General detection and isolation of specific cells by binding of labeled molecules
Inventors
Pedersen, Henrik • Jakobsen, Søren • HADRUP, Sine Reker • BENTZEN, Amalie Kai • JOHANSEN, Kristoffer Haurum
Assignees
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Publication Number
US-11585806-B2
Publication Date
2023-02-21
Expiration Date
Abstract
The present invention relates to detection molecules comprising at least one binding molecule, at least one linker and at least one label, and detection methods making use of same. The invention provides a high-throughput method for detection, isolation and/or identification of specific entities or cells.
Core Innovation
The invention provides a composition for analysis of a cell population that includes from 5 to 1,000,000 sets of detection molecules. Each detection molecule set comprises at least two identical binding molecules coupled to a multimerization domain via separate connector molecules, and the multimerization domain is selected from polysaccharides, dextran moieties, avidins, streptavidins, and streptactin. The binding molecules recognize and/or bind to a T cell receptor or a B cell receptor specific for a given cell or cell type.
The detection molecules further include a first label comprising at least one nucleic acid label coupled with the multimerization domain or with at least one of the identical binding molecules, and a second label selected from fluorophores, chromophores, and peptides. The nucleic acid label is 30-200 nucleotides in length and includes a 5′ first primer region, a barcode region, a 3′ second primer region, and a random nucleotide region. The barcode region is common within the set but uniquely represents the set within the composition, and the random nucleotide region is unique within the composition.
The document describes multimeric MHC display for antigen-responsive T-cell detection and a general framework for detecting, isolating, and identifying specific cell populations using nucleic-acid labels with barcode regions for multiplexing many distinct specificities within one analysis. It also describes DNA-barcoding schemes, dextran-streptavidin-PE detection molecules, and readout of recovered barcodes by qPCR or sequencing-based barcode identification.
Claims Coverage
The consolidated claim coverage is directed to a cell-analysis composition comprising 5 to 1,000,000 sets of detection molecules with multimerization, nucleic-acid barcode labeling, and a second label. The independent claim features are combined with receptor-specific binding to T cell receptors or B cell receptors.
Multispecific detection-molecule sets for cell population analysis
A composition for analysis of a cell population comprising from 5 to 1,000,000 sets of detection molecules that recognize and/or bind to a T cell receptor or a B cell receptor specific for a given cell or cell type.
Multimerization domain with separate connectors on at least two identical binding molecules
Each set includes at least two identical binding molecules coupled to a multimerization domain via separate connector molecules, where the multimerization domain is selected from polysaccharides, dextran moieties, avidins, streptavidins, and streptactin.
Set barcode nucleic-acid first label with common barcode region and unique random nucleotide region
A first label includes at least one nucleic acid label coupled with the multimerization domain or with at least one of the identical binding molecules, where the nucleic acid label is 30-200 nucleotides in length and comprises a 5′ first primer region, a barcode region, a 3′ second primer region, and a random nucleotide region; the barcode region is common within the set but uniquely represents the set within the composition, while the random nucleotide region is unique within the composition.
Second label coupled for detection as fluorophore, chromophore, or peptide
A second label is included in each detection molecule set, where the second label is selected from fluorophores, chromophores, and peptides, and is coupled with the multimerization domain or with at least one of the identical binding molecules.
Overall, the claim coverage centers on a cell-analysis composition that uses multimerized receptor-binding detection molecules tagged with set-specific nucleic-acid barcodes and a second label for detection.
Stated Advantages
Enables high-throughput multiplexing by using nucleic-acid labels with barcode regions for many distinct specificities within a single composition.
Provides contamination control via unique random nucleotide regions in the nucleic-acid labels.
Supports sample encoding using sample-ID barcodes.
Improves stability of labels using HEG/TEG stability modifications.
Multiplex analysis using from 5 to 1,000,000 sets of detection molecules.
Set-wise identification via a barcode region common within the set and uniquely representing the set within the composition.
Composition-wide discrimination via a random nucleotide region unique within the composition.
Compatibility with receptor-specific recognition of cells via binding molecules recognizing and/or binding to T cell receptors or B cell receptors specific for a given cell or cell type.
Documented Applications
Analysis of a cell population using multiplex detection molecules that bind to T cell receptors or B cell receptors specific for a given cell or cell type of the cell population.
Characterization and detection of antigen-responsive T cells using multimeric MHC display with nucle-acid barcode readout.
Discrimination of CMV vs HIV-specific T cells using DNA-tagged dextramers or multimer libraries with unique DNA barcodes.
Multiplex detection of multiple antigen specificities with readout correlating with T-cell frequency.
Detecting, isolating, and identifying specific cell populations using detection-molecule libraries and identification of bound labels by PCR, qPCR, deep sequencing, or other label readouts.
Analyzing mixtures containing CMV-specific T cells by incubating with a large library of MHC multimers and identifying specific bound labels.
Encoding tumor-infiltrating lymphocytes (TIL) samples using sample-ID barcodes.
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