Patent Application
20230235034
The neuropathological hallmarks of Alzheimer's Disease (AD) comprise extracellular amyloid-beta (A3) plaques and intraneuronal tau protein aggregates (inclusions) manifesting as neuritic plaques (NPs), neuropil threads (NTs), and neurofibrillary tangles (NFTs). Tau is a natively unstructured microtubule-associated protein expressed in the central nervous system as six differentially-spliced isoforms containing either 0, 1, or 2 N-terminal acidic exons and 3 or 4 microtubule-binding repeats (MTBRs). The typically unstructured tau protein can adopt a misfolded beta-sheet conformation that aggregates into fibrils with a filament core comprised of the MTBRs and folding that enables contact of the N-terminus with the core domains, to form paired helical filaments (PHFs) that assemble into NFTs. Accumulations of tau protein closely correlate with cognitive decline and neuron death in AD patients more so than the presence of A3 plaques. Although there are no mutations in the gene encoding tau protein associated with AD, mutation of the tau (MAPT) gene results in Frontotemporal Dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Furthermore, tau forms intracellular inclusions in additional neurodegenerative tauopathies such as fronto-temporal lobar degeneration with tau (FLTD-tau) including Pick's disease, progressive supranuclear palsy, and corticobasal degeneration. Therefore, tau plays a central role in the neurodegenerative disease process and presents an attractive target for therapeutic intervention in AD and related tauopathies.
Tau aggregates propagate throughout the brain in a stereotypical spatiotemporal pattern that progresses with disease severity, beginning in the locus coeruleus and transentorhinal cortex, followed by the hippocampus and neocortex, and reaching the visual cortex at the latest disease stages. Growing evidence suggests this process is mediated by cell-to-cell transmission of pathological tau seeds from a neuron containing fibrillar tau species to a normal recipient neuron. Release of monomeric tau and polymeric misfolded tau from neurons may result from cell death or neuronal activity, generating both free extracellular tau and a small percentage of vesicle encapsulated tau. Once taken up into a recipient neuron, pathological tau seeds act as a template to recruit native cellular tau into newly formed oligomers and fibrils. Mouse models have demonstrated that intracerebral injection of either synthetic tau preformed fibrils (PFFs) or human AD-brain-derived pathological tau (AD-tau) can instigate tau pathology in regions of the brain distant from the injection site in either transgenic (Tg) mice expressing mutant human tau or non-transgenic wildtype (WT) mice. Together, these findings provide impetus for the development of anti-tau antibodies as immunotherapeutics for AD based on the hypothesis that antibody binding to extracellular tau prevents spread of pathological tau aggregates throughout the brain.
There is thus a need for novel agents that can be used for treating, ameliorating, and/or preventing AD, based in one aspect on neutralizing pathological tau. This disclosure addresses and meets those needs.
In one aspect, the present disclosure provides an isolated monoclonal antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 23 or 51; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 25 or 53; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 27 or 55, and wherein the VH comprises a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 9 or 37; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 11 or 39; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 13 or 41.
In another aspect, the present disclosure provides a pharmaceutical composition comprising at least one monoclonal antibody of the disclosure and at least one pharmaceutical excipient.
In yet another aspect, the present disclosure provides an isolated polynucleotide comprising at least one of the nucleic acid sequences of SEQ ID NOs: 57, 59, 61, or 63.
In yet another aspect, the present disclosure provides a method of preventing, minimizing, and/or reversing fibrillization of native tau, the method comprising contacting the native tau with an effective amount of at least one isolated monoclonal antibody of the disclosure.
In yet another aspect, the present disclosure provides a method of preventing or minimizing transmission of pathological tau to a cell and/or uptake of pathological tau by a cell, the method comprising contacting the cell with an effective amount of at least one isolated monoclonal antibody of the disclosure.
In yet another aspect, the present disclosure provides a method of reducing and/or preventing further increase in any intraneuronal tau aggregates or inclusions in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one isolated monoclonal antibody of the disclosure.
In yet another aspect, the present disclosure provides a method of treating, preventing, and/or ameliorating a tauopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one isolated monoclonal antibody of the disclosure.
In yet another aspect, the present disclosure provides a method of detecting a tauopathy in a subject, the method comprising administering to the subject at least one labeled isolated monoclonal antibody of the disclosure, and detecting presence or absence of a complex of the at least one labeled isolated monoclonal antibody with any pathological tau present in the subject, wherein, if the complex is detected, the subject has a tauopathy.
In yet another aspect, the present disclosure provides a method of detecting pathological tau in a sample, the method comprising contacting the sample with at least one labeled isolated monoclonal antibody of the disclosure, and detecting presence or absence of a complex of the at least one labeled isolated monoclonal antibody with any pathological tau present in the sample, wherein, if the complex is detected, pathological tau are present in the sample.
In yet another aspect, the present disclosure provides an isolated monoclonal antibody, or fragment thereof, which recognizes a conformational epitope comprising amino acids 151-244 and amino acids 369-441 of pathological tau, and which affinity for pathological tau is higher than for native tau.
In yet another aspect, the present disclosure provides an autonomously replicating or an integrative mammalian cell vector comprising a recombinant nucleic acid encoding an antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 23 or 51; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 25 or 53; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 27 or 55, and wherein the VH comprises a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 9 or 37; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 11 or 39; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 13 or 41.
In yet another aspect, the present disclosure provides an isolated host cell comprising any least one vector of the disclosure.
The following detailed description of specific embodiments of the disclosure will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosure, the drawings show specific embodiments. It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, selected materials and methods are described herein. In describing and claiming the present disclosure, the following terminology will be used. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
As used herein, each of the following terms has the meaning associated with it in this section.
Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, analytical chemistry, immunology, and nucleic acid chemistry and hybridization are those well-known and commonly employed in the art.
Standard techniques or modifications thereof are used for chemical syntheses and chemical analyses.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
As used herein, the term “Tau” or “tau” refers to a protein that promotes microtubule assembly and stability in cells. In certain embodiments, for the sake of illustration, the canonical sequence of tau is represented in SEQ ID NO: 69:
As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a concentration, a temporal duration, and the like, the term “about” is meant to encompass variations of 20% or ±10%, more preferably +5%, even more preferably +1%, and still more preferably +0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
As used herein, the term “affinity” for a molecule towards another refers to the degree (or tightness) of binding between the two molecules. A higher affinity means tighter binding between the two molecules. Affinity can be quantified in terms of dissociation constant (or Kd), where a Kd value that is lower in magnitude (closer to zero) indicates a higher affinity.
An “amino acid” as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids. “Standard amino acid” means any of the twenty L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid residues” means any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or derived from a natural source. As used herein, “synthetic amino acid” also encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and substitutions. Amino acids contained within the peptides, and particularly at the carboxy- or amino-terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change a peptide's circulating half-life without adversely affecting activity of the peptide. Additionally, a disulfide linkage may be present or absent in the peptides.
The term “antibody,” as used herein, refers to an immunoglobulin molecule able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present disclosure may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“intrabodies”), Fv, Fab and F(ab)2, as well as single chain antibodies (scFv), camelid antibodies and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). As used herein, a “neutralizing antibody” is an immunoglobulin molecule that binds to and blocks the biological activity of the antigen.
The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated or synthesized, or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
A “coding region” of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene that are homologous with or complementary to, respectively, the coding region of an mRNA molecule produced by transcription of the gene.
A “coding region” of an mRNA molecule also consists of the nucleotide residues of the mRNA molecule that are matched with an anti-codon region of a transfer RNA molecule during translation of the mRNA molecule or that encode a stop codon. The coding region may thus include nucleotide residues corresponding to amino acid residues not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).
“Complementary” as used herein to refer to a nucleic acid, refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
The term “delivery vehicle” is used herein as a generic reference to any delivery vehicle capable of delivering a compound to a subject, including, but not limited to, dermal delivery vehicles and transdermal delivery vehicles.
The term “DNA” as used herein is defined as deoxyribonucleic acid.
“Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein, effective to achieve a particular biological result. Such results may include, but are not limited to, treatment of a disease or condition as determined by any means suitable in the art.
“Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
As used herein, the term “fragment,” as applied to a protein or peptide, refers to a subsequence of a larger protein or peptide. A “fragment” of a protein or peptide can be at least about 20 amino acids in length; for example at least about 50 amino acids in length; at least about 100 amino acids in length, at least about 200 amino acids in length, at least about 300 amino acids in length, and at least about 400 amino acids in length (and any integer value in between). As used herein, an antibody fragment refers to active fragments thereof, i.e., fragments having the same characteristics that are used for the definition of an antibody according to the disclosure, in certain embodiments higher affinity for pathological tau than for native (non-pathological tau). For convenience when the term antibody is used, fragments thereof exhibiting the same characteristic are also being considered.
As used herein, the term “fragment,” as applied to a nucleic acid, refers to a subsequence of a larger nucleic acid. A “fragment” of a nucleic acid can be at least about 15 nucleotides in length; for example, at least about 50 nucleotides to about 100 nucleotides; at least about 100 to about 500 nucleotides, at least about 500 to about 1000 nucleotides, at least about 1000 nucleotides to about 1500 nucleotides; or about 1500 nucleotides to about 2500 nucleotides; or about 2500 nucleotides (and any integer value in between).
Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5′-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5′-direction.
The direction of 5′ to 3′ addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the “coding strand”; sequences on the DNA strand which are located 5′ to a reference point on the DNA are referred to as “upstream sequences”; sequences on the DNA strand which are 3′ to a reference point on the DNA are referred to as “downstream sequences.”
An “individual”, “patient” or “subject”, as that term is used herein, includes a member of any animal species including, but are not limited to, birds, humans and other primates, and other mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs. Preferably, the subject is a human.
“Instructional material,” as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the composition and/or compound of the disclosure in a kit. The instructional material of the kit may, for example, be affixed to a container that contains the compound and/or composition of the disclosure or be shipped together with a container which contains the compound and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively. Delivery of the instructional material may be, for example, by physical delivery of the publication or other medium of expression communicating the usefulness of the kit, or may alternatively be achieved by electronic transmission, for example by means of a computer, such as by electronic mail, or download from a website.
“Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
An “isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, i.e., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
By “nucleic acid” is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).
Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
The term “oligonucleotide” typically refers to short polynucleotides, generally no greater than about 60 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T.
As used herein, the term “pathological tau” refers to a conformation, phosphorylated form, derivative, analogue, and/or modification of tau that forms and/or induces formation of paired helical filaments (PHFs), neuritic plaques (NPs), neuropil threads (NTs), and/or neurofibrillary tangles (NFTs). In certain embodiments, pathological tau plays a role in development of tauopathies, which are a class of neurodegenerative diseases involving the aggregation of tau protein into neurofibrillary or gliofibrillary tangles in the human brain. Non-limiting examples of Tauopathies include: Alzheimer's Disease, Pick's disease, Corticobasal degeneration, Argyrophilic grain disease (AGD), Primary age-related tauopathy (PART)/Neurofibrillary tangle-predominant senile dementia, Chronic traumatic encephalopathy (CTE), Progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), Lytico-bodig disease (Parkinson-dementia complex of Guam), Ganglioglioma and gangliocytoma, Meningioangiomatosis, Postencephalitic parkinsonism, Subacute sclerosing panencephalitis (SSPE), Lead encephalopathy, Tuberous sclerosis, Pantothenate kinase-associated neurodegeneration, and Lipofuscinosis.
As used herein, the term “pharmaceutical composition” refers to a mixture of at least one compound of the disclosure with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.
“Pharmaceutically acceptable” refers to those properties and/or substances that are acceptable to the patient from a pharmacological/toxicological point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding composition, formulation, stability, patient acceptance and bioavailability. “Pharmaceutically acceptable carrier” refers to a medium that does not interfere with the effectiveness of the biological activity of the active ingredient(s) and is not toxic to the host to which it is administered.
The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.
As used herein, the terms “protein”, “peptide” and “polypeptide” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. The term “peptide bond” means a covalent amide linkage formed by loss of a molecule of water between the carboxyl group of one amino acid and the amino group of a second amino acid. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that may comprise the sequence of a protein or peptide. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Proteins” include, for example, biologically active fragments, substantially homologous proteins, oligopeptides, homodimers, heterodimers, variants of proteins, modified proteins, derivatives, analogs, and fusion proteins, among others. The proteins include natural proteins, recombinant proteins, synthetic proteins, or a combination thereof. A protein may be a receptor or a non-receptor.
The term “recombinant DNA” as used herein is defined as DNA produced by joining pieces of DNA from different sources.
The term “recombinant polypeptide” as used herein is defined as a polypeptide produced by using recombinant DNA methods.
The term “RNA” as used herein is defined as ribonucleic acid.
The term “therapeutic” as used herein means a treatment and/or prophylaxis.
The term to “treat,” as used herein, means reducing the frequency with which symptoms are experienced by a subject or administering an agent or compound to reduce the frequency and/or severity with which symptoms are experienced. As used herein, “alleviate” is used interchangeably with the term “treat.”
As used herein, “treating a disease, disorder or condition” means reducing the frequency or severity with which a symptom of the disease, disorder or condition is experienced by a subject. Treating a disease, disorder or condition may or may not include complete eradication or elimination of the symptom.
The following abbreviations are used herein: Aβ, amyloid beta; AD, Alzheimer's disease; AD-tau, Alzheimer's brain derived tau; CBD, corticobasal degeneration; CDR, complementary-determining region; CNS, central nervous system; CSF, cerebrospinal fluid; DLB, dementia with Lewy bodies; ELISA, enzyme linked immunosorbent assay; IHC, immunohistochemistry; i.p., intraperitoneal; mAbs, monoclonal antibodies; MSA, multiple system atrophy; NFT, neurofibrillary tangles; nM, nanomolar; NP, neuritic plaque; NT, neuropil threads; PHF, paired helical filaments; PiD, Pick's disease; PSP, progressive supranuclear palsy; VH, heavy chain variable region; VL, light chain variable region; TBS, Tris buffered saline; tg, transgenic; WT, wildtype.
Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
Based on the stereotypical pattern of pathological tau spread throughout the brain during AD progression, it is thought that tau undergoes cell-to-cell transmission in which portions of tau PHFs exit a cell and are taken-up into adjacent cells, wherein the seeds recruits naïve tau and templates the misfolded conformation thus propagating tau pathology.
The present disclosure is generally directed to certain anti-tau monoclonal antibodies (mAbs), or immunologically active fragments thereof, that selectively bind to pathological tau protein. In certain embodiments, the antibodies of the disclosure inhibit cell-to-cell transmission (spread) of pathological tau among cells. In certain embodiments, the antibodies of the disclosure can be used as immunotherapies for treatment of AD and FTLD-tau, among other tauopathies.
As described here, monoclonal mouse hybridoma cell lines were generated from murine spleen cells, and human SP2 myeloma cell lines and monoclonal populations were derived. DMR7 and SKT82 were identified as lead mAbs based on ELISA binding to AD PHFs, immunoprecipitation of tau from complex brain extracts, inhibition of seeded aggregation of tau in primary neuron models, and in vivo inhibition of pathological tau spread in mice (5×FAD) injected with AD brain derived tau. Without wishing to be limited by any theory, as a low proportion of peripherally administered IgG crosses the blood brain barrier, the selective targeting of pathological tau with conformation-selective antibodies reduces binding to non-pathological tau species, whereas high-affinity linear epitope tau antibodies can be sequestered by extracellular soluble tau present in the brain interstitial fluid.
In certain embodiments, the antibodies of the disclosure show preferential or selective binding towards the pathological form of tau compared to the native (non-pathological) form. In other embodiments, the antibodies of the disclosure reduce formation of pathological tau. In yet other embodiments, the antibodies of the disclosure detect pathological tau. In yet other embodiments, the antibodies of the disclosure are used as therapeutics for decreasing the development/spread of pathological tau.
In certain embodiments, the antibodies of the disclosure bind to pathological tau with a dissociation constant Kd equal to or less than about 10−6 M, about 10−7 M, about 10−8 M, about 10−9 M, about 10−10 M, about 10−11, about 10−12, or about 10−13 M. In other embodiments, the antibodies of the disclosure bind to pathological tau with an affinity that at least about 1.5 times, 2 times, 2.5 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 30 times, 100 times, 300 times, or 1000 times tighter (lower absolute Kd) than the affinity of the antibodies for native tau. In yet other embodiments, the antibodies of the disclosure bind to native tau with a dissociation constant Kd equal to or higher than about 10−10 M, about 10−9 M, about 10−8 M, about 10−7 M, about 10−6 M, about 10−5 M, about 10−4 M, or about 10−3 M. In yet other embodiments, the antibodies of the disclosure bind with nearly equal affinity to native tau and pathological tau. Binding affinities of the antibodies can be determined by using a variety of methods recognized in the art, including methods described elsewhere herein, such as but not limited to isothermal calorimetry, surface plasmon resonance, immunoassays such as ELISA or RIAs, and the like.
In one aspect, the disclosure comprises isolated monoclonal antibodies that selectively bind pathological tau over native tau.
In certain embodiments, the isolated monoclonal antibody, or fragment thereof, recognizes a conformational epitope comprising amino acids 151-244 and amino acids 369-441 of pathological tau, and its affinity for pathological tau is higher than for native tau. In other embodiments, the isolated monoclonal antibody, or fragment thereof, recognizes amino acids 120-151, 151-244, and 369-441 of pathological tau, and its affinity for pathological tau is higher than for native tau. In other embodiments, the isolated monoclonal antibody, or fragment thereof, recognizes amino acids 120-151 and 369-441 of pathological tau, and its affinity for pathological tau is higher than for native tau.
In certain embodiments, the antibody comprises a heavy chain. In other embodiments, the heavy chain comprises three complementary-determining regions (CDR), namely CDR1, CDR2, and CDR3. In other embodiments, the antibody comprises a light chain. In yet other embodiments, the light chain comprises three complementary-determining regions (CDR), namely CDR1, CDR2, and CDR3.
In certain embodiments, the monoclonal antibody comprises light and heavy variable chains having the sequences shown below:
ATGAAATGCAGCTGGGTCATCTTCTTCCTGATGGCAGTGGTTATAGGAATCAATTCA
GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAGGTCAGGGGCCTCAGTCAAG
TTGTCCTGCACAGCTTCTGGCTTCAACATTAAA
GACTACTATATGCAC
TGGGTTAAG
CAGAGGCCTGAACAGGGCCTGGAGTGGATTGGA
TGGTTTGATCCTGAGAATGGTGAT
GCTGAATATGCCCCGAAGTTCCAGGAC
AAGGCCACTATGACTGCAGACACATCCTCC
AACGCAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTAC
TGTAATGGT
TATCTTTAC
TGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA
MKCSNVIFFIMAVVIGINS
EVQLQQSGAELVRSGASVKLSCTASGFNIK
DYYMH
WVK
QRPEQGLEWIG
WFDPENGDAEYAPKFQD
KATMTADTSSNAAYLQLSSLTSEDTAVYY
CNG
YLY
WGQGTLVTVSS
ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGT
GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCC
TCCATCTCTTGC
AGATCTAGTCAGAACATTGTACATAATAATGGAAACACCTATTTA
AACCGATTTTCT
GGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTC
ACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTACTGC
TTTCAA
GGTTCACATGTTCCGCACACG
TTCGGAGGGGGGACCAGGCTGGAAATAAAA
MKLPVRLLVLMFWIPASSS
DVLMTQTPLSLPVSLGDQASISC
RSSQNIVHNNGNTYL
E
WYLQKPGQSPKLLIY
KVSNRFS
GVPDRFSGSGSGTDFTLKISRVEAEDLGIYYC
FQ
GSHVPHT
FGGGTRLEIK
CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAA
CTGTCCTGCAAGGCTTCT
GGCTACATCTTCACCACCTACTGG
ATGAACTGGGTGAAG
CAGAGGCCTGGACAGGGCCTTGAATGGATTGCTATG
ATTGATCCTTCAGACAGTGAA
ACT
CACTACAATCAAATGTTCAAGGACAAGGCCACATTGACTGTAGACACATCCTCC
AGCACGGCCTACATGCAGCTCAGCGGCCTGACATCTGAAGACTCTGCGGTCTATTAC
TGTGCAAGAGGGGAAGGCTACTGG
GGCCAAGGCACCACTCTCACAGTCTCCTCA
QVQLQQPGAELVRPGASVKLSCKAS
GYIFTTYW
MNWVKQRPGQGLEWIAM
IDPSDSE
T
HYNQMFKDKATLTVDTSSSTAYMQLSGLTSEDSAVYY
CARGEGYW
GQGTTLTVSS
GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCC
TCCATCTCTTGCAAGTCAAGT
CAGAGCCTCTTAGATAGTGATGGAAAGACATAT
TTG
AATTGGTTGTTACAGAGCCCAGGCCAGTCTCCAAAGCGCCTAATCTTC
CTGGTGTCT
AAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTC
ACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTAT
TGCTGGCAA
GGTACACATTTTCCGTGGACGTTC
GGTGGAGGCACCAAGCTGGAAATCAAA
DVVMTQTPLTLSVTIGQPASISCKSS
QSLLDSDGKTY
LNWLLQSPGQSPKRLIF
LVS
KLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYY
CWQGTHFPWTF
GGGTKLEIK
Further combined sequences are provided herein:
In certain embodiments, the antibody comprises an immunoglobulin light chain variable region (VL) comprising the amino acid sequence of SEQ ID NOs: 60 or 64.
In certain embodiments, the antibody comprises an immunoglobulin heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NOs: 58 or 62.
In certain embodiments, the antibody comprises an immunoglobulin light chain variable region (VL) comprising the amino acid sequence of SEQ ID NOs: 60 or 64, and an immunoglobulin heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NOs: 58 or 62.
In certain embodiments, the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 60, and a VH comprising SEQ ID NO: 58. In other embodiments, the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 64, and a VH comprising SEQ ID NO: 62.
In certain embodiments, the antibody comprises a VL comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 23 or 51; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 25 or 53; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 27 or 55.
In certain embodiments, the antibody comprises a VL comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 23; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 25; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 27. In other embodiments, the antibody comprises a VL comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 51; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 53; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 55.
In certain embodiments, the antibody comprises a VH comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 9 or 37; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 11 or 39; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 13 or 41.
In certain embodiments, the antibody comprises a VH comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 9; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 11; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 13. In other embodiments, the antibody comprises a VH comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 37; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 39; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 41.
In certain embodiments, the antibody comprises a VL comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 23; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 25; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 27; and a VH comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 9; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 11; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 13.
In certain embodiments, the antibody comprises a VL comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 51; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 53; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 55; a VH comprising: a CDR1 region comprising the amino acid sequence of SEQ ID NO: 37; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 39; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 41.
The disclosure further provides isolated polynucleotides (including RNA and/or DNA) encoding the antibodies or antigen binding fragments thereof, for example a nucleic acid encoding for one or more CDRs, or a variable heavy chain or variable light chain region of the antibodies of the disclosure. Nucleic acid includes DNA and RNA.
In certain embodiments, the antibody has a VL encoded by the nucleic acid sequence of SEQ ID NOs: 59 or 63. In other embodiments, the antibody has a VH encoded by the nucleic acid sequence of SEQ ID NOs: 57 or 61.
In certain embodiments, the disclosure provides an isolated polynucleotide comprising the nucleic acid sequence of SEQ ID NOs: 59 or 63. In other embodiments, he disclosure provides an isolated polynucleotide comprising the nucleic acid sequence of SEQ ID NOs: 57 or 61.
In certain embodiments, the disclosure provides an isolated polynucleotide comprising a nucleic acid encoding the amino acid sequence of SEQ ID NOs: 58 or 62. In other embodiments, the disclosure provides an isolated polynucleotide comprising a nucleic acid encoding the amino acid sequence of SEQ ID NOs: 60 or 64.
In certain embodiments, the disclosure provides an autonomously replicating or an integrative mammalian cell vector comprising a recombinant nucleic acid of the disclosure. In other embodiments, the disclosure provides a vector comprising a recombinant nucleic acid of the disclosure. In yet other embodiments, the recombinant nucleic acid of the disclosure encodes an antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 23 or 51; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 25 or 53; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 27 or 55, and wherein the VH comprises a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 9 or 37; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 11 or 39; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 13 or 41. In yet other embodiments, the vector comprises a plasmid or virus. In yet other embodiments, the vector comprises a mammalian cell expression vector. The expression vector can comprise nucleic acid sequences that direct and/or control expression of the inserted polynucleotide. Such nucleic acid sequences can include regulatory sequence, including promoter sequences, terminator sequences, polyadenylation sequences, and enhancer sequences. Systems for cloning and expression of a polypeptide in a variety of cells are well known in the art.
The disclosure further provides a host cell comprising the expression vector of the disclosure. In certain embodiments, the host cell is isolated. In other embodiments, the host cell is a non-human cell. In yet other embodiments, the host cell is mammalian.
The antibody of the disclosure can be a mammalian antibody, such as primate, human, rodent, rabbit, ovine, porcine or equine antibody. The antibody can be any class or isotype antibody, for example IgM or IgG. In certain embodiments, the antibody is IgG.
The disclosure further provides a kit comprising an antibody of the disclosure. The antibody may be an intact immunoglobulin molecule or fragment thereof such as Fab, F(ab)2 or Fv fragment. The antibody can be labelled as described elsewhere herein. The kit can be for use in a method of determining whether a subject has a neurodegenerative disease contemplated herein, and/or for treating a subject afflicted or thought to be afflicted with a neurodegenerative disease contemplated herein. The kit can further any other reagent or instrument that is required to implement a method of the disclosure, such as a buffer, an applicator, and the like.
In certain embodiments, the disclosure comprises pharmaceutical compositions comprising antibodies contemplated herein in combination with one or more pharmaceutically acceptable excipients. In some embodiments the pharmaceutical composition is formulated for parenteral delivery. In other embodiments, the antibodies are humanized.
In another aspect, the disclosure provides a method of treating a tauopathy comprising administering a therapeutically effective amount of an isolated monoclonal antibody of the disclosure to a patient. In certain embodiments, the antibody is humanized. In other embodiments, the antibody is administered as a pharmaceutical composition.
The monoclonal antibodies described above may be used to treat, prevent, and/or ameliorate tauopathies by inhibiting cell-to-cell transmission (spread) of pathological tau, as demonstrated herein. In certain embodiments, the tauopathies include but are not limited to Alzheimer's Disease, Pick's disease, Corticobasal degeneration, Argyrophilic grain disease (AGD), Primary age-related tauopathy (PART)/Neurofibrillary tangle-predominant senile dementia, Chronic traumatic encephalopathy (CTE), Progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), Lytico-bodig disease (Parkinson-dementia complex of Guam), Ganglioglioma and gangliocytoma, Meningioangiomatosis, Postencephalitic parkinsonism, Subacute sclerosing panencephalitis (SSPE), Lead encephalopathy, Tuberous sclerosis, Pantothenate kinase-associated neurodegeneration, Lipofuscinosis, and any other pathological tau neurodegenerative disorders. The antibody can be administered systemically or directly to the site where pathological tau is observed or thought to be present. In a non-limiting example, the antibody can be administered by injection into a blood vessel supplying the brain or into the brain itself. The subject can be a mammal, such as a human or a non-human mammal.
In yet another aspect, the disclosure provides methods of detecting pathological tau and/or a tauopathy in a patient. In other embodiments, the antibodies of the disclosure can be used as diagnostic tools for neurodegenerative disorders associated with pathological tau, including but not limited to Alzheimer's Disease, Pick's disease, Corticobasal degeneration, Argyrophilic grain disease (AGD), Primary age-related tauopathy (PART)/Neurofibrillary tangle-predominant senile dementia, Chronic traumatic encephalopathy (CTE), Progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), Lytico-bodig disease (Parkinson-dementia complex of Guam), Ganglioglioma and gangliocytoma, Meningioangiomatosis, Postencephalitic parkinsonism, Subacute sclerosing panencephalitis (SSPE), Lead encephalopathy, Tuberous sclerosis, Pantothenate kinase-associated neurodegeneration, Lipofuscinosis, and any other pathological tau neurodegenerative disorders.
In certain embodiments, the method of detecting pathological tau and/or a tauopathy in a subject comprises the steps of administering a labeled, isolated monoclonal antibody of the disclosure to the subject, and detecting the presence of absence of a complex between any pathological tau in the subject and the antibody. If the complex is present, that indicates that pathological tau exist in the subject. In certain embodiments, if pathological tau is present in the subject, the subject has a neurodegenerative disease. In other embodiments, if pathological tau is not present in the subject, the subject does not have a neurodegenerative disease. In yet other embodiments, if the subject has a neurodegenerative disease, the individual is counseled to undergo therapy and/or pharmacological treatment for the neurodegenerative disease. In yet other embodiments, if the subject has a neurodegenerative disease, the individual is provided therapy and/or pharmacological treatment for the neurodegenerative disease.
In certain embodiments, the method further comprises comparing the level of antibody/pathological tau complex formed in the subject with the level of antibody/pathological tau complex formed in a reference subject. The reference subject can be a subject known not to have pathological tau, a subject known to have detectable pathological tau, and/or a subject known to have a certain level of pathological tau. The reference subject can further be the same subject being treated or evaluated, but corresponding to an earlier pathological tau detection experiment, as a way to evaluate disease progression and/or treatment efficacy in the subject.
In yet another aspect, the disclosure provides methods of detecting pathological tau in a sample. In certain embodiments, the antibodies of the disclosure can be used as diagnostic tools for detecting the presence of pathological tau in a sample.
In certain embodiments, the method of detecting pathological tau in a sample (for example, from a subject) comprises the steps of contacting the sample with a labeled, isolated monoclonal antibody of the disclosure, and detecting the presence or absence of a complex between any pathological tau in the sample and the antibody. If the complex is detected, that indicates the presence of pathological tau in the sample. The sample can be, in non-limiting examples, cerebrospinal fluid (CSF), blood, urine, saliva, or tissues from brain, gut, colon, skin, or salivary gland. In certain embodiments, the sample is a CSF sample and/or a brain tissue sample. In other embodiments, the sample is used as is after being removed from the subject. In other embodiments, the sample is pre-treated being used within the present methods.
In certain embodiments, the method further comprises comparing the level of antibody/pathological tau complex formed in the sample with the level of antibody/pathological tau complex formed in a reference sample. The reference sample can be from a subject known not to have pathological tau, a subject known to have detectable pathological tau, and/or a subject known to have a certain level of pathological tau. The reference sample can further be from the same subject being treated or evaluated, but corresponding to an earlier pathological taudetection, as a way to evaluate disease progression and/or treatment efficacy in the subject.
In certain embodiments, the level of pathological tau detected in a subject or in a sample from a subject correlates with severity or progression of a neurodegenerative disease in the subject. In other embodiments, the methods of the disclosure can be used to monitor severity or progression of a neurodegenerative disease in the subject. In yet other embodiments, the methods of the disclosure can be used to monitor effectiveness of a therapy and/or pharmacological intervention in a subject afflicted or believed to be afflicted with a neurodegenerative disease.
Methods for detecting formation of a complex between the antibody and pathological tau comprise, but are not limited to, radioimmunoassay, enzyme-linked immunosorbant assay (ELISA), sandwich immunoassay, fluorescent immunoassay, precipitation reaction, gel immunodiffusion assay, agglutination assay, protein A immunoassay, immunoelectrophoresis assay, electrophoresis, western blotting, or any other technique known in the art.
The antibodies of the disclosure can be combined with a label and used to detect pathological tau in a patient or in a sample. Methods of labeling antibodies are known in the art and a variety of approaches may be employed. In certain embodiments the label is a radiolabel, such as but not limited to F18, I123, I111, I131, C14, H3, Tc99m, P32, I125, Ga68 and the like. In other embodiments, the label is a fluorescent label, such as but not limited to fluorescein, rhodamine and the like. In yet other embodiments, the label is a contrast agent, such as but not limited to gadolinium (Gd), dysprosium and iron, magnetic agents, and the like. Other labels include nuclear magnetic resonance active labels, positron emitting isotopes detectable by a PET scanner, chemiluminescent and enzymatic markers. Non-limiting imaging techniques include electron microscopy, confocal microscopy, light microscopy, positron emission tomography (PET), gamma-scintigraphy, magnetic resonance imaging (MRI), functional magnetic resonance imaging (FMRI), magnetoencephalography (MEG), and single photon emission computerized tomography (SPECT). In yet other embodiments, the label is on a secondary antibody that binds a primary antibody comprising the above described sequences.
Administration of the compounds and/or compositions of the present disclosure to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treating or preventing a disease or disorder and/or perform an imaging method contemplated in the disclosure. An effective amount of the therapeutic compound necessary for adequate disease or disorder treatment and/or imaging may vary according to factors such as the state of a disease or disorder in the patient; the age, sex, and weight of the patient; and the equipment used to detect the compound of the disclosure. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic and/or imaging compound without undue experimentation.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve successful disease or disorder treatment and/or imaging for a particular patient, composition, and mode of administration, without being toxic to the patient.
In certain embodiments, the compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the disclosure comprise an effective amount of a compound of the disclosure and a pharmaceutically acceptable carrier.
The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
Compounds of the disclosure for administration may be in the range of from about 1 g to about 10,000 mg, about 20 μg to about 9,500 mg, about 40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg to about 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about 6,000 mg, about 500 μg to about 5,000 mg, about 750 g to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.
In certain embodiments, the dose of a compound of the disclosure is from about 1 mg and about 2,500 mg. In certain embodiments, a dose of a compound of the disclosure used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in certain embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like.
Routes of administration of any one of the compositions of the disclosure include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the disclosure may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans) buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.
As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.
Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multidose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In certain embodiments of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen free water) prior to parenteral administration of the reconstituted composition.
The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
Additional dosage forms of this disclosure include dosage forms as described in U.S. Pat. Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos. 2003/0147952; 2003/0104062; 2003/0104053; 2003/0044466; 2003/0039688; and 2002/0051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos. WO 03/35041; WO 03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO 02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO 98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.
The disclosure is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the disclosure should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, practice the claimed methods of the present disclosure. The following working examples therefore, specifically point out selected embodiments of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure.
Postmortem human brain tissue from neuropathologically confirmed AD cases were used for extraction of AD-tau. Briefly, gray matter from frontal cortex was homogenized in high salt buffer (10 mM Tris-HCl pH 7.4, 800 mM NaCl, 1 mM EDTA, 2 mM DTT, with a protease inhibitor cocktail and PMSF as well as 0.1% sarkosyl and 10% sucrose) in a dounce homogenizer in nine volumes of buffer per gram tissue. This was followed by centrifugation at 10,000 g for 10 min at 4° C. Sarkosyl was added to the pooled supernatant up to 1% and rotated 1 h at room temperature (RT) then centrifuged at 300,000 g for 1 h at 4° C. The sarkosyl-insoluble pellet, containing pathological tau, was washed in PBS at 100 μL/g gray matter, then resuspended in PBS by sonicating with 20 pulses at 0.5 sec/pulse using a hand-held QSonica probe and then centrifuged again at 45,000 g for 30 min at 4° C. The pellet was resuspended in one-fifth the previous PBS volume and sonicated with 20-60 pulses and centrifuged 10,000 g for 30 min at 4° C. The final supernatant contained enriched AD-tau at 5-20% purity. Total tau concentration was determined using a Tau5 ELISA assay (Guo & Lee, 2011, J. Biol. Chem. 286(17):15317-31).
Full length tau protein, T40 (2N4R) isoform, and fragments were expressed in BL21(DE3)RIL E. coli and purified by cation exchange chromatography (Li & Lee, 2006, Biochemistry 45(51):15692-701). AD-tau seeded recombinant tau PFFs (i.e., AD-P1 PFFs) were generated by incubation of 36 μM T40 tau (2N4R) with 4 μM AD-tau seeds comprising 10% of the fibrillization reaction with 2 mM DTT in PBS pH 7.0, with shaking at 1000 RPM for 3 days at 37° C. (Guo, et al., 2016, J. Experim. Med. 213(12):2635-54). The reaction mixture was centrifuged at 45,000 g for 30 min and fibrillized tau was collected in the pellet fraction, which was resuspended in the original reaction volume mixture.
DMR7 and SKT82 hybridoma clones were generated as previously described for other tau mAbs (Gibbons, et al., 2018, J. Neuropathol. Experim. Neur. 77(3):216-28). Briefly, mice were injected subcutaneously with AD-tau in Freund's complete adjuvant, and spleens were dissociated and fused with SP2 myeloma cells with PEG treatment. Hybridoma clones were diluted with limiting dilutions and screened for tau antibody binding to T40 tau, AD-P1 PFFs, and AD-tau. Clones with selective binding to AD-P1 PFFs and AD-tau were prioritized and sub-cloned two times to ensure monoclonal populations. Antibodies were purified from hybridoma cell culture media using HiTrap MabSelect SuRe columns (GE Healthcare).
Rabbit polyclonal anti-tau antibody K9JA (Dako) was coated onto 384-well MaxiSorp plates (Fisher Scientific) at 100 ng/well in 0.1 M sodium carbonate buffered to pH 9.6 at 4° C. overnight. Blocking was performed with BlockAce solution (Abd Serotec) at 4° C. overnight. Fibrillized tau antigens AD-P1 PFFs and AD-tau were sonicated, and then all tau antigens including T40 monomer were diluted to 0.2-0.8 μg/mL in 0.2% bovine serum albumin (BSA) in PBS and applied to K9JA-coated plates. Antigens were captured by total tau antibody K9JA at 4° C. overnight. Plates were washed with PBS containing 0.015% Tween-20 (PBST) and the novel mouse tau antibodies or Tau5 total tau control antibody were added to plates for 2 h at 22° C. Plates were washed with PBST and HRP-conjugated anti-mouse (Jackson Immunoresearch) secondary antibodies were applied for 2 h at 22° C. Plates were washed and tetramethylbenzidine peroxidase substrate (KPL laboratories) was added to wells to provide a colorimetric readout, quenched with 10% phosphoric acid and then absorbance measured at 450 nm (Molecular Devices SpectraMax).
T40 tau monomer or AD-tau were chemically and thermally denatured by 1:10 dilution in 8M guanidine hydrochloride and heating at 100° C. for 15 min, and non-denatured controls were diluted in Tris-buffered saline (TBS) at RT. Denatured or non-denatured tau was then diluted 1:50 in TBS and applied to 0.2 μm nitrocellulose membrane using a vacuum apparatus. For western blots, tau isoforms and fragments were diluted in SDS sample buffer, heated 10 min at 100° C., run on 12.5% SDS-PAGE gels and transferred to 0.2 μm nitrocellulose membrane. Immunoblots and dot blots were probed with either total tau control antibody K9JA diluted to 2 μg/mL or conformation-selective mAbs diluted in 5% non-fat milk at 20 μg/mL at 4° C. Infrared dye labeled secondary antibodies (LiCor) were used to detect primary antibody binding, with analysis on a LiCor scanner (LiCor).
All human brain tissue samples used in this study were obtained at autopsy, fixed in ethanol or paraformaldehyde, paraffin-embedded, and cut into 6 μm thick sections and characterized (Arnold, et al., 2013, J. Compar. Neurol. 521(18):4339-55; Toledo, et al., 2014, Alzheimer's & Dementia: J. Alzheimer's Association 10(4):477-84 el). For IHC staining, sections were deparaffinized in xylene and rehydrated in ethanol (100-70%) (Gibbons, et al., 2018, J. Neuropathol. Experim. Neur. 77(3):216-28). Tau antibodies MCi and PHF-1 (gift of Peter Davies) were diluted 1:1,000 and 1:5,000, respectively, while DMR7 and SKT82 were diluted to 2 μg/mL in 2% FBS in 50 mM Tris pH 7.2 and applied overnight to rehydrated tissue sections without antigen retrieval in a humidified chamber at 4° C. Antibody binding was detected by Vectastain Elite ABC Kit (Vector) followed by DAB peroxidase substrate (Vector) and counterstaining with Harris hematoxylin (ThermoFisher).
Primary cortical neurons from E16-19 CD-1 WTmice were cultured in 96-well plates at a density of 17,500 cells/well. After 7 days in vitro (DIV 7), the cells were treated in triplicates with either SKT82, DMR7, or IgG with at concentrations of 3.3-26.7 nM and subsequently transduced with 0.125 μg of AD tau PHF per well. After 7 days of AD-tau and antibody treatment, neurons were maintained in growth factor-rich conditioned media until DIV 21. Cells were washed with PBS and soluble tau was extracted with 0.5% HDTA for 15 min at RT, followed by fixation with 4% PFA and 4% sucrose for 15 min at RT. Immunocytochemistry was performed with the mouse tau-specific R2295M (CNDR) antibody, fluorescently labeled with anti-rabbit secondary antibody, and DAPI to stain nuclei. Imaging was performed on an InCell Analyzer 2200 microscope. The DAPI-positive cell count, and area and density of mouse tau pathology, were analyzed using the InCell Developer Toolbox software. The final quantification was based on density×area of pathological insoluble mouse tau normalized to cell number by DAPI-positive nuclei.
A mixture of Dynabeads conjugated with protein A and protein G were mixed with 10 μg purified DMR7, SKT82, or IgG control antibody and AD-tau containing 2 μg of tau protein and rotated 3 h at RT. A magnetic stand was used to sequester Dynabeads and supernatant was transferred to another tube as the unbound fraction. Beads were washed three times with PBS and bound proteins eluted with SDS sample buffer and heating at 100° C. for 10 min. Equal proportions of unbound reaction and eluted proteins were run on 10% SDS-PAGE gels, transferred to nitrocellulose membranes and immunoblotted with the rabbit polyclonal total tau antibody 17025 (CNDR in-house).
pH-Sensitive Tau Labeling and Cellular Uptake Experiments:
Purified recombinant T40 tau (2N4R) was labeled with the pH-sensitive fluorescent tag, pHrodo red succinimidyl ester (ThermoFisher). Fluorescently-labeled tau was termed pHR-T40 and used in an in vitro seeded fibrillization reaction consisting of 10% pHR-T40, 80% unlabeled T40, and 10% sonicated AD-tau seeds. The reaction was shaken at 1,000 RPM for 4 days at 37° C. and fibrils collected by centrifugation at 100,000 g for 30 min. Pelleted fibrils (AD-P1 pHR-T40), were resuspended in PBS, sonicated, and added to E16-E18 cultured cortical neurons at DIV7-10. After 24 h treatment with AD-P1 pHR-T40, live cells were treated with NucBlue to stain nuclei and internalized AD-P1 pHR-T40 was imaged on a Leica DMI6000 microscope or imaged and evaluated with an InCell2200.
Four month old female 5×FAD mice were stereotaxically-injected with 2 μg AD-tau into the hippocampus (bregma: −2.5 mm; lateral: +2 mm; depth: −2.4 mm from the skull) (Gibbons, et al., 2017, J. Neurosci. 37(47):11485-94). Mice were injected (intraperitoneal or i.p.) with 60 mg/kg of tau mAbs or IgG control mAb 4 days prior to AD-tau injection, on the day of AD-tau injection, and weekly thereafter for 3 months. Upon completion of the dosing period, mice were transcardiallyperfused with 30 mL phosphate buffered saline (PBS) at 120 mL/h and brains were fixed in 4% paraformaldehyde overnight. Fixed brains were embedded into paraffin blocks and then sectioned into 6 μm sections. Every 20th section through the hippocampus was subjected to IHC staining with AT8 mAb (ThermoFisher) diluted at 0.04 μg/mL or alternatively with 50 μM X-34, an amyloid binding dye (Sigma Aldrich). Slides were scanned with a 20× objective using a Lamina slide scanner (PerkinElmer). Brain regions were annotated and AT8 positive area was quantified using Halo software (Indica Labs).
Sequential Extraction of Soluble and Insoluble Tau from Mouse Tissue:
Brains were separated into ipsilateral and contralateral hemispheres and hippocampus was dissected and flash frozen on dry ice and kept at −80° C. Tissue was homogenized by sonication in 9 volumes of HS-RAB buffer (100 mM MES, 1 mM EDTA, 0.5 mM MgSO4, 1 mM DTT, 1 mM PMSF, 0.75 M NaCl, 20 mM NaF, pH 6.8) with protease inhibitors and phosphatase inhibitors. Homogenates were centrifuged at 45,000 g for 30 min at 4° C. and the supernatant was saved as the soluble fraction, while the pellet was resuspended in 9 volumes of HS-RAB buffer containing 1% Triton X-100. Samples were again centrifuged (45,000 g for 30 min at 4° C.) and the supernatants were discarded, while the pellet was resuspended in 9 volumes of HS-RAB buffer containing 1% sarkosyl and then rotated for 1 h at 22° C. Samples were centrifuged at 45,000 g for 45 min at 4° C., the supernatant discarded, and the pellet washed with 500 μL PBS. Samples were centrifuged (45,000 g 30 for min at 4° C.), the supernatant discarded, and the pellet was resuspended in PBS at a volume two-times the mass of the original tissue, and sonicated to resuspend as the insoluble fraction.
Open Field procedures were horizontal and vertical activity recorded with IR beam breaks during a 10 min trial. Data were acquired with a Photobeam Activity System (San Diego Instruments). The percent spontaneous alternation in the Y-maze was calculated as 100×[number of alternations/(total arm entries-2)] where arm entry was defined as all four paws placed inside an arm. For contextual fear conditioning, a mouse was placed in a conditioning chamber (Med Associate) within a sound-attenuating cabinet, and a 2-s, 1.75 mA foot shock was delivered at 148-150 s of a 180-s acquisition trial. Twenty-four hours after acquisition, mice were returned to the conditioning chamber for a 5 min recall trial to assess long-term memory. Fourteen days after acquisition, remote memory was also assessed with a second recall trial. All trials were digitally recorded. Time spent motionless was automatically assessed by Freezescan software (Clever Systems).
Statistical analyses were performed with GraphPad Prism software. Quantification of tau uptake in primary neurons and tau pathology in the seeded aggregation model in primary neurons and slice culture were evaluated by One-way ANOVA with Tukey's post-hoc analysis. Quantification of AT8 positive tau pathology was performed using the mean percentage of AT8 positive area in 3-5 sections per mouse. Initial IgG1 and IgG2a treated groups showed statistically insignificant differences using two-tailed unpaired Student's t tests and were combined for statistical power in analysis of IHC staining and immunoblots of soluble and insoluble tau. DMR7- or SKT82-treated mice were compared against the IgG group by two-tailed unpaired Student's t tests. Contextual fear conditioning measures of % freezing consisted of repeated measurements of the same mice at different time points and were analyzed by two-tailed paired t-test to campre changes from prestimulation to 24 h recall and 24 h recall to 14 day remote recall.
Towards the goal of generating conformation-selective tau mAbs, mice were inoculated with human AD brain-derived extracts enriched in insoluble tau (AD-tau). Hybridoma clones generated from immunized mice were screened by sandwich ELISA to simultaneously assess binding of antibodies to tau monomer, AD-tau seeded recombinant tau preformed fibrils (AD-P1 PFFs), and AD-tau. The pan-tau antibody Tau5 served as a loading control to ensure equivalent capture of the three tau forms by the immobilized pan-tau K9JA antibody (
To assess whether conformational differences between AD-tau and tau monomer contributed to the differences observed by ELISA, AD-tau and tau monomer were chemically and thermally denatured prior to immobilization on dot blots and probing with the DMR7 and SKT82 mAbs (
To determine whether DMR7 and SKT82 detect various forms of pathological tau, brain tissue from several human tauopathies, including AD, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), and Pick's disease (PiD), were analyzed by IHC. Both mAbs detected pathological tau lesions in all of the tauopathy brain sections to a similar extent as the diagnostic standard PHIF1 antibody, which binds tau phosphorylated at Ser396 and Ser404 (
To gain insight into the nature of the conformation selectivity of DMR7 and SKT82, the epitope of these mAbs were investigated. DMR7 and SKT82 were able to detect full-length tau monomer by immunoblot even though they have a relatively low affinity for monomers by ELISA. Therefore, a panel of tau fragments were examined to identify those containing binding epitopes of DMR7 and SKT82 (
To determine whether DMR7 and SKT82 binding to AD-tau would inhibit the seeding of tau aggregates in primary neurons in a previously described assay (Guo, et al., 2016, J. Experim. Med. 213(12):2635-54), WT mouse cortical neurons were treated with AD-tau to template fibrillization of endogenous cellular mouse tau into insoluble aggregates. Both DMR7 and SKT82 significantly reduced the seeding of pathological tau aggregates in a dose-dependent manner (
To elucidate the mechanism by which the conformation-selective tau mAbs inhibit seeding of cellular tau aggregates, recombinant tau protein was covalently-modified with the pH-sensitive pHRodo-red fluorophore, which fluoresces in acidic conditions including late endosomal and lysosomal compartments. Fluorophore-labeled T40 tau (pHRT40) was fibrillized in the presence of 10% AD-tau seeds to template propagation of the AD fibril conformation (pHRT40 AD-P1 PFFs) and pHRodo fluorescence was monitored after pHRT40 AD-P1 PFF addition to primary neurons (
To expand on the primary neuron studies of AD-tau induced pathology, hippocampal slice cultures from WT mice were co-treated with the tau mAbs and AD-tau. Similar to what is observed with primary neurons, AD-tau treatment of hippocampal slice cultures results in recruitment of endogenous mouse tau into insoluble tau aggregates that are detected with the phospho-tau antibody, AT8. The seeding of AT8-positive tau inclusions by AD-tau is significantly inhibited by SKT82 and there is a trend towards reduction of pathology by DMR7 that did not achieve statistical significance (
Given the selective binding of DMR7 and SKT82 to pathological tau, as well as inhibition of AD-tau seeded aggregation in primary neurons and cortical slice cultures, it was evaluated whether these tau mAbs inhibit the transmission of tau pathology in vivo. Intracerebral injection of AD-tau into the 5×FAD mouse model of A3 plaque formation induces NP tau pathology in dystrophic neurites surrounding A3 plaques, recapitulating the hallmark plaque and tau pathology observed in AD brain. In addition, seeded aggregation of tau in 5×FAD mice does not rely on transgenic overexpression of mutant human tau to develop tau pathology, as AD-tau induces fibrillization of endogenous mouse tau that accumulates in plaque-associated neuronal processes. A prevention model of antibody treatment was employed in which 4 month old 5×FAD female mice received i.p. injections of 60 mg/kg of SKT82, DMR7, or isotype-matched IgG controls four days prior to unilateral injection of AD-tau into the hippocampus. Mice subsequently received the same mAb doses on the day of AD-tau injection and weekly thereafter for 3 months. To confirm CNS exposure to the mAbs, the levels of SKT82 and DMR7 were assessed in the CSF and plasma of mice 4 and 7 days post-injection, with nanomolar (nM) levels of antibody detected in CSF and μM levels found in plasma, providing a CSF/Plasma ratio from 0.12-0.31% (Table 1) that is consistent with previously published ratios of IgG molecules.
AD-tau induced pathology in the treated 5×FAD mice was evaluated by IHC and biochemical extraction of soluble and insoluble tau to assess total tau and phospho-tau levels. Both SKT82 and DMR7 significantly inhibited tau pathology in the contralateral hippocampus (
To demonstrate that AT8 positive tau pathology detected by IHC represents insoluble pathological tau aggregates, sequential biochemical extractions of ipsilateral and contralateral hippocampi were performed to examine insoluble and soluble tau levels by immunoblot, probing with the PHIF1 antibody that recognizes pathological tau that is phosphorylated at Ser396/Ser404. SKT82 and DMR7 treatment led to lower insoluble phosphorylated tau and total tau in the ipsilateral and contralateral sides of AD-tau injected 5×FAD mice (
To assess the behavioral phenotypes of 7-month old 5×FAD mice treated for 11-13 weeks with SKT82 and DMR7 after AD-tau injection, open-field, Y-maze, and contextual fear conditioning tests were performed. SKT82 and DMR7 were generally well tolerated and did not influence total motor activity of mice in the open field test (
Here, two novel tau mAbs that bind selectively to the pathological misfolded conformation of tau compared to tau monomer are disclosed, with discontinuous epitopes in the proline-rich central domain of tau and the C-terminal domain that are detectable in multiple human tauopathies. Binding of SKT82 and DMR7 to tau seeds prevents their uptake into primary neurons and inhibits the seeded aggregation of tau in WT mouse primary neurons and hippocampal slice cultures. Utilizing 5×FAD mice that harbor A3 plaques, human brain-derived AD-tau injection induces robust endogenous mouse tau pathology that is inhibited by both SKT82 and DMR7, with SKT82 causing a reduction of both ipsilateral and contralateral tau pathology as assessed by IHC and biochemical extraction of insoluble tau from treated mice.
In certain embodiments, SKT82 and DMR7 were developed against human AD brain-derived insoluble tau extracts, and thus are able to detect pathological tau species present in human disease. As shown herein, DMR7 and SKT82 were evaluated in the context of physiological endogenous mouse tau with AD-tau seeded pathology.
DMR7 and SKT82 have similar binding affinities, selectivity, and epitopes, and yet SKT82 was more effective than DMR7 at inhibiting tau pathology in slice cultures and the ipsilateral hippocampus in vivo. Without wishing to be limited by any theory, one possible explanation is that AD-tau bound by SKT82 may be cleared more actively since SKT82 is a mouse IgG2a isotype that activates microglial FC receptors, whereas DMR7 is mouse IgG1 that does not induce a microglial response in murine models.
The present results demonstrate that DMR7 and SKT82 inhibit seeded aggregation of tau pathology in primary neurons by blocking the uptake of fluorescently-labeled tau seeds. In certain non-limiting embodiments, the epitopes of tau bound by mAbs may influence the ability of mAb to block neuronal uptake of tau.
The present studies did not demonstrate significant changes in the A3 plaque burden upon induction of tau pathology with AD-tau injection or changes upon reduction of tau pathology with SKT82 or DMR7. In certain non-limiting embodiments, this may be caused by the relatively aggressive plaque deposition in 5×FAD mice relative to other mouse models. The study did not show changes in freezing during remote recall of contextual fear conditioning, which in certain embodiments may be due to young mice used in the present study or chronic handling of mice for weekly i.p. injections of mAbs.
Given the great demand for tau immunotherapy candidates, it is crucial to explore tau mAbs with varying affinities, epitopes, and isotypes. The present disclosure provides two novel candidates with unique conformational epitopes that selectively bind AD-tau compared to tau monomer. SKT82 and DMR7 reduce tau pathology in vitro and in an AD model with multiple neuropathological hallmarks of AD.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or sub-combination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
The following enumerated embodiments are provided, the number of which is not to be construed as designating levels of importance.
Embodiment 1 provides an isolated monoclonal antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 23 or 51; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 25 or 53; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 27 or 55, and wherein the VH comprises a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 9 or 37; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 11 or 39; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 13 or 41.
Embodiment 2 provides the monoclonal antibody of Embodiment 1, wherein the VL comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 23; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 25; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 27; and wherein the VH comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 9; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 11; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 13.
Embodiment 3 provides the monoclonal antibody of Embodiment 1, wherein the VL comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 51; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 53; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 55, and wherein the VH comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 37; a CDR2 region comprising the amino acid sequence of SEQ ID NO: 39; and a CDR3 region comprising the amino acid sequence of SEQ ID NO: 41.
Embodiment 4 provides the monoclonal antibody of any one of Embodiments 1-3, wherein the VL comprises the amino acid sequence of SEQ ID NOs: 60 or 64, and wherein the VH comprises the amino acid sequence of SEQ ID NOs: 58 or 62.
Embodiment 5 provides the monoclonal antibody of any one of Embodiments 1-4, wherein: (a) the VL comprises the amino acid sequence of SEQ ID NO: 60, and the VH comprises SEQ ID NO: 58; and (b) the VL comprises the amino acid sequence of SEQ ID NO: 64, and the VH comprises SEQ ID NO: 62.
Embodiment 6 provides the monoclonal antibody of any one of Embodiments 1-5, which is humanized.
Embodiment 7 provides the monoclonal antibody of any one of Embodiments 1-6, which is labeled.
Embodiment 8 provides a pharmaceutical composition comprising the monoclonal antibody of any one of Embodiments 1-7 and at least one pharmaceutical excipient.
Embodiment 9 provides an isolated polynucleotide comprising at least one of the nucleic acid sequences of SEQ ID NOs: 57, 59, 61, or 63.
Embodiment 10 provides the polynucleotide of Embodiment 9, comprising: (a) the nucleic acid sequences of SEQ ID NOs: 57 and 59; or (b) the nucleic acid sequences of SEQ ID NOs: 61 and 63.
Embodiment 11 provides a method of preventing, minimizing, and/or reversing fibrillization of native tau, the method comprising contacting the native tau with an effective amount of the isolated monoclonal antibody of any one of Embodiments 1-7.
Embodiment 12 provides a method of preventing or minimizing transmission of pathological tau to a cell and/or uptake of pathological tau by a cell, the method comprising contacting the cell with an effective amount of the isolated monoclonal antibody of any one of Embodiments 1-7.
Embodiment 13 provides the method of Embodiment 11, wherein the native tau is within a cell.
Embodiment 14 provides the method of any one of Embodiments 12-13, wherein the cell is in vivo.
Embodiment 15 provides the method of any one of Embodiments 12-14, wherein the cell comprises a neuron.
Embodiment 16 provides a method of reducing and/or preventing further increase in any intraneuronal tau aggregates or inclusions in a subject, the method comprising administering to the subject a therapeutically effective amount of the isolated monoclonal antibody of any one of Embodiments 1-7.
Embodiment 17 provides a method of treating, preventing, and/or ameliorating a tauopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of the isolated monoclonal antibody of any one of Embodiments 1-7.
Embodiment 18 provides the method of claim 17, wherein the tauopathy comprises Alzheimer's Disease, Pick's disease, Corticobasal degeneration, Argyrophilic grain disease (AGD), Primary age-related tauopathy (PART)/Neurofibrillary tangle-predominant senile dementia, Chronic traumatic encephalopathy (CTE), Progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), Lytico-bodig disease (Parkinson-dementia complex of Guam), Ganglioglioma and gangliocytoma, Meningioangiomatosis, Postencephalitic parkinsonism, Subacute sclerosing panencephalitis (SSPE), Lead encephalopathy, Tuberous sclerosis, Pantothenate kinase-associated neurodegeneration, and/or Lipofuscinosis.
Embodiment 19 provides the method of any one of Embodiments 17-18, wherein the administering reduces or prevents further increase in any intraneuronal tau aggregates or inclusions in the subject.
Embodiment 20 provides the method of any one of Embodiments 16-19, wherein the antibody is provided to the subject as a pharmaceutical composition.
Embodiment 21 provides the method of any one of Embodiments 16-20, wherein the antibody is administered parenterally to the subject.
Embodiment 22 provides a method of detecting a tauopathy in a subject, the method comprising administering to the subject a labeled isolated monoclonal antibody of any one of Embodiments 1-7, and detecting presence or absence of a complex of the labeled isolated monoclonal antibody with any pathological tau present in the subject, wherein, if the complex is detected, the subject has a tauopathy.
Embodiment 23 provides a method of detecting pathological tau in a sample, the method comprising contacting the sample with a labeled isolated monoclonal antibody of any one of Embodiments 1-7, and detecting presence or absence of a complex of the labeled isolated monoclonal antibody with any pathological tau present in the sample, wherein, if the complex is detected, pathological tau are present in the sample.
Embodiment 24 provides an isolated monoclonal antibody, or fragment thereof, which recognizes a conformational epitope comprising amino acids 151-244 and amino acids 369-441 of pathological tau, and which affinity for pathological tau is higher than for native tau.
Embodiment 25 provides the isolated monoclonal antibody of Embodiment 24, which further recognizes amino acids 120-151 of pathological tau.
Embodiment 26 provides an autonomously replicating or an integrative mammalian cell vector comprising a recombinant nucleic acid encoding an antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 23 or 51; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 25 or 53; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 27 or 55, and wherein the VH comprises a CDR1 region comprising the amino acid sequence of SEQ ID NOs: 9 or 37; a CDR2 region comprising the amino acid sequence of SEQ ID NOs: 11 or 39; and a CDR3 region comprising the amino acid sequence of SEQ ID NOs: 13 or 41.
Embodiment 27 provides the vector of Embodiment 26, which comprises a plasmid or a virus.
Embodiment 28 provides the vector of any one of Embodiments 26-27, which comprises a mammalian cell expression vector.
Embodiment 29 provides the vector of any one of Embodiments 26-28, further comprising at least one nucleic acid sequence that directs and/or controls expression of the antibody.
Embodiment 30 provides an isolated host cell comprising the vector of any one of Embodiments 26-29.
Embodiment 31 provides the cell of Embodiment 30, which is a non-human cell.
Embodiment 32 provides the cell of any one of Embodiments 30-31, which is mammalian.
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.
While this disclosure has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this disclosure may be devised by others skilled in the art without departing from the true spirit and scope of the disclosure. The appended claims are intended to be construed to include all such embodiments and equivalent variations.
This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/029,977, filed May 26, 2020, which is hereby incorporated herein by reference in its entirety.
This invention was made with government support under grant numbers AG053036, AG010124, AG062418, and AG017586 awarded by National Institutes of Aging (NIH). The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/034056 | 5/25/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63029977 | May 2020 | US |
