(314c) Novel Experimental and Computational Approaches for Conformational Antibody Engineering

A common feature of many neurodegenerative disorders is the misfolding of disease-specific peptides and proteins into related types of pre-fibrillar oligomers and amyloid fibrils. Antibodies that recognize protein aggregates with strict conformational specificity are important for mechanistic, diagnostic and therapeutic applications. Despite progress in using in vitro antibody display methods such as yeast surface display for isolating conformational antibodies, a key limitation is that the insoluble nature of many protein aggregates (e.g., amyloid fibrils) prevents their use as soluble antigens and requires their immobilization on magnetic beads for magnetic-activated cell sorting (MACS) of yeast-displayed libraries. The use of conventional MACS methods to sort antibody libraries is undesirable because the binding selections are strongly influenced by the antibody expression level on the yeast surface and avidity effects in addition to the intrinsic antibody affinity, which leads to isolation of sub-optimal antibodies with modest affinity and/or conformational specificity.

Here we report two novel methods for addressing the limitations of conventional MACS methods to isolate high-quality conformational antibodies. First, we have developed a quantitative fluorescence-activated cell sorting (FACS) method for directly selecting high-quality conformational antibodies against multiple types of amyloid fibrils. Our approach uses quantum dots (QDs) functionalized with antibodies to capture amyloid fibrils, and the resulting QD-amyloid conjugates are used in a similar manner as conventional soluble fluorescently-labeled antigens for multiparameter FACS selections. Notably, we find that this approach is robust for isolating high-quality conformational antibodies against tau and a-synuclein fibrils with combinations of high affinity, high conformational specificity and, in some cases, low off-target binding that rival or exceed those of tau (zagotanemab) and a-synuclein (cinpanemab) clinical-stage antibodies. The generality of this approach is expected to simplify the generation and affinity maturation of high-quality conformational antibodies against diverse types of protein aggregates and other insoluble antigens (e.g., membrane proteins) that are compatible with presentation on antibody-functionalized QDs.

Second, we have developed a novel MACS methodology for generating high-quality conformational antibodies against Alzheimer’s Abeta fibrils in the native IgG format. Our directed evolution approach uses targeted mutagenesis, yeast surface display, cell sorting and deep sequencing to identify antibody candidates with optimized binding properties. Notably, we find that this approach results in robust isolation of IgGs with higher affinity, higher conformational specificity and lower off-target binding than multiple clinical-stage Abeta antibodies, including aducanumab and crenezumab. This antibody engineering platform can be readily applied to generate high-quality conformational antibodies against diverse types of peptide and protein aggregates linked to neurodegenerative diseases.