Methods and compositions for assessing antibody specificities
Inventors
Daugherty, Patrick Sean • Kamath, Kathryn Vinaya Louise • Reifert, Jack Ryan
Assignees
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Publication Number
US-10386373-B2
Publication Date
2019-08-20
Expiration Date
Abstract
The present invention provides compositions and methods that can be used to determine a peptide signature for an antibody repertoire in a sample comprising multiple antibodies. The method can be used to characterize a phenotype in a sample, such as providing a diagnosis, prognosis or theranosis of a medical condition.
Core Innovation
The invention provides a Display-seq platform for assessing antibody specificities in mixed antibody samples and for NGS/QC processing of antibody-epitope repertoires and IMUNE motif-discovery for large datasets. A random peptide display library is incubated with a sample, enriched binders are recovered, and the encoding nucleic acids are identified by high-throughput sequencing to generate peptide/epitope signatures.
Motif discovery is performed from the peptide/epitope signatures to derive phenotype-associated peptide motifs and diagnostic motif panels. Motifs are scored via enrichment versus controls using thresholding and standard deviations, optionally via z-score summation for multiplex diagnostic panels, and the workflow includes comparison to the MEME algorithm and reporting motif discovery results on large datasets.
The invention addresses the identification of Lyme disease motifs and motif families, including non-redundant motifs and rules for down-selection based on specificity, sensitivity, and patient coverage. It also describes a depletion reagent library for removing common cross-reactive antibodies using iterative selection and cross-redundancy or motif-overlap quantification (HASRD enrichment), and reports substantial depletion and improved detection of remaining motifs.
Claims Coverage
The provided claim content identifies one independent claim and related array limitations. It covers a peptide probe array comprising an array surface and at least five peptide probes with specified binding motifs, with peptide probes extending from the array surface.
Peptide probe array with motif-selected probes
An array comprising an array surface and at least five peptide probes, each peptide probe comprising a binding motif selected from a specified group of SEQ ID NOs, and wherein the at least five peptide probes extend from the array surface.
Peptide probe array with SEQ ID NO binding motifs
An array comprising an array surface and at least five peptide probes, wherein each of the at least five peptide probes comprises a binding motif selected from the group consisting of SEQ ID NO: 362, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 368, SEQ ID NO: 369, SEQ ID NO: 374, SEQ ID NO: 375, SEQ ID NO: 376, SEQ ID NO: 378, and SEQ ID NO: 383, and wherein the at least five peptide probes extend from the array surface.
Extended probe array surface on an Escherichia coli eCPX scaffold
The array specifies that at least a portion of an Escherichia coli eCPX scaffold comprises the array surface and that the array surface is a biological particle, wherein the biological particle is an Escherichia coli cell.
The claim coverage is directed to an array format where at least five extending peptide probes are defined by selected binding motifs, with further refinement to arrays based on an Escherichia coli eCPX scaffold and Escherichia coli cell-based array surfaces.
Stated Advantages
Reported diagnostic panel performance of 100% sensitivity and 100% specificity.
Substantial depletion of common cross-reactive antibodies.
Improved detection of remaining motifs after depletion.
Documented Applications
Motif discovery and diagnostic motif panel construction for Lyme disease, including mapping motifs to putative Borrelia burgdorferi antigens and reporting stated sensitivity and specificity.
Motif discovery and panel examples for multiple pathogens and conditions, including Toxoplasma, Taenia, EBV, Zika, HIV, Sjogren’s syndrome, rhinovirus, CMV, Streptococcus, Haemophilus influenza, Leishmania, Babesia microti, Ehrlichia, and Anaplasma.
Use of a depletion reagent library for depletion of common antibody specificities to improve remaining motif detection.
Diagnostic applications for multiple diseases or infections, including celiac disease, Chagas, Lyme, Toxoplasma, cysticercosis, EBV mononucleosis, Zika, and HIV.
An example diagnostic context for Borrelia burgdorferi infection.
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