1. Instruct-ERIC network : biophysical characterization of antigen-nanobody complexesClaudia D'Ercole, 2024, published scientific conference contribution abstract Abstract: Forest environments are exposed to multiple stressful factors of both abiotic and biotic nature which may lead to their massive decline [1]. Understanding the molecular mechanisms of specific stress conditions and monitoring the fluctuations of reliable forest plant biomarkers with affordable methods would be instrumental for assessing stress levels over the time. Ascorbate peroxidase (APX) represents a suitable plant biomarker. APX is a hydrogen peroxide-scavenging enzyme the critical role of which has been described in several plants, both herbaceous and woody. Its activity generally increases under oxidative stress during which its peroxide detoxifying function is part of the wider ascorbate-glutathione cycle [2]. The development of reagents to detect such fluctuations would help the evaluation of plant physiological conditions. In this study, nanobodies (Nbs) targeting APX have been identified. Nbs correspond to the variable domain of heavy chain-only antibodies derived from camelids. They are small (15 kDa), stable, and can be easily produced in bacteria fused to different protein tags according to the downstream applications [3]. After their isolation by biopanning against soluble APX, they have been produced and underwent a biophysical characterization in combination with their antigen (APX-Nb complex) to identify the best binders in terms of stability and affinity. The protein complex characterization was supported by Instruct-ERIC and mainly performed at the BIOCEV institute of Prague. Data from Mass Photometry and Dynamic Light scattering evidenced the formation of the protein complexes, whereas the preliminary data of Hydrogen-Deuterium Exchange Mass Spectrometry, performed with the aim of identifying the residues involved in the paratope/epitope interface, were insufficient to clarify the issue and rather suggested that the interaction has low affinity. This indication was then confirmed by ELISA assay. The combination of multiple methods allowed a comprehensive sample characterization which will require further structural analyses to provide a complete picture of the APX-Nb complex.
[1] G. Marie. B. C. M. B. C. Walters, “Forest decline and tree mortality in a southeastern Ohio oak-hickory forest,” Ohio Journal of Science , vol. 97, 1997.
[2] O. Chew, J. Whelan, and A. H. Millar, “Molecular Definition of the Ascorbate-Glutathione Cycle in Arabidopsis Mitochondria Reveals Dual Targeting of Antioxidant Defenses in Plants,” Journal of Biological Chemistry, vol. 278, no. 47, 2003, doi: 10.1074/jbc.M307525200.
[3] S. Muyldermans, “A guide to: generation and design of nanobodies,” FEBS J, vol. 288, no. 7, pp. 2084–2102, Apr. 2021, doi: 10.1111/febs.15515. Keywords: nanobody, ascorbate peroxidase, plant stress, protein complex, biophysical methodologies Published in RUNG: 31.05.2024; Views: 1102; Downloads: 0 This document has many files! More... |
2. Design of nanobody-based bispecific constructs by in silico affinity maturation and umbrella sampling simulationsZixuan Bai, Jiewen Wang, Jiaqi Liu, Haibin Yuan, Ping Wang, Miao Zhang, Yuanhang Feng, Xiangtong Cao, Xiangan Cao, Guangbo Kang, Ario De Marco, He Huang, 2023, original scientific article Keywords: nanobody rational design, bispecific binders, protein activity inhibition Published in RUNG: 03.01.2023; Views: 2112; Downloads: 7 Full text (8,75 MB) This document has many files! More... |
3. Research progress and applications of multivalent, multispecific and modified nanobodies for disease treatmentJiewen Wang, Guangbo Kang, Haibin Yuan, Xiaocang Cao, He Huang, Ario De Marco, 2022, original scientific article Keywords: nanobody multimers, immunomodulation, intrabodies, imaging, nanobody functionalization Published in RUNG: 18.01.2022; Views: 2557; Downloads: 156 Link to full text This document has many files! More... |
4. Effect of Humanizing Mutations on the Stability of the Llama Single-Domain Variable RegionMiguel Soler, Barbara Medagli, Jiewen Wang, Sandra Oloketuyi, Gregor Bajc, He Huang, Sara Fortuna, Ario De Marco, 2021, original scientific article Keywords: nanobody framework, modeling, nanobody humanization, CDR grafting Published in RUNG: 27.01.2021; Views: 3171; Downloads: 68 Full text (2,00 MB) |
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6. Identification of New DR5 Agonistic Nanobodies and Generation of Multivalent Nanobody Constructs for Cancer TreatmentGolnaz Sadeghnezhad, Ema Romao, Robert Alvin Bernedo-Navarro, Sam Massa, Khosro Khajeh, Serge Muyldermans, Sadegh Hassania, 2019, original scientific article Keywords: death receptor 5, nanobody, TRAIL, cancer therapy, apoptosis Published in RUNG: 01.10.2019; Views: 3784; Downloads: 0 This document has many files! More... |
7. Nanobodies: towards rational design of immune-reagentsArio De Marco, 2017, published scientific conference contribution abstract (invited lecture) Abstract: Antibodies are irreplaceable reagents in both research and clinical practice. Despite their relevance, the structural complexity of conventional mono- and polyclonal antibodies (IgG) has always been a limit for their engineering towards reagents optimized for specific applications, such as in vivo diagnostics and therapy. Furthermore, their isolation is time consuming, their production expensive, and their functionalization results often in heterogeneous macromolecule populations. These drawbacks promoted the search for both innovative antibody isolation strategies and alternative scaffolds. In vitro panning of pre-immune collections of recombinant antibody fragments allows for the simple and fast recovery of binders. Since they did not undergo somatic maturation, their affinity for targets can be insufficient but on the other hand they can be rapidly mutated by standard molecular biology techniques to generate second-generation antibodies among which to identify clones with improved characteristics. Both stochastic and rational methods have been proposed for the optimization process. Random mutagenesis followed by panning at stringent conditions has been successful used to select binders with improved physical characteristics. Rational methods try to identify in silico key residues involved in the regulation of specific antibody features, such as stability or binding affinity. The accuracy of these methods usually depends on the calculation resources. In this perspective, smaller molecules can be analyzed “better” than larger because of their restricted number of residues. Nanobodies small dimensions have been long appreciated since enable better tissue penetration, shorter clearance time, higher yields. Now it becomes evident that this characteristic makes them also optimal objects for modeling. Keywords: recombinant antibody modeling, nanobody engineering, molecular dynamics and docking Published in RUNG: 21.03.2018; Views: 5576; Downloads: 0 This document has many files! More... |
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