Anticorps à domaine unique | EPFL Graph Search

vignette |Diagramme en ruban d'un domaine VH de lama.La grande boucle CDR3 est de couleur orangée. Un anticorps à domaine unique (sdAb, de l'single-domain antibody), également connu sous le nom de nanocorps, est un fragment d'anticorps composé d'un seul domaine d'anticorps variable monomère. Comme un anticorps entier, il est capable de se lier sélectivement à un antigène spécifique. Avec un poids moléculaire de seulement 12 à 15 kDa, les anticorps à domaine unique sont beaucoup plus petits que les anticorps communs () qui sont composés de deux chaînes de protéines lourdes et de deux chaînes légères, et même plus petits que les fragments Fab (~ 50 kDa, une chaîne légère et une demi-chaîne lourde) et des fragments variables à chaîne unique (~ 25 kDa, deux domaines variables, l'un issu d'une chaîne légère et l'autre d'une chaîne lourde). Les premiers anticorps à domaine unique ont été conçus à partir d' anticorps à chaîne lourde trouvés chez les camélidés ; ils sont appelés fragment

Systematic analysis of biomolecular binding reactions using microfluidic systems

Amir Shahein

Transcription factor binding to a single binding site on DNA and its functional consequence in a promoter context are beginning to be relatively well understood. However, binding to clusters of sites has yet to be characterized in depth, and the functional relevance of binding site clusters remains uncertain. We employed a high-throughput biochemical method to characterize transcription factor binding to clusters varying across a range of affinities and configurations. We found that transcription factors can bind concurrently to overlapping sites, challenging the notion of binding exclusivity. Furthermore, compared to an individual high-affinity binding site, small clusters with binding sites an order of magnitude lower in affinity give rise to higher mean occupancies at physiologically-relevant transcription factor concentrations in vitro. To assess whether the observed in vitro occupancies translate to transcriptional activation in vivo, we tested low-affinity binding site clusters by inserting them into a synthetic minimal CYC1 and the native PHO5 S. cerevisiae promoter. In the minCYC1 promoter, clusters of low-affinity binding sites can generate transcriptional output comparable to a promoter containing three consensus binding sites. In the PHO5 promoter, replacing the native Pho4 binding sites with clusters of low-affinity binding sites recovered activation of these promoters as well. This systematic characterization demonstrates that clusters of low-affinity binding sites achieve substantial occupancies, and that this occupancy can drive expression in eukaryotic promoters. In the antibody discovery phase, before advancing a particular antibody sequence to clinical trials, a large number of candidates are characterized for properties of therapeutic interest. Above all, this includes their ability to bind to the target antigen. Standard processes currently in use to characterize antibody binding are either only semi-quantitative (ELISA), or low-throughput (SPR). We developed an experimental process to first retrieve DNA encoding promising synthetic antibody variants directly from the output pools of phage and ribosome display methods, followed by expressing these variants on a microfluidic chip for characterization in a fluorescence-based binding assay. We applied our method to characterize the binding of retrieved Sybody variants against the Spike trimer and RBD domain of the SARS-CoV-2 variant. After an initial proof-of-concept, we improved our method by removing bottlenecks, reducing costs, and incorporating a large degree of process automation to enable measuring binding affinities at scale.

EPFL2022

Humoral Responses Against Variants of Concern by COVID-19 mRNA Vaccines in Immunocompromised Patients

Céline Pellaton, Madeleine Suffiotti, Didier Trono

IMPORTANCE There are limited comparative data on the durability of neutralizing antibody (nAb) responses elicited by messenger RNA (mRNA) vaccines against the SARS-CoV-2 variants of concern (VOCs) in immunocompromised patients and healthy controls. OBJECTIVE To assess the humoral responses after vaccination with BNT162b2 (Pfizer- BioNTech) or mRNA-1273 (Moderna) vaccines. DESIGN, SETTING, AND PARTICIPANTS In this prospective, longitudinal monocentric comparative effectiveness study conducted at the Lausanne University Hospital, binding IgG anti-spike antibody and nAb levels were measured at 1 week, 1 month, 3 months, and 6 months after vaccination with mRNA-1273 (24.6% of participants) or BNT162b2 (753% of participants). INTERVENTIONS All participants received 2 doses of either mRNA-1273 or BNT162b2 vaccines 4 to 6 weeks apart. MAIN OUTCOMES AND MEASURES The primary outcome of the study was the persistence of nAb responses against the original, nonvariant SARS-CoV-2 (2019-nCoV) and different VOCs at 6 months after vaccination. Key secondary outcomes were associations of the type of mRNA vaccine, the underlying disease, and the treatment with the response to vaccination. RESULTS Among the 841 participants enrolled between January 14 and August 8, 2021, the patient population comprised 637 participants (mean [SD] age, 61.8 [13.7] years; 386 [60.6%] female), and the healthy control population comprised 204 participants (mean [SD] age, 45.9 [12.0] years; 144 [70.6%] female). There were 399 patients with solid cancers, 101 with hematologic cancers. 38 with solid organ transplants, 99 with autoimmune diseases, and 204 healthy controls. More than 15 000 nAb determinations were performed against the original, nonvariant 2019-nCoV and the Alpha, Beta, Gamma, and Delta variants. The proportions of nAbs and their titers decreased in all study groups at 6 months after vaccination, with the greatest decreases for the Beta and Delta variants. For Beta, the proportion decreased to a median (SE) of 392% (5.5%) in those with hematologic cancers, 44,8% (2.7%) in those with solid cancers, 23.1% (8.3%) in those with solid organ transplants, and 22.7% (4.8%) in those with autoimmune diseases compared with 52.1% (4.2 degrees A.) in healthy controls. For Delta, the proportions decreased to 41.8% (5.6%) in participants with hematologic cancer, 51.9% (2.7%) in those with solid cancers, 26.9% (8.7%) in those with solid organ transplants, and 30.7% (5.3%) in those with autoimmune diseases compared with 56.9% (4.1%) healthy controls. Neutralizing antibody titers decreased 3.5- to 5-fold between month 1 and month 6, and the estimated duration of response was greater and more durable among those participants vaccinated with mRNA-1273. In participants with solid cancers, the estimated duration of nAbs against the Beta variant was 221 days with mRNA-1273 and 146 days with BNT162b2, and against the Delta variant, it was 226 days with mRNA-1273 and 161 with BNT162b2. The estimated duration of nAbs in participants with hematologic cancers was 113 and 127 days against Beta and Delta variants, respectively. CONCLUSIONS AND RELEVANCE This comparative effectiveness study suggests that approximately half of patients with hematologic cancers and solid cancers, about 70% of patients with solid organ transplants or autoimmune diseases, and 40% of healthy controls have lost nAbs against the circulating VOCs at 6 months after vaccination. These findings may be helpful for developing the best boosting vaccination schedule especially in immunocompromised patients.

AMER MEDICAL ASSOC2022

Expression, Biochemistry, and Stabilization with Camel Antibodies of Membrane Proteins: Case Study of the Mouse 5-HT3 Receptor

Cédric Deluz, Luigino Grasso, Ghérici Hassaïne, Rudolf Hovius, Sonja Minniberger, Henning Paul-Julius Stahlberg, Horst Vogel, Romain Wyss

There is growing interest in the use of mammalian protein expression systems, and in the use of antibodyderived chaperones, for structural studies. Here, we describe protocols ranging from the production of recombinant membrane proteins in stable inducible cell lines to biophysical characterization of purified membrane proteins in complex with llama antibody domains. These protocols were used to solve the structure of the mouse 5-HT3 serotonin receptor but are of broad applicability for crystallization or cryoelectron microscopy projects.

Springer New York2017