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@article{pmid41959119,

title = {Structural Basis of Serine Protease Inhibition by Antibodies from Biased Fab Phage-Display Libraries},

author = {Kyle J Anderson and Melody S Lee and Natalia Sevillano and Gang Chen and Michael J Hornsby and Sachdev S Sidhu and Charles S Craik},

doi = {10.64898/2026.03.12.711446},

issn = {2692-8205},

year  = {2026},

date = {2026-03-01},

urldate = {2026-03-01},

journal = {bioRxiv},

abstract = {Biased Fab phage-display libraries were designed to determine whether inhibitory CDR H3 motifs from potent anti-matriptase antibodies could be transferred to target homologous serine proteases. Using reverse-binding and substrate-like H3 motifs from parental clones A11 and E2 as templates, six synthetic libraries with 10 diversity were constructed. Selection against matriptase identified sixteen inhibitors with sub-100 nM potency, representing 100,000-fold improvement over circularized H3 loops alone. Selection against TMPRSS2, a serine protease implicated in viral entry and prostate cancer with 43% sequence identity to matriptase, yielded binders with micromolar inhibitory potency. Selection against urokinase plasminogen activator (uPA, 35% identity) identified binders that adopted a substrate-like CDR H3 binding mode in our structural models. Across all reference structures, including the separately identified uPA inhibitor AB2 (PDB: 9PYF, deposited with this work), benchmarking of five co-folding methods and rigid-body docking showed that co-folding consistently achieved acceptable to high quality DockQ scores, outperforming traditional docking and capturing the recognition of key active site determinants. Ensemble predictions of mutational binding energy changes (ΔΔ) using these models identified key paratope-epitope interactions, with predictions validated through mutagenesis. This work establishes a framework integrating biased antibody libraries with computational structure prediction and analysis for targeting conserved protease epitopes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid41875162,

title = {Dynamic translocation of Inside-Out proteins to the cell surface underlies cellular adaptation to cancer-induced stress},

author = {Tomasz Slezak and Kelly M O'Leary and Tanya Guevara Avella and Natalia Musial and Jinyang Li and Anna Andrzejczak and Elizabeth F Scott and Duc Anh Le and Anthony A Kossiakoff},

doi = {10.1073/pnas.2529493123},

issn = {1091-6490},

year  = {2026},

date = {2026-03-01},

urldate = {2026-03-01},

journal = {Proc Natl Acad Sci U S A},

volume = {123},

number = {13},

pages = {e2529493123},

abstract = {Inside-Out (I-O) protein display, the noncanonical surface localization of intracellular proteins, represents an underexplored feature of tumor cell biology. Here, we map the molecular landscape and trafficking mechanisms that control the presentation of I-O proteins on cancer cell membranes. Employing APEX2-mediated proximity biotinylation and a custom antibody generation and validation platform, we identified approximately 140 high-confidence I-O proteins, primarily ribosomal, proteasomal, chaperone, and translation factors, notably enriched in protein families associated with stress-response pathways. Validation of 500 antibodies encompassing 40 I-O targets across seven tumor cell lines confirmed selective and robust surface localization, while in vivo imaging in mouse xenografts demonstrated pronounced and tumor-specific antibody accumulation. I-O proteins were absent on peripheral blood mononuclear cells (PBMCs) and in normal tissues, indicating cancer cell selectivity. Functional analyses revealed that I-O protein tethering to the membrane is dependent on heparan sulfate interactions; enzymatic removal of these glycans led to the clearance of I-O proteins from the cell surface. Notably, the removed proteins returned to baseline levels within 6 h, indicating a dynamic balance related to Endoplasmic Reticulum (ER)-Golgi trafficking and cellular stress. Nearly half of these I-O proteins overlapped with known stress granule (SG) components; however, stress elements that promote SG formation do not similarly affect surface display of I-O proteins. Furthermore, I-O proteins are present on standard cancer cell lines under lower stress levels needed to induce SG formation, suggesting parallel yet mechanistically distinct aspects of the stress response. These findings position I-O display as a paradigm in protein trafficking, different from traditional secretion pathways and closely linked to stress response.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid41818370,

title = {Autophagolysosomal exocytosis inverts Src kinase onto the cell surface in cancer},

author = {Corleone S Delaveris and Rita P Loudermilk and Apurva Pandey and Soumya G Remesh and Trenton M Peters-Clarke and Snehal D Ganjave and William J N Dougherty and Henry M Delavan and Chunyue Wang and Jesse Ling and Juan Antonio Camara Serrano and Fernando Salangsang and Chien-Kuang Cornelia Ding and Nancy Greenland and Carissa E Chu and Sima Porten and Veronica Steri and Jonathan Chou and Michael J Evans and Kevin K Leung and James A Wells},

doi = {10.1126/science.aec1778},

issn = {1095-9203},

year  = {2026},

date = {2026-03-01},

urldate = {2026-03-01},

journal = {Science},

volume = {391},

number = {6790},

pages = {eaec1778},

abstract = {Overexpression of the proto-oncogene Src is common to a wide variety of cancers. In this work, we found that Src is noncanonically translocated and inverted onto the cell surface in cancer, both in vitro and in vivo. We identified autophagolysosomal exocytosis (ALE) as a secretory mechanism prominent in cancer cell lines. Src represents the prototypical example of a family of membrane-anchored proteins that are transported by this process. Furthermore, this extracellular membrane-associated Src (eSrc) was found in primary tumors, and anti-Src antibody-based therapies mediated tumor cell killing in cell culture systems and in mouse xenograft models. Thus, intracellular -myristoylated proteins, prototypically Src, can be topologically inverted onto the cell surface in cancer and targeted with antibody therapeutics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}