Truillet, C. | bioRxiv Science

bioRxiv2026

Machine Learning-Guided Engineering of High-Affinity Cross-Reactive Antibodies with Minimal Mutations

Antibodies raised against human targets often fail to recognize their animal orthologs, limiting preclinical evaluation in relevant models. We developed a Deep Mutational Scanning (DMS)-coupled deep learning strategy to engineer potent cross-reactive antibodies with minimal sequence divergence. Starting from C4, a fully human anti-PD-L1 antibody with weak recognition of murine PD-L1, DMS identified substitutions that improved binding to both human and mouse antigens. Conventional recombination of beneficial mutations generated highly cross-reactive antibodies but required 13 to 15 substitutions. To reduce this mutational burden, a deep learning model trained on DMS-derived sequence-binding data was used to identify minimal mutation combinations predicted to retain high affinity. This approach yielded variants carrying only 4 to 5 substitutions, with in vitro and cellular binding properties comparable to highly mutated antibodies. Epitope mapping, structural modeling and in vivo assessment further confirmed that these engineered antibodies retained PD-1/PD-L1 blockade and demonstrated therapeutic activity in a mouse tumor model.

molecular biology↗

bioRxiv2025

Endothelin Signaling via EDNRB receptor Reduces Proliferation and Promotes Proneural-to-Mesenchymal Transition in Gliomas

Diffuse gliomas are incurable primary brain tumors encompassing three histo-molecular subtypes: glioblastomas (GB), astrocytomas, and oligodendrogliomas. The latter two harbor IDH1 mutations and exhibit slower progression than glioblastomas. Diffuse gliomas are composed of highly plastic tumor cells capable of transitioning between astrocyte-like, oligodendrocyte-like, progenitor-like, and mesenchymal-like states, driven by genetic alterations and microenvironmental cues. The proneural-to-mesenchymal transition (PMT), associated with increased malignancy, is notably influenced by cytokines in the tumor microenvironment. Endothelin cytokines (ET-1, ET-2, ET-3), primarily secreted by vascular cells, regulate not only vascular tone but also astrocyte and neural stem cell proliferation via the G-protein-coupled receptors EDNRA and EDNRB. Prior studies using serum-cultured glioma lines suggested pro-proliferative effects of endothelins; however, such models poorly recapitulate the in vivo glioma context. In this study, we comprehensively revisited endothelin signaling - covering receptor expression, regulation, downstream pathways, and cellular responses-using eleven serum-free, patient-derived glioma lines (glioblastomas, IDH-wt and IDH-mutant oligodendrogliomas and astrocytomas), along with primary tumor samples. Multi-omics and electrophysiological analyses revealed EDNRB as the predominant receptor, enriched in astrocyte-like cells, upregulated by BMPs or growth factor withdrawal, and downregulated by interferons, IL-6 cytokines, endothelins, and Hippo/YAP activation. In contrast, EDNRA was expressed by a perivascular tumor subpopulation and induced by Notch signaling in glioblastomas but not in IDH1-mutant cells. Functionally, endothelins reduced proliferation across all models while promoting migration and PMT. Mechanistically, EDNRB activation increased intracellular Ca{superscript 2} and activated ERK, STAT3, and apamin-sensitive SK2/SK3 potassium channels. These findings identify endothelin signaling as an important regulator of glioma cell plasticity and behavior. HighlightsO_LIEDNRB is the predominant endothelin receptor expressed in glioma cells, with a small subset of tumor cells expressing EDNRA in close proximity to blood vessels C_LIO_LIEndothelin signaling reduces proliferation while promoting cell migration and Proneural-to-Mesenchymal transition C_LIO_LIEndothelin activates downstream Ca2+, K+, ERK, and STAT3 signaling pathways C_LIO_LIEDNRB expression is both positively and negatively regulated by inflammatory cytokines and the Hippo/YAP1 pathway, whereas EDNRA is upregulated by Notch signaling and hypoxia C_LI

cancer biology↗

bioRxiv2018

Profiling the surface proteome identifies actionable biology for TSC1 mutant cells beyond mTORC1 signaling

Loss of the TSC1/TSC2 complex leads to constitutively high mTORC1 signaling; however, pharmacological inhibition of mTORC1 in this setting produces a broad spectrum of clinical responses. We report herein several cell surface proteins upregulated by inactivation of TSC1 that present therapeutic alternatives or adjuvants to direct mTORC1 inhibition. A proteomics screen revealed that TSC1 loss most dramatically induced the expression of neprilysin (NEP/CD10) and aminopeptidase N (APN/CD13). The survival of TSC1 null human cancer cells was dependent on NEP expression, and TSC1 mutation sensitized cells to biochemical inhibition of APN. Remarkably, NEP and APN upregulation occurred via a TSC2- and mTORC1-independent mechanism; therefore, the antiproliferative effects of mTORC1 inhibition could be augmented by co-suppression of APN activity.\n\nStatement of significanceThese data introduce a non-canonical biological role for TSC1 beyond regulating mTORC1 signaling, which also enabled several immediately translatable therapeutic strategies for clinically problematic cells with TSC1 mutations.

cancer biology↗