Project description:The bacterial pathogen, Acinetobacter baumannii, is a leading cause of drug-resistant infections. Here, we investigated the potential of developing nanobodies that specifically recognize A. baumannii over other Gram-negative bacteria. Through generation and panning of a synthetic nanobody library, we identified several potential lead candidates. We demonstrate how incorporation of next generation sequencing analysis can aid in selection of lead candidates for further characterization. Using monoclonal phage display, we validated the binding of several lead nanobodies to A. baumannii. Subsequent purification and biochemical characterization revealed one particularly robust nanobody that broadly and specifically bound A. baumannii compared to other common drug resistant pathogens. These findings support the potentially for nanobodies to selectively target A. baumannii and the identification of lead candidates for possible future diagnostic and therapeutic development.
Project description:In the rapidly advancing field of synthetic biology, there is a critical need for technology to discover targeting moieties for therapeutic biologics. We developed 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 varies across immune cell subtypes in the tumor, lymph node, and spleen. INSPIRE-seq identified a dendritic cell binding clone that binds PHB2. Single-cell RNA sequencing revealed a connection with cDC1s, and immunofluorescence confirmed nanobody-PHB2 colocalization along cell membranes. Structural modeling and docking studies assisted 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.
Project description:In the rapidly advancing field of synthetic biology, there exists a critical need for technology to discover targeting moieties for therapeutic biologics. We are developing developed 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 varies across immune cell subtypes in the tumor, lymph node, and spleen. INSPIRE-seq identified a dendritic cell binding clone that binds PHB2. Single-cell RNA sequencing revealed a connection with cDC1s, and immunofluorescence confirmed nanobody-PHB2 colocalization along cell membranes. Structural modeling and docking studies assisted 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. microenvironment, which can be distinct from draining lymph nodes. To identify targets, we selected a clone enriched for dendritic cells that binds to PHB2. Using single cell RNA sequencing, we observe PHB2 signaling is associated with activation in cDC1’s. Immunofluorescence confirmed that the nanobody colocalizes with PHB2 in regions along the cell membrane. Structural modeling with AlphaFold2 and antibody docking using Rosetta assist binding site predictions, thus could be used to guide nanobody selection for future aims. This work shows that INSPIRE-seq can interrogate complex microenvironments and may assist in developing therapeutics.
Project description:To identify cell adhesion molecules (CAMs) targeting bacterial membrane proteins within a synthetic bacteria-displayed nanobody library, we present a comprehensive whole-cell screening platform. This involves targeted amplicon sequencing to discover nanobodies targeting the natural adhesin, TraN. Furthermore, we employ deep mutational engineering to enhance the binding affinity of these nanobodies toward TraN.
Project description:Multi-modal measurements of single cell profiles are a powerful tool for characterizing cell states and regulatory mechanisms. While current methods allow profiling of RNA along with either readouts of chromatin or protein, connecting chromatin state to protein levels remains a barrier. Here, we developed PHAGE-ATAC, a method that uses engineered camelid single-domain antibody (‘nanobody’)-displaying phages for simultaneous single-cell measurement of surface proteins, chromatin accessibility profiles, and mtDNA-based clonal tracing through a single-cell and massively parallel droplet-based assay of transposase-accessible chromatin with sequencing (ATAC-seq). We demonstrate PHAGE-ATAC for multimodal analysis in primary human immune cells, for multiplexing, for intracellular protein analysis, and for the detection of SARS-CoV-2 spike protein. Finally, we construct a synthetic high-complexity phage library for selection of novel antigen-specific nanobodies that bind cells of particular molecular profiles, opening a new avenue for protein detection, cell characterization and screening with single-cell genomics.
Project description:Multi-modal measurements of single cell profiles are a powerful tool for characterizing cell states and regulatory mechanisms. While current methods allow profiling of RNA along with either readouts of chromatin or protein, connecting chromatin state to protein levels remains a barrier. Here, we developed PHAGE-ATAC, a method that uses engineered camelid single-domain antibody (‘nanobody’)-displaying phages for simultaneous single-cell measurement of surface proteins, chromatin accessibility profiles, and mtDNA-based clonal tracing through a single-cell and massively parallel droplet-based assay of transposase-accessible chromatin with sequencing (ATAC-seq). We demonstrate PHAGE-ATAC for multimodal analysis in primary human immune cells, for multiplexing, for intracellular protein analysis, and for the detection of SARS-CoV-2 spike protein. Finally, we construct a synthetic high-complexity phage library for selection of novel antigen-specific nanobodies that bind cells of particular molecular profiles, opening a new avenue for protein detection, cell characterization and screening with single-cell genomics.
Project description:Multi-modal measurements of single cell profiles are a powerful tool for characterizing cell states and regulatory mechanisms. While current methods allow profiling of RNA along with either readouts of chromatin or protein, connecting chromatin state to protein levels remains a barrier. Here, we developed PHAGE-ATAC, a method that uses engineered camelid single-domain antibody (‘nanobody’)-displaying phages for simultaneous single-cell measurement of surface proteins, chromatin accessibility profiles, and mtDNA-based clonal tracing through a single-cell and massively parallel droplet-based assay of transposase-accessible chromatin with sequencing (ATAC-seq). We demonstrate PHAGE-ATAC for multimodal analysis in primary human immune cells, for multiplexing, for intracellular protein analysis, and for the detection of SARS-CoV-2 spike protein. Finally, we construct a synthetic high-complexity phage library for selection of novel antigen-specific nanobodies that bind cells of particular molecular profiles, opening a new avenue for protein detection, cell characterization and screening with single-cell genomics.
Project description:Single cell multi-omic readouts of both the cellular transcriptome and proteome have significantly enhanced our ability to comprehensively characterize cellular states. Most approaches in this area rely on oligonucleotide barcode-conjugated antibodies that target cell surface epitopes of interest, enabling their concomitant detection with the transcriptome. However, a similar high-throughput measurement of other cellular modalities such as the epigenome in concert with protein levels have not been described. Moreover, detection of epitopes is limited to antigens for which a specific antibody is available. Here, we introduce PHAGE-ATAC, an approach that enables the scalable and simultaneous detection of protein levels and chromatin accessibility data in single cells using the assay of transposase-accessible chromatin with sequencing (ATAC-seq). Quantitative detection of proteins by PHAGE-ATAC is accomplished through the use of engineerable nanobody-displaying phages that are genetically barcoded within the nanobody-encoding phagemids. We demonstrate the utility of PHAGE-ATAC for multimodal single cell genomic analysis in both cell lines and primary human cells. Analogous to phage display approaches, we further establish a synthetic high-complexity library of nanobody-displaying phages and demonstrate its utility to select novel antigen-specific nanobodies for PHAGE-ATAC.