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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 16, 2023 |
| Title |
Simultaneous selection of nanobodies for accessible epitopes on immune cells in the tumor microenvironment |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
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.
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| Overall design |
CD45 magnetic microbeads enriched cells were isolated form Py8119 bearing mice (three mice per group) two hours after injection of either PBS, insertless phage display, CD45, DCs, or CD8 specific VHHs phage display libraries.
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| Contributor(s) |
Sekar TV, Elghonaimy EA, Swancutt KL, Diegeler S, Gonzalez I, Hamilton C, Leung PQ, Meiler J, Martina CE, Michael M, Aguilera TA |
| Citation(s) |
37978291 |
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| Submission date |
Jan 21, 2023 |
| Last update date |
Dec 08, 2023 |
| Contact name |
Todd Aguilera |
| E-mail(s) |
todd.aguilera@utsouthwestern.edu
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| Phone |
214-648-8935
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| Organization name |
UTSouthwestern- Medical Center
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| Department |
Molecular radiation biology
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| Lab |
Todd A. Aguilera
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| Street address |
2280 Inwood Rd
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| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390 |
| Country |
USA |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (5)
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| Relations |
| BioProject |
PRJNA926133 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE223428_RAW.tar |
58.8 Mb |
(http)(custom) |
TAR (of H5) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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