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    • 专利摘要:
    anti-phf-tau antibodies and their uses. the present invention relates to isolated antibodies that bind to phf-tau comprising an antigen binding site of a heavy chain variable region (vh) of sequence 35 or 37 and an antigen binding site of a light chain variable region (vl) of sequence no .: 36 or 38, as well as refers to methods of production and use thereof.
    公开号:BR112014015323A2
    申请号:R112014015323-0
    申请日:2012-12-19
    公开日:2020-10-27
    发明作者:Christopher Alderfer；Dariusz Janecki；Xuesong Liu；Melissa Murdock；Sheng-Jiun Wu；Marc Mercken；Marc Vandermeeren；Thomas Malia
    申请人:Janssen Biotech, Inc.；
    IPC主号:
    专利说明:

    [001] [001] This application claims the benefit of the Provisional Application under U.S. number 61 / 577,817, filed on December 20, 2011, the entire content of which is hereby incorporated by reference. FIELD OF THE INVENTION
    [002] [002] The present invention relates to anti-PHF-tau antibodies and methods of producing and using them. BACKGROUND OF THE INVENTION
    [003] [003] Alzheimer's disease (AD) is a degenerative brain disorder clinically characterized by progressive loss of memory, cognition, reasoning, judgment and emotional stability that gradually leads to profound mental deterioration and, finally, death. AD is a common cause of progressive mental failure (dementia) in elderly humans and is believed to represent the fourth most common medical cause of death in the United States of America. AD has been observed in ethnic groups around the world and poses a major public health problem now and in the future.
    [004] [004] The brains of individuals affected by AD exhibit characteristic lesions called senile plaques (or amyloid), amyloid angiopathy (amyloid deposits in blood vessels) and neurofibrillary tangles. The large amounts of these lesions, particularly amyloid plaques and neurofibrillary tangles of paired helical filaments, are found, in general, in several areas of the human brain that are important for memory and cognitive function in patients with AD.
    [005] [005] The main protein component of neurofibrillary degeneration in AD and several other neurodegenerative diseases is a hyperphosphorylated form of the tau protein associated with a microtubule. The
    [006] [006] Recently, preclinical evidence has been produced in mouse models of transgenic tau that passive and active immunization for tau may have therapeutic potential (Chai, et al. J Biol Chem 286: 34457-67, 2011, Boutajangout , et al. J Neurochem 118: 658-67, 2011, Boutajangout, et al. J Neurosci 30: 16559-66, 2010, Asuni, et al. J Neurosci 27: 9115-29, 2007). A transmission of tauopathy and spreading hypothesis has been described recently and is based on the Braak stages of progression of tauopathy in the human brain and spreading of tauopathy after injections of tau aggregate in pre-clinical models of tau ( Frost, et al. J Biol Chem 284: 12845-52, 2009, Clavaguera, et al. Nat Cell Biol 11: 909-13, 2009). Therefore, there is a need for therapies to prevent aggregation of tau and progression of tauopathy to treat AD and other neurodegenerative diseases. BRIEF DESCRIPTION OF THE FIGURES
    [007] [007] Figure 1 shows the competition for AT8 labeled by various anti-tau antibodies.
    [008] [008] Figure 2 shows the competition of PT1 labeled by various anti-tau antibodies.
    [009] [009] Figure 3 shows the competition of PT3 labeled by various anti-tau antibodies.
    [0010] [0010] Figure 4 shows the AT100 competition labeled by various anti-tau antibodies.
    [0011] [0011] Figure 5 shows the competition for HT7 labeled by various anti-tau antibodies.
    [0012] [0012] Figure 6 shows (A) analysis of phosphorylated tau in stem brain homogenates (P1 fraction) of 5-month-old female P301L transgenic animals treated with mouse saline, IgG1, PT3 or AT8 as indicated in Figure, or untreated non-transgenic animals (B6). ELISA was performed using AT8 (left panel) or AT100 (right panel) as capture antibodies followed by biotinylated HT7 and avidin HRP. ELISA signals are plotted as a relative amount of DA brain homogenate (ng / ml) providing the same ELISA signal as a mean sample from a non-transgenic animal (B6). Data are plotted individually together with mean +/- S.D. p-values for differences between animals treated by PT3 and IgG1 are indicated. (B) Western blot of P1 fraction of stem brain homogenates of animals treated by IgG1 or PT3 with the use of AT100. The homogenous signal from 10 animals treated with IgG1 (I9G1-1 to IgG1-10) and 7 animals treated with PT3 (PT3-1, PT3-2, PT3-7 to PT3-10 are shown. used as a loading control.
    [0013] [0013] Figure 7 shows levels of total tau in soluble sarcosil (soluble pT4), total tau in insoluble (insoluble pT4) and phosphorylated tau in insoluble (insoluble AT8) cortex homogenates derived from P301L transgenic mice 5 months age treated with PT3 or isotype control (IgG) as indicated in the Figure. The levels are shown as a measure of an ELISA signal plotted individually together with the mean +/- SD. The 1 m inj sample is a positive control sample derived from a P301L mouse brain injected with a tau aggregate. SUMMARY OF THE INVENTION
    [0014] [0014] One aspect of the invention is an isolated antibody that binds PHF-tau which comprises an antigen binding site of a heavy chain variable region (VH) of SEQ ID NO: 35 or 37 and a
    [0015] [0015] Another aspect of the invention is an isolated antibody that binds PHF-tau which comprises certain light and heavy chain complementarity determining regions.
    [0016] [0016] Another aspect of the invention is an isolated antibody that binds PHF-tau comprising an antigen binding site of a VH of SEQ ID NO: 35 and an antigen binding site of a VL of SEQ ID NO: 36.
    [0017] [0017] Another aspect of the invention is an isolated antibody that binds PHF-tau comprising an antigen binding site of a VH of SEQ ID NO: 37 and an antigen binding site of a VL of SEQ ID NO: 38.
    [0018] [0018] Another aspect of the invention is an isolated antibody or fragment that competes for the binding of PHF-tau with a monoclonal antibody that comprises an antigen binding site of a VH of SEQ ID NO: 35 and a site of antigen binding of a VL of SEQ ID NO: 36, or an antigen binding site of a VH of SEQ ID NO: 37 and an antigen binding site of a VL of SEQ ID NO: 36
    [0019] [0019] Another aspect of the invention is the polynucleotides that co-complicate the antibodies of the invention or fragments thereof.
    [0020] [0020] Another aspect of the invention is a vector that comprises the polynucleotides of the invention.
    [0021] [0021] Another aspect of the invention is a host cell that comprises the vector of the invention.
    [0022] [0022] Another aspect of the invention is a method of producing an antibody that binds PHF-tau which comprises culturing the host cell of the invention and recovering the antibody produced by the host cell. DETAILED DESCRIPTION
    [0023] [0023] The term "antibodies", as used here, has a broad meaning and includes immunoglobulin molecules or antibody molecules including polyclonal antibodies, monoclonal antibodies including murine, human, humanized, humanized and chimeric monoclonal antibodies and antibody fragments.
    [0024] [0024] In general, antibodies are proteins or peptide chains that exhibit specificity of binding to a specific antigen. Antibody structures are well known. Immunoglobulins can be assigned to five main classes, namely, IgA, IgD, IgE, IgG and IgM, depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further sub-classified as the IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4 isotypes. Antibody light chains of any vertebrate species can be attributed to one of two clearly distinct types, namely, kappa (K «) and lambda (A), based on the amino acid sequences of their constant domains.
    [0025] [0025] The term "antibody fragments" means with a portion of an antibody intact. Examples of antibody fragments include fragments of Fab, Fab ', F (ab'), and Fv, CDR, antigen binding site, light or heavy chain variable region, diabody, single chain antibody molecules and multispecific antibodies formed from at least two intact antibodies or fragments thereof.
    [0026] [0026] A variable region of heavy or light chain of immunoglobulin consists of a "structure" region interrupted by the three "antigen binding sites". Antigen binding sites are defined using various terms as follows: (i) Complementarity Determining Regions (CDRs) are based on sequence variability (Wu and Kabat J Exp Med 132: 211-50, 1970 ). In general, the antigen binding site has three CDRs in each variable region (HCDR1, HCDR2 and HCDR3 in the heavy chain variable region (VH) and LCDR1, LCDR2 and LCDR3 in the light chain variable region (VL)) ( Kabat et a /., Sequences of Proteins of Immunological Interests, 5th Edition (Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) The term "hypervariable region", "HVR", or "HV" refers to regions of an antibody variable domain that are hypervariable in structure as defined by Chothia and Lesk (Cho- thia and Lesk J Mol Biol 196: 901-17, 1987). In general, the antigen binding site has three hypervariable regions in each VH (H1, H2, H3) and VL (L1, L2, L3). Chothia and Lesk refer to structurally conserved HVs as "canonical structures". The numbering systems as well as annotation of CDRs and HVs were recently reviewed by Abhinandan and Martin (Abhinandan and Martin Mol Immunol 45: 3832-9, 2008). (iii) Another definition of the regions that form the antigen binding site was proposed by Lefranc (Lefranc, et al. Dev Comp Immunol 27:55 to 77, 2003) based on the comparison of V domains of immunoglobulins and receptors for T cell. The International InMunoGeneTics (IMGT) database (http: // www imgt org) provides standardized numbering and definition of these regions. The correspondence between CDRs, HVs and IMGT designs is described in Le francisco et al., Dev. (Iv) The antigen binding site can also be designed based on the Use of Specific Determination Residue (SDRU) (Almagro J Mol Recognit 17: 132-43, 2004), where Specificity-determining residues (SDR), refers to the amino acid residues of an immunoglobulin that are directly involved in antigen contact.
    [0027] [0027] "Frame" or "frame sequence" are the sequences remaining within the variable region of an antibody in addition to those defined to be the antigen-binding site sequences
    [0028] [0028] The term "monoclonal antibody" (mAb), as used herein, means an antibody (or antibody fragment) obtained from a population of substantially homogeneous antibodies. Monoclonal antibodies are highly specific, typically targeting a single antigenic determinant.
    [0029] [0029] The term "epitope" as used here means a portion of an antigen to which an antibody specifically binds. Epitopes typically consist of chemically active surface clusters (such as polar, non-polar or hydrophobic) of portions such as amino acids, phosphorylated amino acids or polysaccharide side chains and may have specific three-dimensional structural characteristics as well as specific charge characteristics. An epitope can be linear in nature or it can be a discontinuous epitope, for example, a conformational epitope, which is formed by a spatial relationship between non-contiguous amino acids of an antigen instead of a linear series of amino acids. A conformational epitope includes epitopes resulting from the folding of an antigen, where the amino acids of portions that differ from the linear sequence of the antigen are in close proximity in 3-dimensional space.
    [0030] [0030] Tau is an abundant central and peripheral nervous system protein that has multiple well-known isoforms. In the human CNS, six major tau isoforms that range in size from 352 to 441 exist due to alternative splicing (Hanger, et al. Trends Mol Med 15: 112-9, 2009). These isoforms are different from each other by the regulated inclusion of N-terminal 0-2 inserts and 3 or 4 microtubule binding repetitions arranged in tandem and are referred to as
    [0031] [0031] Tau binds microtubules and regulates the transport of charge through cells, a process that can be modulated by phosphorylation of tau. In AD and disorders related to abnormal tau phosphorylation is prevalent and it is thought to precede and / or trigger the aggregation of tau in fibrils, called paired helical filaments (PHF). The main constituent of PHF is hyperphosphorylated tau. The term "paired helical filament tau" or "PHF-tau" as used herein refers to well-known tau aggregates in paired helical filaments. Two main regions in the PHF structure are evident in electron microscopy, the flaky coat (fuzzy coat) and the core filament; In which the flaky layer is sensitive to proteolysis and is located outside the filaments and the protease-resistant nucleus of the filaments that form the main structure of PHFs (Wischik, et al. Proc Natl Acad Sci USA 85: 4884-8, 1988 ).
    [0032] [0032] "Antibodies that bind PHF-tau" as used herein refer to antibodies that bind PHF-tau as assessed in western blot. Typically, antibody binding to PHF-tau can be assessed after Coomassie stain of about 500 ng PHF-tau after 1 hour blocking in 5% (weight by volume) skimmed dry milk (NFDM) TBS-T , 0.05% Tween-20. Antibodies that bind PHF-tau optionally do not bind control tau (SEQ ID NO: 6) as measured by western blot when tested under the antigen loading condition where both control tau and PHF-tau are detected equally by tau antibodies that have no preference for PHF-tau epitopes (for example antibody
    [0033] [0033] Well-known one-letter and three-letter conventional amino acid codes are used in this document. COMPOSITIONS OF SUBSTANCES
    [0034] [0034] The present invention relates to anti-PHF-tau antibodies and uses of those antibodies. Such anti-PHF-tau antibodies may have the binding properties of a PHF-tau phosphorylated epitope or binding to a PHF-tau non-phosphorylated epitope. Anti-PHF-tau antibodies can be useful as therapies and as research or diagnostic reagents to detect PHF-tau in biological samples, for example, in tissues or cells.
    [0035] [0035] One embodiment of the invention is an isolated antibody that binds PHF-tau comprising an antigen binding site of a heavy chain variable region (VH) of SEQ ID NO: 35 or 37 or an antigen binding site of a light chain variable region (VL) of SEQ ID NO: 36 or 38. Table 1 shows the antigen binding site residues of the exemplary antibodies of the invention defined according to Kabat or Chothia as well as variable chain regions exemplary light or heavy.
    [0036] [0036] In another embodiment, the VH antigen binding site of the antibodies of the invention comprises heavy chain complementarity determining regions (CDRs) 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs: 7 , 8 and 9 or 13, 14 and 15, respectively, or the VL antigen binding site of the antibodies of the invention comprises light chain CDRs 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3) of SEQ ID NOs: 10, 11 and 12 or 16, 17 and 18, respectively.
    [0037] [0037] Another embodiment of the invention is an isolated antibody that binds PHF-tau comprising an antigen binding site of a heavy chain variable region (VH) of SEQ ID NO: 35 or 37 and an antigen binding site of a light chain variable region (VL) of SEQ ID NO: 36 or 38.
    [0038] [0038] In another embodiment, the VH antigen binding site of the antibodies of the invention comprises heavy chain complementarity determining regions (CDRs) 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs: 7 , 8 and 9 or 13, 14 and 15, respectively, and the VL antigen binding site of the antibodies of the invention comprises light chain CDRs 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3) of SEQ ID NOs: 10, 11 and 12 or 16, 17 and 18, respectively.
    [0039] [0039] Although the modalities illustrated in the examples comprise pairs of variable regions, one of a heavy chain and one of a light chain, an individual skilled in the art will recognize that alternative modalities may comprise unique variable regions of heavy or light chain. The single variable region can be used for screening variable domains capable of forming a two-domain specific antigen-binding fragment capable of, for example, binding to PHF-tau. Screening can be performed by phage display screening methods with the use, by e-
    [0040] [0040] Another embodiment of the invention is an isolated antibody that binds PHF-tau comprising an antigen binding site of a heavy chain variable region (VH) of SEQ ID NO: 35 or and an antigen binding site of a light chain variable region (VL) of SEQ ID NO: 36.
    [0041] [0041] Another embodiment of the invention is an isolated antibody that binds PHF-tau comprising an antigen binding site of a heavy chain variable region (VH) of SEQ ID NO: 37 or and an antigen binding site of a light chain variable region (VL) of SEQ ID NO: 38.
    [0042] [0042] Another embodiment of the invention is an isolated antibody that binds PHF-tau that comprises heavy chain complementarity determining regions (CDRs) 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs: 7 , 8 and 9, respectively and light chain CDRs 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3) of SEQ ID NOs: 10, 11 and 12, respectively.
    [0043] [0043] Another embodiment of the invention is an isolated antibody that binds PHF-tau which comprises heavy chain complementarity determining regions (CDRs) 1 (HCDR1), 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs: 13 , 14 and 15, respectively and light chain CDRs 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3) of SEQ ID NOs: 16, 17 and 18, respectively.
    [0044] [0044] In any of the preceding modalities, the antibody
    [0045] [0045] The antibodies of the present invention can be produced by a variety of techniques, for example by the hybridoma method (Kohler and Milstein Nature 256: 495-7, 1975). Chimeric mAbs containing a light and heavy chain variable region derived from a donor antibody (typically murine) in association with light and heavy chain constant regions derived from an accepting antibody (typically another mammalian species) , such as human) can be prepared by the method presented in US patent No. 4,816,567. CDR-grafted mAbs that have CD Rs derived from an immunoglobulin from a non-human donor (typically murine) and the remaining immunoglobulin-derived parts of the molecule that is derived
    [0046] [0046] of one or more human immunoglobulins can be prepared by techniques known to those skilled in the art such as those disclosed in U.S. Patent No. 5,225,539. Fully human mAbs that lack any non-human sequences can be prepared from transgenic mice with human immunoglobulin by techniques referred to (Lonberg, et al. Nature 368: 856-9, 1994, Fishwild, et al. Nat Biotechnol 14: 845-51, 1996, Mendez, et al. Nat Genet 15: 146-56, 1997). Human mAbs can also be prepared and optimized from phage display libraries (Knappik, et al. J Mol Biol 296: 57 to 86, 2000, Krebs, et al. J Immunol Methods 254: 67 to 84, 2001, Shi, et al. J Mol Biol 397: 385-96, 2010).
    [0047] [0047] Antibody humanization can be performed using well-known methods, such as reconstruction of specificity determination residues (SDRR) (Publication under US No. 2010/0261620), reconstruction (Padlan et al. Mol. Immunol 28: 489-98, 1991), super humanization (International Patent Publication under
    [0048] [0048] The preparation of PHF-tau to be used as an antigen for immunizing antibodies or isolation from phage display libraries can be done with the use of any suitable technique. In an exemplary method, PHF-tau is isolated from the brains of patients who have AD using well-known protocols, such as those described in Greenberg and Davies (Greenberg and Davies Proc Natl Acad Sci USA 87: 5827-31, 1990). PHF-tau can be isolated from the postmortem cortex of an Alzheimer's patient. The isolated PHF-tau is characterized by its purity and hyperphosphorylation with antibodies that are known to react with PHF-tau. In a typical PHF-tau preparation, the hyperphosphorylated bands that migrate around 60, 64, 68 and 72 kDa in western blot (Spillantini and Goertert Trends Neurosci 21: 428-33, 1998) are detected by a AT8 antibody that specifically binds to hisphosphorylated PHF-tau but not to dephosphorylated PHF-tau.
    [0049] [0049] The antibodies of the present invention may have non-binding characteristics of control tau of SEQ ID NO: 6. These antibodies can be generated using the methods described above and testing the antibodies for their lack of binding of control tau in western blots followed by the Coomassie stain as described above. Control tau can be purified and expressed in a way that matches standard methods. The antibodies exemplified
    [0050] [0050] Can the antibodies of the invention target PHF-tau with a constant dissociation (Kp) less than or equal to about 107, 10, 10, 10, 10 or 10 M. The affinity of a given molecule for PHF-tau can be determined experimentally using any suitable method. These methods can use instrumentation, Biacore, ProteOn or KinExA, ELISA or competitive binding tests known to those skilled in the art.
    [0051] [0051] Another aspect of the invention is an isolated antibody or fragment that competes for the binding of PHF-tau with a monoclonal antibody that comprises an antigen binding site of a heavy chain variable region (VH) of SEQ ID NO: 35 and a light chain variable region (VL) antigen binding site of SEQ ID NO: 36, or a heavy chain variable region (VH) antigen binding site of SEQ ID NO: 37 and an antigen binding site of a light chain variable region (VL) of SEQ ID NO: 38.
    [0052] [0052] The competition between binding to PHF-tau can be tested in vitro using well-known methods. For example, the binding of NHS ester labeled antibody with MSD Sulfo-Tag "" to PHF-tau in the presence of an unlabeled antibody can be assessed with the use of immunoassay followed by electrochemiluminescence detection.
    [0053] [0053] Several well-known methodologies in addition to competitive binding can be employed to determine the epitope binding of the antibodies of the invention. For example, when the structures of both individual components are known, protein-protein coupling in silico can be performed in order to identify compatible sites of interaction. The hydrogen-deuterium (H / D) exchange can be performed with the antibody and antigen complex in order to map the regions in the antigen that can be linked through the antibody. Mutagenesis of the antigen point or segment can be used to locate the amino acids important for antibody binding. The cocrystalline structure of the antibody-antigen complex is used to identify residues that contribute to the epitope and paratope.
    [0054] [0054] The antibodies of the invention can be monoclonal antibodies of the IgG, IgD, IgA or IgM isotypes. The antibodies of the invention can be bispecific or multispecific. An exemplifying bispecific antibody can bind two distinct epitopes in PHF-tau or can bind PHF-tau and beta amyloid (AB). Another bispecific exemplifying antibody can bind PHF-tau and an endogenous blood brain barrier transcytosis receptor as an insulin receptor, transfer receptor, insulin-like growth factor 1 receptor and receptor for lipoprotein. An exemplifying antibody is of the I9G1 type.
    [0055] [0055] The immune effector properties of the antibodies of the invention can be enhanced or silenced through Fc modifications by techniques known to those skilled in the art. For example, Fc effector functions such as C1q binding, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytoxicity (ADCC), phagocytosis, downward regulation of cell surface receptors (for example , B cell receptor; BCR), etc. they can be supplied and / or controlled by modifying residues in the Fc responsible for these activities. The pharmacokinetic properties could also be accentuated by the change of residues in the Fc domain that extends the half-life of the body (Strohl Curr Opin Biotechnol 20: 685-91, 2009).
    [0056] [0056] Additionally, the antibodies of the invention can be modified post-translationally by processes such as glycosylation, iso-
    [0057] [0057] Another embodiment of the invention is an isolated polynucleotide that encodes the antibodies of the invention or its complement, or fragments thereof. Exemplary isolated polynucleotides are polynucleotides encoding polypeptides comprising immunoglobulin heavy chain CDRs HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 7, 8 and 9 or 13, 14 and 15, respectively, or polypeptides comprising light chain CDRs of immunoglobulin LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 10, 11 and 12 or 16, 17 and 18, respectively and polynucleotides that have a sequence shown in SEQ ID NOs: 31-34, which encode variable regions of antibody from invention. Other polynucleotides that, given the degeneracy of the genetic code or codon preferences in a given expression system, encode the antibodies of the invention are also within the scope of the invention. Isolated nucleic acids of the present invention can be made using well-known synthetic or recombinant techniques. The DNA encoding the monoclonal antibodies is easily isolated and sequenced using methods known in the art. When a hybridoma is produced, these cells can serve as a source of this DNA. Alternatively, with the use of presentation techniques in which the sequence
    [0058] [0058] Another embodiment of the invention is a vector that comprises at least one polynucleotide of the invention. These vectors can be plasmid vectors, viral vectors, transposon-based vectors or any other vectors suitable for introducing the polynucleotides of the invention into a given organism or genetic background by any means.
    [0059] [0059] Another embodiment of the invention is a host cell that comprises any of the polynucleotides of the invention. Such host cells can be eukaryotic cells, bacterial cells, plant cells or archea cells. Exemplary eukaryotic cells can be of mammalian, insect, bird or other animal origin. Mammalian eukaryotic cells included cell lines immortalized as hybridoma or myeloma cell lines as murine cell lines SP2 / 0 (American Type Culture Collection (ATCC), Manassas, VA, USA CRL-1581), NSO (European Cell Culture Collection (ECACC), Salisbury, Wiltshire, United Kingdom, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580). An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196). Other useful cell lines include those derived from Chinese hamster ovary (CHO) cells such as CHO-K1ISV (Lonza Biologics), CHO-K1 (ATCC CRL-61, Invitrogen) or DG44.
    [0060] [0060] Another embodiment of the invention is a method of producing an antibody that binds PHF-tau which comprises culturing a host cell of the invention and recovering the antibody produced by the host cell. Methods of making antibodies and purifying them are well known in the art. TREATMENT METHODS
    [0061] [0061] The anti-PHF-tau antibodies of the invention or fragments thereof, which include Fab, (Fab ') 2, scFv fragments, or antibodies comprising antigen binding sites of the antibodies of the invention can be used to treat , reduce or avoid symptoms in patients who have a neurodegenerative disease that involves pathological aggregation of tau within the brain, such as patients suffering from AD or any other tauopathy. Although it is not desired to be arrested by any particular theory, the antibodies of the invention can exert their beneficial effect by reducing the aggregation of pathological tau and therefore the amount of PHF-tau in the brain. The antibodies of the invention can be used to treat an animal patient belonging to any classification. Examples of such animals include mammals such as humans, rodents, dogs, cats and farm animals. For example, the antibodies of the invention are also useful in the preparation of a medicament for the treatment of AD, wherein the medicament is prepared for administration in the dosages defined herein.
    [0062] [0062] Another embodiment of the invention is a method of reducing the aggregation of tau in patients in need thereof which comprises administering to the patient a therapeutically effective amount of the isolated antibody of the invention long enough to reduce the aggregation of tau.
    [0063] [0063] Another embodiment of the invention is a method of treating or reducing symptoms of a neurodegenerative disease that involves
    [0064] [0064] In any of the above modalities, the neurodegenerative disease that involves aggregation of tau is a tauopathy.
    [0065] [0065] In any of the above embodiments, the isolated antibody comprises an antibody that binds PHF-tau that comprises an antigen binding site of a VH of SEQ ID NO: 35 and an antigen binding site of a VL of SEQ ID NO: 36.
    [0066] [0066] In any of the above embodiments, the isolated antibody comprises a PHF-tau binding antibody comprising a VH antigen binding site of SEQ ID NO: 37 and a VL antigen binding site SEQ ID NO: 38.
    [0067] [0067] As used herein, a "tauopathy" encompasses any neurodegenerative disease that involves the pathological aggregation of tau within the brain. In addition to familial and sporadic AD, other exemplifying tauopathies are frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, cortical basal degeneration, Pick's diseases, progressive subcortical gliomas, tangle-only dementia, ema - diffuse neurofibrillary grooves with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis parkinsonism dementia complex, Down syndrome, Gerstmann-Strâussler-Scheinker disease, Hallervorden-Spatz disease, concluding body myositis, Creutzfeld disease -Jakob, multiple system atropy, type C Niemann-Pick disease, cerebral prion protein amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guanamian motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism and chronic chronic encephalopathy , such as pugulistic dementia (boxer's disease). (Morris, et al. Neuron 70: 410-26, 2011).
    [0068] [0068] A behavioral phenotype related to tauopathy includes cognitive limitations, early personality change and disinhibition, apathy, abulia, mutism, apraxia, perseveration, stereotyped behaviors / movements, hyperorality, disorganization, inability to plan or organize sequential tasks, selfishness / insensitivity, antisocial traits, a lack of empathy, hesitancy, agrarian speech with frequent phrasing errors but relatively preserved understanding, impaired understanding and word-finding deficits, slow progressive walking instability, re - tropulsions, freezing, frequent falls, axial stiffness not responsive to levodopa, paralysis of the supranuclear gaze, shaken in square waves, slow vertical saccades, pseudobulbar paralysis, limb apraxia, dystonia, corticoid sensory loss and tremor.
    [0069] [0069] Patients favorable for treatment include asymptomatic individuals at risk for AD or other tauopathy, as well as patients who currently show symptoms. Treatment-friendly patients include individuals who have a known genetic risk of AD, such as a family history of AD or the presence of genetic risk factors in the genome. Exemplary risk factors are changes in the amyloid precursor protein (APP), especially in position 717 and positions 670 and 671 (mutations of Hardy and Swedish, respectively). Other risk factors are mutations in the presenilin, PS1 and PS2 and ApoE4 genes, a family history of hypercholesterolemia or atherosclerosis. Individuals currently suffering from AD can be recognized from characteristic dementias by the presence of risk factors described above. In addition, several diagnostic tests are available to identify individuals who have AD. These tests include measuring levels of cerebrospinal fluid tau.
    [0070] The anti-PHF-tau antibodies of the invention are suitable for both therapeutic and prophylactic agents to treat or prevent neurodegenerative diseases involving pathological aggregation of tau, such as AD or other tauopathies. In asymptomatic patients, treatment can begin at any age (for example, about 10, 15, 20, 25, 30 years). Generally, however, it is not necessary to start treatment until a patient is about 40, 50, 60, or 70 years old. Treatment typically involves multiple doses over a period of time. Treatment can be monitored by assessing the antibody, or activated T cell or B cell responses to the therapeutic agent over time. If the response fails, a booster dose is indicated.
    [0071] [0071] In prophylactic applications, pharmaceutical compositions or medications are administered to a patient susceptible to, or otherwise at risk for, AD in an amount sufficient to eliminate or reduce the risk, decrease the severity, or delay the onset disease, including biochemical, histological and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presented during the development of the disease. In therapeutic applications, the compositions or drugs are administered to a patient suspected of, or already suffering from a disease, in an amount sufficient to reduce, stop, or attract any of the symptoms of the disease (biochemical, histological and / or behavioral). Administration of a therapeutic can reduce or eliminate mild cognitive impairment in patients who have not yet developed
    [0072] [0072] Anti-PHF-tau antibodies or fragments thereof of the invention can be administered in combination with other agents that are effective for treating related neurodegenerative diseases. In the case of AD, the antibodies of the invention can be administered in combination with agents that reduce or prevent amyloid-beta (AB) deposition. It is possible that the pathologies of PHF-tau and AB are synergistic. Therefore, combination therapy that aims to clear pathologies related to both PHF-tau and AB and AB at the same time can be more effective than targeting each one individually.
    [0073] [0073] In the case of Parkinson's disease and related neurodegenerative diseases, immune modulation to clear aggregated forms of the α-synuclein protein is also an emerging therapy. A combination therapy that aims to clean both tau and a-synuclein proteins simultaneously can be more effective than targeting any individual protein.
    [0074] [0074] In the methods of the invention, the "therapeutically effective amount" of the antibody in the treatment or alleviation of symptoms of a tauopathy can be determined by standard screening techniques. For example, the dosage of the antibody can be determined by administering the agent to relevant animal models well known in the art.
    [0075] [0075] In addition, in vitro tests can optionally be used to assist in the identification of ideal dosage ranges. The selections of a particular effective dosage can be determined (for example, through clinical tests) by those skilled in the art based on consideration of various factors. Such factors include the disease to be treated or avoided, the symptoms involved, the patient's body mass, the patient's immune status and other factors known to those skilled in the art. The dosage that needs to be used in the formulation will also depend on the route of administration and the severity of the disease and should be decided according to the judgment of the professional and the circumstances of each patient. Effective dosages can be extrapolated from these dosage response curves derived from animal model or in vitro test systems.
    [0076] [0076] The mode of administration for therapeutic use of the antibodies of the invention can be any suitable route that distributes the host to the host. The pharmaceutical compositions of these antibodies are useful for parenteral administration, for example, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal or intracranial or they can be administered in the cerebrospinal fluid of the brain or spine.
    [0077] [0077] The antibodies of the invention can be prepared as a pharmaceutical composition that contains an effective amount of the antibody as an active ingredient in a pharmaceutically acceptable carrier. The term "carrier" refers to a diluent, adjuvant, excipient, or carrier with which the antibody is administered. Such pharmaceutical vehicles can be liquids, such as water and oils, including those derived from petroleum, animal, vegetable or synthetic origin, such as peanut oil, soy oil, mineral oil, germ oil and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and, in general, free of particulate matter. They can be sterilized using well-known sterilization techniques (for example, filtration). The com-
    [0078] [0078] Treatment can be given on a single dose schedule or as a multiple dose schedule in which a primary course of treatment can be with 1 to 10 separate doses, followed by other doses given at subsequent necessary time intervals to maintain and / or reinforce the response, for example, 1 to 4 months for a second dose and, if necessary, a subsequent dose (s) after several months. Examples of suitable treatment schedules include: (i) O, 1 month and 6 months, (ii) 0, 7 days and 1 month, (ii) 0 and 1 month, (iv) O and 6 months, or other schedules sufficient to generate the desired responses that are intended to reduce the symptoms of the disease, or to reduce the severity of the disease.
    [0079] [0079] In this way a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 ml of sterile buffered water, between about 1 ng to about 100 mg, for example, about 50 ng about 30 mg or about 5 mg to about mg of an antagonist antibody of the invention Similarly, a pharmaceutical composition of the invention for intravenous infusion could be prepared to contain about 250 ml of sterile Ringer's solution, and about 1 mg to about 30 mg or about 5 mg about 25 mg of an antibody of the invention. Current methods for preparing parenterally administrable compositions are well known and are described in more detail in, for example, "Remington's Pharmaceutical Science", 15th edition, Mack Publishing Company, Easton, PA, USA.
    [0080] [0080] The antibodies of the invention can be lyophilized for storage and reconstituted in a suitable vehicle before use. This technique has been shown to be effective with antibody and other protein preparations and the reconstitution and lyophilization techniques known in the art can be employed. DIAGNOSTIC METHODS AND KITS
    [0081] [0081] The antibodies of the invention can be used in the methods of diagnosing AD or other tauopathy in a subject. This method involves detecting, in the subject, the presence of PHF-tau with the use of a diagnostic reagent such as an antibody or a fragment thereof.
    [0082] [0082] PHF-tau can be detected in a biological sample from a subject (for example, blood, urine, cerebrospinal fluid) by placing the biological sample in contact with the diagnostic antibody reagent and detecting the binding of the diagnostic antibody reagent to PHF-tau in the subject sample. Assays to perform the deletion include well-known methods such as ELISA, immunohistochemistry, western blot, or in vivo imaging. Exemplary diagnostic antibodies are PT1 and PT3 antibodies of the invention and are of the IgG1, K type.
    [0083] [0083] Diagnostic antibodies or similar reagents can be administered by intravenous injection into the patient's body, or directly into the brain by any suitable route that delivers the agent to the host as exemplified above. The antibody dosage should be within the same ranges for the treatment methods. Typically, the antibody is labeled, although in some methods, the primary antibody with PHF-tau affinity is untagged and a secondary labeling agent is used to bind to the primary antibody. The choice of label depends on the means of detection. For example, a fluorescent label is suitable for optical detection. The use of paramagnetic tags is suitable for tomographic detection without surgical intervention. Radioactive tags can also be detected using PET or SPECT.
    [0084] [0084] The diagnosis is made by comparing the number, size, and / or intensity of labeled PHF-tau, tau aggregates, and / or neurofibrillary tangles in a sample of the subject or in the subject, to line values of corresponding base. Baseline values can represent the average levels in a population of individuals without disease. Baseline values can also represent previous levels determined in the same subject.
    [0085] [0085] The methods of diagnostic methods described above can also be used to monitor a subject's response to therapy by detecting the presence of PHF-tau in a subject before, during or after treatment. A decrease in baseline-related values signals a positive response to treatment. Values may also temporarily increase in biological fluids as pathological tau is being cleared from the brain.
    [0086] [0086] The present invention is additionally directed to a kit to carry out the monitoring and diagnostic methods described above. Typically, these kits contain a diagnostic reagent such as the antibodies of the invention and optionally a detectable label. The diagnostic antibody itself can contain the detectable tag (for example, fluorescent molecule, biotin, etc.) that is directly detectable or detectable through a side reaction (for example, streptavidin reaction). Alternatively, a second reagent containing the detectable label can be used, in which the second
    [0087] [0087] The contents of all cited references (including literature references, granted patents, published patent applications and copending patent applications) cited in this specification are expressly incorporated herein by reference in their entirety. EXAMPLE 1 PURIFICATION OF TYPICAL HELICIDE FILAMENT TAU (PHF-TAU)
    [0088] [0088] PHF-tau was partially purified by a modified method by Greenberg and Davies (Greenberg and Davies Proc Natl Acad Sci USA 87: 5827-31, 1990). Soon, the postmortem tissue from the cortex obtained from a confirmed Alzheimer's patient was partially purified. Typically, 5 mg of frontal cortex was homogenized in 10 vol of cold Buffer H (10 mM Tris, 800 mM NaCl, 1 mM EGTA and 10% sucrose / pH 7.4) using a homogenizer. glass fabric / Teflon Potter paints (IKA Works, Inc; Staufen, Germany) at 1000 rpm. The homogenized material was centrifuged at 264.8 N (27000 g) for 20 minutes in a Sorvall SS34 rotor. The pellet was discarded and the supernatant was adjusted to a final concentration of 1% (weight by volume) of N-lauroylsarcosine and 1% (v / v) of 2-mercapto ethanol and incubated for 2 hours at 37 CT. Subsequently the supernatant was centrifuged at 1059.1 N (108000 g) for 35 minutes at 20% in a Beckman 60Ti rotor. The pellet was washed carefully in PBS and suspended in PBS. The supernatant was centrifuged a second time as described and the final pellet was dissolved, aliquoted and frozen at -80 (C. The quality of the PHF-tau preparations was evaluated on a 12% SDS-PAGE and western blot with anti-tau antibodies AT8 and HT7 (ThermoScientific, Rockford, IL) AT8 detects PHF-tau phosphorylated in S202 / T205, but does not bind to non-phosphorylated PHF-tau or to wild-type tau. non-phosphorylated epitope in amino acids of Tau 159-163 (SEQ ID NO: 6) and recognizes both tau and PHF-tau A good quality PHF-tau preparation is composed of 4 bands that have weights molecular molecules of about 60, 64, 66 and 72 kDa in a Western blot detected with a hyperphosphorylated PHF-tau antibody such as AT8. Two separate PHF-tau preparations with comparable purity and quality were made from the same brain sample Preparation 1 was used for immunization EXAMPLE 2 GENERATION OF MONOCLONAL ANTIBODIES AGAINST PHF-TAU
    [0089] [0089] Anti-PHF-tau antibodies were generated using standard hybridoma technology in normal Balb / c mice (Kohler and Milstein Nature 256: 495-7, 1975). Hybridomas obtained were dried in 96-well plates and sorted after 10 days and a direct 25 ng / well E-LISA coated with PHF-tau as described below. Positive cells were tested by cross-reactivity in 10 ng / well coated with control tau (SEQ ID NO: 6) expressed in E. coli BL21 cells and purified by heat treatment and ammonium sulfate precipitation and were immediately subcloned - positive clones were frozen in liquid nitrogen. All hybridomas were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (Hyclone, Europe), Hybridoma Fusion Cloning Complement (2%) (Roche, Brussels, Belgium) 2% HT (Sigma , USA), 1 mM sodium pyruvate, 2 mM L-glutamine and penicillin (100 U / ml) and streptomycin (50 mg / ml).
    [0090] [0090] The antibody variable regions were cloned to select the hybridoma cells in the IgG1 / I9G2 / K mouse background and expressed and purified using routine methods. DIRECT ELISA FOR ANTIBODY SELECTION
    [0091] [0091] 25 nglow well of PHF-tau was coated overnight at 4 ° C in NUNC Maxisorp (Life Technologies) microtiter plates with 96 wells of high-bonded flat bottom in 50 pl / well of coating buffer ( 10 MM Tris, 10 mM NaCl and 10 mM NaN3, pH 8.5). On the next day, the plates were blocked with 75 ul / well of 0.1% casein in PBS for 60 min at room temperature. Then, 50 µl of hybridoma supernatant was added and incubated for 1 hour at 37 ° C. After washing, the bound monoclonal antibodies were detected with 50 μl / well of mouse anti-IgG Sheep conjugated to horseradish peroxidase for 1 hour at 37 * C (Amersham-Pharmacia Biotech). Both reagents were diluted in 0.1% casein / PBS. The plates were washed and 50 µl of a solution of 0.42 mM 3.5.3 ', 5-tetramethyl-benzidine, 0.003% (v / v) H2O7 in 100 mM citric acid and 100 mM disodium hydrogen phosphate (pH 4.3) was added as the substrate. The reaction was allowed to proceed for a maximum of 15 minutes on a plate shaker at room temperature, after which color development was stopped with 2 NH, SO ;, 50 ul / well and the plates were read in a 450 nm microtiter plate reader (Thermomax, Molecular Devices). SPECIFICITY OF MONOCLONAL ANTIBODIES
    [0092] [0092] The selected antibodies obtained from the hybridoma screening were tested for their cross-reactivity with recombinantly expressed control tau (SEQ ID NO: 6). 500 ng of PHF-tau and 200 ng of control tau were loaded onto a NUPAGEG NexagO Bis-Tris 4 at 12% gel and stained on a nitrogen membrane.
    [0093] [0093] Monoclonal antibodies PT1, PT2, PT3, PT4, PT5, AT8 (ThermoScientific, Rockford, IL), AT100 (ThermoScientific, Rockford, IL) and HT7 (MN1000) (Thermo Scientific, Rockford, IL) were evaluated by binding competitive to PHF-tau or phosphorylated tau pepetids. The antibodies were labeled using MSD & SULFO-TAG NHS Ester (Meso Scale Discovery) according to the manufacturer's instructions.
    [0094] [0094] For competition with labeled PT1, PT3, AT1I00 and HT7,
    [0095] [0095] Non-overlapping epitopes of anti-Tau mAbs HT7, ATI00 and AT8 are described above. Based on the published data, these antibodies were not expected to compete with each other for binding to Tau peptides or proteins.
    [0096] [0096] Based on the competition tests performed, none of the antibodies competed for binding, indicating that they all bind to different epitopes. In each experiment, only self-inhibition was observed. Figures 1 to 5 show results of competition tests with AT8, PT1, PT3, AT100 and HT7 labeled, respectively.
    [0097] [0097] Female P301L mice of 5 months old (Taco-nic, catt002508) were treated once a month with mouse IgG1, saline, PT3 (500 ug / mouse) or AT8 (expressed in ECACC hybridoma) , deposit number 9110086) for 5 months. The mice were anesthetized, perfused with cold PBS and the brains dissected on ice. For each mouse, one brain hemisphere was homogenized in 10 volume of H buffer, followed by centrifugation at 205.9 N (21,000 g) for 20 minutes at 4. The resulting supernatant was further centrifuged at 205.9 N (100,000 g) for 60 minutes. After centrifugation, the pellet (fraction P1) was recovered and resuspended with lysis buffer for Western and ELISA analyzes, as described by Chehai et al. J Biol Chem 286: 34457-67, 2011. For cortex samples, the P1 fraction was additionally treated with 1% (weight by volume) N-lauroylsarcosine and ultra-centrifuged to further enrich PHF-tau in the pellet. It was observed that the male P301L mice have low transgene expression and were not included in the analyzes.
    [0098] [0098] Phosphorylated tau was measured in trunk-head homogenates (P1 fraction) with sandwich ELISA using antibodies AT8 and ATIO0O as capture antibodies followed by detection by biotinylated HT7 and avidin HRP (Figure 6A and 6B) and with AT100 in a western blot (Figure 6C) essentially as described in Chai et al. J Biol Chem 286: 34457-67, 2011. Briefly, the P1 pellet was resuspended with lysis buffer (Cell Signaling). P1 pellet samples were incubated in wells pre-coated with AT8 or AT100 (Thermo Scientific) and the HT-7 antibody labeled with biotin. The samples were then washed 5 times with buffer,
    [0099] [0099] A statistically significant decrease or trend toward significance in phosphorylated tau was seen in transgenic P301L animals treated with PT3 compared to animals administered with isotype control in ELISA assays using AT100 (p = 0.057) or AT8 (p = 0.0475) for the detected phosphorylation (ELISA signal: saline group (1135 + 228.8); group | gG1 (1344 + 245.6); group PT3 (660.5 + 134, 5); group AT8 (127 1 + 274)).
    [00100] [00100] To confirm the data obtained with ELISA, the homogenates of brainstem (P1 fraction) of animals treated by I9G1 or PT3 were analyzed in western blot detecting PHF-tau with the use of AT100 antibody. The filters were stained using anti-actin antibody as a loading control (Figure 6B). Western blots showed the amount of attenuated PHF-tau detected with AT100 antibody compared to animals treated with Ig9G1.
    [00101] [00101] For cortex analysis, both the soluble sarcosil (representing soluble tau) and the insoluble sarcosyl (representing PHF-tau) were analyzed by sandwich ELISA using pan-tau antibody (PT4) or antibody phospho-tau (AT8) for capture followed by a biotin-pan-tau antibody (hTau10) followed by HRP-avidin. PT3-treated animals had similar levels of total tau compared to animals treated with the I9gG1 isotype control. A trend towards lower PHF-tau levels was evident in animals treated with PT3 when compared to isotope control in the insoluble fractions of N-lauroilsarcosine (ELISA capture with PT4: IgG group: 851026 + 261198 and PT3 group: 585639 + 120498; ELI-SA, capture with AT8: IgG group: 1125886 + 286240 (N = 10) and pT3 group: 746582 + 124970 (N = 7)). EXAMPLE 4 CHARACTERIZATION OF ANTI-PHF-TAU ANTIBODIES DETERMINATION OF AFFINITY
    [00102] [00102] The interactions of monoclonal antibodies PT1 and PT3 with soluble recombinant human tau or PHF-tau have been studied by ProteOn. All interactions were studied at 25ºC using PBS pH 7.4, supplemented with 3 mM EDTA and 0.005% Tween-20 as a system or run buffer. Two different experiment formats were used, one for interacting with recombinantly expressed control tau and another for interacting with PHF-tau. In these HT7 experiments (Pierce, cat ZMN1000), a mouse anti-tau antibody was used as a positive control.
    [00103] [00103] To study the interaction with the recombinantly expressed control tau a biosensor surface was prepared by coupling a specific antibody by human or mouse (Ab) anti-IgG Fct + fragment to the surface of an integrated circuit of GLC sensor (ProteOn) using each manufacturer's instructions for amine coupling chemistry (-5000 response units (UK)). The coupling buffer was 10 mM sodium acetate, pH 4.5. Anti-PHF-tau antibodies were diluted in the running buffer and injected to obtain a capture of 60 to 130 RUs. The capture of anti-PFH-Tau mAbs was followed by the injection of recombinantly expressed control tau (Tau-441, Sigma catalogft TO576-50 ug) in solution
    [00104] [00104] For PHF-tau this is the apparent intrinsic affinity in which KD is obtained as the ratio of kff1 / kon-1 derived from an adjustment performed using a bivalent bonding model.
    [00105] [00105] ** No significant connection
    [00106] [00106] *** No connection in 4 out of 5 experiments
    [00107] [00107] To study the interaction with PHF-tau a biosensor surface was prepared by capturing-coupling PHF-tau with the use of HT7 as the capture reagent. Additional preparation of PHF-tau as described previously was required for Proteon to limit the amount of insoluble material that enters the fluids. The PHF-tau as described above was further prepared by centrifuging twice in 49 N (5000 g), € 5, 10 minutes in which the supernatant of the second centrifugation was then diluted 1/20 or 1/40 in running buffer. To prepare the integrated circuit, HT7 was covalently immobilized to the surface of a GLC sensor integrated circuit (ProteOn) using the instructions of each manufacturer for amine coupling chemistry (-3000 response units (RU The coupling buffer was 10 mM sodium acetate, pH 4.5. After immobilizing HT7, PHF-tau was injected and captured (-300 RU) by HT7. After capture, PHF-tau was immobilized from covalent form to the sensor integrated circuit by activating the integrated circuit using each manufacturer's instructions for amine coupling chemistry.The remaining reactive sites were finally blocked by injection of ethanolamine After preparation and stabilization of the modified surface by PHF-tau and reference surface (antigen-free), anti-PHF-tau antibodies were diluted in the running buffer and injected into the solution (0.1 to 75 nM in 5-fold dilutions). association was monitored for 3 minutes (120 ul injected at 40 ul / min). Dissociation was monitored for 10 or 15 minutes. The regeneration of the sensor surface was achieved with 10 mM of Gly pH 2. The data were adjusted using a bivalent bond model in which the apparent intrinsic affinity was reported as the koff-1 / kon-1 ratio.
    [00108] [00108] Based on ProteOn PT1 experiments, it linked PHF-tau with an affinity of 322 pM and showed no connection to the control tau under the conditions tested (Table 2). PT3 bound PHF-tau with an affinity of 18 +2 pM and showed no link to control tau in 4 out of 5 measurements under the conditions tested. One of the 5 Prote-On measurements showed weak binding that could be used to estimate the affinity> 75 nM based on the highest concentration of control tau used.
    [00109] [00109] As the present invention has been fully described, it will be clear to the person skilled in the art that many changes and modifications can be made to it without departing from the spirit and scope of the attached claims.
    权利要求:
    Claims (13)
    [1]
    1. Isolated antibody that binds PHF-tau, characterized by the fact that it comprises an antigen binding site of a heavy chain variable region (VH) of SEQ ID NO: 35 or 37 and an antigen binding site of a light chain variable region (VL) of SEQ ID NO: 36 or 38.
    [2]
    2. Isolated antibody, according to claim 1, characterized by the fact that the VH antigen binding site comprises heavy chain complementarity determining regions (CDRs) 1 (HCDR1), 2 (HCDR2 ) and 3 (HCDR3) of SEQ ID NOs: 7, 8 and 9 or SEQ ID NOs: 13, 14 and 15, respectively, and the VL antigen binding site comprises light chain CDRs 1 (LCDR1), 2 (LCDR2 ) and 3 (LCDR3) of SEQ ID NOs: 10, 11 and 12 or SEQ ID NOs: 16, 17 and 18, respectively.
    [3]
    3. Isolated antibody according to either of Claims 1 or 2, characterized by the fact that it binds PHF-tau comprising an antigen binding site of a VH of SEQ ID NO: 35 and a binding site antigen of a VL of SEQ ID NO: 36.
    [4]
    4. Isolated antibody according to claim 3, characterized by the fact that the VH antigen binding site comprises HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 7, 8 and 9, respectively and the antigen binding site of the VL comprises LCDR1, LCDR 2 and LCDR3 of SEQ ID NOs: 10, 11 and 12, respectively.
    [5]
    5. Isolated antibody according to either of Claims 1 or 2, characterized by the fact that it binds PHF-tau which comprises an antigen binding site of a VH of SEQ ID NO: 37 and a binding site of antigen of a VL of SEQ ID NO:
    38.
    [6]
    6. Isolated antibody according to claim 5, characterized by the fact that the VH antigen binding site comprises
    comprises HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 13, 14 and 15, respectively, and the VL antigen binding site comprises LC-DR1, LCDR2 and LCDR3 of SEQ ID NOs: 16, 17 and 18, respectively.
    [7]
    7. Isolated antibody, according to any of claims 1 to 6, characterized by the fact that the antibody is humanized.
    [8]
    8. Isolated antibody or fragment that competes for the binding of PHF-tau with a monoclonal antibody, characterized by the fact that it comprises an antigen binding site of a VH of SEQ ID NO: 35 and an antigen binding site of a VL of SEQ ID NO: 36, or an antigen binding site of a VH of SEQ ID NO: 37 and an antigen binding site of a VL of SEQ ID NO: 38.
    [9]
    9. Isolated polynucleotide encoding an antibody VH, characterized by the fact that it comprises HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 7, 8 and 9, respectively, or SEQ ID NOs: 13, 14 and 15, respectively.
    [10]
    10. Isolated polynucleotide encoding a VL antibody, characterized by the fact that it comprises LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 11 and 12, respectively, or SEQ ID NOs: 16, 17 and 18, respectively.
    [11]
    11. Vector, characterized by the fact that it comprises a polynucleotide, as defined in claim 9 or 10.
    [12]
    12. Host cell, characterized by the fact that it comprises the vector, as defined in claim 11.
    [13]
    13. Production method an antibody that binds PHF-tau, characterized by the fact that it comprises culturing the host cell, as defined in claim 12, and recovering the antibody produced by the host cell.
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    法律状态:
    2020-11-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2021-01-19| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |
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    优先权:
    申请号 | 申请日 | 专利标题
    US201161577817P| true| 2011-12-20|2011-12-20|
    US61/577,817|2011-12-20|
    PCT/US2012/070486|WO2013096380A2|2011-12-20|2012-12-19|Anti-phf-tau antibodies and their uses|
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