Many neurodegenerative diseases are characterized by the intracellular accumulation of amyloid beta, tau and alpha synuclein fibrils in neurons, resulting in cell damage or death. Conformation-specific antibodies that are capable of recognizing these 3-dimensional disease-associated protein structures are important research tools for diseases such as Alzheimer’s and Parkinson’s. Specifically, these antibodies harbour potential for diagnostic or therapeutic use.

The need for high-quality conformational antibodies has been steadily growing in recent years. Current methods have been largely based on time-intensive and resource-draining trial-and-error processes, and as such, there is a strong demand for an optimized approach. A study by Desai et al., describes a systematic workflow for isolating high-quality antibodies with conformational specificity, which rival clinical-stage antibodies. Selecting variants with strict conformation and sequence specificity, this innovative workflow could transform the engineering of disease-specific conformational antibodies into a highly effective process.

Conformation-specific selection steps in antibody development

Developing conformational antibodies has remained a challenging process. Common challenges include optimizing affinity, ensuring strict conformational specificity and modulating non-specific binding to unrelated targets. To overcome these hurdles, researchers in this study employed a human non-immune single-chain antibody library displayed on the surface of yeast. Antibodies against amyloid fibrils were identified by means of positive and negative sorting steps with amyloid structures conjugated to quantum dots and fluorescence-activated cell sorting (FACS).

The first antibody was targeted to tau fibrils, which accumulate as neurofibrillary tangles in Alzheimer’s disease. StressMarq’s Tau-441 (2N4R) P301S Mutant Pre-formed Fibrils (catalog# SPR-329) were used in the initial discovery rounds to sort yeast cells displaying antibodies. Antibodies specific to tau fibrils were selected, and antibodies that bound to monomeric tau were discarded. Downstream sorting steps utilized StressMarq’s tau fibrils to enrich antibody libraries. Following affinity maturation steps, the resulting high-affinity antibodies rivalled the properties of the clinical-stage conformational tau antibody, Zagotenemab. Furthermore, the generated tau antibodies displayed less off-target binding than Zagotenemab.

Robust development of conformational antibodies with sequence specificity

To demonstrate the robustness and flexibility of the workflow, researchers used the same human antibody library to develop a second conformational antibody. Alpha synuclein was selected as the second target due to its role in neurotoxicity in Parkinson’s disease. Initial sorting steps selected antibodies that bound StressMarq’s Alpha Synuclein Pre-formed Fibrils (catalog# SPR-317). As was previously done with the tau antibodies, following affinity maturation steps the conformation specificity of the generated alpha synuclein fibril-specific antibodies exceeded that of the clinical-grade comparison, Cinpanemab.

Finally, validation testing ensured comprehensive characterisation of the antibodies. One crucial step was ensuring the antibodies bound multiple forms of tau or alpha synuclein, respectively. For this, a variety of StressMarq’s reagents were utilized. Similar to Zagotenemab, the tau antibodies bound different tau variants, including the largest isoform 2N4R, produced in adult brains, Tau-441 (2N4R) fibrils (catalog# SPR-329), and the smallest isoform (0N3R) Tau fibrils (catalog# SPR-491), which are expressed in fetal brains.

Due to differences in mouse and human N-terminal tau sequences, the antibodies were unable to bind mouse tau fibrils (catalog# SPR-475). Similar to the clinical-stage antibody Cinpanemab, the alpha synuclein antibodies generated in this study recognized multiple alpha synuclein constructs including Alpha Synuclein Pre-formed Fibrils (catalog# SPR-317) and Kinetically Stable Alpha Synuclein Oligomers (catalog# SPR-484).

StressMarq products support conformational antibody research

In recent years, the development of conformational antibodies has been hampered by inefficient antibody production systems. However, StressMarq’s repertoire of conformation-, isoform- and species-specific protein reagents have been instrumental to researchers in producing antibodies with high-affinity and high conformational specificity, rivalling the clinical-stage antibodies for tau (Zagotenemab) and alpha synuclein (Cinpanemab).

Making use of StressMarq’s extensive product range for tau and alpha synuclein, Desai et al. describe a workflow that ensures the precise selection of robust conformation-specific antibodies. Such a methodology could have implications for a streamlined approach to conformational antibody development. Moreover, the inclusion of selection steps with amyloid beta fibrils described in this methodology could facilitate the investigation into high-quality conformational antibodies as therapeutic treatments for a variety of amyloid-related neurodegenerative diseases.

Figure 1. Thioflavin T (ThT) fluorescence assay monitoring tau aggregation when Tau Pre-formed Fibrils (catalog# SPR-329) are combined with Tau Monomers (catalog# SPR-327).

Related StressMarq products

As an established provider of highly characterized conformational forms of disease-related proteins, StressMarq has a range of reagents to support antibody production workflows. Visit our website for more details on our range of monomers, oligomers and pre-formed fibrils for alpha synuclein, tau and amyloid beta.

References 

1. Flow cytometric isolation of drug-like conformational antibodies specific for amyloid fibrils. Desai, A. A, et al., bioRxiv. 2023. Note: This is a preprint. The article has not yet been peer-reviewed by a journal.
2. Comparative analysis of aducanumab, zagotenemab, and pioglitazone as targeted treatment strategies for Alzheimer’s disease. Abyadeh, M. et al. Aging Dis. 2021.
3. Trial of cinepanemab in early Parkinson’s disease. Lang, A. E., et al., New England Journal of Medicine. 2022.