Selection of Novel Nanobodies Targeting EGFR
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ABSTRACT:
PROVIDER: PRJEB35883 | ENA |
REPOSITORIES: ENA
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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.
2023-06-21 | GSE202972 | GEO
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.
2023-11-16 | GSE223428 | GEO
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.
2024-01-26 | PXD046363 | Pride
Project description:Selection of activating EGFR mutations from randomly mutated EGFR libraries
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Project description:Jailkhani N, Clauser KR, Mak H, Rickelt S, Tian C, Whittaker CA, Tanabe KK, Purdy SR, Carr SA, and Hynes RO.2022.
Metastases are hard to detect and treat, and cause most cancer-related deaths. The relative lack of therapies targeting metastases represents a major unmet clinical need. The extracellular matrix or ECM forms a major component of the tumor microenvironment in both primary and metastatic tumors and certain ECM proteins can be selectively and abundantly expressed in such tumors. We propose that nanobodies against ECM proteins that show selective abundance in metastases can be used as vehicles for delivery of imaging and therapeutic cargoes. To gain a deeper understanding of the microenvironment of human metastases, we describe here an LC-MS/MS-based ECM signature shared by human TNBC and CRC metastases to different organs and provide evidence that this conserved set of ECM proteins is selectively elevated in other tumors. We also describe phage-display libraries of nanobodies raised against ECM-enriched preparations from human metastases from TNBC and CRC. As proof of concept, we developed selective and specific, high-affinity nanobodies against an example signature protein, Tenascin-C (TNC), known to be abundant in many tumor types and to play a role in metastases. We report that TNC is abundantly expressed in patient metastases and widely expressed across diverse metastatic sites originating from several primary tumor types. Using immuno-PET/CT we showed that anti-TNC nanobodies bind TNBC tumors and metastases with excellent specificity. We propose that such generic anti-tumor nanobodies are promising cancer-agnostic, in vivo tools for the delivery of therapeutics to tumor and metastatic ECM.
2022-03-28 | MSV000089136 | MassIVE
Project description:Simultaneous selection of nanobodies for accessible epitopes on immune cells in the tumor microenvironment
| PRJNA926133 | ENA
Project description:DNA sequencing for Round 5 of the pHLA-A2 yeast display library selection for MA2 Fab
2022-10-09 | GSE210479 | GEO
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.
2021-10-21 | GSE172923 | GEO
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.
2021-10-21 | GSE172921 | GEO
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.
2021-10-21 | GSE172920 | GEO