Project description
High-throughput strategy for the design, synthesis and screening of camelid antibodies
Camelid single-domain antibodies (nanobodies or VHHs) are derived from the Camelidae mammals (llamas, camels and alpacas). Camelid antibodies lack a light chain and are composed of two identical heavy chains. They are very specific and display strict monomeric behaviour, high thermostability and solubility. The EU-funded AutoCAb project aims to develop a high-throughput strategy to design, synthesise and screen repertoires of VHHs that target functional sites on specific proteins. Each VHH will be individually designed for high stability and target site affinity. Researchers will employ innovative methodologies to achieve custom-DNA oligo synthesis, and machine learning methods to obtain stable, high-affinity, site-targeted antibodies for basic and applied biomedical research.
Objective
We propose to develop the first high-throughput strategy to design, synthesize, and screen repertoires comprising millions of single-domain camelid antibodies (VHH) that target desired protein surfaces. Each VHH will be individually designed for high stability and target-site affinity. We will leverage recent methods developed by our lab for designing stable, specific, and accurate backbones at interfaces, the advent of massive and affordable custom-DNA oligo synthesis, and machine learning methods to accomplish the following aims:
Aim 1: Establish a completely automated computational pipeline that uses Rosetta to design millions of VHHs targeting desired protein surfaces. The variable regions in each design will be encoded in DNA oligo pools, which will be assembled to generate the entire site-targeted repertoire. We will then use high-throughput binding screens followed by deep sequencing to characterize the designs’ target-site affinity and isolate high-affinity binders.
Aim 2: Develop an epitope-focusing strategy that designs several variants of a target antigen, each of which encodes dozens of radical surface mutations outside the target site to disrupt potential off-target site binding. The designs will be used to isolate site-targeting binders from repertoires of Aim 1.
Each high-throughput screen will provide unprecedented experimental data on target-site affinity in millions of individually designed VHHs.
Aim 3: Use machine learning methods to infer combinations of molecular features that distinguish high-affinity binders from non binders. These will be encoded in subsequent designed repertoires, leading to a continuous “learning loop” of methods for high-affinity, site-targeted binding.
AutoCAb’s interdisciplinary strategy will thus lead to deeper understanding of and new general methods for designing stable, high-affinity, site-targeted antibodies, potentially revolutionizing binder and inhibitor discovery in basic and applied biomedical research.
Fields of science
Programme(s)
Funding Scheme
ERC-COG - Consolidator GrantHost institution
7610001 Rehovot
Israel