Discovery and Engineering of Nanobodies Directed Toward Complex Targets
Creators: name: Zupancic, Jennifer
Publisher: name: My University
Year: 2023
Subjects: subject: Nanobody
subject: Antibody engineering
subject: Directed evolution
subject: Multivalent engineering
subject: Protein engineering
subject: Conformational antibody
subject: Chemical Engineering
subject: FOS: Chemical engineering
subject: Engineering
subject: Science

description: Antibodies are widely used in numerous critical biotechnology applications, including as therapeutics and diagnostic agents. Among their many desirable properties, their ability to recognize a target, termed an antigen, with both high affinity and high specificity, is the most important. Single-domain antibodies, often termed nanobodies or VHHs, represent the smallest antibody fragments with antigen-binding ability and have generated great interest for their abilities beyond those of conventional antibodies, such as their ability to penetrate deeper into tissues for drug delivery and recognize specific conformations for structural biology studies. However, both the development of methods to generate nanobodies and the isolation of nanobodies specific for complex antigens has lagged behind those available for conventional antibodies. Therefore, we have sought to develop new methods for the systematic, predictable isolation of nanobodies that recognize complex targets, including the SARS-CoV-2 virus and tau aggregates associated with Alzheimer’s disease. First, we demonstrate a simple approach for the in vitro isolation of high-affinity nanobodies from a synthetic nanobody library using the wildtype SARS-CoV-2 receptor-binding domain as a model antigen. We demonstrate that swapping the complementarity-determining regions (CDRs) between low affinity lead clones produces large increases in both affinity and neutralization activity. A nanobody generated in this manner demonstrates greater neutralization activity towards the live virus than a potent neutralizing human antibody isolated after infection (CB6) and a potent neutralizing nanobody isolated after immunization (Ty1). We further demonstrate that systematic CDR swapping can be incorporated into the sorting process through simple PCR-based methods, allowing identification of high affinity, potent neutralizing nanobodies without the need for initial lead clone selection and affinity maturation. Second, we have developed novel multivalent nanobody constructs that increase the neutralization activity of a nanobody specific for multiple SARS-CoV-2 variants. Methods to rapidly generate potent, broadly neutralizing antibodies and nanobodies are needed to quickly respond to the emergence of infectious variants during disease outbreaks. We demonstrate that a cross-reactive nanobody (VHH-72) with moderate affinity and neutralization activity as a bivalent Fc-fusion can be produced in tetravalent and hexavalent formats. This multivalent engineering approach produces large, synergistic increases in the neutralization activity of VHH-72 toward the wildtype SARS-CoV-2 virus. Further, we demonstrate that hexavalent VHH-72 potently neutralizes multiple SARS-CoV-2 variants (B.1.1.7 and B.1.351), demonstrating that this approach may be applied to rapidly increase the neutralization activity of promising cross-reactive lead nanobodies without the need for sequence modification and further sorting. Third, we have developed a novel method for the rapid and predictable isolation of nanobodies and antibodies with conformational specificity for amyloid aggregates. Developing nanobodies which bind specifically to aggregated proteins in a predictable manner is challenging due to the insoluble nature of such aggregates. Our method immobilizes amyloid fibrils of the Alzheimer’s tau protein on the surface of fluorescent quantum dots. This immobilization approach both enables fibrils to be used similarly to conventional soluble antigens and serves as a detection method for performing quantitative, multiparameter selections (target binding and nanobody expression) using fluorescence-activated cell sorting. Nanobodies isolated using this method show conformational recognition of tau aggregates formed in transgenic mouse models and human diseased brains. We expect that the nanobodies developed using these novel methods will further the study of their target proteins and associated diseases and that our methods can be applied to discover nanobodies for diverse complex targets relevant to human health.