Methods and compositions for the production and use of humanized conformation-specific phosphorylated tau antibodies

Abstract

The present disclosure generally relates to a conformation-specific antibody that can bind to phosphorylated threonine 231-tau protein (pT231-tau) and neutralize its activity.The antibody of the present technology is useful in a method for treating neurological diseases associated with elevated expression of cis-pT231-tau protein in a subject in need thereof.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 583,850, filed on 9 November 2017, the disclosures of which are incorporated herein by reference in their entirety for any purpose whatsoever. This technology generally relates to the preparation and use of conformation-specific antibodies that specifically bind to phosphorylated threonine 231-tau protein (pT231-tau). In particular, this technology relates to cis-pT231-tau neutralizing antibodies and their use in the treatment of neurological disorders involving elevated cis-pT231-tau protein expression. [Background technology]

[0002] The following explanation is provided to aid the reader's understanding. None of the information provided or the references cited are acknowledged to be prior art to this method. The brains of patients with Alzheimer's disease (AD) and several other central nervous system disorders, such as frontotemporal dementia, Pick's disease, corticobasal degeneration, traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), and progressive supranuclear palsy, contain neurofibrillary tangles (NFTs) that include tau protein. This common pathological feature gives rise to these various neurodegenerative diseases collectively known as "tauopathies." Tau-related pathology is well correlated with the progressive loss of neurons and memory in AD. A very early event in tauopathies in AD is tau hyperphosphorylation, particularly at the Ser / Thr-Pro motif, which causes microtubule disruption and neurotoxicity. The phosphorylated Thr231-Pro motif in tau (pT231-tau) is known to exist in two distinct cis- and transconformations. The cis-conformation of the pT231-tau protein is primarily expressed in the brains of AD patients. Since neuronal dysfunction occurs well before tangle formation, the main challenge is the development of immunotherapies that exclusively target early pathogenic events leading to tauopathy and memory loss in Alzheimer's disease. [Overview of the project]

[0003] This technology generally relates to a conformation-specific antibody that specifically binds to and neutralizes the activity of phosphorylated threonine 231-tau protein (pT231-tau). The antibody developed using this technology is useful in methods for treating neurological disorders accompanied by elevated cis-pT231-tau protein expression. In one embodiment, the technology provides antibodies comprising the heavy chain immunoglobulin variable domain sequence of SEQ ID NOs. 1-4 or 7-14 or a variant thereof having one or more conserved amino acid substitutions, and the light chain immunoglobulin variable domain sequence of SEQ ID NOs. 41, 42 or 45-48 or a variant thereof having one or more conserved amino acid substitutions. In some embodiments, the antibodies are humanized antibodies. In another embodiment, the technology provides an antigen-binding fragment of an antibody, the antigen-binding fragment being selected from the group consisting of Fab, F(ab')2, Fab', scFv, and Fv. In any of the embodiments described above, the antibody binds to an epitope of phosphorylated threonine 231-tau protein containing the amino acid sequence KVAVVRTPPKSPS (SEQ ID NO: 56). In some embodiments, the antibody specifically binds to the cis-conformation of phosphorylated threonine 231-tau protein (pT231-tau). In some embodiments, the antibody has an isotype selected from the group consisting of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM. In one embodiment, the technology provides a recombinant nucleic acid sequence encoding an anti-cis-pT231-tau antibody disclosed herein. In yet another embodiment, the technology provides a host cell or vector containing a recombinant nucleic acid encoding an anti-cis-pT231-tau antibody described herein. In one embodiment, the Technology provides a composition comprising an anti-cis-pT231-tau antibody and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises an anti-cis-pT231-tau antibody comprising the heavy chain immunoglobulin variable domain sequence of SEQ ID NOs. 1-4 or 7-14 or a variant thereof having one or more conserved amino acid substitutions, and which may also comprise the light chain immunoglobulin variable domain sequence of SEQ ID NOs. 41, 42 or 45-48 or a variant thereof having one or more conserved amino acid substitutions.

[0004] In another embodiment, the present technology provides a method for treating neurological disorders involving elevated cis-pT231-tau protein expression in subjects requiring such treatment, comprising administering to a subject an effective amount of an antibody comprising the heavy chain immunoglobulin variable domain sequence of SEQ ID NOs. 1-4 or 7-14 or a variant thereof having one or more conserved amino acid substitutions, wherein the antibody specifically binds to and neutralizes the cis-pT231-tau protein. In certain embodiments of the method, the antibody further comprises the light chain immunoglobulin variable domain sequence of SEQ ID NOs. 41, 42, or 45-48, or a variant thereof having one or more conserved amino acid substitutions. In some embodiments of the method, the neurological disorder is tauopathy, traumatic brain injury (TBI), or stroke. In further embodiments, tauopathy is selected from the group consisting of progressive supranuclear palsy (PSP), chronic traumatic encephalopathy (CTE), frontotemporal dementia (FTD), frontotemporal lobar degeneration, Ritiko-Bodig disease, Tangle-predominant dementia, meningeal hemangioma, subacute sclerosing panencephalitis, Pick's disease, corticobasal degeneration, and Alzheimer's disease (AD).

[0005] In certain embodiments, the subject is in the early stages of the tauopathy. In one embodiment, the early stages of the tauopathy are determined by an increase in the level of cis-pT231-tau or an increase in the cis:trans-pT231-tau ratio in a sample obtained from the subject. In some embodiments, the method further includes determining the levels of CSF t-tau, pT181-tau, αβ42, or ApoE4 in a sample obtained from the subject. In certain embodiments, the sample is selected from the group consisting of urine, blood, serum, plasma, saliva, amniotic fluid, and cerebrospinal fluid (CSF). In some embodiments, the subjects have a history of repeated traumatic brain injury. Furthermore, or in some embodiments of the method, the antibody is administered to the subject sequentially or simultaneously with any additional therapeutic agent. In some embodiments, the additional therapeutic agent is one or more of donepezil, rivastigmine, galantamine, memantine, and lithium chloride.

[0006] In one embodiment, the present technology provides a method for selecting a subject for treatment using an anti-cis-pT231-tau antibody disclosed herein, comprising: (a) detecting an increase in the level of cis-pT231-tau protein or an increase in the cis:trans-pT231-tau protein ratio in a sample obtained from the subject compared to that observed in a reference sample; and (b) selecting the subject for treatment using an anti-cis-pT231-tau antibody disclosed herein. In some embodiments, the sample obtained from the subject is selected from the group consisting of urine, blood, serum, plasma, saliva, amniotic fluid, and cerebrospinal fluid (CSF). In certain embodiments, the reference sample is obtained from a healthy control subject. In another embodiment, the technology provides a kit for treating neurological disorders involving elevated cis-pT231-tau protein expression in subjects requiring it, comprising an antibody that specifically binds to and neutralizes cis-pT231-tau protein, and instructions for using the antibody, wherein the antibody comprises variants thereof having the heavy chain immunoglobulin variable domain sequence of SEQ ID NOs. 1-4 or 7-14 or one or more conserved amino acid substitutions. In some embodiments of the kit, the antibody further comprises variants thereof having the light chain immunoglobulin variable domain sequence of SEQ ID NOs. 41, 42 or 45-48 or one or more conserved amino acid substitutions.

[0007] In one embodiment, the technology provides a kit for detecting cis-pT231-tau protein in a sample, comprising an antibody that specifically binds to cis-pT231-tau protein and instructions for using the antibody, wherein the antibody comprises variants thereof having the heavy chain immunoglobulin variable domain sequence of SEQ ID NOs. 1-4 or 7-14 or one or more conserved amino acid substitutions. In some embodiments of the kit, the antibody further comprises variants thereof having the light chain immunoglobulin variable domain sequence of SEQ ID NOs. 41, 42 or 45-48 or one or more conserved amino acid substitutions. In certain embodiments, the antibody is coupled to one or more detectable labels. In one embodiment, the one or more detectable labels include radioactive labels, fluorescent labels, or chromogenic labels. Furthermore, or in some embodiments, the kit further comprises a secondary antibody that specifically binds to the cis-pT231-tau protein antibody. In some embodiments, the secondary antibody is coupled to at least one detectable label selected from the group consisting of radiolabeling, fluorescent labeling, or chromogenic labeling. [Brief explanation of the drawing]

[0008] [Figure 1]Figure 1 shows the nucleotide and amino acid sequences of the heavy chain immunoglobulin variable (VH) domain of the mouse antibody PT-113. Amino acid residues are indicated by single-letter codes. A portion of the CH1 sequence is included in the figure. [Figure 2] Figure 2 shows the nucleotide and amino acid sequences of the variable light chain immunoglobulin (VL) domain of the mouse antibody PT-113. Amino acid residues are indicated by single-letter codes. A portion of the Cκ sequence is included in the figure. [Figure 3] Figure 3 shows the amino acid sequences of the mature VH domain of the mouse PT-113 antibody, aligned with its putative parental germline V segment IGHV1S127*01 and JH2 segments, using single-letter codes. Residue numbers are assigned according to Kabat et al., Sequences of Proteins of Immunological Interests, NIH Publication No. 91-3242, US Department of Health and Human Services (5th ed., 1991). The CDR sequence of the PT-113 VH domain is underlined. Asterisks indicate differences between the PT-113 VH and IGHV1S127*01 amino acid sequences. [Figure 4] Figure 4 shows the amino acid sequences of the mature VL domain of the mouse PT-113 antibody, aligned with its putative parental germline V segment IGKV1-110*02 and Jk1 segment, using single-letter codes. Residue numbers are assigned according to Kabat et al. (1991). The CDR sequence of the PT-113 VL domain is underlined. Asterisks indicate differences between the PT-113 VL and IGKV1-110*02 amino acid sequences. [Figure 5]Figure 5 shows the nucleotide sequence of the synthetic mouse PT-113VH gene, flanked by the SpeI and HindIII sites (underlined), along with the predicted amino acid sequence. Amino acid residues are shown with single-letter codes. Signal peptide sequences are italicized. The N-terminal amino acid residue (Q) of the mature VH domain is double-underlined. CDR sequences, according to the definition of Kabat et al. (1991), are underlined. Intron sequences consecutive to the HindIII site are italicized. [Figure 6] Figure 6 shows the nucleotide sequence of the synthetic mouse PT-113VL gene, flanked by the NheI and EcoRI sites (underlined), along with the predicted amino acid sequence. Amino acid residues are shown with single-letter codes. Signal peptide sequences are italicized. The N-terminal amino acid residue (D) of the mature VL domain is double-underlined. CDR sequences, according to the definition of Kabat et al. (1991), are underlined. Intron sequences consecutive to the EcoRI site are italicized. [Figure 7]Figure 7 shows the schematic structures of the pChPT-113, pHuPT-113A, pHuPT-113B, pHuPT-113C, pHuPT-113D, and pHuPT-113D-AA vectors (collectively expressed vectors). Starting from the SalI site and proceeding clockwise, the plasmid contains a heavy chain transcription unit beginning with the human cytomegalovirus (CMV) major pre-initial promoter and enhancer (CMV-P) to initiate transcription of the antibody heavy chain gene. The CMV promoter is followed by a genome sequence containing the human gamma-1 heavy chain constant region, which includes the VH exon, CH1, hinge, CH2, and CH3 exons along with intervening introns, followed by a polyadenylation site after the CH3 exon. Following the heavy chain gene sequence, the light chain transcription unit begins with the CMV promoter and contains a genome sequence containing the human kappa chain constant region exon (CL) along with the VL exon and preceding intron region, followed by a polyadenylation site after the CL exon. The light chain gene is followed by the SV40 initial promoter (SV40-P), the puromycin N-acetyl-transferase gene (puro) for resistance to puromycin, and a segment (SV40-A) containing the SV40 polyadenylation site. Finally, the plasmid contains a portion of plasmid pUC19, which includes the bacterial origin of replication (pUC ori) and the beta-lactamase gene (β-lactamase). In pHuPT-113D-AA, the CH2 region retains the Leu-to-Ala amino acid substitution at positions 234 and 235 (Eu numbering in Kabat et al. (1)). The locations of the relevant restriction enzyme sites are shown in the figure. [Figure 8]Figure 8 shows the amino acid sequence alignment of the mouse PT-113VH domain, four versions of the humanized PT-113VH (HuPT-113 VH1, VH2, VH3, and VH4) domains, and the human acceptor AF174092 VH domain. Amino acid residues are indicated by single-letter codes. The numbers above the sequences indicate the position according to Kabat et al. (1991). The CDR sequence of the mouse PT-113VH domain is underlined. The CDR residue in the AF174092VH domain is omitted from the figure. The underlined residues in HuPT-113VH1, VH2, VH3, and VH4 are predicted to play an important role in the formation of antigen-binding sites, and therefore the corresponding mouse residues were retained at these positions. [Figure 9] Figure 9 shows the amino acid sequence alignment of the mouse PT-113VL domain, the humanized PT-113VL (HuPT-113 VL1) domain, and the human acceptor M99608 VL domain. Amino acid residues are indicated by single-letter codes. The numbers above the sequences indicate the position according to Kabat et al. (1991). The CDR sequence of the mouse PT-113VL domain is underlined. The CDR residue in the M99608VL domain is omitted from the figure. Framework amino acid substitutions were not necessary when designing HuPT-113 VL1. [Figure 10] Figure 10 shows the nucleotide sequence of the HuPT-113VH1 gene, flanked by the SpeI and HindIII sites (underlined), along with the predicted amino acid sequence. Amino acid residues are shown with single-letter codes. Signal peptide sequences are italicized. The N-terminal amino acid residue (Q) of the mature VH domain is double-underlined. CDR sequences, according to the definition of Kabat et al. (1991), are underlined. Intron sequences consecutive to the HindIII site are italicized. [Figure 11]Figure 11 shows the nucleotide sequence of the HuPT-113VH2 gene, flanked by the SpeI and HindIII sites (underlined), along with the predicted amino acid sequence. Amino acid residues are shown with single-letter codes. Signal peptide sequences are italicized. The N-terminal amino acid residue (Q) of the mature VH domain is double-underlined. CDR sequences, according to the definition of Kabat et al. (1991), are underlined. Intron sequences consecutive to the HindIII site are italicized. [Figure 12] Figure 12 shows the nucleotide sequence of the HuPT-113VH3 gene, flanked by the SpeI and HindIII sites (underlined), along with the predicted amino acid sequence. Amino acid residues are indicated by single-letter codes. Signal peptide sequences are italicized. The N-terminal amino acid residue (Q) of the mature VH domain is double-underlined. CDR sequences, according to the definition of Kabat et al. (1991), are underlined. Intron sequences consecutive to the HindIII site are italicized. [Figure 13] Figure 13 shows the nucleotide sequence of the HuPT-113VH4 gene, flanked by the SpeI and HindIII sites (underlined), along with the predicted amino acid sequence. Amino acid residues are shown with single-letter codes. Signal peptide sequences are italicized. The N-terminal amino acid residue (Q) of the mature VH domain is double-underlined. CDR sequences, according to the definition of Kabat et al. (1991), are underlined. Intron sequences consecutive to the HindIII site are italicized. [Figure 14]Figure 14 shows the nucleotide sequence of the HuPT-113VL1 gene, flanked by the NheI and EcoRI sites (underlined), along with the predicted amino acid sequence. Amino acid residues are shown with single-letter codes. Signal peptide sequences are italicized. The N-terminal amino acid residue (D) of the mature VL domain is double-underlined. CDR sequences, according to the definition of Kabat et al. (1991), are underlined. Intron sequences consecutive to the EcoRI site are italicized. [Figure 15] Figure 15 shows the binding of transiently expressed ChPT-113, HuPT-113A, HuPT-113B, HuPT-113C, and HuPT-113D IgG1(I) antibodies to the pT231-Dmp-tau peptide disclosed herein. [Figure 16] Figure 16 shows the SDS-PAGE analysis of ChPT-113 IgG1, HuPT-113D IgG1(I), and HuPT-113D IgG1-AA(I) antibodies. Antibodies (5 μg each) were run on 4-12% SDS-PAGE gels in MES buffer (Thermo Fisher Scientific) under reducing conditions. PageRuler Prestained Protein Ladder (Thermo Fisher Scientific) was used as the molecular weight standard (lane 4). Lane 1: ChPT-113 IgG1; Lane 2: HuPT-113D IgG1(I); Lane 3: HuPT-113D IgG1-AA(I). H and L represent the heavy and light chains of each test antibody, respectively. [Figure 17] Figure 17 shows the sequences of oligonucleotides used for PCR amplification and sequencing of ChPT-113 IgG1, HuPT-113D IgG1(I), and HuPT-113D IgG1-AA(I) heavy and light chain cDNAs. [Figure 18]Figure 18 shows the nucleotide sequences of the coding regions (variable and constant regions) of the gamma-1 heavy chain encoded by the pChPT-113 construct. Amino acid residues are indicated by single-letter codes. Stop codons are represented by "·". [Figure 19] Figure 19 shows the nucleotide sequences of the coding regions (variable and constant regions) of the kappa light chain encoded by the pChPT-113 construct. Amino acid residues are shown with single-letter codes. Stop codons are represented by "·". [Figure 20] Figure 20 shows the nucleotide sequences of the coding regions (variable and constant regions) of the gamma-1 heavy chain encoded by the pHuPT-113D construct. Amino acid residues are indicated by single-letter codes. Stop codons are represented by "·". [Figure 21] Figure 21 shows the nucleotide sequences of the coding regions (variable and constant regions) of the kappa light chain encoded by the pHuPT-113D and pHuPT-113D-AA constructs. Amino acid residues are indicated by single-letter codes. Stop codons are represented by "·". [Figure 22] Figure 22 shows the nucleotide sequences of the coding regions (variable and constant regions) of the gamma-1 heavy chain encoded by the pHuPT-113D-AA construct. Amino acid residues are indicated by single-letter codes. Stop codons are represented by "·". [Figure 23] Figure 23 shows the ELISA analysis of the binding of the mouse PT-113 IgG2b antibody to four different tau peptides. [Figure 24] Figure 24 shows the ELISA analysis of the binding of ChPT-113 IgG1 antibody to four different tau peptides. [Figure 25] Figure 25 shows the binding of (i) biotinylated mouse PT-113 IgG2b and (ii) ChPT-113 IgG1 antibodies to the pThr231-Dmp tau peptide in the presence of various concentrations of competing antibodies. [Figure 26A]Figure 26A shows the ELISA analysis of the binding of ChPT-113 IgG1, HuPT-113D IgG1(I), and HuPT-113D IgG1-AA(I) antibodies to the pThr231-Dmp tau peptide. [Figure 26B] Figure 26B shows the ELISA analysis of the binding of ChPT-113 IgG1, HuPT-113D IgG1(I), and HuPT-113D IgG1-AA(I) antibodies to the pThr231-Pro tau peptide. [Figure 26C] Figure 26C shows the ELISA analysis of the binding of ChPT-113 IgG1, HuPT-113D IgG1(I), and HuPT-113D IgG1-AA(I) antibodies to the np-Thr231-Pro tau peptide. [Figure 26D] Figure 26D shows the ELISA analysis of the binding of ChPT-113 IgG1, HuPT-113D IgG1(I), and HuPT-113D IgG1-AA(I) antibodies to the pThr231-Ala tau peptide. [Figure 27A] Figure 27A shows the nucleotide sequence of the heavy chain variable strand of the HuPT-113D IgG1-AA(II) construct (SEQ ID NO: 128). [Figure 27B] Figure 27B shows the amino acid sequence (sequence number 39) encoded by sequence number 128. [Figure 27C] Figure 27C shows the nucleotide sequence of the kappa light chain of the HuPT-113D IgG1-AA(II) construct (SEQ ID NO: 129). [Figure 27D] Figure 27D shows the amino acid sequence (sequence number 55) encoded by sequence number 129. [Figure 28A] Figure 28A shows the nucleotide sequence of the heavy chain variable strand of the HuPT-113D IgG4(II) construct (SEQ ID NO: 130). [Figure 28B] Figure 28B shows the amino acid sequence (sequence number 40) encoded by sequence number 130. [Figure 28C]Figure 28C shows the nucleotide sequence of the kappa light chain of the HuPT-113D IgG4(II) construct (SEQ ID NO: 129). [Figure 28D] Figure 28D shows the amino acid sequence (sequence number 55) encoded by sequence number 129. [Figure 29A] Figure 29A shows the nucleotide sequence of the heavy chain variable strand of the HuPT-113D IgG1(II) construct (SEQ ID NO: 131). [Figure 29B] Figure 29B shows the amino acid sequence (sequence number 132) encoded by sequence number 131. [Figure 29C] Figure 29C shows the nucleotide sequence of the kappa light chain of the HuPT-113D IgG1(II) construct (SEQ ID NO: 129). [Figure 29D] Figure 29D shows the amino acid sequence (sequence number 55) encoded by sequence number 129. [Figure 30A] Figure 30A is a histogram plot showing CD16 staining in THP-1 cells. Red indicates unstained cells, and blue indicates cells stained with anti-CD16 antibody. [Figure 30B] Figure 30B is a histogram plot showing CD32 staining in THP-1 cells. Red indicates unstained cells, and blue indicates cells stained with anti-CD32 antibody. [Figure 30C] Figure 30C is a histogram plot showing CD64 staining in THP-1 cells. Red indicates unstained cells, and blue indicates cells stained with anti-CD64 antibody. [Figure 31A] Figure 31A is a graph showing the effects of ovalbumin immune complex (IC) coating density and cell number (5,000 cells per well) on THP-1 cell activation, as measured by TNFα secretion. The ovalbumin ICs contained rabbit polyclonal anti-ovalbumin antibodies. [Figure 31B]Figure 31B is a graph showing the effects of ovalbumin immune complex (IC) coating density and cell number (50,000 cells per well) on THP-1 cell activation, as measured by TNFα secretion. The ovalbumin ICs contained rabbit polyclonal anti-ovalbumin antibodies. [Figure 31C] Figure 31C is a graph showing the effect of ovalbumin immune complex (IC) coating density and cell number (5,000 cells per well) on THP-1 cell activation, as measured by IL-6 secretion. The ovalbumin IC contained rabbit polyclonal anti-ovalbumin antibody. [Figure 31D] Figure 31D is a graph showing the effects of ovalbumin immune complex (IC) coating density and cell number (50,000 cells per well) on THP-1 cell activation, as measured by IL-6 secretion. The ovalbumin ICs contained rabbit polyclonal anti-ovalbumin antibodies. [Figure 32A] Figure 32A is a graph showing the effects of ovalbumin immune complex (IC) coating density (0.1 μg / ml coating) and cell number (50,000 cells per well) on THP-1 cell activation, as measured by TNFα secretion. The ovalbumin IC contained mouse monoclonal anti-ovalbumin antibody. [Figure 32B] Figure 32B is a graph showing the effects of ovalbumin immune complex (IC) coating density (10 μg / ml coating) and cell number (50,000 cells per well) on THP-1 cell activation, as measured by TNFα secretion. The ovalbumin IC contained mouse monoclonal anti-ovalbumin antibody. [Figure 32C]Figure 32C is a graph showing the effects of ovalbumin immune complex (IC) coating density (0.1 μg / ml coating) and cell number (150,000 cells per well) on THP-1 cell activation, as measured by TNFα secretion. The ovalbumin IC contained mouse monoclonal anti-ovalbumin antibody. [Figure 32D] Figure 32D is a graph showing the effects of ovalbumin immune complex (IC) coating density (10 μg / ml coating) and cell number (150,000 cells per well) on THP-1 cell activation, as measured by TNFα secretion. The ovalbumin IC contained mouse monoclonal anti-ovalbumin antibody. [Figure 33A] Figure 33A is a graph showing the effects of ovalbumin immune complex (IC) coating density (0.1 mg / ml coating) and cell number (50,000 cells per well) on THP-1 cell activation, as measured by IL-6 secretion. The ovalbumin IC contained mouse monoclonal anti-ovalbumin antibody. [Figure 33B] Figure 33B is a graph showing the effects of ovalbumin immune complex (IC) coating density (10 μg / ml coating) and cell number (50,000 cells per well) on THP-1 cell activation, as measured by IL-6 secretion. The ovalbumin IC contained mouse monoclonal anti-ovalbumin antibody. [Figure 33C] Figure 33C is a graph showing the effects of ovalbumin immune complex (IC) coating density (0.1 μg / ml coating) and cell number (150,000 cells per well) on THP-1 cell activation, as measured by IL-6 secretion. The ovalbumin IC contained mouse monoclonal anti-ovalbumin antibody. [Figure 33D]Figure 33D is a graph showing the effects of ovalbumin immune complex (IC) coating density (10 μg / ml coating) and cell number (150,000 cells per well) on THP-1 cell activation, as measured by IL-6 secretion. The ovalbumin IC contained mouse monoclonal anti-ovalbumin antibody. [Figure 34A] Figure 34A is a graph showing the effect of ovalbumin IC concentration and Jurkat cell number (50,000 cells per well) on FcR activation. Ovalbumin IC was generated from a range of concentrations of ovalbumin-coated wells (10 μg / mL) and both rabbit polyclonal and mouse monoclonal anti-ovalbumin antibodies. [Figure 34B] Figure 34B is a graph showing the effect of ovalbumin IC concentration and Jurkat cell number (100,000 cells per well) on FcR activation. Ovalbumin IC was generated from a range of concentrations of ovalbumin-coated wells (10 μg / mL) and both rabbit polyclonal and mouse monoclonal anti-ovalbumin antibodies. [Figure 35] Figure 35 is a graph showing the binding curves of each antibody (HuPT-113D IgG1-AA(II)(IgG1-AA), HuPT-113D IgG4(II)(IgG4), and HuPT-113D IgG1(II)(IgG1)) at each pThr-Dmp peptide coating concentration (1 μg / ml, 10 μg / ml, and 100 μg / ml). [Figure 36A] Figure 36A is a graph showing TNFα secretion in THP-1 cells in response to pThr-Dmp peptide diluted with 2,2,2-trifluoroethanol (TFE) or DPBS. [Figure 36B] Figure 36B is a graph showing IL-6 secretion in THP-1 cells in response to pThr-Dmp peptide diluted with 2,2,2-trifluoroethanol (TFE) or DPBS. [Figure 37]Figure 37 is a graph showing the level of FcγRIIIa activation in Jurkat cells coated with pThr-Dmp peptide diluted with either 2,2,2-trifluoroethanol (TFE) or DPBS at a concentration of 1 μg / ml. [Figure 38A] Figure 38A is a graph showing the induction of TNFα release from THP-1 cells contacted with the natural pThr-Dmp peptide IC and HuPT-113D antibodies (HuPT-113D IgG1(II) (IgG1; blue), HuPT-113D IgG-AA(II) (IgG-AA; red), or HuPT-113D IgG4(II) (IgG4; green)). [Figure 38B] Figure 38B is a graph showing the induction of IL-1β release from THP-1 cells contacted with the natural pThr-Dmp peptide IC and HuPT-113D antibodies (HuPT-113D IgG1(II) (IgG1; blue), HuPT-113D IgG-AA(II) (IgG-AA; red), or HuPT-113D IgG4(II) (IgG4; green)). [Figure 38C] Figure 38C is a graph showing the induction of IL-6 release from THP-1 cells contacted with the natural pThr-Dmp peptide IC and HuPT-113D antibodies (HuPT-113D IgG1(II) (IgG1; blue), HuPT-113D IgG-AA(II) (IgG-AA; red), or HuPT-113D IgG4(II) (IgG4; green)), respectively. [Figure 38D] Figure 38D is a graph showing the induction of TNFα release from THP-1 cells contacted with biotinylated pThr-Dmp peptide IC and HuPT-113D antibodies (HuPT-113D IgG1(II) (IgG1; blue), HuPT-113D IgG-AA(II) (IgG-AA; red), or HuPT-113D IgG4(II) (IgG4; green)). [Figure 38E]Figure 38E is a graph showing the induction of IL-1β release from THP-1 cells contacted with biotinylated pThr-Dmp peptide IC and HuPT-113D antibodies (HuPT-113D IgG1(II) (IgG1; blue), HuPT-113D IgG-AA(II) (IgG-AA; red), or HuPT-113D IgG4(II) (IgG4; green)). [Figure 38F] Figure 38F is a graph showing the induction of IL-6 release from THP-1 cells contacted with biotinylated pThr-Dmp peptide IC and HuPT-113D antibodies (HuPT-113D IgG1(II) (IgG1; blue), HuPT-113D IgG-AA(II) (IgG-AA; red), or HuPT-113D IgG4(II) (IgG4; green)). [Figure 39] Figure 39 is a graph showing the EC50 values ​​for HuPT-113D IgG1(II) (IgG1; blue), HuPT-113D IgG-AA(II) (IgG-AA; red), and HuPT-113D IgG4(II) (IgG4; green), obtained from Figures 38A-C. [Figure 40] Figure 40 is a graph showing the activation of FcγRIIIa in Jurkat cells treated with HuPT-113D IgG1(II)(IgG1), HuPT-113D IgG-AA(II)(IgG-AA), or HuPT-113D IgG4(II)(IgG4). The wells were coated with the natural pThr-Dmp peptide IC at 1, 10, or 100 μg / ml. [Figure 41A] Figure 41A is a graph showing the induction of TNFα release from THP-1 cells contacted with the natural pThr-Dmp peptide IC and HuPT-113D antibodies (HuPT-113D IgG1(II) (IgG1; blue), HuPT-113D IgG-AA(II) (IgG-AA; red), or HuPT-113D IgG4(II) (IgG4; green)). [Figure 41B]Figure 41B is a graph showing the induction of IL-1β release from THP-1 cells contacted with the natural pThr-Dmp peptide IC and HuPT-113D antibodies (HuPT-113D IgG1(II) (IgG1; blue), HuPT-113D IgG-AA(II) (IgG-AA; red), or HuPT-113D IgG4(II) (IgG4; green)). [Figure 41C] Figure 41C is a graph showing the induction of IL-6 release from THP-1 cells contacted with the natural pThr-Dmp peptide IC and HuPT-113D antibodies (HuPT-113D IgG1(II) (IgG1; blue), HuPT-113D IgG-AA(II) (IgG-AA; red), or HuPT-113D IgG4(II) (IgG4; green)), respectively. [Figure 42] Figure 42 is a graph showing the EC50 values ​​for HuPT-113D IgG1(II) (IgG1; blue), HuPT-113D IgG-AA(II) (IgG-AA; red), and HuPT-113D IgG4(II) (IgG4; green)) obtained from Figures 41A-C. [Figure 43] Figure 43 is a graph showing the activation of FcγRIIIa in Jurkat cells treated with HuPT-113D IgG1(II)(IgG1), HuPT-113D IgG-AA(II)(IgG-AA), or HuPT-113D IgG4(II)(IgG4). [Figure 44A] Figure 44A is a graph showing the activation of FcγRIIIa V158 in Jurkat cells treated with HuPT-113D IgG1(II)(IgG1), HuPT-113D IgG-AA(II)(IgG-AA), or HuPT-113D IgG4(II)(IgG4). The wells were coated with the natural pThr-Dmp peptide IC at a 1 μg / ml coating in TFE. Proliferating cells were used. [Figure 44B]Figure 44B is a graph showing the activation of FcγRIIIa V158 in Jurkat cells treated with HuPT-113D IgG1(II)(IgG1), HuPT-113D IgG-AA(II)(IgG-AA), or HuPT-113D IgG4(II)(IgG4). The wells were coated with natural pThr-Dmp peptide IC at a 1 μg / ml coating in TFE. Thawed and used cells were utilized. [Figure 45A] Figure 45A is a graph showing the activation of FcγRIIIa V158 in Jurkat cells treated with HuPT-113D IgG1(II)(IgG1), HuPT-113D IgG-AA(II)(IgG-AA), or HuPT-113D IgG4(II)(IgG4). The wells were coated with natural pThr-Dmp peptide IC using a 1 μg / ml coating in TFE. Thawed and used cells were employed. [Figure 45B] Figure 45B is a graph showing the activation of FcγRIIIa F158 in Jurkat cells treated with HuPT-113D IgG1(II)(IgG1), HuPT-113D IgG-AA(II)(IgG-AA), or HuPT-113D IgG4(II)(IgG4). The wells were coated with natural pThr-Dmp peptide IC1 in TFE at a rate of 1 μg / ml. Thawed and used cells were utilized. [Figure 45C] Figure 45C is a graph showing the activation of FcγRIIa H131 in Jurkat cells treated with HuPT-113D IgG1(II)(IgG1), HuPT-113D IgG-AA(II)(IgG-AA), or HuPT-113D IgG4(II)(IgG4). The wells were coated with natural pThr-Dmp peptide IC at a 1 μg / ml coating in TFE. Thawed and used cells were utilized. [Figure 46]Figure 46 is a graph showing the activation of FcγRIIIa V158 in Jurkat cells treated with HuPT-113D IgG1(II)(IgG1), HuPT-113D IgG-AA(II)(IgG-AA), or HuPT-113D IgG4(II)(IgG4). The wells were coated with pTau at a 10 μg / ml coating in DBPS. [Figure 47] Figure 47 shows the SDS-PAGE analysis of purified HuPT-113D IgG-AA(II)(IgG-AA). [Modes for carrying out the invention]

[0009] Naturally, in order to provide a substantial understanding of this technology, specific aspects, styles, embodiments, variations, and features of this method are described below at various levels of detail. This disclosure generally provides a conformation-specific antibody that specifically binds to and neutralizes the activity of phosphorylated threonine 231-tau protein (pT231-tau). The antibody of this technology is useful in methods for treating neurological disorders involving elevated cis-pT231-tau protein expression in subjects requiring it. Accordingly, various aspects of this method relate to the preparation, characterization, and manufacture of anti-cis-pT231-tau antibodies. The antibody of this technology is useful alone or in combination with further therapeutic agents for treating tauopathy. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is coupled to a detectable label. In other embodiments, the kit includes a secondary antibody coupled to a detectable label.

[0010] The implementation of this method utilizes numerous conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA. For example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; US Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds.See (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds. (1996) Weir's Handbook of Experimental Immunology. Methods for detecting and measuring polypeptide gene expression levels (i.e., gene translation levels) are well known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to the extent of this art. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the content clearly indicates otherwise. For example, a reference to “cell” includes combinations of two or more cells, etc. Generally, the nomenclature used herein, as well as experimental procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry, and the hybridizations described below, are well known and commonly used in the art. All references cited herein are incorporated herein by reference in the same way that individual publications, patents, or patent applications are incorporated in their entirety by reference in any way that individual publications, patents, or patent applications are incorporated in their entirety by reference in any way.

[0012] In this specification, the term “about” in relation to numbers is generally interpreted to include numbers that fall within the range of 1%, 5%, or 10% in either direction (greater or less) unless otherwise stated or the context makes clear (except when such numbers are less than 0% or greater than 100% of a possible value). In this specification, “administration” of a drug or substance to a subject includes any route through which the compound is introduced or delivered to the subject in order to exert its intended function. Administration may be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, or topically. Administration includes self-administration and administration by another person. An "adjuvant" refers to one or more substances that stimulate the immune system. In this context, adjuvants are used to enhance the immune response to one or more vaccine antigens or antibodies. Adjuvants may be administered to a subject before, in combination with, or after the administration of a vaccine. Examples of chemical compounds used as adjuvants include aluminum compounds, oils, block polymers, immunostimulatory complexes, vitamins and minerals (e.g., vitamin E, vitamin A, selenium, and vitamin B12), Quil A (saponin), bacterial and fungal cell wall components (e.g., lipopolysaccharides, lipoproteins, and glycoproteins), hormones, cytokines, and co-stimulators.

[0013] As used herein, the term "antibody" collectively refers to immunoglobulins or immunoglobulin-like molecules including, by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice, as well as in non-mammalian species such as shark immunoglobulins. The term "antibody" includes intact immunoglobulins and "antibody fragments" or "antigen-binding fragments" that specifically bind to a subject molecule (or a group of highly similar molecules of the subject) to such an extent that binding to other molecules is substantially excluded (e.g., at least 10, greater than binding to other molecules in a biological sample 3 M -1 greater than, at least 10 4 M -1 greater than or at least 10 5 M -1 greater than, antibodies and antibody fragments that have a binding constant for the subject molecule). The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3 rd Ed., W.H. Freeman & Co., New York, 1997Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3 rd Ed., W.H. Freeman & Co., New York, 1997. See also.

[0014] More specifically, an antibody refers to a polypeptide ligand that comprises at least a light chain immunoglobulin variable region or a heavy chain immunoglobulin variable region, and specifically recognizes and binds an epitope of an antigen. An antibody is composed of heavy and light chains, each of which has a heavy chain variable (V H) region and light chain variable (V L It has a variable region called the ) region. In short, V H Region and V L The region is involved in binding to the antigen recognized by the antibody. Typically, immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each heavy and light chain contains a constant region and a variable region (the region is also known as a "domain"). In combination, the heavy and light chain variable regions bind specifically to the antigen. The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called the "complementarity-determining region" or "CDR." The extent of the framework region and CDR is defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, incorporated herein by reference). The Kabat database is currently maintained online. The sequences of various light chain or heavy chain framework regions are relatively conserved within a species. The framework region of an antibody, which is a combined framework region of component light and heavy chains, primarily adopts a β-sheet conformation, and the CDR connects with the β-sheet structure, forming loops that in some cases form part of the β-sheet structure. Thus, the framework region works to form a scaffold that positions the CDR in the correct orientation through interchain, non-covalent interactions.

[0015] CDRs are primarily involved in binding to the antigen's epitope. The CDRs on each strand are usually called CDR1, CDR2, and CDR3, sequentially numbered starting from the N-terminus, and are also typically identified by the strand on which each individual CDR is located. Therefore, V HCDR3 is located in the variable domain of the antibody heavy chain where it is found, V L CDR1 is a CDR1 derived from the variable domain of the light chain of the antibody in which it is found. Antibodies that bind to the cis-pT231-tau protein are specific V H Region and V L A regional sequence, and therefore a specific CDR sequence, is present. Antibodies with different specificities (i.e., different combination sites for different antigens) have different CDRs. Although each antibody has a different CDR, only a limited number of amino acid positions within the CDR are directly involved in antigen binding. These positions within the CDR are called specificity-determining residues (SDRs). Examples of antibodies include monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, and antibody fragments. Antibodies may be, for example, conformation-specific antibodies (e.g., antibodies that bind to cis or transconformations of the Xaa-Pro motif where Xaa is an amino acid). Antibodies bind specifically to antigens.

[0016] In this specification, the term "antibody-related polypeptide" means an antigen-binding antibody fragment containing a single-chain antibody that may contain a variable region alone or in combination with all or some of the following polypeptide elements: the hinge region, CH1, CH2, and CH3 domains of the antibody molecule. Any combination of the variable region and the hinge region, CH1, CH2, and CH3 domains is also included in this technology. Antibody-related molecules useful in this method include, but are not limited to, Fab, Fab', and F(ab')2, Fd, single-chain Fv(scFv), single-chain antibodies, disulfide-bonded Fv(sdFv), and V L or V H A fragment containing any of the domains. For example, (i) Fab fragment, V L , V H , C L (ii) a monovalent fragment consisting of the CH1 domain, (ii) a bivalent fragment containing the F(ab')2 fragment and two Fab fragments linked by a disulfide bridge in the hinge region, (iii) V Hand an Fd fragment consisting of the CH1 domain, (iv) V of a single arm of the antibody L and V H Fv fragment consisting of domains, (v)V H Examples include dAb fragments consisting of domains (Ward et al., Nature 341: 544-546, 1989) and (vi) isolated complementarity-determining regions (CDRs). Such “antibody fragments” may include a portion of a full-length antibody, generally its antigen-binding or variable region. Examples of “antibody fragments” include Fab, Fab', F(ab')2 and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0017] In this specification, the term "diabody" refers to a small antibody fragment having two antigen-binding sites, and the fragment contains a light chain variable domain (V) within the same polypeptide chain. L ) connected to the heavy chain variable domain (V H ) including (V H V L ). By using a linker that is too short to allow pairing between two domains on the same chain, the domains are paired with a complementary domain on another chain, generating two antigen-binding sites. Diabodies are well described, for example, by EP404,097, WO93 / 11161 and 30 Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0018] In this specification, the terms "single-chain antibody" or "single-chain Fv (scFv)" refer to the Fv fragment, V L and V H This refers to an antibody fusion molecule of two domains. A single-chain antibody molecule may include a polymer having several individual molecules, such as a dimer, trimer, or other polymer. Furthermore, F v Fragment, V L and V H The two domains are encoded by separate genes, but they are V L and VH The regions form pairs to form a monovalent molecule (single-chain F v (scF v Such single-chain antibodies can be linked using recombination methods by synthetic linkers, which allow them to be produced as single protein chains forming what are known as single-chain antibodies. (Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883). Such single-chain antibodies can be prepared by recombination techniques or by enzymatic or chemical cleavage of intact antibodies. All of the above antibody fragments are obtained using prior art known to those skilled in the art, and the fragments are screened for binding specificity and neutralizing activity in the same manner as intact antibodies. In this specification, “antigen” refers to a molecule to which an antibody can selectively bind. Target antigens may be proteins (e.g., antigenic peptides), carbohydrates, nucleic acids, lipids, haptens, or other naturally occurring or synthetic compounds. Target antigens may be polypeptides (e.g., polypeptides containing a Xaa-Pro motif (e.g., phosphorylated or unphosphorylated Ser / Thr-Pro motif)) or peptide mimes (e.g., polypeptides containing a Xaa-homoproline motif (e.g., phosphorylated or unphosphorylated Ser / Thr-homoproline motif)). Antigens may be administered to animals to induce an immune response in animals.

[0019] "Binding affinity" refers to the strength of the combined non-covalent interaction between a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigenic peptide) at a single binding site. The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (K). d Affinity can be expressed by ( ). Affinity can be measured by standard methods known in the art, including those described herein. Low-affinity conjugates generally contain antibodies that tend to dissociate easily from the antigen, while high-affinity conjugates generally contain antibodies that tend to remain bound to the antigen for extended periods.

[0020] In this specification, the term “biological sample” means sample material derived from living cells. A biological sample may include tissues, cells, cellular proteins or membrane extracts and biological fluids (e.g., ascites or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells, and fluids present within the subject. Examples of biological samples in this technique include, but are not limited to, samples taken from breast tissue, kidney tissue, cervix, endometrium, head or neck, gallbladder, parotid gland tissue, prostate, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovarian tissue, adrenal gland tissue, testicular tissue, tonsils, thymus, blood, hair, buccal tissue, skin, serum, plasma, CSF, semen, prostatic fluid, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsies of internal organs or cancer. Biological samples can be obtained from subjects for diagnostic or research purposes, or from unaffected individuals as controls or for basic research. Samples can be obtained by standard methods, including, for example, venous aspiration and surgical biopsy. In certain embodiments, the biological sample is a breast, lung, colon, or prostate tissue sample obtained by needle biopsy. In this specification, the term "CDR graft antibody" means an antibody in which at least one CDR of the "acceptor" antibody is replaced by a CDR "graft" derived from a "donor" antibody having the desired antigen specificity.

[0021] In this specification, the term "chimeric antibody" means an antibody in which the Fc constant region of a monoclonal antibody derived from one species (e.g., mouse Fc constant region) is replaced with the Fc constant region of an antibody derived from another species (e.g., human Fc constant region) using recombinant DNA technology. In general, Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., WO86 / 01533; Cabilly et al. al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application No. 125,023; Better et al., Science 240: 1041-1043, 1988; Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443, 1987; Liu et al., J. Immunol 139: 3521-3526, 1987; Sun et al., Proc. See Natl. Acad. Sci. USA 84: 214-218, 1987; Nishimura et al., Cancer Res 47: 999-1005, 1987; Wood et al., Nature 314: 446-449, 1885; and Shaw et al., J. Natl. Cancer Inst. 80: 1553-1559, 1988. A "conformation-specific antibody" is an antibody or fragment thereof that recognizes and specifically binds to a specific conformation (e.g., a conformational isomer or conformational isomer) of its complementary antigen. For example, as described herein, a conformation-specific antibody may specifically bind to the cis-conformation of the Xaa-Pro motif (e.g., cis-pT231-tau), but not to the trans-conformation of the Xaa-Pro motif where Xaa is serine or threonine (e.g., trans-pT231-tau). In certain embodiments, the Ser / Thr-Pro motif may be phosphorylated (i.e., pSer / Thr-Pro). In this case, the conformation-specific antibody has, for example, at least 10 to 100 times greater affinity for the cis-conformation of the Xaa-Pro motif than for the trans-conformation.

[0022] In this specification, the term "consensus FR" refers to the framework (FR) antibody region in a consensus immunoglobulin sequence. The FR region of the antibody does not come into contact with the antigen. In this specification, the term “effective dose” or “therapeutably effective dose” of a composition means an amount sufficient to achieve the desired therapeutic effect, for example, an amount that results in a reduction of symptoms associated with the disease being treated, e.g., a disease or medical condition associated with the target polypeptide (e.g., tauopathy, traumatic brain injury (TBI), stroke). The amount of the composition of this technology administered to a subject varies depending on the type and severity of the disease, as well as individual characteristics, e.g., overall health, age, sex, weight, and tolerance to the drug. It varies depending on the degree, severity, and type of the disease. Those skilled in the art can determine an appropriate dose depending on these and other factors. The composition of this technology may also be administered in combination with one or more further therapeutic compounds. In some embodiments, the effective dose refers to the amount of the anti-cis-pT231-tau antibody of this technology that is partially or fully effective in neutralizing the activity of the cis-pT231-tau protein.

[0023] In this specification, the term “effector cell” means an immune cell involved in the effector phase of the immune response, as opposed to the cognitive and activation phases of the immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Effector cells express specific Fc receptors and possess specific immune functions. Effector cells can induce antibody-dependent cell-mediated cytotoxicity (ADCC), such as ADCC-inducible neutrophils. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes expressing FcαR are involved in the specific death of target cells, presenting antigens to other components of the immune system, or binding to antigen-presenting cells.

[0024] In this specification, the term “epitope” means a protein determinant that enables specific binding to an antibody. Epitopes typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural and charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former is lost in the presence of a denaturing solvent, while binding to the latter is not. In some embodiments, the “epitope” of the cis-conformational pT231-tau protein is a region of the protein to which the anti-cis-pT231-tau antibody of this technology specifically binds. To screen for anti-cis-pT231-tau antibodies that bind to an epitope, a standard cross-blocking assay, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), may be performed. This assay can be used to determine whether the anti-tau antibody binds to the same site or epitope as the anti-cis-pT231-tau antibody of this technology. Alternatively, epitope mapping may be performed by methods known in the art. For example, the antibody sequence may be mutated by alanine scanning or the like to identify contact residues. Alternatively, a peptide corresponding to a different region of the pT231-tau protein may be used in a competitive assay using a test antibody, or using a test antibody and an antibody having a characterized or known epitope. In this specification, “expression” includes, where necessary for proper expression and function, one or more of the following: transcription of a gene into precursor mRNA; splicing and other processing of precursor mRNA to produce mature mRNA; mRNA stability; translation of mature mRNA into protein (including codon use and tRNA utilization); and glycosylation and / or other modifications of the translation product.

[0025] In this specification, “increased expression” means an increase in gene or protein expression compared to a control or reference sample (e.g., an increase of at least 2 times, about 2 times to about 150 times, 5 times to 150 times, 5 times to 100 times, 10 times to 150 times, 10 times to 100 times, 50 times to 150 times, 50 times to 100 times, 75 times to 150 times, or 75 times to 100 times compared to a control or normal reference sample). “Decreased expression” means an overall decrease in gene or protein expression compared to a control or reference sample (e.g., 20% or more, 50% or more, or 75%, 80%, 85%, 90%, 95% or more). Increases or decreases in gene or protein expression can be determined using any useful method known in the art or described herein (e.g., ELISA). For therapeutic applications, "reduction" may refer to a reduction in the level of polypeptides or proteins associated with a disorder (e.g., tauopathy, TBI, or stroke). For diagnostic or monitoring applications, "reduction" refers to a reduction in the level of proteins or nucleic acids detected by a diagnostic or monitoring assay.

[0026] In this specification, the term “gene” means a segment of DNA containing all the information about the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other non-translational regions that control expression. In this specification, the term "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. For the most part, a humanized antibody is a human immunoglobulin in which the hypervariable region residues of the recipient are replaced by hypervariable region residues derived from a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having desired specificity, affinity, and capabilities. In some embodiments, the Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody may contain residues not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance, such as binding affinity. Generally, humanized antibodies contain substantially all of at least one, usually two, variable domains (e.g., Fab, Fab', F(ab')2, or Fv), all or substantially all of the hypervariable loops corresponding to those of non-human immunoglobulins, and all or substantially all of the FR region being from the human immunoglobulin consensus FR sequence, although the FR region may contain one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR is usually 6 or less in the H chain and 3 or less in the L chain. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), usually from human immunoglobulins. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0027] In this specification, the term "hypervariable region" refers to amino acid residues of an antibody involved in antigen binding. The hypervariable region is generally derived from amino acid residues of the "complementarity-determining region" or "CDR" (e.g., V L Approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3), as well as V HApproximately 31-35B(H1), 50-65(H2), and 95-102(H3) within (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues derived from the "hypervariable loop" (e.g., V L The residues within are 26-32 (L1), 50-52 (L2), and 91-96 (L3), as well as V H This includes 26-32(H1), 52A-55(H2), and 96-101(H3) (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0028] In this specification, the term “identical” or “identical” percentage is used in relation to two or more nucleic acid or polypeptide sequences (when compared and aligned to achieve maximum correspondence across a comparison window or specified region, or by manual alignment and visual inspection, such as as measured by the NCBI website), and whether the amino acid residues are identical or identical across a specified region (e.g., the nucleotide sequence encoding the antibody described herein or the amino acid sequence of the antibody described herein) when compared and aligned to achieve maximum correspondence across a comparison window or specified region. This refers to two or more sequences or subsequences having a specified percentage of nucleotides (i.e., approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity). Such sequences are then said to be "substantially identical." This term may also refer to or apply to the complement of a test sequence. The term also includes sequences with deletions and / or additions, as well as those with substitutions. In some embodiments, identity exists over a region having at least approximately 25 amino acids or nucleotide lengths or 50 to 100 amino acids or nucleotide lengths.

[0029] In this specification, the term "immunologically cross-reactive" means an antigen that reacts with an antibody produced using a different antigen. In this specification, the term “intact antibody” means an antibody having at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Each heavy chain has a heavy chain variable region (HCVR or V in this specification). H It consists of a heavy chain constant region (abbreviated as LCVR or V). The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (LCVR or V in this specification). L It consists of a (abbreviated as) and a light chain constant region. The light chain constant region is one domain, CL It consists of V. H and V L The region is called the Complementarity Determination Region (CDR), and can be further subdivided into hypervariable regions, which are dispersed into more conserved regions called the Framework Region (FR). H and V L It consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complementation system.

[0030] With respect to antigenic peptides, the term "mimicking" refers to a synthetic chemical compound having substantially identical structural and / or functional characteristics to the antigenic peptide KVAVVRTPPKSPS (SEQ ID NO: 56). Mimickings may consist entirely of synthetic non-natural analogs of amino acids, or they may be chimeric molecules of natural amino acids and non-natural analogs of amino acids. Mimickings may also incorporate any amount of conserved substitutions, provided that such substitutions substantially alter the structure or activity of the mimicking. In this specification, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies within the population are identical except for naturally occurring mutations that may exist in small amounts. For example, a monoclonal antibody may be derived from a single clone, including any eukaryotic cell, prokaryotic cell, or phage clone, and not from the method by which it is produced. Monoclonal antibody compositions exhibit single-binding specificity and affinity for a particular epitope. Monoclonal antibodies are highly specific and target a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically contain various antibodies against various determinants (epitopes), each monoclonal antibody targets a single determinant on an antigen. The modifying phrase “monoclonal” indicates an antibody characteristic such as being obtained from a substantially homogeneous population and should not be interpreted as requiring the production of the antibody by any particular method. Monoclonal antibodies may be prepared using a variety of techniques known in the art, including, but not limited to, hybridoma, recombinant, and phage display techniques. For example, the monoclonal antibodies to be used according to this method may be prepared by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). The “monoclonal antibodies” may also be isolated from a phage antibody library using, for example, the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0031] In this specification, “neurological disorder” means a disruption of the structure or function of the nervous system resulting from developmental abnormalities, disorders, injuries, or toxins, such as mild cognitive impairment (MCI), Parkinson’s disease (PD), traumatic brain injury, progressive supranuclear palsy, chronic traumatic encephalopathy (CTE), frontotemporal dementia, frontotemporal lobar degeneration, Ritiko-Bodig disease, Tangle-predominant dementia, meningeal hemangioma, subacute sclerosing panencephalitis, Pick’s disease, corticobasal degeneration, and Alzheimer’s disease. In this specification, the term “pharmaceutically acceptable carrier” is intended to include any solvent, dispersion medium, coating, antimicrobial and antifungal compound, isotonic and absorption-delaying compound, etc., that is compatible with pharmaceutically acceptable administration. pharmaceutically acceptable carriers and their formulations are known to those skilled in the art, for example, Remington's Pharmaceutical Sciences (20 th This is described in edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).

[0032] In this specification, the term “polyclonal antibody” means an antibody preparation derived from at least two (2) different antibody-producing cell lines. The use of this term includes an antibody preparation containing at least two (2) antibodies that specifically bind to different epitopes or regions of an antigen. In this specification, the terms “polynucleotide” or “nucleic acid” mean any RNA or DNA, which may be unmodified or modified RNA or DNA. Examples of polynucleotides include, but are not limited to, single-stranded and double-stranded DNA, DNA which is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA which is a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA which may be single-stranded or, more commonly, double-stranded or a mixture of single-stranded and double-stranded regions. Furthermore, a polynucleotide refers to a triple-stranded region containing RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases and DNA or RNA having a modified backbone for stability or other reasons.

[0033] In this specification, the terms “polypeptide,” “peptide,” and “protein” are used synonymously to mean polymers containing two or more amino acids linked to one another by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptides generally refer to both short chains called peptides, glycopeptides, or oligomers, and long chains generally called proteins. Polypeptides may contain amino acids other than those encoded by 20 genes. Polypeptides may contain amino acid sequences modified by natural processes such as post-translational processing, or by chemical modification techniques well known in the art. Such modifications are well described in basic textbooks and more detailed laboratories, as well as in a large amount of research literature.

[0034] Polypeptides are sometimes referred to as “isolated” or “substantially pure” if physical, mechanical, or chemical methods are used to remove them from cellular components. “Isolated polypeptides” (e.g., isolated antibodies), “substantially pure polypeptides,” or “substantially pure, isolated polypeptides” are typically substantially pure if they are removed from cellular components and contain at least 60% by mass of the proteins and naturally occurring organic molecules that naturally accompany them. Polypeptides can be at least 75% by mass, 80% by mass, 85% by mass, 90% by mass, 95% by mass, or 99% by mass pure. Substantially pure polypeptides (e.g., substantially pure antibodies or fragments thereof) can be obtained by standard techniques, for example, by the expression of recombinant nucleic acids encoding polypeptides, or by the chemical synthesis of polypeptides. Purity can be measured by any suitable method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0035] In this specification, the term “recombinant” means, for example, when used in relation to cells, nucleic acids, proteins, or vectors, that the cells, nucleic acids, proteins, or vectors have been modified by the introduction of heterologous nucleic acids or proteins or by alteration of native nucleic acids or proteins, or that the material originates from cells that have been thus modified. For example, recombinant cells express genes not found in the native (non-recombinant) form of the cell, or express native genes that would otherwise be abnormally expressed, underexpressed, or not expressed at all. In this specification, the term “separate” in therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients via different routes. In this specification, the term “sequential” therapeutic use refers to the administration of at least two active ingredients at different times, either via the same or different routes of administration. More specifically, sequential use refers to the total administration of one active ingredient prior to the commencement of the administration of another(s). Thus, it is possible to administer one active ingredient over several minutes, hours, or days, and then administer the other(s). In this case, there is no simultaneous treatment. In this specification, the term “simultaneous” therapeutic use means the administration of at least two active ingredients via the same route, either simultaneously or substantially simultaneously.

[0036] In this specification, the terms “subject,” “individual,” and “patient” are used synonymously and refer to humans or non-human animals, such as domesticated animals (e.g., dogs, cats, etc.), livestock (e.g., cattle, sheep, pigs, horses, etc.), wild animals (e.g., bats, raccoons, foxes, skunks, squirrels, chipmunks, mice, rabbits, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.). In this specification, the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a target that requires it. The amino acid sequence modifications of anti-cis-pT231-tau antibodies described herein are considered. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of anti-cis-pT231-tau antibodies are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions is performed to obtain the target antibody, as long as the resulting antibody has the desired properties. Modifications also include changes in the glycosylation pattern of the protein. The hypervariable region is the largest target site for substitutional mutagenesis, but FR changes are also considered. "Conservative substitutions" are shown in the table below.

[0037] [Table 1]

[0038] One of the substitutional variants involves substituting one or more hypervariable region residues of the parent antibody. A convenient method for producing such substitutional variants involves affinity maturation using phage display. Specifically, several hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino acid substitutions at each site. The antibody variant thus produced is presented in a monovalent manner from filamentous phage particles as a fusion to the M13 gene III product packaged within each particle. The variant presented to the phage is then screened for its biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis may be performed to identify hypervariable region residues that significantly contribute to antigen binding. Once such variants are produced, a panel of variants may be screened as described herein, and antibodies with similar or superior properties in one or more relevant assays may be selected for further development.

[0039] In this specification, “reference sample” refers to any sample used for comparison purposes. A “normal reference sample” may be a pre-sample taken from the same subject before the onset of a disorder (e.g., tauopathy, traumatic brain injury (TBI), or stroke), a sample obtained from a non-disordered subject that has been successfully treated for the disorder, or a sample of a purified reference polypeptide at a known normal concentration. The terms “reference standard” or “reference level” refer to the value, number, or expression pattern of one or more genes or proteins derived from a reference sample. A normal reference standard or level may also be a value, number, or expression pattern derived from a normal subject that corresponds to the subject's sample by at least one of the following criteria: age, weight, disease stage, and overall health. For example, a normal reference level for a disordered polypeptide or conformation of a disordered polypeptide might be less than 5 ng / ml in a serum sample, less than 4 ng / ml, less than 3 ng / ml, less than 2 ng / ml, or less than 1 ng / ml in a serum sample. A “positive reference” sample, standard, or value is a sample, standard, value, or number derived from a subject known to have a disorder (e.g., tauopathy, TBI, or stroke) that corresponds to the subject sample by at least one of the following criteria: age, weight, disease stage, and overall health. For example, a positive reference value for a polypeptide indicating a disorder might be greater than 5 ng / ml serum, greater than 10 ng / ml serum, greater than 20 ng / ml, greater than 30 ng / ml, greater than 40 ng / ml, or greater than 50 ng / ml serum.

[0040] In this specification, “specifically bind” means a molecule that recognizes and binds to another molecule (e.g., an antigen) but substantially does not recognize or bind to other molecules (e.g., an antibody). For example, an antibody that specifically binds to the cis-conformation of pT231-tau does not specifically bind to the trans-conformation of pT231-tau. In this specification, the terms “specifically bind,” “specifically bind to,” or “specific to” a particular molecule (e.g., a polypeptide, an epitope on a polypeptide, or a conformation of a polypeptide) are used, for example, at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 The K of the binding molecule is M or larger. d This can be indicated by a molecule having a specific binding property. The term "specifically binding" may also refer to a binding property where a molecule (e.g., an antibody) binds to a specific polypeptide (e.g., a polypeptide containing the Xaa-Pro motif (where Xaa is serine or threonine)), an epitope on a specific polypeptide, or a specific polypeptide conformation (e.g., a cis-conformation of the Xaa-Pro motif, e.g., cis-pT231-tau) and substantially does not bind to any other polypeptide, polypeptide epitope, or polypeptide conformation (e.g., a trans-conformation of the Xaa-Pro motif, e.g., trans-pT231-tau). For example, a conformation-specific antibody has, for example, an affinity (e.g., 10 to 100 times greater) for one conformation of the Ser / Thr-Pro motif (e.g., cis-conformation) than for another conformation (e.g., trans-conformation). 1 -, 10 2 -, 10 3 -, 10 4 -, 10 5 -, 10 6 -, 107 -, 10 8 -, 10 9 - or 10 10 It may have an affinity that is - times greater.

[0041] In this specification, “test sample” means a biological sample obtained from the subject of the study. For example, a test sample may be a biological fluid (e.g., serum), a cell or tissue sample, a sample obtained from a culture or growth medium, or isolated nucleic acids or polypeptides derived therefrom. "To treat" or "treatment" as used herein refers to the treatment of a disease or disorder described herein in an object such as a human, and includes (i) inhibiting the disease or disorder, i.e., stopping its occurrence; (ii) reducing the disease or disorder, i.e., causing regression of the disorder; (iii) slowing the progression of the disorder; and / or (iv) inhibiting, reducing, or slowing the progression of one or more symptoms of the disease or disorder. "To treat tauopathy, TBI or stroke" means that the symptoms associated with tauopathy, TBI or stroke are, for example, alleviated, reduced, cured or put into remission. It should be understood that the various forms of treatment for neurological disorders described herein include not only complete treatment but also less complete treatment, and mean "substantial" treatment in which several biological or medically relevant outcomes are achieved. Treatment may be a series of long-term treatments for chronic diseases or a single or two- or three-dose administration for acute conditions.

[0042] I. Composition of this technology This technology describes methods and compositions for the production and use of conformation-specific antibodies or fragments thereof. Conformation-specific antibodies can, for example, specifically bind to cis- or trans-conformations of specific polypeptides. In some embodiments, the conformation-specific antibodies of this technology bind to the cis-conformation of the phosphorylated Ser / Thr-Pro motif of a polypeptide (e.g., cis-pT231-tau). The binding of conformation-specific antibodies to their antigen (e.g., pT231-tau) may be useful in the treatment, diagnosis, or monitoring of the progression of neurological diseases accompanied by increased cis-pT231-tau protein expression.

[0043] Conformation-specific antibodies of this technology can be produced, for example, using immunogenic antigens containing an immobilized phosphorylated Xaa-Pro motif (where Xaa is serine or threonine) in a specific conformation (e.g., cis- or trans-conformation) or in a mixed cis- and trans-conformation. For example, the cis or trans content of the antigenic peptide of this technology containing phosphorylated Ser / Thr-Pro can be fixed by stereoselective synthesis of (Z)- and (E)-alkene mimes by the Still-Wittig and Ireland-Claisen rearrangement (incorporated herein by reference, J. Org. Chem. 68: 2343-2349 (2003)). Alternatively, the cis or trans content of the phosphorylated Ser / Thr-Pro-containing antigenic peptide of this technology can be increased or fixed by substituting proline amino acid residues with proline analogs. Examples of proline analogs, but not limited to, include homoproline, pipecolic acid (Pip), dimethylproline (Dmp), azetidine-2-carboxylic acid (Aze), t-butyl-L-proline (TBP), trans-4-fluoro-L-proline (t-4F-Pro), and cis-4-fluoro-L-proline (c-4F-Pro). The cis or trans content of a given antigen can be analyzed, for example, by nuclear magnetic resonance (NMR) analysis.

[0044] The antigenic peptide of this technology may contain a cis / trans isomerizable phosphorylated or unphosphorylated Xaa-Pro motif (wherein Xaa is serine or threonine). In some embodiments, the antigenic peptide may contain amino acid residues of the Ser / Thr-Pro motif of tau protein in which the proline residue is substituted with a proline analog. The antigenic peptide may also contain amino acid residues of the Ser / Thr-Pro motif of the full-length tau polypeptide. The antigenic peptide may further contain additional residues surrounding the Ser / Thr-Pro motif of the full-length tau polypeptide. For example, the antigenic peptide may contain 3 to 10 amino acid residues at the N-terminus of the Ser / Thr residue of the full-length tau polypeptide and 3 to 10 amino acid residues at the C-terminus of the proline of the full-length tau polypeptide. The antigenic peptide of this technology may be, for example, at least 4, 5, 6, 7, or 8 amino acid residues in length. Antigenic peptides can be as long as 8 to 20 amino acid residues (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues), but may also be longer than 20 amino acid residues.

[0045] Such antigens can be produced and purified by any of the various methods known to those skilled in the art. Antigenic peptides can be produced and purified, for example, by solid-phase chemical synthesis, in vitro transcription / translation, or by recombinant techniques. Antigenic peptides may be chemically coupled to a carrier protein, or the peptides may be prepared as fusion proteins to enhance antigenicity. Antigenic peptides can be screened based on their ability to induce the production of conformation-specific antibodies. In this regard, such screening techniques include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), immunoprecipitation, or other immunoassays. Exemplary antigens useful in the production of conformation-specific antibodies include antigens containing phosphorylated or unphosphorylated Ser / Thr-homoproline, Ser / Thr-Pip, Ser / Thr-Dmp, Ser / Thr-Aze, Ser / Thr-TBP, Ser / Thr-t-4F-Pro, or Ser / Thr-c-4F-Pro motifs. Specific examples of such antigens include, for example, the pT231-Pip tau peptide and the pT231-Dmp tau peptide (KVAVVR-pT231-Pro-PKSPS). Such peptides can be used, for example, as antigens for producing polyclonal or monoclonal antibodies (e.g., rabbit or mouse monoclonal antibodies).

[0046] In one embodiment, the present disclosure provides an isolated antibody containing a heavy chain immunoglobulin variable domain, wherein the heavy chain immunoglobulin variable domain has the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRATLTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVSS(Sequence ID 1) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVSS(Sequence ID 2) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRATMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVSS(Sequence ID 3); QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVSS(Sequence ID 4); or a variant thereof having one or more conservative amino acid substitutions.

[0047] In another embodiment, the Disclosure provides an isolated antibody comprising a heavy chain immunoglobulin variable domain, the heavy chain immunoglobulin variable domain comprising a signal sequence and a heavy chain immunoglobulin variable domain amino acid sequence as described herein. For example, but not limited to, in some embodiments, the signal sequence is MRWSCIILFLVATATGVNS (SEQ ID NO: 5) or MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 6).

[0048] For example, but not limited to, in some embodiments, the isolated antibody includes a signal sequence (underlined) and an amino acid sequence selected from the following: MRWSCIILFLVATATGVNS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRATLTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVSS(Sequence ID 7); MRWSCIILFLVATATGVNS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVSS(Sequence ID 8); MRWSCIILFLVATATGVNS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRATMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVSS(Sequence ID 9); MRWSCIILFLVATATGVNS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVSS(Sequence ID 10);

[0049] MEWSWVFLFFLSVTTGVHS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRATLTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVSS(Sequence ID 11); MEWSWVFLFFLSVTTGVHS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVSS(Sequence ID 12); MEWSWVFLFFLSVTTGVHS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRATMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVSS(Sequence ID 13); MEWSWVFLFFLSVTTGVHS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVSS(Sequence ID 14); or comprising a heavy chain immunoglobulin variable domain having a variant thereof having one or more conservative amino acid substitutions.

[0050] In another embodiment, the present disclosure provides isolated antibodies comprising heavy chain immunoglobulin variable domain amino acid sequences and heavy chain immunoglobulin constant domains as described herein. For example, but not limited to, in some embodiments, the heavy chain immunoglobulin constant domain is an amino acid sequence selected from the group consisting of: PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence No. 15); and PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (Sequence ID 16) It holds.

[0051] For example, but not limited to, in some embodiments, the separated antibody containing a single-chain immunoglobulin variable domain and a heavy-chain immunoglobulin constant domain (bold font) has an amino acid sequence selected from the following: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRATLTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence No. 17); QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRATLTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(Sequence ID 18);

[0052] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence No. 19);

[0053] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(Sequence ID 20);

[0054] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRATMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence No. 21); QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRATMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(Sequence ID 22);

[0055] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence No. 23);

[0056] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(Sequence code 24); or a variant thereof having one or more conservative amino acid substitutions.

[0057] In another embodiment, the Disclosure provides an isolated antibody comprising a heavy immunoglobulin variable domain, the heavy immunoglobulin variable domain comprising a signal sequence, a heavy immunoglobulin variable domain amino acid sequence, and a heavy immunoglobulin constant domain. For example, but not limited to, in some embodiments, the isolated antibody comprises a heavy immunoglobulin variable domain, the heavy immunoglobulin variable domain comprising a signal sequence, the heavy immunoglobulin variable domain amino acid sequence, and the heavy immunoglobulin constant domain comprising an amino acid sequence selected from the following (the signal sequence is underlined and the heavy immunoglobulin constant domain is in bold): MRWSCIILFLVATATGVNS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRATLTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence No. 25);

[0058] MRWSCIILFLVATATGVNSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRATLTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(Sequence code 26);

[0059] MRWSCIILFLVATATGVNS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence No. 27);

[0060] MRWSCIILFLVATATGVNS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(Sequence code 28); MRWSCIILFLVATATGVNS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRATMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence No. 29);

[0061] MRWSCIILFLVATATGVNSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRATMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(Sequence ID 30);

[0062] MRWSCIILFLVATATGVNS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence No. 31);

[0063] MRWSCIILFLVATATGVNS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(Sequence ID 32);

[0064] MEWSWVFLFFLSVTTGVHS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRATLTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 33);

[0065] MEWSWVFLFFLSVTTGVHS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRATLTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(Sequence ID 34);

[0066] MEWSWVFLFFLSVTTGVHS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 35); MEWSWVFLFFLSVTTGVHS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(Sequence ID 36);

[0067] MEWSWVFLFFLSVTTGVHS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRATMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence No. 37);

[0068] MEWSWVFLFFLSVTTGVHS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRATMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(Sequence ID 38);

[0069] MEWSWVFLFFLSVTTGVHS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence No. 39); MEWSWVFLFFLSVTTGVHS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(Sequence ID 40);

[0070] MRWSCIILFLVATATGVNS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 132)

[0071] MEWSWVFLFFLSVTTGVHS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIHWVRQAPGQGLEWIGVIDPSDSYTRYNQKFKGRVTMTVDTSTSTAYMELRSLRSDDTAVYYCTTWEVDYWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 133) or a variant thereof having one or more conservative amino acid substitutions.

[0072] Furthermore, or in some embodiments, the isolated antibody of the present technology contains a light chain immunoglobulin variable domain, the light chain immunoglobulin variable domain having the following amino acid sequence: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSDGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYFCSQSTHVPWTFGGGTKLEIK (Sequence ID 41), DIVMTQSPLSLPVTPGEPASISCRSSQSLVHSDGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRT (Sequence ID 42), or a variant thereof having one or more conservative amino acid substitutions. Furthermore, or in another embodiment, the present disclosure provides an isolated antibody comprising a light chain immunoglobulin variable domain, the light chain immunoglobulin variable domain comprising a signal sequence and a light chain immunoglobulin variable domain amino acid sequence. For example, but not limited to, in some embodiments, the signal sequence is MKLPVRLLVLMFWIPASNS (SEQ ID NO: 43) or MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 44).

[0073] For example, but not limited to, in some embodiments, the isolated antibody includes a signal sequence and an amino acid sequence selected from the following (signal sequences are underlined): MKLPVRLLVLMFWIPASNS DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSDGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYFCSQSTHVPWTFGGGTKLEIK(Sequence ID 45); MSVPTQVLGLLLLWLTDARC DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSDGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYFCSQSTHVPWTFGGGTKLEIK(Sequence ID 46); MKLPVRLLVLMFWIPASNS DIVMTQSPLSLPVTPGEPASISCRSSQSLVHSDGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRT(Sequence ID 47); MSVPTQVLGLLLLWLTDARC DIVMTQSPLSLPVTPGEPASISCRSSQSLVHSDGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRT(Sequence ID 48); or comprising a light chain immunoglobulin variable domain having a variant thereof having one or more conservative amino acid substitutions.

[0074] Furthermore, or in other embodiments, the present disclosure provides isolated antibodies comprising a light chain immunoglobulin variable domain and a light chain immunoglobulin constant domain. For example, but not limited to, in some embodiments, the light chain immunoglobulin constant domain is VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 49) It has the following amino acid sequence.

[0075] For example, but not limited to, in some embodiments, isolated antibodies containing a light chain immunoglobulin variable domain and a light chain immunoglobulin constant domain are selected from the following amino acid sequences (the light chain immunoglobulin constant domain is in bold): DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSDGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYFCSQSTHVPWTFGGGTKLEIKVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 50) DIVMTQSPLSLPVTPGEPASISCRSSQSLVHSDGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 51); or a variant thereof having one or more conservative amino acid substitutions.

[0076] Furthermore, or in another embodiment, the present disclosure provides an isolated antibody comprising a light chain immunoglobulin variable domain, the light chain immunoglobulin variable domain comprising a signal sequence, a light chain immunoglobulin variable domain amino acid sequence, and a light chain immunoglobulin constant domain. For example, but not limited to, in some embodiments, the isolated antibody comprises a light chain immunoglobulin variable domain, the light chain immunoglobulin variable domain comprising a signal sequence, a light chain immunoglobulin variable domain amino acid sequence, and the light chain immunoglobulin constant domain comprising an amino acid sequence selected from the following (the signal sequence is underlined and the light chain immunoglobulin constant domain is bold): MKLPVRLLVLMFWIPASNS DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSDGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYFCSQSTHVPWTFGGGTKLEIKVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 52); MSVPTQVLGLLLLWLTDARC DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSDGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYFCSQSTHVPWTFGGGTKLEIKVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 53);

[0077] MKLPVRLLVLMFWIPASNS DIVMTQSPLSLPVTPGEPASISCRSSQSLVHSDGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 54); MSVPTQVLGLLLLWLTDARC DIVMTQSPLSLPVTPGEPASISCRSSQSLVHSDGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 55); or a variant thereof having one or more conservative amino acid substitutions.

[0078] In some embodiments, the isolated antibody of the technology comprises at least one heavy chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4, 7-14, 17-40, and 132-133. In some embodiments, the isolated antibody of the technology comprises at least one light chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-42, 45-48, 50-54, and 55. In some embodiments, the isolated antibody of the technology comprises at least one heavy chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4, 7-14, 17-40, and 132-133, and at least one light chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-42, 45-48, 50-54, and 55. In some embodiments, the heavy and light chain immunoglobulin variable domain sequences form antigen-binding sites that bind to epitopes of the human cis-pT231-tau protein, including the amino acid sequence KVAVVRTPPKSPS (SEQ ID NO: 56) or its mimicry.

[0079] In some embodiments of the antibody, the heavy chain and light chain immunoglobulin variable domain sequences are components of the same polypeptide chain. In some embodiments of the antibody, the heavy chain and light chain immunoglobulin variable domain sequences are components of different polypeptide chains. In certain embodiments, the antibody is a full-length antibody. In another aspect, the present disclosure provides antigen-binding fragments of antibodies disclosed herein, wherein the antigen-binding fragments are Fab, F(ab)'2, Fab', scF v and F v It is selected from the group consisting of the following. In some embodiments, the anti-cis-pT231-tau antibody of the present technology specifically binds to the cis-pT231-tau protein. In some embodiments, the antibody of the present technology binds to the cis-pT231-tau protein. -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 Dissociation constant less than M (K D The antibody is bound via ). In certain embodiments, the antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody. In some embodiments, the antibody includes a human antibody framework region.

[0080] Anti-cis-pT231-tau antibodies within the scope of this technology include, but are not limited to, monoclonal, chimeric, humanized, and diabolic antibodies that specifically bind to target polypeptides, their homologs, derivatives, or fragments. Antibodies useful for the methods disclosed herein may include Fc domains of any isotype, for example, but are not limited to, IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM and IgY. Examples of constant region sequences, not limited to these, include:

[0081] Human IgD constant region, Uniprot:P01880 SEQ ID NO:134 APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFV VGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK Human IgG1 constant region, Uniprot:P01857 SEQ ID NO:135 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG2 constant region, Uniprot:P01859 SEQ ID NO:136 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0082] Human IgG3 constant region, Uniprot:P01860 SEQ ID NO:137 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0083] Human IgM constant region, Uniprot:P01871 Sequence ID: 138 GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKV SVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFA IPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPARE QLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0084] Human IgG4 constant region, Uniprot:P01861 SEQ ID NO:139 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Human IgA1 constant region, Uniprot:P01876 SEQ ID NO:140 ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPE RDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

[0085] Human IgA2 constant region, Uniprot:P01877 SEQ ID NO:141 ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCY SVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY Human Ig kappa constant region, Uniprot:P01834 SEQ ID NO:142 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC In some embodiments, the anti-cis-pT231-tau antibody of the present technology includes a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NOs. 134-140 or 141. Furthermore, in some embodiments, the anti-cis-pT231-tau antibody of the present technology includes a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NOs. 142.

[0086] The heavy chain (HC) and light chain (LC) immunoglobulin variable domain sequences of the HuPT-113A, HuPT-113B, HuPT-113C, and HuPT-113D antibodies (including the leader sequence) are provided below: [Table 2]

[0087] The heavy chain (HC) and light chain (LC) immunoglobulin sequences of the HuPT-113D IgG1-AA(II), HuPT-113D IgG4(II), and HuPT-113D IgG1(II) antibodies are provided below. [Table 3]

[0088] In some embodiments of the antibody, the HC variable domain sequence includes the variable domain sequence of HuPT-113A, and the LC variable domain sequence includes the variable domain sequence of HuPT-113A. In certain embodiments, the antibody further includes an HC constant region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. In certain embodiments, the antibody includes an IgG1 constant region containing an amino acid substitution from Leu to Ala at positions 234 and 235. In some embodiments of the antibody, the HC variable domain sequence includes the variable domain sequence of HuPT-113B, and the LC variable domain sequence includes the variable domain sequence of HuPT-113B. In certain embodiments, the antibody further includes an HC constant region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. In certain embodiments, the antibody includes an IgG1 constant region containing an amino acid substitution from Leu to Ala at positions 234 and 235.

[0089] In some embodiments of the antibody, the HC variable domain sequence includes the variable domain sequence of HuPT-113C, and the LC variable domain sequence includes the variable domain sequence of HuPT-113C. In certain embodiments, the antibody further includes an HC constant region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. In certain embodiments, the antibody includes an IgG1 constant region containing an amino acid substitution from Leu to Ala at positions 234 and 235. In some embodiments of the antibody, the HC variable domain sequence includes the variable domain sequence of HuPT-113D, and the LC variable domain sequence includes the variable domain sequence of HuPT-113D. In certain embodiments, the antibody further includes an HC constant region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. In certain embodiments, the antibody includes an IgG1 constant region containing an amino acid substitution from Leu to Ala at positions 234 and 235.

[0090] In certain embodiments, the isolated antibody comprises one or more of the following features: (a) the light chain immunoglobulin variable domain sequence is at least 85% identical to SEQ ID NO: 45 and / or (b) the heavy chain immunoglobulin variable domain sequence is at least 85% identical to any one of SEQ ID NOs: 7-10. In another embodiment, one or more amino acid residues in the antibody provided herein are substituted with other amino acids. The substitution may be a “conservative substitution” as defined herein. In some embodiments of the antibodies provided herein, the antibodies contain structural modifications to promote rapid binding and cellular uptake and / or sustained release. In some embodiments, the cis-pT231-tau antibody contains a deletion in the CH2 constant antibody heavy chain region of the antibody to promote rapid binding and cellular uptake and / or sustained release. In some embodiments, a Fab fragment is used to promote rapid binding and cellular uptake and / or sustained release. In some embodiments, an F(ab)'2 fragment is used to promote rapid binding and cellular uptake and / or sustained release. In some embodiments, the Technology provides a nucleic acid sequence or fragment thereof encoding the cis-pT231-tau neutralizing antibody of the Technology. In some embodiments, the Technology provides a host cell expressing the nucleic acid sequence encoding the cis-pT231-tau neutralizing antibody of the Technology.

[0091] Antibodies in this technology may be monospecific, bispecific, triplicate, or more. Multispecific antibodies may be specific to different epitopes of the cis-pT231-tau protein in this technology, or they may be specific to both the cis-pT231-tau protein and heterogeneous compositions, such as heterogeneous polypeptides or solid support materials. See, for example, WO93 / 17715, WO92 / 08802, WO91 / 00360, WO92 / 05793, Tutt et al., J. Immunol. 147: 60-69 (1991), U.S. Patent Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648, and 6,106,835, and Kostelny et al., J. Immunol. 148: 1547-1553 (1992). In some embodiments, the antibody is a chimeric form. In certain embodiments, the antibody is humanized. The antibodies of this technology may be further recombinantly fused to a heterologous polypeptide at the N-terminus or C-terminus, or chemically conjugated to a polypeptide or other composition (including covalent and non-covalent conjugation). For example, the antibodies of this technology may be recombinantly fused to or conjugated to molecules and effector molecules useful as labels in detection assays, such as heterologous polypeptides, drugs, or toxins. See, for example, WO92 / 08495, WO91 / 14438, WO89 / 12624, U.S. Patent No. 5,314,995, and EP0396387.

[0092] A. Method for preparing an anti-cis-pT231-tau antibody using this technology. General Overview. First, a target polypeptide from which antibodies of this technology can be produced is selected. For example, antibodies can be produced against full-length tau protein or against a portion of tau protein containing phosphorylated threonine at position 231 (pT231). Techniques for producing antibodies against such target polypeptides are well known to those skilled in the art. Examples of such techniques include, but are not limited to, display libraries, heterologous or human mice, hybridomas, etc. Target polypeptides within the scope of this technology include any polypeptide derived from tau protein containing pT231 that can induce an immune response. The preparation of antibodies specific to cis-pT231-tau protein is illustrated in Examples 2-8. It should be understood that recombinantly genetically engineered antibodies and antibody fragments, such as antibody-related polypeptides, directed at the cis-pT231-tau protein and its fragments are suitable for use in accordance with this disclosure.

[0093] Anti-cis-pT231-tau antibodies that can be applied to the techniques described herein include monoclonal and polyclonal antibodies and antibody fragments, e.g., Fab, Fab', F(ab')2, Fd, scFv, diabodies, antibody light chains, antibody heavy chains, and / or antibody fragments. Methods useful for high-yield production of antibody Fv-containing polypeptides, e.g., Fab' and F(ab')2 antibody fragments, are described. See U.S. Patent No. 5,648,237. Generally, antibodies are obtained from the original species. More specifically, the nucleic acid or amino acid sequence of the light chain, heavy chain, or variable portion of the original species antibody that is specific to the target polypeptide antigen is obtained. The original species is any species that has been useful in producing the antibody or antibody library of this technology, such as rats, mice, rabbits, chickens, monkeys, or humans. Phage or phagemid display techniques are useful for deriving antibodies for this technology. Techniques for producing and cloning monoclonal antibodies are well known to those skilled in the art. Expression of sequences encoding antibodies for this technology can be carried out in Escherichia coli (E. coli).

[0094] For the degeneracy of the sequence-encoding nucleic acid, other sequences encoding amino acid sequences substantially identical to those of naturally occurring proteins may be used in the implementation of this technique. These include, but are not limited to, nucleic acid sequences containing all or part of the nucleic acid sequences encoding the polypeptides described above, which are modified by substitutions of different codons encoding functionally equivalent amino acid residues within the sequence, thus resulting in silent changes. It is understood that the nucleotide sequences of the immunoglobulins in this technique may tolerate up to 25% sequence homology variation, as calculated by standard methods ("Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1998, Alan R. Liss, Inc.), as long as such variants form activating antibodies that recognize the cis-pT231-tau protein. For example, one or more amino acid residues within the polypeptide sequence may be substituted with other amino acids of similar polarity that act as functional equivalents, resulting in silent changes. Amino acid substitutions within the sequence may be selected from other members of the class to which the amino acids belong. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar natural amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Proteins or fragments or derivatives that are modified differently during or after translation, such as by glycosylation, cleavage by proteolysis, or linkage with antibody molecules or other cellular ligands, are also included within the scope of this technology.Furthermore, nucleic acid sequences encoding immunoglobulins can be mutated in vitro or in vivo to further promote in vitro modifications, such as creating and / or disrupting translation, start and / or stop sequences, thereby creating variations in the coding region, and / or forming novel restriction endonuclease sites, or disrupting existing ones. However, any mutagenesis techniques known in the art, including but not limited to in vitro site-directed mutagenesis, J. Biol. Chem. 253:6551, use of Tab linkers (Pharmacia), etc., may be used.

[0095] Preparation of polyclonal antisera and immunogens. The method for producing antibodies or antibody fragments of this technique involves immunizing a subject (generally a non-human subject such as a mouse or rabbit) with purified cis-pT231-tau protein or a fragment thereof, or with cells expressing cis-pT231-tau protein or a fragment thereof. Suitable immunogenic preparations may contain recombinantly expressed cis-pT231-tau protein or chemically synthesized cis-pT231-tau peptides. The cis-conformation of pT231-tau protein or a portion or fragment thereof may be used as an immunogen for producing anti-cis-pT231-tau antibodies that conjugate to cis-pT231-tau protein or a portion or fragment thereof using standard techniques for polyclonal and monoclonal antibody preparation.

[0096] The full-length pT231-tau protein or its fragments are useful as immunogen fragments. In some embodiments, the pT231-tau fragment contains at least eight amino acid residues of the amino acid sequence KVAVVRTPPKSPS (SEQ ID NO: 56) or its mimicry, encompassing the epitope of the pT231-tau protein, and as a result, antibodies produced against the peptide form specific immune complexes with the cis-pT231-tau protein. In some embodiments, the antigenic tau peptide containing pT231 comprises at least 5, 8, 10, 15, 20, or 30 amino acid residues. Longer antigenic peptides are sometimes preferable to shorter ones, depending on the application, according to methods well known to those skilled in the art. A polymer of a given epitope is sometimes more effective than a monomer.

[0097] If necessary, the immunogenicity of the cis-pT231-tau protein (or fragment thereof) may be increased by fusion or conjugation with a hapten such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Numerous such haptens are known in the art. To enhance the target immune response against the polypeptide, the cis-pT231-tau protein may be combined with a conventional adjuvant, such as Freund's complete or incomplete adjuvant. Various adjuvants used to enhance immunological responses include, but are not limited to, Freund's (complete and imperfect), inorganic gels (e.g., aluminum hydroxide), surfactants (e.g., lysolecithin, pluronic polyols, polyanions, peptides or oil emulsions, dinitrophenol, etc.), human adjuvants, such as Calmette-Guérin bacilli and Corynebacterium parvum or similar immunostimulatory compounds. These techniques are standard in the art.

[0098] In this description of the technology, immune responses may be described as “primary” or “secondary” immune responses. A primary immune response, also described as a “protective” immune response, refers to an immune response that occurs in an individual as a result of several initial exposures (e.g., initial “immunotherapy”) to a particular antigen, such as the cis-pT231-tau protein. In some embodiments, immunization may occur as a result of vaccinating an individual with a vaccine containing the antigen. For example, the vaccine may be a cis-pT231-tau vaccine containing one or more antigens derived from the cis-pT231-tau protein. A primary immune response may become attenuated or weakened over time, may even disappear, or may become attenuated to the point of being undetectable. Thus, the technology also relates to a “secondary” immune response, also described herein as a “memory immune response.” The term secondary immune response refers to an immune response that is induced in an individual after a primary immune response has already occurred. Therefore, a secondary immune response may be induced to enhance an existing immune response that has become weakened or attenuated, or to reconstruct a previous immune response that has disappeared or is no longer detectable. A secondary or memory immune response can be either a humoral (antibody) response or a cellular response. A secondary or memory humoral response occurs in response to the stimulation of memory B cells that were produced by the initial presentation of the antigen. A delayed-type hypersensitivity (DTH) response is CD4 + DTH is a type of cellular secondary or memory immune response mediated by T cells. Initial exposure to an antigen stimulates the immune system, and further exposure leads to DTH.

[0099] After appropriate immunotherapy, anti-cis-pT231-tau antibodies can be prepared from the serum of the subject. If necessary, antibody molecules directed against the cis-pT231-tau protein can be isolated from mammals (e.g., from blood) by known techniques, such as polypeptide A chromatography, to obtain the IgG fraction, and further purified. Monoclonal antibody. In one embodiment of this technology, the antibody is an anti-cis-pT231-tau monoclonal antibody. For example, in some embodiments, the anti-cis-pT231-tau monoclonal antibody may be a human or mouse anti-cis-pT231-tau monoclonal antibody. Any technique that provides the production of antibody molecules by serial cell line culture may be used for the preparation of monoclonal antibodies against the cis-conformation or derivatives, fragments, analogs or homologs of the pT231-tau protein. Examples of such technologies, though not limited to these, include hybridoma technology (see, e.g., Kohler & Milstein, 1975. Nature 256: 495-497), trioma technology, human B-cell hybridoma technology (see, e.g., Kozbor, et al., 1983. Immunol. Today 4: 72), and EBV hybridoma technology for producing human monoclonal antibodies (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). In implementing this technology, human monoclonal antibodies may be used and may be manufactured using human hybridomas (see, e.g., Cote, et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by transforming human B cells with Epstein-Barr virus in vitro (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). For example, a population of nucleic acids encoding a region of the antibody may be isolated. PCR is used to amplify the sequence encoding a portion of the antibody from the population, using primers derived from the sequence encoding a conserved region of the antibody, and then the antibody or a fragment thereof, for example, DNA encoding a variable domain, is reconstructed from the amplified sequence.Such amplified sequences can also be fused with DNA encoding other proteins, such as bacteriophage coats or bacterial cell surface proteins, for expression and display of fusion polypeptides in phages or bacteria. The amplified sequences are then expressed and may be further selected or isolated based on the affinity of the expressed antibody or fragment against an antigen or epitope present on the cis-conformation of the pT231-tau protein, for example. Alternatively, hybridomas expressing anti-cis-pT231-tau monoclonal antibodies may be prepared by immunizing a subject and then isolating the hybridoma from the subject's spleen using routine methods. See, for example, Milstein et al., (Galfre and Milstein, Methods Enzymol (1981) 73: 3-46). Screening hybridomas using standard methods yields monoclonal antibodies with varying specificity (i.e., against various epitopes) and affinity. A selected monoclonal antibody having a desired property, such as a cis-pT231-tau linkage, may be used as if expressed by a hybridoma, or it may be conjugated to a molecule such as polyethylene glycol (PEG) to modify its properties, or the cDNA encoding it may be isolated, sequenced, or manipulated in various ways. To enhance the immunogenicity of the cis-pT231-tau protein, a synthetic dendromer tree may be attached to a reactive amino acid side chain, such as lysine. CPG-dinucleotide technology may also be used to enhance the immunogenicity of the cis-pT231-tau protein. Other manipulations include substituting or deleting specific aminoacyl residues that contribute to antibody instability during storage or after administration to a subject, and affinity maturation techniques to improve the affinity of the antibody to the cis-pT231-tau protein.

[0100] Hybridoma technology. In some embodiments, the antibodies of this technology are anti-cis-pT231-tau monoclonal antibodies produced by hybridomas containing B cells obtained from transgenic non-human animals, such as transgenic mice, which have a genome containing human heavy chain and light chain transgenes fused to immortalized cells. Hybridoma technology includes those known in the art, as taught in Harlow et al., Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988); and Hammerling et al., Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981). Other methods for producing hybridomas and monoclonal antibodies are well known to those skilled in the art.

[0101] Phage display technology. As described above, antibodies of this technology can be produced by applying recombinant DNA and phage display technology. For example, anti-cis-pT231-tau antibodies can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that hold the encoding polynucleotide sequence. Phages with desired binding properties are selected from a repertoire or combinatorial antibody library (e.g., human or mouse) by direct selection using antigens, usually bound to or captured on a solid surface or beads. The phages used in these methods are typically filamentous phages containing fd and M13 having Fab, Fv, or disulfide-stabilized Fv antibody domains recombinantly fused to either phage gene III or gene VIII protein. Furthermore, the method can be applied to construct a Fab expression library to enable the rapid and effective identification of monoclonal Fab fragments having desired specificity for cis-pT231-tau polypeptides, such as polypeptides or their derivatives, fragments, analogs, or homologs (see, for example, Huse, et al., Science 246: 1275-1281, 1989).Other examples of phage display methods that can be used to produce antibodies using this technology include: Huston et al., Proc. Natl. Acad. Sci USA, 85: 5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci USA, 87: 1066-1070, 1990; Brinkman et al., J. Immunol. Methods 182: 41-50, 1995; Ames et al., J. Immunol. Methods 184: 177-186, 1995; Kettleborough et al., Eur. J. Immunol. 24: 952-958, 1994; Persic et al., Gene 187: 9-18, 1997; Burton et al., Advances in Immunology 57: 191-280, 1994, PCT / GB91 / 01134, WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, WO96 / 06213, WO92 / 01047 (Medical Research Council Examples include those disclosed in (al.), WO97 / 08320 (Morphosys), WO92 / 01047 (CAT / MRC), WO91 / 17271 (Affymax), and U.S. Patents No. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, and 5,733,743. A useful method for displaying polypeptides on the surface of bacteriophage particles by attracting them via disulfide bonds is described by Lohning, U.S. Patent No. 6,753,136.As described in the references above, after phage selection, the antibody coding region obtained from the phage is isolated and used to produce a whole antibody containing human antibodies or any other desired antigen-binding fragment, which can be expressed in any desired host, including any mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques for recombinant production of Fab, Fab', and F(ab')2 fragments can also be used using methods known in the art, such as those disclosed in WO92 / 22324; Mullinax et al., BioTechniques 12: 864-869, 1992; and Sawai et al., AJRI 34: 26-34, 1995; and Better et al., Science 240: 1041-1043, 1988.

[0102] Generally, hybrid antibodies or hybrid antibody fragments cloned into display vectors can be selected for suitable antigens to identify variants that maintain good binding activity, because the antibodies or antibody fragments reside on the surface of phage or phagemid particles. See, for example, Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). However, other vector formats, such as cloning antibody fragment libraries into soluble phage vectors (modified T7 or lambda Zap systems), can be used in this process for selection and / or screening.

[0103] Expression of Recombinant Anti-Cis-pT231-Tau Antibodies. As described above, the antibodies of this technology can be produced by the application of recombinant DNA technology. The recombinant polynucleotide construct encoding the anti-cis-pT231-Tau antibody of this technology typically includes an expression control sequence operably ligated to the coding sequence of the anti-cis-pT231-Tau antibody chain, which usually includes naturally occurring or heterologous promoter regions. As such, another aspect of this technology includes a vector containing one or more nucleic acid sequences encoding the anti-cis-pT231-Tau antibody of this technology. For recombinant expression of one or more polypeptides of this technology, the nucleic acid containing all or part of the nucleotide sequences encoding the anti-cis-pT231-Tau antibody is inserted into a suitable cloning vector or expression vector (i.e., a vector containing the elements necessary for the transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA technology, which is well known in the art and is detailed below. Methods for producing diverse populations of vectors are described by Lerner et al., U.S. Patents 6,291,160 and 6,680,192.

[0104] Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In this disclosure, “plasmid” and “vector” may be used synonymously, as plasmids are the most common form of vectors. However, this technology is intended to include other forms of expression vectors that are not technically plasmids but perform equivalent functions, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses). Such viral vectors enable infection of a target and expression of a construct in that target. In some embodiments, the expression regulatory sequence is a eukaryotic cell promoter system in a vector that can transform or transfect eukaryotic cell host cells. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of a nucleotide sequence encoding an anti-cis-pT231-tau antibody, as well as for the collection and purification of the anti-cis-pT231-tau antibody, e.g., a cross-reactive anti-cis-pT231-tau antibody. For general information, see U.S. Patent Application Publication 2002 / 0199213. These expression vectors are typically replicable in host organisms either as episomes or as an integral part of host chromosomal DNA. Generally, expression vectors contain a selection marker, such as ampicillin resistance or hygromycin resistance, to enable the detection of cells transformed with the desired DNA sequence. The vector may also encode a signal peptide, such as pectinate lyase, useful for directing the secretion of extracellular antibody fragments. See U.S. Patent No. 5,576,195.

[0105] The recombinant expression vector of this technology contains a nucleic acid encoding a protein having cis-pT231-tau binding properties in a form suitable for nucleic acid expression in host cells, meaning that the recombinant expression vector contains one or more regulatory sequences selected based on the host cell to be used for expression, which are operably ligated to the nucleic acid sequence to be expressed. Within the recombinant expression vector, "operably ligated" means that the nucleotide sequence of interest is ligated to the regulatory sequence in a manner that enables the expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system, or in the host cell if the vector is introduced into a host cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct the constitutive expression of nucleotide sequences in many types of host cells and those that direct the expression of nucleotide sequences only in specific host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will understand that the design of expression vectors can vary depending on factors such as the selection of host cells to be transformed and the desired level of polypeptide expression. Common regulatory sequences useful as promoters for recombinant polypeptide expression (e.g., anti-cis-pT231-tau antibody) include, but are not limited to, promoters of 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters derived from alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization. In one embodiment, the polynucleotide encoding the anti-cis-pT231-tau antibody of this technology is operably ligated to the ara B promoter and expressible in host cells. See U.S. Patent No. 5,028,530.The expression vector of this technology can be introduced into host cells to produce polypeptides or peptides (e.g., anti-cis-pT231-tau antibodies) containing fusion polypeptides encoded by nucleic acids as described herein.

[0106] Another aspect of this technology relates to host cells expressing anti-cis-pT231-tau antibodies, comprising nucleic acids encoding one or more anti-cis-pT231-tau antibodies. Recombinant expression vectors of this technology may be designed for the expression of anti-cis-pT231-tau antibodies in prokaryotic or eukaryotic cells. For example, anti-cis-pT231-tau antibodies may be expressed in Escherichia coli, insect cells (using baculovirus expression vectors), fungal cells, and bacterial cells such as yeast, yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, recombinant expression vectors may be transcribed and translated using, for example, a T7 promoter regulatory sequence and T7 polymerase. Methods useful for the preparation and screening of polypeptides with specific properties, such as anti-cis-pT231-tau antibodies, by probabilistically generated polynucleotide sequence expression have been described. See U.S. Patents 5,763,192, 5,723,323, 5,814,476, 5,817,483, 5,824,514, 5,976,862, 6,492,107, and 6,569,641.

[0107] Polypeptide expression in prokaryotes is most commonly carried out using vectors containing constitutive or inductive promoters that direct the expression of either fused or non-fused polypeptides in Escherichia coli (E. coli). Fusion vectors typically add several amino acids to the recombinant polypeptide, usually at its amino terminus, to the polypeptide encoded herein. Such fusion vectors usually serve three purposes: (i) to increase the expression of the recombinant polypeptide, (ii) to increase the solubility of the recombinant polypeptide, and (iii) to assist in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Fusion expression vectors often introduce a proteolytic cleavage site at the junction of the fusion site and the recombinant polypeptide to allow for the separation of the recombinant polypeptide from the fusion site after the purification of the fusion polypeptide. Examples of such enzymes and their homologous recognition sequences include factor X, thrombin, and enterokinase. Common fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Massachusetts), and pRIT5 (Pharmacia, Piscataway, New Jersey), which fuse glutathione S-transferase (GST), maltose E-binding polypeptide, or polypeptide A to the target recombinant polypeptide, respectively.

[0108] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). Methods for targeted assembly of separate active peptide or protein domains to obtain multifunctional polypeptides by polypeptide fusion are described by Pack et al., U.S. Patents 6,294,353 and 6,692,935. One strategy for maximizing recombinant polypeptide expression in E. coli, e.g., anti-cis-pT231-tau antibody, is to express the polypeptide in a host bacterium whose ability to proteolytically cleave the recombinant polypeptide is impaired. See, for example, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to modify the nucleic acid sequence of the nucleic acid to be inserted into the expression vector so that each individual codon of each amino acid is preferentially utilized in the expression host, e. coli (see, for example, Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such modification of the nucleic acid sequence in this technique can be carried out by standard DNA synthesis techniques.

[0109] In another embodiment, the anti-cis-pT231-tau antibody expression vector is a yeast expression vector. Examples of vectors for expression in the yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, Cell 30: 933-943, 1982), pJRY88 (Schultz et al., Gene 54: 113-123, 1987), pYES2 (Invitrogen Corporation, California, San Diego), and picZ (Invitrogen Corp, California, San Diego). Alternatively, the anti-cis-pT231-tau antibody may be expressed in insect cells using a baculovirus expression vector. Examples of baculovirus vectors available for the expression of polypeptides, such as anti-cis-pT231-tau antibodies, in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., Mol. Cell. Biol. 3: 2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

[0110] In yet another embodiment, the nucleic acid encoding the anti-cis-pT231-tau antibody of this technology is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, Nature 329: 840, 1987) and pMT2PC (Kaufman, et al., EMBO J. 6: 187-195, 1987). When used in mammalian cells, the regulatory function of the expression vector is often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells that are suitable for expressing the anti-cis-pT231-tau antibody of this technology, see, for example, Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0111] In another embodiment, recombinant mammalian expression vectors can direct the expression of nucleic acids in specific cell types (e.g., tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Suitable tissue-specific promoters, though not limited to these examples, include albumin promoters (liver-specific; Pinkert, et al., Genes Dev. 1: 268-277, 1987), lymphocyte-specific promoters (Calame and Eaton, Adv. Immunol. 43: 235-275, 1988), T cell receptor promoters (Winoto and Baltimore, EMBO J. 8: 729-733, 1989), and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, Cell 33: 741-748, 1983.), and neuron-specific promoters (e.g., neuronal filament promoters; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477). Examples include pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916) and mammary gland-specific promoters (e.g., whey promoter; U.S. Patent No. 4,873,316 and European Patent Application Publication No. 264,166). Promoter regulated by development is also included, e.g., mouse hox promoter (Kessel and Gruss, Science 249: 374-379, 1990) and α-fetal protein promoter (Campes and Tilghman, Genes Dev. 3: 537-546, 1989). Another aspect of this method relates to host cells into which the recombinant expression vector of this technology has been introduced. The terms “host cell” and “recombinant host cell” are used synonymously herein. It is understood that such terms refer not only to specific target cells but also to their progeny or potential progeny. Since certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not be identical to the parent cells in fact, but they are still included within the scope of the terms herein.

[0112] The host cell can be any prokaryotic or eukaryotic cell. For example, the anti-cis-pT231-tau antibody can be expressed in bacterial cells such as Escherichia coli, insect cells, yeast, or mammalian cells. Mammalian cells are suitable hosts for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes To Clones (VCH Publishers, NY, 1987). Several suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells, and myeloma cell lines. In some embodiments, the cells are non-human. Expression vectors for these cells may contain expression regulatory sequences, e.g., origin of replication, promoter, enhancer, and necessary processing information sites, e.g., ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Queen et al., Immunol. Rev. 89: 49, 1986. Exemplary regulatory sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, etc. (Co et al., J Immunol. 148: 1149, 1992). Other suitable host cells are known to those skilled in the art.

[0113] Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. In this specification, the terms “transformation” and “transfection” are intended to refer to various technically recognized techniques for introducing foreign nucleic acids (e.g., DNA) into host cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran mediated transfection, lipofection, electroporation, particulate gun, or virus-based transfection. Other methods used to transform mammalian cells include the use of polyblens, protoplast fusion, liposomes, electroporation, and microinjection (see Sambrook et al., Molecular Cloning, for general information). Suitable methods for transducing or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals. Vectors containing the target DNA segment can be transferred into host cells by known methods, depending on the type of cell host.

[0114] For stable transfection of mammalian cells, it is known that, depending on the expression vector and transfection technique used, only small fractions of cells can incorporate foreign DNA into their genome. To identify and select these incorporates, a gene encoding a selection marker (e.g., antibiotic resistance) is generally introduced into the host cell along with the target gene. Examples of selection markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. The nucleic acid encoding the selection marker can be introduced into the host cell using the same vector as that encoding the anti-cis-pT231-tau antibody, or it can be introduced using a different vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells incorporating the selection marker gene survive, while other cells die). Host cells containing the anti-cis-pT231-tau antibody of this technology, such as prokaryotic or eukaryotic host cells in culture, can be used to produce (i.e., express) recombinant anti-cis-pT231-tau antibody. In one embodiment, the method comprises culturing host cells (introduced with a recombinant expression vector encoding the cis-pT231-tau antibody) in a suitable medium such that the anti-cis-pT231-tau antibody is produced. In another embodiment, the method further comprises the step of isolating the anti-cis-pT231-tau antibody from the medium or host cells. Once expressed, the collection of the anti-cis-pT231-tau antibody, e.g., anti-cis-pT231-tau antibody or anti-cis-pT231-tau antibody-related polypeptide, is purified from the culture medium and host cells. The anti-cis-pT231-tau antibody may be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, etc. In one embodiment, the anti-cis-pT231-tau antibody is produced in a host organism by the method of Boss et al., U.S. Patent No. 4,816,397. Typically, the anti-cis-pT231-tau antibody chain is expressed together with a signal sequence and therefore released into the culture medium. However, if the anti-cis-pT231-tau antibody chain is not spontaneously secreted by the host cell, it can be released by treatment with a mild washing agent. Purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography, column chromatography, ion exchange purification, and gel electrophoresis (see Scopes, Protein Purification (Springer-Verlag, NY, 1982) for general information).

[0115] Polynucleotides encoding anti-cis-pT231-tau antibodies, such as anti-cis-pT231-tau antibody coding sequences, can be incorporated into transgenes for introduction into the genome of transgenic animals and subsequent expression in the milk of transgenic animals. See, for example, U.S. Patents 5,741,957, 5,304,489, and 5,849,992. Suitable transgenes include light and / or heavy chain coding sequences in ligations operable with promoters and enhancers derived from casein or β-lactoglobulin, for example, mammary gland-specific genes. For the production of transgenic animals, the transgene may be microinjected into fertilized oocytes or incorporated into the genome of embryonic stem cells, the nuclei of such cells being transferred to enucleated oocytes.

[0116] Single-chain antibody. In one embodiment, the anti-cis-pT231-tau antibody of the present technology is a single-chain anti-cis-pT231-tau antibody. According to the present technology, the technology can be employed for the production of single-chain antibodies specific to the cis-pT231-tau protein (see, for example, U.S. Patent No. 4,946,778). Examples of technologies that can be used to produce single-chain Fv and antibodies of the present technology include those described in U.S. Patents No. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203: 46-88, 1991; Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90: 7995-7999, 1993; and Skerra et al., Science 240: 1038-1040, 1988.

[0117] Chimeric and humanized antibodies. In one embodiment, the anti-cis-pT231-tau antibody of this technology is a chimeric anti-cis-pT231-tau antibody. In one embodiment, the anti-cis-pT231-tau antibody of this technology is a humanized anti-cis-pT231-tau antibody. In one embodiment of this technology, the donor and acceptor antibodies are monoclonal antibodies derived from different species. For example, the acceptor antibody is a human antibody (to minimize its antigenicity in humans), in which case the resulting CDR-transplanted antibody is called a "humanized" antibody. Recombinant anti-cis-pT231-tau antibodies, such as chimeric and humanized monoclonal antibodies containing both human and non-human portions, can be produced using standard recombinant DNA techniques and are within the scope of this technique. Chimeric or humanized anti-cis-pT231-tau antibodies may be used for several applications, including in vivo use of anti-cis-pT231-tau antibodies of this technique in humans and use of these substances in in vitro detection assays. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art. Such useful methods include, for example, but are not limited to, International Application No. PCT / US86 / 02269, U.S. Patent No. 5,225,539, European Patent No. 184187, European Patent No. 171496, European Patent No. 173494, PCT International Publication No. WO86 / 01533, U.S. Patent No. 4,816,567, U.S. Patent No. 5,225,539, European Patent No. 125023, Better, et al., 1988. Science 240: 1041-1043; Liu, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, et al., 1987. J. Immunol. 139: 3521-3526; Sun, et al., 1987. Proc. Methods described in Natl. Acad. Sci. USA 84: 214-218; Nishimura, et al., 1987. Cancer Res. 47: 999-1005; Wood, et al., 1985. Nature 314: 446-449; Shaw, et al., 1988. J. Natl. Cancer Inst. 80: 1553-1559; Morrison (1985) Science 229: 1202-1207; Oi, et al. (1986) BioTechniques 4: 214; Jones, et al., 1986. Nature 321: 552-525; Verhoeyan, et al., 1988 are examples.For example, antibodies can be humanized using various techniques, including CDR transplantation (EP0239400, WO91 / 09967, U.S. Patent Nos. 5,530,101, 5,585,089, 5,859,205, 6,248,516, EP460167), veneering or resurfacing (EP0592106, EP0519596, Padlan EA, Molecular Immunology, 28: 489-498, 1991; Studnicka et al., Protein Engineering 7: 805-814, 1994; Roguska et al., PNAS 91: 969-973, 1994) and chain shuffling (U.S. Patent No. 5,565,332).In one embodiment, the cDNA encoding a mouse anti-cis-pT231-tau monoclonal antibody is digested using a restriction enzyme specially selected to remove the sequence encoding the Fc constant region, and the equivalent portion of the human Fc constant region of the cDNA is replaced (Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., WO86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application No. 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et al. (1987) J Immunol 139: 3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et al. (1987) Cancer Res 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559; U.S. Patent No. 6,180,370; U.S. Patent No. 6,300,064;

[0118] In one embodiment, the present technology provides the construction of humanized anti-cis-pT231-tau antibodies that are less likely to induce a human anti-mouse antibody (hereinafter referred to as "HAMA" herein) response while still retaining effective antibody effector functions. As used herein in relation to antibodies, the terms "human" and "humanized" refer to any antibody that is predicted to elicit a therapeutically acceptable weak immunogenic response in a human subject. In one embodiment, the present technology provides humanized anti-cis-pT231-tau antibodies, heavy chain and light chain immunoglobulins.

[0119] CDR antibody. In some embodiments, the anti-cis-pT231-tau antibody of the present technology is an anti-cis-pT231-tau CDR antibody. In general, the donor and acceptor antibodies used to generate anti-cis-pT231-tau CDR antibodies are monoclonal antibodies from different species; typically, the acceptor antibody is a human antibody (to minimize antigenicity in humans), in which case the resulting CDR-grafted antibody is referred to as a "humanized" antibody. Grafting can be of a single V H or V L of a single CDR (or even a portion of a single CDR) within, or can be of multiple CDRs (or portions thereof) within one or both of V H and V L . It is only necessary to replace the number required to enable appropriate binding of the resulting CDR-grafted antibody to the cis-pT231-tau protein, but all three CDRs of all variable domains of the acceptor antibody are often replaced. Methods for generating CDR-grafted and humanized antibodies are taught by Queen et al. U.S. Patent No. 5,585,089; U.S. Patent No. 5,693,761; U.S. Patent No. 5,693,762; and Winter U.S. Patent No. 5,225,539; and EP0682040. V H and V LUseful methods for preparing polypeptides are taught by Winter et al. U.S. Patent Nos. 4,816,397; 6,291,158; 6,291,159; 6,291,161; 6,545,142; EP0368684; EP0451216; and EP0120694.

[0120] After selecting a suitable framework region candidate from the same family and / or family members, either or both of the heavy chain and light chain variable regions are produced by transplanting the CDR from the original species into the hybrid framework region. With respect to any of the above embodiments, an assembly of a hybrid antibody or hybrid antibody fragment having a hybrid variable chain region can be achieved using conventional methods known to those skilled in the art. For example, the DNA sequences encoding the hybrid variable domains described herein (i.e., CDR-based frameworks derived from the target species and the original species) can be produced by oligonucleotide synthesis and / or PCR. The nucleic acids encoding the CDR region can also be isolated from the antibody of the original species using a suitable restriction enzyme and ligated into the target species framework using a suitable ligation enzyme. Alternatively, the framework region of the variable chain of the antibody of the original species can be modified by site-directed mutagenesis.

[0121] Since hybrids are constructed by selecting from multiple candidates corresponding to each framework domain, there are numerous combinations of sequences that accept construction according to the principles described herein. Therefore, libraries of hybrids having members with different combinations of individual framework domains can be assembled. Such libraries may be electronic or physical database collections of hybrid sequences. This process typically does not alter the FR of the acceptor antibody that franks the transplanted CDR. However, those skilled in the art may sometimes improve the antigen-binding affinity of the resulting anti-cis-pT231-tau CDR transplant antibody by substituting specific residues of a given FR to create a similar FR using the corresponding FR of the donor antibody. Suitable sites for substitution include amino acid residues adjacent to or interacting with the CDR (see, e.g., US5,585,089, particularly columns 12-16). Alternatively, those skilled in the art may start with a donor FR and modify it to be more similar to the acceptor FR or human consensus FR. Techniques for performing these modifications are known in the art. In particular, if the resulting FR is compatible with the human consensus FR at its position, or at least 90% or more identical to such consensus FR, doing so may not significantly increase the antigenicity of the resulting modified anti-cis-pT231-tau CDR transplant antibody compared to an identical antibody with a complete human FR.

[0122] Fusion protein. In one embodiment, the anti-cis-pT231-tau antibody of this technology is a fusion protein. The anti-cis-pT231-tau antibody of this technology can be used as an antigenic tag when fused to a second protein. Examples of domains that can be fused to a polypeptide include not only heterologous signal sequences but also other heterologous functional regions. Fusion does not necessarily have to be direct and may occur via a linker sequence. Furthermore, the fusion protein of this technology can also be genetically engineered to improve the characteristics of the anti-cis-pT231-tau antibody. For example, additional amino acids, particularly regions of charged amino acids, may be added to the N-terminus of the anti-cis-pT231-tau antibody to improve stability and persistence during purification from host cells or subsequent handling and storage. Alternatively, a peptide portion may be added to the anti-cis-pT231-tau antibody to facilitate purification. Such regions may be removed prior to the final preparation of the anti-cis-pT231-tau antibody. The addition of peptide moieties to facilitate polypeptide handling is a well-known and routine technique in the art. The anti-cis-pT231-tau antibody of this technology may be fused to a marker sequence such as a peptide, which facilitates the purification of the fused polypeptide. In a selected embodiment, the marker amino acid sequence is a hexahistidine peptide, for example, a tag provided in many commercially available pQE vectors (QIAGEN, Inc., Chatsworth, California). Hexahistidine, for example, provides convenient purification of the fusion protein, as described in Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989. Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from influenza hemagglutinin protein. Wilson et al., Cell 37: 767, 1984.

[0123] Therefore, any of these fusion proteins described herein can be genetically engineered using polynucleotides or polypeptides of the present technology. In addition, in some embodiments, the fusion proteins described herein exhibit an increased half-life in vivo. Fusion proteins with a disulfide-linked dimeric structure (mediated by IgG) may be more effective at binding to and neutralizing other molecules compared to proteins or protein fragments secreted as monomers alone. Fountoulakis et al., J. Biochem. 270: 3958-3964, 1995.

[0124] Similarly, EP-AO 464533 (Canadian equivalent 2045869) discloses fusion proteins containing various portions of the constant region of an immunoglobulin molecule, along with another human protein or fragment thereof. Often, the Fc portion in the fusion protein is beneficial in therapy and diagnosis, and therefore may result in improvements to pharmacokinetic properties, for example. See EP-A0232262. Alternatively, it may be desirable to delete the Fc portion after the fusion protein has been expressed, detected, and purified. For example, if the fusion protein is used as an antigen in immunotherapy, the Fc portion may interfere with therapy and diagnosis. In drug discovery, for example, human proteins such as hIL-5 are fused with Fc portions for high-throughput screening assays to identify hIL-5 antagonists. Bennett et al., J. Molecular Recognition 8: 52-58, 1995; Johanson et al., J. Biol. Chem., 270: 9459-9471, 1995.

[0125] A labeled anti-cis-pT231-tau antibody. In one embodiment, the anti-cis-pT231-tau antibody of the present technology is coupled to a labeling moiety, that is, a detectable group. The specific label or detectable group conjugated to the anti-cis-pT231-tau antibody is not a critical aspect of the technology, as long as it does not significantly interfere with the specific binding of the anti-cis-pT231-tau antibody of the present technology to the cis-pT231-tau protein. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the fields of immunoassays and imaging. In general, almost any label useful in such methods can be applied to the present technology. Accordingly, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels useful in the practice of the present technology include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, etc.), radiolabels (e.g., 3 H, 14 C, 35 S, 125 I, 121 I, 131 I, 112 In, 99 mTc), other contrast agents such as microbubbles (for ultrasound imaging), 18 F, 11 C, 15 O (for positron emission tomography), 99m TC, 111Examples of calorimetric labels or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads include those for single-photon emission computed tomography (SMT), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and others commonly used in ELISA), and colloidal gold. Patents describing the use of such labels include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, each incorporated herein by reference in its entirety for all purposes. Also, see Handbook of Fluorescent Probes and Research Chemicals (6 th See also Ed., Molecular Probes, Inc., Eugene OR.

[0126] The label is coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As shown above, a wide range of labels may be used, and the choice of label depends on factors such as required sensitivity, ease of conjugation with the compound, stability requirements, available measurement methods, and disposal regulations. Non-radioactive labels are often attached by indirect means. Generally, ligand molecules (e.g., biotin) are covalently bound to the molecule. The ligand then binds to an antiligand (e.g., streptavidin) molecule that is either inherently detectable or covalently bound to a signaling system such as a detectable enzyme, fluorescent compound, or chemiluminescent compound. Several ligands and antiligands may be used. If the ligand has a neutral antiligand, e.g., biotin, thyroxine, and cortisol, it can be used with a labeled, naturally occurring antiligand. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody, e.g., an anti-cis-pT231-tau antibody.

[0127] Molecules can also be conjugated directly to signal-generating compounds, for example, by conjugation with enzymes or fluorophores. The enzymes targeted as labels are primarily hydrolases, particularly phosphatases, esterases, and glycosidases, or oxidoreductases, particularly peroxidases. Useful fluorescent compounds as labeling moieties include, but are not limited to, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, and umbelliferone. Useful chemiluminescent compounds as labeling moieties include, but are not limited to, luciferin and 2,3-dihydrophthalazinedione, such as luminol. For an overview of the various labeling or signal-generating systems that can be used, see U.S. Patent No. 4,391,904.

[0128] Means for detecting labels are well known to those skilled in the art. For example, if the label is a radioactive label, means of detection include scintillation counters or photographic film, such as in autoradiography. If the label is a fluorescent label, it can be detected by exciting a fluorescent dye with an appropriate wavelength of light and detecting the resulting fluorescence. Fluorescence can be detected visually by photographic film using an electron detector such as a charge-coupled device (CCD) or photomultiplier tube. Similarly, enzymatic labels can be detected by providing an appropriate substance to an enzyme and detecting the resulting reaction product. Finally, simple colorimetric labels can be easily detected by observing the color associated with the label. Thus, in various urine test strip assays, conjugated gold appears pink, while various conjugated beads appear as the color of the bead. Some assay formats do not require the use of labeled components. For example, an agglutination assay can be used to detect the presence of a labeled antibody, such as an anti-cis-pT231-tau antibody. In this case, particles coated with the antigen are agglutinated by a sample containing the target antibody. In this format, there are no components that need to be labeled, and the presence of the labeled antibody can be detected by a simple visual inspection.

[0129] B. Identification and characterization of the anti-cis-pT231-tau antibody of this technology Method for identifying and / or screening anti-cis-pT231-tau antibodies of this technology. Useful methods for identifying and screening antibodies against cis-pT231-tau polypeptides that have desired specificity to the cis-pT231-tau protein include any immunologically mediated techniques known in the art. Components of the immune response can be detected in vitro by various methods well known to those skilled in the art. For example, (1) cytotoxic T lymphocytes are incubated with radioactively labeled target cells, and the lysis of these target cells can be detected by the release of radioactivity; (2) helper T lymphocytes are incubated with antigens and antigen-presenting cells, and cytokine synthesis and secretion can be measured by standard methods (Windhagen A et al., Immunity, 2: 373-80, 1995); (3) antigen-presenting cells are incubated with whole protein antigens, and the presentation of their antigens on MHC can be detected by either a T lymphocyte activation assay or a biophysical method (Harding et al., Proc. Natl. Acad. Sci., 86: 4230-4, 1989); (4) mast cells are incubated with reagents that crosslink their Fc-epsilon receptors, and histamine release can be measured by an enzyme immunoassay (Siraganian et al., TIPS, 4: 432-437). 1983) and (5) Enzyme-linked immunosorbent assay (ELISA).

[0130] Similarly, the products of an immune response in either a model organism (e.g., a mouse) or a human subject can also be detected by various methods well known to those skilled in the art. For example, (1) the production of antibodies in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, e.g., ELISA; (2) the migration of immune cells to the site of inflammation can be detected by scratching the surface of the skin and placing a sterile container to capture the migrating cells across the scratched area (Peters et al., Blood, 72: 1310-5, 1988); (3) the proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reactants, 3 (4) Phagocytosis of granulocytes, macrophages and other phagocytic cells in PBMCs can be measured using H-thymidine by placing PBMCs in wells together with labeled particles (Peters et al., Blood, 72: 1310-5, 1988) and (5) differentiation of immune system cells can be measured by labeling PBMCs with antibodies against CD molecules such as CD4 and CD8 and measuring fractions of PBMCs expressing these markers.

[0131] In one embodiment, the anti-cis-pT231-tau antibody of the present technology is selected by displaying the cis-pT231-tau peptide on the surface of a replicable gene package. See, for example, U.S. Patents 5,514,548, 5,837,500, 5,871,907, 5,885,793, 5,969,108, 6,225,447, 6,291,650, 6,492,160, EP585287, EP605522, EP616640, EP1024191, EP589877, EP774511, and EP844306. A useful method for producing / selecting fibrous bacteriophage particles containing a phagemid genome encoding a binding molecule with desired specificity is described. See, for example, EP774511, US5871907, US5969108, US6225447, US6291650, and US6492160. In some embodiments, the anti-cis-pT231-tau antibody of this technology is selected using the display of cis-pT231-tau peptides on the surface of yeast host cells. A useful method for isolating Fv polypeptides by yeast surface display is described by Kieke et al., Protein Eng. 1997 Nov; 10(11): 1303-10. In some embodiments, the anti-cis-pT231-tau antibody of this technology is selected using ribosome display. Useful methods for identifying ligands in peptide libraries using ribosome display are described by Mattheakis et al., Proc. Natl. Acad. Sci. USA 91: 9022-26, 1994 and Hanes et al., Proc. Natl. Acad. Sci. USA 94: 4937-42, 1997.

[0132] In certain embodiments, the anti-cis-pT231-tau antibody of this technology is selected using tRNA display of the cis-pT231-tau peptide. A useful method for in vitro ligand selection using tRNA display is described by Merryman et al., Chem. Biol., 9: 741-46, 2002. In one embodiment, the anti-cis-pT231-tau antibody of this technology is selected using RNA display. Useful methods for selecting peptides and proteins using RNA display libraries are described by Roberts et al. Proc. Natl. Acad. Sci. USA, 94: 12297-302, 1997 and Nemoto et al., FEBS Lett., 414: 405-8, 1997. Useful methods for selecting peptides and proteins using non-natural RNA display libraries are described by Frankel et al., Curr. Opin. Struct. Biol., 13: 506-12, 2003.

[0133] In some embodiments, the anti-cis-pT231-tau antibody of this technology is expressed in the periplasm of Gram-negative bacteria and mixed with labeled cis-pT231-tau protein. See WO02 / 34886. As described in Harvey et al., Proc. Natl. Acad. Sci. 22: 9193-98 2004 and U.S. Patent Publication No. 2004 / 0058403, in clones expressing a recombinant polypeptide having affinity for cis-pT231-tau protein, the concentration of labeled cis-pT231-tau protein conjugated to the anti-cis-pT231-tau antibody is increased, allowing the cells to be isolated from the rest of the library. After selecting a desired anti-cis-pT231-tau antibody, it is considered that the antibody may be produced in large quantities by any technique known to those skilled in the art, such as prokaryotic or eukaryotic cell expression. For example, but not limited to, an anti-cis-pT231-tau hybrid antibody or an anti-cis-pT231-tau antibody fragment may be produced using a prior art for constructing an expression vector encoding an antibody heavy chain, wherein the CDR and, optionally, the minimum portion of the variable region framework necessary to preserve the antibody-binding specificity of the original species (since it is genetically engineered according to the techniques described herein) are derived from the antibody of the original species, and the remainder of the antibody is derived from a target species immunoglobulin engineered as described herein, thereby generating a vector for the expression of the hybrid antibody heavy chain.

[0134] Measurement of cis-pT231-tau binding. In some embodiments, a cis-pT231-tau binding assay refers to an assay format in which cis-pT231-tau protein and anti-cis-pT231-tau antibody are mixed under conditions suitable for binding between the cis-pT231-tau protein and the anti-cis-pT231-tau antibody, and the amount of binding between the cis-pT231-tau protein and the anti-cis-pT231-tau antibody is evaluated. The amount of binding is compared to the amount of binding in the absence of cis-pT231-tau protein, the amount of binding in the presence of a nonspecific immunoglobulin composition, or both, with a suitable control. The amount of binding can be evaluated by any suitable method. Examples of binding assay methods include ELISA, radioimmunoassay, scintillation proximity assay, fluorescence energy transfer assay, liquid chromatography, and membrane filtration assay. Biophysical assays for the direct measurement of cis-pT231-tau protein binding with anti-cis-pT231-tau antibodies include, for example, nuclear magnetic resonance, fluorescence, fluorescence polarization, and surface plasmon resonance (BIACORE chip). Specific binding is determined by standard assays known in the art, such as radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), and mass spectrometry. If the specific binding of a candidate anti-cis-pT231-tau antibody is at least 1 percent greater than the binding observed in the absence of the candidate anti-cis-pT231-tau antibody, then the candidate anti-cis-pT231-tau antibody is useful as an anti-cis-pT231-tau antibody in this technology. Measurement of cis-pT231-tau neutralization. As used herein, “cis-pT231-tau neutralization” refers to the reduction of the activity and / or expression of the cis-pT231-tau protein by binding to an anti-cis-pT231-tau antibody. The ability of the anti-cis-pT231-tau antibody of this technology to neutralize cis-pT231-tau activity / expression can be evaluated in vitro or in vivo using methods known in the art. See, for example, WO2014152157; Yanamandra et al., Ann Clin Transl Neurol. 2(3): 278-288 (2015).

[0135] II. Use of anti-cis-pT231-tau antibody in this technology General. The anti-cis-pT231-tau antibody of this technology is useful in methods known in the art for the localization and / or quantification of cis-pT231-tau protein (e.g., for use in measuring the level of cis-pT231-tau protein within a suitable range). The antibody of this technology is useful for isolating cis-pT231-tau protein by standard techniques such as affinity chromatography or immunoprecipitation. The anti-cis-pT231-tau antibody of this technology can facilitate the purification of innate immunoreactive cis-pT231-tau protein from biological samples, e.g., mammalian serum or cells, as well as the purification of immunoreactive cis-pT231-tau protein produced by recombination expressed in a host system. Furthermore, the anti-cis-pT231-tau antibody can be used to detect immunoreactive cis-pT231-tau protein (e.g., in plasma, cell lysates or cell supernatants) to assess the amount and pattern of immunoreactive polypeptide expression. The anti-cis-pT231-tau antibody of this technology may be used diagnostically, for example, to assess the effectiveness of a given treatment plan, to monitor immunoreactive cis-pT231-tau protein levels in tissues as part of a clinical trial procedure. As described above, detection may be facilitated by coupling (i.e., physically linking) the anti-cis-pT231-tau antibody of this technology to a detectable substance.

[0136] Detection of cis-pT231-tau protein. An exemplary method for detecting the presence or absence of immunoreactive cis-pT231-tau protein in a biological sample includes obtaining a biological sample from a test subject and contacting the biological sample with an anti-cis-pT231-tau antibody of the present technology that is detectable for immunoreactive cis-pT231-tau protein, thereby detecting the presence of immunoreactive cis-pT231-tau protein in the biological sample. Detection may be achieved by a detectable label attached to the antibody. The term "labeled" is intended to encompass both direct labeling of an antibody by coupling (i.e., physically linking) a detectable substance to the antibody, and indirect labeling of an antibody by reactivity with another directly labeled compound, such as a secondary antibody. Examples of indirect labeling include the detection of a primary antibody using a fluorescently labeled secondary antibody and the end labeling of a DNA probe using biotin, which can be detected using fluorescently labeled streptavidin.

[0137] The detection methods of this technology can be used to detect immunoreactive cis-pT231-tau protein in vitro and in vivo in biological samples. In vitro techniques for detecting immunoreactive cis-pT231-tau protein include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, radioimmunoassay, and immunofluorescence. Furthermore, in vivo techniques for detecting immunoreactive cis-pT231-tau protein include introducing a labeled anti-cis-pT231-tau antibody into the subject. For example, the anti-cis-pT231-tau antibody may be labeled with a radioactive marker whose presence and location in the subject can be detected by standard imaging techniques. In one embodiment, the biological sample contains cis-pT231-tau protein molecules obtained from a test subject.

[0138] Immunoassays and imaging. The anti-cis-pT231-tau antibodies of the present technology can be used to assay immunoreactive cis-pT231-tau protein levels in biological samples (e.g., human plasma) using antibody-based technologies. For example, protein expression in tissues can be studied using classical immunohistological methods. Jalkanen, M. et al., J. Cell. Biol. 101: 976-985, 1985; Jalkanen, M. et al., J. Cell. Biol. 105: 3087-3096, 1987. Other antibody-based methods useful for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art, including enzyme labels such as glucose oxidase and radioisotopes or other radioactive substances, for example, iodine ( 125 I, 121 I, 131 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In) and technetium ( 99 mTc), as well as fluorescent labels, for example, fluorescein, rhodamine and green fluorescent protein (GFP), and biotin.

[0139] In addition to assaying immunoreactive cis-pT231-tau protein levels in biological samples, the anti-cis-pT231-tau antibodies of this technology can be used for in vivo imaging of cis-pT231-tau. Suitable antibodies for this method include those detectable by XX-ray imaging, NMR, or ESR. For XX-ray imaging, suitable labels include radioisotopes such as barium or cesium that emit detectable radiation but are not explicitly harmful to the target. Suitable markers for NMR and ESR include those with detectable characteristic spins, such as deuterium, which can be incorporated into the anti-cis-pT231-tau antibody by labeling the nutrients of the associated scFv clone. A suitable detectable imaging region, for example, a radioactive isotope (e.g., 131 I, 112 In, 99 An anti-cis-pT231-tau antibody, labeled with a radiopaque material or a material detectable by nuclear magnetic resonance (mTc), is introduced into the subject (e.g., parenterally, subcutaneously, or intraperitoneally). It will be understood in the art that the size of the subject and the imaging system used will affect the amount of imaging portion required to create a diagnostic image. In the case of radioisotope portions, for human subjects, the amount of radioactivity injected is usually about 5-20 millicuries. 99 This falls within the mTc range. The labeled anti-cis-pT231-tau antibody then accumulates at the location of cells containing the specific target polypeptide. For example, the labeled anti-cis-pT231-tau antibody of this technology accumulates within the target in cells and tissues where the cis-pT231-tau protein is localized.

[0140] Accordingly, the present technology provides a method for diagnosing a medical condition, comprising: (a) assaying the expression of immunoreactive cis-pT231-tau protein by measuring the binding of the anti-cis-pT231-tau antibody of the present technology in cells or body fluid of an individual; and (b) comparing the amount of the immunoreactive cis-pT231-tau protein present in the sample with a standard reference, wherein an increase or decrease in the level of the immunoreactive cis-pT231-tau protein compared to the standard is indicative of the medical condition. Affinity purification. The anti-cis-pT231-tau antibody of the present technology can be used to purify the immunoreactive cis-pT231-tau protein from a sample. In some embodiments, the antibody is immobilized on a solid phase support. Examples of such solid phase supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid phase supports are well known in the art (Weir et al., "Handbook of Experimental Immunology" 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby et al., Meth. Enzym. 34 Academic Press, N.Y. (1974)). The simplest method for binding an antigen to an antibody-support matrix is ​​to collect beads in a column and pass the antigen solution through the column. The efficiency of this method depends on the contact time between the immobilized antibody and the antigen, which can be extended by using a low flow rate. The immobilized antibody captures the antigen as it flows past. Alternatively, the antigen may be brought into contact with the antibody-support matrix by mixing the antigen solution with a support (e.g., beads) and rotating or shaking the slurry, thereby allowing for maximum contact between the antigen and the immobilized antibody. After the binding reaction is complete, the slurry is passed through the column for bead recovery. The beads are washed using a suitable washing buffer, and then pure or substantially pure antigen is eluted.

[0141] The antibody or polypeptide of interest may be conjugated to a solid support such as a bead. Furthermore, the first solid support, such as a bead, may also be conjugated to a second solid support, which may be a second bead or support, by any suitable means, including those disclosed herein for conjugation with a polypeptide support, as necessary. Thus, any of the conjugation methods and means disclosed herein with respect to conjugation of a polypeptide with a solid support may be applied to the conjugation of the first support with a second support, and the first and second solid supports may be the same or different.

[0142] Suitable linkers that can be used as crosslinking agents for conjugating polypeptides with solid supports include various substances that can react with functional groups present on the surface of the support, or with the polypeptide, or both. Useful reagents as crosslinking agents include homobifunctional, and especially heterobifunctional, reagents. Useful bifunctional crosslinking agents include, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC, and 6-HYNIC. The crosslinking agent may be selected to provide a selectively cleavable bond between the polypeptide and the solid support. For example, photosensitive crosslinking agents, such as 3-amino-(2-nitrophenyl)propionic acid, can be used as a means of cleaving polypeptides from solid supports (Brown et al., Mol. Divers, pp, 4-12 (1995); Rothschild et al., Nucl. Acids Res., 24:351-66 (1996) and U.S. Patent No. 5,643,722). Other crosslinking reagents are well known in the art (see, for example, Wong (1991), op. cit., and Hermanson (1996), op. cit.).

[0143] Antibodies or polypeptides can be immobilized on a solid support, such as beads, by covalent amide bonds formed between carboxyl-functionalized beads and the amino terminus of the polypeptide, or conversely, by covalent amide bonds formed between amino-functionalized beads and the carboxyl terminus of the polypeptide. Furthermore, bifunctional trityl linkers can be attached to a support, such as a 4-nitrophenyl active ester on a resin like Wang resin, by amino or carboxyl groups on the resin via an amino resin. Using the bifunctional trityl approach, the solid support may require treatment with a volatile acid, such as formic acid or trifluoroacetic acid, to ensure that the polypeptide can be cleaved and removed. In such cases, the polypeptide may be deposited at the bottom of the wells of the solid support or as bead-free patches on a flat surface of the solid support. After the addition of the matrix solution, the polypeptide can be released into the MS. Hydrophobic trityl linkers can also be used as acid-unstable linkers by using a volatile acid or a suitable matrix solution, such as a matrix solution containing 3-HPA, to cleave the amino-linked trityl group from the polypeptide. Acid instability can also be modified. For example, trityl, monomethoxytrityl, dimethoxytrityl, or trimethoxytrityl may be replaced with a suitable p-substituted or more acid-unstable tritylamine derivative of the polypeptide, i.e., trityl ether and tritylamine bonds are made to the polypeptide. Thus, the polypeptide can be removed from the hydrophobic linker, for example, by breaking the hydrophobic attraction, or, if necessary, by cleaving the trityl ether or tritylamine bond under acidic conditions, including normal MS conditions where a matrix such as 3-HPA acts as an acid.

[0144] Orthogonally cleavable linkers may also be useful for binding a first solid support, e.g., beads to a second solid support, or for binding a polypeptide of interest to a solid support. Using such linkers, the first solid support, e.g., beads, can be selectively cleaved from the second solid support without cleaving the polypeptide from the support, and then, at a later time, the polypeptide can be cleaved from the beads. For example, a disulfide linker that can be cleaved using a reducing agent such as DTT may be used to bind the beads to the second solid support, or an acid-cleavable bifunctional trityl group may be used to immobilize the polypeptide to the support. If necessary, for example, the linkage between the first and second supports may remain intact while the linkage of the polypeptide to the solid support is cleaved first. Trityl linkers may provide covalent or hydrophobic conjugations, but regardless of the nature of the conjugation, the trityl group is readily cleaved under acidic conditions.

[0145] For example, beads may be bonded to a second support by linking groups that can be selected to have length and chemical properties that promote high-density bonding of the beads to a solid support, or high-density bonding of polypeptides to beads. Such linking groups may have, for example, a "tree-like" structure, thereby providing a variety of functional groups per attachment site on the solid support. Examples of such linking groups include polylysine, polyglutamic acid, pentaerythrol, and tris-hydroxy-aminomethane. Non-covalent interactions. By non-covalent interactions, an antibody or polypeptide can be conjugated to a solid support, or a first solid support can also be conjugated to a second solid support. For example, magnetic beads made of a ferromagnetic material that can be magnetized can be attracted to a magnetic solid support and released from the support by the removal of the magnetic field. Alternatively, the solid support may provide an ionic or hydrophobic moiety, which can enable interaction of the ionic or hydrophobic moiety with a polypeptide, such as a polypeptide containing an attached trityl group, or with a hydrophobic second solid support, respectively.

[0146] The solid support may also be provided with a member of a specific binding pair and thus can be conjugated to a polypeptide containing a complementary binding moiety or to a second solid support. For example, beads coated with avidin or streptavidin can be conjugated to a polypeptide incorporating a biotin moiety, or to a second solid support coated with biotin or a biotin derivative, such as iminobiotin. It should be understood that any of the binding members disclosed herein, or otherwise known in the art, may be reversed. Thus, biotin may be incorporated into either a polypeptide or a solid support, for example, and conversely, avidin or other biotin-binding moieties may be incorporated into the support or polypeptide, respectively. Other specific binding pairs considered for use herein, but not limited to, hormones and their receptors, enzymes and their substrates, nucleotide sequences and their complementary sequences, antibodies and antigens that specifically interact with them, and other such pairs known to those skilled in the art. A. Diagnostic use of the anti-cis-pT231-tau antibody of this technology General. The anti-cis-pT231-tau antibody of this technology is useful in diagnostic methods. As such, this technology provides a method for using the antibody in the diagnosis of cis-pT231-tau activity in a subject. The anti-cis-pT231-tau antibody of this technology can be selected to have any level of epitope binding specificity and extremely high binding affinity to the cis-pT231-tau protein. Generally, in immunoassays, the higher the binding affinity of the antibody, the more stringent washing conditions can be performed to remove nonspecifically bound material without removing the target polypeptide. Therefore, the anti-cis-pT231-tau antibody of this technology, which is useful in diagnostic assays, is usually at least 10 8 , 10 9 , 10 10 , 10 11 or 10 12 M -1 It has binding affinity. Furthermore, it is desirable that the anti-cis-pT231-tau used as a diagnostic reagent has a sufficient kinetic binding rate (on-rate) to reach equilibrium under standard conditions in at least 12 hours, at least 5(5) hours, or at least 1(1) hours.

[0147] Anti-cis-pT231-tau antibodies can be used to detect immunoreactive cis-pT231-tau protein in various standard assay formats. These formats include immunoprecipitation, Western blotting, ELISA, radioimmunoassay, and immunoassay. (Harlow & Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York)) See U.S. Patent Nos. 3,791,932, 3,839,153, 3,850,752, 3,879,262, 4,034,074, 3,791,932, 3,817,837, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074 and 4,098,876 (1988). Biological samples can be obtained from any tissue or body fluid of the subject. In certain embodiments, the subject is in the early stages of the tauopathy. In one embodiment, the early stages of the tauopathy are determined by an increase in the level of cis-pT231-tau or an increase in the cis:trans-pT231-tau ratio in the sample obtained from the subject. In some embodiments, the method further includes determining the levels of CSF t-tau, pT181-tau, αβ42, or ApoE4 in the sample obtained from the subject. In certain embodiments, the sample is selected from the group consisting of urine, blood, serum, plasma, saliva, amniotic fluid, and cerebrospinal fluid (CSF). In some embodiments, the subject has a history of repeated traumatic brain injury.

[0148] Immunoassays or sandwich assays are one form of diagnostic methods for this technology. See U.S. Patents 4,376,110, 4,486,530, 5,914,241, and 5,965,375. Such assays use one type of antibody, e.g., an anti-cis-pT231-tau antibody or a population of anti-cis-pT231-tau antibodies immobilized on a solid phase, and another anti-cis-pT231-tau antibody or a population of anti-cis-pT231-tau antibodies in solution. Typically, the solution anti-cis-pT231-tau antibody or the population of anti-cis-pT231-tau antibodies is labeled. When antibody populations are used, the population may contain antibodies that bind to different epitope specificities within the target polypeptide. Thus, the same population may be used for both solid-phase and solution antibodies. When anti-cis-pT231-tau monoclonal antibodies are used, first and second cis-pT231-tau monoclonal antibodies with different binding specificities to the solid and solution phases are used. The solid-phase (also called "capture") and solution (also called "detection") antibodies can be contacted with the target antigen in either order or simultaneously. If the solid-phase antibody is contacted first, the assay is called a forward assay. Conversely, if the solution antibody is contacted first, the assay is called a reverse assay. If the target is contacted with both antibodies simultaneously, the assay is called a simultaneous assay. After contacting the cis-pT231-tau protein with the anti-cis-pT231-tau antibody, the sample is incubated for a period of typically about 1 hour, which typically varies from about 10 minutes to about 24 hours. A washing step is then performed to remove any components of the sample that are not specifically bound to the anti-cis-pT231-tau antibody used as the diagnostic reagent. If the solid-phase and solution antibodies are conjugated in separate steps, washing may be performed after either or both of the conjugation steps. After washing, conjugation is usually quantified by detecting the label linked to the solid phase by the conjugation of the labeled solution antibody. Typically, a calibration curve is prepared from a sample containing a known concentration of the target antigen for a given pair of antibodies or populations of antibodies and given reaction conditions.The concentration of immunoreactive cis-pT231-tau protein in the sample being tested is then read from the calibration curve (i.e., the standard curve) by interpolation. The analyte can be measured from the amount of bound labeled solution antibody at equilibrium, or by kinetic measurements of the bound labeled solution antibody at a series of time points before equilibrium is reached. The slope of such a curve is a measure of the concentration of cis-pT231-tau protein in the sample.

[0149] Suitable supports for use in the above method include, for example, nitrocellulose membranes, nylon membranes and derivatized nylon membranes, as well as particles such as agarose, dextran-based gels, urine test strips, fine particles, microspheres, magnetic particles, test tubes, microtiter wells, and SEPHADEX® (Amersham Pharmacia Biotech, Piscataway, New Jersey). Immobilization may be by absorption or by covalent attachment. The anti-cis-pT231-tau antibody may be linked to a linker molecule such as biotin for attachment to a linker bound to a surface such as avidin.

[0150] B. Therapeutic use of anti-cis-pT231-tau antibodies of this technology The antibodies of this technology are useful for treating neurological disorders characterized by pathologically high levels of cis-conformation of phosphorylated tau protein. Such treatment may be used in patients identified with pathologically high levels of cis-conformation of phosphorylated tau (e.g., diagnosed by the methods described herein) or in patients diagnosed with diseases known to be associated with such pathological levels. Examples of neurological disorders that can be treated with the antibodies of this technology include Alzheimer's disease (AD), mild cognitive impairment (MCI), Parkinson's disease (PD), corticobasal degeneration, Pick's disease, stroke, traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy, frontotemporal lobar degeneration, Ritiko-Bodiegg disease, Tangle-predominant dementia, meningeal hemangiomatosis, and subacute sclerosing panencephalitis.

[0151] The compositions of this technology may be used in conjunction with other therapeutic agents useful in treating neurological disorders associated with elevated cis-pT231-tau protein expression. For example, the antibodies of this technology may be administered separately, sequentially, or concurrently with at least one further therapeutic agent selected from the group consisting of donepezil, rivastigmine, galantamine, memantine, and lithium chloride. The composition of this technology may be administered as a single bolus to a subject requiring it. Alternatively, the dosing regimen may include multiple doses administered at various time intervals after the appearance of tau NFTs. Administration may be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intracranially, subarachnoidally, or topically. Administration may include self-administration and administration by another person. It should also be understood that the various forms of treatment for medical conditions as described include not only complete treatment but also less complete treatment, and mean “substantial” treatment in which some biological or medically relevant outcome is achieved. In some embodiments, the antibodies of this technology include pharmaceutical formulations that can be administered in single or multiple doses to subjects requiring them. The administration schedule can be adjusted to provide a desired response (e.g., a therapeutic response).

[0152] Typically, the effective amount of the antibody composition of this technology sufficient to achieve a therapeutic effect is in the range of approximately 0.000001 mg / kg of body weight / day to approximately 10,000 mg / kg of body weight / day. The typical dosage range is approximately 0.0001 mg / kg of body weight / day to approximately 100 mg / kg of body weight / day. For the administration of anti-cis-pT231-tau antibody, the dosage is in the range of approximately 0.0001 to 100 mg / kg of the subject's body weight, more commonly 0.01 to 5 mg / kg weekly, every two weeks, or every three weeks. For example, the dosage may be in the range of 1 mg / kg of body weight or 10 mg / kg weekly, every two weeks, or every three weeks, or 1 to 10 mg / kg weekly, every two weeks, or every three weeks. In one embodiment, the single dose of antibody is in the range of 0.1 to 10,000 micrograms / kg of body weight. In one embodiment, the antibody concentration in the carrier ranges from 0.2 to 2000 micrograms per milliliter delivered. Exemplary treatment dosing regimens involve administration every two weeks, once a month, or once every three to six months. Anti-cis-pT231-tau antibody may be administered multiple times. Intervals between single doses may be hourly, daily, weekly, monthly, or yearly. Intervals may be irregular, as they are indicated by measuring blood antibody levels in the subject. In some methods, doses are adjusted to achieve serum antibody concentrations in the subject of approximately 75 μg / mL to 125 μg / mL, 100 μg / mL to 150 μg / mL, 125 μg / mL to 175 μg / mL, or 150 μg / mL to 200 μg / mL. Alternatively, anti-cis-pT231-tau antibody may be administered as a sustained-release formulation, in which case less frequent administration is required. The dosage and frequency of administration vary depending on the half-life of the antibody in the subject. The dosage and frequency of administration also vary depending on whether the treatment is prophylactic or therapeutic. For prophylactic use, relatively low doses are administered over a long period at relatively infrequent intervals. For therapeutic use, relatively high doses at relatively short intervals are sometimes required until disease progression is reduced or terminated, or until the subject achieves partial or complete remission of disease symptoms. Thereafter, the patient may be administered a prophylactic treatment plan.

[0153] Toxicity. Optimally, an effective amount (e.g., dose) of the anti-cis-pT231-tau antibody described herein provides therapeutic benefits without causing substantial toxicity to the subject. The toxicity of the anti-cis-pT231-tau antibody described herein is determined by standard pharmaceutical procedures in cell culture or experimental animals, for example, by LD 50 (Lethal dose for 50% of the population) or LD 100 This can be determined by determining the lethal dose (for 100% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic coefficient. Data obtained from these cell culture assays and animal studies may be used in formulating non-toxic dose ranges for use in humans. The doses of anti-cis-pT231-tau antibodies described herein are within the range of circulating concentrations that include effective doses with little to no toxicity. Dosages may vary within this range depending on the dosage form used and the route of administration utilized. The exact prescription, route of administration, and dose may be selected by individual physicians in consideration of the patient's condition. See, for example, Fingl et al., In: The Pharmacological Basis of Therapeutics, Ch. 1 (1975).

[0154] Formulation of pharmaceutical compositions. According to the methods of this technology, anti-cis-pT231-tau antibodies can be incorporated into pharmaceutical compositions suitable for administration. Pharmaceutical compositions generally contain recombinant or substantially purified natural antibodies and pharmaceutically acceptable carriers in a form suitable for administration to a target. The pharmaceutically acceptable carrier is determined by the portion of the individual composition to be administered, and by the individual method used to administer the composition. Therefore, there are various suitable formulations of pharmaceutical compositions for administering antibody compositions (e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18). th (See ed., 1990). Pharmaceutical compositions are generally formulated to be sterile and substantially isotonic, in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0155] The terms “medically acceptable,” “physiologically acceptable,” and their grammatical variations are used synonymously when referring to compositions, carriers, diluents, and reagents, indicating that the material is administerable to a subject without producing undesirable physiological effects to the extent that it interferes with the administration of the composition. For example, “medically acceptable excipients” generally means excipients useful in the preparation of safe, non-toxic, and desirable pharmaceutical compositions, and include excipients acceptable for veterinary use and for human pharmaceutical use. Such excipients may be solids, liquids, semi-solids, or, in the case of aerosol compositions, gases. “Medicinally acceptable salts and esters” means salts and esters that are medically acceptable and possess desired pharmacological properties. Such salts include those that can be formed when acidic protons present in the composition are reactive with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals, such as sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with amine bases, such as ethanolamine, diethanolamine, triethanolamine, tromethamine, and N-methylglucamine. Other examples of such salts include acid addition salts formed with inorganic acids (e.g., hydrochloric acid and hydrobromic acid) and organic acids (e.g., acetic acid, citric acid, maleic acid, and alkane- and allene-sulfonic acids, such as methanesulfonic acid and benzenesulfonic acid). Pharmacopoecitable esters include esters formed from carboxy, sulfonyloxy, and phosphooxy groups present in anti-cis-pT231-tau antibodies, such as C 1-6Alkyl esters are examples. When two acidic groups are present, the pharmaceutically acceptable salt or ester may be a mono-acid-mono-salt or ester or a di-salt or ester; similarly, when two or more acidic groups are present, some or all of such groups may be chlorinated or esterified. Anti-cis-pT231-tau antibodies named in this art may exist in unchlorinated or unesterified forms, or in chlorinated and / or esterified forms, and the naming of such anti-cis-pT231-tau antibodies is intended to include both the original (unchlorinated and unesterified) compound and its pharmaceutically acceptable salts and esters. Furthermore, certain embodiments of this art may exist in two or more stereoisomers, and the naming of such anti-cis-pT231-tau antibodies is intended to include all single stereoisomers and all mixtures (whether racemic or not) of such stereoisomers. Those skilled in the art will have no difficulty determining the appropriate timing, order, and dosage for administering specific drugs and compositions of this art.

[0156] Examples of such carriers or diluents include, but are not limited to, water, physiological saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and hydrogenated oils may also be used. The use of such media and compounds for pharmaceutically active substances is well known in the art. Any conventional media or compound is considered for use in a composition unless it is unsuitable for the anti-cis-pT231-tau antibody. Complementary active compounds are also incorporated into the composition.

[0157] The pharmaceutical compositions of the present technology are formulated to be compatible with their intended routes of administration. The anti-cis-pT231-tau antibody compositions of the present technology can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial, intrathecal, intraperitoneal, intranasal or intramuscular routes, or as an inhalant. The anti-cis-pT231-tau antibody may be administered in combination with other substances that are at least partially effective in the treatment of various diseases, including various actin- or microfibril-associated diseases. As solutions or suspensions used for parenteral, intradermal or subcutaneous application, the following components may be mentioned: sterile diluents such as water for injection, physiological saline solution, hardened oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, antibacterial compounds such as benzyl alcohol or methylparaben, antioxidant substances such as ascorbic acid or sodium bisulfite, chelating compounds such as ethylenediaminetetraacetic acid (EDTA), buffers such as acetic acid, citric acid or phosphoric acid, and compounds for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, disposable syringes or multiple-dose vials made of glass or plastic.

[0158] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, Cremophor EL® (BASF, New Jersey, Parsippany), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to be readily injectable. It must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. Carriers may be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal compounds, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is desirable to include isotonic compounds in the composition, such as sugars, polyhydric alcohols like mannitol and sorbitol, and sodium chloride. Extending the absorption of the injectable composition can be achieved by including absorption-delaying compounds in the composition, such as aluminum monostearate and gelatin.

[0159] Sterile injectable solutions can be prepared by incorporating the anti-cis-pT231-tau antibody, along with one or a combination of the components listed above as needed, in the required amount in a suitable solvent, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the anti-cis-pT231-tau antibody into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preparation method is vacuum drying and lyophilization, from which a powder of the active ingredient and any further desired components is obtained from the solution that has been previously sterile filtered. Antibodies of this technology can be administered in the form of depot injections or indwelling preparations, which can be formulated in a manner that allows for sustained or pulsatile release of the active ingredient.

[0160] Oral compositions generally contain an inert diluent or food carrier. They may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, anti-cis-pT231-tau antibodies may be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, in which the compounds in the fluid carrier are applied orally, quickly swished, and then spit out or swallowed. Pharmaceutically compatible conjugate compounds and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds of similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrating compounds such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; flow enhancers such as colloidal silicon dioxide; sweetening compounds such as sucrose or saccharin; or flavoring compounds such as peppermint, methyl salicylate, or orange flavor. For administration by inhalation, the anti-cis-pT231-tau antibody is delivered in the form of an aerosol spray from a pressurized container or dispenser or sprayer containing a suitable propellant, such as a gas like carbon dioxide.

[0161] Systemic administration may be by mucosal or transdermal means. For mucosal or transdermal administration, an appropriate penetrating agent is used for the barrier to which the formulation is to be penetrated. Such penetrating agents are generally known in the art and include, for example, cleansing agents, bile salts, and fusidic acid derivatives for mucosal administration. Mucosal administration can be achieved by the use of nasal sprays or suppositories. For transdermal administration, the anti-cis-pT231-tau antibody is formulated in ointments, salves, gels, or creams, as is generally known in the art. Anti-cis-pT231-tau antibodies can also be prepared as pharmaceutical compositions in the form of suppositories (e.g., using conventional suppository bases such as cocoa butter and other glycerides) or retained enemas for rectal delivery.

[0162] In one embodiment, anti-cis-pT231-tau antibodies are prepared using carriers that protect the anti-cis-pT231-tau antibodies from rapid elimination from the body, such as controlled-release formulations including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes targeting infected cells having monoclonal antibodies against viral antigens) may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, such as those described in, for example, U.S. Patent No. 4,522,811.

[0163] 1.C. Kit The present technology provides a kit for the diagnosis and / or treatment of neurological disorders involving elevated cis-pT231-tau protein expression, comprising at least one antibody of the present technology or a functional variant thereof (e.g., a substitution variant). The above components of the kit of the present technology may be packaged in suitable containers and labeled for the diagnosis and / or treatment of neurological disorders involving elevated cis-pT231-tau protein expression. The above components may be stored in unit or multi-dose containers, e.g., sealed ampoules, vials, bottles, syringes and test tubes, as aqueous, optionally sterile solutions, or as lyophilized, optionally sterile formulations for reconstitution. The kit may further include a second container for holding a diluent suitable for diluting the pharmaceutical composition to a larger volume. Suitable diluents include, but are not limited to, pharmaceutically acceptable excipients of the pharmaceutical composition and saline solutions. Furthermore, the kit may include instructions for diluting the pharmaceutical composition and / or instructions for administering the pharmaceutical composition, whether diluted or not. The containers may be formed from various materials such as glass or plastic and may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be punctured by a subcutaneous injection needle). The kit may further include more containers containing pharmaceutically acceptable buffers such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes and culture media for one or more suitable hosts. The kit may also include instructions that are customarily included in the commercial packaging of therapeutic or diagnostic agents, which may contain information relating to such therapeutic or diagnostic agents, such as indications, usage, dosage, manufacturing, administration, contraindications and / or warnings relating to use. The kit is useful for detecting the presence of immunoreactive cis-pT231-tau protein in any body fluid, including, but not limited to, serum, plasma, lymph, cystic fluid, urine, feces, cerebrospinal fluid, ascites, or blood, and biopsy samples of body tissue. For example, the kit may include one or more anti-cis-pT231-tau antibodies (or their antigen-binding fragments) of the present technology capable of binding to cis-pT231-tau protein in the biological sample, means for determining the amount of cis-pT231-tau protein in the sample, and means for comparing the amount of immunoreactive cis-pT231-tau protein in the sample to a standard. One or more anti-cis-pT231-tau antibodies may be labeled. The kit components (e.g., reagents) may be packed in suitable containers. The kit may further include instructions for using the kit to detect immunoreactive cis-pT231-tau protein.

[0164] For antibody-based kits, the kit may include, for example, 1) a cis-pT231-tau antibody of the technology attached to a solid support that conjugates to a first antibody, e.g., a cis-pT231-tau protein, and optionally, 2) a second different antibody conjugated to a detectable label that conjugates to either the cis-pT231-tau protein or the first antibody. The kit may also include, for example, buffers, preservatives, or protein stabilizers. The kit may further include components necessary for detecting a detectable label, such as enzymes or substrates. The kit may also contain a control sample or a set of control samples that can be assayed and compared with the test sample. Each component of the kit may be sealed in an individual container, and all of the various containers may be in a single package along with instructions for interpreting the results of assays performed using the kit. The kit of this technology may contain a written product on or inside the kit container. The written product describes the use of the reagents contained in the kit for, for example, the detection of cis-pT231-tau protein in vitro or in vivo, for the treatment of neurological disorders involving elevated cis-pT231-tau protein expression in subjects requiring it. In certain embodiments, the use of the reagents may follow the methods of this technology. [Examples]

[0165] The technology is further illustrated by the following examples, but this should not be construed as limiting. The following examples demonstrate the preparation, characterization, and use of exemplary conformation-specific phosphorylated tau (pT231-tau) antibodies of the technology. Examples 1-12 demonstrate the production of chimeric and humanized antibodies of the technology and the characterization of their binding specificity.

[0166] (Example 1) Sequence of the variable region of the mouse PT-113 monoclonal antibody Using TRIzol reagent (Invitrogen, Carlsbad, California) according to the supplier's protocol, approximately 10 6Total RNA was extracted from 1 mouse PT-113 hybridoma cells (see WO2014152157). Oligo-dT priming cDNA for 5'-RACE was synthesized using the SMARTer RACE cDNA amplification kit (Clontech, Mountain View, California) according to the supplier's protocol. Variable domain cDNAs of the heavy and light chains were amplified by polymerase chain reaction (PCR) using Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, Massachusetts) with 3' primers that specifically anneal to the mouse heavy and light chain constant regions and the 5'-RACE primers provided in the SMARTer RACE cDNA amplification kit. MCG2b (5'-GCCAGTGGATAGACTGATGG-3') was used as a gene-specific 3' primer for VH amplification. MCK (5'-GATGGATACAGTTGGTGCAGC-3') was used for VL amplification. The amplified VH and VL cDNAs were subcloned into the pJet1.2 vector (Thermo Fisher Scientific) for sequencing. DNA sequencing was performed at Eurofins Genomics (Louisville, Kentucky) using JetRev (5'-AAGAACATCGATTTTCCATGGCAG-3') as a primer. Several heavy chain and light chain clones were sequenced, and unique sequences homologous to the normal mouse heavy chain and light chain variable regions were identified. Figures 1 and 2 show the nucleotide sequences of the heavy chain and light chain variable regions (VH and VL, respectively) of the mouse PT-113 IgG2b / kappa monoclonal antibody, alongside their predicted amino acid sequences.

[0167] Figure 3 shows the positions of each amino acid residue and the arrangement of the complementarity-determining region (CDR) of the mouse PT-113 VH domain, based on the Kabat definition. Figure 3 also shows the amino acid sequence alignment between the PT-113 VH domain and its predicted mouse parent germline V segment IGHV1S127*01 and its JH2 segment. Asterisks indicate differences between the PT-113VH and IGHV1S127*01 amino acid sequences, which are most likely due to somatic high-frequency mutations. Figure 4 shows the positions of each amino acid residue in the mouse PT-113 VL domain and the arrangement of the CDRs, based on the Kabat definition. Figure 4 also shows the amino acid sequence alignment between the PT-113 VL domain and its predicted mouse parent germline V segment IGKV1-110*02(2) and its Jκ1 segment. Asterisks indicate differences between the PT-113 VL and IGKV1-110*02 amino acid sequences, which are most likely due to somatic high-frequency mutations.

[0168] (Example 2) Construction of a chimeric PT-113 IgG1 antibody The gene encoding the VH domain of the mouse PT-113 antibody was designed as an exon containing a splice donor signal, a SpeI site at the 5' end, and a HindIII site at the 3' end after the coding region (Figure 5). Similarly, the gene encoding the VL domain of the mouse PT-113 antibody was designed as an exon containing a splice donor signal, a NheI site at the 5' end, and an EcoRI site at the 3' end after the coding region (Figure 6). In the PT-113VH and VL exons, codons rarely used in mammals were replaced with their frequently used corresponding codons. The PT-113 VH and VL genes were synthesized using Eurofins MWG Operon (Huntsville, Alabama). After digestion with SpeI and HindIII (for the VH gene) or NheI and EcoRI (for the VL gene), the resulting PT-113 VH and VL fragments were cloned into mammalian expression vectors retaining human gamma-1 and kappa constant regions for the production of chimeric PT-113 (ChPT-113)IgG1 / kappa antibodies. The schematic structure of the resulting expression vector, pChPT-113, is shown in Figure 7.

[0169] (Example 3) Design of humanized PT-113 VH and VL genes The humanized PT-113 VH and VL amino acid sequences were designed as described below. Human VH sequences homologous to the mouse PT-113 VH framework were queried within the GenBank database, and the VH sequence encoded by human AF174092 cDNA (GenBank accession number: AF174092, described in Wang & Stollar, Clin. Immunol. 93:132-142 (1999)) was selected as the acceptor for humanization. The CDR sequence of PT-113 VH was first moved to the corresponding position in AF174092 VH. Then, at framework positions 48, 67, 69, 71, 93, and 94, where the three-dimensional model of the PT-113 variable domain showed significant contact with the CDR, amino acid residues of AF174092 VH were replaced with corresponding residues of mouse PT-113 VH. The amino acid sequence of the obtained humanized VH domain, HuPT-113 VH1, is shown in Figure 8, along with the mouse PT-113 and human AF174092 VH sequences.

[0170] While Ala at position 67 and Leu at position 69 in mouse PT-113 VH are positioned close to the CDR and are predicted to be important for the formation of the CDR structure, detailed structural analysis of PT-113 CDR suggested that either or possibly both of the two amino acid residues at positions 67 and 69 in HuPT-113 VH1 may be replaced by the corresponding residues in the human acceptor AF174092 VH sequence. To further reduce the possibility of immunogenicity, three further humanized VH domains (HuPT-113 VH2, HuPT-113 VH3, and HuPT-113 VH4) were designed. In HuPT-113 VH2, Ala at position 67 in HuPT-113 VH1 was replaced with Val. In HuPT-113 VH3, Leu at position 69 in HuPT-113 VH1 was replaced with Met. In HuPT-113 VH4, the Ala at position 67 and the Leu at position 69 in HuPT-113 VH1 were replaced with Val and Met, respectively. The amino acid sequences of HuPT-113 VH2, VH3, and VH4 are shown in Figure 8. Based on homology searches using the mouse PT-113 VL framework sequence, the human Vκ region encoded by M99608 cDNA (GenBank accession number: M99608, described in Weng et al. J. Immuno. 149:2518-2529 (1992)) was selected as the acceptor for humanization. The CDR sequence of PT-113 VL was first moved to the corresponding position in M99608 VL. No amino acid substitutions with mouse residues were required in the framework region. The amino acid sequence of the resulting humanized VL, HuPT-113 VL1, is shown in Figure 9 along with the mouse PT-113 and human M99608 VL sequences.

[0171] (Example 4) Construction of humanized PT-113 VH and VL genes The gene encoding HuPT-113 VH1 was designed as an exon containing a signal peptide, splice donor signal, 5' SpeI site, and 3' HindIII site (Figure 10). The signal peptide sequence and splice donor signal from the mouse PT-113 VH exon (Figure 5) were used for HuPT-113 VH1. The gene encoding HuPT-113 VL1 was similarly designed as an exon containing a signal peptide, splice donor signal, 5' NheI site, and 3' EcoRI site (Figure 14). The signal peptide sequence and splice donor signal in the mouse PT-113 VL exon (Figure 6) were used for HuPT-113 VL1.

[0172] The HuPT-113 VH1 and VL1 genes were synthesized using Eurofins MWG Operon. After digestion using SpeI and HindIII (for VH) or NheI and EcoRI (for VL), the HuPT-113 VH1 and HuPT-113 VL1 genes were subcloned to corresponding sites in mammalian expression vectors for production in human IgG1 / kappa form. The resulting expression vector pHuPT-113A expresses humanized PT-113 IgG1 / kappa antibodies containing HuPT-113 VH1 and VL1 (HuPT-113A). The genes encoding the HuPT-113 VH2, VH3, and VH4 exons (Figure 8) were generated by site-directed mutagenesis of the HuPT-113 VH1 gene using the double-extension PCR method described in Higuchi, R., Using PCR to Engineer DNA. In PCR Technology: Principles and Applications for DNA Amplification. 61-67 (HA Erlich, ed., New York, NY (1989)). The nucleotide sequences of HuPT-113 VH2, VH3, and VH4, along with their predicted amino acid sequences, are shown in Figures 11, 12, and 13, respectively. The HuPT-113 VH1 gene in the pHuPT-113A expression vector was replaced with HuPT-113 VH2, VH3, and VH4 to generate pHuPT-113B, pHuPT-113C, and pHuPT-113D, respectively. The HuPT-113B, HuPT-113C, and HuPT-113D IgG1(I) antibodies are expressed from the pHuPT-113B, pHuPT-113C, and pHuPT-113D vectors, respectively.

[0173] (Example 5) Characterization of ChPT-113, HuPT-113A, HuPT-113B, HuPT-113C, and HuPT-113D IgG1(I) antibodies for antigen binding. The binding of ChPT-113, HuPT-113A, HuPT-113B, HuPT-113C, and HuPT-113D IgG1(I) antibodies to the pThr-Dmp peptide (H-KVAVVRT(PO3H2)XPKSPS-OH, X=5,5-dimethyl-L-proline) was characterized by ELISA. For the preparation of these five antibodies, the pChPT-113, pHuPT-113A, pHuPT-113B, pHuPT-113C, and pHuPT-113D vectors were individually transfected into the human embryonic kidney cell line, HEK293, using Lipofectamine2000 reagent (Invitrogen, Carlsbad, California) according to the supplier's protocol. HEK293 cells were grown in DME medium containing 10% fetal bovine serum (FBS; HyClone, Logan, Utah) in a 7.5% CO2 incubator at 37°C.

[0174] Antibody expression in the culture supernatant of transiently transfected HEK293 cells was analyzed by ELISA. ELISA plates were coated overnight at 4°C with 1 / 2,000 dilution of goat anti-human IgG, Fcγ-specific polyclonal antibody (Sigma-Aldrich, St. Louis, Missouri) in PBS at 100 μl / well. The plates were washed with washing buffer (PBS containing 0.05% Tween 20) and blocked with 300 μl / well blocking buffer (PBS containing 2% skim milk and 0.05% Tween 20). After washing with washing buffer, 100 μl / well test samples, appropriately diluted with binding buffer (PBS containing 1% skim milk and 0.025% Tween 20), were applied to the ELISA plates. A suitable humanized IgG1 / kappa antibody was used as a standard. ELISA plates were incubated at room temperature for 1 hour, washed with wash buffer, and the conjugated antibody was detected using 100 μl / well of 1 / 2,000 dilution of HRP-conjugated goat anti-human kappa chain polyclonal antibody (SouthernBiotech, Birmingham, Alabama). After incubation at room temperature for 0.5 hours and washing with wash buffer, color development was initiated by adding 100 μl / well of ABTS substrate (Sigma-Aldrich), and stopped with 100 μl / well of 2% oxalic acid. Absorbance was read at 405 nm.

[0175] The binding of ChPT-113, HuPT-113A, HuPT-113B, HuPT-113C, and HuPT-113D IgG1(I) antibodies to pThr-Dmp was tested by ELISA. For coating MaxiSorp ELISA plates (Thermo Fisher Scientific, Waltham, Massachusetts), 50 μl of 1 μg / ml pThr-Dmp (lot number 1556880, AnaSpec, Fremont, CA) in 2,2,2-trifluoroethanol was applied to each well. After drying the peptide solutions overnight at 37°C, the wells were blocked with ELISA buffer (10 mM TrisHCl (pH 7.6) containing 150 mM NaCl, 5% skim milk, 0.4% bovine serum albumin, and 0.05% Tween 20). Various concentrations of test antibodies (50 μl / well) were loaded into the ELISA buffer and incubated at room temperature for 2 hours. After washing the wells with a washing buffer (10 mM TrisHCl (pH 7.6) containing 150 mM NaCl, 0.4% bovine serum albumin, and 0.05% Tween 20), 50 μl / well of HRP-conjugated goat anti-human kappa chain antibody (diluted 1 / 2,000 in ELISA buffer) was applied to each well. After incubation at room temperature for 1 hour, the wells were washed with the washing buffer. Color development was initiated with 50 μl / well of TMB substrate (1-Step Ultra TMB-ELISA, catalog no. 34028, Thermo Fisher Scientific) and stopped with 50 μl / well of 2N H2SO4. Absorbance was measured at 450 nm.

[0176] The binding patterns of ChPT-113, HuPT-113A, HuPT-113B, HuPT-113C, and HuPT-113D IgG1(I) antibodies to the pThr-Dmp peptide are shown in Figure 15. No significant differences in the pThr-Dmp binding patterns were observed among these five antibodies. For further analysis, HuPT-113D IgG1(I), which retains the smallest number of mouse framework residues among the four humanized PT-113 antibodies, was selected.

[0177] (Example 6) Construction of HuPT-113D IgG1-AA(I) antibody To eliminate the potential cytotoxic activity of the HuPT-113D IgG1(I) antibody, amino acid substitutions from Leu to Ala (Kabat's Eu-numbering) at positions 234 and 235, known to eliminate the effector function of the IgG antibody (Xu et al., Cell Immunol. 200:16-26 (2000); Hezareh et al., J. Virol. 75:12161-12168 (2001)), were introduced into the CH2 constant region of pHuPT-113D by site-directed mutagenesis using overlapping extension PCR (Higuchi, R (1989), see above). The resulting plasmid pHuPT-113D-AA expresses the HuPT-113D IgG1-AA(I) antibody.

[0178] (Example 7) Preparation of CHO-K1 stable transfections yielding ChPT-113 IgG1, HuPT-113D IgG1(I), and HuPT-113D IgG1-AA(I) antibodies. To obtain cell lines that stably produce ChPT-113 IgG1, HuPT-113D IgG1(I), and HuPT-113D IgG1-AA(I) antibodies, the expression vectors pChPT-113, pHuPT-113D, and pHuPT-113D-AA were introduced into the chromosomes of the Chinese hamster ovary cell line CHO-K1 (ATCC, Virginia, Manassas), respectively. CHO-K1 cells were grown in SFM4CHO medium (HyClone, Utah, Logan) at 37°C in a 7.5% CO2 incubator. Stable transfection of CHO-K1 was performed by electroporation. Prior to transfection, each expression vector was linearized using FspI. 6Cells were transfected with 20 μg of linearized plasmid, suspended in SFM4CHO medium, and plated into several 96-well plates after appropriate cell dilution. After 48 hours, 10 μg / ml of puromycin was added for the isolation of stable transderm. Approximately 10 days after the start of selection, the culture supernatant of the transderm was assayed for antibody production by sandwich ELISA as described above. Stable CHO-K1 transderm producing high levels of ChPT-113 IgG1, HuPT-113D IgG1(I), and HuPT-113D-AA(I) antibodies (CHO-K1-ChPT-113 1D11, CHO-K1-HuPT-113D 1D6, and CHO-K1-HuPT-113D-AA 1D2, respectively) were tested using a PCR mycoplasma detection kit (Takara Bio USA, Madison, Wisconsin) and found to be negative for the presence of mycoplasma.

[0179] (Example 8) Purification of ChPT-113 and HuPT-113D IgG1 / Kappa(I) antibodies Each of the CHO-K1-ChPT-113 1D11, CHO-K1-HuPT-113D 1D6, and CHO-K1-HuPT-113D-AA 1D2 cells was incubated in a roller bottle with SFM4CHO for approximately 10 minutes. 6Cells were grown to a density of cells / ml, then given 1 / 10 volume of 35 mg / ml Cell Boost 4 (HyClone, Logan, Utah), and further grown until cell viability fell below 50%. After centrifugation and filtration, the culture supernatant was loaded onto a Protein A Sepharose column (HiTrap MabSelect SuRe, GE Healthcare, Piscataway, NJ). The column was washed with PBS, and then the antibody was eluted with 0.1 M glycine-HCl (pH 3.0). After neutralization with 1 M Tris-HCl (pH 8), the buffer of the eluted antibody was converted to PBS by dialysis. The antibody concentration was determined by measuring the absorbance at 280 nm (1.4 OD = 1 mg / ml). The yields from 500 ml of culture supernatant were 20 mg for ChPT-113 IgG1, 38 mg for HuPT-113D IgG1(I), and 16 mg for HuPT-113D IgG1-AA(I).

[0180] Purified ChPT-113 IgG1, HuPT-113D IgG1(I), and HuPT-113D IgG1-AA(I) antibodies were characterized by SDS-PAGE according to standard procedures. Analysis under reducing conditions showed that each of these antibodies consisted of a heavy chain with a molecular weight of approximately 50 kDa and a light chain with a molecular weight of approximately 25 kDa (Figure 16). The purity of each antibody appeared to be higher than 95%.

[0181] (Example 9) cDNA analysis of heavy and light chain mRNA sequences isolated from CHO-K1-ChPT-113 1D11, CHO-K1-HuPT-113D 1D6, and CHO-K1-HuPT-113D-AA 1D2 cells. The correctness of the heavy and light chains produced by CHO-K1-ChPT-113 1D11, CHO-K1-HuPT-113D 1D6, and CHO-K1-HuPT-113D-AA 1D2 cells was confirmed by cDNA sequencing. Total RNA was extracted from these cells using TRIzol reagent, and oligo-dT priming cDNA was synthesized using the ProtoScript M-MuLV First Strand cDNA synthesis kit (New England Biolabs, Ipswich, Massachusetts) according to the supplier's protocol. The coding region of the gamma heavy chain was amplified by PCR using CMV2 and JNT098 as primers and Phusion DNA polymerase (Thermo Fisher Scientific). The PCR fragments were gel-purified and subjected to sequencing using CMV2 and JNT098 as primers. Similarly, the coding region of the kappa light chain was amplified using CMV2 and JNT026 (Figure 17). The gel-purified DNA fragments were subjected to sequencing using CMV2 and JNT026 as primers. The obtained nucleotide sequences of the coding regions of the ChPT-113 IgG1 heavy chain, ChPT-113 IgG1 light chain, HuPT-113D IgG1(I) heavy chain, HuPT-113D IgG1(I) light chain, HuPT-113D IgG1-AA(I) heavy chain, and HuPT-113D IgG1-AA(I) light chain all corresponded perfectly to the corresponding sequences in the pChPT-113, pHuPT-113D, and pHuPT-113D-AA vectors (Figures 18-22).

[0182] (Example 10) Antigen binding of mouse PT-113 IgG2b and ChPT-113 IgG1 antibodies. The binding of purified mouse PT-113 IgG2b and ChPT-113 IgG1 antibodies to the following four Thr231-tau peptides (synthesized by AnaSpec, Fremont, California) was analyzed by ELISA: pThr-Dmp(H-KVAVVRT(PO3H2)XPKSPS-OH, X=5,5-dimethyl-L-proline) (Lot number 1556880) pThr-Pro(H-KVAVVRT(PO3H2)PPKSPS-OH) (Lot number 1556878) pThr-Ala(H-KVAVVRT(PO3H2)APKSPS-OH) (Lot number 1556881) np-Thr-Pro(H-KVAVVRTPPKSPS-OH)(Lot number 1556879)

[0183] The wells of the MaxiSorp plate were coated with 50 μl / well of each peptide dissolved in 1 μg / ml 2,2,2-trifluoroethanol. The subsequent ELISA procedure is described in Example 5, except that the detection of mouse PT-113 IgG2b was performed using HRP-conjugated goat anti-mouse kappa chain antibody (SouthernBiotech, Birmingham, Alabama). The binding patterns of mouse PT-113 IgG2b and ChPT-113 IgG1 with four peptides are shown in Figures 23 and 24, respectively. The overall binding trends with the four tested T231-tau peptides were similar between these two antibodies. In particular, both mouse PT-113 IgG2b and ChPT-113 IgG1 bound strongly to the pThr-Dmp-tau peptide (locked in cis-conformation) and poorly to the pThr-Ala-tau peptide (locked in trans-conformation). Furthermore, Figures 23 and 24 show that the binding of ChPT-113 IgG1 to the np-Thr-Pro-tau peptide (i.e., unphosphorylated T231-tau peptide) was weaker than that of mouse PT-113. These results demonstrate that the ChPT-113 IgG1 antibody shows less immunological cross-reactivity with the non-phosphorylated T231-tau peptide compared to that observed with the mouse PT-113 antibody.

[0184] (Example 11) Competitive binding assay using mouse PT-113 IgG2b and ChPT-113 IgG1 antibodies The affinity of mouse and chimeric PT-113 antibodies was evaluated by a competitive binding assay. Wells of a MaxiSorp plate were coated with pThr-Dmp-tau peptide and blocked with ELISA buffer as described in Example 5. After washing the wells, 50 μl of a mixture of 2 μg / ml biotinylated mouse PT-113 IgG2b antibody and various concentrations of competing antibodies (10 μg / ml and serially 3-fold dilutions) in ELISA buffer was applied to each well. After incubation at room temperature for 2 hours and washing with wash buffer, 50 μl of HRP-conjugated streptavidin (diluted 1 / 2,000 in ELISA buffer; SouthernBiotech, Birmingham, Alabama) was applied to each well for incubation at room temperature for 1 hour. After washing with wash buffer, color development was initiated with 50 μl / well of TMB substrate and stopped with 50 μl / well of 2N H2SO4. Absorbance was measured at 450 nm. The results of the competitive binding ELISA are shown in Figure 25. The competitive pattern when using mouse PT-113 IgG2b was similar to that when using ChPT-113 IgG1, indicating that the affinity for binding to the pThr-Dmp-tau peptide (locked in cis-conformation) is very similar between these two antibodies. However, complete blocking of biotinylated PT-113 binding was not observed even at the highest concentration used (10 μg / ml).

[0185] (Example 12) Analysis of HuPT-113D IgG1(I) and HuPT-113D IgG1-AA(I) antibodies regarding antigen binding. The antigen binding of purified HuPT-113D IgG1(I) and HuPT-113D IgG1-AA(I) antibodies to each of the four T231-tau peptides, pThr-Dmp, pThr-Pro, pThr-Ala, and np-Thr-Pro, was compared with that of ChPT-113 IgG1 by ELISA following the procedures described in Examples 5 and 10. As shown in Figure 26, these three antibodies showed similar binding patterns when using each of the four T231-tau peptides. In particular, binding to the pThr-Dmp-tau peptide (locked in cis-conformation) was almost indistinguishable among the ChPT-113 IgG1, HuPT-113D IgG1(I), and HuPT-113D IgG1-AA(I) antibodies. These results indicate that the antibody of this technology specifically binds to the cis-conformation of phosphorylated threonine 231-tau protein (cis-pT231-tau), and is therefore useful in methods for treating neurological diseases involving elevated cis-pT231-tau protein expression in subjects requiring it.

[0186] (Example 13) HuPT-113D(II) antibody Transient transfection. Using 12 mg of purified dual gene vector, 2 × 10⁶ transfections were performed. 9 3 × 10¹ GS-KO CHO cells were transfected. The transfected cells were placed in medium supplemented with the proprietary feed (II) and 1 mM glutamine, 3 × 10¹⁶ cells. 6 Cells were seeded at individual cells / ml and cultured in a 5 L (Generon, 931116) shaking flask at 31°C, 5% CO2, 85% humidity, and 140 rpm. Primary recovery. The 2L culture was recovered by centrifugation at 8000rpm, sterilized by 0.22μm filtration, and then purified of protein A. Protein A assay using Octet QKe. Clarified cell culture supernatant samples were analyzed using Octet QKe with Protein A Biosensors (ForteBio, 18-5010). 200 μL aliquots of the supernatant sample, filtered through a 0.22 μm filter, were loaded into 96-well plates and quantified against an 8-point standard curve. Samples with concentrations outside the standard curve range were re-analyzed after dilution.

[0187] Protein A affinity chromatography. For transient culture, the clarified supernatant was purified using one of four 5 mL HiTrap MabSelect SuRE columns on an AKT A purifier (running at 10 mL / min). In all cases, the column was equilibrated with 50 mM sodium phosphate, 125 mM sodium chloride, pH 7.0, washed with 50 mM sodium phosphate and 1 M sodium chloride, pH 7.0, then re-equilibrium was introduced, and elution was performed. Molecules were eluted with 10 mM sodium formate, pH 3.5. The eluted fraction was immediately pH-adjusted by neutralization with 2 × PBS buffer, pH 7.4, and titrated to approximately pH 7.2 by adding dilute sodium hydroxide solution. SE-HPLC. Dual samples were analyzed by SE-HPLC on an Agilent 1200 series HPLC system using a Zorbax GF-250 9.4 mm ID × 25 cm column (Agilent). 80 μl aliquots of 1 mg / ml sample (or stock concentration if the sample <1 mg / mL) were injected and run for 15 minutes at 1 mL / min in 50 mM sodium phosphate, 150 mM sodium chloride, 500 mM arginine, pH 6.0. Soluble aggregate levels were analyzed using Chemstation software. Signals from buffer components were analyzed by blank buffer injection and were omitted from data analysis unless otherwise indicated.

[0188] SDS-PAGE analysis. Reduced samples for analysis were prepared by mixing with NuPage 4x LOS sample buffer (Life Technologies, NP0007) and NuPage I Ox sample reducing agent (Life Technologies, NP0009), and incubated at 70°C for 10 minutes. For non-reduced samples, the reducing agent and heat incubation were omitted. Samples were electrophoresed on 1.5 mm NuPage 4-12% Bis-Tris Novex precast gels (Life Technologies, NP0315 / 6) under denaturing conditions using NuPage MES SOS running buffer. 10 μl aliquots of SeeBlue Plus 2 pre-stained molecular weight standard (Life Technologies, LC5925) and 1 mg / ml of control antibody were added to the gel. 1 μg of each sample was loaded onto the gel. After electrophoresis, the gels were stained with InstantBlue (TripleRed, ISBO 1 L) at room temperature for 30 minutes. Images of the stained gels were analyzed using the BioSpectrum Imaging System (UVP). Protein A purification results. Initial purification was performed using a single 5 mL column. This resulted in the recovery of 60.8 mg of material, corresponding to approximately 25% according to Octet measurements, and an antibody binding capacity of approximately 12 mg per gram of resin.

[0189] Subsequent purification was performed using four tandem 5 mL MabSelect SURE columns, yielding binding capacities exceeding the product titers for all three antibodies (i.e., HuPT-113D IgG1-AA(II), HuPT-113D IgG4(II), and HuPT-113D IgG1(II)). Purification of all three antibody products resulted in the elution of a single, clearly defined peak. Octet analysis of the unbound fractions indicated that all products were recovered. Figure 47 confirms the presence of antibody products with good levels of purity. The antibody products are well comparable to the control IgG1 antibody (lanes 10 and 11). For all products, two bands were observed under reducing conditions (lanes 5, 7, and 9) that corresponded to the sizes of the heavy chain (>49 kDa) and light chain (<28 kDa), and were comparable to the bands observed for the control antibody (lane 11). The interassay control IgG1 antibody (lanes 10 and 11) results were as predicted. Under non-reducing conditions, all products (lanes 2, 4, 6, and 8) showed protein species bands between 98 kDa and 198 kDa, comparable to the control IgG1 antibody (lane 10) run under the same conditions, and were consistent with the predicted molecular weight of the full-length antibody. For batch 455-030417-01 of HuPT-113D IgG1-AA(II), a band of approximately 198 kDa was observed in the non-reduced sample, indicating the presence of aggregates, which is consistent with the SE-HPLC results.

[0190] (Example 14) FcR activation by HuPT-113D antibody This study demonstrates that HuPT-113D IgG1-AA(II) and HuPT-113D IgG4(II) do not activate FcR (i.e., antibodies in this technology do not induce activation of immune cells expressing different Fc receptors). Methods and materials Ovalbumin was purchased from Sigma-Aldrich and stored at +2 to 8°C until use. Anti-ovalbumin (rabbit polyclonal) was purchased from Sigma-Aldrich and stored at -20°C until use. Anti-ovalbumin (mouse monoclonal IgG2a) was purchased from Biolegend and stored at +2 to 8°C until use. The following HuPT-113D antibodies were used: HuPT-113D IgG1-AA(II), HuPT-113D IgG4(II), and HuPT-113D IgG1(II).

[0191] The following peptides derived from human tau protein were used: 1) WT-pThr-Pro (KVAVVRpTPPKSPS), a 10-amino acid sequence derived from human tau. 2) pThr-Dmp(KVAVVRpT(5,5-dimethyl-L-proline)PKSPS), a 10-amino acid "cis-locked" sequence similar to the WT peptide. Both peptides (≥10 mg per peptide) were freeze-dried and stored at -80°C until reconstitution. The peptides were reconstituted in 2,2,2-trifluoroethanol (Fisher Scientific) at a concentration of 1 mg / mL, stored in multiple aliquots at -80°C, and the number of freeze-thaw cycles was minimized. The pThr-Dmp peptide was also biotinylated using the Lightening-Link® Rapid Biotin Kit (Innova Biosciences) and stored at -20°C until use.

[0192] Cell lines. THP-1 cells (DSMZ) were cultured in THP-1 culture medium (RPMI 1640(II) + 10% thermo-inactivated (hi)FBS((II))) and passaged three times a week. Jurkat cells (Promega) expressing the FcγRIIIa V158 variant and luciferase gene were cultured in Jurkat culture medium (RPMI 1640((II)), 10% hiFBS((II)), 100 μg / mL hygromycin (Life Technologies), 250 μg / mL G-418 sulfate solution (Promega), 1 mM sodium pyruvate (Life Technologies), 0.1 mM MEM non-essential amino acids (Life Technologies)) and passaged three times a week. Cell line FcR expression. THP-1 or Jurkat cells were harvested, washed with cold CellWASH solution (BD), and stained for cell surface expression of CD16 (FcγRIII), CD32 (FcγRII), and CD64 (FcγR1). An FcR blocking step (Biolegend) was also included before the addition of staining antibodies to minimize nonspecific binding. FcR expression was examined by flow cytometry using anti-CD16 A647, anti-CD32 PE, and anti-CD64 A488 (Biolegend) and analyzed using the Guava® easyCyte 8HT system (Merck Millipore).

[0193] HuPT-113D enzyme-linked immunosorbent assay. Wells of a MaxiSorp plate (Fisher Scientific) were coated overnight at +4°C with 1, 10, or 100 μL / well of pThr-Dmp peptide dissolved in 2,2,2-trifluoroethanol or DPBS((II)). The wells were then washed with washing buffer (DPBS + 0.05% Tween-20 (Sigma-Aldrich)) and blocked at room temperature for 60 minutes with DPBS + 1% BSA (Miltenyi Biotec). The wells were washed with washing buffer, and 100 μL / well of one of the following HuPT-113D antibodies (e.g., HuPT-113D IgG1-AA(II), HuPT-113D IgG4(II), or HuPT-113D IgG1(II)) was added in a two-strand configuration at room temperature for 60 minutes. After washing the wells with washing buffer, the bound antibodies were detected using biotinylated anti-human Fc antibody and streptavidin-HRP, and the signals were detected using 100 μL of TMB substrate (Sigma-Aldrich). The reaction was stopped by adding 50 μL of TMB stop solution (Sigma-Aldrich), and the absorbance was measured at 450 nm.

[0194] THP-1 cytokine assay. Assay plates (Lumitrac 600, Greiner Bio-One) were coated overnight at +4°C with 100 μL / well of ovalbumin or 2,2,2-trifluoroethanol or pThr-Dmp peptide dissolved in DPBS in DPBS. The wells were then washed with DPBS and blocked at room temperature for 60 minutes with 200 μL of blocking medium (RPMI + 5% very low IgG serum (GE Life Sciences)). After blocking, 100 μL of antibody (anti-ovalbumin or HuPT-113D antibody (e.g., HuPT-113D IgG1-AA(II), HuPT-113D IgG4(II), or HuPT-113D IgG1(II))) per well was added at room temperature for 60 minutes. In some assays, unbound antibodies were removed by washing with a washing buffer, and THP-1 cells were added in 100 μl assay medium (RPMI + 0.5% ultra-low IgG serum) per well and incubated at 37°C, 5% CO2, and a humidified atmosphere for 24 hours. The supernatant samples were collected and centrifuged at 300x G for 10 minutes to collect any cells, which were then stored at -80°C for cytokine analysis. Cytokine levels in the supernatant (TNFα, IL-1β, and IL-6) were assayed using the Milliplex® assay (Merck Millipore) on a MAGPIX® multiplexing platform (Luminex).

[0195] Jurkat FcRIIIa activation assay. Assay plates were coated overnight at +4°C with 100 μL / well of ovalbumin or 2,2,2-trifluoroethanol or pThr-Dmp peptide dissolved in DPBS in DPBS. The wells were then washed with DPBS and blocked at room temperature for 60 minutes with 200 μL of blocking medium (RPMI + 5% very low IgG serum (GE Life Sciences)). After blocking, 100 μL of antibody (anti-ovalbumin or HuPT-113D antibody (e.g., HuPT-113D IgG1-AA(II), HuPT-113D IgG4(II), or HuPT-113D IgG1(II))) per well was added at room temperature for 60 minutes. In some assays, unbound antibodies were removed by washing with a washing buffer, and Jurkat cells were added in 100 μL of assay medium (RPMI + 0.5% ultra-low IgG serum) per well and incubated for 24 hours at 37°C, 5% CO2, in a humidified atmosphere. FcγRIIIa activation was assessed by adding BioGlo® reagent for 10 minutes, and RLU was measured by a GloMax® luminometer (Promega).

[0196] result THP-1 cell Fc receptor expression. THP-1 cells were stained with antibodies against three major Fc receptors (CD16, CD32, and CD64) to evaluate cell surface expression. Figures 30A-C show histogram plots of each FcR, with unstained cells in red and cells stained with anti-FcR antibodies in blue. THP-1 cells appear to express high levels of CD32 (FCγRII) and CD64 (FCγRI), but little to no expression of CD16 (FCγRIII). This indicates that the THP-1 assay primarily evaluated HuPT-113D IgG1-AA(II), HuPT-113D IgG4(II), and HuPT-113D IgG1(II) binding and activation at CD32 and CD64, while the Jurkat assay evaluated HuPT-113D IgG1-AA(II), HuPT-113D IgG4(II), and HuPT-113D IgG1(II) binding and activation at CD16 only. Jurkat FcγRIIIa(CD16)V158 cells were also evaluated by flow cytometry, but no FcR expression was detected (data not shown). This was predictable, as CD16 levels are extremely low in Jurkat, and signaling is normally detected by luciferase reporter gene assays. The manufacturer provides data showing inhibition of signaling when an anti-CD16 blocking antibody is added, which indicates that luciferase signaling is CD16-dependent. Activation of THP-1 cells by an immune complex (IC) induced by a positive control. Immobilization of ICs (by coating plates with antigens) increased cross-linking and subsequent activation of FcRs. ICs contain ovalbumin protein and anti-ovalbumin antibodies. ICs were prepared using two different anti-ovalbumin antibodies separately: one mouse monoclonal antibody and one rabbit polyclonal antibody. While rabbit polyclonal antibodies are known to stimulate immune cells, mouse monoclonal antibodies were not tested in these assays. The mouse monoclonal antibody was included to mimic the monoclonal nature of the HuPT-113D antibody.

[0197] Figures 31A–D show the effects of ovalbumin IC coating density and cell number on THP-1 cell activation. Ovalbumin IC was prepared using ovalbumin-coated wells and rabbit polyclonal anti-ovalbumin antibody (the wells were not washed after adding the antibody to the ovalbumin-coated wells, so some free antibody may be present under some conditions). The two upper plots show TNFα secretion in response to incremental amounts of IC (0.1, 1, and 10 μg / mL ovalbumin coating and 50 μg / mL fixed amount of rabbit antibody) and THP-1 cells (5,000 and 50,000 cells / well), while the two lower plots show IL-6 secretion. Increasing the amount of ovalbumin IC and the number of THP-1 cells led to increased levels of TNFα and IL-6 secreted during the assay.

[0198] Figures 32A-D (TNFα release) and 33A-D (IL-6 release) show further optimization of IC concentration and THP-1 cell number using mouse monoclonal antibodies to produce ovalbumin ICs. Figures 32A-B and 33A-B use 50,000 THP-1 cells / well, and Figures 32C-D and 33C-D use 150,000 THP-1 cells / well. Figures 32A and C, and 33A and C, show data when 0.1 μg / mL ovalbumin is used to coat the wells. Figures 32B and D, and 33B and D, show data when 10 μg / mL ovalbumin is used to coat the wells. Figures 32A-D and 33A-D show that using 150,000 THP-1 cells / well and ovalbumin coated with 10 μg / mL resulted in the highest levels of cytokine release, and that THP-1 cell activation was dose-dependent on the antibody. The data demonstrate that the THP-1 cytokine assay can be used to assess the ability of antibodies to bind to cells after binding to immobilized protein antigens, and subsequently to activate them.

[0199] Activation of Jurkat cells induced by a positive control. This study demonstrated FcR activation in Jurkat cells by a well-characterized immune complex (IC). Immobilization of the IC (by coating plates with antigen) increased crosslinking and subsequent FcR activation. The IC contains ovalbumin protein and anti-ovalbumin antibody. Two different anti-ovalbumin antibodies were used separately to construct the IC; one mouse monoclonal antibody and one rabbit polyclonal antibody. Antibodies had not been tested in these assays to date. The mouse monoclonal antibody was included to mimic the monoclonality of the HuPT-113D antibodies used in this study (e.g., HuPT-113D IgG1-AA(II), HuPT-113D IgG4(II), and HuPT-113D IgG1(II)). Figures 34A and 34B show the effects of ovalbumin IC concentration and Jurkat cell number on FcR activation. Ovalbumin IC was prepared using ovalbumin-coated wells (10 μg / mL) and a range of concentrations of both rabbit polyclonal and mouse monoclonal anti-ovalbumin antibodies (since the wells were not washed after adding the antibodies to the ovalbumin-coated wells, some free antibody may be present under some conditions). Figure 34A shows FcR activation using 50,000 Jurkat cells / well, and Figure 34B shows activation using 100,000 Jurkat cells / well. Both plots show FcR activation with rabbit polyclonal antibody but not with mouse monoclonal antibody, and 100,000 Jurkat cells produced a stronger response than 50,000 Jurkat cells / well.

[0200] The data demonstrate that the Jurkat FcR activation assay can be used to assess the ability of antibodies to bind to and subsequently activate FcγRIIIa after binding to an immobilized protein antigen. The data also suggest that mouse antibodies are not as potent as rabbit antibodies in activating FcγRIIIa. HuPT-113D peptide-binding ELISA. All three HuPT-113D antibodies (HuPT-113D IgG1-AA(II), HuPT-113D IgG4(II), and HuPT-113D IgG1(II)) successfully formed ICs, and the binding of HuPT-113D antibodies to pThr-Dmp peptides was evaluated using binding ELISA. Dose-response data for each HuPT-113D antibody were prepared using ELISA plates coated with 1, 10, and 100 μg / mL of pThr-Dmp peptide in 2,2,2-trifluoroethanol.

[0201] Figure 35 shows the binding curves for each antibody at each pThr-Dmp peptide coating concentration. All three HuPT-113D antibodies yielded remarkably similar binding curves, with no effect from increasing pThr-Dmp peptide coating concentration, suggesting that a concentration of 1 μg / mL already saturated the wells. The EC50 value for all conditions was approximately 21 ng / mL (140 pM). Optimization of HuPT-113D pThr-Dmp peptide coating in a THP-1 cytokine assay. This study was conducted to investigate whether 2,2,2-trifluoroethanol has any effect on THP-1 cells. The pThr-Dmp peptide was diluted to 1 μg / mL with either 2,2,2-trifluoroethanol or DPBS and used to coat the wells of the assay plate. After coating, the wells were washed with DPBS, and a range of HuPT-113D IgG1(II) concentrations were added to induce IC formation, after which THP-1 cells were added (150, 1000 cells / well). After 24 hours, TNFα and IL-6 levels were assessed. Figure 36A shows the level of TNFα secretion, and Figure 36B shows the level of IL-6 secretion. The data show that the secretion of both cytokines increased in a dose-dependent manner, and there was no significant difference between the 2,2,2-trifluoroethanol and DPBS peptide coating conditions.

[0202] Optimization of HuPT-113D pThr-Dmp peptide coating in a Jurkat FcR activation assay. This study was conducted to investigate whether 2,2,2-trifluoroethanol has any effect on Jurkat cells. The pThr-Dmp peptide was diluted to 1 μg / mL with either 2,2,2-trifluoroethanol or DPBS and used to coat the wells of the assay plate. After coating, the wells were washed with DPBS, and a range of HuPT-113D IgG1(II) concentrations were added to induce IC formation, after which Jurkat cells were added (100,000 cells / well). After 24 hours, FcγRIIIa activation was evaluated using BioGlo® reagent. Figure 37 shows the level of FcγRIIIa activation and each peptide coating condition. There was no significant difference between the 2,2,2-trifluoroethanol and DPBS peptide coating conditions, suggesting that neither could activate FcγRIIIa.

[0203] HuPT-113D THP-1 Cytokine Assay 1. ICs were prepared using both natural and biotinylated pThr-Dmp peptides to compare the ability of each HuPT-113D antibody (e.g., HuPT-113D IgG1-AA(II), HuPT-113D IgG4(II), and HuPT-113D IgG1(II)) to induce cytokine secretion in a THP-1 cell assay. Wells of assay plates were coated with 1 μg / mL of the natural peptide (pThr-Dmp peptide), and wells of streptavidin pre-coated plates (Greiner Bio-one) were coated with the biotinylated peptide. After overnight coating with the peptides, both plates were washed, and serial dilutions of HuPT-113D IgG1-AA(II), HuPT-113D IgG4(II), or HuPT-113D IgG1(II) were added to the plates in triplicates. To ensure that only ICs were present when THP-1 cells were added, a further washing step was included after the addition of the HuPT-113D antibody. This further washing step removed all free HuPT-113D antibodies that could act as blocking antibodies and interfere with ICs immobilized on the plate that crosslink FcRs.

[0204] Figures 38A-C show the induction of cytokine release (TNFα, IL-1β, and IL-6) by each HuPT-113D antibody (e.g., HuPT-113D IgG1-AA(II), HuPT-113D IgG4(II), and HuPT-113D IgG1(II)) and the natural pThr-Dmp peptide. Figures 38D-F show the induction of cytokine release (TNFα, IL-1β, and IL-6) by each HuPT-113D antibody and the biotinylated pThr-Dmp peptide. HuPT-113D IgG1(II) is shown in blue, HuPT-113D IgG1-AA(II) in red, and HuPT-113D IgG4(II) in green. All three HuPT-113D antibodies induced cytokine release in a dose-dependent manner, but each antibody tended to produce different curves in some cases, with the IgG1 antibody in particular resulting in lower maximum cytokine release levels. However, when natural peptides were used to construct ICs, a similar pattern was observed for all three cytokines, with IgG1 antibody showing the maximum efficacy and IgG1-AA showing the lowest efficacy for cytokine induction. The biotinylated peptide plot showed a similar pattern, with IgG1-AA being the least potent stimulator of cytokine release.

[0205] Direct comparison of results with natural and biotinylated peptides showed that biotinylated peptides increased the background level of cytokine release. This is likely due to streptavidin used to coat the plates, rather than the biotinylated peptides themselves. To keep the background level of THP-1 cell activation as low as possible, subsequent assays coated plates with natural pThr-Dmp peptides and generated ICs. Figure 39 shows the EC50 values ​​calculated for three types of HuPT-113D antibodies (natural peptides). The IgG1 antibody had the lowest EC50 value for all three cytokines, while IgG1-AA had the highest, suggesting that the IgG1-AA antibody had the lowest ability to induce cytokine release from THP-1 cells. HuPT-113D Jurkat FcR Activation Assay 1. To compare the ability of each HuPT-113D antibody (e.g., HuPT-113D IgG1-AA(II), HuPT-113D IgG4(II), and HuPT-113D IgG1(II)) to activate FcγRIIIa in a Jurkat cell assay, ICs were generated using the native pThr-Dmp peptide (wells of the assay plate were coated with the native peptide at 1, 10, or 100 μg / mL). The plates were then washed, and serial dilutions of HuPT-113D IgG1-AA(II), HuPT-113D IgG4(II), and HuPT-113D IgG1(II) were added to the plates in triplicates. A further washing step was included after the addition of the HuPT-113D antibodies to ensure that only ICs were present when Jurkat cells were added. This further washing step removed all free HuPT-113D antibodies that could act as blocking antibodies and interfere with IC immobilized on the plate to crosslink FcγRIIIa. Figure 39 shows the activation of FcγRIIIa by each of the HuPT-113D antibodies. The IgG1 antibody was able to activate Jurkat cells in a dose-dependent manner, generating similar curves for each peptide coating concentration. The IgG1-AA and IgG4 antibodies showed no activation of Jurkat cells at all.

[0206] HuPT-113D THP-1 Cytokine Assay 2. The THP-1 cytokine assay was repeated to confirm that HuPT-113D IgG1-AA(II) and HuPT-113D IgG4(II) antibodies are indeed less potent inducers of cytokine release. Plates were coated with 1 μg / mL of native peptide to include a wider dose range for each antibody. Any unbound antibodies were washed off before adding THP-1 cells to ensure that only ICs were present. Figures 41A-C show plots of each HuPT-113D antibody for each of the three cytokines. The plots are similar to those seen above in Figures 38A-F, where IgG1 is the most potent inducer of cytokine release in THP-1 cells, and IgG1-AA is the least potent inducer.

[0207] Figure 42 shows the EC50 values ​​calculated for the three HuPT-113D antibodies. The IgG1 antibody had lower EC50 values ​​for all three cytokines than the IgG1-AA and IgG4 antibodies, suggesting that the IgG1-AA and IgG4 antibodies have a lower ability to induce cytokine release from THP-1 cells. HuPT-113D Jurkat FcR Activation Assay. The Jurkat FcR activation assay was repeated to confirm that IgG1-AA and IgG4 antibodies are indeed less potent inducers of FcR activation. Plates were coated with 1 μg / mL of native peptide to include a larger dose range for each antibody. Any unbound antibodies were washed off before adding Jurkat cells to ensure that only ICs were present. Figure 43 shows the plots for each HuPT-113D antibody. The plots are similar to those seen above in Figure 40, showing that only the IgG1 antibody can activate FcγRIIIa.

[0208] The results also show that HuPT-113D IgG1(II) induced dose-dependent responses to all three cytokines (TNFα, IL-1β, and IL-6) in the THP-1 cytokine assay. The results also indicate that the IgG1-AA and IgG4 versions are less potent cytokine inducers and have EC50 values ​​up to 30 times higher than those of IgG1. The results indicate that HuPT-113D IgG1(II) induced a dose-dependent response in the Jurkat FcγRIIIa activation assay. No activation was observed for HuPT-113D IgG1-AA(II) and HuPT-113D IgG4(II) antibodies. The results indicate that HuPT-113D IgG1-AA(II) and HuPT-113D IgG4(II) exhibit a lower ability to bind to and activate immune cells via their FcR, and should therefore be considered to pose a lower risk of inducing unwanted immune responses in the target population. These results demonstrate that the HuPT-113D antibodies of this technology (e.g., HuPT-113D IgG1-AA(II) and HuPT-113D IgG4(II)) do not activate FcγRIIIa, indicating that the antibodies of this technology do not induce a cytotoxic response in the target organism.

[0209] (Example 15) Jurkat FcγRIIIa V158 Activation Assay - Thawing and Use Kit vs. Proliferating Cell Line This study evaluated the ability of the antibody of this technology to activate FcRIIIa V158 cells. Methods. Wells were coated with 1 μg / ml of pThr-Dmp peptide in TFE. HuPT-113D IgG1-AA(II), HuPT-113D IgG4(II), or HuPT-113D IgG1(II) was added for 60 minutes. Jurkat cells were added for 24 hours. After 24 hours, BioGlo® reagent was added, and luminescence was read using GloMax®. Results: Only the HuPT-113D IgG1(II) antibody was able to activate the cells. See Figures 44A-B. The results indicate that activation was not achieved by HuPT-113D IgG1-AA(II) and HuPT-113D IgG4(II) antibodies. See Figures 44A-B. Cells grown, thawed, and used showed similar results. These results demonstrate that the HuPT-113D antibodies of this technology (e.g., HuPT-113D IgG1-AA(II) and HuPT-113D IgG4(II)) do not activate FcγRIIIa, indicating that the antibodies of this technology do not induce a cytotoxic response in the target organism.

[0210] (Example 16) Jurkat FcγRIIIa and FcγRIIa activation using HuPT-113D antibody This study evaluated the ability of the antibody of this technology to activate FcγRIIIa V158, FcγRIIIa F158, and FcγRIIa H131. method Wells were coated with 1 μg / ml of pThr-Dmp peptide in TFE. HuPT-113D IgG1-AA(II), HuPT-113D IgG4(II), or HuPT-113D IgG1(II) was added for 60 minutes. Jurkat cells were added for 24 hours. After 24 hours, BioGlo® reagent was added, and luminescence was read using GloMax®.

[0211] result HuPT-113D IgG1(II) activated FcγRIIIa V158 and FcγRIIa H131 cells. See Figures 45A and C. HuPT-113D IgG4(II) showed low levels of activation in FcγRIIa H131 cells and no activation at all in FcγRIIIa V158 cells. See Figures 45A and C. HuPT-113D IgG1-AA(II) did not activate FcγRIIIa V158 or FcγRIIa H131 cells. See Figures 45A and C. No antibody activated FcγRIIIa F158. See Figure 45B. These results demonstrate that the HuPT-113D antibodies of this technology (e.g., HuPT-113D IgG1-AA(II) and HuPT-113D IgG4(II)) do not activate FcγRIIIa, indicating that the antibodies of this technology do not induce a cytotoxic response in the target organism.

[0212] (Example 17) Jurkat FcγRIIIa V158 Activation Assay - Proliferating Cells This study evaluated HuPT-113D activation in FcγRIIIa V158 cells. method Wells were coated with 10 μg / ml pTau in DPBS. HuPT-113D IgG1-AA(II), HuPT-113D IgG4(II), or HuPT-113D IgG1(II) was added for 60 minutes. Jurkat cells were added for 24 hours. After 24 hours, BioGlo® reagent was added, and luminescence was read using GloMax®. result No antibody induced activation of FcγRIIIa V158 cells. See Figure 46. These results demonstrate that the HuPT-113D antibodies of this technology (e.g., HuPT-113D IgG1-AA(II) and HuPT-113D IgG4(II)) do not activate FcγRIIIa, indicating that the antibodies of this technology do not induce a cytotoxic response in the target organism.

[0213] (Example 18) Prevention of traumatic brain injury (TBI) using HuPT-113D antibodies This study demonstrates that antibodies of this technology (e.g., HuPT-113A, HuPT-113B, HuPT-113C, HuPT-113D, HuPT-113D IgG1-AA(II), and HuPT-113D IgG4(II)) are useful in preventing TBI. method wtC57 / Bl6 mice were treated with a single dose of HuPT-113D antibody or a control antibody (200 μg ip = approximately 8 mg / kg) three days prior to TBI. The TBI model was selected from the following: single severe TBI (blast; ssTBI), recurrent mild TBI (impact; rmTBI), or single mild TBI (impact; smTBI). Fifteen minutes prior to trauma, all mice were treated with a single dose of HuPT-113D antibody or a control antibody (20 μg / 5 μl) by intraventricular administration. In some embodiments, mice were treated with three dose levels, e.g., 3, 10, and 30 mg / kg.

[0214] In some embodiments, a sham group of mice (i.e., without TBI) is used as a control. The first cohort (mice pre-treated for 12 days with 8 mg / kg ip every 4 days (total of 3 doses)) were sacrificed 14 days after trauma for ELISA / Western blot analysis (e.g., to evaluate cystau and total (intermediate domain) tau in the brain and CSF) and fEPSP. A second cohort (mice treated as described above but with continued treatment, 8 mg / kg ip weekly for an additional 6 weeks) will be sacrificed 2 months after trauma. Behavioral endpoints will be evaluated before sacrifice (e.g., EPM, MWM, and spontaneous movement tests (rotorod)). A third cohort (mice that continued to receive 8 mg / kg ip weekly for up to 6 months post-traumatic stress) will be sacrificed, and tau aggregation (e.g., cystau and total tau) and neuronal atrophy will be evaluated. Other endpoints include assays for IHC and assays using ALZ50, MC1, and / or AT8. result Mice treated with antibodies using this technology are predicted to show one or more of the following compared to untreated control mice: reduced cystauosis, reduced incidence and spread of tauopathy, reduced neurodegeneration, and improved histopathological and functional outcomes. These results indicate that antibodies based on this technology (e.g., HuPT-113A, HuPT-113B, HuPT-113C, HuPT-113D, HuPT-113D IgG1-AA(II), and HuPT-113D IgG4(II)) are useful in preventing neurological diseases such as TBI that involve increased cis-pT231-tau protein expression.

[0215] (Example 19) Prevention of neurodegenerative states using HuPT-113D antibody in a TMHT mouse model of Alzheimer's disease This study demonstrates that antibodies based on this technology (e.g., HuPT-113A, HuPT-113B, HuPT-113C, HuPT-113D, HuPT-113D IgG1-AA(II), and HuPT-113D IgG4(II)) are useful in preventing neurodegenerative conditions such as Alzheimer's disease. method TMHT (Thy-1 mutant human tau) transgenic mice (Flunkert et al., 2013) are treated with HuPT-113D antibody or a control antibody (between 1 and 100 mg / kg) starting at 1 month of age. Mice are treated weekly with HuPT-113D antibody or the control antibody by intraperitoneal (IP) injection, subcutaneous (SC) injection, or intravenous (IV) administration. Alternative assays may use different dose frequencies, but are not limited to daily, every 2 days, every 3 days, every 4 days, every other week, every 3 weeks, and monthly. Wild-type (WT) mice are used as controls.

[0216] The first cohort (mice treated with 1–100 mg / kg IP weekly for 2 months) were sacrificed at 3 months of age for ELISA / Western blot analysis (e.g., to evaluate cystau and total (intermediate domain) tau in the brain and CSF). The second cohort (mice treated with 1–100 mg / kg IP weekly for 4 months) will be sacrificed at 5 months of age. Behavioral endpoints will be evaluated before sacrifice (e.g., general indicators of spontaneous motility, including MWM, elevated cruciform maze, novel object recognition, rotational ability, and olfaction). A third cohort (mice treated with 1–100 mg / kg IP weekly for 8 months until 9 months of age) will be sacrificed, and tau aggregation (e.g., cystau and total tau) and neuronal atrophy will be evaluated. Other endpoints include assays for IHC and assays using ALZ50, MC1, and / or AT8. result Mice treated with antibodies using this technology are predicted to show one or more of the following compared to untreated control mice or mice treated with control antibodies that do not recognize cystau: reduced cystauosis, reduced overall tauopathy incidence and spread, reduced neurodegeneration, and improved histopathological and functional outcomes. These results indicate that antibodies based on this technology (e.g., HuPT-113A, HuPT-113B, HuPT-113C, HuPT-113D, HuPT-113D IgG1-AA(II), and HuPT-113D IgG4(II)) are useful in preventing neurological diseases such as Alzheimer's disease that involve elevated cis-pT231-tau protein expression.

[0217] (Example 20) Treatment of neurodegenerative conditions using HuPT-113D antibody in a THMT mouse model of Alzheimer's disease This study demonstrates that antibodies of this technology (e.g., HuPT-113A, HuPT-113B, HuPT-113C, HuPT-113D, HuPT-113D IgG1-AA(II), and HuPT-113D IgG4(II)) are useful in treating neurodegenerative conditions such as Alzheimer's disease. method TMHT (Thy-1 mutant human tau) transgenic mice (Flunkert et al., 2013) were treated with HuPT-113D antibody or a control antibody (200 μg ip = approximately 8 mg / kg) starting at 5 months of age. Wild-type mice were used as controls.

[0218] Mice are treated with 8 mg / kg ip every 4 days for 12 days (a total of 3 treatments). Behavioral endpoints (e.g., MWM, olfaction) are evaluated before sacrifice. Mice are then sacrificed for ELISA / Western blot analysis (e.g., to evaluate cystau and total (intermediate domain) tau in the brain and CSF). In an alternative assay, mice are treated with three different escalating dose levels for each treatment, e.g., 3, 10, and 30 mg / kg. result Mice treated with antibodies using this technology are predicted to show one or more of the following compared to control antibody-treated mice and untreated control mice: reduced cystauosis, reduced tauopathy incidence and spread, reduced neurodegeneration, and improved histopathological and functional outcomes. These results indicate that antibodies of this technology (e.g., HuPT-113A, HuPT-113B, HuPT-113C, HuPT-113D, HuPT-113D IgG1-AA(II), and HuPT-113D IgG4(II)) are useful for treating neurological diseases accompanied by elevated cis-pT231-tau protein expression, such as Alzheimer's disease.

[0219] (Example 21) Prevention of neurodegenerative states using HuPT-113D antibody in an hTau mouse model of Alzheimer's disease This study demonstrates that antibodies based on this technology (e.g., HuPT-113A, HuPT-113B, HuPT-113C, HuPT-113D, HuPT-113D IgG1-AA(II), and HuPT-113D IgG4(II)) are useful in preventing neurodegenerative conditions such as Alzheimer's disease. method hTau transgenic mice (Andorfer et al., 2003) were treated with HuPT-113D antibody or a control antibody (200 μg ip = approximately 8 mg / kg) starting at 1 month of age. Wild-type mice were used as controls.

[0220] The first cohort (mice treated with 8 mg / kg ip every 4 days for 5 months) was sacrificed at 6 months of age for ELISA / Western blot analysis (e.g., to evaluate cystau and total (intermediate domain) tau in the brain and CSF). A second cohort (mice receiving 8 mg / kg ip weekly for 11 months, continuing until 12 months of age). Behavioral endpoints (e.g., MWM, novel object recognition) will be assessed before sacrifice. Tau aggregation (e.g., cystau and total tau) and neuronal atrophy will be assessed. Other endpoints include assays for IHC and assays using ALZ50, MC1 and / or AT8. result Mice treated with antibodies using this technology are predicted to show one or more of the following compared to control antibody-treated mice and untreated control mice: reduced cystauosis, reduced tauopathy incidence and spread, reduced neurodegeneration, and improved histopathological and functional outcomes. These results indicate that antibodies based on this technology (e.g., HuPT-113A, HuPT-113B, HuPT-113C, HuPT-113D, HuPT-113D IgG1-AA(II), and HuPT-113D IgG4(II)) are useful for preventing neurological diseases such as Alzheimer's disease that involve elevated cis-pT231-tau protein expression.

[0221] (Example 22) Treatment of neurodegenerative conditions using HuPT-113D antibody in an hTau mouse model of Alzheimer's disease This study demonstrates that antibodies of this technology (e.g., HuPT-113A, HuPT-113B, HuPT-113C, HuPT-113D, HuPT-113D IgG1-AA(II), and HuPT-113D IgG4(II)) are useful in treating neurodegenerative conditions such as Alzheimer's disease. method hTau transgenic mice (Andorfer et al., 2003) were treated with HuPT-113D antibody or a control antibody (200 μg ip = approximately 8 mg / kg) starting at 12 months of age. Wild-type mice were used as controls. Mice are treated with 8 mg / kg ip every 4 days for 12 days (total of 3 doses). Behavioral endpoints are evaluated before sacrifice (e.g., MWM, novel object recognition). Mice are sacrificed at 3 months of age for ELISA / Western blot analysis (e.g., to evaluate cystau and total (intermediate domain) tau in the brain and CSF). In an alternative assay, mice are treated with three different escalating dose levels for each treatment, e.g., 3, 10, and 30 mg / kg.

[0222] result Mice treated with antibodies using this technology are predicted to show one or more of the following compared to control antibody-treated mice and untreated control mice: reduced cystauosis, reduced tauopathy incidence and spread, reduced neurodegeneration, and improved histopathological and functional outcomes. These results indicate that antibodies of this technology (e.g., HuPT-113A, HuPT-113B, HuPT-113C, HuPT-113D, HuPT-113D IgG1-AA(II), and HuPT-113D IgG4(II)) are useful for treating neurological diseases accompanied by elevated cis-pT231-tau protein expression, such as Alzheimer's disease.

[0223] (Example 23) Treatment of traumatic brain injury (TBI) using HuPT-113D antibody This study demonstrates that antibodies of this technology (e.g., HuPT-113A, HuPT-113B, HuPT-113C, HuPT-113D, HuPT-113D IgG1-AA(II), and HuPT-113D IgG4(II)) are useful for treating TBI. method wtC57 / Bl6 mice are treated immediately after TBI with a single dose of HuPT-113D antibody or a control antibody (200 μg ip = approximately 8 mg / kg). The TBI model is selected from the following: single severe TBI (blast; ssTBI), recurrent mild TBI (impact; rmTBI), or single mild TBI (impact; smTBI). Fifteen minutes after trauma, all mice are treated by intraventricular administration with a single dose of HuPT-113D antibody or a control antibody (20 μg / 5 μl). In some embodiments, mice are treated with three dose levels, e.g., 3, 10, and 30 mg / kg. In some embodiments, a sham group of mice (i.e., without TBI) is used as a control. The first cohort (mice treated with 8 mg / kg ip every 4 days for 12 days (total of 3 doses)) were sacrificed 14 days after trauma for ELISA / Western blot analysis (e.g., to evaluate cystau and total (intermediate domain) tau in the brain and CSF) and fEPSP. A second cohort (mice treated as described above but with continued treatment, 8 mg / kg ip weekly for an additional 6 weeks) will be sacrificed 2 months after trauma. Behavioral endpoints will be evaluated before sacrifice (e.g., EPM, MWM, and spontaneous movement tests (rotorod)).

[0224] A third cohort (mice that continued to receive 8 mg / kg ip weekly for up to 6 months post-traumatic stress) will be sacrificed, and tau aggregation (e.g., cystau and total tau) and neuronal atrophy will be evaluated. Other endpoints include assays for IHC and assays using ALZ50, MC1, and / or AT8. result Mice treated with antibodies using this technology are predicted to show one or more of the following compared to untreated control mice: reduced cystauosis, reduced incidence and spread of tauopathy, reduced neurodegeneration, and improved histopathological and functional outcomes. These results indicate that antibodies of this technology (e.g., HuPT-113A, HuPT-113B, HuPT-113C, HuPT-113D, HuPT-113D IgG1-AA(II), and HuPT-113D IgG4(II)) are useful in treating neurological disorders accompanied by elevated cis-pT231-tau protein expression, such as TBI.

[0225] Equal portions This technology should not be limited to the specific embodiments described in this application, but is intended as a single example of individual aspects of this technology. As will be apparent to those skilled in the art, many modifications and alterations of this technology can be made without departing from its spirit and scope. In addition to those enumerated herein, functionally equivalent methods and apparatus within the scope of this technology will be apparent to those skilled in the art from the foregoing description. Such modifications and alterations are intended to fall within the scope of the appended claims. This technology should be limited only by the terminology of the appended claims and the entire scope of equivalents to which such claims are given. It should be understood that this technology is not limited to specific methods, reagents, compound compositions or biological systems, which are of course subject to change. It should also be understood that the terminology used herein is intended solely to describe specific embodiments and is not intended to be limiting.

[0226] Furthermore, if any feature or aspect of this disclosure is described from the perspective of the Markush Group, a person skilled in the art will recognize that the disclosure may also be described from the perspective of any individual member or subgroup of a member of the Markush Group. As will be understood by those skilled in the art, and for all purposes, particularly in terms of providing written explanations, all scopes disclosed herein also encompass all possible subscopes and combinations thereof. The enumerated scopes are readily recognizable as being adequately described and effective when the same scope is broken down into at least equal halves, thirds, quarters, fifths, tenths, etc., etc., by each scope discussed herein can be readily broken down into the lower third, middle third, upper third, etc. Also, as will be understood by those skilled in the art, all language such as “up to,” “at least,” “greater than,” and “less than” includes the enumerated numbers and refers to scopes that can subsequently be broken down into the subscopes discussed above. Finally, as will be understood by those skilled in the art, a scope includes each individual member. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc.

[0227] All patents, patent applications, provisional applications, and publications referenced or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent that they do not conflict with the express teachings herein. Other embodiments are shown in the following claims.

Claims

1. An antibody comprising the heavy chain sequence shown in SEQ ID NO: 24 and the light chain sequence shown in SEQ ID NO: 51, It binds to the epitope of phosphorylated threonine 231-tau protein (i.e., pT231-tau protein) containing the amino acid sequence KVAVVRTPPKSPS (SEQ ID NO: 56), The antibody preferentially binds to pT231-tau protein locked in cis-conformation compared to pT231-tau protein locked in trans-conformation.

2. The antibody according to claim 1, which specifically binds to the cis-conformation of the pT231-tau protein.

3. Recombinant nucleic acid encoding the antibody according to claim 1.

4. A host cell or vector comprising the recombinant nucleic acid described in claim 3.

5. A composition comprising the antibody described in claim 1 and a pharmaceutically acceptable carrier.

6. A composition for the treatment of neurological disorders accompanied by elevated cis-pT231-tau protein expression in subjects requiring treatment of such disorders, comprising an antibody containing the heavy chain immunoglobulin variable domain sequences of SEQ ID NOs. 2-4 and the light chain immunoglobulin variable domain sequences of SEQ ID NOs. 41, 42, The antibody specifically binds to the cis-pT231-tau protein, The composition wherein the treatment for the neurological disorder comprises preferential binding of the antibody to a cis-conformation-locked pT231-tau protein compared to binding to a trans-conformation-locked pT231-tau protein.

7. The composition according to claim 6, wherein the neurological disorder is tauopathy, traumatic brain injury (TBI), or stroke.

8. The composition according to claim 7, wherein the tauopathy is selected from the group consisting of progressive supranuclear palsy (PSP), chronic traumatic encephalopathy (CTE), frontotemporal dementia (FTD), frontotemporal lobar degeneration, Liticor-Bodig disease, Tangle-predominant dementia, meningeal hemangioma, subacute sclerosing panencephalitis, Pick's disease, corticobasal degeneration, and Alzheimer's disease (AD).

9. The composition according to claim 8, wherein the target is the initial stage of the tauopathy.

10. The composition according to claim 9, wherein the initial stage of the tauopathy is determined by an increase in the level of cis-pT231-tau or an increase in the cis:trans-pT231-tau ratio in a sample obtained from the subject.

11. The composition according to claim 10, wherein the level of CSF t-tau, pT181-tau, αβ42, or ApoE4 is measured in a sample obtained from the subject.

12. The composition according to claim 11, wherein the sample is selected from the group consisting of urine, blood, serum, plasma, saliva, amniotic fluid, and cerebrospinal fluid (CSF).

13. The composition according to claim 6, wherein the subject has a medical history of repeated traumatic brain injury.

14. The composition according to claim 6, wherein the antibody is administered to the target sequentially or simultaneously, separately from further therapeutic agents.

15. The composition according to claim 14, wherein the further therapeutic agent is one or more of donepezil, rivastigmine, galantamine, memantine, and lithium chloride.

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