Precise epitope determination of therapeutic antibodies is of great value as it allows for further comprehension of mechanism of action, therapeutic responsiveness prediction, avoidance of unwanted cross reactivity, and vaccine design. The golden standard for discontinuous epitope determination is the laborious X-ray crystallography method. Here, we present a combinatorial method for rapid mapping of discontinuous epitopes by mammalian antigen display, eliminating the need for protein expression and purification. The method is facilitated by automated workflows and tailored software for antigen analysis and oligonucleotide design. These oligos are used in automated mutagenesis to generate an antigen receptor library displayed on mammalian cells for direct binding analysis by flow cytometry. Through automated analysis of 33930 primers an optimized single condition cloning reaction was defined allowing for mutation of all surface-exposed residues of the receptor binding domain of SARS-CoV-2. All variants were functionally expressed, and two reference binders validated the method. Furthermore, epitopes of three novel therapeutic antibodies were successfully determined followed by evaluation of binding also towards SARS-CoV-2 Omicron BA.2. We find the method to be highly relevant for rapid construction of antigen libraries and determination of antibody epitopes, especially for the development of therapeutic interventions against novel pathogens.
© 2024. The Author(s).

The entry of the SARS-CoV-2 virus into a human host cell begins with the interaction between the viral spike protein (S protein) and human angiotensin-converting enzyme 2 (hACE2). Therefore, a possible strategy for the treatment of this infection is based on inhibiting the interaction of the two abovementioned proteins. Compounds that bind to the SARS-CoV-2 S protein at the interface with the alpha-1/alpha-2 helices of ACE2 PD Subdomain I are of particular interest. We present a stepwise optimisation of helical peptide foldamers containing trans-2-aminocylopentanecarboxylic acid residues as the folding-inducing unit. Four rounds of optimisation led to the discovery of an 18-amino-acid peptide with high affinity for the SARS-CoV-2 S protein (Kd = 650 nM) that inhibits this protein-protein interaction with IC50 = 1.3 µM. Circular dichroism and nuclear magnetic resonance studies indicated the helical conformation of this peptide in solution.

In order to circumvent the limited access and donor variability of human primary alveolar cells, directed differentiation of human pluripotent stem cells (hiPSCs) into alveolar-like cells, provides a promising tool for respiratory disease modeling and drug discovery assays. In this work, a unique, miniaturized 96-Transwell microplate system is described where hiPSC-derived alveolar-like cells were cultured at an air-liquid interface (ALI). To this end, hiPSCs were differentiated into lung epithelial progenitor cells (LPCs) and subsequently matured into a functional alveolar type 2 (AT2)-like epithelium with monolayer-like morphology. AT2-like cells cultured at the physiological ALI conditions displayed characteristics of AT2 cells with classical alveolar surfactant protein expressions and lamellar-body like structures. The integrity of the epithelial barriers between the AT2-like cells was confirmed by applying a custom-made device for 96-parallelized transepithelial electric resistance (TEER) measurements. In order to generate an IPF disease-like phenotype in vitro, the functional AT2-like cells were stimulated with cytokines and growth factors present in the alveolar tissue of IPF patients. The cytokines stimulated the secretion of pro-fibrotic biomarker proteins both on the mRNA (messenger ribonucleic acid) and protein level. Thus, the hiPSC-derived and cellular model system enables the recapitulation of certain IPF hallmarks, while paving the route towards a miniaturized medium throughput approach of pharmaceutical drug discovery.
© 2021. The Author(s).

In order to overcome the challenges associated with a limited number of airway epithelial cells that can be obtained from clinical sampling and their restrained capacity to divide ex vivo, miniaturization of respiratory drug discovery assays is of pivotal importance. Thus, a 96-well microplate system was developed where primary human small airway epithelial (hSAE) cells were cultured at an air-liquid interface (ALI). After four weeks of ALI culture, a pseudostratified epithelium containing basal, club, goblet and ciliated cells was produced. The 96-well ALI cultures displayed a cellular composition, ciliary beating frequency, and intercellular tight junctions similar to 24-well conditions. A novel custom-made device for 96-parallelized transepithelial electric resistance (TEER) measurements, together with dextran permeability measurements, confirmed that the 96-well culture developed a tight barrier function during ALI differentiation. 96-well hSAE cultures were responsive to transforming growth factor β1 (TGF-β1) and tumor necrosis factor α (TNF-α) in a concentration dependent manner. Thus, the miniaturized cellular model system enables the recapitulation of a physiologically responsive, differentiated small airway epithelium, and a robotic integration provides a medium throughput approach towards pharmaceutical drug discovery, for instance, in respect of fibrotic distal airway/lung diseases.

The role of brain-derived neurotrophic factor (BDNF) signaling in chronic pain has been well documented. Given the important central role of BDNF in long term plasticity and memory, we sought to engineer a high affinity, peripherally-restricted monoclonal antibody against BDNF to modulate pain. BDNF shares 100% sequence homology across human and rodents; thus, we selected chickens as an alternative immune host for initial antibody generation. Here, we describe the affinity optimization of complementarity-determining region-grafted, chicken-derived R3bH01, an anti-BDNF antibody specifically blocking the TrkB receptor interaction. Antibody optimization led to the identification of B30, which has a > 300-fold improvement in affinity based on BIAcore, an 800-fold improvement in potency in a cell-based pERK assay and demonstrates exquisite selectivity over related neurotrophins. Affinity improvements measured in vitro translated to in vivo pharmacological activity, with B30 demonstrating a 30-fold improvement in potency over parental R3bH01 in a peripheral nerve injury model. We further demonstrate that peripheral BDNF plays a role in maintaining the plasticity of sensory neurons following nerve damage, with B30 reversing neuron hyperexcitability associated with heat and mechanical stimuli in a dose-dependent fashion. In summary, our data demonstrate that effective sequestration of BDNF via a high affinity neutralizing antibody has potential utility in modulating the pathophysiological mechanisms that drive chronic pain states.