Biology subjects

Martineau, P.

Publications and source records attributed to Martineau, P..

7 recordsLinked to original sources

bioRxiv2026

Selective targeting of RalA with intrabodies impairs Triple Negative Breast Cancer metastasis

This study successfully developed and validated isoform-specific intrabodies targeting the highly homologous Ral oncoproteins, key effectors in cancer progression. Phage display was used to isolate single-chain variable fragment (scFv) clones that recognize specifically RalA (C1-A, G5-A), RalB (F6-B), or both paralogs (A12-AB). Using lentiviral transduction, these intrabodies were stably expressed as GFP fusions in murine breast cancer 4T1 cells. The anti-RalA clones C1-A and A12-AB demonstrated clear colocalization with RalA, confirming their binding activity inside the cell. We further confirmed their activity in cells by analyzing Ral-dependent pathways. All intrabodies targeting RalA (C1-A, G5-A, A12-AB) but not the one specific to RalB inhibited mitochondrial fission, a known RalA function. All clones, except the pan-Ral one, altered the endo-lysosome pathway by decreasing lysosome number. Furthermore, the RalA-specific C1-A clone reduced lysosomes size, and uniquely and strongly reduced extracellular vesicle secretion, highlighting its distinct inhibitory potential. In an orthotopic Triple-Negative Breast Cancer (TNBC) mouse model, The C1-A RalA specific clone significantly but weakly reduced primary tumor growth, but exerted a powerful anti-metastatic effect, dramatically reducing lung metastases, with a complete abolition of metastases observed in 2/5 mice. In summary, these potent, isoform-specific Ral intrabodies act as effective intracellular inhibitors, successfully modulating RalA-specific functions in cells and offering a promising therapeutic strategy for significantly suppressing tumor growth and metastasis in vivo.

cancer biology↗

bioRxiv2026

Characterization of the p38α MAPK allosteric inhibition by a single chain Fv antibody

Most of the available p38 kinase inhibitors target the highly conserved ATP-binding site, thus explaining their off-target effects and toxicity. Here, we report the use of single-chain fragment variable (scFv) antibodies to identify new highly specific inhibitors. Using phage display, we selected five anti-p38 scFv antibodies among which one fully inhibited p38 kinase activity in vitro. We showed that this scFv did not affect ATP binding, while still acting as a competitive inhibitor of ATP hydrolysis. When expressed as intrabody in the nucleus of the THP-1 human monocytic cell line, the scFv inhibited the function of endogenous p38 and induced a significant decrease in lipopolysaccharide-induced tumor necrosis factor production. To gain insight into the mechanisms by which the scFv induced p38 inhibition, the scFv epitope was mapped by deep mutational scanning of p38 displayed on yeast surface and high-throughput selection by flow cytometry of p38 mutants no longer bound by the scFv. By this approach, we showed that the inhibitory scFv interacts with two helices within the C-terminal lobe of the kinase distant from the ATP-binding pocket and the docking groove. Altogether, our data show that the scFv antibody behaves as an allosteric inhibitor, constraining and maintaining p38 in an inactive form.

biochemistry↗

bioRxiv2025

scFv intrabody targeting wildtype TDP-43 presents protective effects in a cellular model of TDP-43 proteinopathy

TDP-43 proteinopathies are a set of neurological disorders characterized by the abnormal accumulation and mislocalization of TDP-43 in the cytoplasm, leading to the disruption of the normal function of the protein. In most of the cases, it is the wildtype (wt) form of the protein that is involved. An untargeted high-throughput screen of a single-chain variable fragment (scFv) library was performed using phage display against human full-length wt TDP-43. Two scFvs (B1 and D7) were retained following cellular expression (then termed intrabodies) and colocalization with cytoplasmic TDP-43 in vitro. We generated a 3D structure of full length wt TDP-43 in silico, and used it for epitope mapping. In a cellular model of TDP-43 proteinopathy, D7 enhanced the proteasomal degradation of the insoluble 35-kDa C-terminal fragment TDP-43 and reversed some TDP-43-induced metabolomic alterations, particularly relating to the lipid metabolism. Our findings offer a new scFv intrabody that bind to human wtTDP-43 and modify cellular pathways associated with TDP-43 proteinopathies.

neuroscience↗

bioRxiv2024

Investigation of Lipid-PEG Anchorage of Proteins onto Mesenchymal Stem Cell Derived Extracellular Vesicles for Intracellular Delivery: A Quantitative Assessment

Despite biomolecule delivery is a natural function of Extracellular Vesicles (EVs), low loading of exogenous macromolecules such as proteins into EVs limits their interest as convincing protein delivery systems for health applications. In this context, lipid-anchorage of exogenous cargo into EV membrane recently emerged as a promising option to enable their vectorization into cells. Nevertheless, this option was not explored for protein intracellular delivery, and further characterization of critical parameters governing the association of a lipid-anchored cargo protein to EVs stills needed to confirm the relevance of this anchorage strategy. Therefore, we sought to identify these parameters in a precise and quantitative manner, using bulk and single nanoparticle analysis methods to identify protein loading capacity and subsequent intracellular delivery. We identified incubation temperature, cargo concentration, Lipid Anchor (LA) structure (lipid nature, linker) and EV origin as critical factors influencing maximal EV loading capacity. Precise control of these parameters enabled to load cargo protein close to EV saturation without hindering cellular delivery. Structural properties of LA influenced not only cargo protein/EV association, but also intracellular delivery into different carcinoma cell lines. By thoroughly characterizing Lipid-PEG-protein anchorage, this study evidences the interest of this tunable and controllable approach for efficient EV protein delivery. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC="FIGDIR/small/610030v3_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1fed681org.highwire.dtl.DTLVardef@1b4d728org.highwire.dtl.DTLVardef@1998e3aorg.highwire.dtl.DTLVardef@488be3_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Activation of peripheral neuronal FLT3 promotes exaggerated sensorial and emotional pain-related behaviors facilitating the transition from acute to chronic pain

Acute pain events have been associated with persistent pain sensitization of nociceptive pathways increasing the risk of transition from acute to chronic pain. However, it is unclear whether injuryinduced persistent pain sensitization can promote long-term mood disorders. The receptor tyrosine kinase FLT3 is causally required for pain chronification after peripheral nerve injury, questioning its role in the development of pain-induced mood alterations. Here, we evaluated the emotional and sensorial components of pain after a single (SI) or double paw incision (DI). We then investigated the role of FLT3 either by inhibition using transgenic knock-out mice and functional antibodies or by activation with FLT3 ligand (FL) administrations. DI mice showed significant anxiodepressive-like and spontaneous pain behaviors as opposed to SI mice. DI also promoted and extended mechanical pain hypersensitivity compared to SI. This emotional and sensorial pain exaggeration correlated with spinal changes especially by increased microglia activation after DI versus SI. Intrathecal minocycline, a microglial inhibitor, specifically reversed DI inducedmechanical hypersensitivity in males. Repeated treatment with the microglia proliferation inhibitor GW2580 not only eliminated the exaggerated pain hypersensitivity produced by DI but also prevented anxiodepressive-related behaviors in DI animals. Finally, FL injections in naive animals provoked mechanical allodynia and anxiodepressive-like disorders concomitant with a strong microglial activation while Flt3 silencing in a genetic mouse line or FLT3 blocking via functional antibodies, blunted the development of persistent pain and depression after DI. Altogether our results show that the repetition of peripheral lesions facilitate not only exaggerated nociceptive behaviors but also induced anxiodepressive disorders supported by spinal central changes. The inhibition of FLT3 could thus become a promising therapy in the management of pain sensitization and related mood alterations.

neuroscience↗

bioRxiv2019

Biasing HER4 Tyrosine Kinase Signaling with Antibodies: Induction of Cell Death by Antibody-Dependent HER4 Intracellular Domain Trafficking

HER4 isoforms have oncogenic or tumor suppressor functions depending on their susceptibility to proteolytic cleavage and HER4 Intracellular Domain (4ICD) translocation. Here, we report that the NRG1 tumor suppressor mechanism through the HER4 JMa/CYT1 isoform can be mimicked by the agonist anti-HER4 antibody C6. NRG1 induced cleavage of poly(ADP-ribose) polymerase (PARP) and sub-G1 DNA fragmentation, and also reduced the metabolic activity of HER3-negative/HER4-positive cervical (C-33A) and ovarian (COV318) cancer cells. This effect was confirmed in HER4 JMa/CYT1-, but not JMa/CYT2-transfected BT549 triple-negative breast cancer cells. NRG1 favored 4ICD cleavage and retention in mitochondria in JMa/CYT1-transfected BT549 cells, leading to Reactive Oxygen Species (ROS) production through mitochondrial depolarization. Similarly, the anti-HER4 antibody C6, which binds to a conformational epitope located on aa 575-592 and 605-620 of HER4 domain IV, induced 4ICD cleavage and retention in mitochondria, and mimicked NRG1-mediated effects on PARP cleavage, ROS production, and mitochondrial membrane depolarization in cancer cells. In vivo, C6 reduced growth of COV434 and HCC187 tumor cell xenografts in nude mice. Biasing 4ICD trafficking to mitochondria with anti-HER4 antibodies to mimic NRG1 suppressor functions could be an alternative anti-cancer strategy.

cancer biology↗

bioRxiv2017

A Low-Cost Multiplex Biomarker Assay Stratifies Colorectal Cancer Patient Samples into Clinically-Relevant Subtypes

Previously, we classified colorectal cancers (CRCs) into five CRCA subtypes with different prognoses and potential treatment responses, using a 786-gene signature. We merged our subtypes and those described by five other groups into four consensus molecular subtypes (CMS) that are similar to CRCA subtypes. Here we demonstrate the analytical development and application of a custom NanoString platform-based biomarker assay to stratify CRC into subtypes. To reduce costs, we switched from the standard protocol to a custom modified protocol (NanoCRCA) with a high Pearson correlation coefficient (>0.88) between protocols. Technical replicates were highly correlated (>0.96). The assay included a reduced robust 38-gene panel from the 786-gene signature that was selected using an in-laboratory developed computational pipeline of class prediction methods. We applied our NanoCRCA assay to untreated CRCs including fresh-frozen and formalin-fixed paraffin-embedded (FFPE) samples (n=81) with matched microarray or RNA-Seq profiles. We further compared the assay results with CMS classification, different platforms (microarrays/RNA-Seq) and gene-set classifiers (38 and 786 genes). NanoCRCA classified fresh-frozen samples (n=39; not including those showing a mixture of subtypes) into all five CRCA subtypes with overall high concordance across platforms (89.7%) and with CMS subtypes (84.6%), independent of tumour cellularity. This analytical validation of the assay shows the association of subtypes with their known molecular, mutational and clinical characteristics. Overall, our modified NanoCRCA assay with further clinical assessment may facilitate prospective validation of CRC subtypes in clinical trials and beyond.\n\nNovelty and ImpactWe previously identified five gene expression-based CRC subtypes with prognostic and potential predictive differences using a 786-gene signature and microarray platform. Subtype-driven clinical trials require a validated assay suitable for routine clinical use. This study demonstrates, for the first time, how molecular CRCA subtype can be detected using NanoString Technology-based biomarker assay (NanoCRCA) suitable for clinical validation. NanoCRCA is suitable for analysing FFPE samples, and this assay may facilitate patient stratification within clinical trials.

cancer biology↗