Methods and compositions comprising tau oligomers
Inventors
Moe, James G. • Davidowitz, Eliot J.
Assignees
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Publication Number
US-11104710-B2
Publication Date
2021-08-31
Expiration Date
Abstract
Tau protein has a causative role in Alzheimer's disease and multiple other neurodegenerative disorders exhibiting tau histopathology collectively termed tauopathies. The primary function of tau protein is to facilitate assembly and maintenance of microtubules in neuronal axons. In the disease process tau protein becomes modified, loses its affinity to microtubules and accumulates in the cell body where it forms aggregates. The large neurofibrillary tangles formed from tau protein assembled into filaments were thought to be the pathological structure of tau. However, more recent work indicates that smaller, soluble oligomeric forms of tau are best associated with neuron loss and memory impairment. Here, novel compositions of tau oligomers and novel mechanisms for tau oligomer nucleation, extension and termination are taught. Methods for producing and purifying these structures for the development of small molecule and immunotherapeutics as well as antibodies for biomarkers of neurodegenerative diseases are taught.
Core Innovation
The invention relates to stabilized, substantially purified tau oligomers, including tau dimers through dodecamers and specific stabilized tau trimeric forms, formed by a disulfide-mediated, cross-beta-sheet nucleation and extension model with a termination model. The stabilized tau oligomers include disulfide-linked tau oligomer species in which covalent attachment occurs between tau monomers through intermolecular disulfide linkages between specified cysteines, with the N-terminal and C-terminal of each monomer in a parallel orientation relative to each other in the trimeric form.
The invention further defines tau oligomer structure using human recombinant tau and specified 3R and 4R tau components, including microtubule binding repeats (R1–R4) and R′, together with PHF6 and PHF6* motifs and cysteine residues such as cysteine 291 and cysteine 322. The disulfide-mediated model is described as enabling nucleation, extension, and termination of tau oligomer formation consistent with a cross-beta-sheet structure and staggered alignment.
The tau oligomer is formed without the addition of heparin or other polyanions. The document additionally describes stability concepts including thermal stability, salt stability, and redox dependence, and uses conformation-specific detection such as immunoassays with mAb T46 and pAb A11 and immunoblotting with mAb HT7.
Claims Coverage
The independent claims cover three inventive feature sets relating to non-naturally occurring, purified and stabilized human recombinant tau oligomers in buffer, with specified disulfide-linked trimeric forms, at least 90% purification, and formation without heparin or other polyanions.
Stabilized disulfide-linked tau trimer in neutral-pH buffer without heparin or polyanions
A composition comprising a non-naturally occurring purified and/or isolated and stabilized tau oligomer and a buffer having a neutral pH, wherein the tau oligomer comprises human recombinant tau protein produced from eukaryotic cell culture using cell lines in which human tau is not naturally found comprising yeast, Sf9 insect cells, or Chinese hamster ovary (CHO) cells; the tau oligomer is in a trimeric form with covalently attached monomers via intermolecular disulfide linkage between specified cysteines; each monomer has an N-terminal and a C-terminal in a parallel orientation; and the tau oligomer is at least 90% purified and formed without the addition of heparin or other polyanions.
Bacterial production of stabilized disulfide-linked tau trimer in Tris or Tris-HCl buffer as a monoclonal antibody antigen
A composition comprising a non-naturally occurring purified and/or isolated and stabilized tau oligomer and a buffer, wherein the tau oligomer comprises human recombinant tau protein and is produced in bacteria; the buffer being a Tris buffer or a Tris-HCl buffer; the tau oligomer is in a trimeric form with covalently attached monomers via intermolecular disulfide linkage between specified cysteines; each monomer has an N-terminal and a C-terminal in a parallel orientation; and the tau oligomer is at least 90% purified and formed without the addition of heparin or other polyanions, wherein the tau oligomer can be used as an antigen to make monoclonal antibodies.
Bacterial production of stabilized disulfide-linked tau trimer in neutral-pH buffer with a monoclonal antibody epitope
A composition comprising a non-naturally occurring purified and/or isolated and stabilized tau oligomer and a buffer, wherein the tau oligomer comprises human recombinant tau protein and is produced in bacteria in the buffer having a neutral pH; the tau oligomer is in a trimeric form with covalently attached monomers via an intermolecular disulfide linkage between specified cysteines; each monomer has an N-terminal and a C-terminal in a parallel orientation; and the tau oligomer is at least 90% purified and formed without the addition of heparin or other polyanions, wherein the tau oligomer comprises an epitope for making monoclonal antibodies.
Overall, the independent-claim coverage centers on non-naturally occurring stabilized and substantially purified disulfide-linked tau trimer compositions in neutral-pH buffer (or Tris/Tris-HCl in the bacterial embodiment), with specific covalent disulfide linkages between cysteine-defined monomers and parallel N-/C-terminal orientation, formed without heparin or other polyanions, and configured to function as an antigen or epitope for monoclonal antibody generation.
Stated Advantages
Provides compositions of stabilized and substantially purified non-naturally occurring tau oligomers.
Provides tau oligomers formed without the addition of heparin or other polyanions.
Supports use as an antigen to make monoclonal antibodies and comprises an epitope for monoclonal antibody making.
Enables measurement of tau oligomer levels in CSF as biomarkers for diagnosing, stratifying, and monitoring Alzheimer’s disease and tauopathies.
Describes tau oligomers as immunotherapeutic targets and drug targets, including small-molecule screening to modulate or disrupt tau oligomer formation.
Documented Applications
Diagnosing, stratifying, and monitoring Alzheimer’s disease and tauopathies using tau oligomer levels in cerebrospinal fluid (CSF) as biomarkers.
Immunotherapeutic targeting of tau oligomers, including use as immunotherapeutic targets and making monoclonal antibodies using the tau oligomer as an antigen or epitope.
Drug target use, including screening small molecules to modulate or disrupt tau oligomer formation.
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