Bacteriophage gene 3 protein compositions and use as amyloid binding agents
Inventors
Krishnan, Rajaraman • Fisher, Richard
Assignees
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Publication Number
US-9688728-B2
Publication Date
2017-06-27
Expiration Date
Abstract
The invention relates to agents and to pharmaceutical compositions for reducing the formation of amyloid and/or for promoting the disaggregation of amyloid proteins. The compositions may also be used to detect amyloid.
Core Innovation
The invention relates to g3p-Ig amyloid-binding Fc fusion therapeutics, including bacteriophage g3p-derived amyloid-binding agents and constructs such as rs-g3p(N1N2)-hIgG1-Fc and related hIgG4-Fc/IgG1-Fc variants. The fusion proteins comprise an amyloid binding fragment of g3p fused to an Fc fragment of an immunoglobulin, and the g3p portion is defined by specific amino-acid ranges from SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:31, or by mutants or variants that are at least 95% identical and capable of binding to amyloid.
The constructs bind aggregated amyloid targets and affect amyloid oligomer and fiber states, including reduction of amyloid formation, disaggregation of preformed amyloid fibers, inhibition of amyloid aggregation, and prevention or removal of toxic oligomers. The structural basis for amyloid binding and disaggregation is described in terms of g3p amyloid binding and unfolding of a hinge region associated with N1/N2 domains, including a melting temperature related to hinge unfolding.
The content describes engagement of amyloid-associated disease pathology including amyloid beta, tau fibers, alpha-synuclein fibers, prion protein, SOD-1 fibers, and aggregated transthyretin. Reported characterization includes surface plasmon resonance, Thioflavin T assays, filter trap assay, TEM, and NMR (H/D exchange), together with stated therapeutic and behavioral improvement outcomes in Alzheimer’s mouse models and other disease models.
Claims Coverage
The independent claim coverage centers on a fusion protein and associated methods using g3p-Ig amyloid-binding Fc fusion therapeutics. The inventive features supported across the items are 5.
G3p amyloid binding fragment fused to immunoglobulin Fc
A fusion protein comprising an amyloid binding fragment of g3p and an Fc fragment of an immunoglobulin, including the specified SEQ ID NO-derived amino-acid ranges or a mutant or variant that is at least 95% identical and capable of binding to amyloid.
Up to five substitutions in the g3p amyloid binding fragment
The amyloid binding fragment of g3p contains up to 5 amino acid substitutions.
Reduced immunogenicity in humans
The fusion protein is less immunogenic in humans than the specified reference sequences.
Treatment by reducing amyloid, inhibiting formation or aggregation, and removing toxic oligomers
A method for treating a patient with a disease or condition linked to misfolded and/or aggregated amyloid beta, tau, and/or alpha-synuclein, and/or prion protein by administering the fusion protein to reduce amyloid, inhibit amyloid formation and/or inhibit amyloid aggregation, and/or remove or prevent the formation of toxic oligomers.
Biomarker-positive selection using florbetapir PET
The patient is positive for the biomarker florbetapir when florbetapir is used as an imaging agent in positron emission tomography (PET).
The inventive features are the defined g3p amyloid-binding Fc fusion protein, sequence-limited variants and substitutions, reduced immunogenicity in humans, treatment by reducing amyloid and toxic oligomers, and biomarker-positive patient selection using florbetapir PET.
Stated Advantages
Reduces amyloid formation.
Disaggregates preformed amyloid fibers.
Prevents formation of toxic oligomers.
Inhibits amyloid formation and/or amyloid aggregation.
Removes or prevents the formation of toxic oligomers.
Provides therapeutic and behavioral improvement outcomes in Alzheimer’s mouse models.
Enables biomarker-guided patient selection using florbetapir PET.
Fusion proteins are described as less immunogenic in humans than specified reference sequences.
Increases synaptophysin with no GFAP elevation in Tg2576 mice.
Inhibits/disaggregates Aβ oligomer toxicity.
Binds/disaggregates tau and reduces Aβ plaque and tau in aged 3×Tg and tau(P301L) mice with improved motor behavior.
Inhibits PrPSc formation in N2a22LSc prion cells in a dose-dependent manner.
Engages fAβ42 hydrophobic core residues and disaggregates preformed fibers.
Extends disease-model efficacy across ALS, Parkinson’s disease, and amyloidosis models, including reduction of α-synuclein aggregates and effects associated with aggregated transthyretin binding.
Documented Applications
Therapeutic treatment of diseases or conditions linked to misfolded and/or aggregated amyloid beta, tau, alpha-synuclein, and/or prion protein.
Treatment of Alzheimer’s disease, including early onset Alzheimer’s disease, late onset Alzheimer’s disease, and presymptomatic Alzheimer’s disease.
Treatment of Parkinson’s disease.
Treatment of bovine spongiform encephalitis (BSE).
Diagnostic or selection use via florbetapir PET for biomarker-positive patient selection.
In vivo evaluation in Tg2576 mice for Aβ plaque-related readouts including synaptophysin and GFAP markers.
In vivo evaluation in 3×Tg and tau(P301L) mice for tau pathology, including motor behavior outcomes.
Cell-based evaluation of PrPSc formation using N2a22LSc cells.
Amyloid-fiber engagement and disaggregation characterization of fAβ42 fibers using NMR and TEM.
Disease-model application in ALS using SOD-1 fiber assembly inhibition.
Disease-model application in Parkinson’s disease using reduced α-synuclein aggregates with increased tyrosine hydroxylase and behavioral readouts.
Disease-model application in amyloidosis using selective binding to aggregated transthyretin and verification of fiber binding.
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