generalizable method for optical control of kinases would enable fine spatiotemporal interrogation or manipulation of these various functions. We report the design and application of single-chain cofactor-free kinases with photoswitchable activity. We engineered a dimeric protein, pdDronpa, that dissociates in cyan light and reassociates in violet light. Attaching two pdDronpa domains at rationally selected locations in the kinase domain, we created the photoswitchable kinases psRaf1, psMEK1, psMEK2, and psCDK5. Using these photoswitchable kinases, we established an all-optical cell-based assay for screening inhibitors, uncovered a direct and rapid inhibitory feedback loop from ERK to MEK1, and mediated developmental changes and synaptic vesicle transport in vivo using light.

Zhou, X., Pan, M., Lin, M.‡, Investigating neuronal function with optically controllable proteins. Frontiers in Molecular Neuroscience, 8: 37, 2015.

Zhou, X., Lin, M.‡, Photoswitchable fluorescent proteins: ten years of colorful chemistry and exciting applications. Curr. Opin. Chem. Biol. 17: 682 – 690, 2013.

Zhou, X., Chung, H., Lam, A., Lin, M.‡, Optical control of protein activity by fluorescent protein domains. Science, 338: 810 – 814, 2012.

Fluorescent proteins (FPs) are widely used as optical sensors, whereas other light-absorbing domains have been used for optical control of protein localization or activity. Here, we describe light-dependent dissociation and association in a mutant of the photochromic FP Dronpa, and we used it to control protein activities with light. We created a fluorescent light-inducible protein design in which Dronpa domains are fused to both termini of an enzyme domain. In the dark, the Dronpa domains associate and cage the protein, but light induces Dronpa dissociation and activates the protein. This method enabled optical control over guanine nucleotide exchange factor and protease domains without extensive screening. Our findings extend the applications of FPs from exclusively sensing functions to also encompass optogenetic control.

Biosensor Design

Elledge, S., Eigl, I., Phelps, M., McClinton, K., Zhou, X., Leung, K., Tata, C., Wells, J.‡, Using split luminescent biosensors for SARS-CoV-2 antibody detection in serum, plasma, and blood samples. Curr Protoc, 2(10): e521, 2022.

Elledge, S.*, Zhou, X.*, Byrnes, J., Martinko, A., Lui, I., Pance, K., Lim, S., Glasgow, J., Glasgow, A., Turcios, K., Iyer, N., Torres, L., Peluso, M., Henrich, T., Wang, T., Tato, C., Leung, K., Greenhouse, B., Wells, J.‡, Engineering luminescent biosensors for point-of-care SARS-CoV-2 antibody detection. Nat. Biotechnol. 39: 928–935, 2021.

Current serology tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies mainly take the form of enzyme-linked immunosorbent assays, chemiluminescent microparticle immunoassays or lateral flow assays, which are either laborious, expensive or lacking sufficient sensitivity and scalability. Here we present the development and validation of a rapid, low-cost, solution-based assay to detect antibodies in serum, plasma, whole blood and to a lesser extent saliva, using rationally designed split luciferase antibody biosensors. This new assay, which generates quantitative results in 30 min, substantially reduces the complexity and improves the scalability of coronavirus disease 2019 (COVID-19) antibody tests. This assay is well-suited for point-of-care, broad population testing, and applications in low-resource settings, for monitoring host humoral responses to vaccination or viral infection.

Peluso, M., Takahashi, S., Hakim, J., Kelly, J., Torres, L., Iyer, N., Turcios, K., Janson, O., Munter, S., Thanh, C., Nixon, C., Hoh, R., Tai, V., Fehrman, E., Hernandez, Y., Spinelli, M., Gandhi, M., Palafox, M., Vallari, A., Rodgers, M., Prostko, J., Hackett, J., Trinh, L., Wrin, T., Petroplolous, C., Chiu, C., Norris, P., DiGermanio, C., Stone, M., Busch, M., Elledge, S., Zhou, X., Wells, J., Shu, A., Kurtz, T., Pak, J., Wu, W., Burbelo, P., Cohen, J., Rutishauser, R., Martin, J., Deeks, S., Henrich, T., Rodriguez-Barraquer, I., Greenhouse, B.‡, SARS-CoV-2 antibody magnitude and detectability are driven by disease severity, timing, and assay. Science Advances, 7(31): eabh3409, 2021.

Byrnes, J., Zhou, X., Lui, I., Elledge, S., Glasgow, J., Lim, S., Loudermilk, R., Chiu, C., Wang, T., Wilson, M., Leung, K., Wells, J.‡, A competitive SARS-CoV-2 serological assay reveals a majority of patient antibodies targeting the spike receptor binding domain compete for ACE2 binding. mSphere, 5(5): e00802-20, 2020.

Zamecnik, C., Rajan, J., Yamauchi, K., Mann, S., Loudermilk, R., Sowa, G., Zorn, K., Alvarenga, B., Gaebler, C., Caskey, M., Stone, M., Norris, P., Gu, W., Chiu, C., Ng, D., Byrnes, J., Zhou, X., Wells, J., Robbiani, DF., Nussenzweig, M., DeRisi, J., Wilson, M.‡, ReScan, a Multiplex Diagnostic Pipeline, Pans Human Sera for SARS-CoV-2 Antigens. Cell Rep. Med. 1(7):100123, 2020.

A complete list of publications:

https://www.ncbi.nlm.nih.gov/myncbi/xin.zhou.8/bibliography/public/

*Co-first author; ‡Corresponding author; Bolded: Present or former members of the Zhou Lab.