Nanobody Discovery Summer School:
Current & Future Methods

Bor Krajnik^1,2, Uroš Petrovič^1,2 — ¹Jožef Stefan Institute, Department of Molecular and Biomedical Sciences, Ljubljana, Slovenia; ²University of Ljubljana, Biotechnical Faculty, Ljubljana, Slovenia

Intracellular binders provide a powerful approach for perturbing protein function. Targeting functional sites, such as active sites and protein–protein interaction interfaces, enables a more nuanced understanding of protein function than gene knockout or knockdown. Nanobodies are attractive scaffolds for such applications as their single-domain architecture and high stability make them well suited for intracellular use. A major limitation in nanobody discovery is the lack of control over the targeted epitope. Advances in deep-learning based structure prediction and protein design have enabled the design of epitope specific nanobodies, addressing this limitation. Using BoltzGen, a generative model for binder design, we have designed nanobodies against a panel of human intracellular protein targets involved in diverse cellular processes, including proteostasis, nuclear transport and translation. The resulting designs will be tested for target binding and functional perturbation as intrabodies in the yeast Saccharomyces cerevisiae. By integrating nanobody design with intracellular assays in yeast we aim to expand the number of intrabodies against human proteins.

Background and Objectives. This project focuses on the generation and development of single-domain antibody fragments (nanobodies / VHH) as novel tools for the diagnosis and therapy of major human diseases, including cancer and Alzheimer's disease. Due to their small size, high stability, and excellent tissue penetration, these nanobodies offer significant advantages over conventional antibodies for targeted biomedical applications.

Current Progress & Methodology. The current phase of the project is dedicated to targeting Galectin-3 (Gal3), a key biomarker associated with disease progression. The project has successfully advanced through the following stages: an immune/synthetic nanobody library was generated (Library Generation & Selection); using the phage display technique, the library was screened against Gal3, leading to the successful selection and isolation of 4 distinct nanobody clusters (Phage Display Screening).

Current and Future Work. We are currently in the biophysical characterization phase of these selected nanobodies to evaluate their binding affinity, stability, and specificity toward Gal3. The ultimate goal of this research is to validate these candidates for downstream applications in advanced diagnostics or as therapeutic agents.

Chaeyeon Park¹, Rajib Schubert^1,* — ¹Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea

Tau is a soluble microtubule-associated protein essential for maintaining axonal integrity and cytoskeletal stability in healthy neurons. In pathological states such as Alzheimer's disease, hereditary mutations and conformational shifts induce soluble tau to form various pathogenic tau variants and toxic, insoluble filamentous aggregates. Accurate visualization and non-invasive detection of these pathological tau species are crucial for early diagnosis, tracking disease progression, and monitoring therapeutic responses. Although monoclonal antibody-based imaging agents are widely explored, their diagnostic utility is frequently constrained by high molecular weight, poor blood-brain barrier permeability, and prolonged systemic circulation times that lower target-to-background contrast. To overcome these biophysical limitations, we propose a novel molecular imaging strategy utilizing a Designed Ankyrin Repeat Protein (DARPin)-based probe platform. Characterized by high thermal stability, a small molecular footprint, rapid clearance kinetics, and high modularity, DARPins offer an exceptional scaffold for target-specific molecular imaging. In this study, we systematically engineer the hypervariable binding surfaces of the DARPin scaffold to generate high-affinity candidates capable of recognizing general tau and distinct pathological tau variants. We evaluate these engineered DARPin probes in Alzheimer's disease mouse models to assess their brain permeability, binding specificity, and imaging efficacy in vivo. This modular platform represents a promising, highly bioavailable diagnostic tool to visualize pathological tau dynamics and advance precision imaging in Alzheimer's disease.

Secondary metabolites of natural origin represent an important source of bioactive compounds with diverse biological effects, among which antimicrobial activity is particularly prominent. In my PhD project we have so far isolated several groups of secondary metabolites — primarily tannins and carotenoids — from various natural sources, including bacteria and plants. These compounds are systematically investigated for their antimicrobial activity as well as their ability to bind to target proteins.

A central focus of our research is the elucidation of the molecular mechanisms underlying interactions between these isolated compounds and proteins. However, the precise binding sites and structural characteristics of the involved protein motifs remain unknown.

So far, we are analysing the binding of these compounds and model proteins using circular dichroism (CD) spectroscopy, which allows monitoring of changes in the secondary and tertiary structure of proteins upon ligand interaction, and nuclear magnetic resonance (NMR), which allows for determination of binding constants even for weak binders. Using this approach, we will evaluate the binding between proteins and secondary metabolites and assess its impact on protein structure, including thermal stability and denaturation temperature, thereby contributing to a deeper understanding of the molecular mechanisms underlying their biological effects. Based on the preliminary data we can see that the strength of the interactions of these ligands varies based on the surface properties of the model proteins. Next, we want to characterise the structural motifs that are crucial for ligand binding and determine which mutations improve the binding affinity.

To this end we would like to try the use of nanobodies, which represent a powerful tool for the specific recognition and binding of target protein structures, especially since they are small enough to be studied by NMR — in combination with strategies based on affinity chromatography principles, mutated nanobodies can serve as selective ligands for the isolation of proteins with the highest affinity for the studied compounds. This approach enables the enrichment of proteins sharing common structural motifs that are hypothesized to be optimal binding sites for specific secondary metabolites.

Mariagrazia Scarano^1,2, Elian Dupré², Idir Malki², Justine Mortelecque², Céline Reverdy¹, Isabelle Landrieu², Jean-Christophe Rain¹ — ¹Hybrigenics Services, 1 rue Pierre Fontaine, Evry; ²Univ. Lille, CNRS UMR9031 – BSI, Integrative Structural Biology, Institut Pasteur de Lille, F-59000 Lille, France

The protein tyrosine kinase (PTK) Fyn is involved in cell proliferation and alternative splicing. Its dysregulation has been linked to carcinogenesis and the generation of abnormally phosphorylated Tau, a hallmark of several neurodegenerative disorders including Alzheimer's disease. The interaction between Tau and the SH3 domain of Fyn has emerged as a potential therapeutic target, as it is implicated in the pathological cascade linking Tau to neuronal damage.

We previously identified H6-1, a VHH against the Fyn SH3 domain, through three rounds of phage display using a synthetic VHH library against full-length mouse Fyn. Epitope mapping by NMR and X-ray crystallography revealed that H6-1 targets the RT loop and N-Src loop of the Fyn SH3 domain, the same region engaged by Tau proline-rich motifs and Sam68. However, H6-1 displayed moderate affinity toward the Fyn SH3 domain, as demonstrated by both yeast surface display and SPR measurements, motivating an affinity maturation campaign focused on CDR3 engineering to identify variants with improved binding properties.

To improve binding properties, a CDR3-focused library was constructed by NNK randomization and screened by yeast surface display over four rounds of FACS-based selection. Among the affinity-matured variants, clone F12 showed a two to 3-fold improvement in binding affinity by SPR, high thermostability, and better intracellular interaction with both Fyn's SH3 and full-length Fyn in yeast two-hybrid assays. NMR competition experiments further demonstrated that F12 prevents the Tau-Fyn SH3 interaction without directly binding Tau, confirming its potential as an intracellular inhibitor of this pathological interaction.

1. Kurochkina, N. & Guha, U. SH3 domains: modules of protein-protein interactions. Biophys. Rev. 5, 29-39 (2012).
2. Bielli, P., Busà, R., Paronetto, M. P. & Sette, C. The RNA-binding protein Sam68 is a multifunctional player in human cancer. Endocr. Relat. Cancer 18, R91–R102 (2011).
3. Lee, G. et al. Phosphorylation of Tau by Fyn: Implications for Alzheimer's Disease. J. Neurosci. 24, 2304–2312 (2004).

Microcystins are toxic cyanobacterial metabolites that can contaminate freshwater systems, aquaculture environments, and seafood, posing significant risks to environmental and food safety. The development of sensitive and specific detection methods requires robust biorecognition elements with high affinity for these toxins. This work aims to develop recombinant nanobodies targeting microcystin through an integrated pipeline combining computational modelling, phage display, recombinant expression, and assay development. Initially, in silico approaches, including de novo binder design, molecular docking, and molecular simulations, will be used to investigate nanobody–microcystin interactions and identify promising candidate binders. A nanobody phage display library will subsequently undergo biopanning against relevant microcystin antigens, followed by amplification, preliminary ELISA screening, and sequencing of positive clones. The identified nanobody sequences will be further characterized through physicochemical analysis and structural prediction to evaluate properties such as molecular weight, isoelectric point, stability, hydrophobicity, and three-dimensional structure. Promising nanobody candidates will then be subcloned, recombinantly expressed in Escherichia coli, purified using affinity chromatography, and characterized by SDS-PAGE. Finally, the selected nanobodies will be evaluated for their applicability in the development of a lateral flow assay for rapid microcystin detection. The developed nanobody-based lateral flow platform could support sensitive and rapid monitoring of microcystin in water, aquaculture environments, and seafood.

The blood brain barrier (BBB) is a major obstacle to the delivery of therapeutic biologics to the central nervous system. Receptor-mediated transcytosis, including approaches targeting transferrin receptor 1 (TfR1), offers a potential strategy for facilitating the transport of large therapeutic molecules across the BBB. We are exploring phage display as an approach to identify TfR1-binding Fab fragments that could potentially serve as BBB shuttle modules. TfR1 protein has been prepared as the target antigen, and our planned workflow involves iterative biopanning, phage ELISA, sequence analysis, and recombinant Fab production.

Vicky Chouhan, PhD Researcher, University of Nova Gorica, Slovenia

Coniferous trees are long-lived organisms that continuously experience environmental stresses such as drought, temperature fluctuations, salinity, and pathogen pressure. Their ability to survive and adapt to these changing conditions is influenced not only by genetic variation but also by epigenetic mechanisms. Plant epigenetics involves heritable or reversible changes in gene regulation without alterations in the underlying DNA sequence and includes DNA methylation, histone modifications, chromatin remodeling, and regulation by non-coding RNAs. This research aims to investigate stress-associated epigenetic changes in selected conifer species and to identify reliable molecular biomarkers associated with different stress conditions. The study will combine molecular and epigenomic approaches to examine changes in DNA methylation, histone modifications, chromatin structure, and the expression of stress-responsive genes. Comparative analysis of stressed and control plants will be used to identify epigenetic signatures that correlate with specific environmental conditions. The identified biomarkers may provide valuable information about the molecular mechanisms underlying stress adaptation and resilience in long-lived conifers. In the longer term, such biomarkers could contribute to early detection of environmental stress, improved monitoring of forest health, and the development of strategies for conserving and managing conifer populations under changing climatic conditions.

Keywords: Plant epigenetics; conifers; environmental stress; DNA methylation; histone modification; chromatin remodeling; epigenetic biomarkers; stress adaptation.