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-12259396-B2
Publication Date
2025-03-25
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 relates to identifying disease-specific peptide motifs from large antibody epitope repertoires using Display-seq methods, IMUNE, together with NGS processing. The approach performs motif discovery and clustering of enriched short patterns into motif families, derives peptide signatures and peptide motif panels, and links NGS-derived motif information to antigen candidates.
The disclosure further addresses non-specific binding by optionally depleting antibodies or peptides, including physical and computational depletion concepts, and by using peptide libraries to enrich binding peptides for removal of common, cross-reactive antibodies. Enrichment calculations, z-score aggregation, threshold and cutoff logic, MEME, IMUNE, PAM30 similarity scoring, Scanprosite, Swiss-Prot/TrEMBL, and HASRD are referenced in support of motif discovery and mapping.
Array-based antibody detection is described using short peptides that contain specified binding motif sequences presented on a solid surface or by engineered biological particles that display heterologous peptide sequences on an outer surface via a display scaffold. The document positions these peptide and motif outputs for diagnostic detection of antibodies associated with Borrelia burgdorferi infection and for phenotypes such as disease status and prognosis/theranosis.
Claims Coverage
The partial content includes four independent claims. Across these claims, the main inventive features concern presenting short, motif-constrained peptides of 30 amino acids or less on an array surface, either on a solid surface or via engineered biological particles displaying heterologous peptides on outer surfaces, including arrays with one motif or two specified motif peptides.
Short peptide motif array for antibody binding on an array surface
An array comprising a peptide comprising a binding motif sequence [LI]xxA[ILV]xxRG, [DA]DPTN, or KETPPALN, wherein a length of the peptide comprising the binding motif sequence is 30 amino acids or less, and wherein the peptide is operably linked to an array surface to present the peptide for binding by an antibody, wherein the array surface comprises a solid surface with the peptide attached, or a biological particle engineered to express the peptide operably linked to a display scaffold configured to display the peptide on the outer surface of the biological particle, and wherein the amino acid sequence of the peptide is heterologous to the biological particle.
Two-peptide binding motif array using solid or engineered particle presentation
An array comprising a first peptide with a binding motif sequence [LI]xxA[ILV]xxRG, [DA]DPTN, or KETPPALN of 30 amino acids or less; and a second peptide comprising a second binding motif selected from LXGM[RQ]K, QEG[IV]Q, Q[TI]EQxxxxxK, VxxYFxx[LV]xK, PFx[AP]YxK, DxSP[IL]E, KxVDxDR, and [DN][AS]A[AG]F; wherein both peptides are operably linked to an array surface to present the peptides for binding by one or more antibodies, with either both peptides attached to the same solid surface, or a first and a second biological particle engineered to express each respective peptide operably linked to a display scaffold configured to display the respective peptides on the outer surface of the respective biological particle, with the amino acid sequence of the peptide heterologous to the respective biological particle.
Overall, the independent claims cover peptide arrays using specified motif-containing peptides of 30 amino acids or less presented either on solid surfaces or via heterologous peptide display on engineered biological particles. The partial content also ties the array to antibody detection for Borrelia burgdorferi infection.
Stated Advantages
Improved detection of non-depletion motifs after depletion reagent overlap reduction.
Reported depletion reagent validation includes substantial enrichment reduction for common cross-reactive binding and improved detection outcomes.
Lyme diagnostic performance is described using sensitivity/specificity thresholds, including specificity at least 99% as recited in dependent claim coverage.
Documented Applications
Detecting antibodies associated with a Borrelia burgdorferi infection by contacting a biological sample with an array, incubating for binding, measuring peptide binding, and indicating positivity when antibodies associated with Borrelia burgdorferi infection are detected.
Disease motif discovery and diagnostic panel construction from large antibody epitope repertoires, with documented motif discovery examples including acute toxoplasmosis, cysticercosis, EBV, Zika, HIV, Sjogren’s syndrome, rhinovirus, CMV, Streptococcus, Haemophilus influenza, Leishmania, Babesia, Ehrlichia, Anaplasma, Toxocara, and related motif panels.
Mapping discovered motifs to putative Borrelia burgdorferi antigens using Scanprosite and Swiss-Prot/TrEMBL, and querying motif overlap/enrichment concepts using HASRD.
Depletion reagent use with peptide library display to remove common cross-reactive antibodies by enriching and quantifying overlap between depletion tracks and motif overlap outcomes.
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