Biology subjects

CIANFERANI, S.

Publications and source records attributed to CIANFERANI, S..

8 recordsLinked to original sources

bioRxiv2026

Hydrogel confinement enables subcutaneous delivery of vesicant antibody-drug conjugates

Antibody-drug conjugates (ADCs) deliver cytotoxic payloads to tumors with antibody selectivity, yet all approved ADCs are administered by intravenous (IV) infusion despite a strong patient and clinical preference for subcutaneous (SC) delivery. SC administration would reduce treatment burden, but many ADC payloads are vesicants that cause tissue necrosis upon local release, a liability amplified, not mitigated, by the dispersion-enhancing excipients used for SC antibody formulations. We developed an injectable diacetyl-L-tartaric anhydride-functionalized chitosan hydrogel (TACT) that addresses this conflict by confining ADCs within a protective SC depot. TACT is compatible with clinically approved ADC formulations without drug-product modification and provides drug-to-antibody ratio (DAR)-dependent release kinetics that support a quantitative relationship with in vivo absorption timing. In direct comparison, recombinant human hyaluronidase (rHuPH20) co-formulated with vesicant ADCs caused severe tissue necrosis, whereas TACT prevented macroscopic injury while preserving antitumor efficacy comparable to intravenous dosing. TUNEL staining of injection sites showed that TACT attenuated peri-depot apoptotic injury 3-fold relative to T-DM1 alone and 2-fold relative to rHuPH20 co-formulation. In non-human primates, SC TACT achieved 78% relative bioavailability for total trastuzumab, reduced peak circulating T-DM1 catabolite (free DM1) exposure 7.6-fold compared to IV administration and produced only transient, self-resolving cutaneous reactions. These results identify depot-mediated confinement as a viable alternative to excipient-mediated dispersion for SC delivery of vesicant ADCs, demonstrated here for trastuzumab-based conjugates across two approved ADC drug products (T-DM1 and T-DXd, with non-cleavable MCC and cleavable peptide linkers), with supporting validation in a custom cleavable monomethyl auristatin E (MMAE) series. Additional validation with enfortumab vedotin (EV), a Nectin-4-targeting MMAE ADC, supported the applicability of this strategy beyond trastuzumab-based conjugates.

cancer biology↗

bioRxiv2026

Effects of polystyrene and polylactide nanoparticles on macrophages under a repeated exposure mode

Micro and nanoplastics are pollutants which concentration in different biotopes increases continuously over time, which poses the question of their potential effects on health. In animals, these micro and nanoplastics are recognized as particulate materials and thus handled by macrophages, which are therefore a key cell type to study. Most studies have used an experimental scheme in which the cells are exposed to a single dose of plastics, with a readout made immediately after exposure. However, this classical experimental scheme does not take into account the impact of biopersistence, nor the potential cellular adaptation that may take place when cells are exposed repeatedly to a low dose of plastics. We thus used a repeated exposure scheme, in order to better take into account these phenomena. Within this frame, we compared the macrophages responses to a persistent nanoplastic, i.e. polystyrene nanoparticles and to a biodegradable nanoplastic, i.e. polylactide, by a combination of proteomic and targeted experiments. Our results show that under this repeated exposure scheme, the proteome changes were of a lesser (for PS) or similar (for PLA) extent than under the acute exposure mode, indicating cell adaptation. However, PLA particles induced mitochondrial dysfunction and depression of response to bacterial molecules perceived as danger signals, such as lipopolysaccharide. Polystyrene nanoparticles also induced a slight alteration of the immune functions of macrophages. This indicates harmful effects even in the repeated exposure scheme.

pharmacology and toxicology↗

bioRxiv2025

Toward Site-Specific Characterization of Structural Perturbations on Glycosylated Fc Using NMR at Natural Abundance

Monoclonal antibodies (mAbs) are leading therapeutic agents due to their high specificity and limited side effects. Ensuring their structural integrity under stress and maintaining batch consistency require robust quality control. Methyl 2D NMR has emerged as a powerful tool to probe mAb structure at natural isotopic abundance, enabling spectral fingerprint comparisons across production batches to detect subtle structural changes. However, extracting atomic-level structural information requires assignment of methyl resonances to their amino acids. While such assignments are available for several antigen-binding fragments (Fabs), no comprehensive assignment has been reported for the crystallisable fragment (Fc). In this study, we present the methyl group assignment of the 50-kDa Fc fragment of an immunoglobulin G1 (IgG1) antibody. Using cell-free expression, strategic isotopic labelling, and high-quality 2D and 3D NMR experiments, we successfully assigned 94% of methyl resonances of a non-glycosylated Fc. Given that therapeutic mAbs are typically produced in Chinese Hamster Ovary (CHO) cells, we transferred this assignment to the methyl spectrum of a glycosylated Fc fragment obtained by the enzymatic cleavage of a CHO-produced mAb at natural abundance, achieving 83% assignment coverage. This assignment was then used to investigate the impact of methionine oxidation on Fc structure at atomic resolution using NMR. The methyl group assignment transforms 2D methyl NMR fingerprinting into a powerful tool for quality control. It enables the direct comparison of spectra acquired on mAbs produced at natural abundance, allowing the detection and localisation of chemical modifications and structural changes without the need for isotopic labelling. This approach offers a robust solution for monitoring the structural integrity of therapeutic antibodies throughout development and manufacturing.

biochemistry↗

bioRxiv2025

Effects of true to life polyethylene terephthalate and polycaprolactone nanoparticles on macrophages under a repeated exposure mode.

Micro and nanoplastics are pollutants which concentration in different biotopes increases continuously over time, which poses the question of their potential effects on health. In animals, these micro and nanoplastics are recognized as particulate materials and thus handled by macrophages, either directly in the case of lung exposure, or after the particles have crossed the epithelial barriers in case of oral or dermal exposure. It is thus important to study the potential effects of micro and nanoplastics on macrophages. Most studies have used an experimental scheme in which the cells of interest are exposed to a single dose of plastics, and where the readout of the studied parameters is made immediately after exposure. However, this classical experimental scheme does not take into account the impact of biopersistence, nor the potential cellular adaptation that may take place when cells are exposed repeatedly to a low dose of plastics. We thus used a repeated exposure scheme, in order to better take into account these phenomena. Within this frame, we compared the macrophages responses to a persistent nanoplastic, i.e. true-to-life polyethylene terephthalate nanoparticles and to a biodegradable nanoplastic, i.e. polycaprolactone, by a combination of proteomic and targeted experiments. Our results show that under this repeated exposure scheme, the proteome changes were of a lesser extent than under the acute exposure mode, indicating cell adaptation. However, polyethylene terephthalate nanoparticles induced oxidative stress and a pro-inflammatory response, while polycaprolactone nanoparticles induced a depression of macrophages functions, indicating harmful effects even in the repeated exposure scheme.

pharmacology and toxicology↗

bioRxiv2024

Biobased, Biodegradable but not bio-neutral: about the effects of polylactic acid nanoparticles on macrophages

Plastics are persistent pollutants, because of their slow degradation, which suggests that they may lead to cumulative and/or delayed adverse effects due to their progressive accumulation over time. Macroplastics produced by human activity are released in the environment, where they degrade into micro and nanoplastics that are very easily uptaken by a wide variety of organisms, including humans. Microplastics and nanoplastics being particulates, they are handled in the body by specialized cells such as macrophages (or their evolutionary counterparts), where they can elicit a variety of responses. One solution to alleviate the problems due to biopersistence, such as accumulation over life, would be to use biodegradable plastics. One of the emerging biodegradable plastics being polylactide, we decided to test the responses of macrophages to polylactide nanoparticles, using a combination of untargeted proteomics and targeted validation experiments. Proteomics showed important adaptive changes in the proteome in response to exposure to polylactide nanoparticles. These changes affected for example mitochondrial, cytoskeletal and lysosomal proteins, but also proteins implicated in immune functions or redox homeostasis. Validation experiments showed that many of these changes were homeostatic, with no induced oxidative stress and no gross perturbation of the mitochondrial function. However, polylactide particles altered the immune functions such as phagocytosis (-20%) or cytokine production (2-fold increase for TNF production), which may translate into a decreased ability to macrophages to respond to bacterial infections. Furthermore, polylactide particles also induced moderate cross-toxicity with some quinones such as phenanthrene quinone, a combustion by-product that is a suspected carcinogen.

pharmacology and toxicology↗

bioRxiv2021

Large-scale conformational changes of FhaC provide insights into the two-partner secretion mechanism

The Two-Partner secretion pathway mediates protein transport across the outer membrane of Gram-negative bacteria. TpsB transporters belong to the Omp85 superfamily, whose members catalyze protein insertion into, or translocation across membranes without external energy sources. They are composed of a transmembrane {beta} barrel preceded by two periplasmic POTRA domains that bind the incoming protein substrate. Here we used an integrative approach combining in vivo assays, mass spectrometry, nuclear magnetic resonance and electron paramagnetic resonance techniques suitable to detect minor states in heterogeneous populations, to explore transient conformers of the TpsB transporter FhaC. This revealed substantial, spontaneous conformational changes with a portion of the POTRA2 domain coming close to the lipid bilayer and surface loops. Specifically, the amphipathic {beta} hairpin immediately preceding the first barrel strand can insert into the {beta} barrel. We propose that these motions enlarge the channel and hoist the substrate into it for secretion. An anchor region at the interface of the {beta} barrel and the POTRA2 domain stabilizes the transporter in the course of secretion. Our data propose a solution to the conundrum how these transporters mediate protein secretion without the need for cofactors, by utilizing intrinsic protein dynamics.

biophysics↗

bioRxiv2020

An original structural fold underlies the multitask P1, a silencing suppressor encoded by the Rice yellow mottle virus

ABSTRACTThe Rice Yellow Mottle sobemovirus (RYMV) belongs to the most damaging pathogens devastating rice fields in Africa. P1, a key protein for RYMV, was reported as a potent RNAi suppressor counteracting RNA silencing in plant reporter systems. Here we describe the complete 3D structure and dynamics of P1. Its N-terminal region contains ZnF1, a structural CCCC-type zinc finger strongly affine to zinc and a prominent short helix, rendering this region poorly amenable to structural changes. P1 C-terminal region contains ZnF2, an atypical HCHC-type ZnF that does not belong to any existing class of Zn finger proteins. ZnF2 appeared much less affine to zinc and more sensitive to oxidizing environments than ZnF1, and may serve as a sensor of plant redox status. The structure helped us to identify key residues essential for RYMV infectivity and spread in rice tissues through their participation in P1 oligomerization and folding. Altogether, our results provide the first complete structure of an antiviral silencing suppressor encoded by a virus infecting rice and highlight P1 structural and dynamical properties that may serve RYMV functions to infect and invade its host plant.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pathology↗

bioRxiv2020

Middle-level IM-MS and CIU experiments for improved therapeutic immunoglobulin isotype fingerprinting

Currently approved therapeutic monoclonal antibodies (mAbs) are based on immunoglobulin G (IgG) 1, 2 or 4 isotypes, which differ in their specific inter-chains disulfide bridge connectivities. Different analytical techniques have been reported for mAb isotyping, among which native ion mobility mass spectrometry (IM-MS) and collision induced unfolding (CIU) experiments. However, mAb isotyping by these approaches is based on detection of subtle differences and thus remains challenging at the intactlevel. We report here on middle-level (after IdeS digestion) IM-MS and CIU approaches to afford better differentiation of mAb isotypes. Our method provides simultaneously CIU patterns of F(ab)2 and Fc domains within a single run. Middle-level CIU patterns of F(ab)2 domains enable more reliable classification of mAb isotypes compared to intact level CIU, while CIU fingerprints of Fc domains are overall less informative for mAb isotyping. F(ab)2 regions can thus be considered as diagnostic domains providing specific CIU signatures for mAb isotyping. Benefits of middle-level IM-MS and CIU approaches are further illustrated on the hybrid IgG2/IgG4 eculizumab. While classical analytical techniques led to controversial results, middle-level CIU uniquely allowed to face the challenge of eculizumab << hybridicity >>, highlighting that its F(ab)2 and Fc CIU patterns corresponds to an IgG2 and an IgG4, respectively. Altogether, the middle-level CIU approach is more clear-cut, accurate and straightforward for canonical but also more complex, engineered next generation mAb formats isotyping. Middle-level CIU thus constitutes a real breakthrough for therapeutic protein analysis, paving the way for its implementation in R&D laboratories.

biophysics↗