Simultaneous selection of nanobodies for accessible epitopes on immune cells in the tumor microenvironment

Abstract

In the rapidly advancing field of synthetic biology, there exists a critical need for technology to discover targeting moieties for therapeutic biologics. Here we present INSPIRE-seq, an approach that utilizes a nanobody library and next-generation sequencing to identify nanobodies selected for complex environments. INSPIRE-seq enables the parallel enrichment of immune cell-binding nanobodies that penetrate the tumor microenvironment. Clone enrichment and specificity vary across immune cell subtypes in the tumor, lymph node, and spleen. INSPIRE-seq identifies a dendritic cell binding clone that binds PHB2. Single-cell RNA sequencing reveals a connection with cDC1s, and immunofluorescence confirms nanobody-PHB2 colocalization along cell membranes. Structural modeling and docking studies assist binding predictions and will guide nanobody selection. In this work, we demonstrate that INSPIRE-seq offers an unbiased approach to examine complex microenvironments and assist in the development of nanobodies, which could serve as active drugs, modified to become drugs, or used as targeting moieties.

Nanobody Engineering: Computational Modelling and Design for Biomedical and Therapeutic Applications

“…HDOCK [110] was found to be the most suitable program for docking of novel nanobodies to the receptorbinding domain of SARS-CoV-2 with high accuracy [111]. While RosettaAntibody [112,113] is also being used to predict nanobody-antigen complexes [114][115][116], there is ongoing research aimed at improving its accuracy and speed of CDR-H3 loop modeling [113]. PatchDock, which employs an algorithm capable of automatically detecting the CDRs of the antibody and restricts the search to these specific regions [117], has been integrated into a computational dock-anddesign workflow [79].…”