WO2020180819A1 - Antibodies recognizing tau - Google Patents

Antibodies recognizing tau Download PDF

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Publication number
WO2020180819A1
WO2020180819A1 PCT/US2020/020704 US2020020704W WO2020180819A1 WO 2020180819 A1 WO2020180819 A1 WO 2020180819A1 US 2020020704 W US2020020704 W US 2020020704W WO 2020180819 A1 WO2020180819 A1 WO 2020180819A1
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WIPO (PCT)
Prior art keywords
seq
antibody
antibodies
tau
amino acid
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PCT/US2020/020704
Other languages
French (fr)
Inventor
Tarlochan S. NIJJAR
Robin Barbour
Philip James DOLAN, III
Yue Liu
Svetlana Alexander
Mark Renz
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Prothena Biosciences Limited
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Publication date
Priority to BR112021016947A priority Critical patent/BR112021016947A2/en
Priority to SG11202108414UA priority patent/SG11202108414UA/en
Application filed by Prothena Biosciences Limited filed Critical Prothena Biosciences Limited
Priority to MX2021010517A priority patent/MX2021010517A/en
Priority to AU2020231366A priority patent/AU2020231366A1/en
Priority to CN202080018265.8A priority patent/CN113544149A/en
Priority to KR1020217031410A priority patent/KR20210134943A/en
Priority to JP2021552257A priority patent/JP2022524588A/en
Priority to CU2021000073A priority patent/CU20210073A7/en
Priority to PE2021001439A priority patent/PE20212324A1/en
Priority to CA3131531A priority patent/CA3131531A1/en
Priority to EP20766551.4A priority patent/EP3935083A4/en
Priority to EA202192179A priority patent/EA202192179A1/en
Publication of WO2020180819A1 publication Critical patent/WO2020180819A1/en
Priority to IL285973A priority patent/IL285973A/en
Priority to CONC2021/0011644A priority patent/CO2021011644A2/en
Priority to ZA2021/07214A priority patent/ZA202107214B/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • This application includes an electronic sequence listing in a file named
  • Tau is a well-known human protein that can exist in phosphorylated forms (see, e.g., Goedert, Proc. Natl. Acad. Sci. U.S.A. 85:4051-4055(1988); Goedert, EMBO J. 8:393- 399(1989); Lee, Neuron 2: 1615-1624(1989); Goedert, Neuron 3:519-526(1989); Andreadis, Biochemistry 31 : 10626-10633(1992). Tau has been reported to have a role in stabilizing microtubules, particularly in the central nervous system.
  • Total tau t-tau, i.e., phosphorylated and unphosphorylated forms
  • phospho-tau i.e., phosphorylated tau
  • Tau is the principal constituent of neurofibrillary tangles, which together with plaques are a hallmark characteristic of Alzheimer’s disease.
  • the tangles constitute abnormal fibrils measuring 10 nm in diameter occurring in pairs wound in a helical fashion with a regular periodicity of 80 nm.
  • the tau within neurofibrillary tangles is abnormally phosphorylated (hyperphosphorylated) with phosphate groups attached to specific sites on the molecule.
  • Severe involvement of neurofibrillary tangles is seen in the layer II neurons of the entorhinal cortex, the CA1 and subicular regions of the hippocampus, the amygdala, and the deeper layers (layers III, V, and superficial VI) of the neocortex in Alzheimer’s disease. Hyperphosphorylated tau has also been reported to interfere with microtubule assembly, which may promote neuronal network breakdown.
  • Tau inclusions are part of the defining neurophathology of several neurodegenerative diseases including Alzheimer’s disease, frontotemporal lobar degeneration, progressive supranuclear palsy and Pick’s disease.
  • the invention provides an isolated antibody specifically binding to human tau, comprising a mature heavy chain variable region comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9 or SEQ ID NO: 149, and CDR-H3 comprising SEQ ID NO: 10, wherein the heavy chain variable region is at least 90% identical to SEQ ID NO: 18; and a mature light chain variable region comprising CDR-L1 comprising SEQ ID NO: 12, CDR-L2 comprising SEQ ID NO: 150, 151, 153, 156, 158, 159, 160, 163, 165, 166, 167, 168, 169, 170, 171, 172, 173 or 174, and CDR-L3 comprising SEQ ID NO: 14, wherein the light chain variable region is at least 90% identical to SEQ ID NO: 122.
  • At least one of positions H12, H13, H17, H24, H40, H43, H48, H66, H67, H76, H80, H81, and H91 can be occupied by V, K, T, A, R, Q, I, R, A, D, L, Q, and F, respectively, and at least one of positions L2, L12, L15, L37, L39, L45, L60 and L100 can be occupied by V, P, L, Q, R, R, D and Q respectively.
  • CDR-L2 comprises SEQ ID NO: 150, 151, 163, 167, 168, or 169.
  • the heavy chain variable region comprises SEQ ID NO: 18 and the light chain variable region comprises SEQ ID NO: 110, 121, 122 or 123.
  • the light chain variable region comprises SEQ ID NO: 110. In some antibodies, the light chain variable region comprises SEQ ID NO: 121. In some antibodies, the light chain variable region comprises SEQ ID NO: 122. In some antibodies, the light chain variable region comprises SEQ ID NO: 123. [0010] In some antibodies, the heavy chain variable region comprises SEQ ID NO: 146 and the light chain variable region comprises SEQ ID NO: 94 or 122. In some antibodies, the light chain variable region comprises SEQ ID NO:94. In some antibodies, the light chain variable region comprises SEQ ID NO: 122.
  • the heavy chain variable region comprises SEQ ID NO: 18 or 146 and the light chain variable region comprises SEQ ID NO: 122.
  • the invention provides an antibody specifically binding to human tau, comprising a mature heavy chain variable region comprising CDRs HI, H2 and H3 comprising SEQ ID NOs:8, 9, and 10, respectively except that position H28 can be occupied by N or T, H54 can be occupied by N or D, H56 can be occupied by D or E, position H58 occupied by V or I, and position H60 can be occupied by D or E, and a mature light chain variable region comprising CDRs LI, L2 and L3 comprising SEQ ID NOs: 12, 13, and 14 respectively, except that position L24 can be occupied by K or R, position L50 can be occupied by L, E.
  • position L52 can be occupied by S or G
  • position L54 can be occupied by L, D, G, N, E, Q, K, R, T, V, or S, wherein at least one of the following positions is occupied by the amino acid as specified: HI is occupied by Q, H5 is occupied by Q, HI 1 is occupied by L, H20 is occupied by L, H23 is occupied by T, H38 is occupied by K, H75 is occupied by S, H56 is occupied by E, H58 is occupied by I, H60 is occupied by E, H82c is occupied by V, L10 is occupied by T, L17 is occupied by E, L24 is occupied by R, L37 is occupied by Q, L47 is occupied by G, N, D, E, P, T, S, or A, L48 is occupied by G or D, L49 is occupied by E, L50 is occupied by E, D, G, or V, L52 is occupied by G
  • the invention provides an isolated monoclonal antibody that binds human tau comprising three light chain CDRs and three heavy chain CDRs of monoclonal antibody 3D6, wherein 3D6 is a mouse antibody characterized by a heavy chain variable region having an amino acid sequence comprising SEQ ID NO:7 and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 11, except that position H27 can be occupied by F or Y, position H28 can be occupied by N or T, position H29 can be occupied by I or F, position H30 can be occupied by K or T, position H51 can be occupied by I or V, position H54 can be occupied by N or D, position H60 can be occupied by D, A, or E, position H61 can be occupied by P or E, position HI 02 can be occupied by F or Y, position L50 can be occupied by L, E.
  • 3D6 is a mouse antibody characterized by a heavy chain variable region having an amino acid sequence comprising SEQ ID NO:7 and
  • position L52 can be occupied by S or G
  • position L54 can be occupied by L, D, G, N, E, Q, K, R, T, V, or S, wherein at least one of the following positions is occupied by the amino acid as specified: L37 is occupied by Q, L47 is occupied by G, N, D, E, P, T, S, or A, L48 is occupied by G or D, L49 is occupied by E, L50 is occupied by E, D, G, or V, L52 is occupied by G, L54 is occupied by D, G, N. E, Q, K, R, T, V, or S, L100 is occupied by Q, H60 is occupied by E, H82c is occupied by V.
  • CDR-H1 has an amino acid sequence comprising SEQ ID NO:86.
  • CDR-H2 has an amino acid sequence comprising SEQ ID NO: 87, SEQ ID NO:88, SEQ ID NO:92, or SEQ ID NO: 149.
  • CDR-L2 has an amino acid sequence comprising any of SEQ ID NOs: 150 - 175.
  • CDR-L1 has an amino acid sequence comprising SEQ ID NO:89.
  • CDR-H1 has an amino acid sequence comprising SEQ ID NO:86 and CDR-H2 has an amino acid sequence comprising SEQ ID NO:87.
  • CDR-H1 has an amino acid sequence comprising SEQ ID NO:86 and CDR-H2 has an amino acid sequence comprising SEQ ID NO:88. In some antibodies, CDR-H1 has an amino acid sequence comprising SEQ ID NO:86 and CDR-H2 has an amino acid sequence comprising SEQ ID NO:92. In some antibodies, CDR-H1 has an amino acid sequence comprising SEQ ID NO:42, SEQ ID NO:58, SEQ ID NO:59, or SEQ ID NO:60. In some antibodies, CDR-H2 has an amino acid sequence comprising SEQ ID NO:43, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, or SEQ ID NO: 149. In some antibodies, CDR-H3 has an amino acid sequence comprising SEQ ID NO:65. In some antibodies, CDR-L2 has an amino acid sequence comprising any of SEQ ID NOs: 150 - 175.
  • Some antibodies are a humanized antibody, veneered antibody, or chimeric antibody.
  • Some antibodies comprise a humanized mature heavy chain variable region having an amino acid sequence at least 95% identical to any one of SEQ ID NOs:76-80 and SEQ ID NOs: 90-91, and a humanized mature light chain variable region having an amino acid sequence at least 90% identical to any one of SEQ ID NOs:83-85.
  • Some antibodies comprise a a humanized mature heavy chain variable region having an amino acid sequence at least 95% identical to any one of SEQ ID NO:18 and SEQ ID NOs: 146- 148, and a humanized mature light chain variable region having an amino acid sequence at least 90% identical to any one of SEQ ID NOs:93-145.
  • At least one of the following positions in the VH region is occupied by the amino acid as specified: H93 is occupied by S and H94 is occupied by T. In some antibodies, positions H93 and H94 are occupied by S and T, respectively. In some antibodies position H91 in the VH region is occupied by F.
  • At least one of the following positions in the VH region is occupied by the amino acid as specified: HI is occupied by E, H5 is occupied by V, HI 1 is occupied by V, H20 is occupied I, H23 is occupied by K, H38 is occupied by R, H42 is occupied by G, H43 is occupied by K, H66 is occupied by R, H75 is occupied by T, H76 is occupied by D, H81 is occupied by E, HI 08 is occupied by L, HI 09 is occupied by V.
  • positions HI, H5, HI 1, H20, H23, H38, H42, H43, H66, H75, H76, H81, H108, and H109 in the VH region are occupied by E, V, V, I, K, R, G, K, R, T, D, E, L, and V, respectively.
  • At least one of the following positions in the VH region is occupied by the amino acid as specified: HI 7 is occupied by T, H80 is occupied by M, H83 is occupied by R. In some antibodies, positions HI 7, H80, and H83 in the VH region are occupied by T, M, and R, respectively.
  • position H58 in the VH region is occupied by I.
  • At least one of the following positions in the VH region is occupied by the amino acid as specified: H28 is occupied by T, H67 is occupied by V. In some antibodies, positions H28 and H67 in the VH region are occupied by T and V, respectively.
  • H54 is occupied by D
  • H56 is occupied by E
  • positions H54 and H56 in the VH region are occupied by D and E, respectively.
  • HI is occupied by Q or E
  • H5 is occupied by Q or V
  • HI 1 is occupied by L or V
  • HI 7 is occupied by S or T
  • H20 is occupied by L or I
  • H23 is occupied by T or K
  • H28 is occupied by N or T
  • H38 is occupied by K or R
  • H42 is occupied by E or G
  • H43 is occupied by Q or K
  • H54 is occupied by N or D
  • H56 is occupied by D or E
  • H58 is occupied by V or I
  • H60 is occupied by D or E
  • H66 is occupied by K or R
  • H67 is occupied by A or V
  • H75 is occupied by S or T
  • H76 is occupied by N or D
  • H80 is occupied by L or M
  • H81 is occupied by Q or E
  • H82c is occupied by L or V
  • H83 is occupied by T or R ,
  • At least one of the following positions in the VH region is occupied by the amino acid as specified: H10 is occupied by E or D, H12 is occupied by K or V, H13 is occupied by K or R, HI 7 is occupied by T, L or S, H24 is occupied by V or A, H27 is occupied by F or Y, H28 is occupied by N or T, H29 is occupied by I or F, H30 is occupied by K or T,
  • H38 is occupied by Q or R
  • H40 is occupied by A or R
  • H42 is occupied by G or E
  • H43 is occupied by K or Q
  • H48 is occupied by M or I
  • H51 is occupied by V or I
  • H54 is occupied by N or D
  • H60 is occupied by D
  • H61 is occupied by P or E
  • H66 is occupied by R or K
  • H67 is occupied by V or A
  • H76 is occupied by D or N
  • H80 is occupied by M or L
  • H81 is occupied by E or Q
  • H82a is occupied by S or G
  • H82c is occupied by L or V
  • H83 is occupied by T or R
  • H91 is occupied by Y or F
  • H93 is occupied by A or S
  • HI 02 is occupied by F or Y
  • HI 08 is occupied by T or L
  • HI 09 is occupied by L or V.
  • positions H91, H93, and H94 in the VH region are occupied by F, S, and T, respectively.
  • positions HI, H5, HI 1, H20, H23, H38, H42, H43, H66, H75, H76, H81, H91, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, I, K, R, G, K, R, T, D, E, F, S, T,L, and V, respectively.
  • positions HI, H5, HI 1, H17, H20, H23, H38, H42, H43, H58, H66, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T,I, K, R, G, K, I, R, T, D, M, E, R, S, T, L, and V, respectively.
  • positions HI, H5, HI 1, H17, H20, H23, H28, H38, H42, H43, H58, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, I, R, V, T, D, M, E, R, S, T, L, and V, respectively.
  • positions HI, H5, HI 1, H17, H20, H23, H28, H38, H42, H43, H54, H56, H58, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, I, R, V, T, D, M, E, R, S, T, L, and V, respectively.
  • positions HI, H5, HI 1, H17, H20, H23, H28, H38, H42, H43, H54, H56, H66, H67, H75, H76, H80, H81, H83, H91, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, R, V, T, D, M, E, R, F, S, T, L, and V, respectively.
  • positions HI, H5, HI 1, H17, H20, H23, H28, H38, H42, H43, H54, H56, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, R, V, T, D, M, E, R, S , T, L, and V, respectively.
  • position H60 is occupied by E.
  • position H82C is occupied by V.
  • positions H60, H80, H81, H82c, and H83 are occupied by E, M, E, V, and R, respectively.
  • At least one of the following positions in the VL region is occupied by the amino acid as specified: L7 is occupied by S, L10 is occupied by S, LI 5 is occupied by L, L83 is occupied by V, L86 is occupied by Y, and LI 06 is occupied by I. In some antibodies, positions L7, L10, L15, L83, L86, and L106 are occupied by S, S, L, V, Y, and Y, respectively.
  • L7 is T or S
  • L10 is T or S
  • L15 is I or L
  • L17 is Q or E
  • L24 is K or R
  • L37 is L or Q
  • L45 is K or R
  • L47 is L
  • L48 is I, G, or D
  • L49 is Y or E
  • L50 is L
  • L52 is S or G
  • L54 is L, D, G, N, E, Q, K, R, T, V, or S
  • L83 is L or V
  • L86 is H or Y
  • L100 is A or Q
  • L106 is L or I.
  • L2 is V or I
  • L7 is S or T
  • L12 is P or S
  • L15 is L or I
  • L36 is L
  • L37 is L or Q
  • L45 is R or K
  • L47 is L, G, N, D, E, P, T, S, or A
  • L48 is I, G, or D
  • L49 is Y or E
  • L50 is L, E, D, G, or V
  • L52 is S or G
  • L100 is G or Q.
  • positions L7, L10, L15, L83, L86, and L106 in the VL region are occupied by S, S, L, V, Y, and I, respectively.
  • positions L7, L10, L15, L17, L24, L37, L45, L83, L86, L100, and L106 in the VL region are occupied by S, S, L, E, R, Q, R, V, Y, Q, and I, respectively.
  • position L54 is occupied by D. In some antibodies, position L54 is occupied by G. In some antibodies, position L54 is occupied by N. In some antibodies, position L54 is occupied by E. In some antibodies, position L50 is occupied by E. In some antibodies, position L54 is occupied by Q. In some antibodies, position L50 is occupied by D. In some antibodies, position L54 is occupied by K. In some antibodies, position L54 is occupied by R In some antibodies, position L54 is occupied by T. In some antibodies, position L50 is occupied by G. In some antibodies, position L48 is occupied by G. In some antibodies, position L48 is occupied by D. In some antibodies, position L47 is occupied by G. In some antibodies, position L49 is occupied by E. In some antibodies, position L54 is occupied by V. In some antibodies, position L54 is occupied by S.
  • position L52 is occupied by G.
  • position L47 is occupied by N.
  • position L47 is occupied by D.
  • position L47 is occupied by E.
  • position L47 is occupied by P.
  • position L47 is occupied by T.
  • position L47 is occupied by S.
  • position L47 is occupied by A.
  • position L50 is occupied by V.
  • positions L37, L50, and L54 are occupied by Q, G, and R,
  • positions L37, L50, and L54 are occupied by Q, G, and G, respectively.
  • positions L37, L52, and L54 are occupied by Q, G, and G, respectively.
  • positions L37, L52, and L54 are occupied by Q, G, and R, respectively.
  • positions L37, L52, and L54 are occupied by Q, G, and T, respectively.
  • positions L37, L52, and L54 are occupied by Q, G, and D, respectively.
  • positions L37 and L54 are occupied Q and R, respectively. [0037] In some antibodies, positions L37 and L54 are occupied by Q and G, respectively.
  • positions L37 and L54 are occupied by Q and D, respectively.
  • positions L37 and L50 are occupied by Q and G, respectively.
  • positions L37 and L50 are occupied by Q and D, respectively.
  • positions L37 and L54 are occupied by Q and T, respectively.
  • positions L37 and L52 are occupied by Q and G, respectively.
  • positions L37 and L54 are occupied by Q and E, respectively.
  • positions L37, L50, and L54 are occupied by Q, D, and G, respectively.
  • positions L37, L50, and L54 are occupied by Q, D, and R, respectively.
  • positions L37, L50, and L54 are occupied by Q, E, and G, respectively. In some antibodies, positions L37, L50, and L54 are occupied by Q, E, and R, respectively. In some antibodies, positions L37, L50, L54, and L100 are occupied by Q, G, R, and Q, respectively. In some antibodies, positions L37, L50, L54, and L100 are occupied by Q, G, G, and Q, respectively. In some antibodies, positions L37, L52, L54, and LI 00 are occupied by Q, G, R, and Q, respectively. In some antibodies, positions L37, L52, L54, and L100 are occupied by Q, G, D, and Q, respectively. In some antibodies, positions L37, L50, L54, and L100 are occupied by Q, D, G, and Q, respectively. In some antibodies, positions L37, L50, L54, and L100 are occupied by Q, D, G, and Q, respectively. In some antibodies, positions L37, L50,
  • L54, and L100 are occupied by Q, D, R, and Q, respectively.
  • positions L37, L50, L54, and L100 are occupied by Q, V, D, and Q, respectively.
  • position L37 is occupied by Q.
  • position L100 is occupied by Q.
  • the mature heavy chain variable region has an amino acid sequence of any one of SEQ ID NOs:76-80 and SEQ ID NOs:90-91 and the mature light chain variable region has an amino acid sequence of any one of SEQ ID NO:83-85.
  • the mature heavy chain variable region has an amino acid sequence of any one of SEQ ID NO: 18 and SEQ ID NOs: 146-148 and the mature light chain variable region has an amino acid sequence of any one of SEQ ID NOs:93-145.
  • the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:76 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:76 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:84. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:76 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:85.
  • the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:77 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:77 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:84. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:77 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:85.
  • the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:78 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:78 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:84. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:78 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:85.
  • the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:79 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:79 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:84. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:79 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:85.
  • the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 80 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 83.
  • he mature heavy chain variable region has an amino acid sequence of SEQ ID NO:80 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:84.
  • the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 80 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:85.
  • the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 90 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:90 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:84. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 90 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:85.
  • the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:91 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:91 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:84. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:91 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:85.
  • the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 18 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 122. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 18 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 123.
  • the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 146 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 122.
  • the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 18 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 121. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 18 and the mature light chain variable region has an amino acid sequence of SEQ ID NO : 110.
  • the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 146 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:94.
  • the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 18 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 103.
  • the antibody can be a chimeric, veneered, or humanized antibody.
  • the antibody can be an intact chimeric, veneered or humanized antibody or a binding fragment, single-chain antibody Fab fragment, Fab’2 fragment, or single chain Fv.
  • Some of the antibodies have a human IgGl isotype, while others may have a human IgG2 or IgG4 isotype.
  • Some antibodies have the mature light chain variable region fused to a light chain constant region and the mature heavy chain variable region fused to a heavy chain constant region.
  • the heavy chain constant region of some antibodies is a mutant form of a natural human heavy chain constant region which has reduced binding to a Fey receptor relative to the natural human heavy chain constant region.
  • the heavy chain constant region has an amino acid sequence of SEQ ID NO: 176.
  • the mature heavy chain variable region fused to a heavy chain constant region has an amino acid sequence of SEQ ID NO: 178.
  • Some antibodies further comprise a signal peptide fused to the mature heavy and/or light chain variable region.
  • the heavy chain has an amino acid sequence of SEQ ID NO: 180.
  • the light chain constant region has an amino acid sequence of SEQ ID NO: 177.
  • the mature light chain variable region is fused to a light chain constant region has an amino acid sequence of SEQ ID NO: 179.
  • the light chain has an amino acid sequence of SEQ ID NO: 181.
  • the heavy chain has an amino acid sequence of SEQ ID NO: 178 and the light chain has an amino acid sequence of SEQ ID NO: 179. In some antibodies, the heavy chain has an amino acid sequence of SEQ ID NO: 180 and the light chain has an amino acid sequence of SEQ ID NO: 181.
  • Some antibodies may have at least one mutation in the constant region, such as a mutation that reduces complement fixation or activation by the constant region, for example, a mutation at one or more of positions 241, 264, 265, 270, 296, 297, 318, 320, 322, 329 and 331 by EU numbering. Some antibodies have an alanine at positions 318, 320 and 322. Some antibodies can be at least 95% w/w pure.
  • the antibody can be conjugated to a therapeutic, cytotoxic, cytostatic, neurotrophic, or neuroprotective agent.
  • the invention provides a pharmaceutical composition comprising any of the antibodies disclosed herein and a pharmaceutically-acceptable carrier.
  • the invention provides a nucleic acid encoding the heavy chain and/or light chain of any of the antibodies disclosed herein, a recombinant expression vector comprising the nucleic acid and a host cell transformed with the recombinant expression vector.
  • the heavy chain is encoded by a sequence comprising SEQ ID NO: 182 and the light chain is encoded by a sequence comprising SEQ ID NO: 183.
  • Methods of producing antibodies such as a humanized, chimeric or veneered antibody, for example humanized, chimeric or veneered forms of 3D6, are also provided.
  • cells transformed with nucleic acids encoding the heavy and light chains of the antibody are cultured so that the cells secrete the antibody.
  • the antibody can then be purified from the cell culture media.
  • Cell lines producing any of the antibodies disclosed herein can be produced by introducing a vector encoding heavy and light chains of the antibody and a selectable marker into cells, propagating the cells under conditions to select for cells having increased copy number of the vector, isolating single cells from the selected cells; and banking cells cloned from a single cell selected based on yield of antibody.
  • the invention comprises a vector comprising a nucleic acid encoding a mature heavy chain variable region and a mature light chain variable region operably linked to one or more regulatory sequences to effect expression in a mammalian cell of any of the antibodies disclosed herein.
  • the expressed antibody is a scFv or an Fab fragment.
  • the one or more regulatory sequences include one or more of a promoter, enhancer, ribosome binding site, and transcription termination signal.
  • the nucleic acid further encodes signal peptides fused to the mature heavy and light chain variable regions.
  • the nucleic acid is codon-optimized for expression in a host cell.
  • the one or more regulatory sequences include a eukaryotic promoter.
  • the nucleic acid further encodes a selectable gene.
  • the invention provides methods of expressing an antibody in a mammalian cell comprising incorporating the nucleic acids disclosed herein into the genome of a transgenic animal, whereby the antibody is expressed.
  • the invention provides first and second vectors respectively comprising nucleic acids encoding a mature heavy chain variable region and a mature light chain variable region, each operably linked to one or more regulatory sequences to effect expression of any of the antibodies disclosed herein, and a host cell comprising the nucleic acids.
  • the nucleic acids respectively further encodes a heavy chain constant region fused to the mature heavy chain variable region and a light chain constant region fused to the mature light chain variable region.
  • the heavy chain constant region has the sequence of SEQ ID NO: 176 with or without the C-terminal lysine and the light chain constant region has the sequence of SEQ ID NO: 177.
  • the invention provides methods of expressing an antibody in a mammalian cell comprising incorporating any of the nucleic acids disclosed herein into the genome of a transgenic animal, whereby the antibody is expressed.
  • Methods of producing antibodies are also provided.
  • cells cells transformed with nucleic acids encoding the heavy and light chains of the antibody of any of the antibodies disclosed herein are cultured so that the cells secrete the antibody.
  • the antibody can then be purified from the cell culture media.
  • Cell lines producing any of the antibodies disclosed herein can be produced by introducing a vector encoding heavy and light chains of the antibody of claim 1 and a selectable marker into cells, propagating the cells under conditions to select for cells having increased copy number of the vector, isolating single cells from the selected cells, and banking cells cloned from a single cell selected based on yield of antibody.
  • Some cells can be propagated under selective conditions and screened for cell lines naturally expressing and secreting at least 100 mg/L/10 6 cells/24 hours. Single cells can be isolated from the selected cells. Cells cloned from a single cell can then be banked. Single cells can be selected based on desirable properties, such as the yield of the antibody. Exemplary cell lines are cell lines expressing 3D6 or humanized versions of 3D6.
  • the invention also provides methods of inhibiting or reducing aggregation of tau in a subject having or at risk of developing a tau-mediated amyloidosis, comprising administering to the subject an effective regime of an antibody disclosed herein, thereby inhibiting or reducing aggregation of tau in the subject.
  • exemplary antibodies include humanized versions of 3D6.
  • a tau-related disease comprising administering an effective regime of an antibody disclosed herein and thereby treating or effecting prophylaxis of the disease.
  • Alzheimer’s disease Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia,
  • CBD Alzheimer disease degeneration
  • LDV D Lewy body variant of Alzheimer disease
  • CTE chronic traumatic encephalopathy
  • GTT globular glial tauopathy
  • PSP progressive supranuclear palsy
  • the tau-related disease is Alzheimer’s disease.
  • the patient is an ApoE4 carrier.
  • Also provided are methods of reducing aberrant transmission of tau comprising administering an effective regime of an antibody disclosed herein and thereby reducing transmission of tau.
  • methods of inducing phagocytosis of tau comprising administering an effective regime of an antibody disclosed herein and thereby inducing phagocytosis of tau.
  • Also provided are methods of inhibiting tau aggregation or deposition comprising administering an effective regime of an antibody disclosed herein thereby inhibiting tau aggregation or deposition.
  • Also provided are methods of inhibiting formation of tau tangles comprising administering an effective regime of an antibody disclosed herein.
  • the invention also provides a method of detecting tau protein deposits in a subject having or at risk of a disease associated with tau aggregation or deposition comprising administering to a subject an antibody disclosed herein, and detecting the antibody bound to tau in the subject.
  • Alzheimer’s disease Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBV D), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).
  • CBD corticobasal degeneration
  • LBV D Alzheimer disease
  • CTE chronic traumatic encephalopathy
  • GTT progressive supranuclear palsy
  • the antibody is administered by intravenous injection into the body of the subject. In some embodiments, the antibody is administered directly to the brain of the subject by intracranial injection or by drilling a hole through the skull of the subject. In some embodiments, the antibody is labeled. In some embodiments, the antibody is labeled with a fluorescent label, a paramagnetic label, or a radioactive label. In some embodiments, the radioactive label is detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • the invention also provides a method of measuring efficacy of treatment in a subject being treated for a disease associated with tau aggregation or deposition, comprising measuring a first level of tau protein deposits in the subject prior to treatment by administering to a subject an antibody disclosed herein, and detecting a first amount of the antibody bound to tau in the subject, administering the treatment to the subject, measuring a second level of tau protein deposits in the in subject after treatment by administering to a subject the antibody, and detecting the antibody bound to tau in the subject, wherein a decrease in the level of tau protein deposits indicates a positive response to treatment.
  • the invention also provides a method of measuring efficacy of treatment in a subject being treated for a disease associated with tau aggregation or deposition, comprising measuring a first level of tau protein deposits in the subject prior to treatment by administering to a subject an antibody disclosed herein, and detecting a first amount of antibody bound to tau in the subject, administering the treatment to the subject, measuring a second level of tau protein deposits in the in subject after treatment by administering to a subject the antibody, and detecting a second amount of antibody bound to tau in the subject, wherein no change in the level of tau protein deposits or a small increase in tau protein deposits indicates a positive response to treatment.
  • the invention provides methods of producing an antibody that specifically binds to human tau at an epitope within a motif of the formula KXXSXXNX(K/H)H (SEQ ID NO: 191) or KIGSLDNITH (SEQ ID NO: 194) comprising immunizing an animal with human tau or a fragment thereof to produce antibodies and screening for an antibody of the produced antibodies that specifically binds within the motif.
  • the invention provides methods of producing an antibody that specifically binds to a peptide consisting of residues KXXSXXNX(K/H)H (SEQ ID NO: 191) or KIGSLDNITH (SEQ ID NO: 194) comprising immunizing an animal with human tau or a fragment thereof to produce antibodies and screening for an antibody of the produced antibodies that specifically binds to the peptide.
  • the invention provides methods of producing an antibody that specifically binds to an epitope comprising KXXSXXNX(K/H)H (SEQ ID NO: 191) comprising immunizing an animal with tau or a fragment thereof, and screening for antibodies that specifically bind to the epitope.
  • the animal is immunized with 383 amino acid human tau (4R0N).
  • the human tau contains a P301S mutation.
  • the human tau is recombinant N-terminally His-tagged.
  • the screening determines specific binding between the antibodies and one or more peptides of up to 15 amino acids comprising KIGSTENLKH (SEQ ID NO: 188), KCGSKDNIKH (SEQ ID NO: 192), KCGSLGNIHH (SEQ ID NO: 193) respectively or any other consensus motif represented by KXXSXXNX(K/H)H (SEQ ID NO: 191).
  • the one or more peptides comprise KIGSTENLKH (SEQ ID NO: 188) or KCGSKDNIKH (SEQ ID NO: 192) or KCGSLGNIHH (SEQ ID NO: 193) respectively.
  • the animal is immunized with a tau fragment of up to 15 amino acids comprising KXXSXXNX(K/H)H (SEQ ID NO: 191), linked to a carrier.
  • the peptide is KIGSTENLKH (SEQ ID NO: 188) or KCGSKDNIKH (SEQ ID NO: 192) or KCGSLGNIHH (SEQ ID NO: 193).
  • Figure 1 depicts the results of experiments designed to map the epitope(s) bound by the murine 3D6 monoclonal antibody.
  • Figure 2 depicts an alignment of heavy chain variable regions of the mouse 3D6 antibody (SEQ ID NO:7) and humanized versions of the 3D6 antibody (hu3D6VHvbl, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7) with human germline heavy chain variable region sequence IGHVl-69-2*01 (SEQ ID NO:25) and with human acceptor heavy chain variable region sequence 2RCS VH hFrwk (SEQ ID NO:75).
  • hu3D6VHvbl is SEQ ID NO:76
  • hu3D6VHvb2 is SEQ ID NO:77
  • hu3D6VHvb3 is SEQ ID NO:78
  • hu3D6VHvb4 is SEQ ID NO:79
  • hu3D6VHvb5 is SEQ ID NO:80
  • hu3D6VHvb6 is SEQ ID NO:90
  • hu3D6VHvb7 is SEQ ID NO:91.
  • the CDRs as defined by Kabat/Chothia Composite are in boldface.
  • Figure 3 depicts an alignment of light chain variable regions of the mouse 3D6 antibody (SEQ ID NO: 11) and humanized versions of the 3D6 antibody (hu3D6VLvbl, hu3D6VLvb2, and hu3D6VLvb3) with human germline light chain variable region sequence IGKV2-30*02 (SEQ ID NO:27) and with human acceptor ARX71335_VL_hFrwk (SEQ ID NO:82).
  • hu3D6VLvbl is SEQ ID NO:83
  • hu3D6VLvb2 is SEQ ID NO:84
  • hu3D6VLvb3 is SEQ ID NO:85.
  • the CDRs as defined by Kabat are in boldface.
  • Figures 4A, 4B, and 4C depict results of ELISA screening assays for selected mouse monoclonal anti-tau antibodies.
  • Figure 5 depicts binding kinetics for selected mouse monoclonal anti-tau antibodies to recombinant human tau.
  • Figure 6 depicts results of functional blocking assays for selected mouse monoclonal anti-tau antibodies.
  • Figure 7 depicts results of disaggregation assays for selected mouse monoclonal anti- tau antibodies.
  • Figure 8 depicts results of experiments showing that 3D6 and 5G8 immunocapture tau from human Alzheimer's disease tissue.
  • Figures 9A and 9B show alignments of the heavy chain variable region of murine 3D6 (SEQ ID NO:7) and of humanized versions of the 3D6 antibody (hu3D6VHvbl, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, hu3D6VHvb7, hu3D6VHvlbAl 1, h3D6VHvb8, and h3D6VHvb9) with human germline heavy chain variable region sequence IGHVl-69-2*01 (SEQ ID NO:25) and with human acceptor heavy chain variable region sequence 2RCS VH hFrwk (SEQ ID NO:75).
  • hu3D6VHvbl is SEQ ID NO:76
  • hu3D6VHvb2 is SEQ ID NO:77
  • hu3D6VHvb3 is SEQ ID NO:78
  • hu3D6VHvb4 is SEQ ID NO:79
  • hu3D6VHvb5 is SEQ ID NO:80
  • hu3D6VHvb6 is SEQ ID NO:90
  • hu3D6VHvb7 is SEQ ID NO:91
  • hu3D6VHvlbAl l is SEQ ID NO: 18
  • h3D6VHvb8 is SEQ ID NO: 146
  • h3D6VHvb9 is SEQ ID NO: 148.
  • Residues identical to those of the heavy chain variable region of murine 3D6 (SEQ ID NO:7) are noted with a“”
  • the CDRs as defined by Kabat/Chothia Composite are in boldface.
  • hu3D6VLv2 L37Q SEQ ID NO: 143
  • hu3D6VLv2 L50G SEQ ID NO: 103
  • hu3D6VLv2 S52G SEQ ID NO:
  • L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), and hu3D6VLv2 L37Q_S52G_L54R_G100Q (SEQ ID NO: 138).
  • Residues identical to those of the light chain variable region of hu3D6VLv2 (SEQ ID NO:21) are noted with a The CDRs as defined by Rabat are in boldface.
  • Figure 11 shows results of tau internalization assay for selected humanized 3D6 variants.
  • Figures 12A and 12B show results of subsitution mapping microarray experiments.
  • Figure 12A shows a plot of subsitution effect
  • Figure 12B shows an alignment of portions of the tau microtubule binding repeats with important residues for binding 3D6 highlighted aa 255-271 are SEQ ID NO: 184, aa 286-302 are SEQ ID NO: 185, aa 317-333 are SEQ ID NO: 186, and aa 349-365 are SEQ ID NO: 187.
  • Figure 13 depicts results of immunohistochemistry experiments showing 3D6 binds tau in normal (top panel) and Alzheimer’s disease tissue (middle and bottom panels).
  • Figure 14 shows results of mass spectrometry experiments to assess binding
  • Figure 15 shows that 3D6 interrupts tau seeding in an in vivo diseae model of
  • Figure 16 shows that 3D6 humanized variants hu3D6VHvlbAl l/hu3D6VLv2 and hu3D6VHvlbAl 1/L2-DIM4 block interaction between tau and heparin.
  • Figure 17 shows that 3D6 humanized variants hu3D6VHvlbAl l/hu3D6VLv2 and hu3D6VHvlbAl 1/L2-DIM4 bind fibrillar forms of tau.
  • SEQ ID NO: 1 sets forth the amino acid sequence of an isoform of human tau (Swiss- Prot P10636-8).
  • SEQ ID NO:2 sets forth the amino acid sequence of an isoform of human tau (Swiss- Prot PI 0636-7).
  • SEQ ID NO:3 sets forth the amino acid sequence of an isoform of human tau (Swiss- Prot PI 0636-6), (4R0N human tau).
  • SEQ ID NO:4 sets forth the amino acid sequence of an isoform of human tau (Swiss- Prot PI 0636-5)
  • SEQ ID NO:5 sets forth the amino acid sequence of an isoform of human tau (Swiss- Prot PI 0636-4).
  • SEQ ID NO:6 sets forth the amino acid sequence of an isoform of human tau (Swiss- Prot PI 0636-2).
  • SEQ ID NO:7 sets forth the amino acid sequence of the heavy chain variable region of the mouse 3D6 antibody.
  • SEQ ID NO:8 sets forth the amino acid sequence of Kabat/Chothia composite CDR-H1 of the mouse 3D6 antibody.
  • SEQ ID NO:9 sets forth the amino acid sequence of Rabat CDR-H2 of the mouse 3D6 antibody.
  • SEQ ID NO: 10 sets forth the amino acid sequence of Rabat CDR-H3 of the mouse 3D6 antibody.
  • SEQ ID NO: 11 sets forth the amino acid sequence of the light chain variable region of the mouse 3D6 antibody and of the mouse 6A10 antibody.
  • SEQ ID NO: 12 sets forth the amino acid sequence of Kabat CDR-L1 of the mouse 3D6 antibody and of the mouse 6A10 antibody.
  • SEQ ID NO: 13 sets forth the amino acid sequence of Kabat CDR-L2 of the mouse 3D6 antibody and of the mouse 6A10 antibody.
  • SEQ ID NO: 14 sets forth the amino acid sequence of Kabat CDR-L3 of the mouse 3D6 antibody and of the mouse 6A10 antibody.
  • SEQ ID NO: 15 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvl.
  • SEQ ID NO: 16 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHv2.
  • SEQ ID NO: 17 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvlb.
  • SEQ ID NO: 18 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvlbAl 1.
  • SEQ ID NO: 19 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHv5:
  • SEQ ID NO:20 sets forth the amino acid sequence of the light chain variable region of the humanized 3D6 antibody hu3D6VLvl.
  • SEQ ID NO:21 sets forth the amino acid sequence of the light chain variable region of the humanized 3D6 antibody hu3D6VLv2.
  • SEQ ID NO:22 sets forth the amino acid sequence of the light chain variable region of the humanized 3D6 antibody hu3D6VLv3.
  • SEQ ID NO:23 sets forth the amino acid sequence of the light chain variable region of the humanized 3D6 antibody hu3D6VLv4.
  • SEQ ID NO:24 sets forth the amino acid sequence of the heavy chain variable acceptor Acc.# BAC01986.1.
  • SEQ ID NO:25 sets forth the amino acid sequence of the heavy chain variable acceptor Acc # IMGT# IGHV1 -69-2*01.
  • SEQ ID NO:26 sets forth the amino acid sequence of the heavy chain variable acceptor Acc # IMGT#IGKJ 1*01.
  • SEQ ID NO:27 sets forth the amino acid sequence of the light chain variable acceptor Acc. # IMGT#IGKV2-30*02
  • SEQ ID NO:28 sets forth the amino acid sequence of the light chain variable acceptor Acc. # IMGT#IGKJ2*01.
  • SEQ ID NO:29 sets forth the amino acid sequence of the light chain variable acceptor Acc. # AAZ09048.1.
  • SEQ ID NO:30 sets forth a nucleic acid sequence encoding the heavy chain variable region of the mouse 3D6 antibody.
  • SEQ ID NO:31 sets forth a nucleic acid sequence encoding the light chain variable region of the mouse 3D6 antibody.
  • SEQ ID NO:32 sets forth the amino acid sequence of Kabat CDR-H1 of the mouse 3D6 antibody.
  • SEQ ID NO:33 sets forth the amino acid sequence of Chothia CDR-H1 of the mouse 3D6 antibody.
  • SEQ ID NO:34 sets forth the amino acid sequence of Chothia CDR-H2 of the mouse 3D6 antibody.
  • SEQ ID NO:35 sets forth the amino acid sequence of AbM CDR-H2 of the mouse 3D6 antibody.
  • SEQ ID NO:36 sets forth the amino acid sequence of Contact CDR-L1 of the mouse 3D6 antibody.
  • SEQ ID NO:37 sets forth the amino acid sequence of Contact CDR-L2 of the mouse 3D6 antibody.
  • SEQ ID NO:38 sets forth the amino acid sequence of Contact CDR-L3 of the mouse 3D6 antibody.
  • SEQ ID NO:39 sets forth the amino acid sequence of Contact CDR-H1 of the mouse 3D6 antibody.
  • SEQ ID NO:40 sets forth the amino acid sequence of Contact CDR-H2 of the mouse 3D6 antibody.
  • SEQ ID NO:41 sets forth the amino acid sequence of Contact CDR-H3 of the mouse 3D6 antibody.
  • SEQ ID NO:42 sets forth the amino acid sequence of an alternate Kabat-Chothia Composite CDR-H1 of a humanized 3D6 antibody (as in hu3D6VHv5, hu3D6VHvlbAl 1B6G2, hu3D6VHvlbAl 1B6H3, hu3D6VHvle, and hu3D6VHvlf).
  • SEQ ID NO:43 sets forth the amino acid sequence of an alternate Rabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHv5 and hu3D6VHvlbAl 1B6H3).
  • SEQ ID NO:44 sets forth the consensus amino acid sequence among the heavy chain variable regions of the mouse 3D6 and selected humanized 3D6 antibodies (VHvl, VHv2, VHvlb, VHvlbAl l, and VHv5) (labeled“Majority’ in Figure 2 of PCT/IB2017/052544.
  • SEQ ID NO:45 sets forth the consensus amino acid sequence between the light chain variable regions of the mouse 3D6 and selected humanized 3D6 antibodies (labeled“Majority’ in Figure 3 of PCT/IB2017/052544).
  • SEQ ID NO:46 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvlbAl 1B6G2.
  • SEQ ID NO:47 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvlbAl 1B6H3.
  • SEQ ID NO:48 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvlc.
  • SEQ ID NO:49 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvld.
  • SEQ ID NO:50 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvle.
  • SEQ ID NO:51 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvlf.
  • SEQ ID NO:52 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHv3.
  • SEQ ID NO:53 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHv3b.
  • SEQ ID NO:54 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHv3c.
  • SEQ ID NO:55 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHv4.
  • SEQ ID NO:56 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHv4b.
  • SEQ ID NO:57 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHv4c.
  • SEQ ID NO:58 sets forth the amino acid sequence of an alternate Kabat-Chothia Composite CDR-H1 of a humanized 3D6 antibody (as in hu3D6VHlc).
  • SEQ ID NO:59 sets forth the amino acid sequence of an alternate Kabat-Chothia Composite CDR-H1 of a humanized 3D6 antibody (as in hu3D6VHvld, hu3D6VHv3c, and hu3D6VHv4c).
  • SEQ ID NO:60 sets forth the amino acid sequence of an alternate Kabat-Chothia Composite CDR-H1 of a humanized 3D6 antibody (as in hu3D6VHv3b and hu3D6VHv4b).
  • SEQ ID NO:61 sets forth the amino acid sequence of an alternate Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvlbAl 1B6G2).
  • SEQ ID NO:62 sets forth the amino acid sequence of an alternate Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvlc, hu3D6VHv3b, AND hu3D6VHv4b.
  • SEQ ID NO:63 sets forth the amino acid sequence of an alternate Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvld, hu3D6VHvlf, hu3D6VHv3c, and hu3D6VHv4c).
  • SEQ ID NO:64 sets forth the amino acid sequence of an alternate Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvle).
  • SEQ ID NO:65 sets forth the amino acid sequence of an alternate Kabat CDR-H3 of a humanized 3D6 antibody (as in hu3D6VHvlf).
  • SEQ ID NO:66 sets forth the amino acid sequence of the heavy chain variable region of the mouse 6A10 antibody.
  • SEQ ID NO:67 sets forth the amino acid sequence of Kabat/Chothia composite CDR- H1 of the mouse 6A10 antibody.
  • SEQ ID NO:68 sets forth the amino acid sequence of Kabat CDR-H2 of the mouse 6A10 antibody.
  • SEQ ID NO:69 sets forth the amino acid sequence of Kabat CDR-H3 of the mouse 6A10 antibody.
  • SEQ ID NO:70 sets for the amino acid sequence of the VH region of mouse antibody (pdb code 1CR9) used as a structure template for heavy chain humanization.
  • SEQ ID NO:71 sets forth the consensus amino acid sequence among the heavy chain variable regions of the selected humanized 3D6 antibodies (VHvl, VHvlb, VHvlbAl 1, VHvlbAl 1B6G2, VHvlbAl 1B6H3, VHvlc, VHvld, VHvle, VHvlf, VHv2, VHv3, VHv3b, VHv3c, VHv4, VHv4b, VHv4c, and VHv5) (labeled“Majority’ in Figures 4A and 4B of PCT/IB2017/052544).
  • SEQ ID NO:72 sets forth the amino acid sequence of the heavy chain of a chimeric 3D6 antibody.
  • SEQ ID NO:73 sets forth the amino acid sequence of the light chain of a chimeric 3D6 antibody.
  • SEQ ID NO:74 sets forth the amino acid sequence of heavy chain variable structural model Acc.# 5MYX-VH_mSt.
  • SEQ ID NO:75 sets forth the amino acid sequence of heavy chain variable acceptor Acc.# 2RCS-VH_huFrwk.
  • SEQ ID NO:76 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvbl.
  • SEQ ID NO:77 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb2.
  • SEQ ID NO:78 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb3.
  • SEQ ID NO:79 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb4.
  • SEQ ID NO:80 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb5.
  • SEQ ID NO:81 sets forth the amino acid sequence of light chain variable structural model Acc.# 5MYX-VL_mSt.
  • SEQ ID NO:82 sets forth the amino acid sequence of light chain variable acceptor Acc.# ARX71335 - VL_huF rwk.
  • SEQ ID NO:83 sets forth the amino acid sequence of light chain variable region of the humanized 3D6 antibody hu3D6VLvbl.
  • SEQ ID NO:84 sets forth the amino acid sequence of light chain variable region of the humanized 3D6 antibody hu3D6VLvb2.
  • SEQ ID NO:85 sets forth the amino acid sequence of light chain variable region of the humanized 3D6 antibody hu3D6VLvb3.
  • SEQ ID NO:86 sets forth the amino acid sequence of an alternate Kabat-Chothia Composite CDR-H1 of a humanized 3D6 antibody (as in hu3D6VHvb4 and hu3D6VHvb5).
  • SEQ ID NO:87 sets forth the amino acid sequence of an alternate Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvb3 and hu3D6VHvb4).
  • SEQ ID NO:88 sets forth the amino acid sequence of an alternate Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvb5).
  • SEQ ID NO:89 sets forth the amino acid sequence of an alternate Kabat CDR-L1 of a humanized 3D6 antibody (as in hu3D6VLvb3).
  • SEQ ID NO:90 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb6.
  • SEQ ID NO:91 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb7.
  • SEQ ID NO:92 sets forth the amino acid sequence of an alternate Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvb6 and hu3D6VHvb7).
  • SEQ ID NO:93 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L54D.
  • SEQ ID NO:94 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L54G.
  • SEQ ID NO:95 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L45N.
  • SEQ ID NO:96 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L54E.
  • SEQ ID NO:97 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L50E.
  • SEQ ID NO:98 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L54Q.
  • SEQ ID NO:99 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L50D.
  • SEQ ID NO: 100 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L54K.
  • SEQ ID NO: 101 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L54R.
  • SEQ ID NO: 102 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L54T.
  • SEQ ID NO: 103 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L50G.
  • SEQ ID NO: 104 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant I48G.
  • SEQ ID NO: 105 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant I48D.
  • SEQ ID NO: 106 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L47G.
  • SEQ ID NO: 107 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant Y49E.
  • SEQ ID NO: 108 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L54V.
  • SEQ ID NO: 109 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L54S.
  • SEQ ID NO: 110 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant S52G.
  • SEQ ID NO: 111 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L47N.
  • SEQ ID NO: 112 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L47D.
  • SEQ ID NO: 113 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L47E.
  • SEQ ID NO: 114 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L47P.
  • SEQ ID NO: 115 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L47T.
  • SEQ ID NO: 116 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L47S.
  • SEQ ID NO: 117 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L47A.
  • SEQ ID NO: 118 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L50V.
  • SEQ ID NO: 119 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q L50G L54R.
  • SEQ ID NO: 120 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q L50G L54G.
  • SEQ ID NO: 121 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q_S52G_L54G.
  • SEQ ID NO: 122 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q S52G L54R.
  • SEQ ID NO: 123 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q S52G L54T.
  • SEQ ID NO: 124 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q_S52G_L54D.
  • SEQ ID NO: 125 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q L54R.
  • SEQ ID NO: 126 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37QJL54G.
  • SEQ ID NO: 127 sets forth the amino acid sequence of light chain vari able region of a hu3D6VLv2 variant L37QJL54D.
  • SEQ ID NO: 128 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q L50G.
  • SEQ ID NO: 129 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q L50D.
  • SEQ ID NO: 130 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q_L54T.
  • SEQ ID NO: 131 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q_S52G.
  • SEQ ID NO: 132 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q L50D L54G.
  • SEQ ID NO: 133 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q_L50D_L54R.
  • SEQ ID NO: 134 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q L50E L54G.
  • SEQ ID NO: 135 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q L50E L54R.
  • SEQ ID NO: 136 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q_L50G_L54R_G100Q.
  • SEQ ID NO: 137 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q L50G L54G G100Q.
  • SEQ ID NO: 138 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q_S52G_L54R_G100Q.
  • SEQ ID NO: 139 sets forth the amino acid sequence of light chain vari able region of a hu3D6VLv2 variant L37Q_S52G_L54D_G100Q.
  • SEQ ID NO: 140 sets forth the amino acid sequence of light chain variable region of a Hu3D6VLv2 variant L37Q _L50D _L54G _G10QQ.
  • SEQ ID NO: 141 sets forth the amino acid sequence of light chain variable region of a Hu3D6VLv2 variant L37Q L50D L54R G100Q.
  • SEQ ID NO: 142 sets forth the amino acid sequence of light chain variable region of a Hu3D6VLv2 variant L37Q_L50V_L54D_G100Q.
  • SEQ ID NO: 143 sets forth the amino acid sequence of light chain variable region of a Hu3D6VLv2 variant L37Q.
  • SEQ ID NO: 144 sets forth the amino acid sequence of light chain variable region of a Hu3D6VLv2 variant G100Q.
  • SEQ ID NO: 145 sets forth the amino acid sequence of light chain variable region of a Hu3D6VLv2 variant L37QJL54E.
  • SEQ ID NO: 146 sets forth the amino acid sequence of heavy chain variable region of a hu3D6VHvlbAl 1 variant D60E, also known as h3D6VHvb8.
  • SEQ ID NO: 147 sets forth the amino acid sequence of heavy chain variable region of a hu3D6VHv 1 bAl 1 variant L82cV.
  • SEQ ID NO: 148 sets forth the amino acid sequence of heavy chain vari able region of a hu3D6VHvl bAl 1 variant D60E_L80M_Q81E_L82cV_T83R, also known as h3D6VHvb9.
  • SEQ ID NO: 149 sets forth the amino acid sequence of an alternate Kabat CDR-H2 of a humanized 3D6 antibody (as in h3D6VHvb8 and in h3D6VHvb9).
  • SEQ ID NO: 150 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54D and in hu3D6VLv2 L37Q L54D).
  • SEQ ID NO: 151 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54G and in hu3D6VLv2 L37Q L54G).
  • SEQ ID NO: 152 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54N).
  • SEQ ID NO: 153 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54E and in hu3D6VLv2 L37Q L54E).
  • SEQ ID NO: 154 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L50E).
  • SEQ ID NO: 155 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54Q).
  • SEQ ID NO: 156 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L50D and in hu3D6VLv2 L37Q L50D).
  • SEQ ID NO: 157 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54K).
  • SEQ ID NO: 158 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54R and in hu3D6VLv2 L37Q L54R).
  • SEQ ID NO: 159 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54T and in hu3D6VLv2 L37Q L54T).
  • SEQ ID NO: 160 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L50G and in hu3D6VLv2 L37Q L50G).
  • SEQ ID NO: 161 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54V).
  • SEQ ID NO: 162 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54S).
  • SEQ ID NO: 163 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 S52G and in hu3D6VLv2 L37Q S52G).
  • SEQ ID NO: 164 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L50V).
  • SEQ ID NO: 165 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q L50G L54R and hu3D6VLv2
  • SEQ ID NO: 166 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q_L50G_L54G and in and in hu3D6VLv2 L37Q_L50G_L54G_G100Q).
  • SEQ ID NO: 167 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q S52G L54G).
  • SEQ ID NO: 168 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q S52G L54R and in and in hu3D6VLv2 L37Q_S52G_L54R_G100Q).
  • SEQ ID NO: 169 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q S52G L54T).
  • SEQ ID NO: 170 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q S52G L54D and in hu3D6VLv2
  • SEQ ID NO: 171 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q L50D L54G and in hu3D6VLv2
  • SEQ ID NO: 172 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q L50D L54R and in hu3D6VLv2
  • SEQ ID NO: 173 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q_L50E_L54G).
  • SEQ ID NO: 174 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q_L50E_L54R).
  • SEQ ID NO: 175 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q_L50V_L54D_G100Q).
  • SEQ ID NO: 176 sets forth the amino acid sequence of a heavy chain constant region (IgGl : allotype Glml7,l)
  • SEQ ID NO: 177 sets forth the amino acid sequence of a light chain constant region (kappa)
  • SEQ ID NO: 178 sets forth the amino acid sequence of a mature heavy chain of a 3D6 humanized variant (hu3D6VHvlbAl 1 IgGl Glml7 allotype)
  • SEQ ID NO: 179 sets forth the amino acid sequence of a mature light chain of a 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4 kappa)
  • SEQ ID NO: 180 sets forth the amino acid sequence of a heavy chain of a 3D6 humanized variant (hu3D6VHvlbAl 1 IgGl Glml7 allotype) with bovine alpha-lactalbumin signal peptide at the N-terminus
  • SEQ ID NO: 181 sets forth the amino acid sequence of a light chain of a 3D6 humanized variant (hu3D6VLv2 variant L37Q S 52G L54R, L2-DIM4 kappa) with bovine alpha- lactalbumin signal peptide at the N-terminus.
  • SEQ ID NO: 182 sets forth the nucleotide sequence encoding a heavy chain of a 3D6 humanized variant (hu3D6VHvlbAl 1 IgGl Glml7 allotype) with bovine alpha-lactalbumin signal peptide at the N-terminus
  • SEQ ID NO: 183 sets forth the nucleotide sequence encoding a light chain of a 3D6 humanized variant (hu3D6VLv2 variant L37Q S52G L54R, L2-DIM4 kappa) with bovine alpha-lactalbumin signal peptide at the N-terminus
  • SEQ ID NO: 184 sets forth the amino acid sequence of a region of tau microtubule binding repeat 1 (amino acid residues 255-271 of SEQ ID NO: l)
  • SEQ ID NO: 185 sets forth the amino acid sequence of of a region of tau microtubule binding repeat 2 (amino acid residues 286-302 of SEQ ID NO: 1)
  • SEQ ID NO: 186 sets forth the amino acid sequence of of a region of tau microtubule binding repeat 3 (amino acid residues 317-333 of SEQ ID NO: 1)
  • SEQ ID NO: 187 sets forth the amino acid sequence of of a region of tau microtubule binding repeat 4 (amino acid residues 349-365 of SEQ ID NO: 1)
  • SEQ ID NO: 188 sets forth the amino acid sequence of a core motif of tau in MBTR 1 bound by 3D6
  • SEQ ID NO: 189 sets forth the amino acid sequence of tau sequence N-terminal to core motif of tau in MBTR 1 bound by 3D6
  • SEQ ID NO: 190 sets forth the amino acid sequence of tau sequence C-terminal to core motif of tau in MBTR lbound by 3D6
  • SEQ ID NO: 191 sets forth the amino acid sequence of epitope of 3D6
  • SEQ ID NO: 192 sets forth the amino acid sequence of a core motif of tau in MBTR 2 bound by 3D6
  • SEQ ID NO: 193 sets forth the amino acid sequence of a core motif of tau in MBTR 3 bound by 3D6
  • SEQ ID NO: 194 sets forth the amino acid sequence of a core motif of tau in MBTR 4 bound by 3D6
  • Monoclonal antibodies or other biological entities are typically provided in isolated form. This means that an antibody or other biologically entity is typically at least 50% w/w pure of interfering proteins and other contaminants arising from its production or purification but does not exclude the possibility that the monoclonal antibody is combined with an excess of pharmaceutically acceptable carrier(s) or other vehicle intended to facilitate its use. Sometimes monoclonal antibodies are at least 60%, 70%, 80%, 90%, 95% or 99% w/w pure of interfering proteins and contaminants from production or purification. Often an isolated monoclonal antibody or other biological entity is the predominant macromolecular species remaining after its purification.
  • Specific binding of an antibody to its target antigen means an affinity and/or avidity of at least 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 M 1 . Specific binding is detectably higher in magnitude and distinguishable from non-specific binding occurring to at least one unrelated target. Specific binding can be the result of formation of bonds between particular functional groups or particular spatial fit ( e.g ., lock and key type) whereas nonspecific binding is usually the result of van der Waals forces. Specific binding does not however necessarily imply that an antibody binds one and only one target.
  • the basic antibody structural unit is a tetramer of subunits.
  • Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy" chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed linked to a cleavable signal peptide.
  • the variable region without the signal peptide is sometimes referred to as a mature variable region.
  • a light chain mature variable region means a light chain variable region without the light chain signal peptide.
  • the carboxy -terminal portion of each chain defines a constant region primarily responsible for effector function.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 or more amino acids. See generally , Fundamental Immunology , Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989, Ch. 7 (incorporated by reference in its entirety for all purposes).
  • An immunoglobulin light or heavy chain variable region (also referred to herein as a “light chain variable domain” (“VL domain”) or“heavy chain variable domain” (“VH domain”), respectively) consists of a“framework” region interrupted by three“complementarity determining regions” or“CDRs.”
  • the framework regions serve to align the CDRs for specific binding to an epitope of an antigen.
  • the CDRs include the amino acid residues of an antibody that are primarily responsible for antigen binding. From amino-terminus to carboxyl-terminus, both VL and VH domains comprise the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • CDRs 1, 2, and 3 of a VL domain are also referred to herein, respectively, as CDR-L1, CDR-L2, and CDR-L3; CDRs 1, 2, and 3 of a VH domain are also referred to herein, respectively, as CDR-H1, CDR-H2, and CDR-H3.
  • R the R can alternatively be considered as being the N-terminal residue of the light chain constant region.
  • the application should also be understood as disclosing the VL sequence without the C-terminal R.
  • an antibody comprising CDRs defined by Rabat includes among other possibilities, an antibody in which the CDRs contain Rabat CDR residues and no other CDR residues, and an antibody in which CDR HI is a composite Chothia-Rabat CDR HI and other CDRs contain Rabat CDR residues and no additional CDR residues based on other definitions. [0299] Table 1
  • antibody includes intact antibodies and binding fragments thereof.
  • fragments compete with the intact antibody from which they were derived for specific binding to the target including separate heavy chains, light chains Fab, Fab', F(ab')2, F(ab)c, Dabs, nanobodies, and Fv. Fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins.
  • the term“antibody” also includes a bispecific antibody and/or a humanized antibody.
  • a bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy /light chain pairs and two different binding sites (see, e.g., Songsivilai and Lachmann, Clin. Exp.
  • the two different heavy /light chain pairs include a humanized 3D6 heavy chain/light chain pair and a heavy chain/light chain pair specific for a different epitope on tau than that bound by 3D6.
  • one heavy chain/light chain pair is a humanized 3D6 antibody as further disclosed below and the other heavy chain/light chain pair is from an antibody that binds to a receptor expressed on the blood brain barrier, such as an insulin receptor, an insulin-like growth factor (IGF) receptor, a leptin receptor, or a lipoprotein receptor, or a transferrin receptor (Friden e/ a/., Proc. Natl. Acad. Sci. USA 88:4771-4775, 1991; Friden et al., Science 259:373-377, 1993).
  • a bispecific antibody can be transferred cross the blood brain barrier by receptor-mediated transcytosis.
  • Brain uptake of the bispecific antibody can be further enhanced by engineering the bi-specific antibody to reduce its affinity to the blood brain barrier receptor. Reduced affinity for the receptor resulted in a broader distribution in the brain (see, e.g ., Atwal et al., Sci. Trans. Med. 3, 84ra43, 2011; Yu et al., Sci. Trans. Med. 3, 84ra44, 2011).
  • Exemplary bispecific antibodies can also be: (1) a dual -variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-IgTM) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (2) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (3) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (4) a so-called “dock and lock” molecule, based on the "dimerization and docking domain" in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked
  • bispecific antibodies examples include BiTE (Micromet), DART (MacroGenics), Fcab and Mab2 (F-star), Fc-engineered IgGl (Xencor) or DuoBody (based on Fab arm exchange, Genmab).
  • epitope refers to a site on an antigen to which an antibody binds.
  • An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
  • Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).
  • Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay showing the ability of one antibody to compete with the binding of another antibody to a target antigen.
  • the epitope of an antibody can also be defined X-ray
  • two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
  • Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
  • Competition between antibodies is determined by an assay in which an antibody under test inhibits specific binding of a reference antibody to a common antigen (see, e.g. , Junghans et al., Cancer Res. 50: 1495, 1990).
  • a test antibody competes with a reference antibody if an excess of a test antibody (e.g, at least 2x, 5x, lOx, 20x or lOOx) inhibits binding of the reference antibody by at least 50% as measured in a competitive binding assay.
  • Some test antibodies inhibit binding of the references antibody by at least 75%, 90% or 99%.
  • Antibodies identified by competition assay include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur.
  • pharmaceutically acceptable means that the carrier, diluent, excipient, or auxiliary is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.
  • patient includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
  • An individual is at increased risk of a disease if the subject has at least one known risk- factor (e.g, genetic, biochemical, family history, and situational exposure) placing individuals with that risk factor at a statistically significant greater risk of developing the disease than individuals without the risk factor.
  • risk- factor e.g, genetic, biochemical, family history, and situational exposure
  • biological sample refers to a sample of biological material within or obtainable from a biological source, for example a human or mammalian subject. Such samples can be organs, organelles, tissues, sections of tissues, bodily fluids, peripheral blood, blood plasma, blood serum, cells, molecules such as proteins and peptides, and any parts or combinations derived therefrom.
  • biological sample can also encompass any material derived by processing the sample. Derived material can include cells or their progeny.
  • Processing of the biological sample may involve one or more of filtration, distillation, extraction, concentration, fixation, inactivation of interfering components, and the like.
  • control sample refers to a biological sample not known or suspected to include tau-related disease-affected regions, or at least not known or suspect to include diseased regions of a given type.
  • Control samples can be obtained from individuals not afflicted with the tau-related disease.
  • control samples can be obtained from patients afflicted with the tau-related disease.
  • Such samples can be obtained at the same time as a biological sample thought to comprise the tau-related disease or on a different occasion.
  • a biological sample and a control sample can both be obtained from the same tissue.
  • control samples consist essentially or entirely of normal, healthy regions and can be used in comparison to a biological sample thought to comprise tau-related disease-affected regions.
  • the tissue in the control sample is the same type as the tissue in the biological sample.
  • the tau-related disease-affected cells thought to be in the biological sample arise from the same cell type (e.g ., neurons or glia ) as the type of cells in the control sample.
  • disease refers to any abnormal condition that impairs physiological function.
  • the term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired, irrespective of the nature of the etiology.
  • symptom refers to a subjective evidence of a disease, such as altered gait, as perceived by the subject.
  • a "sign” refers to objective evidence of a disease as observed by a physician.
  • positive response to treatment refers to a more favorable response in an individual patient or average response in a population of patients relative to an average response in a control population not receiving treatment.
  • amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gin, his, lys, arg; Group V (residues influencing chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids in the same class. Non conservative substitutions constitute exchanging a member of one of these classes for a member of another.
  • Percentage sequence identities are determined with antibody sequences maximally aligned by the Kabat numbering convention. After alignment, if a subject antibody region (e.g ., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage.
  • a subject antibody region e.g ., the entire mature variable region of a heavy or light chain
  • compositions or methods“comprising” or“including” one or more recited elements may include other elements not specifically recited.
  • a composition that “comprises” or“includes” an antibody may contain the antibody alone or in combination with other ingredients.
  • the disclosure refers to a feature comprising specified elements, the disclosure should alternative be understood as referring to the feature consisting essentially of or consisting of the specified elements.
  • Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.
  • the invention provides antibodies that bind to tau. Some antibodies specifically bind to an epitope or epitopes within the microtubule binding region (MTBR) region of human tau defined by approximately residues 244 to 372 of SEQ ID NO: 1. Because the MTBR region contains sequence repeats, a single antibody can bind at multiple repeated sites within this region. Some antibodies specifically bind to an epitope of KXXSXXNX(K/H)H (SEQ ID NO: 191). Some antibodies bind within residues 199-213 and/or 262-276 of SEQ ID NO:3 (corresponding to residues 257-271 or 320-334, respectively, of SEQ ID NO: 1).
  • MTBR microtubule binding region
  • Some antibodies bind within residues 259-268 and/or 290-299 and/or 321-330 and/or 353-362 of SEQ ID NO: 1. In some antibodies, binding to the MTBR 4 region at residues 353-362 is weaker than binding to MTBRs 1, 2 and 3. Some antibodies bind to tau irrespective of phosphorylation state. Some antibodies of the invention serve to inhibit or delay tau-associated pathologies and associated symptomatic deterioration.
  • a reduction in toxicity may occur as a result of the antibody inducing phagocytosis of tau, inhibiting tau from inter or intramolecular aggregation, or from binding to other molecules, by stabilizing a non-toxic conformation, by inhibiting intercellular or intracellular transmission of pathogenic tau forms, by blockade of tau phosphorylation, by preventing binding of tau to cells, or by inducing proteolytic cleavage of tau, among other mechanisms.
  • the antibodies of the invention or agents that induce such antibodies can be used in methods of treating or effecting prophylaxis of Alzheimer’s and other diseases associated with tau.
  • tau means a natural human form of tau including all isoforms irrespective of whether posttranslational modification (e.g ., phosphorylation, gly cation, or acetylation) is present.
  • posttranslational modification e.g ., phosphorylation, gly cation, or acetylation
  • reference to a phosphorylation at position 404 means position 404 of the 441 isoform, or corresponding position of any other isoform when maximally aligned with the 441 isoform.
  • the amino acid sequences of the isoforms and Swiss-Prot numbers are indicated below.
  • P10636-6 4R0N human tau (SEQ ID NO : 3 )
  • PKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTP PTREPKKVAV VRTPPKSPSS
  • PKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTP PTREPKKVAV VRTPPKSPSS
  • Reference to tau includes known natural variations about 30 of which are listed in the Swiss-Prot database and permutations thereof, as well as mutations associated with tau pathologies, such as dementia, Pick’s disease, supranuclear palsy, etc. (see, e.g ., Swiss-Prot database and Poorkaj, et al. Ann Neurol. 43:815-825 (1998)).
  • tau mutations numbered by the 441 isoform are a lysine to threonine mutation at amino acid residue 257 (K257T), an isoleucine to valine mutation at amino acid position 260 (I260V); a glycine to valine mutation at amino acid position 272 (G272V); an asparagine to lysine mutation at amino acid position 279 (N279K); an asparagine to histidine mutation at amino acid position 296 (N296H); a proline to serine mutation at amino acid position 301 (P301S); a proline to leucine mutation at amino acid 301 (P301L); a glycine to valine mutation at amino acid position 303 (G303V); a serine to asparagine mutation at position 305 (S305N); a glycine to serine mutation at amino acid position 335 (G335S); a valine to methionine mutation at position 337 (V337M); a glutamic acid
  • Tau can be phosphorylated at one or more amino acid residues including tyrosine at amino acid positions 18, 29, 97, 310, and 394 serine at amino acid positions 184, 185, 198, 199, 202, 208, 214, 235, 237, 238, 262, 293, 324, 356, 396, 400, 404, 409, 412, 413, and 422; and threonine at amino acids positions 175, 181, 205, 212, 217, 231, and 403.
  • reference to tau, or their fragments includes the natural human amino acid sequences including isoforms, mutants, and allelic variants thereof.
  • the invention provides antibodies that bind to tau. Some antibodies specifically bind to an epitope within KXXSXXNX(K/H)H (SEQ ID NO: 191). Some antibodies bind to a peptide comprising, consisting essentially of, or consisting of amino acid residues 259-268 of 441 amino acid tau protein (SEQ ID NO: 1). Some antibodies bind to a peptide comprising, consisting essentially of, or consisting of amino acid residues 290-299 of 441 amino acid tau protein (SEQ ID NO: 1). Some antibodies bind to a peptide comprising, consisting essentially of, or consisting of amino acid residues 321-330 of 441 amino acid tau protein (SEQ ID NO: 1).
  • Some antibodies bind to a peptide comprising, consisting essentially of or consisting of amino acid residues 353- 362 of 441 amino acid tau protein (SEQ ID NO: 1). Some antibodies bind to two, three or all four of these peptides. Some antibodies specifically bind to an epitope within residues 199-213 of 383 amino acid 4R0N human tau protein (SEQ ID NO:3) (corresponding to residues 257-271 of SEQ ID NO: 1). Some antibodies specifically bind to an epitope within residues 262-276 of 383 amino acid 4RON human tau protein (SEQ ID NO:3) (corresponding to residues 320-334 of SEQ ID NO: 1).
  • Some antibodies of the invention specifically bind to a peptide consisting of residues 257-271 of 441 amino acid tau protein (SEQ ID NO: 1). Some antibodies of the invention specifically bind to a peptide consisting of residues 320-334 of 441 amino acid tau protein (SEQ ID NO: 1). Some antibodies of the invention specifically bind to a peptide consisting of residues 259-268 of 441 amino acid tau protein SEQ ID NO: l, namely
  • KIGSTENLKH SEQ ID NO: 188.
  • Some antibodies of the invention specifically bind to a peptide consisting of residues 290-299 of 441 amino acid tau protein SEQ ID NO: l, namely KCGSKDNIKH (SEQ ID NO: 192).
  • Some antibodies of the invention specifically bind to a peptide consisting of residues 321-330 of 441 amino acid tau protein SEQ ID NO: l, namely KCGSLGNHm (SEQ ID NO: 193).
  • Some antibodies of the invention specifically bind to a peptide consisting of residues 353-362 of 441 amino acid tau protein SEQ ID NO: l, namely KIGSLDNITH (SEQ ID NO: 194).
  • Some antibodies of the invention specifically bind to a peptide consisting of the consensus motif KXXSXXNX(K/H)H (SEQ ID NO: 191). Some antibodies bind to an epitope comprising residues 259, 262, 265, 267, 268, residues 290, 293, 296, 298, 299, residues 321, 324, 327, 329, 330, or residues 353, 356, 359, 362 of 441 amino acid tau protein SEQ ID NO: 1. Some antibodies bind to tau irrespective of phosphorylation state. Some antibodies bind to an epitope not including a residue subject to phosphorylation. These antibodies can be obtained by immunizing with a tau polypeptide purified from a natural source or recombinantly expressed. Antibodies can be screened for binding tau in
  • unphosphorylated form as well as a form in which one or more residues susceptible to phosphorylation are phosphorylated.
  • Such antibodies preferably bind with indistinguishable affinities or at least within a factor of 1.5, 2 or 3 -fold to phosphorylated tau compared to non- phosphorylated tau (i.e., are“pan-specific”).
  • 3D6 is an example of a pan-specific monoclonal antibody.
  • the invention also provides antibodies binding to the same epitope as any of the foregoing antibodies, such as, for example, the epitope of 3D6. Also included are antibodies competing for binding to tau with any of the foregoing antibodies, such as, for example, competing with 3D6.
  • the above-mentioned antibodies can be generated de novo by immunizing with a peptide including, consisting essentially of or consisting of residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to residues 257-271 or 320-334, respectively, of SEQ ID NO: 1) or by immunizing with a peptide including, consisting essentially of or consisting of residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO: 1, or by immunizing with a full length tau polypeptide or fragment thereof comprising such residues and screening for specific binding to a peptide including such residues.
  • Such peptides are preferably attached to a heterologous conjugate molecule that helps elicit an antibody response to the peptide. Attachment can be direct or via a spacer peptide or amino acid. Cysteine is used as a spacer amino acid because its free SH group facilitates attachment of a carrier molecule. A polyglycine linker (e.g ., 2-6 glycines), with or without a cysteine residue between the glycines and the peptide can also be used.
  • the carrier molecule serves to provide a T-cell epitope that helps elicit an antibody response against the peptide.
  • KLH keyhole limpet hemocyanin
  • ovalbumin ovalbumin
  • bovine serum albumin BSA
  • Peptide spacers can be added to peptide immunogen as part of solid phase peptide synthesis.
  • Carriers are typically added by chemical cross-linking.
  • Some examples of chemical crosslinkers that can be used include cross-N- maleimido-6-aminocaproyl ester or m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (see for example, Harlow, E. et ah, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1988; Sinigaglia et ah, Nature, 336:778-780 (1988); Chicz et ah, J. Exp.
  • the carrier and spacer if present can be attached to either end of the immunogen.
  • a peptide with optional spacer and carrier can be used to immunize laboratory animals or B-cells as described in more detail below.
  • Hybridoma supernatants can be tested for ability to bind one or more peptides including, consisting essentially of or consisting of residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to residues 257-271 or 320-334, respectively, of SEQ ID NO: l), or including, consisting essentially of or consisting of residues 259-268, 290- 299, 321-330, or 353-362 of SEQ ID NO: l, and/or phosphorylated and non-phosphorylated forms of tau, such as, for example, a full-length isoform of tau with position 404 in
  • the peptide can be attached to a carrier or other tag to facilitate the screening assay.
  • the carrier or tag is preferentially different than the combination of spacer and carrier molecule used for immunization to eliminate antibodies specific for the spacer or carrier rather than the tau peptide. Any of the tau isoforms can be used.
  • the invention provides monoclonal antibodies binding to epitopes within tau.
  • An antibody designated 3D6 is one such exemplary mouse antibody. Unless otherwise apparent from context, reference to 3D6 should be understood as referring to any of the mouse, chimeric, veneered, and humanized forms of this antibody.
  • the antibody has been deposited as [DEPOSIT NUMBER] This antibody specifically binds to an epitope of KXXSXXNX(K/H)H (SEQ ID NO: 191).
  • This antibody specifically binds within amino acid residues 199-213 and/or 262-276 of the 383 amino acid 4R0N human tau protein (SEQ ID NO:3) (corresponding to amino acid residues 257-271 and/or 320-334, respectively, of SEQ ID NO: l).
  • the antibody specifically binds within amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: l, and combinations of any 2, 3 or all four thereof.
  • This antibody is further characterized by its ability to bind both phosphorylated and unphosphorylated tau, both non-pathological and pathological forms and conformations of tau, and misfolded/aggregated forms of tau.
  • An antibody designated 6A10 is one such exemplary mouse antibody.
  • reference to 6A10 should be understood as referring to any of the mouse, chimeric, veneered, and humanized forms of this antibody.
  • Kabat/Chothia Composite CDRs of the heavy chain of 6A10 are designated SEQ ID NOs:67, 68, and 69, respectively, and Rabat CDRs of the light chain of 6A10 are designated SEQ ID NOs: 12, 13, and 14, respectively.
  • Mouse 6A10 shares 82.1% of VH sequence identity and 100% VL sequence identity with the VH chain and VL chain, respectively, of mouse 3D6.
  • Some antibodies of the invention bind to the same or overlapping epitope as an antibody designated 3D6.
  • the sequences of the heavy and light chain mature variable regions of this antibody are designated SEQ ID NOs:7 and 11, respectively.
  • Other antibodies having such a binding specificity can be produced by immunizing mice with tau or a portion thereof including, consisting essentially of or consisting of the desired epitope (e.g.
  • Fragments of tau including the desired epitope can be linked to a carrier that helps elicit an antibody response to the fragment and/or be combined with an adjuvant the helps elicit such a response.
  • Such antibodies can be screened for differential binding to tau or a fragment thereof compared with mutants of specified residues. Screening against such mutants more precisely defines the binding specificity to allow identification of antibodies whose binding is inhibited by mutagenesis of particular residues and which are likely to share the functional properties of other exemplified antibodies.
  • the mutations can be systematic replacement substitution with alanine (or serine if an alanine is present already) one residue at a time, or more broadly spaced intervals, throughout the target or throughout a section thereof in which an epitope is known to reside. If the same set of mutations significantly reduces the binding of two antibodies, the two antibodies bind the same epitope.
  • Antibodies having the binding specificity of a selected murine antibody can also be produced using a variant of the phage display method. See Winter, WO 92/20791. This method is particularly suitable for producing human antibodies. In this method, either the heavy or light chain variable region of the selected murine antibody is used as a starting material. If, for example, a light chain variable region is selected as the starting material, a phage library is constructed in which members display the same light chain variable region (i.e., the murine starting material) and a different heavy chain variable region. The heavy chain variable regions can for example be obtained from a library of rearranged human heavy chain variable regions.
  • a phage showing strong specific binding for tau or a fragment thereof (e.g, at least 10 8 and preferably at least 10 9 M 1 ) is selected.
  • the heavy chain variable region from this phage then serves as a starting material for constructing a further phage library.
  • each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) and a different light chain variable region.
  • the light chain variable regions can be obtained for example from a library of rearranged human variable light chain regions.
  • phage showing strong specific binding for tau or a fragment thereof are selected.
  • the resulting antibodies usually have the same or similar epitope specificity as the murine starting material.
  • Table 2 indicates the 3D6 CDRs as defined by Kabat, Chothia, Composite of Chothia and Kabat (also referred to herein as“Kabat/Chothia Composite”), AbM, and Contact.
  • Table 2 3D6 CDRs as defined by Kabat, Chothia, Composite of Chothia and Kabat,
  • Other antibodies can be obtained by mutagenesis of cDNA encoding the heavy and light chains of an exemplary antibody, such as 3D6.
  • Monoclonal antibodies that are at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to 3D6 in amino acid sequence of the mature heavy and/or light chain variable regions and maintain its functional properties, and/or which differ from the respective antibody by a small number of functionally inconsequential amino acid substitutions (e.g ., conservative substitutions), deletions, or insertions are also included in the invention.
  • Monoclonal antibodies having at least one or all six CDR(s) as defined by any conventional definition, but preferably Kabat, that are 90%, 95%, 99% or 100% identical to corresponding CDRs of 3D6 are also included.
  • the invention also provides antibodies having some or all (e.g ., 3, 4, 5, and 6) CDRs entirely or substantially from 3D6.
  • Such antibodies can include a heavy chain variable region that has at least two, and usually all three, CDRs entirely or substantially from the heavy chain variable region of 3D6 and/or a light chain variable region having at least two, and usually all three, CDRs entirely or substantially from the light chain variable region of3D6.
  • the antibodies can include both heavy and light chains.
  • a CDR is substantially from a corresponding 3D6 CDR when it contains no more than 4, 3, 2, or 1 substitutions, insertions, or deletions, except that CDR-H2 (when defined by Rabat) can have no more than 6, 5, 4, 3, 2, or 1 substitutions, insertions, or deletions.
  • Such antibodies can have at least 70%, 80%, 90%, 95%, 96%, 97%,
  • Some antibodies identified by such assays can bind to monomeric, misfolded, aggregated, phosphorylated, or unphosphorylated forms of tau or otherwise. Likewise, some antibodies are immunoreactive on non-pathological and pathological forms and conformations of tau.
  • non-human antibodies e.g, murine, guinea pig, primate, rabbit or rat, against tau or a fragment thereof (e.g., amino acid residues 199-213 or 262-276 of SEQ ID NO:3, corresponding to amino acid residues 257-271 or 320-334, respectively, of SEQ ID NO: l; or amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: l) can be accomplished by, for example, immunizing the animal with tau or a fragment thereof . See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes).
  • Such an immunogen can be obtained from a natural source, by peptide synthesis, or by recombinant expression.
  • the immunogen can be administered fused or otherwise complexed with a carrier protein.
  • the immunogen can be administered with an adjuvant.
  • adjuvant Several types of adjuvant can be used as described below. Complete Freund’s adjuvant followed by incomplete adjuvant is preferred for immunization of laboratory animals. Rabbits or guinea pigs are typically used for making polyclonal antibodies. Mice are typically used for making monoclonal antibodies.
  • Antibodies are screened for specific binding to tau or an epitope within tau (e.g., an epitope comprising one or more of amino acid residues 199-213 or 262-276 of SEQ ID NO:3; corresponding to amino acid residues 257-271 or 320-334, respectively, of SEQ ID NO: 1 or an epitope comprising one or more of amino acid residues 259- 268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: 1).
  • an epitope within tau e.g., an epitope comprising one or more of amino acid residues 199-213 or 262-276 of SEQ ID NO:3; corresponding to amino acid residues 257-271 or 320-334, respectively, of SEQ ID NO: 1 or an epitope comprising one or more of amino acid residues 259- 268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: 1).
  • Such screening can be accomplished by determining binding of an antibody to a collection of tau variants, such as tau variants containing amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or 320-334, respectively, of SEQ ID NO: 1) or tau variants containing amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: l, or mutations within these residues, and determining which tau variants bind to the antibody. Binding can be assessed, for example, by Western blot, FACS or ELISA.
  • a humanized antibody is a genetically engineered antibody in which CDRs from a non human“donor” antibody are grafted into human“acceptor” antibody sequences (see, e.g.,
  • the acceptor antibody sequences can be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence.
  • a humanized antibody is an antibody having at least three, four, five or all CDRs entirely or substantially from a donor antibody and variable region framework sequences and constant regions, if present, entirely or substantially from human antibody sequences.
  • a humanized heavy chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody heavy chain, and a heavy chain variable region framework sequence and heavy chain constant region, if present, substantially from human heavy chain variable region framework and constant region sequences.
  • a humanized light chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody light chain, and a light chain variable region framework sequence and light chain constant region, if present, substantially from human light chain variable region framework and constant region sequences.
  • a humanized antibody comprises a humanized heavy chain and a humanized light chain.
  • a CDR in a humanized antibody is substantially from a corresponding CDR in a non-human antibody when at least 85%, 90%, 95% or 100% of corresponding residues (as defined by any conventional definition but preferably defined by Rabat) are identical between the respective CDRs.
  • the variable region framework sequences of an antibody chain or the constant region of an antibody chain are substantially from a human variable region framework sequence or human constant region respectively when at least 85%, 90%, 95% or 100% of corresponding residues defined by Kabat are identical.
  • WHO World Health Organization
  • INN International non-proprietary names
  • humanized as used herein is not intended to be limited to the 2014 WHO INN definition of humanized antibodies.
  • Some of the humanized antibodies provided herein have at least 85% sequence identity to human germline sequences and some of the humanized antibodies provided herein have less than 85% sequence identity to human germline sequences.
  • Some of the heavy chains of the humanized antibodies provided herein have from about 60% to 100% sequence identity to human germ line sequences, such as, for example, in the range of about 60% to 69%, 70% to 79%, 80% to 84%, or 85% to 89%.
  • Some heavy chains fall below the 2014 WHO INN definition and have, for example, about 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, or 82%, 83%, or 84% sequence identity to human germ line sequences, while other heavy chains meet the 2014 WHO INN definition and have about 85%, 86%, 87%, 88%, 89% or greater sequence identity to human germ line sequences.
  • Some of the light chains of the humanized antibodies provided herein have from about 60% to 100% sequence identity to human germ line sequences, such as, for example, in the range of about 80% to 84% or 85% to 89%.
  • Some humanized antibodies provided herein that are "chimeric" under the 2014 WHO INN definition have heavy chains with less than 85% identity to human germ line sequences paired with light chains having less than 85% identity to human germ line sequences.
  • Some humanized antibodies provided herein are "mixed" under the 2014 WHO INN definition, for example, having a heavy chain with at least 85% sequence identity to human germ line sequences paired with a light chain having less than 85% sequence identity to human germ line sequences, or vice versa.
  • Some humanized antibodies provided herein meet the 2014 WHO INN definition of "humanized” and have a heavy chain with at least 85% sequence identity to human germ line sequences paired with a light chain having at least 85% sequence identity to human germ line sequences.
  • Additional humanized antibodies of the invention meet the 2014 WHO INN definition of“mixed.”
  • humanized antibodies often incorporate all six CDRs (defined by any conventional definition but preferably as defined by Kabat) from a mouse antibody, they can also be made with less than all CDRs (e.g ., at least 3, 4, or 5 CDRs) from a mouse antibody (e.g, Pascalis et a/., J. Immunol. 169:3076, 2002; Vajdos et al., J. of Mol. Biol., 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36: 1079-1091, 1999; Tamura et al, J. Immunol., 164: 1432-1441, 2000).
  • CDRs defined by any conventional definition but preferably as defined by Kabat
  • CDR residues not contacting antigen and not in the SDRs can be identified based on previous studies (for example residues H60-H65 in CDR H2 are often not required), from regions of Kabat CDRs lying outside Chothia hypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987), by molecular modeling and/or empirically, or as described in Gonzales et al., Mol. Immunol. 41 : 863, 2004.
  • the amino acid occupying the position can be an amino acid occupying the corresponding position (by Kabat numbering) in the acceptor antibody sequence.
  • the number of such substitutions of acceptor for donor amino acids in the CDRs to include reflects a balance of competing considerations.
  • Such substitutions are potentially advantageous in decreasing the number of mouse amino acids in a humanized antibody and consequently decreasing potential immunogenicity and/or for meeting the WHO INN definition of“humanized”.
  • substitutions can also cause changes of affinity, and significant reductions in affinity are preferably avoided.
  • Positions for substitution within CDRs and amino acids to substitute can also be selected empirically.
  • the human acceptor antibody sequences can optionally be selected from among the many known human antibody sequences to provide a high degree of sequence identity (e.g ., 65- 85% identity) between a human acceptor sequence variable region frameworks and
  • variable region frameworks of a donor antibody chain are corresponding variable region frameworks of a donor antibody chain.
  • Some humanized and chimeric antibodies have the same (within experimental error) or improved functional properties, e.g., binding affinity for human tau, inhibition of tau
  • some humanized and chimeric antibodies have a binding affinity within a factor of 3, 2 or 1 of the murine antibody from which they were derived or an affinity indistinguishable within experimental error.
  • Some humanized and chimeric antibodies inhibit tau internalization into neurons as described in the examples within a factor of 3, 2 or 1 of the murine antibody from which they were derived or inhibit the same within experimental error as the mouse antibody from which they were derived.
  • hu3D6VHvlbAl l/L2-DIM4 demonstrated improved affinity, as evidenced by on-rate, off-rate, and Kd numbers, over parental hu3D6VHvlbAl 1/ hu3D6VLv2.
  • hu3D6VHvlbAl 1/L2-DIM4 demonstrated higher thermostability and titer over parental hu3D6VHvlbAl 1/ hu3D6VLv2.
  • An example of an acceptor sequence for the heavy chain is the human mature heavy chain variable region of humanized 48G7 Fab with PDB accession code 2RCS-VH_huFrwk (SEQ ID NO:75). The variable domains of 3D6 and 48G7 Fab also share identical lengths for the CDR-H1, H2 loops.
  • An example of an acceptor sequence for the heavy chain is the human mature heavy chain variable region IMGT# IGHVl-69-2*01 (SEQ ID NO:25). IMGT# IGHV1- 69-2*01 (SEQ ID NO:25) shares the canonical form of mouse 3D6 heavy chain CDR-H1 and H2.
  • IMGT# IGHVl-69-2*01 belongs to human heavy chain subgroup 1.
  • An example of an acceptor sequence for the light chain is the human mature light chain variable region with PDB accession code human antibody ARX71335 VL (SEQ ID NO:82). The variable light domain of 3D6 and ARX71335 antibody also share identical lengths for the CDR- LI, L2 and L3 loops.
  • An example of an acceptor sequence for the light chain is the human mature light chain variable region with IMGT#IGKV2-30*02 (SEQ ID NO:27).
  • IMGT#IGKV2- 30*02 (SEQ ID NO:27) has the same canonical classes for CDR-L1, CDR-L2 and L3 as mouse 3D6.
  • IMGT#IGKV2-30*02 belongs to human kappa subgroup 2.
  • a composite or hybrid of those acceptors can be used, and the amino acids used at different positions in the humanized light chain and heavy chain variable regions can be taken from any of the human acceptor antibody sequences used.
  • the human mature heavy chain variable regions of IMGT# IGHV1 -69-2*01 (SEQ ID NO:25) and PDB accession code # 2RCS-VH_huFrwk (SEQ ID NO: 75) were used as acceptor sequences for humanization of the 3D6 mature heavy chain variable region.
  • An example of a positions in which these two acceptors differ is position HI 7 (T or S).
  • Humanized versions of the 3D6 heavy chain variable region can include either amino acid at this position.
  • the human mature light chain variable regions IMGT# IGKV2-30*02 (SEQ ID NO:27) and PDB code # ARX71335-VL_huFrwk (SEQ ID NO:82) were used as acceptor sequences for humanization of the 3D6 mature light chain variable region.
  • An example of a position in which these two acceptors differ is position LI 00 (Q or A).
  • Humanized versions of the3D6 light chain variable region can include either amino acid at this position.
  • Certain amino acids from the human variable region framework residues can be selected for substitution based on their possible influence on CDR conformation and/or binding to antigen. Investigation of such possible influences is by modeling, examination of the characteristics of the amino acids at particular locations, or empirical observation of the effects of substitution or mutagenesis of particular amino acids.
  • the human framework amino acid when an amino acid differs between a murine variable region framework residue and a selected human variable region framework residue, the human framework amino acid can be substituted by the equivalent framework amino acid from the mouse antibody when it is reasonably expected that the amino acid:
  • humanized sequences are generated using a two-stage PCR protocol that allows introduction of multiple mutations, deletions, and insertions using QuikChange site- directed mutagenesis [Wang, W. and Malcolm, B.A. (1999) BioTechniques 26:680-682)].
  • framework residues that are candidates for substitution are residues creating a potential glycosylation site. Still other candidates for substitution are acceptor human framework amino acids that are unusual for a human immunoglobulin at that position. These amino acids can be substituted with amino acids from the equivalent position of the mouse donor antibody or from the equivalent positions of more typical human immunoglobulins.
  • N-terminal glutamine residues (Q) that may be replaced with glutamic acid (E) to minimize potential for pyroglutamate conversion
  • E glutamic acid
  • Glutamic acid (E) conversion to pyroglutamate (pE) occurs more slowly than from glutamine (Q). Because of the loss of a primary amine in the glutamine to pE conversion, antibodies become more acidic. Incomplete conversion produces heterogeneity in the antibody that can be observed as multiple peaks using charge-based analytical methods. Heterogeneity differences may indicate a lack of process control.
  • Exemplary humanized antibodies are humanized forms of the mouse 3D6, designated Hu3D6.
  • the mouse antibody 3D6 comprises mature heavy and light chain variable regions having amino acid sequences comprising SEQ ID NO:7 and SEQ ID NO: 11, respectively.
  • the invention provides humanized forms of the murine 3D6 antibody including 10 exemplified humanized heavy chain mature variable regions (hu3D6VHvbl (SEQ ID NO:76), hu3D6VHvb2 (SEQ ID NO: 77), hu3D6VHvb3 (SEQ ID NO: 78), hu3D6VHvb4 (SEQ ID NO: 79),
  • hu3D6VHvb5 (SEQ ID NO:80), hu3D6VHvb6 (SEQ ID NO:90), hu3D6VHvb7 (SEQ ID NO:91), hu3D6VHvlbAl l D60E (h3D6VHvb8, SEQ ID NO: 146), hu3D6VHvlbAl 1 L82cV (SEQ ID NO: 147), and hu3D6VHvlbAl 1 D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9, SEQ ID NO: 148)) and 56 exemplified humanized light chain mature variable regions
  • hu3D6VLv2 L54N (SEQ ID NO:95), hu3D6VLv2 L54E (SEQ ID NO:96), hu3D6VLv2 L50E (SEQ ID NO:97), hu3D6VLv2 L54Q (SEQ ID NO:98), hu3D6VLv2 L50D (SEQ ID NO:99), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 I48G (SEQ ID NO: 104), hu3D6VLv2 I48D (SEQ ID NO: 105), hu3D6VLv2 L47G (SEQ ID NO:
  • L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), and hu3D6VLv2 G100Q (SEQ ID NO: 141).
  • Figures 2 and 3 show alignments of the heavy chain variable region and light chain variable region, respectively, of murine 3D6 and various humanized antibodies.
  • Figures 9A and 9B show alignment of the heavy chain variable region of the murine 3D6 with the heavy chain variable region of various humanized antibodies.
  • Figures 10 A, 10B, IOC, and 10D show alignment of the light chain variable region of hu3D6VLv2 with the light chain variable region of various humanized antibodies.
  • variable region framework positions were considered as candidates for substitutions in the 56 exemplified human mature light chain variable regions and the 10 exemplified human mature heavy chain variable regions, as further specified in the examples: L7 (T7S), L10 (T10S), L15 (I15L), L17 (Q17E), L37 (L37Q), L45 (K45R), L47 (L47G, L47N, L47D, L47E, L47P, L47T, L47S, or L47A) , L48 (I48G or I48D), L49 (Y49E), L83 (L83V), L86 (H86Y), L100 (A100Q
  • L82cV H83 (T83R), H91 (Y91F), H93 (A93S), H94 (S94T), H108 (T108L), and H109 (LI 09V).
  • the following 9 variable region CDR positions were considered as candidates for substitutions in the 56 exemplified human mature light chain variable regions and 10 exemplified human mature heavy chain variable regions, as further specified in the examples: L24 (K24R), L50 (L50E, L50D, L50G, or L50V), L52 (S52G), L54 (L54D, L54G, L54N, L54E, L54Q, L54K, L54R, L54T, L54V, or L54S), H28 (N28T), H54 (N54D), H56 (D56E), H58 (V58I), and H60 (D60E).
  • Rabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 87.
  • Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 149.
  • Kabat-Chothia Composite CDR-H1 has an amino acid sequence comprising SEQ ID NO:86
  • Kabat CDR- H2 has an amino acid sequence comprising SEQ ID NO:87.
  • Kabat-Chothia Composite CDR-H1 has an amino acid sequence comprising SEQ ID NO:86 and Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO:88.
  • Kabat-Chothia Composite CDR-H1 has an amino acid sequence comprising SEQ ID NO:86 and Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO:92.
  • Kabat CDR-L1 has an amino acid sequence comprising SEQ ID NO:89.
  • Kabat CDR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 150-175.
  • the first-mentioned residue is the residue of a humanized antibody formed by grafting Kabat CDRs or a composite Chothia-Kabat CDR in the case of CDR-H1 into a human acceptor framework, and the second-mentioned residue is a residue being considered for replacing such residue.
  • the first mentioned residue is human
  • the first mentioned residue is mouse.
  • Exemplified antibodies include any permutations or combinations of the exemplified mature heavy and light chain variable regions VHvbl/VLvbl, VHvbl/VLvb2, VHvbl/VLvb3, VHvb2/VLvbl, VHvb2/VLvb2, VHvb2/VLvb3, VHvb3/VLvbl, VHvb3/VLvb2,
  • VHvb5/VLvb2 VHvb5/VLvb3, VHvb6/VLvbl, VHvb6/VLvb2, VHvb6/VLvb3,
  • Exemplified antibodies include any amino acids, VHvb7/VLvbl, VHvb7/VLvb2, VHvb7/VLvb3.
  • Exemplified antibodies include any amino acids, VHvb7/VLvbl, VHvb7/VLvb2, VHvb7/VLvb3.
  • Exemplified antibodies include any amino acids, VHvb7/VLvbl, VHvb7/VLvb2, VHvb7/VLvb3.
  • Exemplified antibodies include any amino acids, VHvb7/VLvbl, VHvb7/VLvb2, VHvb7/VLvb3.
  • Exemplified antibodies include any amino acids, VHvb7/VLvbl, VHvb7/VLvb2, VHvb7/VLvb3.
  • L37Q L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q L50D L54G (SEQ ID NO: 132), hu3D6VLv2 L37Q L50D L54R (SEQ ID NO: 133), hu3D6VLv
  • L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), and hu3D6VLv2 G100Q (SEQ ID NO: 144).
  • Exemplified antibodies include any permutations or combinations of the exemplified mature heavy chain variable regions hu3D6VHvb l (SEQ ID NO:76), hu3D6VHvb2 (SEQ ID NO:77), hu3D6VHvb3 (SEQ ID NO:78), hu3D6VHvb4 (SEQ ID NO:79), hu3D6Hvb5 (SEQ ID NO: 80), hu3D6VHvb6 (SEQ ID NO: 90), hu3D6VHvb7 (SEQ ID NO:91), hu3D6VHvb7 (SEQ ID NO:91), hu3D6VHvlbAl l D60E (h3D6VHvb8, SEQ ID NO: 146), hu3D6VHvlbAl 1 L82cV (SEQ ID NO: 147), and hu3D6VHvl
  • hu3D6VLv4 (SEQ ID NO:22).
  • Exemplified antibodies include any permutations or
  • L37Q L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q L50D L54G (SEQ ID NO: 132), hu3D6VLv2 L37Q L50D L54R (SEQ ID NO: 133), hu3D6VLv
  • the invention provides an antibody in which humanized heavy chain variable region hu3D6VHvlbAl 1, also known as h3D6Hu5, (SEQ ID NO: 18) is combined with humanized light chain variable region hu3D6VLv2 L37Q S52G L54R (L2-DIM4, SEQ ID NO: 122).
  • the invention provides an antibody in which humanized heavy chain variable region
  • hu3D6VHvlbAl also known as h3D6Hu5, (SEQ ID NO: 18) is combined with humanized light chain variable region hu3D6VLv2 L37Q S52G L54T (L2-DIM5, SEQ ID NO: 123).
  • the invention provides an antibody in which humanized heavy chain variable region h3D6VHvb8 (SEQ ID NO: 146) is combined with humanized light chain variable region hu3D6VLv2
  • L37Q S52G L54R L37Q S52G L54R (L2-DIM4, SEQ ID NO: 122).
  • the invention provides an antibody in which humanized heavy chain variable region hu3D6VHvlbAl 1, also known as h3D6Hu5, (SEQ ID NO: 18) is combined with humanized light chain variable region hu3D6VLv2
  • the invention provides an antibody in which humanized heavy chain variable region hu3D6VHvlbAl 1, also known as h3D6Hu5, (SEQ ID NO: 18) is combined with humanized light chain variable region hu3D6VLv2 S52G (L2-DIM9, SEQ ID NO: 110).
  • the invention provides an antibody in which humanized heavy chain variable region h3D6VHvb8 (SEQ ID NO: 146) is combined with humanized light chain variable region hu3D6VLv2 L54G (L2-DIM7, SEQ ID NO:94).
  • the invention provides an antibody in which humanized heavy chain variable region hu3D6VHvlbAl 1, also known as h3D6Hu5, (SEQ ID NO: 18) is combined with humanized light chain variable region hu3D6VLv2 L50G (L2-DIM22, SEQ ID NO: 103).
  • the invention provides an antibody in which any one of the exemplified humanized heavy chain variable regions is combined with a human heavy chain constant region.
  • An exemplary human heavy chain constant region is provided as SEQ ID NO: 176 (IgGl : allotype Glml7,l).
  • SEQ ID NO: 178 sets forth the amino acid sequence of a mature heavy chain of a 3D6 humanized variant (hu3D6VHvlbAl 1 IgGl Glml7 allotype).
  • SEQ ID NO: 178 sets forth the amino acid sequence of a mature heavy chain of a 3D6 humanized variant (hu3D6VHvlbAl 1 IgGl Glml7 allotype).
  • SEQ ID NO: 180 sets forth the amino acid sequence of a heavy chain of a 3D6 humanized variant (hu3D6VHvlbAl 1 IgGl Glml7 allotype) bovine alpha-lactalbumin signal peptide at the N- terminus.
  • the invention provides an antibody in which any one of the exemplified humanized light chain variable regions is combined with a light chain constant region.
  • An exemplary light chain constant region is provided as SEQ ID NO: 177 (kappa).
  • SEQ ID NO: 179 sets forth the amino acid sequence of a mature light chain of a 3D6 humanized variant
  • SEQ ID NO: 181 sets forth the amino acid sequence of a light chain of a 3D6 humanized variant (hu3D6VLv2 variant L37Q__S52G__L54R, L2-DIM4 kappa) with bovine alpha-lactalbumin signal peptide at the N- terminus.
  • the invention provides variants of the 3D6 humanized antibody in which the humanized mature heavy chain variable region shows at least 90%, 95%, 96%, 97%, 98%, or 99% identity to hu3D6VHvbl (SEQ ID NO:76), hu3D6VHvb2 (SEQ ID NO:77), hu3D6VHvb3 (SEQ ID NO:78), hu3D6VHvb4 (SEQ ID NO:79), hu3D6Hvb5 (SEQ ID NO:80), hu3D6VHvb6 (SEQ ID NO:90), hu3D6VHvb7 (SEQ ID NO:91), hu3D6VHvb7 (SEQ ID NO:91),
  • hu3D6VHvlbAl l D60E (h3D6VHvb8, SEQ ID NO: 146), hu3D6VHvlbAl 1 L82cV (SEQ ID NO: 147), or hu3D6VHvlbAl 1 D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9, SEQ ID NO: 148) and the humanized mature light chain variable region shows at least 90%, 95%, 96%, 97%, 98%, or 99% identity to hu3D6VLvbl (SEQ ID NO:83), hu3D6VLvb2 (SEQ ID NO:84), hu3D6VLvb3 (SEQ ID NO:85), hu3D6VLv2 L54D (SEQ ID NO:93), hu3D6VLv2 L54G (SEQ ID NO: 94), hu3D6V
  • L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2 L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2 L37Q_S52G_L54R_G100Q (SEQ ID NO: 138), hu3D6VLv2
  • L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), or hu3D6VLv2 G100Q (SEQ ID NO: 144).
  • hu3D6VLv2 L37Q (SEQ ID NO: 143)
  • hu3D6VLv2 G100Q (SEQ ID NO: 144).
  • In some such antibodies at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
  • H93 is occupied by S and H94 is occupied by T.
  • positions H93 and H94 are occupied by S and T, respectively.
  • position H91 in the VH region is occupied by F.
  • At least one of the following positions in the VH region is occupied by the amino acid as specified: HI is occupied by E, H5 is occupied by V,
  • HI 1 is occupied by V
  • H20 is occupied I
  • H23 is occupied by K
  • H38 is occupied by R
  • H42 is occupied by G
  • H43 is occupied by K
  • H66 is occupied by R
  • H75 is occupied by T
  • H76 is occupied by D
  • H81 is occupied by E
  • HI 08 is occupied by L
  • HI 09 is occupied by V.
  • positions HI, H5, HI 1, H20, H23, H38, H42, H43, H66, H75, H76, H81, HI 08, and HI 09 in the VH region are occupied by E, V, V, I, K, R, G, K, R, T, D, E, L, and V, respectively.
  • HI 7 is occupied by T
  • H80 is occupied by M
  • H83 is occupied by R.
  • positions HI 7, H80, and H83 in the VH region are occupied by T, M, and R, respectively.
  • position H58 in the VH region is occupied by I.
  • H28 is occupied by T
  • H67 is occupied by V
  • positions H28 and H67 in the VH region are occupied by T and V, respectively.
  • H54 is occupied by D
  • H56 is occupied by E
  • positions H54 and H56 in the VH region are occupied by D and E, respectively.
  • HI is occupied by Q or E
  • H5 is occupied by Q or V
  • HI 1 is occupied by L or V
  • HI 7 is occupied by S or T
  • H20 is occupied by L or I
  • H23 is occupied by T or K
  • H28 is occupied by N or T
  • H38 is occupied by K or R
  • H42 is occupied by E or G
  • H43 is occupied by Q or K
  • H54 is occupied by N or D
  • H56 is occupied by D or E
  • H58 is occupied by V or I
  • H66 is occupied by K or R
  • H67 is occupied by A or V
  • H75 is occupied by S or T
  • H76 is occupied by N or D
  • H80 is occupied by L or M
  • H81 is occupied by Q or E
  • H83 is occupied by T or R
  • H91 is occupied by F or Y
  • H93 is occupied by occupied by
  • positions H91, H93, and H94 in the VH region are occupied by F, S, and T, respectively, as in huVHvbl .
  • positions HI, H5, Hl l, H20, H23, H38, H42, H43, H66, H75, H76, H81, H91, H93, H94, H108, and HI 09 in the VH region are occupied by E, V, V, I, K, R, G, K, R, T, D, E, F, S, T,L, and V, respectively, as in huVHvb2.
  • positions HI, H5, Hl l, HI 7, H20, H23, H38, H42, H43, H58, H66, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T,I, K, R, G, K, I, R, T, D, M, E, R, S, T, L, and V, respectively, as in huVHvb3.
  • positions HI, H5, HI 1, HI 7, H20, H23, H28, H38, H42, H43, H58, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108, and HI 09 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, I, R, V, T, D, M, E, R, S, T, L, and V, respectively, as in huVHvb4.
  • positions HI, H5, HI 1, HI 7, H20, H23, H28, H38, H42, H43, H58, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108, and HI 09 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, I, R, V, T, D, M, E, R, S, T, L, and
  • H5 HI 1, H17, H20, H23, H28, H38, H42, H43, H54, H56, H58, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, I, R, V, T, D, M, E, R, S, T, L, and V, respectively, as in huVHvb5.
  • positions HI, H5, HI 1, H17, H20, H23, H28, H38, H42, H43, H54, H56, H66, H67, H75, H76, H80, H81, H83, H91, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, R, V, T, D, M, E, R, F, S, T, L, and V, respectively, as in huVHvb6.
  • HI 08, and HI 09 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, R, V, T, D, M, E, R, S, T, L, and V, respectively, as in huVHvb7.
  • position H60 is occupied by E, as in
  • hu3D6VHvlbAl 1 D60E hu3D6VHvb8.
  • position H82C is occupied by V, as in hu3D6VHvlbAl 1 L82cV.
  • positions H60, H80, H81, H82c, and H83 are occupied by E, M, E, V, and R, as in hu3D6VHvlbAl 1 D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9).
  • the heavy chain variable region of any of the above-referenced antibodies can be modified to further reduce immunogenicity.
  • position H80 is occupied by M and/or position H82c is occupied by V.
  • At least one of the following positions in the VL region is occupied by the amino acid as specified: L7 is occupied by S, L10 is occupied by S,
  • L15 is occupied by L
  • L83 is occupied by V
  • L86 is occupied by Y
  • LI 06 is occupied by I.
  • positions L7, L10, L15, L83, L86, and L106 are occupied by S, S, L, V, Y, and Y, respectively.
  • L7 is T or S
  • L10 is T or S
  • L15 is I or L
  • L17 is Q or E
  • L24 is K or R
  • L37 is L or Q
  • L45 is K or R
  • L83 is L or V
  • L86 is H or Y
  • L100 is A or Q
  • L106 is L or I.
  • positions L7, L10, L15, L83, L86, and L106 in the VL region are occupied by S, S, L, V, Y, and I, respectively, as in huVLvb2.
  • positions L7, L10, L15, L17, L24, L37, L45, L83, L86, L100, and LI 06 in the VL region are occupied by S, S, L, E, R, Q, R, V, Y, Q, and I, respectively, as in huVLvb3.
  • the light chain variable region of any of the above referenced antibodies can be modified to further reduce immunogenicity.
  • position L47 is occupied by G, N, D, E, P, T, S or A;
  • position L48 is occupied by G or D;
  • the heavy chain variable region of any of the above-referenced antibodies can be modified to further reduce immunogenicity.
  • position H80 is occupied by M and/or position H82c is occupied by V.
  • position L54 is occupied by D, as in hu3D6VLv2 L54D. In some humanized 3D6 antibodies, position L54 is occupied by G, as in hu3D6VLv2 L54G. In some humanized 3D6 antibodies, position L54 is occupied by N, as in hu3D6VLv2 L54N, In some humanized 3D6 antibodies, position L54 is occupied by E, as in hu3D6VLv2 L54E. In some humanized 3D6 antibodies, position L50 is occupied by E, as in hu3D6VLv2 L50E.
  • position L54 is occupied by Q, as in hu3D6VLv2 L54Q.
  • position L50 is occupied by D, as in hu3D6VLv2 L50D.
  • position L54 is occupied by K, as in hu3D6VLv2 L54K.
  • position L54 is occupied by R, as in hu3D6VLv2 L54R.
  • position L54 is occupied by T, as in hu3D6VLv2 L54T.
  • position L50 is occupied by G, as in hu3D6VLv2 L50G.
  • position L48 is occupied by G, as in hu3D6VLv2 I48G.
  • position L48 is occupied by D, as in hu3D6VLv2 I48D.
  • position L47 is occupied by G, as in hu3D6VLv2 L47G.
  • position L49 is occupied by E, as in hu3D6VLv2 Y49E.
  • position L54 is occupied by V, as in hu3D6VLv2 L54V. In some humanized 3D6 antibodies, position L54 is occupied by S, as in hu3D6VLv2 L54S. In some humanized 3D6 antibodies, position L52 is occupied by G, as in hu3D6VLv2 S52G. In some humanized 3D6 antibodies, position L47 is occupied by N, as in hu3D6VLv2 L47N. In some humanized 3D6 antibodies, position L47 is occupied by D, as in hu3D6VLv2 L47D.
  • position L47 is occupied by E, as in hu3D6VLv2 L47E. In some humanized 3D6 antibodies, position L47 is occupied by P, as in hu3D6VLv2 L47P. In some humanized 3D6 antibodies, position L47 is occupied by T, as in hu3D6VLv2 L47T. In some humanized 3D6 antibodies, position L47 is occupied by S, as in hu3D6VLv2 L47S. In some humanized 3D6 antibodies, position L47 is occupied by A, as in hu3D6VLv2 L47A. In some humanized 3D6 antibodies, position L50 is occupied by V, as in hu3D6VLv2 L50V.
  • positions L37, L50, and L54 are occupied by Q, G, and R, respectively, as in hu3D6VLv2 L37Q_L50G_L54R.
  • positions L37, L50, and L54 are occupied by Q, G, and G, respectively, as in hu3D6VLv2 L37Q_L50G_L54G.
  • positions L37, L52, and L54 are occupied by Q, G, and G, respectively, as in hu3D6VLv2 L37Q S52G L54G.
  • positions L37, L52, and L54 are occupied by Q, G, and R, respectively, as in hu3D6VLv2 L37Q S52G L54R. In some humanized 3D6 antibodies, positions L37, L52, and L54 are occupied by Q, G, and T, respectively, as in hu3D6VLv2 L37Q S52G L54T. In some humanized 3D6 antibodies, positions L37, L52, and L54 are occupied by Q, G, and D, respectively, as in hu3D6VLv2 L37Q S52G L54D.
  • positions L37 and L54 are occupied Q and R, respectively, as in hu3D6VLv2 L37Q L54R. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and G, respectively, as in hu3D6VLv2 L37Q L54G. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and D, respectively, as in hu3D6VLv2 L37Q L54D. In some humanized 3D6 antibodies, positions L37 and L50 are occupied by Q and G, respectively, as in hu3D6VLv2 L37Q L50G.
  • positions L37 and L50 are occupied by Q and D, respectively, as in hu3D6VLv2 L37Q L50D.
  • positions L37 and L54 are occupied by Q and T, respectively, as in hu3D6VLv2 L37Q L54T.
  • positions L37 and L52 are occupied by Q and G, respectively, as in hu3D6VLv2 L37Q S52G.
  • positions L37 and L54 are occupied by Q and E, respectively, as in hu3D6VLv2 L37Q L54E.
  • positions L37, L50, and L54 are occupied by Q, D, and G, respectively, as in hu3D6VLv2 L37Q L50D L54G.
  • positions L37, L50, and L54 are occupied by Q, D, and R, respectively, as in hu3D6VLv2 L37Q L50D L54R.
  • positions L37, L50, and L54 are occupied by Q, E, and G, respectively, as in hu3D6VLv2 L37Q L50E L54G.
  • positions L37, L50, and L54 are occupied by Q, E, and R, respectively, as in hu3D6VLv2 L37Q_L50E_L54R.
  • positions L37, L50, L54, and L100 are occupied by Q, G, R, and Q, respectively, as in hu3D6VLv2 L37Q_L50G_L54R_G100Q.
  • positions L37, L50, L54, and L100 are occupied by Q, G, G, and Q, respectively, as in hu3D6VLv2 L37Q_L50G_L54G_G100Q.
  • positions L37, L52, L54, and LI 00 are occupied by Q, G, R, and Q, respectively, as in hu3D6VLv2 L37Q_S52G_L54R_G100Q.
  • positions L37, L52, L54, and LI 00 are occupied by Q, G, D, and Q, respectively, as in hu3D6VLv2
  • positions L37, L50, L54, and LI 00 are occupied by Q, D, G, and Q, respectively, as in hu3D6VLv2
  • positions L37, L50, L54, and LI 00 are occupied by Q, D, R, and Q, respectively, as in hu3D6VLv2
  • positions L37, L50, L54, and LI 00 are occupied by Q, V, D, and Q, respectively, as in hu3D6VLv2
  • position L37 is occupied by Q, as in hu3D6VLv2 L37Q.
  • position LI 00 is occupied by Q as in hu3D6VLv2 G100Q.
  • Some humanized 3D6 antibodies comprise a mature heavy chain variable region comprising CDRs HI, H2 and H3 comprising SEQ ID NOs:8, 9, and 10, respectively except that position H28 can be occupied by N or T, H54 can be occupied by N or D, H56 can be occupied by D or E, position H58 occupied by V or I, and position H60 can be occupied by D or E, and a mature light chain variable region comprising CDRs LI, L2 and L3 comprising SEQ ID NOs.:
  • position L24 can be occupied by K or R
  • position L50 can be occupied by L, E. D, G, or V
  • position L52 can be occupied by S or G
  • position L54 can be occupied by L, D, G, N, E, Q, K, R, T, V, or S, wherein at least one of the following positions is occupied by the amino acid as specified: HI is occupied by Q, H5 is occupied by Q, HI 1 is occupied by L, H20 is occupied by L, H23 is occupied by T, H38 is occupied by K, H75 is occupied by S, H56 is occupied by E, H58 is occupied by I, H60 is occupied by E, H82c is occupied by V, L10 is occupied by T, L17 is occupied by E, L24 is occupied by R, L37 is occupied by Q, L47 is occupied by G, N, D, E, P, T, S, or A, L48 is occupied by G or
  • L83 is occupied by L
  • L86 is occupied by H
  • LI 00 is occupied by Q
  • L106 is occupied by L.
  • Some humanized 3D6 antibodies comprise three light chain CDRs and three heavy chain CDRs of monoclonal antibody 3D6, wherein 3D6 is a mouse antibody characterized by a heavy chain variable region having an amino acid sequence comprising SEQ ID NO:7 and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 11, except that position H27 can be occupied by F or Y, position H28 can be occupied by N or T, position H29 can be occupied by I or F, position H30 can be occupied by K or T, position H51 can be occupied by I or V, position H54 can be occupied by N or D, position H60 can be occupied by D, A, or E, position H61 can be occupied by P or E, position HI 02 can be occupied by F or Y, position L50 can be occupied by L, E.
  • 3D6 is a mouse antibody characterized by a heavy chain variable region having an amino acid sequence comprising SEQ ID NO:7 and a light chain variable region having an amino acid sequence
  • position L52 can be occupied by S or G
  • position L54 can be occupied by L, D, G, N, E, Q, K, R, T, V, or S, wherein at least one of the following positions is occupied by the amino acid as specified: L37 is occupied by Q, L47 is occupied by G, N, D, E, P, T, S, or A, L48 is occupied by G or D, L49 is occupied by E, L50 is occupied by E, D, G, or V, L52 is occupied by G, L54 is occupied by D, G, N. E, Q, K, R, T, V, or S, LI 00 is occupied by Q, H60 is occupied by E, H82c is occupied by V.
  • variable heavy chain has > 85% identity to human sequence.
  • variable light chain has > 85% identity to human sequence.
  • each of the variable heavy chain and variable light chain has > 85% identity to human germline sequence.
  • the three heavy chain CDRs are as defined by Kabat/Chothia Composite (SEQ ID NOs:8, 9, and 10) and the three light chain CDRs are as defined by
  • Kabat/Chothia Composite (SEQ ID NOs: 12, 13, and 14); provided that position H28 is occupied by N or T, position H54 is occupied by N or D, position H56 is occupied by D or E, position H58 is occupied by V or I, position H60 is occupied by D or E, position L24 is occupied by K or R, position L50 is occupied by L, E, D, G, or V, position L52 is occupied by S or G, and position L54 is occupied by L, D, G, N, E, Q, K, R, T, V, or S.
  • Kabat/Chothia Composite CDR-H1 has an amino acid sequence comprising SEQ ID NO:86.
  • Rabat CDR-H2 has an amino acid sequence comprising SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:92, or SEQ ID NO: 149.
  • Rabat CDR-L1 has an amino acid sequence comprising SEQ ID NO:89.
  • Rabat CDR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 150-175.
  • the CDR regions of such humanized antibodies can be identical or substantially identical to the CDR regions of 3D6,
  • the CDR regions can be defined by any conventional definition (e.g., Chothia, or composite of Chothia and Rabat) but are preferably as defined by Rabat.
  • Variable regions framework positions are in accordance with Rabat numbering unless otherwise stated.
  • Other such variants typically differ from the sequences of the exemplified Hu3D6 heavy and light chains by a small number (e.g., typically no more than 1, 2, 3, 5, 10, or 15) of replacements, deletions or insertions. Such differences are usually in the framework but can also occur in the CDRs.,
  • variable region frameworks A possibility for additional variation in humanized 3D6 variants is additional backmutations in the variable region frameworks. Many of the framework residues not in contact with the CDRs in the humanized mAb can accommodate substitutions of amino acids from the corresponding positions of the donor mouse mAb or other mouse or human antibodies, and even many potential CDR-contact residues are also amenable to substitution. Even amino acids within the CDRs may be altered, for example, with residues found at the corresponding position of the human acceptor sequence used to supply variable region frameworks. In addition, alternate human acceptor sequences can be used, for example, for the heavy and/or light chain.
  • the humanized 3D6 antibodies are further characterized by their ability to bind both phosphorylated and unphosphorylated tau and misfolded/aggregated forms of tau.
  • the invention further provides chimeric and veneered forms of non-human antibodies, particularly the 3D6 antibodies of the examples.
  • a chimeric antibody is an antibody in which the mature variable regions of light and heavy chains of a non-human antibody (e.g ., a mouse) are combined with human light and heavy chain constant regions. Such antibodies substantially or entirely retain the binding specificity of the mouse antibody, and are about two-thirds human sequence.
  • a chimeric 3D6 antibody has a heavy chain amino acid sequence of SEQ ID NO: 72 and a light chain amino acid sequence of SEQ ID NO: 73.
  • a veneered antibody is a type of humanized antibody that retains some and usually all of the CDRs and some of the non-human variable region framework residues of a non-human antibody but replaces other variable region framework residues that may contribute to B- or T- cell epitopes, for example exposed residues (Padlan, Mol. Immunol. 28:489, 1991) with residues from the corresponding positions of a human antibody sequence.
  • the result is an antibody in which the CDRs are entirely or substantially from a non-human antibody and the variable region frameworks of the non-human antibody are made more human-like by the substitutions.
  • Veneered forms of the 3D6 antibody are included in the invention.
  • Human antibodies against tau or a fragment thereof e.g., amino acid residues 199-213 and/or 262-276 of SEQ ID NO:3, corresponding to amino acid residues 257-271 and/or 320-334, respectively, of SEQ ID NO: l or amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: 1 or any combination of 2, 3 or all 4 thereof
  • Some human antibodies are selected by competitive binding experiments, by the phage display method of Winter, above, or otherwise, to have the same epitope specificity as a particular mouse antibody, such as one of the mouse monoclonal antibodies described in the examples.
  • Human antibodies can also be screened for a particular epitope specificity by using only a fragment of tau, such as a tau fragment containing only amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257- 271 or 320-334, respectively, of SEQ ID NO: l) or containing only amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: 1, as the target antigen, and/or by screening antibodies against a collection of tau variants, such as tau variants containing various mutations within amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or 320-334, respectively, of SEQ ID NO: 1), or within amino acid residues 259- 268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: l.
  • a fragment of tau such as a tau fragment containing only amino acid residues 199-213 or 262-2
  • Methods for producing human antibodies include the trioma method of Oestberg et al ., Hybridoma 2:361-367 (1983); Oestberg, U.S. Patent No. 4,634,664; and Engleman et al., US Patent 4,634,666, use of transgenic mice including human immunoglobulin genes (see, e.g., Lonberg et al, W093/12227 (1993); US 5,877,397; US 5,874,299; US 5,814,318; US 5,789,650; US 5,770,429; US 5,661,016; US 5,633,425; US 5,625,126; US 5,569,825; US 5,545,806;
  • phage display methods see, e.g., Dower et al., WO 91/17271; McCafferty et al., WO 92/01047; US 5,877,218; US 5,871,907; US 5,858,657; US 5,837,242; US 5,733,743; and US 5,565,332); and methods described in WO 2008/081008 (e.g., immortalizing memory B cells isolated from humans, e.g., with EBV, screening for desired properties, and cloning and expressing
  • the heavy and light chain variable regions of chimeric, veneered or humanized antibodies can be linked to at least a portion of a human constant region.
  • the choice of constant region depends, in part, whether antibody-dependent cell-mediated cytotoxicity, antibody dependent cellular phagocytosis and/or complement dependent cytotoxicity are desired.
  • human isotypes IgGl and IgG3 have complement-dependent cytotoxicity and human isotypes IgG2 and IgG4 do not.
  • Human IgGl and IgG3 also induce stronger cell mediated effector functions than human IgG2 and IgG4.
  • Light chain constant regions can be lambda or kappa. Numbering conventions for constant regions include EU numbering (Edelman, G.M.
  • One or several amino acids at the amino or carboxy terminus of the light and/or heavy chain may be missing or derivatized in a proportion or all of the molecules. Substitutions can be made in the constant regions to reduce or increase effector function such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., US Patent No. 5,624,821; Tso et al., US Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol.
  • substitutions include a Gin at position 250 and/or a Leu at position 428 (EU numbering is used in this paragraph for the constant region) for increasing the half-life of an antibody. Substitution at any or all of positions 234, 235, 236 and/or 237 reduce affinity for Fey receptors, particularly FcyRI receptor (see, e.g., US
  • An alanine substitution at positions 234, 235, and 237 of human IgGl can be used for reducing effector functions.
  • Some antibodies have alanine substitution at positions 234, 235 and 237 of human IgGl for reducing effector functions.
  • positions 234, 236 and/or 237 in human IgG2 are substituted with alanine and position 235 with glutamine (see, e.g., US
  • a mutation at one or more of positions 241, 264, 265, 270, 296, 297, 322, 329, and 331 by EU numbering of human IgGl is used.
  • a mutation at one or more of positions 318, 320, and 322 by EU numbering of human IgGl is used.
  • positions 234 and/or 235 are substituted with alanine and/or position 329 is substituted with glycine.
  • positions 234 and 235 are substituted with alanine.
  • the isotype is human IgG2 or IgG4.
  • Antibodies can be expressed as tetramers containing two light and two heavy chains, as separate heavy chains, light chains, as Fab, Fab', F(ab')2, and Fv, or as single chain antibodies in which heavy and light chain mature variable domains are linked through a spacer.
  • Human constant regions show allotypic variation and isoallotypic variation between different individuals, that is, the constant regions can differ in different individuals at one or more polymorphic positions. Isoallotypes differ from allotypes in that sera recognizing an isoallotype bind to a non-polymorphic region of a one or more other isotypes.
  • another heavy chain constant region is of IgGl Glm3 with or without the C-terminal lysine.
  • Reference to a human constant region includes a constant region with any natural allotype or any permutation of residues occupying positions in natural allotypes.
  • An exemplary heavy chain constant region is SEQ ID NO: 176, with or without the C-terminal lysine, and an exemplary light chain constant region is SEQ ID NO: 177.
  • a number of methods are known for producing chimeric and humanized antibodies using an antibody-expressing cell line (e.g ., hybridoma).
  • an antibody-expressing cell line e.g ., hybridoma
  • the immunoglobulin variable regions of antibodies can be cloned and sequenced using well known methods.
  • the heavy chain variable VH region is cloned by RT-PCR using mRNA prepared from hybridoma cells. Consensus primers are employed to the VH region leader peptide
  • sequences from multiple, independently derived clones can be compared to ensure no changes are introduced during amplification.
  • sequence of the VH region can also be determined or confirmed by sequencing a VH fragment obtained by 5' RACE RT-PCR methodology and the 3' g2b specific primer.
  • the light chain variable VL region can be cloned in an analogous manner.
  • a consensus primer set is designed for amplification of VL regions using a 5’ primer designed to hybridize to the VL region encompassing the translation initiation codon and a 3' primer specific for the Ck region downstream of the V-J joining region.
  • 5'RACE RT-PCR methodology is employed to clone a VL encoding cDNA. Exemplary primers are described in Schenk, supra. The cloned sequences are then combined with sequences encoding human (or other non-human species) constant regions.
  • the heavy and light chain variable regions are re-engineered to encode splice donor sequences downstream of the respective VDJ or VJ junctions and are cloned into a mammalian expression vector, such as pCMV-hyl for the heavy chain and pCMV-Mcl for the light chain. These vectors encode human g ⁇ and Ck constant regions as exonic fragments downstream of the inserted variable region cassette.
  • the heavy chain and light chain expression vectors can be co-transfected into CHO cells to produce chimeric antibodies. Conditioned media is collected 48 hours post-transfection and assayed by western blot analysis for antibody production or ELISA for antigen binding.
  • the chimeric antibodies are humanized as described above.
  • Chimeric, veneered, humanized, and human antibodies are typically produced by recombinant expression.
  • Recombinant polynucleotide constructs typically include an expression control sequence operably linked to the coding sequences of antibody chains, including naturally associated or heterologous expression control elements, such as a promoter.
  • the expression control sequences can be promoter systems in vectors capable of transforming or transfecting eukaryotic or prokaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences and the collection and purification of the crossreacting antibodies.
  • expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, e.g ., ampicillin resistance or hygromycin resistance, to permit detection of those cells transformed with the desired DNA sequences.
  • selection markers e.g ., ampicillin resistance or hygromycin resistance
  • E. coli is one prokaryotic host useful for expressing antibodies, particularly antibody fragments.
  • Microbes such as yeast, are also useful for expression.
  • Saccharomyces is a yeast host with suitable vectors having expression control sequences, an origin of replication, termination sequences, and the like as desired.
  • Typical promoters include 3 -phosphogly cerate kinase and other glycolytic enzymes.
  • Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.
  • Mammalian cells can be used for expressing nucleotide segments encoding
  • suitable host cell lines capable of secreting intact heterologous proteins have been developed, and include CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas including Sp2/0 and NS0.
  • the cells can be nonhuman.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al ., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • Expression control sequences can include promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. See Co et al., J.
  • antibody coding sequences can be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g ., U.S. Pat. No. 5,741,957; U.S. Pat. No. 5,304,489; and U.S. Pat. No. 5,849,992).
  • Suitable transgenes include coding sequences for light and/or heavy chains operably linked with a promoter and enhancer from a mammary gland specific gene, such as casein or beta lactoglobulin.
  • the vectors containing the DNA segments of interest can be transferred into the host cell by methods depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics, or viral-based transfection can be used for other cellular hosts. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection.
  • transgenes can be microinjected into fertilized oocytes or can be incorporated into the genome of embryonic stem cells or induced pluripotent stem cells (iPSCs), and the nuclei of such cells transferred into enucleated oocytes.
  • iPSCs induced pluripotent stem cells
  • cell pools can be screened for growth productivity and product quality in serum -free media. Top-producing cell pools can then be subjected of FACS-based single-cell cloning to generate monoclonal lines. Specific productivities above 50 pg or 100 pg per cell per day, which correspond to product titers of greater than 7.5 g/L culture, can be used. Antibodies produced by single cell clones can also be tested for turbidity, filtration properties, PAGE, IEF, UV scan, HP- SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and binding assay, such as ELISA or Biacore. A selected clone can then be banked in multiple vials and stored frozen for subsequent use.
  • antibodies can be purified according to standard procedures of the art, including protein A capture, HPLC purification, column chromatography, gel electrophoresis and the like (see generally , Scopes, Protein Purification (Springer-Verlag, NY, 1982)).
  • Methodology for commercial production of antibodies can be employed, including codon optimization, selection of promoters, selection of transcription elements, selection of terminators, serum-free single cell cloning, cell banking, use of selection markers for
  • Agent used for active immunization serves to induce in a patient the same types of antibody described in connection with passive immunization above.
  • Agents used for active immunization can be the same types of immunogens used for generating monoclonal antibodies in laboratory animals, e.g ., a peptide of 3-15 or 3-12 or 5-12, or 5-8 contiguous amino acids from a region of tau corresponding to residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to residues 257-271 or 320-334, respectively, of SEQ ID NO: l), such as, for example, a peptide including residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to residues 257-271 or 320-334, respectively, of SEQ ID NO: 1) or from a region of tau corresponding to residues 259- 268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: l, such as for example, a peptide including residues
  • the epitope specificity of these antibodies can be mapped (e.g, by testing binding to a series of overlapping peptides spanning tau).
  • a fragment of tau consisting of or including or overlapping the epitope can then be used as an immunogen. Such fragments are typically used in unphosphorylated form.
  • the heterologous carrier and adjuvant may be the same as used for generating monoclonal antibody, but may also be selected for better pharmaceutical suitability for use in humans.
  • Suitable carriers include serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, or a toxoid from other pathogenic bacteria, such as diphtheria (e.g, CRM 197), E. coli, cholera, or H. pylori, or an attenuated toxin derivative.
  • T cell epitopes are also suitable carrier molecules.
  • conjugates can be formed by linking agents of the invention to an immunostimulatory polymer molecule (e.g, tripalmitoyl- S-glycerine cysteine (Par Cys), mannan (a mannose polymer), or glucan (a b 1— >2 polymer)), cytokines (e.g, IL-1, IL-1 alpha and b peptides, IL-2, g-INF, IL-10, GM-CSF), and chemokines (e.g, MIPl-a and b, and RANTES).
  • Immunogens may be linked to the carriers with or without spacers amino acids (e.g, gly-gly). Additional carriers include virus-like particles.
  • VLPs Virus-like particles
  • pseudovirions represent subunit structures composed of multiple copies of a viral capsid and/or envelope protein capable of self-assembly into VLPs of defined spherical symmetry in vivo.
  • peptide immunogens can be linked to at least one artificial T-cell epitope capable of binding a large proportion of MHC Class II molecules., such as the pan DR epitope ("PADRE").
  • PADRE is described in US 5,736,142, WO 95/07707, and Alexander J et al, Immunity, 1 :751-761 (1994).
  • Active immunogens can be presented in multimeric form in which multiple copies of an immunogen and/or its carrier are presented as a single covalent molecule.
  • Fragments are often administered with pharmaceutically acceptable adjuvants.
  • the adjuvant increases the titer of induced antibodies and/or the binding affinity of induced antibodies relative to the situation if the peptide were used alone.
  • a variety of adjuvants can be used in combination with an immunogenic fragment of tau to elicit an immune response.
  • Preferred adjuvants augment the intrinsic response to an immunogen without causing
  • Preferred adjuvants include aluminum salts, such as aluminum hydroxide and aluminum phosphate, 3 De-O-acylated monophosphoryl lipid A (MPLTM) ( see GB 2220211 (RIBI).
  • MPLTM 3 De-O-acylated monophosphoryl lipid A
  • StimulonTM QS-21 is a triterpene glycoside or saponin isolated from the bark of the Quillaja Saponaria Molina tree found in South America (see Kensil et al. , in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); US 5,057,540), (Aquila
  • Adjuvants are oil in water emulsions (such as squalene or peanut oil), optionally in combination with immune stimulants, such as monophosphoryl lipid A (see Stoute et al. , N. Engl. J. Med. 336, 86- 91 (1997)), pluronic polymers, and killed mycobacteria.
  • Ribi adjuvants are oil-in-water emulsions.
  • Ribi contains a metabolizable oil (squalene) emulsified with saline containing Tween 80.
  • Ribi also contains refined mycobacterial products which act as immunostimulants and bacterial monophosphoryl lipid A.
  • Another adjuvant is CpG (WO 98/40100).
  • Adjuvants can be administered as a component of a therapeutic composition with an active agent or can be administered separately, before, concurrently with, or after administration of the therapeutic agent.
  • Analogs of natural fragments of tau that induce antibodies against tau can also be used.
  • one or more or all L-amino acids can be substituted with D amino acids in such peptides.
  • the order of amino acids can be reversed (retro peptide).
  • a peptide includes all D-amino acids in reverse order (retro-inverso peptide).
  • Peptides and other compounds that do not necessarily have a significant amino acid sequence similarity with tau peptides but nevertheless serve as mimetics of tau peptides and induce a similar immune response.
  • Anti- idiotypic antibodies against monoclonal antibodies to tau as described above can also be used. Such anti-id antibodies mimic the antigen and generate an immune response to it (see Essential Immunology, Roit ed., Blackwell Scientific Publications, Palo Alto, CA 6th ed., p. 181).
  • Peptides can also be administered in the form of a nucleic acid encoding the peptide and expressed in situ in a patient.
  • a nucleic acid segment encoding an immunogen is typically linked to regulatory elements, such as a promoter and enhancer that allow expression of the DNA segment in the intended target cells of a patient.
  • regulatory elements such as a promoter and enhancer that allow expression of the DNA segment in the intended target cells of a patient.
  • promoter and enhancer elements from light or heavy chain immunoglobulin genes or the CMV major intermediate early promoter and enhancer are suitable to direct expression.
  • the linked regulatory elements and coding sequences are often cloned into a vector.
  • Antibodies can also be administered in the form of nucleic acids encoding the antibody heavy and/or light chains. If both heavy and light chains are present, the chains are preferably linked as a single chain antibody.
  • Antibodies for passive administration can also be prepared e.g ., by affinity chromatography from sera of patients treated with peptide immunogens.
  • the DNA can be delivered in naked form (z.e., without colloidal or encapsulating materials).
  • a number of viral vector systems can be used including retroviral systems (see, e.g. , Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993));
  • adenoviral vectors see, e.g. , Bett et al, J. Virol. 67, 591 1 (1993)
  • retrovirus derived vectors such MMLV, HIV-1, and ALV
  • adeno-associated virus vectors see, e.g. , Zhou et al., J. Exp. Med. 179, 1867 (1994)
  • lentiviral vectors such as those based on HIV or FIV gag sequences
  • viral vectors from the alpha virus genus such as those derived from Sindbis and Semliki Forest Viruses (see, e.g.
  • DNA encoding an immunogen, or encoding the antibody heavy and/or light chains, or a vector containing the same, can be packaged into liposomes. Suitable lipids and related analogs are described by US 5,208,036, US 5,264,618, US 5,279,833, and US 5,283,185.
  • Vectors and DNA encoding an immunogen or encoding the antibody heavy and/or light chains can also be adsorbed to or associated with particulate carriers, examples of which include polymethyl methacrylate polymers and polylactides and poly(lactide-co-glycolides), (see, e.g ., McGee et al., J. Micro Encap. 1996).
  • Vectors or segments therefrom encoding the antibody heavy and/or light chains can be incorporated in cells ex vivo, for example to cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy ) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the transgenes (see, e.g., WO 2017/091512).
  • Exemplary patient-derived cells include patient derived induced pluripotent stem cells (iPSCs) or other types of stem cells (embryonic, hematopoietic, neural, or mesenchymal).
  • a vector or segment therefrom encoding the antibody heavy and/or light chains can be introduced into any region of interest in cells ex vivo, such as an albumin gene or other safe harbor gene.
  • Cells incorporating the vector can be implanted with or without prior
  • Cells can be implanted into a specific tissue, such as a secretory tissue or a location of pathology, or systemically, such as by infusion into the blood.
  • a specific tissue such as a secretory tissue or a location of pathology
  • cells can be implanted into a secretory tissue of a patient, such as the liver, optionally with prior differentiation to cells present in that tissue, such as hepatocytes in the case of a liver.
  • Expression of the antibody in the liver results in secretion of the antibody to the blood.
  • Antibodies can be initially screened for the intended binding specificity as described above. Active immunogens can likewise be screened for capacity to induce antibodies with such binding specificity. In this case, an active immunogen is used to immunize a laboratory animal and the resulting sera tested for the appropriate binding specificity.
  • Antibodies having the desired binding specificity can then be tested in cellular and animal models.
  • the cells used for such screening are preferentially neuronal cells.
  • a cellular model of tau pathology has been reported in which neuroblastoma cells are transfected with a four-repeat domain of tau, optionally with a mutation associated with tau pathology (e.g ., delta K280, see Khlistunova, Current Alzheimer Research 4, 544-546 (2007)).
  • tau is induced in the neuroblastoma N2a cell line by the addition of doxycyclin.
  • the cell models enable one to study the toxicity of tau to cells in the soluble or aggregated state, the appearance of tau aggregates after switching on tau gene expression, the dissolution of tau aggregates after switching the gene expression off again, and the efficiency of antibodies in inhibiting formation of tau aggregates or disaggregating them.
  • Antibodies or active immunogens can also be screened in transgenic animal models of diseases associated with tau.
  • Such transgenic animals can include a tau transgene (e.g., any of the human isoforms) and optionally a human APP transgene among others, such as a kinase that phosphorylates tau, ApoE, presenilin or alpha synuclein.
  • Such transgenic animals are disposed to develop at least one sign or symptom of a disease associated with tau.
  • An exemplary transgenic animal is the K3 line of mice (Itner et al., Proc. Natl. Acad. Sci. USA 105(41): 15997-6002 (2008)). These mice have a human tau transgene with a K 369 I mutation (the mutation is associated with Pick’s disease) and a Thy 1.2 promoter. This model shows a rapid course of neurodegeneration, motor deficit and degeneration of afferent fibers and cerebellar granule cells.
  • Another exemplary animal is the JNPL3 line of mice.
  • mice have a human tau transgene with a P301L mutation (the mutation is associated with frontotemporal dementia) and a Thy 1.2 promoter (Taconic, Germantown, N.Y., Lewis, et al., Nat Genet.
  • mice have a more gradual course of neurodegeneration.
  • the mice develop neurofibrillary tangles in several brain regions and spinal cord, which is hereby incorporated by reference in its entirety). This is an excellent model to study the consequences of tangle development and for screening therapy that may inhibit the generation of these aggregates.
  • Another advantage of these animals is the relatively early onset of pathology. In the homozygous line, behavioral abnormalities associated with tau pathology can be observed at least as early as 3 months, but the animals remain relatively healthy at least until 8 months of age. In other words, at 8 months, the animals ambulate, feed themselves, and can perform the behavioral tasks sufficiently well to allow the treatment effect to be monitored.
  • Active immunization of these mice for 6-13 months with - Al wl KLH-PHF-1 generated titers of about 1,000 and showed fewer neurofibrillary tangles, less pSer422, and reduced weight loss relative to untreated control ice.
  • the activity of antibodies or active agents can be assessed by various criteria including reduction in amount of total tau or phosphorylated tau, reduction in other pathological characteristics, such as amyloid deposits of Ab, and inhibition or delay or behavioral deficits.
  • Active immunogens can also be tested for induction of antibodies in the sera. Both passive and active immunogens can be tested for passage of antibodies across the blood brain barrier into the brain of a transgenic animal.
  • Antibodies or fragments inducing an antibody can also be tested in non-human primates that naturally or through induction develop symptoms of diseases characterized by tau. Tests on an antibody or active agent are usually performed in conjunction with a control in which a parallel experiment is conduct except that the antibody or active agent is absent ( e.g ., replaced by vehicle). Reduction, delay or inhibition of signs or symptoms disease attributable to an antibody or active agent under test can then be assessed relative to the control. V. Patients Amenable to Treatment
  • neurofibrillary tangles have been found in several diseases including Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia,
  • CBD dementia with Lewy bodies
  • LVAD Lewy body variant of Alzheimer disease
  • CTE chronic traumatic encephalopathy
  • GTT globular glial tauopathy
  • PGP progressive supranuclear palsy
  • the present regimes can be used in treatment or prophylaxis of any subject showing elevated levels of tau or phosphorylated tau (e.g., in the CSF) compared with a mean value in individuals without neurological disease.
  • the present regimes can also be used in treatment or prophylaxis of neurological disease in individuals having a mutation in tau associated with neurological disease.
  • the present methods are particularly suitable for treatment or prophylaxis of Alzheimer’s disease, and especially in patients.
  • Patients amenable to treatment include individuals at risk of disease but not showing symptoms, as well as patients presently showing symptoms.
  • Patients at risk of disease include those having a known genetic risk of disease. Such individuals include those having relatives who have experienced this disease, and those whose risk is determined by analysis of genetic or biochemical markers.
  • Genetic markers of risk include mutations in tau, such as those discussed above, as well as mutations in other genes associated with neurological disease. For example, the ApoE4 allele in heterozygous and even more so in homozygous form is associated with risk of Alzheimer’s disease.
  • markers of risk of Alzheimer’s disease include mutations in the APP gene, particularly mutations at position 717 and positions 670 and 671 referred to as the Hardy and Swedish mutations respectively, mutations in the presenilin genes, PS1 and PS2, a family history of AD, hypercholesterolemia or atherosclerosis.
  • Individuals presently suffering from Alzheimer’s disease can be recognized by PET imaging, from characteristic dementia, as well as the presence of risk factors described above.
  • a number of diagnostic tests are available for identifying individuals who have AD. These include measurement of CSF tau or phospho-tau and Ab42 levels. Elevated tau or phospho-tau and decreased Ab42 levels signify the presence of AD.
  • Individuals can also be diagnosed with any of the neurological diseases mentioned above by the criteria of the DSM IV TR.
  • treatment can begin at any age ( e.g ., 10, 20, 30). Usually, however, it is not necessary to begin treatment until a patient reaches 40, 50, 60 or 70 years of age. Treatment typically entails multiple dosages over a period of time. Treatment can be monitored by assaying antibody levels over time. If the response falls, a booster dosage is indicated. In the case of potential Down’s syndrome patients, treatment can begin antenatally by administering therapeutic agent to the mother or shortly after birth.
  • the invention further provides nucleic acids encoding any of the heavy and light chains described above (e.g., SEQ ID NO:7, SEQ ID NO: l l, SEQ ID NOs: 76-80, SEQ ID NOs:90-91, SEQ ID NOs: 146-148, SEQ ID NOs:83-85, SEQ ID NOs:93-145, and SEQ ID NOs: 178-181).
  • An exemplary nucleic acid encoding a heavy chain of the invention is SEQ ID NO: 182
  • an exemplary nucleic acid encoding a light chain of the invention is SEQ ID NO: 183.
  • nucleic acids further encode a signal peptide and can be expressed with the signal peptide linked to the variable region.
  • Coding sequences of nucleic acids can be operably linked with regulatory sequences to ensure expression of the coding sequences, such as a promoter, enhancer, ribosome binding site, transcription termination signal, and the like.
  • the regulatory sequences can include a promoter, for example, a prokaryotic promoter or a eukaryotic promoter.
  • the nucleic acids encoding heavy or light chains can be codon-optimized for expression in a host cell.
  • the nucleic acids encoding heavy and light chains can encode a selectable gene.
  • the nucleic acids encoding heavy and light chains can occur in isolated form or can be cloned into one or more vectors.
  • the nucleic acids can be synthesized by, for example, solid state synthesis or PCR of overlapping oligonucleotides.
  • Nucleic acids encoding heavy and light chains can be joined as one contiguous nucleic acid, e.g., within an expression vector, or can be separate, e.g., each cloned into its own expression vector.
  • Conjugated antibodies that specifically bind to antigens, such as tau are useful in detecting the presence of tau; monitoring and evaluating the efficacy of therapeutic agents being used to treat patients diagnosed with Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP); inhibiting or reducing aggregation of tau; inhibiting or reducing tau
  • conformations of tau or treating or effecting prophylaxis of Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann- Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies,
  • LVAD Lewy body variant of Alzheimer disease
  • CTE chronic traumatic encephalopathy
  • GTT globular glial tauopathy
  • PSP progressive supranuclear palsy
  • such antibodies can be conjugated with other therapeutic moieties, other proteins, other antibodies, and/or detectable labels. See WO 03/057838; US 8,455,622.
  • Such therapeutic moieties can be any agent that can be used to treat, combat, ameliorate, prevent, or improve an unwanted condition or disease in a patient, such as Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).
  • CBD corticobasal degeneration
  • LVAD dementia with Lewy bodies
  • LVAD chronic traumatic
  • Conjugated therapeutic moieties can include cytotoxic agents, cytostatic agents, neurotrophic agents, neuroprotective agents, radiotherapeutic agents, immunomodulators, or any biologically active agents that facilitate or enhance the activity of the antibody.
  • a cytotoxic agent can be any agent that is toxic to a cell.
  • a cytostatic agent can be any agent that inhibits cell proliferation.
  • a neurotrophic agent can be any agent, including chemical or proteinaceous agents, that promotes neuron maintenance, growth, or differentiation.
  • a neuroprotective agent can be agent, including chemical or proteinaceous agents, that protects neurons from acute insult or degenerative processes.
  • An immunomodulator can be any agent that stimulates or inhibits the development or maintenance of an immunologic response.
  • a radiotherapeutic agent can be any molecule or compound that emits radiation.
  • the coupled therapeutic moieties will have a specific affinity for tau-related disease-affected cells over normal cells. Consequently, administration of the conjugated antibodies directly targets cancer cells with minimal damage to surrounding normal, healthy tissue. This can be particularly useful for therapeutic moieties that are too toxic to be administered on their own. In addition, smaller quantities of the therapeutic moieties can be used.
  • ricin a cellular toxin derived from plants
  • S-acetylmercaptosuccinic anhydride for the antibody
  • succinimidyl 3-(2-pyridyldithio)propionate for ricin.
  • saporin an inhibitor of ribosomal assembly
  • saporin an inhibitor of ribosomal assembly
  • radioisotopes examples include, for example, yttrium 90 (90Y), indium 111 (l l lln), 131 I, "mTc, radiosilver-111, radiosilver- 199, and Bismuth 213 .
  • Linkage of radioisotopes to antibodies may be performed with conventional bifunction chelates.
  • sulfur-based linkers may be used for radiosilver- 111 and radiosilver- 199 linkage. See Hazra et al., Cell Biophys. 24-25:1-7 (1994).
  • Linkage of silver radioisotopes may involve reducing the immunoglobulin with ascorbic acid.
  • ibritumomab tiuxetan For radioisotopes such as 11 lln and 90Y, ibritumomab tiuxetan can be used and will react with such isotopes to form 11 lln-ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol ., 48 Suppl LS91-S95 (2001).
  • antibodies can be linked to other therapeutic moieties.
  • therapeutic moieties can be, for example, cytotoxic, cytostatic, neurotrophic, or neuroprotective.
  • therapeutic moieties can be, for example, cytotoxic, cytostatic, neurotrophic, or neuroprotective.
  • antibodies can be conjugated with toxic chemotherapeutic drugs such as maytansine, geldanamycin, tubulin inhibitors such as tubulin binding agents (e.g ., auristatins), or minor groove binding agents such as calicheamicin.
  • Other representative therapeutic moieties include agents known to be useful for treatment, management, or amelioration of Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann- Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).
  • CBD corticobasal degeneration
  • LVAD dementia with Lewy bodies
  • LVAD chronic traumatic encephalopathy
  • GTT progressive supranuclear palsy
  • Antibodies can also be coupled with other proteins.
  • antibodies can be coupled with Fynomers.
  • Fynomers are small binding proteins (e.g., 7 kDa) derived from the human Fyn SH3 domain. They can be stable and soluble, and they can lack cysteine residues and disulfide bonds.
  • Fynomers can be engineered to bind to target molecules with the same affinity and specificity as antibodies. They are suitable for creating multi-specific fusion proteins based on antibodies.
  • Fynomers can be fused to N-terminal and/or C- terminal ends of antibodies to create bi- and tri-specific FynomAbs with different architectures.
  • Fynomers can be selected using Fynomer libraries through screening technologies using FACS, Biacore, and cell-based assays that allow efficient selection of Fynomers with optimal properties. Examples of Fynomers are disclosed in Grabulovski et al, ./. Biol. Chem. 282:3196-3204 (2007); Bertschinger et al., Protein Eng. Des. Sel. 20:57-68 (2007); Schlatter et al., MAhs. 4:497-508 (2011); Banner et al., Acta. Crystallogr. D. Biol. Crystallogr. 69(Pt6): 1124-1137 (2013); and Brack et al, Mol. Cancer Ther. 13:2030-2039 (2014).
  • the antibodies disclosed herein can also be coupled or conjugated to one or more other antibodies (e.g ., to form antibody heteroconjugates). Such other antibodies can bind to different epitopes within tau or can bind to a different target antigen.
  • Antibodies can also be coupled with a detectable label. Such antibodies can be used, for example, for diagnosing Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP), and/or for assessing efficacy of treatment.
  • CBD corticobasal degeneration
  • LVAD dementia with Lewy bodies
  • LVAD chronic
  • Such antibodies are particularly useful for performing such determinations in subjects having or being susceptible to Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age- related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP), or in appropriate biological samples obtained from such subjects.
  • CBD corticobasal degeneration
  • LVAD dementia with Lewy bodies
  • LVAD chronic traumatic encephalopathy
  • GTT glob
  • Linkage of radioisotopes to antibodies may be performed with conventional bifunction chelates.
  • sulfur-based linkers may be used for radiosilver- 1 1 1 and radiosilver- 199 linkage. See Hazra et al., Cell Biophys. 24-25 : 1 -7 (1994).
  • Linkage of silver radioisotopes may involve reducing the immunoglobulin with ascorbic acid.
  • ibritumomab tiuxetan For radioisotopes such as 1 1 lln and 90Y, ibritumomab tiuxetan can be used and will react with such isotopes to form 1 1 lln-ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother.
  • Therapeutic moieties, other proteins, other antibodies, and/or detectable labels may be coupled or conjugated, directly or indirectly through an intermediate (e.g ., a linker), to an antibody of the invention.
  • an intermediate e.g ., a linker
  • Therapeutic moieties, other proteins, other antibodies, and/or detectable labels may be coupled or conjugated, directly or indirectly through an intermediate (e.g ., a linker), to an antibody of the invention.
  • an intermediate e.g ., a linker
  • Suitable linkers include, for example, cleavable and non- cleavable linkers. Different linkers that release the coupled therapeutic moieties, proteins, antibodies, and/or detectable labels under acidic or reducing conditions, on exposure to specific proteases, or under other defined conditions can be employed.
  • an antibody or agent for inducing an antibody or a pharmaceutical composition the same is administered to a patient susceptible to, or otherwise at risk of a disease (e.g ., Alzheimer’s disease) in regime (dose, frequency and route of a disease).
  • a disease e.g ., Alzheimer’s disease
  • the regime is preferably effective to inhibit or delay tau or phospho-tau and paired filaments formed from it in the brain, and/or inhibit or delay its toxic effects and/or inhibit/or delay development of behavioral deficits.
  • an antibody or agent to induce an antibody is administered to a patient suspected of, or already suffering from a disease (e.g., Alzheimer’s disease) in a regime (dose, frequency and route of administration) effective to ameliorate or at least inhibit further deterioration of at least one sign or symptom of the disease.
  • the regime is preferably effective to reduce or at least inhibit further increase of levels of tau, phosphor-tau, or paired filaments formed from it , associated toxi cities and/or behavioral deficits.
  • a regime is considered therapeutically or prophylactically effective if an individual treated patient achieves an outcome more favorable than the mean outcome in a control population of comparable patients not treated by methods of the invention, or if a more favorable outcome is demonstrated in treated patients versus control patients in a controlled clinical trial (e.g, a phase II, phase II/III or phase III trial) at the p ⁇ 0.05 or 0.01 or even 0.001 level.
  • a controlled clinical trial e.g, a phase II, phase II/III or phase III trial
  • Effective doses of vary depending on many different factors, such as means of administration, target site, physiological state of the patient, whether the patient is an ApoE carrier, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
  • Exemplary dosage ranges for antibodies are from about 0.01 to 60 mg/kg, or from about 0.1 to 3 mg/kg or 0.15-2 mg/kg or 0.15-1.5 mg/kg, of patient body weight.
  • Antibody can be administered such doses daily, on alternative days, weekly, fortnightly, monthly, quarterly, or according to any other schedule determined by empirical analysis.
  • An exemplary treatment entails administration in multiple dosages over a prolonged period, for example, of at least six months. Additional exemplary treatment regimes entail administration once per every two weeks or once a month or once every 3 to 6 months.
  • the amount of an agent for active administration varies from 0.1-500 pg per patient and more usually from 1-100 or 1-10 pg per injection for human administration.
  • the timing of injections can vary significantly from once a day, to once a year, to once a decade.
  • a typical regimen consists of an immunization followed by booster injections at time intervals, such as 6 week intervals or two months.
  • Another regimen consists of an immunization followed by booster injections 1, 2 and 12 months later.
  • Another regimen entails an injection every two months for life.
  • booster injections can be on an irregular basis as indicated by monitoring of immune response.
  • Antibodies or agents for inducing antibodies are preferably administered via a peripheral route (i.e., one in which an administered or induced antibody crosses the blood brain barrier to reach an intended site in the brain.
  • Routes of administration include topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, intranasal, intraocular, or intramuscular.
  • Preferred routes for administration of antibodies are intravenous and subcutaneous.
  • Preferred routes for active immunization are subcutaneous and intramuscular. This type of injection is most typically performed in the arm or leg muscles.
  • agents are injected directly into a particular tissue where deposits have accumulated, for example intracranial injection.
  • compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions.
  • Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration).
  • compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries.
  • the formulation depends on the route of
  • antibodies can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline or acetate buffer (to reduce discomfort at the site of injection).
  • physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline or acetate buffer (to reduce discomfort at the site of injection).
  • the solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g, sterile pyrogen-free water, before use.
  • a suitable vehicle e.g, sterile pyrogen-free water
  • the present regimes can be administered in combination with another agent effective in treatment or prophylaxis of the disease being treated.
  • the present regimes can be combined with immunotherapy against Ab
  • cholinesterase inhibitors or memantine or in the case of Parkinson’s disease immunotherapy against alpha synuclein WO/2008/103472 Levodopa, dopamine agonists, COMT inhibitors, MAO-B inhibitors, Amantadine, or anticholinergic agents.
  • Antibodies are administered in an effective regime meaning a dosage, route of administration and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration, and/or ameliorates at least one sign or symptom of a disorder being treated.
  • the regime can be referred to as a therapeutically effective regime.
  • the patient is at elevated risk of the disorder relative to the general population but is not yet experiencing symptoms, the regime can be referred to as a prophylactically effective regime.
  • therapeutic or prophylactic efficacy can be observed in an individual patient relative to historical controls or past experience in the same patient.
  • therapeutic or prophylactic efficacy can be demonstrated in a preclinical or clinical trial in a population of treated patients relative to a control population of untreated patients.
  • Exemplary dosages for an antibody are 0.1-60 mg/kg (e.g., 0.5, 3, 10, 30, or 60 mg/kg), or 0.5-5 mg/kg body weight (e.g., 0.5, 1, 2, 3, 4 or 5 mg/kg) or 10-4000 mg or 10-1500 mg as a fixed dosage.
  • the dosage depends on the condition of the patient and response to prior treatment, if any, whether the treatment is prophylactic or therapeutic and whether the disorder is acute or chronic, among other factors.
  • Administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular.
  • Some antibodies can be administered into the systemic circulation by intravenous or subcutaneous administration.
  • Intravenous administration can be, for example, by infusion over a period such as 30-90 min.
  • the frequency of administration depends on the half-life of the antibody in the circulation, the condition of the patient and the route of administration among other factors.
  • the frequency can be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the patient’s condition or progression of the disorder being treated.
  • An exemplary frequency for intravenous administration is between weekly and quarterly over a continuous cause of treatment, although more or less frequent dosing is also possible.
  • an exemplary dosing frequency is daily to monthly, although more or less frequent dosing is also possible.
  • the number of dosages administered depends on whether the disorder is acute or chronic and the response of the disorder to the treatment. For acute disorders or acute exacerbations of a chronic disorder, between 1 and 10 doses are often sufficient. Sometimes a single bolus dose, optionally in divided form, is sufficient for an acute disorder or acute exacerbation of a chronic disorder. Treatment can be repeated for recurrence of an acute disorder or acute exacerbation. For chronic disorders, an antibody can be administered at regular intervals, e.g ., weekly, fortnightly, monthly, quarterly, every six months for at least 1, 5 or 10 years, or the life of the patient.
  • the invention provides methods of in vivo imaging tau protein deposits (e.g, neurofibrillary tangles and tau inclusions) in a patient.
  • the methods work by administering a reagent, such as antibody that binds tau (e.g, a mouse, humanized, chimeric or veneered 3D6 antibody), to the patient and then detecting the agent after it has bound.
  • a reagent such as antibody that binds tau (e.g, a mouse, humanized, chimeric or veneered 3D6 antibody)
  • Antibodies binding to an epitope of tau within amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or 320-334, respectively, of SEQ ID NO: 1) or within amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: l, are preferred.
  • the antibody binds to an epitope within amino acid residues 199-213 of SEQ ID NO: 3 (corresponding to amino acid residues 257-271 of SEQ ID NO: 1) , or within amino acids 262- 276 of SEQ ID NO:3 (corresponding to amino acid residues 320-334 of SEQ ID NO: 1).
  • the antibody binds to an epitope within amino acid residues 259-268 of SEQ ID NO: 1, within amino acids 290-299 of SEQ ID NO: l, within amino acids 321-330 of SEQ ID NOl, or within amin acids 353-362 of SEQ ID NO: 1.
  • a clearing response to the administered antibodies can be avoided or reduced by using antibody fragments lacking a full-length constant region, such as Fabs.
  • the same antibody can serve as both a treatment and diagnostic reagent.
  • Diagnostic reagents can be administered by intravenous injection into the body of the patient, or directly into the brain by intracranial injection or by drilling a hole through the skull.
  • the dosage of reagent should be within the same ranges as for treatment methods.
  • the reagent is labeled, although in some methods, the primary reagent with affinity for tau is unlabeled and a secondary labeling agent is used to bind to the primary reagent.
  • the choice of label depends on the means of detection. For example, a fluorescent label is suitable for optical detection. Use of paramagnetic labels is suitable for tomographic detection without surgical intervention. Radioactive labels can also be detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • the methods of in vivo imaging of tau protein deposits are useful to diagnose or confirm diagnosis of a tauopathy, such as Alzheimer's disease, frontotemporal lobar
  • the methods can be used on a patient presenting with symptoms of dementia. If the patient has abnormal neurofibrillary tangles, then the patient is likely suffering from Alzheimer's disease. Alternatively, if the patient has abnormal tau inclusions, then depending on the location of the inclusions, the patient may be suffering from frontotemporal lobar degeneration.
  • the methods can also be used on asymptomatic patients. Presence of abnormal tau protein deposits indicates susceptibility to future symptomatic disease. The methods are also useful for monitoring disease progression and/or response to treatment in patients who have been previously diagnosed with a tau-related disease.
  • Diagnosis can be performed by comparing the number, size, and/or intensity of labeled loci, to corresponding baseline values.
  • the base line values can represent the mean levels in a population of undiseased individuals. Baseline values can also represent previous levels determined in the same patient. For example, baseline values can be determined in a patient before beginning tau immunotherapy treatment, and measured values thereafter compared with the baseline values. A decrease in values relative to baseline signals a positive response to treatment.
  • a PET scan can be performed using, for example, a conventional PET imager and auxiliary equipment.
  • the scan typically includes one or more regions of the brain known in general to be associated with tau protein deposits and one or more regions in which few if any deposits are generally present to serve as controls.
  • the signal detected in a PET scan can be represented as a multidimensional image.
  • the multidimensional image can be in two dimensions representing a cross-section through the brain, in three dimensions, representing the three dimensional brain, or in four dimensions representing changes in the three dimensional brain over time.
  • a color scale can be used with different colors indicating different amounts of label and, inferentially, tau protein deposit detected.
  • the results of the scan can also be presented numerically, with numbers relating to the amount of label detected and consequently amount of tau protein deposits.
  • Alzheimer's disease can be compared with the label present in a region known not to be associated with deposits to provide a ratio indicative of the extent of deposits within the former region.
  • a ratio indicative of the extent of deposits within the former region For the same radiolabeled ligand, such ratios provide a comparable measure of tau protein deposits and changes thereof between different patients.
  • a PET scan is performed concurrent with or in the same patient visit as an MRI or CAT scan.
  • An MRI or CAT scan provides more anatomical detail of the brain than a PET scan.
  • the image from a PET scan can be superimposed on an MRI or CAT scan image more precisely indicating the location of PET ligand and inferentially tau deposits relative to anatomical structures in the brain.
  • Suitable PET ligands include radiolabeled antibodies of the invention (e.g., a mouse, humanized, chimeric or veneered 3D6 antibody).
  • the radioisotope used can be, for example,
  • the interval between administering the PET ligand and performing the scan can depend on the PET ligand and particularly its rate of uptake and clearing into the brain, and the half- life of its radiolabel.
  • PET scans can also be performed as a prophylactic measure in asymptomatic patients or in patients who have symptoms of mild cognitive impairment but have not yet been diagnosed with a tauopathy but are at elevated risk of developing a tauopathy.
  • scans are particularly useful for individuals considered at elevated risk of tauopathy because of a family history, genetic or biochemical risk factors, or mature age.
  • Prophylactic scans can commence for example, at a patient age between 45 and 75 years. In some patients, a first scan is performed at age 50 years.
  • Prophylactic scans can be performed at intervals of for example, between six months and ten years, preferably between 1-5 years. In some patients, prophylactic scans are performed annually.
  • a PET scan performed as a prophylactic measure indicates abnormally high levels of tau protein deposits
  • immunotherapy can be commenced and subsequent PET scans performed as in patients diagnosed with a tauopathy.
  • a PET scanned performed as a prophylactic measure indicates levels of tau protein deposits within normal levels
  • further PET scans can performed at intervals of between six months and 10 years, and preferably 1-5 years, as before, or in response to appearance of signs and symptoms of a tauopathy or mild cognitive impairment.
  • levels of tau protein deposits can be reduced to, or closer to, normal levels, or at least inhibited from increasing further, and the patient can remain free of the tauopathy for a longer period than if not receiving prophylactic scans and tau-directed immunotherapy (e.g ., at least 5, 10, 15 or 20 years, or for the rest of the patient's life).
  • Normal levels of tau protein deposits can be determined by the amount of
  • neurofibrillary tangles or tau inclusions in the brains of a representative sample of individuals in the general population who have not been diagnosed with a particular tauopathy e.g.,
  • Alzheimer's disease and are not considered at elevated risk of developing such disease (e.g, a representative sample of disease-free individuals under 50 years of age).
  • a normal level can be recognized in an individual patient if the PET signal according to the present methods in a region of the brain in which tau protein deposits are known to develop is not different (within the accuracy of measurement) from the signal from a region of the brain in which it is known that such deposits do not normally develop.
  • An elevated level in an individual can be recognized by comparison to the normal levels (e.g, outside mean and variance of a standard deviation) or simply from an elevated signal beyond experimental error in a region of the brain associated with tau protein deposits compared with a region not known to be associated with deposits.
  • the tau protein deposits should preferably be determined in the same region(s) of the brain, these regions including at least one region in which tau protein deposits associated with a particular tauopathy (e.g, Alzheimer's disease) are known to form.
  • a patient having an elevated level of tau protein deposits is a candidate for commencing immunotherapy.
  • a decrease in the level of tau protein deposits can be first seen as an indication that the treatment is having the desired effect.
  • the observed decrease can be, for example, in the range of 1-100%, 1-50%, or 1-25% of the baseline value.
  • Such effects can be measured in one or more regions of the brain in which deposits are known to form or can be measured from an average of such regions.
  • the total effect of treatment can be approximated by adding the percentage reduction relative to baseline to the increase in tau protein deposits that would otherwise occur in an average untreated patient.
  • PET monitoring provides an indication of the nature and extent of response to treatment. Then a determination can be made whether to adjust treatment and if desired treatment can be adjusted in response to the PET monitoring.
  • PET monitoring thus allows for tau-directed immunotherapy or other treatment regime to be adjusted before other biomarkers, MRI or cognitive measures have detectably responded.
  • a significant change means that comparison of the value of a parameter after treatment relative to basement provides some evidence that treatment has or has not resulted in a beneficial effect.
  • a change of values of a parameter in a patient itself provides evidence that treatment has or has not resulted in a beneficial effect.
  • the change of values, if any, in a patient is compared with the change of values, if any, in a representative control population of patients not undergoing immunotherapy.
  • a difference in response in a particular patient from the normal response in the control patient e.g., mean plus variance of a standard deviation
  • monitoring indicates a detectable decline in tau protein deposits but that the level of tau protein deposits remains above normal.
  • the treatment regime can be continued as is or even increased in frequency of administration and/or dose if not already at the maximum recommended dose.
  • the immunotherapy regime can be adjusted from one of induction (i.e., that reduces the level of tau protein deposits) to one of maintenance (i.e . , that maintains tau protein deposits at an approximately constant level).
  • induction i.e., that reduces the level of tau protein deposits
  • maintenance i.e . , that maintains tau protein deposits at an approximately constant level.
  • Such a regime can be affected by reducing the dose and or frequency of administering
  • monitoring can indicate that immunotherapy is having some beneficial effect but a suboptimal effect.
  • An optimal effect can be defined as a percentage reduction in the level of tau protein deposits within the top half or quartile of the change in tau protein deposits (measured or calculated over the whole brain or representative region(s) thereof in which tau protein deposits are known to form) experienced by a representative sample of tauopathy patients undergoing immunotherapy at a given time point after commencing therapy.
  • a patient experiencing a smaller decline or a patient whose tau protein deposits remains constant or even increases, but to a lesser extent than expected in the absence of immunotherapy can be classified as experiencing a positive but suboptimal response.
  • Such patients can optionally be subject to an adjustment of regime in which the dose and or frequency of administration of an agent is increased.
  • tau protein deposits may increase in similar or greater fashion to tau deposits in patients not receiving immunotherapy. If such increases persist over a period of time, such as 18 months or 2 years, even after any increase in the frequency or dose of agents, immunotherapy can if desired be discontinued in favor of other treatments.
  • the methods can be used to monitor a course of therapeutic and prophylactic treatment with the agents provided herein.
  • the antibody profile following passive immunization typically shows an immediate peak in antibody concentration followed by an exponential decay. Without a further dose, the decay approaches pretreatment levels within a period of days to months depending on the half-life of the antibody administered. For example, the half-life of some human antibodies is of the order of 20 days.
  • a baseline measurement of antibody to tau in the subject is made before administration, a second measurement is made soon thereafter to determine the peak antibody level, and one or more further measurements are made at intervals to monitor decay of antibody levels.
  • a predetermined percentage of the peak less baseline e.g, 50%, 25% or 10%
  • administration of a further dose of antibody is administered.
  • peak or subsequent measured levels less background are compared with reference levels previously determined to constitute a beneficial prophylactic or therapeutic treatment regime in other subjects. If the measured antibody level is significantly less than a reference level (e.g, less than the mean minus one or, preferably, two standard deviations of the reference value in a population of subjects benefiting from treatment) administration of an additional dose of antibody is indicated.
  • Also provided are methods of detecting tau in a subject for example, by measuring tau in a sample from a subject or by in vivo imaging of tau in a subject. Such methods are useful to diagnose or confirm diagnosis of diseases associated with tau, or susceptibility thereto. The methods can also be used on asymptomatic subjects. The presence of tau indicates susceptibility to future symptomatic disease.
  • the methods are also useful for monitoring disease progression and/or response to treatment in subjects who have been previously diagnosed with Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).
  • CBD corticobasal degeneration
  • LVAD dementia with Lewy bodies
  • LVAD chronic traumatic encephalopathy
  • GTT progressive supranuclear
  • CBD cerebral spastic disease
  • LBD dementia with Lewy bodies
  • LVAD Lewy body variant of Alzheimer disease
  • CTE chronic traumatic encephalopathy
  • GTT globular glial tauopathy
  • PSP progressive supranuclear palsy
  • levels of tau in such subjects may be compared to those present in healthy subjects.
  • levels of tau in such subjects receiving treatment for the disease may be compared to those of subjects who have not been treated for Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).
  • Some such tests involve a biopsy of tissue obtained from such subjects.
  • ELISA assays may also be useful methods, for example, for assessing tau in fluid samples.
  • kits comprising an antibody disclosed herein and related materials, such as instructions for use (e.g., package insert).
  • the instructions for use may contain, for example, instructions for administration of the antibody and optionally one or more additional agents.
  • the containers of antibody may be unit doses, bulk packages (e.g, multi-dose packages), or sub-unit doses.
  • Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage,
  • Kits can also include a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It can also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • the antibodies can be used for detecting tau, or fragments thereof, in the context of clinical diagnosis or treatment or in research.
  • the antibodies can be used to detect the presence of tau in a biological sample as an indication that the biological sample comprises tau deposits. Binding of the antibodies to the biological sample can be compared to binding of the antibodies to a control sample.
  • the control sample and the biological sample can comprise cells of the same tissue origin. Control samples and biological samples can be obtained from the same individual or different individuals and on the same occasion or on different occasions. If desired, multiple biological samples and multiple control samples are evaluated on multiple occasions to protect against random variation independent of the differences between the samples.
  • a direct comparison can then be made between the biological sample(s) and the control sample(s) to determine whether antibody binding (z.e., the presence of tau) to the biological sample(s) is increased, decreased, or the same relative to antibody binding to the control sample(s).
  • Increased binding of the antibody to the biological sample(s) relative to the control sample(s) indicates the presence of tau in the biological sample(s).
  • the increased antibody binding is statistically significant.
  • antibody binding to the biological sample is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, or 100-fold higher than antibody binding to the control sample.
  • the antibodies can be used to detect the presence of the tau in a biological sample to monitor and evaluate the efficacy of a therapeutic agent being used to treat a patient diagnosed with Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).
  • CBD corticobasal degeneration
  • LVAD dementia with Lewy bodies
  • LVAD chronic
  • CBD cerebral spastic disease
  • LBD dementia with Lewy bodies
  • LVAD Lewy body variant of Alzheimer disease
  • CTE chronic traumatic encephalopathy
  • GTT globular glial tauopathy
  • PSP progressive supranuclear palsy
  • a therapeutic agent is then administered in a regime.
  • the regime may include multiple administrations of the agent over a period of time.
  • binding of the antibodies is evaluated on multiple occasions in multiple biological samples from the patient, both to establish a measure of random variation and to show a trend in response to immunotherapy.
  • the various assessments of antibody binding to the biological samples are then compared. If only two assessments are made, a direct comparison can be made between the two assessments to determine whether antibody binding (i.e., presence of tau) has increased, decreased, or remained the same between the two assessments. If more than two measurements are made, the measurements can be analyzed as a time course starting before treatment with the therapeutic agent and proceeding through the course of therapy.
  • the therapeutic agent was effective in treating the Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP) in the patient.
  • CBD corticobasal degeneration
  • LVAD dementia with Lewy bodies
  • LVAD chronic traumatic encephalopathy
  • GTT progressive supranuclear palsy
  • the decrease in antibody binding can be statistically significant.
  • binding decreases by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
  • Assessment of antibody binding can be made in conjunction with assessing other signs and symptoms of Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supran
  • the antibodies can also be used as research reagents for laboratory research in detecting tau, or fragments thereof.
  • antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes, and can be provided in the form of kit with all the necessary reagents to perform the detection assay.
  • the antibodies can also be used to purify tau, or binding partners of tau, e.g. , by affinity chromatography.
  • Monoclonal antibodies against tau were generated as follows. Immunizations were performed with either recombinant N-terminally His-tagged 383 a.a. human tau (4R0N), containing a P301S mutation [immunogen A] or recombinant 383 a.a. human tau (4R0N), containing a P301S mutation, lacking an N-terminal His-tag [immunogen B] Immunogens were emulsified in RIBI adjuvant.
  • mice Five week old female Balb/c mice were intraperitoneally immunized with 25 pg of immunogen A on day 0, and 10pg of immunogen A each on days 7, 14, 21, 27, 34, 48, 55, and 62. Mice were immunized with 10pg of immunogen B on days 76 and 90. On days 43 and 98, mice were bled and titered against immunogen A; on day 101 the animals with highest titers were boosted with a terminal immunization of 50pg immunogen B, which was delivered 1 ⁇ 2 intraperitoneally and 1 ⁇ 2 intravenously. Fused hybridomas were screened via ELISA against both immunogens, and positives with the highest signal were epitope mapped (see Example 2).
  • a range of overlapping biotinylated peptides spanning the entire 383aa 4R0N human tau protein were used for mapping the murine 3D6 antibody. Additional peptides were used to model potential post-translational modifications of the C- and N-terminal ends of the protein.
  • Biotinylated peptides were bound to separate wells of a streptavidin-coated ELISA plate. The plate was blocked and treated with murine 3D6, followed by incubation with a horseradish peroxidase-conjugated anti-mouse antibody. After thorough washing, OPD was applied to the plate and allowed to develop. The plate was read at 450nm absorbance.
  • the starting point or donor antibody for humanization was the mouse antibody 3D6.
  • the heavy chain variable amino acid sequence of mature m3D6 is provided as SEQ ID NO:7.
  • the light chain variable amino acid sequence of mature m3D6 is provided as SEQ ID NO: 11.
  • the heavy chain Kabat/Chothia Composite CDR1, CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOs:8-10, respectively.
  • the light chain Rabat CDR1, CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOsl2-14 respectively. Rabat numbering is used throughout.
  • variable kappa (Vk) of 3D6 belongs to mouse Rabat subgroup 2 which corresponds to human Rabat subgroup 2 and the variable heavy (Vh) to mouse Rabat subgroup 2c which corresponds to human Rabat subgroup 1
  • Vk variable kappa
  • Vh variable heavy
  • 16 residue Chothia CDR- L1 belongs to canonical class 4, 7 residue Chothia CDR-L2 to class 1, 9 residue Chothia CDR- L3 to class 1 in Vk [Martin A.C, and Thornton J.M. (1996) J. Mol. Biol. 263:800-15.
  • the frameworks of 3D6 VH share a high degree of sequence similarity with the corresponding regions of humanized 48G7 Fab PDB: 2RCS, designed by Wedemayer, G. J., et ak (1997; Science 276: 1665-1669).
  • the variable domains of 3D6 and 48G7 fab also share identical lengths for the CDR-H1, H2 loops.
  • the frameworks of 3D6 VL share a high degree of sequence similarity with the corresponding regions of human antibody ARX71335 VL, cloned by Dafferner, A. J., et ak (2017; . Direct submission).
  • the variable light domain of 3D6 and ARX71335 antibody also share identical lengths for the CDR-L1, L2 and L3 loops.
  • framework regions of 48G7 VH (2RCS-VH) and ARX71335 VL were chosen as the acceptor sequences for the CDRs of 3D6.
  • a model of the 3D6 CDRs grafted onto the respective human frame-works for VH and VL was built and used as a guidance for further backmutations.
  • - are occupied by residues that are predicted to clash with CDRs, according to the model of 3D6 CDRs grafted onto VH and VL frameworks, or - are occupied by residues that are rare among sequenced human antibodies, where either the parental mouse 3D6 residue or some other residue is much more prevalent within human antibody repertoire.
  • hu3D6VHvbl hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6Hvb5, hu3D6VHvb6, or hu3D6VHvb7 (SEQ ID NOs: 76-80 and 90-91, respectively); and hu3D6VLvbl, hu3D6VLvb2, or hu3D6VLvb3 (SEQ ID NOs:83-85, respectively) (Tables 3 and 4).
  • Tables 3 and 4 The exemplary humanized Vk and Vh designs, with backmutations and other mutations based on selected human frameworks, are shown in Tables 3 and 4, respectively.
  • Tables 3 and 4 indicate the CDRs as defined by Kabat/Chothia Composite.
  • SEQ ID NOs:76-80 and SEQ ID NOs:90-91 contain backmutations and other mutations as shown in Table 5.
  • the amino acids at positions in hu3D6VHvbl, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7 are listed in Table 6.
  • the amino acids at positions in hu3D6VLvbl, hu3D6VLvb2, and hu3D6VLvb3 are listed in Table 7.
  • Table 6 Kabat Numbering of Framework (or CDR) Residues (based on Kabat/Chothia Composite CDRs) for Backmutations and Other Mutations in Heavy Chains of Humanized 3D6 Antibodies
  • Positions at which canonical, vernier, or interface residues differ between mouse and human acceptor sequences are candidates for substitution.
  • canonical/CDR interacting residues include Rabat residues H54 and H94 in Table 3.
  • vernier residues include Rabat residues H28, H67, H93, and H94 in Table 3.
  • VH+VL residues include Rabat residues H91 and H93 in Table 3.
  • 3D6-VH_vb2 incorporates backmutations or substitutions Q1E, Q5V, LI IV, L20I, T23K, K38R, E42G, Q43K, K66R, S75T, N76D, Q81E,Y91F, A93S, S94T T108L, and LI 09 V, to enable assessment of these positions’ contributions to antigen-binding affinity and immunogenicity.
  • hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7 consists of further substitutions and either add to antibody stability and/or for optimization of glycosylation, aggregation, N-term heterogeneity, thermostability, surface exposed charged patches, surface exposed charge patches, deamination, and proteinase susceptibility.
  • Q1E is a stability enhancing mutation to mitigate pyroglutamate formation potential (Liu, supra.)
  • Q5V is a frequency -based and germ line-aligning mutation. Val is most frequent in human sequences at this position. Val is in human germ line gene IMGT# IGHV1 -69-2*01 (SEQ (ID NO: 25) at this position.
  • LI IV is a germ line-aligning mutation. Val is in human germ line gene IMGT# IGHV1- 69-2*01 (SEQ (ID NO: 25) at this position.
  • S17T is a germ line-aligning mutation. Thr is in human germ line gene IMGT# IGHV1- 69-2*01 (SEQ (ID NO: 25) at this position.
  • L20I is a germ line-aligning mutation. He is in human germ line gene IMGT# IGHV1- 69-2*01 (SEQ (ID NO: 25) at this position.
  • T23K is a frequency -based and germ line-aligning mutation. Lys is more frequent at this position. Lys is in human germ line gene IMGT# IGHVl-69-2*01 (SEQ (ID NO: 25) at this position.
  • N28T This is a CDR-H1 residue substitution to Thr.
  • K38R is a frequency -based back-mutation. Arg is most frequent at this position. Arg at this position is predicted to make two H-bonds with Glu 46 in addition to one H-bond each with Asp86 and Tyr 90 in heavy chain; therefore, Arg substitution may enhance stability over Lys at this position.
  • E42G is a frequency -based and germ line-aligning mutation. Gly is in human germ line gene IMGT# IGHVl-69-2*01 (SEQ (ID NO: 25) at this position. Gly is most frequent at this position. Gly substitution is predicted not to affect stability.
  • Q43K Lys side chain at this position is predicted to make H-bond with G42 besides main chain making H-bonds with Gin 39 and Arg 40, thereby Lys substitution may enhance stability over Q at this position.
  • N54D and D56E are substitutions of CDR residues and are predicted to be non-antigen contact positions as per homology model. N54D and D56E substitutions are predicted to stabilize antibody structure.
  • V58I is a substitution of a CDR-H2 residue.
  • Germline gene IMGT# IGHVl-69-2*01 SEQ (ID NO: 25) has He at this position. This residue is predicted not to contact antigen.
  • K66R Arg at this position is predicted to make H-bonds with Ser 82a and Thr 83 in addition to making H-bond and salt-bridge with Asp 86.
  • A67V is a substitution of a Vernier zone residue.
  • Germ line gene IMGT# IGHV1-69- 2*01 SEQ (ID NO: 25) has Val at this position.
  • S75T Ser at this position is predicted to make H-bond with Asp 72 and Tyr 76. Thr at this position is predicted to also make these contacts but being surface exposed residue Thr may enhance antibody stability.
  • N76D Asp is a germ line-aligning mutation. Asp is in human germ line gene IMGT# IGHV1 -69-2*01 (SEQ (ID NO: 25) at this position.
  • L80M Met is a germ line-aligning mutation. Met is in human germ line gene IMGT# IGHV1 -69-2*01 (SEQ (ID NO: 25) at this position.
  • Q81E Glu is predicted to make H-bond plus salt-bridge with K19 hence Glu at this position make enhance antibody stability.
  • T83R to enhance thermostability and to increase humanness.
  • Arg is a germ-line aligning mutation. Arg is in human germ line gene IMGT# IGHVl-69-2*01 (SEQ (ID NO: 25) at this position. Arg is most frequent at this position.
  • F91Y is mutation of an interface residue, and is a frequency-based back-mutation. Tyr is typically at this position, and may enhance antibody stability.
  • A93S is a back-mutation of a Vernier zone and interface zone residue.
  • S94T is a back-mutation of a Chothia defined canonical structural residue and vernier residue.
  • T108L Leu is a germ line-aligning mutation. Leu is in human germ line gene IMGT# IGHVl-69-2*01 (SEQ (ID NO: 25) at this position. Leu at this position is predicted to make the antibody less immunogenic and to have no impact on antibody stability.
  • L109V is a frequency -based mutation. Val is most frequent at this position.
  • Hu3D6- VLvb2 & Hu3D6-VLvb3 also include substitutions that contribute to structural stability hu3D6- VL_vb2 incorporates backmutations T7S, I15L, L83V, H86Y and L106I, to enable assessment of these positions’ contributions to antigen-binding affinity and immunogenicity.
  • Hu3D6-VL_vb3 all substitutions mentioned for vb2 along with additional changes at Q17E, K24R, L37Q, K45R, and LI 061
  • T7S is a germ line-aligning mutation. Ser is in human germ line gene IGKV2-30*02 (SEQ (ID NO: 27) at this position.
  • T10S is a frequency-based and germ line-aligning mutation. Ser is frequent at this position. Ser is in human germ line gene IGKV2-30*02 (SEQ (ID NO: 27) at this position.
  • Q17E Glu at this position is predicted to make H-bond with T14 and salt-bridge with Lys 107, both light chain residues, and to enhance antibody stability.
  • K24R is a mutation of a CDR residue. Both Lys and Arg are predicted to make H-bond and salt-bridge with Asp 70 in the light chain. Arg is predicted to fit better in the conformation. Arg is also a germ line-aligning mutation. Arg is in human germ line gene IGKV2-30*02 (SEQ (ID NO: 27) at this position.
  • L37Q This is predicted to be a deep buried residue, Leu is not predicted to interact with surrounding residues, whereas, Gin is predicted to make H-bonds with Q38 and Asp 82 in the light chain. Gin is also a germ line-aligning mutation. Gin is in human germ line gene IGKV2- 30*02 (SEQ (ID NO: 27) at this position.
  • K45R Although, Lys is predicted to make H-bonds with S56 and Gly 57; Arg’s predicted interaction with neighboring residues is much more extensive as it is predicted to form salt-bridges with D55, H-bond with Arg46 and double H-bonds with S56. Arg is also a germ line-aligning mutation. Arg is in human germ line gene IGKV2-30*02 (SEQ (ID NO: 27) at this position.
  • L83V This is a frequency -based mutation of a residue predicted to be surface exposed. Val is also a germ line-aligning mutation. Val is in human germ line gene IGKV2-30*02 (SEQ (ID NO: 27) at this position.
  • H86Y Murine 3D6 VL has Tyr at this position. Tyr is also the most frequent residue at this position.
  • A100Q Ala is rare at this position. Ala is predicted to be surface-exposed residue and is not predicted to interact surrounding residues. Gin is most frequent at this position and is also a germline-aligning mutation. Gin is in human germ line gene IGKV2-30*02 (SEQ (ID NO: 27) at this position. Gin is predicted to make H-bond with Ser 7 stabilizing intra-chain.
  • L106I is a frequency-based and germ line-aligning mutation. He is most frequent at this position. lie is in human germ line gene IGKV2-30*02 (SEQ (ID NO: 27) at this position.
  • Sandwich ELISA 96-well polystyrene plates were coated with anti-mouse antibodies in lxPBS for 2 hr at RT or 16 hr at 4°C. Coating was removed, and plates were blocked for 1 hr with 1%BSA in lxPBS. The plate was next incubated with the Indicated antibodies at identical concentrations, diluted in 0.1% BSA in lxPBS. Plates were successively treated with human tau, polyclonal rabbit anti-tau (Dako #A0024), and HRP-conjugated goat anti-rabbit antibody, all diluted in 0.1%BSA in PBS with washes occurring between each step. Streptavidin-HRP was added, plates were developed with TMB, and A450 was measured with a plate reader. See Figure 4C.
  • Data were double-referenced to both an irrelevant sensor not containing antibody ligand, and 0 nM analyte concentration to account for the dissociation of ligand from the capture moiety. Data was then analyzed using a global 1 : 1 fit.
  • Example 6 Mouse monoclonal antibodies prevent binding of human tau to the surface of immortalized neuronal cells
  • step 2 remove blocking buffer from wells by inverting plate and gently tapping and wash plate 2x with PBS using a multichannel pipette. Ensure excess buffer is completely removed. Cool the plated cells to 4°C prior to adding the Tau: anti-Tau complexes.
  • step 5 Add 50pL of cooled complex, step 3, to the plated cells and incubate on ice for 30 minutes.
  • Antibodies tested were anti -tau antibodies 3D6, 16G7, 3H9, 4C5, and 5G8, and isotype control.
  • Varying molar ratios of 3D6 (triangles), isotype control (circles) and 16G7 (squares) were incubated with amyloid-containing tau fibrils for 96 hours. At the end of this period, the extent of aggregation was assessed by binding to Thioflavin T. 3D6 preferentially decreases the Thioflavin T signal present in the sample, compared to both an isotype control antibody as well as to 16G7, an anti-tau antibody that binds to a different region of tau.
  • Example 8 3D6 and 5G8 immunocapture tau from human disease tissue.
  • High-salt soluble protein fractions were prepared to 1 mg/ml. For each immunoprecipitation, 200 pg of sample was used. 10pg of the indicated antibody (either an isotype control, 3D6, or anti-tau antibody 5G8) was added to the high-salt sample preparations, and incubated for 2 hr. Protein G magnetic beads were then added to the mixtures, and incubated for a further hour to capture antibody/antigen complexes. Samples were thoroughly washed with lxPBS, and beads were boiled in reducing/denaturing sample buffer to release captured proteins. Resulting samples were resolved by SDS-PAGE and Western blotting was performed using a polyclonal anti-tau antibody (Dako, #A0024).
  • Frontotemporal cortices were obtained from patients without neurodegenerative disease or with Alzheimer disease, which was confirmed upon post-mortem assessment.
  • cryosections All staining steps were performed using a Leica BOND Rx autostainer, using Leica consumables. Either murine or a human form of 3D6 was incubated with tissue sections followed by addition of species-appropriate secondary antibodies conjugated to an HRP polymer. To prevent non-specific binding of endogenous immunoglobulin when using humanized antibodies on human tissue, the antibodies were non-covalently labeled with a biotin-conjugated anti-human monovalent Fab fragment in vitro before incubation on tissue. Tissue labeled with the primary antibody -biotin Fab fragment complex was further amplified using an avi din-biotin amplification system (Vector Laboratories, Burlingame, CA). The staining was visualized with a DAB chromogen, which produced a brown deposit. Negative control consisted of performing the entire immunohistochemical procedure on adjacent sections with an IgG isotype control antibody.
  • Antibodies tested were murine CD6, chimeric 3D6 (which contained VH and VL from the murine antibody with human constant regions, heavy chain SEQ ID NO: 72 and light chain SEQ ID NO:73), and humanized variant hu3D6VHv5/hu3D6VLv2.
  • Staining performed with murine, chimeric, and humanized forms of 3D6 were qualitatively compared and assessed for the strength and intensity of staining, as well as localization of immunoreactivity. Intensity of staining was similar for chimeric and humanized forms of 3D6, and displayed similar localization patterns compared with the murine form of the antibody. Tau was detected in neurofibrillary tangles, fibrils, neuropil threads, and in degenerating axons. There was also notable somal staining detected.
  • Methods Indirect ELISA 96-well polystyrene plates are coated with human recombinant tau suspended in lxPBS for 2h at RT or 16h at 4°C. Coating is removed, and plates are blocked for lh with 1%BSA in lxPBS. Humanized variant antibodies at 1 pg/mL in 0.1% BSA in lxPBS are added to plates for 1 hour followed by washing, and HRP-conjugated goat anti-human antibody are added. Plates are developed with TMB, and A450 is measured with a plate reader.
  • Sandwich ELISA 96-well polystyrene plates are coated with anti-human antibodies in lxPBS for 2 hr at RT or 16 hr at 4°C. Coating is removed, and plates are blocked for 1 hr with 1%BSA in lxPBS. Humanized variant antibodies at varying concentrations diluted in 0.1% BSA in lxPBS are added to plates for 1 hour followed by washing, and biotinylated recombinant human tau diluted in 0.1% BSA in lxPBS is added. After washing, Streptavidin-HRP is added, plates are developed with TMB, and A450 is measured with a plate reader.
  • SPR analysis is performed using a Biacore T200 to determine the binding kinetics of h3D6-VHv5-L2 to recombinant human tau.
  • anti-human antibody GE Life Sciences
  • CM5 sensor chip
  • humanized variant antibody is captured at a level to ensure maximum binding of 50 RU.
  • concentrations of recombinant tau ranging from 10-0.14 nM are passed over the captured ligand at a flow rate of 50 pL/min in running buffer (HBS + 0.05% P-20, 1 mg/mL BSA), for 180s association and 900s dissociation.
  • Data are double-referenced to both an irrelevant sensor not containing antibody ligand, and 0 nM analyte concentration to account for the dissociation of ligand from the capture moiety. Data is then analyzed using a global 1 : 1 fit.
  • L37Q is a mutation to increase humanness of the sequence. Gin is a germline-aligning mutation. Gin is in human germline gene IGKV2-30*02 (SEQ (ID NO: 27) at this position.
  • G100Q is a mutation to increase humanness of the sequence.
  • Gin is a germline- aligning mutation. Gin is in human germline gene IGKV2-30*02 (SEQ (ID NO: 27) at this position.
  • hu3D6VLv2 L37Q SEQ ID NO: 143
  • hu3D6VLv2 L50G SEQ ID NO: 103
  • hu3D6VLv2 S52G SEQ ID NO:
  • L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), and hu3D6VLv2 L37Q_S52G_L54R_G100Q (SEQ ID NO: 138).
  • Figures 9A and 9B show alignments of the heavy chain variable region of murine 3D6 (SEQ ID NO:7) and of humanized versions of the 3D6 antibody (hu3D6VHvbl, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, hu3D6VHvb7, hu3D6VHvlbAl 1, h3D6VHvb8, and h3D6VHvb9) with human germline heavy chain variable region sequence IGHVl-69-2*01 (SEQ ID NO:25) and with human acceptor heavy chain variable region sequence 2RCS VH hFrwk (SEQ ID NO:75).
  • hu3D6VHvbl is SEQ ID NO:76
  • hu3D6VHvb2 is SEQ ID NO:77
  • hu3D6VHvb3 is SEQ ID NO:78
  • hu3D6VHvb4 is SEQ ID NO:79
  • hu3D6VHvb5 is SEQ ID NO:80
  • hu3D6VHvb6 is SEQ ID NO:90
  • hu3D6VHvb7 is SEQ ID NO:91
  • hu3D6VHvlbAl l is SEQ ID NO: 18
  • h3D6VHvb8 is SEQ ID NO: 146
  • h3D6VHvb9 is SEQ ID NO: 148.
  • D60E to reduce potential proteolysis.
  • Asp-Pro motifs are known to be potential proteolytic cleavage sites.
  • Glu substitution at position 60 is considered as conformation conserving. Therefore, D60E substitution is made to reduce potential proteolysis.
  • Glu is a germ-line aligning mutation. Glu is in human germline gene IMGT# IGHV1 -69-2*01 (SEQ (ID NO: 25) at this position.
  • L82cV to reduce immunogenicity.
  • immunogenicity analysis tool predicted a potential immunogenic peptide that included L82c.
  • L82cV substitution is designed to reduce immunogenicity. Valine substitution at this position conserves loop conformation.
  • Met is a germline aligning mutation. Met is in human germ line gene IMGT# IGHVl-69-2*01 (SEQ (ID NO: 25) at this position. [0597] T83R to enhance thermostability and to increase humanness. Arg is a germline aligning mutation. Arg is in human germ line gene IMGT# IGHVl-69-2*01 (SEQ (ID NO: 25) at this position. Arg is most frequent at this position.
  • thermostability results were compared with the parental sequence, hu3D6VHvlbAl 1/ hu3D6VLv2 to determine if any loss of activity or stability was noted.
  • Target binding analysis was performed using a Biacore T200 to compare the binding affinity of humanized 3D6 variants to recombinant human 4R0N tau.
  • Anti -Human Fc antibody was immobilized on sensor chip CM3 via amine coupling, and humanized 3D6 variants were captured to equivalent levels.
  • Various concentrations of recombinant 4R0N human tau (ranging from 0.02 nM to 12.5 nM) were passed over the captured ligand at 50 pL/min in running buffer (HBS + 0.05% P-20, 1 mg/mL BSA) for 180 seconds association/420 seconds dissociation as a single cycle. Data was blank subtracted to both an irrelevant sensor not containing antibody and 0 nM analyte concentration. Analysis was performed using a global 1 : 1 fit with Biacore
  • Affinity determination revealed a number of deimmunized variants that retained the affinity of the parental hu3D6VHvlbAl 1/ hu3D6VLv2, which was determined by comparison of the KD of each antibody to that of parental hu3D6VHvlbAl 1/ hu3D6VLv2.
  • Antibodies determined to have KD comparable within 4-fold of hu3D6VHvlbAl 1/ hu3D6VLv2 include hu3D6VHvlbAl 1/L2-DIM4, hu3D6VHvlbAl 1/L2-DIM9, hu3D6VHvlbAl 1/L2- DIM5, hu3D6VHvlbAl 1/L2-DIM3, h3D6VHvb8/L2-DIM4, hu3D6VHvlbAl 1/L2-DIM7, h3D6VHvb8/L2-DIM7, h3D6VHvb8/L2-DIM8, hu3D6VHvlbAl 1/L2-DIM12,
  • thermostability and titer were analyzed for all deimmunized variants. Thermostability and titer levels were compared for antibodies that were comparable to hu3D6VHvlbAl 1/ hu3D6VLv2 based on affinity measurements, and antibodies in Table 16 are listed in order based on variance from T m of hu3D6VHvlbAl 1/ hu3D6VLv2.
  • Thermostability values were determined using differential scanning calorimetry (DSC). All DSC scans were performed using a VP-Capillary DSC system (Malvern). All samples were prepared to 0.5 mg/mL inlxPBS, and referenced to lxPBS. Approximately 0.5 mL of protein solution and buffer were introduced into the sample and reference cell. Calorimetric scan rates were carried out at scan rates of 60°C/hour, from 25°C to 110°C under constant pressure.
  • Titer was determined as follows. After expression in 293 suspension cells, antibodies were purified using Protein A chromatography utilizing standard methods. Following
  • hu3D6VHvlbAl 1/L2-DIM4 demonstrated higher thermostability and titer over parental hu3D6VHvlbAl 1/ hu3D6VLv2. Thermostability and transient expression levels (titer) are both important considerations for drug development.
  • Soluble tau aggregates were generated by incubation of recombinant full length tau with equimolar amounts of low molecular weight heparin for 3d at 37°C. After incubation, insoluble and soluble tau was separated by centrifugation at 1 O,OOOc# for 15 minutes. The supernatant was then resolved by preparative size exclusion chromatography, and aggregate peaks (greater than 100 kDa) were collected and concentrated. To measure internalization, the soluble aggregate fraction was labeled with pHrodo Red succinimidyl ester, which fluoresces when internalized into the endolysosomal pathway.
  • pHrodo-labeled 4R0N human tau P301L soluble oligomer (1.5 pg/ml final concentration) was preincubated with anti-tau antibodies (dose titration: 80 pg/ml starting concentration followed by 4-fold serial dilutions) for 30 min at room temperature in cell culture media.
  • Tau/antibody mixture was then added to B 103 neuroblastoma cell lines at 500,000 cells/ml final concentration and incubated for 3-4 hrs at 37°C in a tissue culture incubator (5% CO2). Cells were then washed 3x with culture media, followed by 10 minutes culture media incubation, and washed 2x with FACS buffer (1% FBS in PBS).
  • substitution mapping via microarray was performed using the peptide NVKSKIGSTENLKHQPG (SEQ ID NO: 184), which is the sequence for residues 255-271 using the numbering of the full length tau isoform.
  • the assay exchanged each of the amino acids in the peptide sequence with all 20 amino acids, to determine with a high degree of accuracy the core epitope for 3D6.
  • the microarray was printed with all peptides in triplicate, along with HA control peptides to ensure assay performance.
  • the array was incubated with 3D6 (lOug/ml) in blocking buffer, washed with PBS-T, followed by incubation with a goat anti-mouse secondary antibody conjugated to DyLight-680.
  • the microarray was incubated with anti-HA antibody 12CA5 directly conjugated to DyLight-800. Microarrays were scanned using a LiCor scanner, and intensities of triplicates were averaged.

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Abstract

The invention provides antibodies that specifically bind tau. The antibodies inhibit or delay tau-associated pathologies and associated symptomatic deterioration.

Description

ANTIBODIES RECOGNIZING TAU
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to US Provisional Application No. 62/813,126, filed March 3, 2019, to US Provisional Application No. 62/813,137, filed March 3, 2019, and to US Provisional Application No. 62/838,159, filed April 24, 2019, each of which is incorporated by reference in its entirety for all purposes.
REFERENCE TO A SEQUENCE LISTING
[0002] This application includes an electronic sequence listing in a file named
536323WO_ST25.TXT, created on February 24, 2020 and containing 168,875 bytes, which is hereby incorporated by reference in its entirety for all purposes.
BACKGROUND OF THE INVENTION
[0003] Tau is a well-known human protein that can exist in phosphorylated forms (see, e.g., Goedert, Proc. Natl. Acad. Sci. U.S.A. 85:4051-4055(1988); Goedert, EMBO J. 8:393- 399(1989); Lee, Neuron 2: 1615-1624(1989); Goedert, Neuron 3:519-526(1989); Andreadis, Biochemistry 31 : 10626-10633(1992). Tau has been reported to have a role in stabilizing microtubules, particularly in the central nervous system. Total tau (t-tau, i.e., phosphorylated and unphosphorylated forms) and phospho-tau (p-tau, i.e., phosphorylated tau) are released by the brain in response to neuronal injury and neurodegeneration and have been reported to occur at increased levels in the CSF of Alzheimer’s patients relative to the general population (Jack et ah, Lancet Neurol 9: 119-28 (2010)).
[0004] Tau is the principal constituent of neurofibrillary tangles, which together with plaques are a hallmark characteristic of Alzheimer’s disease. The tangles constitute abnormal fibrils measuring 10 nm in diameter occurring in pairs wound in a helical fashion with a regular periodicity of 80 nm. The tau within neurofibrillary tangles is abnormally phosphorylated (hyperphosphorylated) with phosphate groups attached to specific sites on the molecule. Severe involvement of neurofibrillary tangles is seen in the layer II neurons of the entorhinal cortex, the CA1 and subicular regions of the hippocampus, the amygdala, and the deeper layers (layers III, V, and superficial VI) of the neocortex in Alzheimer’s disease. Hyperphosphorylated tau has also been reported to interfere with microtubule assembly, which may promote neuronal network breakdown.
[0005] Tau inclusions are part of the defining neurophathology of several neurodegenerative diseases including Alzheimer’s disease, frontotemporal lobar degeneration, progressive supranuclear palsy and Pick’s disease.
BRIEF SUMMARY OF THE CLAIMED INVENTION
[0006] In one aspect, the invention provides an isolated antibody specifically binding to human tau, comprising a mature heavy chain variable region comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9 or SEQ ID NO: 149, and CDR-H3 comprising SEQ ID NO: 10, wherein the heavy chain variable region is at least 90% identical to SEQ ID NO: 18; and a mature light chain variable region comprising CDR-L1 comprising SEQ ID NO: 12, CDR-L2 comprising SEQ ID NO: 150, 151, 153, 156, 158, 159, 160, 163, 165, 166, 167, 168, 169, 170, 171, 172, 173 or 174, and CDR-L3 comprising SEQ ID NO: 14, wherein the light chain variable region is at least 90% identical to SEQ ID NO: 122.
[0007] In some antibodies, at least one of positions H12, H13, H17, H24, H40, H43, H48, H66, H67, H76, H80, H81, and H91 can be occupied by V, K, T, A, R, Q, I, R, A, D, L, Q, and F, respectively, and at least one of positions L2, L12, L15, L37, L39, L45, L60 and L100 can be occupied by V, P, L, Q, R, R, D and Q respectively.
[0008] In some antibodies, CDR-L2 comprises SEQ ID NO: 150, 151, 163, 167, 168, or 169. In some antibodies, the heavy chain variable region comprises SEQ ID NO: 18 and the light chain variable region comprises SEQ ID NO: 110, 121, 122 or 123.
[0009] In some antibodies, the light chain variable region comprises SEQ ID NO: 110. In some antibodies, the light chain variable region comprises SEQ ID NO: 121. In some antibodies, the light chain variable region comprises SEQ ID NO: 122. In some antibodies, the light chain variable region comprises SEQ ID NO: 123. [0010] In some antibodies, the heavy chain variable region comprises SEQ ID NO: 146 and the light chain variable region comprises SEQ ID NO: 94 or 122. In some antibodies, the light chain variable region comprises SEQ ID NO:94. In some antibodies, the light chain variable region comprises SEQ ID NO: 122.
[0011] In some antibodies, the heavy chain variable region comprises SEQ ID NO: 18 or 146 and the light chain variable region comprises SEQ ID NO: 122.
[0012] In another aspect, the invention provides an antibody specifically binding to human tau, comprising a mature heavy chain variable region comprising CDRs HI, H2 and H3 comprising SEQ ID NOs:8, 9, and 10, respectively except that position H28 can be occupied by N or T, H54 can be occupied by N or D, H56 can be occupied by D or E, position H58 occupied by V or I, and position H60 can be occupied by D or E, and a mature light chain variable region comprising CDRs LI, L2 and L3 comprising SEQ ID NOs: 12, 13, and 14 respectively, except that position L24 can be occupied by K or R, position L50 can be occupied by L, E. D, G, or V, position L52 can be occupied by S or G, and position L54 can be occupied by L, D, G, N, E, Q, K, R, T, V, or S, wherein at least one of the following positions is occupied by the amino acid as specified: HI is occupied by Q, H5 is occupied by Q, HI 1 is occupied by L, H20 is occupied by L, H23 is occupied by T, H38 is occupied by K, H75 is occupied by S, H56 is occupied by E, H58 is occupied by I, H60 is occupied by E, H82c is occupied by V, L10 is occupied by T, L17 is occupied by E, L24 is occupied by R, L37 is occupied by Q, L47 is occupied by G, N, D, E, P, T, S, or A, L48 is occupied by G or D, L49 is occupied by E, L50 is occupied by E, D, G, or V, L52 is occupied by G, L54 is occupied by D, G, N. E, Q, K, R, T, V, or S, L83 is occupied by L, L86 is occupied by H, LI 00 is occupied by Q, LI 06 is occupied by L.
[0013] In another aspect, the invention provides an isolated monoclonal antibody that binds human tau comprising three light chain CDRs and three heavy chain CDRs of monoclonal antibody 3D6, wherein 3D6 is a mouse antibody characterized by a heavy chain variable region having an amino acid sequence comprising SEQ ID NO:7 and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 11, except that position H27 can be occupied by F or Y, position H28 can be occupied by N or T, position H29 can be occupied by I or F, position H30 can be occupied by K or T, position H51 can be occupied by I or V, position H54 can be occupied by N or D, position H60 can be occupied by D, A, or E, position H61 can be occupied by P or E, position HI 02 can be occupied by F or Y, position L50 can be occupied by L, E. D, G, or V, position L52 can be occupied by S or G, and position L54 can be occupied by L, D, G, N, E, Q, K, R, T, V, or S, wherein at least one of the following positions is occupied by the amino acid as specified: L37 is occupied by Q, L47 is occupied by G, N, D, E, P, T, S, or A, L48 is occupied by G or D, L49 is occupied by E, L50 is occupied by E, D, G, or V, L52 is occupied by G, L54 is occupied by D, G, N. E, Q, K, R, T, V, or S, L100 is occupied by Q, H60 is occupied by E, H82c is occupied by V.
[0014] In some antibodies, CDR-H1 has an amino acid sequence comprising SEQ ID NO:86. In some antibodies, CDR-H2 has an amino acid sequence comprising SEQ ID NO: 87, SEQ ID NO:88, SEQ ID NO:92, or SEQ ID NO: 149. In some antibodies, CDR-L2 has an amino acid sequence comprising any of SEQ ID NOs: 150 - 175. In some antibodies, CDR-L1 has an amino acid sequence comprising SEQ ID NO:89. In some antibodies, CDR-H1 has an amino acid sequence comprising SEQ ID NO:86 and CDR-H2 has an amino acid sequence comprising SEQ ID NO:87. In some antibodies, CDR-H1 has an amino acid sequence comprising SEQ ID NO:86 and CDR-H2 has an amino acid sequence comprising SEQ ID NO:88. In some antibodies, CDR-H1 has an amino acid sequence comprising SEQ ID NO:86 and CDR-H2 has an amino acid sequence comprising SEQ ID NO:92. In some antibodies, CDR-H1 has an amino acid sequence comprising SEQ ID NO:42, SEQ ID NO:58, SEQ ID NO:59, or SEQ ID NO:60. In some antibodies, CDR-H2 has an amino acid sequence comprising SEQ ID NO:43, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, or SEQ ID NO: 149. In some antibodies, CDR-H3 has an amino acid sequence comprising SEQ ID NO:65. In some antibodies, CDR-L2 has an amino acid sequence comprising any of SEQ ID NOs: 150 - 175.
[0015] Some antibodies are a humanized antibody, veneered antibody, or chimeric antibody.
[0016] Some antibodies comprise a humanized mature heavy chain variable region having an amino acid sequence at least 95% identical to any one of SEQ ID NOs:76-80 and SEQ ID NOs: 90-91, and a humanized mature light chain variable region having an amino acid sequence at least 90% identical to any one of SEQ ID NOs:83-85. [0017] Some antibodies comprise a a humanized mature heavy chain variable region having an amino acid sequence at least 95% identical to any one of SEQ ID NO:18 and SEQ ID NOs: 146- 148, and a humanized mature light chain variable region having an amino acid sequence at least 90% identical to any one of SEQ ID NOs:93-145.
[0018] In some antibodies, at least one of the following positions in the VH region is occupied by the amino acid as specified: H93 is occupied by S and H94 is occupied by T. In some antibodies, positions H93 and H94 are occupied by S and T, respectively. In some antibodies position H91 in the VH region is occupied by F.
[0019] In some antibodies, at least one of the following positions in the VH region is occupied by the amino acid as specified: HI is occupied by E, H5 is occupied by V, HI 1 is occupied by V, H20 is occupied I, H23 is occupied by K, H38 is occupied by R, H42 is occupied by G, H43 is occupied by K, H66 is occupied by R, H75 is occupied by T, H76 is occupied by D, H81 is occupied by E, HI 08 is occupied by L, HI 09 is occupied by V. In some antibodies, positions HI, H5, HI 1, H20, H23, H38, H42, H43, H66, H75, H76, H81, H108, and H109 in the VH region are occupied by E, V, V, I, K, R, G, K, R, T, D, E, L, and V, respectively.
[0020] In some antibodies, at least one of the following positions in the VH region is occupied by the amino acid as specified: HI 7 is occupied by T, H80 is occupied by M, H83 is occupied by R. In some antibodies, positions HI 7, H80, and H83 in the VH region are occupied by T, M, and R, respectively.
[0021] In some antibodies, position H58 in the VH region is occupied by I.
[0022] In some antibodies, at least one of the following positions in the VH region is occupied by the amino acid as specified: H28 is occupied by T, H67 is occupied by V. In some antibodies, positions H28 and H67 in the VH region are occupied by T and V, respectively.
[0023] In some antibodies, at least one of the following positions in the VH region is occupied by the amino acid as specified: H54 is occupied by D, H56 is occupied by E. In some antibodies, positions H54 and H56 in the VH region are occupied by D and E, respectively. [0024] In some antibodies, at least one of the following positions in the VH region is occupied by the amino acid as specified: HI is occupied by Q or E, H5 is occupied by Q or V, HI 1 is occupied by L or V , HI 7 is occupied by S or T, H20 is occupied by L or I, H23 is occupied by T or K, H28 is occupied by N or T, H38 is occupied by K or R, H42 is occupied by E or G, H43 is occupied by Q or K, H54 is occupied by N or D, H56 is occupied by D or E, H58 is occupied by V or I, H60 is occupied by D or E, H66 is occupied by K or R, H67 is occupied by A or V, H75 is occupied by S or T, H76 is occupied by N or D, H80 is occupied by L or M, H81 is occupied by Q or E, H82c is occupied by L or V, H83 is occupied by T or R , H91 is occupied by F or Y, H93 is occupied by S, H94 is occupied by T, HI 08 is occupied by T or L, HI 09 is occupied by L or V.
[0025] In some antibodies, at least one of the following positions in the VH region is occupied by the amino acid as specified: H10 is occupied by E or D, H12 is occupied by K or V, H13 is occupied by K or R, HI 7 is occupied by T, L or S, H24 is occupied by V or A, H27 is occupied by F or Y, H28 is occupied by N or T, H29 is occupied by I or F, H30 is occupied by K or T,
H38 is occupied by Q or R, H40 is occupied by A or R, H42 is occupied by G or E, H43 is occupied by K or Q, H48 is occupied by M or I, H51 is occupied by V or I, H54 is occupied by N or D, H60 is occupied by D, A, or E, H61 is occupied by P or E, H66 is occupied by R or K, H67 is occupied by V or A, H76 is occupied by D or N, H80 is occupied by M or L, H81 is occupied by E or Q, H82a is occupied by S or G, H82c is occupied by L or V, H83 is occupied by T or R, H91 is occupied by Y or F, H93 is occupied by A or S, HI 02 is occupied by F or Y, HI 08 is occupied by T or L, HI 09 is occupied by L or V.
[0026]In some antibodies, positions H91, H93, and H94 in the VH region are occupied by F, S, and T, respectively.
[0027] In some antibodies, positions HI, H5, HI 1, H20, H23, H38, H42, H43, H66, H75, H76, H81, H91, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, I, K, R, G, K, R, T, D, E, F, S, T,L, and V, respectively. In some antibodies, positions HI, H5, HI 1, H17, H20, H23, H38, H42, H43, H58, H66, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T,I, K, R, G, K, I, R, T, D, M, E, R, S, T, L, and V, respectively. In some antibodies, positions HI, H5, HI 1, H17, H20, H23, H28, H38, H42, H43, H58, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, I, R, V, T, D, M, E, R, S, T, L, and V, respectively. In some antibodies, positions HI, H5, HI 1, H17, H20, H23, H28, H38, H42, H43, H54, H56, H58, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, I, R, V, T, D, M, E, R, S, T, L, and V, respectively.
[0028] In some antibodies, positions HI, H5, HI 1, H17, H20, H23, H28, H38, H42, H43, H54, H56, H66, H67, H75, H76, H80, H81, H83, H91, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, R, V, T, D, M, E, R, F, S, T, L, and V, respectively. In some antibodies, positions HI, H5, HI 1, H17, H20, H23, H28, H38, H42, H43, H54, H56, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, R, V, T, D, M, E, R, S , T, L, and V, respectively.
[0029] In some antibodies, position H60 is occupied by E. In some antibodies, position H82C is occupied by V. In some antibodies, positions H60, H80, H81, H82c, and H83 are occupied by E, M, E, V, and R, respectively.
[0030] In some antibodies, at least one of the following positions in the VL region is occupied by the amino acid as specified: L7 is occupied by S, L10 is occupied by S, LI 5 is occupied by L, L83 is occupied by V, L86 is occupied by Y, and LI 06 is occupied by I. In some antibodies, positions L7, L10, L15, L83, L86, and L106 are occupied by S, S, L, V, Y, and Y, respectively.
[0031] In some antibodies, at least one of the following positions in the VL region is occupied by the amino acid as specified: L7 is T or S, L10 is T or S, L15 is I or L, L17 is Q or E, L24 is K or R, L37 is L or Q, L45 is K or R, L47 is L, G, N, D, E, P, T, S, or A, L48 is I, G, or D, L49 is Y or E, L50 is L, E, D, G, or V, L52 is S or G, L54 is L, D, G, N, E, Q, K, R, T, V, or S, L83 is L or V, L86 is H or Y, L100 is A or Q, L106 is L or I.
[0032] In some antibodies, at least one of the following positions in the VL region is occupied by the amino acid as specified: L2 is V or I, L7 is S or T, L12 is P or S, L15 is L or I, L36 is L, L37 is L or Q, L45 is R or K, L47 is L, G, N, D, E, P, T, S, or A, L48 is I, G, or D, L49 is Y or E, L50 is L, E, D, G, or V, L52 is S or G, L54 is L, D, G, N, E, Q, K, R, T, V, L60 is D or S, L100 is G or Q.
[0033] In some antibodies, positions L7, L10, L15, L83, L86, and L106 in the VL region are occupied by S, S, L, V, Y, and I, respectively. In some antibodies, positions L7, L10, L15, L17, L24, L37, L45, L83, L86, L100, and L106 in the VL region are occupied by S, S, L, E, R, Q, R, V, Y, Q, and I, respectively.
[0034] In some antibodies, position L54 is occupied by D. In some antibodies, position L54 is occupied by G. In some antibodies, position L54 is occupied by N. In some antibodies, position L54 is occupied by E. In some antibodies, position L50 is occupied by E. In some antibodies, position L54 is occupied by Q. In some antibodies, position L50 is occupied by D. In some antibodies, position L54 is occupied by K. In some antibodies, position L54 is occupied by R In some antibodies, position L54 is occupied by T. In some antibodies, position L50 is occupied by G. In some antibodies, position L48 is occupied by G. In some antibodies, position L48 is occupied by D. In some antibodies, position L47 is occupied by G. In some antibodies, position L49 is occupied by E. In some antibodies, position L54 is occupied by V. In some antibodies, position L54 is occupied by S.
[0035] In some antibodies, position L52 is occupied by G. In some antibodies, position L47 is occupied by N. In some antibodies, position L47 is occupied by D. In some antibodies, position L47 is occupied by E. In some antibodies, position L47 is occupied by P. In some antibodies, position L47 is occupied by T. In some antibodies, position L47 is occupied by S. In some antibodies, position L47 is occupied by A. In some antibodies, position L50 is occupied by V.
[0036] In some antibodies, positions L37, L50, and L54 are occupied by Q, G, and R,
respectively. In some antibodies, positions L37, L50, and L54 are occupied by Q, G, and G, respectively. In some antibodies, positions L37, L52, and L54 are occupied by Q, G, and G, respectively. In some antibodies, positions L37, L52, and L54 are occupied by Q, G, and R, respectively. In some antibodies, positions L37, L52, and L54 are occupied by Q, G, and T, respectively. In some antibodies, positions L37, L52, and L54 are occupied by Q, G, and D, respectively. In some antibodies, L37 and L54 are occupied Q and R, respectively. [0037] In some antibodies, positions L37 and L54 are occupied by Q and G, respectively. In some antibodies, positions L37 and L54 are occupied by Q and D, respectively. In some antibodies, positions L37 and L50 are occupied by Q and G, respectively. In some antibodies, positions L37 and L50 are occupied by Q and D, respectively. In some antibodies, positions L37 and L54 are occupied by Q and T, respectively. In some antibodies, positions L37 and L52 are occupied by Q and G, respectively. In some antibodies, positions L37 and L54 are occupied by Q and E, respectively. In some antibodies, In some antibodies, positions L37, L50, and L54 are occupied by Q, D, and G, respectively. In some antibodies, positions L37, L50, and L54 are occupied by Q, D, and R, respectively.
[0038] In some antibodies, positions L37, L50, and L54 are occupied by Q, E, and G, respectively. In some antibodies, positions L37, L50, and L54 are occupied by Q, E, and R, respectively. In some antibodies, positions L37, L50, L54, and L100 are occupied by Q, G, R, and Q, respectively. In some antibodies, positions L37, L50, L54, and L100 are occupied by Q, G, G, and Q, respectively. In some antibodies, positions L37, L52, L54, and LI 00 are occupied by Q, G, R, and Q, respectively. In some antibodies, positions L37, L52, L54, and L100 are occupied by Q, G, D, and Q, respectively. In some antibodies, positions L37, L50, L54, and L100 are occupied by Q, D, G, and Q, respectively. In some antibodies, positions L37, L50,
L54, and L100 are occupied by Q, D, R, and Q, respectively.
[0039] In some antibodies, positions L37, L50, L54, and L100 are occupied by Q, V, D, and Q, respectively. In some antibodies, position L37 is occupied by Q. In some antibodies, position L100 is occupied by Q.
[0040] In some antibodies, the mature heavy chain variable region has an amino acid sequence of any one of SEQ ID NOs:76-80 and SEQ ID NOs:90-91 and the mature light chain variable region has an amino acid sequence of any one of SEQ ID NO:83-85. In some antibodies, the mature heavy chain variable region has an amino acid sequence of any one of SEQ ID NO: 18 and SEQ ID NOs: 146-148 and the mature light chain variable region has an amino acid sequence of any one of SEQ ID NOs:93-145.
[0041] In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:76 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:76 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:84. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:76 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:85.
[0042] In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:77 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:77 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:84. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:77 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:85.
[0043] In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:78 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:78 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:84. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:78 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:85.
[0044] In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:79 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:79 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:84. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:79 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:85.
[0045] In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 80 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 83. In some antibodies, he mature heavy chain variable region has an amino acid sequence of SEQ ID NO:80 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:84. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 80 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:85.
[0046] In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 90 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:90 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:84. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 90 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:85.
[0047] In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:91 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:91 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:84. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO:91 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:85.
[0048] In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 18 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 122. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 18 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 123.
[0049] In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 146 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 122.
[0050] In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 18 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 121. In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 18 and the mature light chain variable region has an amino acid sequence of SEQ ID NO : 110.
[0051] In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 146 and the mature light chain variable region has an amino acid sequence of SEQ ID NO:94.
[0052] In some antibodies, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 18 and the mature light chain variable region has an amino acid sequence of SEQ ID NO: 103.
[0053] For example, the antibody can be a chimeric, veneered, or humanized antibody.
[0054] The antibody can be an intact chimeric, veneered or humanized antibody or a binding fragment, single-chain antibody Fab fragment, Fab’2 fragment, or single chain Fv. Some of the antibodies have a human IgGl isotype, while others may have a human IgG2 or IgG4 isotype. Some antibodies have the mature light chain variable region fused to a light chain constant region and the mature heavy chain variable region fused to a heavy chain constant region. The heavy chain constant region of some antibodies is a mutant form of a natural human heavy chain constant region which has reduced binding to a Fey receptor relative to the natural human heavy chain constant region.
[0055] In some antibodies, the heavy chain constant region has an amino acid sequence of SEQ ID NO: 176. In some antibodies, the mature heavy chain variable region fused to a heavy chain constant region has an amino acid sequence of SEQ ID NO: 178. Some antibodies further comprise a signal peptide fused to the mature heavy and/or light chain variable region. In some antibodies, the heavy chain has an amino acid sequence of SEQ ID NO: 180. In some antibodies, the light chain constant region has an amino acid sequence of SEQ ID NO: 177. In some antibodies, the mature light chain variable region is fused to a light chain constant region has an amino acid sequence of SEQ ID NO: 179. In some antibodies, the light chain has an amino acid sequence of SEQ ID NO: 181. In some antibodies, the heavy chain has an amino acid sequence of SEQ ID NO: 178 and the light chain has an amino acid sequence of SEQ ID NO: 179. In some antibodies, the heavy chain has an amino acid sequence of SEQ ID NO: 180 and the light chain has an amino acid sequence of SEQ ID NO: 181.
[0056] Some antibodies may have at least one mutation in the constant region, such as a mutation that reduces complement fixation or activation by the constant region, for example, a mutation at one or more of positions 241, 264, 265, 270, 296, 297, 318, 320, 322, 329 and 331 by EU numbering. Some antibodies have an alanine at positions 318, 320 and 322. Some antibodies can be at least 95% w/w pure. The antibody can be conjugated to a therapeutic, cytotoxic, cytostatic, neurotrophic, or neuroprotective agent.
[0057] In another aspect, the invention provides a pharmaceutical composition comprising any of the antibodies disclosed herein and a pharmaceutically-acceptable carrier.
[0058] In another aspect, the invention provides a nucleic acid encoding the heavy chain and/or light chain of any of the antibodies disclosed herein, a recombinant expression vector comprising the nucleic acid and a host cell transformed with the recombinant expression vector.
[0059] In some nucleic acids, the heavy chain is encoded by a sequence comprising SEQ ID NO: 182 and the light chain is encoded by a sequence comprising SEQ ID NO: 183.
[0060] Methods of producing antibodies, such as a humanized, chimeric or veneered antibody, for example humanized, chimeric or veneered forms of 3D6, are also provided. In such methods, cells transformed with nucleic acids encoding the heavy and light chains of the antibody are cultured so that the cells secrete the antibody. The antibody can then be purified from the cell culture media.
[0061] Cell lines producing any of the antibodies disclosed herein can be produced by introducing a vector encoding heavy and light chains of the antibody and a selectable marker into cells, propagating the cells under conditions to select for cells having increased copy number of the vector, isolating single cells from the selected cells; and banking cells cloned from a single cell selected based on yield of antibody.
[0062] In another aspect, the invention comprises a vector comprising a nucleic acid encoding a mature heavy chain variable region and a mature light chain variable region operably linked to one or more regulatory sequences to effect expression in a mammalian cell of any of the antibodies disclosed herein. In some vectors, the expressed antibody is a scFv or an Fab fragment. In some vectors, the one or more regulatory sequences include one or more of a promoter, enhancer, ribosome binding site, and transcription termination signal. In some vectors, the nucleic acid further encodes signal peptides fused to the mature heavy and light chain variable regions. In some vectors, the nucleic acid is codon-optimized for expression in a host cell. In some vectors, the one or more regulatory sequences include a eukaryotic promoter. In some vectors, the nucleic acid further encodes a selectable gene.
[0063] [0064] In yet another aspect, the invention provides methods of expressing an antibody in a mammalian cell comprising incorporating the nucleic acids disclosed herein into the genome of a transgenic animal, whereby the antibody is expressed.
[0065] In yet another aspect, the invention provides first and second vectors respectively comprising nucleic acids encoding a mature heavy chain variable region and a mature light chain variable region, each operably linked to one or more regulatory sequences to effect expression of any of the antibodies disclosed herein, and a host cell comprising the nucleic acids.. In some first and second vectors, the nucleic acids respectively further encodes a heavy chain constant region fused to the mature heavy chain variable region and a light chain constant region fused to the mature light chain variable region. In some first and second vectors, the heavy chain constant region has the sequence of SEQ ID NO: 176 with or without the C-terminal lysine and the light chain constant region has the sequence of SEQ ID NO: 177.
[0066] In yet another aspect, the invention provides methods of expressing an antibody in a mammalian cell comprising incorporating any of the nucleic acids disclosed herein into the genome of a transgenic animal, whereby the antibody is expressed.
[0067] Methods of producing antibodies, such as a humanized, chimeric, or veneered antibody, are also provided. In such methods, cells cells transformed with nucleic acids encoding the heavy and light chains of the antibody of any of the antibodies disclosed herein are cultured so that the cells secrete the antibody. The antibody can then be purified from the cell culture media. [0068] Cell lines producing any of the antibodies disclosed herein can be produced by introducing a vector encoding heavy and light chains of the antibody of claim 1 and a selectable marker into cells, propagating the cells under conditions to select for cells having increased copy number of the vector, isolating single cells from the selected cells, and banking cells cloned from a single cell selected based on yield of antibody.
[0069] Some cells can be propagated under selective conditions and screened for cell lines naturally expressing and secreting at least 100 mg/L/106 cells/24 hours. Single cells can be isolated from the selected cells. Cells cloned from a single cell can then be banked. Single cells can be selected based on desirable properties, such as the yield of the antibody. Exemplary cell lines are cell lines expressing 3D6 or humanized versions of 3D6.
[0070] The invention also provides methods of inhibiting or reducing aggregation of tau in a subject having or at risk of developing a tau-mediated amyloidosis, comprising administering to the subject an effective regime of an antibody disclosed herein, thereby inhibiting or reducing aggregation of tau in the subject. Exemplary antibodies include humanized versions of 3D6.
[0071] Also provided are methods of treating or effecting prophylaxis of a tau-related disease in a subject, comprising administering an effective regime of an antibody disclosed herein and thereby treating or effecting prophylaxis of the disease. Examples of such a disease are
Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia,
frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal
degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBV D), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). In some methods, the tau-related disease is Alzheimer’s disease. In some methods the patient is an ApoE4 carrier.
[0072] Also provided are methods of reducing aberrant transmission of tau comprising administering an effective regime of an antibody disclosed herein and thereby reducing transmission of tau. [0073] Also provided are methods of inducing phagocytosis of tau comprising administering an effective regime of an antibody disclosed herein and thereby inducing phagocytosis of tau.
[0074] Also provided are methods of inhibiting tau aggregation or deposition comprising administering an effective regime of an antibody disclosed herein thereby inhibiting tau aggregation or deposition.
[0075] Also provided are methods of inhibiting formation of tau tangles comprising administering an effective regime of an antibody disclosed herein.
[0076] The invention also provides a method of detecting tau protein deposits in a subject having or at risk of a disease associated with tau aggregation or deposition comprising administering to a subject an antibody disclosed herein, and detecting the antibody bound to tau in the subject. Examples of such a disease are Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBV D), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). In some embodiments, the antibody is administered by intravenous injection into the body of the subject. In some embodiments, the antibody is administered directly to the brain of the subject by intracranial injection or by drilling a hole through the skull of the subject. In some embodiments, the antibody is labeled. In some embodiments, the antibody is labeled with a fluorescent label, a paramagnetic label, or a radioactive label. In some embodiments, the radioactive label is detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).
[0077] The invention also provides a method of measuring efficacy of treatment in a subject being treated for a disease associated with tau aggregation or deposition, comprising measuring a first level of tau protein deposits in the subject prior to treatment by administering to a subject an antibody disclosed herein, and detecting a first amount of the antibody bound to tau in the subject, administering the treatment to the subject, measuring a second level of tau protein deposits in the in subject after treatment by administering to a subject the antibody, and detecting the antibody bound to tau in the subject, wherein a decrease in the level of tau protein deposits indicates a positive response to treatment.
[0078] The invention also provides a method of measuring efficacy of treatment in a subject being treated for a disease associated with tau aggregation or deposition, comprising measuring a first level of tau protein deposits in the subject prior to treatment by administering to a subject an antibody disclosed herein, and detecting a first amount of antibody bound to tau in the subject, administering the treatment to the subject, measuring a second level of tau protein deposits in the in subject after treatment by administering to a subject the antibody, and detecting a second amount of antibody bound to tau in the subject, wherein no change in the level of tau protein deposits or a small increase in tau protein deposits indicates a positive response to treatment.
[0079] In another aspect, the invention provides methods of producing an antibody that specifically binds to human tau at an epitope within a motif of the formula KXXSXXNX(K/H)H (SEQ ID NO: 191) or KIGSLDNITH (SEQ ID NO: 194) comprising immunizing an animal with human tau or a fragment thereof to produce antibodies and screening for an antibody of the produced antibodies that specifically binds within the motif. In another aspect, the invention provides methods of producing an antibody that specifically binds to a peptide consisting of residues KXXSXXNX(K/H)H (SEQ ID NO: 191) or KIGSLDNITH (SEQ ID NO: 194) comprising immunizing an animal with human tau or a fragment thereof to produce antibodies and screening for an antibody of the produced antibodies that specifically binds to the peptide.
In another aspect, the invention provides methods of producing an antibody that specifically binds to an epitope comprising KXXSXXNX(K/H)H (SEQ ID NO: 191) comprising immunizing an animal with tau or a fragment thereof, and screening for antibodies that specifically bind to the epitope.
[0080] In some methods, the animal is immunized with 383 amino acid human tau (4R0N). In some methods, the human tau contains a P301S mutation. In some methods, the human tau is recombinant N-terminally His-tagged.
[0081] In some methods, the screening determines specific binding between the antibodies and one or more peptides of up to 15 amino acids comprising KIGSTENLKH (SEQ ID NO: 188), KCGSKDNIKH (SEQ ID NO: 192), KCGSLGNIHH (SEQ ID NO: 193) respectively or any other consensus motif represented by KXXSXXNX(K/H)H (SEQ ID NO: 191). In some methods, the one or more peptides comprise KIGSTENLKH (SEQ ID NO: 188) or KCGSKDNIKH (SEQ ID NO: 192) or KCGSLGNIHH (SEQ ID NO: 193) respectively. In some methods, the animal is immunized with a tau fragment of up to 15 amino acids comprising KXXSXXNX(K/H)H (SEQ ID NO: 191), linked to a carrier. In some methods, the peptide is KIGSTENLKH (SEQ ID NO: 188) or KCGSKDNIKH (SEQ ID NO: 192) or KCGSLGNIHH (SEQ ID NO: 193).
BRIEF DESCRIPTION OF THE DRAWINGS
[0082] Figure 1 depicts the results of experiments designed to map the epitope(s) bound by the murine 3D6 monoclonal antibody.
[0083] Figure 2 depicts an alignment of heavy chain variable regions of the mouse 3D6 antibody (SEQ ID NO:7) and humanized versions of the 3D6 antibody (hu3D6VHvbl, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7) with human germline heavy chain variable region sequence IGHVl-69-2*01 (SEQ ID NO:25) and with human acceptor heavy chain variable region sequence 2RCS VH hFrwk (SEQ ID NO:75). hu3D6VHvbl is SEQ ID NO:76, hu3D6VHvb2 is SEQ ID NO:77, hu3D6VHvb3 is SEQ ID NO:78, hu3D6VHvb4 is SEQ ID NO:79, hu3D6VHvb5 is SEQ ID NO:80,
hu3D6VHvb6 is SEQ ID NO:90, and hu3D6VHvb7 is SEQ ID NO:91. The CDRs as defined by Kabat/Chothia Composite are in boldface.
[0084] Figure 3 depicts an alignment of light chain variable regions of the mouse 3D6 antibody (SEQ ID NO: 11) and humanized versions of the 3D6 antibody (hu3D6VLvbl, hu3D6VLvb2, and hu3D6VLvb3) with human germline light chain variable region sequence IGKV2-30*02 (SEQ ID NO:27) and with human acceptor ARX71335_VL_hFrwk (SEQ ID NO:82). hu3D6VLvbl is SEQ ID NO:83, hu3D6VLvb2 is SEQ ID NO:84, and hu3D6VLvb3 is SEQ ID NO:85. The CDRs as defined by Kabat are in boldface.
[0085] Figures 4A, 4B, and 4C depict results of ELISA screening assays for selected mouse monoclonal anti-tau antibodies. [0086] Figure 5 depicts binding kinetics for selected mouse monoclonal anti-tau antibodies to recombinant human tau.
[0087] Figure 6 depicts results of functional blocking assays for selected mouse monoclonal anti-tau antibodies.
[0088] Figure 7 depicts results of disaggregation assays for selected mouse monoclonal anti- tau antibodies.
[0089] Figure 8 depicts results of experiments showing that 3D6 and 5G8 immunocapture tau from human Alzheimer's disease tissue.
[0090] Figures 9A and 9B show alignments of the heavy chain variable region of murine 3D6 (SEQ ID NO:7) and of humanized versions of the 3D6 antibody (hu3D6VHvbl, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, hu3D6VHvb7, hu3D6VHvlbAl 1, h3D6VHvb8, and h3D6VHvb9) with human germline heavy chain variable region sequence IGHVl-69-2*01 (SEQ ID NO:25) and with human acceptor heavy chain variable region sequence 2RCS VH hFrwk (SEQ ID NO:75). hu3D6VHvbl is SEQ ID NO:76, hu3D6VHvb2 is SEQ ID NO:77, hu3D6VHvb3 is SEQ ID NO:78, hu3D6VHvb4 is SEQ ID NO:79,
hu3D6VHvb5 is SEQ ID NO:80, hu3D6VHvb6 is SEQ ID NO:90, hu3D6VHvb7 is SEQ ID NO:91, hu3D6VHvlbAl l is SEQ ID NO: 18, h3D6VHvb8 is SEQ ID NO: 146, and h3D6VHvb9 is SEQ ID NO: 148. Residues identical to those of the heavy chain variable region of murine 3D6 (SEQ ID NO:7) are noted with a“” The CDRs as defined by Kabat/Chothia Composite are in boldface.
[0091] Figures 10A, 10B, IOC, and 10D show alignments of the light chain variable region of humanized versions of the 3D6 antibody: hu3D6VLv2 (SEQ ID NO:21), hu3D6VLv2 L37Q (SEQ ID NO: 143), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 S52G (SEQ ID
NO: 110), hu3D6VLv2 L54G (SEQ ID NO:94), hu3D6VLv2 L54D (SEQ ID NO:93), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L37Q L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q L50G L54R (SEQ ID NO: 119), hu3D6VLv2
L37Q L50G L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q L50D L54R (SEQ ID NO: 133), hu3D6VLv2 L37Q L50D L54G (SEQ ID NO: 132), hu3D6VLv2 L37Q S52G L54D (SEQ ID NO: 124), hu3D6VLv2 L37Q S52G L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q S52G L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q S52G L54R (SEQ ID NO: 122), hu3D6VLv2
L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2 L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2
L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), and hu3D6VLv2 L37Q_S52G_L54R_G100Q (SEQ ID NO: 138). Residues identical to those of the light chain variable region of hu3D6VLv2 (SEQ ID NO:21) are noted with a The CDRs as defined by Rabat are in boldface.
[0092] Figure 11 shows results of tau internalization assay for selected humanized 3D6 variants.
[0093] Figures 12A and 12B show results of subsitution mapping microarray experiments. Figure 12A shows a plot of subsitution effect, and Figure 12B shows an alignment of portions of the tau microtubule binding repeats with important residues for binding 3D6 highlighted aa 255-271 are SEQ ID NO: 184, aa 286-302 are SEQ ID NO: 185, aa 317-333 are SEQ ID NO: 186, and aa 349-365 are SEQ ID NO: 187.
[0094] Figure 13 depicts results of immunohistochemistry experiments showing 3D6 binds tau in normal (top panel) and Alzheimer’s disease tissue (middle and bottom panels).
[0095] Figure 14 shows results of mass spectrometry experiments to assess binding
stoichiometry of 3D6.
[0096] Figure 15 shows that 3D6 interrupts tau seeding in an in vivo diseae model of
Alzheimer’s disease.
[0097] Figure 16 shows that 3D6 humanized variants hu3D6VHvlbAl l/hu3D6VLv2 and hu3D6VHvlbAl 1/L2-DIM4 block interaction between tau and heparin. [0098] Figure 17 shows that 3D6 humanized variants hu3D6VHvlbAl l/hu3D6VLv2 and hu3D6VHvlbAl 1/L2-DIM4 bind fibrillar forms of tau.
BRIEF DESCRIPTION OF THE SEQUENCES
[0099] SEQ ID NO: 1 sets forth the amino acid sequence of an isoform of human tau (Swiss- Prot P10636-8).
[0100] SEQ ID NO:2 sets forth the amino acid sequence of an isoform of human tau (Swiss- Prot PI 0636-7).
[0101] SEQ ID NO:3 sets forth the amino acid sequence of an isoform of human tau (Swiss- Prot PI 0636-6), (4R0N human tau).
[0102] SEQ ID NO:4 sets forth the amino acid sequence of an isoform of human tau (Swiss- Prot PI 0636-5)
[0103] SEQ ID NO:5 sets forth the amino acid sequence of an isoform of human tau (Swiss- Prot PI 0636-4).
[0104] SEQ ID NO:6 sets forth the amino acid sequence of an isoform of human tau (Swiss- Prot PI 0636-2).
[0105] SEQ ID NO:7 sets forth the amino acid sequence of the heavy chain variable region of the mouse 3D6 antibody.
[0106] SEQ ID NO:8 sets forth the amino acid sequence of Kabat/Chothia composite CDR-H1 of the mouse 3D6 antibody.
[0107] SEQ ID NO:9 sets forth the amino acid sequence of Rabat CDR-H2 of the mouse 3D6 antibody.
[0108] SEQ ID NO: 10 sets forth the amino acid sequence of Rabat CDR-H3 of the mouse 3D6 antibody. [0109] SEQ ID NO: 11 sets forth the amino acid sequence of the light chain variable region of the mouse 3D6 antibody and of the mouse 6A10 antibody.
[0110] SEQ ID NO: 12 sets forth the amino acid sequence of Kabat CDR-L1 of the mouse 3D6 antibody and of the mouse 6A10 antibody.
[0111] SEQ ID NO: 13 sets forth the amino acid sequence of Kabat CDR-L2 of the mouse 3D6 antibody and of the mouse 6A10 antibody.
[0112] SEQ ID NO: 14 sets forth the amino acid sequence of Kabat CDR-L3 of the mouse 3D6 antibody and of the mouse 6A10 antibody.
[0113] SEQ ID NO: 15 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvl.
[0114] SEQ ID NO: 16 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHv2.
[0115] SEQ ID NO: 17 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvlb.
[0116] SEQ ID NO: 18 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvlbAl 1.
[0117] SEQ ID NO: 19 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHv5:
[0118] SEQ ID NO:20 sets forth the amino acid sequence of the light chain variable region of the humanized 3D6 antibody hu3D6VLvl.
[0119] SEQ ID NO:21 sets forth the amino acid sequence of the light chain variable region of the humanized 3D6 antibody hu3D6VLv2.
[0120] SEQ ID NO:22 sets forth the amino acid sequence of the light chain variable region of the humanized 3D6 antibody hu3D6VLv3. [0121] SEQ ID NO:23 sets forth the amino acid sequence of the light chain variable region of the humanized 3D6 antibody hu3D6VLv4.
[0122] SEQ ID NO:24 sets forth the amino acid sequence of the heavy chain variable acceptor Acc.# BAC01986.1.
[0123] SEQ ID NO:25 sets forth the amino acid sequence of the heavy chain variable acceptor Acc # IMGT# IGHV1 -69-2*01.
[0124] SEQ ID NO:26 sets forth the amino acid sequence of the heavy chain variable acceptor Acc # IMGT#IGKJ 1*01.
[0125] SEQ ID NO:27 sets forth the amino acid sequence of the light chain variable acceptor Acc. # IMGT#IGKV2-30*02
[0126] SEQ ID NO:28 sets forth the amino acid sequence of the light chain variable acceptor Acc. # IMGT#IGKJ2*01.
[0127] SEQ ID NO:29 sets forth the amino acid sequence of the light chain variable acceptor Acc. # AAZ09048.1.
[0128] SEQ ID NO:30 sets forth a nucleic acid sequence encoding the heavy chain variable region of the mouse 3D6 antibody.
[0129] SEQ ID NO:31 sets forth a nucleic acid sequence encoding the light chain variable region of the mouse 3D6 antibody.
[0130] SEQ ID NO:32 sets forth the amino acid sequence of Kabat CDR-H1 of the mouse 3D6 antibody.
[0131] SEQ ID NO:33 sets forth the amino acid sequence of Chothia CDR-H1 of the mouse 3D6 antibody.
[0132] SEQ ID NO:34 sets forth the amino acid sequence of Chothia CDR-H2 of the mouse 3D6 antibody. [0133] SEQ ID NO:35 sets forth the amino acid sequence of AbM CDR-H2 of the mouse 3D6 antibody.
[0134] SEQ ID NO:36 sets forth the amino acid sequence of Contact CDR-L1 of the mouse 3D6 antibody.
[0135] SEQ ID NO:37 sets forth the amino acid sequence of Contact CDR-L2 of the mouse 3D6 antibody.
[0136] SEQ ID NO:38 sets forth the amino acid sequence of Contact CDR-L3 of the mouse 3D6 antibody.
[0137] SEQ ID NO:39 sets forth the amino acid sequence of Contact CDR-H1 of the mouse 3D6 antibody.
[0138] SEQ ID NO:40 sets forth the amino acid sequence of Contact CDR-H2 of the mouse 3D6 antibody.
[0139] SEQ ID NO:41 sets forth the amino acid sequence of Contact CDR-H3 of the mouse 3D6 antibody.
[0140] SEQ ID NO:42 sets forth the amino acid sequence of an alternate Kabat-Chothia Composite CDR-H1 of a humanized 3D6 antibody (as in hu3D6VHv5, hu3D6VHvlbAl 1B6G2, hu3D6VHvlbAl 1B6H3, hu3D6VHvle, and hu3D6VHvlf).
[0141] SEQ ID NO:43 sets forth the amino acid sequence of an alternate Rabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHv5 and hu3D6VHvlbAl 1B6H3).
[0142] SEQ ID NO:44 sets forth the consensus amino acid sequence among the heavy chain variable regions of the mouse 3D6 and selected humanized 3D6 antibodies (VHvl, VHv2, VHvlb, VHvlbAl l, and VHv5) (labeled“Majority’ in Figure 2 of PCT/IB2017/052544.
[0143] SEQ ID NO:45 sets forth the consensus amino acid sequence between the light chain variable regions of the mouse 3D6 and selected humanized 3D6 antibodies (labeled“Majority’ in Figure 3 of PCT/IB2017/052544). [0144] SEQ ID NO:46 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvlbAl 1B6G2.
[0145] SEQ ID NO:47 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvlbAl 1B6H3.
[0146] SEQ ID NO:48 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvlc.
[0147] SEQ ID NO:49 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvld.
[0148] SEQ ID NO:50 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvle.
[0149] SEQ ID NO:51 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvlf.
[0150] SEQ ID NO:52 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHv3.
[0151] SEQ ID NO:53 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHv3b.
[0152] SEQ ID NO:54 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHv3c.
[0153] SEQ ID NO:55 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHv4.
[0154] SEQ ID NO:56 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHv4b.
[0155] SEQ ID NO:57 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHv4c. [0156] SEQ ID NO:58 sets forth the amino acid sequence of an alternate Kabat-Chothia Composite CDR-H1 of a humanized 3D6 antibody (as in hu3D6VHlc).
[0157] SEQ ID NO:59 sets forth the amino acid sequence of an alternate Kabat-Chothia Composite CDR-H1 of a humanized 3D6 antibody (as in hu3D6VHvld, hu3D6VHv3c, and hu3D6VHv4c).
[0158] SEQ ID NO:60 sets forth the amino acid sequence of an alternate Kabat-Chothia Composite CDR-H1 of a humanized 3D6 antibody (as in hu3D6VHv3b and hu3D6VHv4b).
[0159] SEQ ID NO:61 sets forth the amino acid sequence of an alternate Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvlbAl 1B6G2).
[0160] SEQ ID NO:62 sets forth the amino acid sequence of an alternate Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvlc, hu3D6VHv3b, AND hu3D6VHv4b.
[0161] SEQ ID NO:63 sets forth the amino acid sequence of an alternate Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvld, hu3D6VHvlf, hu3D6VHv3c, and hu3D6VHv4c).
[0162] SEQ ID NO:64 sets forth the amino acid sequence of an alternate Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvle).
[0163] SEQ ID NO:65 sets forth the amino acid sequence of an alternate Kabat CDR-H3 of a humanized 3D6 antibody (as in hu3D6VHvlf).
[0164] SEQ ID NO:66 sets forth the amino acid sequence of the heavy chain variable region of the mouse 6A10 antibody.
[0165] SEQ ID NO:67 sets forth the amino acid sequence of Kabat/Chothia composite CDR- H1 of the mouse 6A10 antibody.
[0166] SEQ ID NO:68 sets forth the amino acid sequence of Kabat CDR-H2 of the mouse 6A10 antibody.
[0167] SEQ ID NO:69 sets forth the amino acid sequence of Kabat CDR-H3 of the mouse 6A10 antibody. [0168] SEQ ID NO:70 sets for the amino acid sequence of the VH region of mouse antibody (pdb code 1CR9) used as a structure template for heavy chain humanization.
[0169] SEQ ID NO:71 sets forth the consensus amino acid sequence among the heavy chain variable regions of the selected humanized 3D6 antibodies (VHvl, VHvlb, VHvlbAl 1, VHvlbAl 1B6G2, VHvlbAl 1B6H3, VHvlc, VHvld, VHvle, VHvlf, VHv2, VHv3, VHv3b, VHv3c, VHv4, VHv4b, VHv4c, and VHv5) (labeled“Majority’ in Figures 4A and 4B of PCT/IB2017/052544).
[0170] SEQ ID NO:72 sets forth the amino acid sequence of the heavy chain of a chimeric 3D6 antibody.
[0171] SEQ ID NO:73 sets forth the amino acid sequence of the light chain of a chimeric 3D6 antibody.
[0172] SEQ ID NO:74 sets forth the amino acid sequence of heavy chain variable structural model Acc.# 5MYX-VH_mSt.
[0173] SEQ ID NO:75 sets forth the amino acid sequence of heavy chain variable acceptor Acc.# 2RCS-VH_huFrwk.
[0174] SEQ ID NO:76 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvbl.
[0175] SEQ ID NO:77 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb2.
[0176] SEQ ID NO:78 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb3.
[0177] SEQ ID NO:79 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb4.
[0178] SEQ ID NO:80 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb5. [0179] SEQ ID NO:81 sets forth the amino acid sequence of light chain variable structural model Acc.# 5MYX-VL_mSt.
[0180] SEQ ID NO:82 sets forth the amino acid sequence of light chain variable acceptor Acc.# ARX71335 - VL_huF rwk.
[0181] SEQ ID NO:83 sets forth the amino acid sequence of light chain variable region of the humanized 3D6 antibody hu3D6VLvbl.
[0182] SEQ ID NO:84 sets forth the amino acid sequence of light chain variable region of the humanized 3D6 antibody hu3D6VLvb2.
[0183] SEQ ID NO:85 sets forth the amino acid sequence of light chain variable region of the humanized 3D6 antibody hu3D6VLvb3.
[0184] SEQ ID NO:86 sets forth the amino acid sequence of an alternate Kabat-Chothia Composite CDR-H1 of a humanized 3D6 antibody (as in hu3D6VHvb4 and hu3D6VHvb5).
[0185] SEQ ID NO:87 sets forth the amino acid sequence of an alternate Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvb3 and hu3D6VHvb4).
[0186] SEQ ID NO:88 sets forth the amino acid sequence of an alternate Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvb5).
[0187] SEQ ID NO:89 sets forth the amino acid sequence of an alternate Kabat CDR-L1 of a humanized 3D6 antibody (as in hu3D6VLvb3).
[0188] SEQ ID NO:90 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb6.
[0189] SEQ ID NO:91 sets forth the amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb7.
[0190] SEQ ID NO:92 sets forth the amino acid sequence of an alternate Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvb6 and hu3D6VHvb7). [0191] SEQ ID NO:93 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L54D.
[0192] SEQ ID NO:94 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L54G.
[0193] SEQ ID NO:95 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L45N.
[0194] SEQ ID NO:96 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L54E.
[0195] SEQ ID NO:97 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L50E.
[0196] SEQ ID NO:98 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L54Q.
[0197] SEQ ID NO:99 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L50D.
[0198] SEQ ID NO: 100 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L54K.
[0199] SEQ ID NO: 101 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L54R.
[0200] SEQ ID NO: 102 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L54T.
[0201] SEQ ID NO: 103 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L50G.
[0202] SEQ ID NO: 104 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant I48G. [0203] SEQ ID NO: 105 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant I48D.
[0204] SEQ ID NO: 106 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L47G.
[0205] SEQ ID NO: 107 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant Y49E.
[0206] SEQ ID NO: 108 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L54V.
[0207] SEQ ID NO: 109 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L54S.
[0208] SEQ ID NO: 110 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant S52G.
[0209] SEQ ID NO: 111 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L47N.
[0210] SEQ ID NO: 112 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L47D.
[0211] SEQ ID NO: 113 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L47E.
[0212] SEQ ID NO: 114 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L47P.
[0213] SEQ ID NO: 115 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L47T.
[0214] SEQ ID NO: 116 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L47S. [0215] SEQ ID NO: 117 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L47A.
[0216] SEQ ID NO: 118 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L50V.
[0217] SEQ ID NO: 119 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q L50G L54R.
[0218] SEQ ID NO: 120 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q L50G L54G.
[0219] SEQ ID NO: 121 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q_S52G_L54G.
[0220] SEQ ID NO: 122 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q S52G L54R.
[0221] SEQ ID NO: 123 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q S52G L54T.
[0222] SEQ ID NO: 124 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q_S52G_L54D.
[0223] SEQ ID NO: 125 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q L54R.
[0224] SEQ ID NO: 126 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37QJL54G.
[0225] SEQ ID NO: 127 sets forth the amino acid sequence of light chain vari able region of a hu3D6VLv2 variant L37QJL54D.
[0226] SEQ ID NO: 128 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q L50G. [0227] SEQ ID NO: 129 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q L50D.
[0228] SEQ ID NO: 130 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q_L54T.
[0229] SEQ ID NO: 131 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q_S52G.
[0230] SEQ ID NO: 132 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q L50D L54G.
[0231] SEQ ID NO: 133 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q_L50D_L54R.
[0232] SEQ ID NO: 134 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q L50E L54G.
[0233] SEQ ID NO: 135 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q L50E L54R.
[0234] SEQ ID NO: 136 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q_L50G_L54R_G100Q.
[0235] SEQ ID NO: 137 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q L50G L54G G100Q.
[0236] SEQ ID NO: 138 sets forth the amino acid sequence of light chain variable region of a hu3D6VLv2 variant L37Q_S52G_L54R_G100Q.
[0237] SEQ ID NO: 139 sets forth the amino acid sequence of light chain vari able region of a hu3D6VLv2 variant L37Q_S52G_L54D_G100Q.
[0238] SEQ ID NO: 140 sets forth the amino acid sequence of light chain variable region of a Hu3D6VLv2 variant L37Q _L50D _L54G _G10QQ. [0239] SEQ ID NO: 141 sets forth the amino acid sequence of light chain variable region of a Hu3D6VLv2 variant L37Q L50D L54R G100Q.
[0240] SEQ ID NO: 142 sets forth the amino acid sequence of light chain variable region of a Hu3D6VLv2 variant L37Q_L50V_L54D_G100Q.
[0241] SEQ ID NO: 143 sets forth the amino acid sequence of light chain variable region of a Hu3D6VLv2 variant L37Q.
[0242] SEQ ID NO: 144 sets forth the amino acid sequence of light chain variable region of a Hu3D6VLv2 variant G100Q.
[0243] SEQ ID NO: 145 sets forth the amino acid sequence of light chain variable region of a Hu3D6VLv2 variant L37QJL54E.
[0244] SEQ ID NO: 146 sets forth the amino acid sequence of heavy chain variable region of a hu3D6VHvlbAl 1 variant D60E, also known as h3D6VHvb8.
[0245] SEQ ID NO: 147 sets forth the amino acid sequence of heavy chain variable region of a hu3D6VHv 1 bAl 1 variant L82cV.
[0246] SEQ ID NO: 148 sets forth the amino acid sequence of heavy chain vari able region of a hu3D6VHvl bAl 1 variant D60E_L80M_Q81E_L82cV_T83R, also known as h3D6VHvb9.
[0247] SEQ ID NO: 149 sets forth the amino acid sequence of an alternate Kabat CDR-H2 of a humanized 3D6 antibody (as in h3D6VHvb8 and in h3D6VHvb9).
[0248] SEQ ID NO: 150 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54D and in hu3D6VLv2 L37Q L54D).
[0249] SEQ ID NO: 151 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54G and in hu3D6VLv2 L37Q L54G).
[0250] SEQ ID NO: 152 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54N). [0251] SEQ ID NO: 153 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54E and in hu3D6VLv2 L37Q L54E).
[0252] SEQ ID NO: 154 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L50E).
[0253] SEQ ID NO: 155 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54Q).
[0254] SEQ ID NO: 156 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L50D and in hu3D6VLv2 L37Q L50D).
[0255] SEQ ID NO: 157 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54K).
[0256] SEQ ID NO: 158 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54R and in hu3D6VLv2 L37Q L54R).
[0257] SEQ ID NO: 159 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54T and in hu3D6VLv2 L37Q L54T).
[0258] SEQ ID NO: 160 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L50G and in hu3D6VLv2 L37Q L50G).
[0259] SEQ ID NO: 161 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54V).
[0260] SEQ ID NO: 162 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54S).
[0261] SEQ ID NO: 163 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 S52G and in hu3D6VLv2 L37Q S52G).
[0262] SEQ ID NO: 164 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L50V). [0263] SEQ ID NO: 165 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q L50G L54R and hu3D6VLv2
L37Q_L50G_L54R_G100Q).
[0264] SEQ ID NO: 166 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q_L50G_L54G and in and in hu3D6VLv2 L37Q_L50G_L54G_G100Q).
[0265] SEQ ID NO: 167 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q S52G L54G).
[0266] SEQ ID NO: 168 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q S52G L54R and in and in hu3D6VLv2 L37Q_S52G_L54R_G100Q).
[0267] SEQ ID NO: 169 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q S52G L54T).
[0268] SEQ ID NO: 170 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q S52G L54D and in hu3D6VLv2
L37Q_S52G_L54D_G100Q).
[0269] SEQ ID NO: 171 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q L50D L54G and in hu3D6VLv2
L37Q_L50D_L54G_G100Q).
[0270] SEQ ID NO: 172 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q L50D L54R and in hu3D6VLv2
L37Q_L50D_L54R_G100Q).
[0271] SEQ ID NO: 173 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q_L50E_L54G).
[0272] SEQ ID NO: 174 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q_L50E_L54R). [0273] SEQ ID NO: 175 sets forth the amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q_L50V_L54D_G100Q).
[0274] SEQ ID NO: 176 sets forth the amino acid sequence of a heavy chain constant region (IgGl : allotype Glml7,l)
[0275] SEQ ID NO: 177 sets forth the amino acid sequence of a light chain constant region (kappa)
[0276] SEQ ID NO: 178 sets forth the amino acid sequence of a mature heavy chain of a 3D6 humanized variant (hu3D6VHvlbAl 1 IgGl Glml7 allotype)
[0277] SEQ ID NO: 179 sets forth the amino acid sequence of a mature light chain of a 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4 kappa)
[0278] SEQ ID NO: 180 sets forth the amino acid sequence of a heavy chain of a 3D6 humanized variant (hu3D6VHvlbAl 1 IgGl Glml7 allotype) with bovine alpha-lactalbumin signal peptide at the N-terminus
[0279] SEQ ID NO: 181 sets forth the amino acid sequence of a light chain of a 3D6 humanized variant (hu3D6VLv2 variant L37Q S 52G L54R, L2-DIM4 kappa) with bovine alpha- lactalbumin signal peptide at the N-terminus.
[0280] SEQ ID NO: 182 sets forth the nucleotide sequence encoding a heavy chain of a 3D6 humanized variant (hu3D6VHvlbAl 1 IgGl Glml7 allotype) with bovine alpha-lactalbumin signal peptide at the N-terminus
[0281] SEQ ID NO: 183 sets forth the nucleotide sequence encoding a light chain of a 3D6 humanized variant (hu3D6VLv2 variant L37Q S52G L54R, L2-DIM4 kappa) with bovine alpha-lactalbumin signal peptide at the N-terminus
[0282] SEQ ID NO: 184 sets forth the amino acid sequence of a region of tau microtubule binding repeat 1 (amino acid residues 255-271 of SEQ ID NO: l) [0283] SEQ ID NO: 185 sets forth the amino acid sequence of of a region of tau microtubule binding repeat 2 (amino acid residues 286-302 of SEQ ID NO: 1)
[0284] SEQ ID NO: 186 sets forth the amino acid sequence of of a region of tau microtubule binding repeat 3 (amino acid residues 317-333 of SEQ ID NO: 1)
[0285] SEQ ID NO: 187 sets forth the amino acid sequence of of a region of tau microtubule binding repeat 4 (amino acid residues 349-365 of SEQ ID NO: 1)
[0286] SEQ ID NO: 188 sets forth the amino acid sequence of a core motif of tau in MBTR 1 bound by 3D6
[0287] SEQ ID NO: 189 sets forth the amino acid sequence of tau sequence N-terminal to core motif of tau in MBTR 1 bound by 3D6
[0288] SEQ ID NO: 190 sets forth the amino acid sequence of tau sequence C-terminal to core motif of tau in MBTR lbound by 3D6
[0289] SEQ ID NO: 191 sets forth the amino acid sequence of epitope of 3D6
[0290] SEQ ID NO: 192 sets forth the amino acid sequence of a core motif of tau in MBTR 2 bound by 3D6
[0291] SEQ ID NO: 193 sets forth the amino acid sequence of a core motif of tau in MBTR 3 bound by 3D6
[0292] SEQ ID NO: 194 sets forth the amino acid sequence of a core motif of tau in MBTR 4 bound by 3D6
DEFINITIONS
[0293] Monoclonal antibodies or other biological entities are typically provided in isolated form. This means that an antibody or other biologically entity is typically at least 50% w/w pure of interfering proteins and other contaminants arising from its production or purification but does not exclude the possibility that the monoclonal antibody is combined with an excess of pharmaceutically acceptable carrier(s) or other vehicle intended to facilitate its use. Sometimes monoclonal antibodies are at least 60%, 70%, 80%, 90%, 95% or 99% w/w pure of interfering proteins and contaminants from production or purification. Often an isolated monoclonal antibody or other biological entity is the predominant macromolecular species remaining after its purification.
[0294] Specific binding of an antibody to its target antigen means an affinity and/or avidity of at least 106, 107, 108, 109, 1010, 1011, or 1012 M 1. Specific binding is detectably higher in magnitude and distinguishable from non-specific binding occurring to at least one unrelated target. Specific binding can be the result of formation of bonds between particular functional groups or particular spatial fit ( e.g ., lock and key type) whereas nonspecific binding is usually the result of van der Waals forces. Specific binding does not however necessarily imply that an antibody binds one and only one target.
[0295] The basic antibody structural unit is a tetramer of subunits. Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed linked to a cleavable signal peptide. The variable region without the signal peptide is sometimes referred to as a mature variable region. Thus, for example, a light chain mature variable region means a light chain variable region without the light chain signal peptide. The carboxy -terminal portion of each chain defines a constant region primarily responsible for effector function.
[0296] Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids. See generally , Fundamental Immunology , Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989, Ch. 7 (incorporated by reference in its entirety for all purposes).
[0297] An immunoglobulin light or heavy chain variable region (also referred to herein as a “light chain variable domain” (“VL domain”) or“heavy chain variable domain” (“VH domain”), respectively) consists of a“framework” region interrupted by three“complementarity determining regions” or“CDRs.” The framework regions serve to align the CDRs for specific binding to an epitope of an antigen. The CDRs include the amino acid residues of an antibody that are primarily responsible for antigen binding. From amino-terminus to carboxyl-terminus, both VL and VH domains comprise the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs 1, 2, and 3 of a VL domain are also referred to herein, respectively, as CDR-L1, CDR-L2, and CDR-L3; CDRs 1, 2, and 3 of a VH domain are also referred to herein, respectively, as CDR-H1, CDR-H2, and CDR-H3. When the application discloses a VL sequence with R as the C-terminal residue, the R can alternatively be considered as being the N-terminal residue of the light chain constant region. Thus, the application should also be understood as disclosing the VL sequence without the C-terminal R.
[0298] The assignment of amino acids to each VL and VH domain is in accordance with any conventional definition of CDRs. Conventional definitions include, the Rabat definition (Rabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, VI D, 1987 and 1991), the Chothia definition (Chothia & Lesk, J Mol. Biol. 196:901-917, 1987;
Chothia et al. , Nature 342:878-883, 1989); a composite of Chothia Rabat CDR in which CDR- H1 is a composite of Chothia and Rabat CDRs; the AbM definition used by Oxford Molecular’s antibody modelling software; and, the contact definition of Martin et al (bioinfo.org.uk/abs) (see Table 1). Rabat provides a widely used numbering convention (Rabat numbering) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. When an antibody is said to comprise CDRs by a certain definition of CDRs (e.g., Rabat) that definition specifies the minimum number of CDR residues present in the antibody (i.e., the Rabat CDRs). It does not exclude that other residues falling within another conventional CDR definition but outside the specified definition are also present. For example, an antibody comprising CDRs defined by Rabat includes among other possibilities, an antibody in which the CDRs contain Rabat CDR residues and no other CDR residues, and an antibody in which CDR HI is a composite Chothia-Rabat CDR HI and other CDRs contain Rabat CDR residues and no additional CDR residues based on other definitions. [0299] Table 1
Conventional Definitions of CDRs Using Kabat Numbering
Figure imgf000042_0001
*CDR-H1 by Chothia can end at H32, H33, or H34 (depending on the length of the loop). This is because the Kabat numbering scheme places insertions of extra residues at 35A and 35B, whereas Chothia numbering places them at 31 A and
3 IB. If neither H35A nor H35B (Kabat numbering) is present, the Chothia CDR- H1 loop ends at H32. If only H35A is present, it ends at H33. If both H35A and H35B are present, it ends at H34.
[0300] The term“antibody” includes intact antibodies and binding fragments thereof.
Typically, fragments compete with the intact antibody from which they were derived for specific binding to the target including separate heavy chains, light chains Fab, Fab', F(ab')2, F(ab)c, Dabs, nanobodies, and Fv. Fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins. The term“antibody” also includes a bispecific antibody and/or a humanized antibody. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy /light chain pairs and two different binding sites (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol ., 79:315-321 (1990); Kostelny el al ., J. Immunol ., 148: 1547-53 (1992)). In some bispecific antibodies, the two different heavy /light chain pairs include a humanized 3D6 heavy chain/light chain pair and a heavy chain/light chain pair specific for a different epitope on tau than that bound by 3D6.
[0301] In some bispecific antibodies, one heavy chain/light chain pair is a humanized 3D6 antibody as further disclosed below and the other heavy chain/light chain pair is from an antibody that binds to a receptor expressed on the blood brain barrier, such as an insulin receptor, an insulin-like growth factor (IGF) receptor, a leptin receptor, or a lipoprotein receptor, or a transferrin receptor (Friden e/ a/., Proc. Natl. Acad. Sci. USA 88:4771-4775, 1991; Friden et al., Science 259:373-377, 1993). Such a bispecific antibody can be transferred cross the blood brain barrier by receptor-mediated transcytosis. Brain uptake of the bispecific antibody can be further enhanced by engineering the bi-specific antibody to reduce its affinity to the blood brain barrier receptor. Reduced affinity for the receptor resulted in a broader distribution in the brain (see, e.g ., Atwal et al., Sci. Trans. Med. 3, 84ra43, 2011; Yu et al., Sci. Trans. Med. 3, 84ra44, 2011).
[0302] Exemplary bispecific antibodies can also be: (1) a dual -variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (2) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (3) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (4) a so-called "dock and lock" molecule, based on the "dimerization and docking domain" in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; or (5) a so-called Scorpion molecule, comprising, e.g, two scFvs fused to both termini of a human Fc-region. Examples of platforms useful for preparing bispecific antibodies include BiTE (Micromet), DART (MacroGenics), Fcab and Mab2 (F-star), Fc-engineered IgGl (Xencor) or DuoBody (based on Fab arm exchange, Genmab).
[0303] The term“epitope” refers to a site on an antigen to which an antibody binds. An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).
[0304] Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay showing the ability of one antibody to compete with the binding of another antibody to a target antigen. The epitope of an antibody can also be defined X-ray
crystallography of the antibody bound to its antigen to identify contact residues. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
[0305] Competition between antibodies is determined by an assay in which an antibody under test inhibits specific binding of a reference antibody to a common antigen (see, e.g. , Junghans et al., Cancer Res. 50: 1495, 1990). A test antibody competes with a reference antibody if an excess of a test antibody (e.g, at least 2x, 5x, lOx, 20x or lOOx) inhibits binding of the reference antibody by at least 50% as measured in a competitive binding assay. Some test antibodies inhibit binding of the references antibody by at least 75%, 90% or 99%. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur.
[0306] The term“pharmaceutically acceptable” means that the carrier, diluent, excipient, or auxiliary is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.
[0307] The term“patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
[0308] An individual is at increased risk of a disease if the subject has at least one known risk- factor (e.g, genetic, biochemical, family history, and situational exposure) placing individuals with that risk factor at a statistically significant greater risk of developing the disease than individuals without the risk factor.
[0309] The term“biological sample” refers to a sample of biological material within or obtainable from a biological source, for example a human or mammalian subject. Such samples can be organs, organelles, tissues, sections of tissues, bodily fluids, peripheral blood, blood plasma, blood serum, cells, molecules such as proteins and peptides, and any parts or combinations derived therefrom. The term biological sample can also encompass any material derived by processing the sample. Derived material can include cells or their progeny.
Processing of the biological sample may involve one or more of filtration, distillation, extraction, concentration, fixation, inactivation of interfering components, and the like.
[0310] The term“control sample” refers to a biological sample not known or suspected to include tau-related disease-affected regions, or at least not known or suspect to include diseased regions of a given type. Control samples can be obtained from individuals not afflicted with the tau-related disease. Alternatively, control samples can be obtained from patients afflicted with the tau-related disease. Such samples can be obtained at the same time as a biological sample thought to comprise the tau-related disease or on a different occasion. A biological sample and a control sample can both be obtained from the same tissue. Preferably, control samples consist essentially or entirely of normal, healthy regions and can be used in comparison to a biological sample thought to comprise tau-related disease-affected regions. Preferably, the tissue in the control sample is the same type as the tissue in the biological sample. Preferably, the tau-related disease-affected cells thought to be in the biological sample arise from the same cell type ( e.g ., neurons or glia ) as the type of cells in the control sample.
[0311] The term“disease” refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired, irrespective of the nature of the etiology.
[0312] The term“symptom” refers to a subjective evidence of a disease, such as altered gait, as perceived by the subject. A "sign" refers to objective evidence of a disease as observed by a physician.
[0313] The term“positive response to treatment” refers to a more favorable response in an individual patient or average response in a population of patients relative to an average response in a control population not receiving treatment.
[0314] For purposes of classifying amino acids substitutions as conservative or
nonconservative, amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gin, his, lys, arg; Group V (residues influencing chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids in the same class. Non conservative substitutions constitute exchanging a member of one of these classes for a member of another.
[0315] Percentage sequence identities are determined with antibody sequences maximally aligned by the Kabat numbering convention. After alignment, if a subject antibody region ( e.g ., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage.
[0316] Compositions or methods“comprising” or“including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or“includes” an antibody may contain the antibody alone or in combination with other ingredients. When the disclosure refers to a feature comprising specified elements, the disclosure should alternative be understood as referring to the feature consisting essentially of or consisting of the specified elements.
[0317] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.
[0318] Unless otherwise apparent from the context, the term“about” encompasses
insubstantial variations, such as values within a standard margin of error of measurement (e.g., SEM) of a stated value.
[0319] Statistical significance means p<0.05.
[0320] The singular forms of the articles“a,”“an,” and“the” include plural references unless the context clearly dictates otherwise. For example, the term“a compound” or“at least one compound” can include a plurality of compounds, including mixtures thereof. DETAILED DESCRIPTION
I. General
[0321] The invention provides antibodies that bind to tau. Some antibodies specifically bind to an epitope or epitopes within the microtubule binding region (MTBR) region of human tau defined by approximately residues 244 to 372 of SEQ ID NO: 1. Because the MTBR region contains sequence repeats, a single antibody can bind at multiple repeated sites within this region. Some antibodies specifically bind to an epitope of KXXSXXNX(K/H)H (SEQ ID NO: 191). Some antibodies bind within residues 199-213 and/or 262-276 of SEQ ID NO:3 (corresponding to residues 257-271 or 320-334, respectively, of SEQ ID NO: 1). Some antibodies bind within residues 259-268 and/or 290-299 and/or 321-330 and/or 353-362 of SEQ ID NO: 1. In some antibodies, binding to the MTBR 4 region at residues 353-362 is weaker than binding to MTBRs 1, 2 and 3. Some antibodies bind to tau irrespective of phosphorylation state. Some antibodies of the invention serve to inhibit or delay tau-associated pathologies and associated symptomatic deterioration. Although an understanding of mechanism is not required for practice of the invention, a reduction in toxicity may occur as a result of the antibody inducing phagocytosis of tau, inhibiting tau from inter or intramolecular aggregation, or from binding to other molecules, by stabilizing a non-toxic conformation, by inhibiting intercellular or intracellular transmission of pathogenic tau forms, by blockade of tau phosphorylation, by preventing binding of tau to cells, or by inducing proteolytic cleavage of tau, among other mechanisms. The antibodies of the invention or agents that induce such antibodies can be used in methods of treating or effecting prophylaxis of Alzheimer’s and other diseases associated with tau.
II. Target Molecules
[0322] Unless otherwise apparent from the context, reference to tau means a natural human form of tau including all isoforms irrespective of whether posttranslational modification ( e.g ., phosphorylation, gly cation, or acetylation) is present. There are six major isoforms (splice variants) of tau occurring in the human brain. The longest of these variants has 441 amino acids, of which the initial met residue is cleaved. Residues are numbered according to the 441 isoform. Thus, for example, reference to a phosphorylation at position 404 means position 404 of the 441 isoform, or corresponding position of any other isoform when maximally aligned with the 441 isoform. The amino acid sequences of the isoforms and Swiss-Prot numbers are indicated below.
P10636-8 (SEQ ID NO : 1 )
10 20 30 40 50 60
MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
70 80 90 100 110 120
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
130 140 150 160 170 180
HVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPK
190 200 210 220 230 240
TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAWR TPPKSPSSAK
250 260 270 280 290 300
SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINK KLDLSNVQSK CGSKDNIKHV
310 320 330 340 350 360
PGGGSVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI
370 380 390 400 410 420
THVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPWS GDTSPRHLSN VSSTGSIDMV
430 440
DSPQLATLAD EVSASLAKQG L
P10636-7 (SEQ ID NO : 2 )
10 20 30 40 50 60
MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
70 80 90 100 110 120
SETSDAKSTP TAEAEEAGIG DTPSLEDEAA GHVTQARMVS KSKDGTGSDD KKAKGADGKT
130 140 150 160 170 180
KIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR
190 200 210 220 230 240
SRTPSLPTPP TREPKKVAW RTPPKSPSSA KSRLQTAPVP MPDLKNVKSK IGSTENLKHQ
250 260 270 280 290 300
PGGGKVQIIN KKLDLSNVQS KCGSKDNIKH VPGGGSVQIV YKPVDLSKVT SKCGSLGNIH
310 320 330 340 350 360
HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN ITHVPGGGNK KIETHKLTFR ENAKAKTDHG
370 380 390 400 410
AEIVYKSPW SGDTSPRHLS NVSSTGSIDM VDSPQLATLA DEVSASLAKQ GL
P10636-6 ( 4R0N human tau) (SEQ ID NO : 3 )
10 20 30 40 50 60
MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA
70 80 90 100 110 120
AGHVTQARMV SKSKDGTGSD DKKAKGADGK TKIATPRGAA PPGQKGQANA TRIPAKTPPA
130 140 150 160 170 180
PKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTP PTREPKKVAV VRTPPKSPSS
190 200 210 220 230 240
AKSRLQTAPV PMPDLKNVKS KIGSTENLKH QPGGGKVQII NKKLDLSNVQ SKCGSKDNIK
250 260 270 280 290 300
HVPGGGSVQI VYKPVDLSKV TSKCGSLGNI HHKPGGGQVE VKSEKLDFKD RVQSKIGSLD
310 320 330 340 350 360
NITHVPGGGN KKIETHKLTF RENAKAKTDH GAEIVYKSPV VSGDTSPRHL SNVSSTGSID
370 380
MVDSPQLATL ADEVSASLAK QGL P10636-5 (SEQ ID NO : 4 )
10 20 30 40 50 60
MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
70 80 90 100 110 120
SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG
130 140 150 160 170 180
HVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPK
190 200 210 220 230 240
TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAWR TPPKSPSSAK
250 260 270 280 290 300
SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIVYK PVDLSKVTSK CGSLGNIHHK
310 320 330 340 350 360
PGGGQVEVKS EKLDFKDRVQ SKIGSLDNIT HVPGGGNKKI ETHKLTFREN AKAKTDHGAE
370 380 390 400 410
IVYKSPWSG DTSPRHLSNV SSTGSIDMVD SPQLATLADE VSASLAKQGL
P10636-4 (SEQ ID NO : 5 )
10 20 30 40 50 60
MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG
70 80 90 100 110 120
SETSDAKSTP TAEAEEAGIG DTPSLEDEAA GHVTQARMVS KSKDGTGSDD KKAKGADGET
130 140 150 160 170 180
KIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR
190 200 210 220 230 240
SRTPSLPTPP TREPKKVAW RTPPKSPSSA KSRLQTAPVP MPDLKNVKSK IGSTENLKHQ
250 260 270 280 290 300
PGGGKVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI
310 320 330 340 350 360
THVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPWS GDTSPRHLSN VSSTGSIDMV
370 380
DSPQLATLAD EVSASLAKQG L
P10636-2 (SEQ ID NO: 6)
10 20 30 40 50 60
MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA
70 80 90 100 110 120
AGHVTQARMV SKSKDGTGSD DKKAKGADGK TKIATPRGAA PPGQKGQANA TRIPAKTPPA
130 140 150 160 170 180
PKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTP PTREPKKVAV VRTPPKSPSS
190 200 210 220 230 240
AKSRLQTAPV PMPDLKNVKS KIGSTENLKH QPGGGKVQIV YKPVDLSKVT SKCGSLGNIH
250 260 270 280 290 300
HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN ITHVPGGGNK KIETHKLTFR ENAKAKTDHG
310 320 330 340 350
AEIVYKSPW SGDTSPRHLS NVSSTGSIDM VDSPQLATLA DEVSASLAKQ GL [0323] Reference to tau includes known natural variations about 30 of which are listed in the Swiss-Prot database and permutations thereof, as well as mutations associated with tau pathologies, such as dementia, Pick’s disease, supranuclear palsy, etc. (see, e.g ., Swiss-Prot database and Poorkaj, et al. Ann Neurol. 43:815-825 (1998)). Some examples of tau mutations numbered by the 441 isoform are a lysine to threonine mutation at amino acid residue 257 (K257T), an isoleucine to valine mutation at amino acid position 260 (I260V); a glycine to valine mutation at amino acid position 272 (G272V); an asparagine to lysine mutation at amino acid position 279 (N279K); an asparagine to histidine mutation at amino acid position 296 (N296H); a proline to serine mutation at amino acid position 301 (P301S); a proline to leucine mutation at amino acid 301 (P301L); a glycine to valine mutation at amino acid position 303 (G303V); a serine to asparagine mutation at position 305 (S305N); a glycine to serine mutation at amino acid position 335 (G335S); a valine to methionine mutation at position 337 (V337M); a glutamic acid to valine mutation at position 342 (E342V); a lysine to isoleucine mutation at amino acid position 369 (K3691); a glycine to arginine mutation at amino acid position 389 (G389R); and an arginine to tryptophan mutation at amino acid position 406 (R406W).
[0324] Tau can be phosphorylated at one or more amino acid residues including tyrosine at amino acid positions 18, 29, 97, 310, and 394 serine at amino acid positions 184, 185, 198, 199, 202, 208, 214, 235, 237, 238, 262, 293, 324, 356, 396, 400, 404, 409, 412, 413, and 422; and threonine at amino acids positions 175, 181, 205, 212, 217, 231, and 403. Unless otherwise apparent from context, reference to tau, or their fragments includes the natural human amino acid sequences including isoforms, mutants, and allelic variants thereof.
III. Antibodies
A. Binding Specificity and Functional Properties
[0325] The invention provides antibodies that bind to tau. Some antibodies specifically bind to an epitope within KXXSXXNX(K/H)H (SEQ ID NO: 191). Some antibodies bind to a peptide comprising, consisting essentially of, or consisting of amino acid residues 259-268 of 441 amino acid tau protein (SEQ ID NO: 1). Some antibodies bind to a peptide comprising, consisting essentially of, or consisting of amino acid residues 290-299 of 441 amino acid tau protein (SEQ ID NO: 1). Some antibodies bind to a peptide comprising, consisting essentially of, or consisting of amino acid residues 321-330 of 441 amino acid tau protein (SEQ ID NO: 1). Some antibodies bind to a peptide comprising, consisting essentially of or consisting of amino acid residues 353- 362 of 441 amino acid tau protein (SEQ ID NO: 1). Some antibodies bind to two, three or all four of these peptides. Some antibodies specifically bind to an epitope within residues 199-213 of 383 amino acid 4R0N human tau protein (SEQ ID NO:3) (corresponding to residues 257-271 of SEQ ID NO: 1). Some antibodies specifically bind to an epitope within residues 262-276 of 383 amino acid 4RON human tau protein (SEQ ID NO:3) (corresponding to residues 320-334 of SEQ ID NO: 1). Some antibodies of the invention specifically bind to a peptide consisting of residues 257-271 of 441 amino acid tau protein (SEQ ID NO: 1). Some antibodies of the invention specifically bind to a peptide consisting of residues 320-334 of 441 amino acid tau protein (SEQ ID NO: 1). Some antibodies of the invention specifically bind to a peptide consisting of residues 259-268 of 441 amino acid tau protein SEQ ID NO: l, namely
KIGSTENLKH (SEQ ID NO: 188). Some antibodies of the invention specifically bind to a peptide consisting of residues 290-299 of 441 amino acid tau protein SEQ ID NO: l, namely KCGSKDNIKH (SEQ ID NO: 192). Some antibodies of the invention specifically bind to a peptide consisting of residues 321-330 of 441 amino acid tau protein SEQ ID NO: l, namely KCGSLGNHm (SEQ ID NO: 193). Some antibodies of the invention specifically bind to a peptide consisting of residues 353-362 of 441 amino acid tau protein SEQ ID NO: l, namely KIGSLDNITH (SEQ ID NO: 194). Some antibodies of the invention specifically bind to a peptide consisting of the consensus motif KXXSXXNX(K/H)H (SEQ ID NO: 191). Some antibodies bind to an epitope comprising residues 259, 262, 265, 267, 268, residues 290, 293, 296, 298, 299, residues 321, 324, 327, 329, 330, or residues 353, 356, 359, 362 of 441 amino acid tau protein SEQ ID NO: 1. Some antibodies bind to tau irrespective of phosphorylation state. Some antibodies bind to an epitope not including a residue subject to phosphorylation. These antibodies can be obtained by immunizing with a tau polypeptide purified from a natural source or recombinantly expressed. Antibodies can be screened for binding tau in
unphosphorylated form as well as a form in which one or more residues susceptible to phosphorylation are phosphorylated. Such antibodies preferably bind with indistinguishable affinities or at least within a factor of 1.5, 2 or 3 -fold to phosphorylated tau compared to non- phosphorylated tau (i.e., are“pan-specific”). 3D6 is an example of a pan-specific monoclonal antibody. The invention also provides antibodies binding to the same epitope as any of the foregoing antibodies, such as, for example, the epitope of 3D6. Also included are antibodies competing for binding to tau with any of the foregoing antibodies, such as, for example, competing with 3D6.
[0326] The above-mentioned antibodies can be generated de novo by immunizing with a peptide including, consisting essentially of or consisting of residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to residues 257-271 or 320-334, respectively, of SEQ ID NO: 1) or by immunizing with a peptide including, consisting essentially of or consisting of residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO: 1, or by immunizing with a full length tau polypeptide or fragment thereof comprising such residues and screening for specific binding to a peptide including such residues. Such peptides are preferably attached to a heterologous conjugate molecule that helps elicit an antibody response to the peptide. Attachment can be direct or via a spacer peptide or amino acid. Cysteine is used as a spacer amino acid because its free SH group facilitates attachment of a carrier molecule. A polyglycine linker ( e.g ., 2-6 glycines), with or without a cysteine residue between the glycines and the peptide can also be used. The carrier molecule serves to provide a T-cell epitope that helps elicit an antibody response against the peptide. Several carriers are commonly used particularly keyhole limpet hemocyanin (KLH), ovalbumin and bovine serum albumin (BSA). Peptide spacers can be added to peptide immunogen as part of solid phase peptide synthesis. Carriers are typically added by chemical cross-linking. Some examples of chemical crosslinkers that can be used include cross-N- maleimido-6-aminocaproyl ester or m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (see for example, Harlow, E. et ah, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1988; Sinigaglia et ah, Nature, 336:778-780 (1988); Chicz et ah, J. Exp. Med., 178:27-47 (1993); Hammer et ah, Cell 74: 197-203 (1993); Falk K. et ah, Immunogenetics, 39:230-242 (1994); WO 98/23635; and, Southwood et al. J. Immunology, 160:3363-3373 (1998)). The carrier and spacer if present can be attached to either end of the immunogen.
[0327] A peptide with optional spacer and carrier can be used to immunize laboratory animals or B-cells as described in more detail below. Hybridoma supernatants can be tested for ability to bind one or more peptides including, consisting essentially of or consisting of residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to residues 257-271 or 320-334, respectively, of SEQ ID NO: l), or including, consisting essentially of or consisting of residues 259-268, 290- 299, 321-330, or 353-362 of SEQ ID NO: l, and/or phosphorylated and non-phosphorylated forms of tau, such as, for example, a full-length isoform of tau with position 404 in
phosphorylated form. The peptide can be attached to a carrier or other tag to facilitate the screening assay. In this case, the carrier or tag is preferentially different than the combination of spacer and carrier molecule used for immunization to eliminate antibodies specific for the spacer or carrier rather than the tau peptide. Any of the tau isoforms can be used.
[0328] The invention provides monoclonal antibodies binding to epitopes within tau. An antibody designated 3D6 is one such exemplary mouse antibody. Unless otherwise apparent from context, reference to 3D6 should be understood as referring to any of the mouse, chimeric, veneered, and humanized forms of this antibody. The antibody has been deposited as [DEPOSIT NUMBER] This antibody specifically binds to an epitope of KXXSXXNX(K/H)H (SEQ ID NO: 191). This antibody specifically binds within amino acid residues 199-213 and/or 262-276 of the 383 amino acid 4R0N human tau protein (SEQ ID NO:3) (corresponding to amino acid residues 257-271 and/or 320-334, respectively, of SEQ ID NO: l). The antibody specifically binds within amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: l, and combinations of any 2, 3 or all four thereof. This antibody is further characterized by its ability to bind both phosphorylated and unphosphorylated tau, both non-pathological and pathological forms and conformations of tau, and misfolded/aggregated forms of tau. An antibody designated 6A10 is one such exemplary mouse antibody. Unless otherwise apparent from context, reference to 6A10 should be understood as referring to any of the mouse, chimeric, veneered, and humanized forms of this antibody. Kabat/Chothia Composite CDRs of the heavy chain of 6A10 are designated SEQ ID NOs:67, 68, and 69, respectively, and Rabat CDRs of the light chain of 6A10 are designated SEQ ID NOs: 12, 13, and 14, respectively. Mouse 6A10 shares 82.1% of VH sequence identity and 100% VL sequence identity with the VH chain and VL chain, respectively, of mouse 3D6.
[0329] Some antibodies of the invention bind to the same or overlapping epitope as an antibody designated 3D6. The sequences of the heavy and light chain mature variable regions of this antibody are designated SEQ ID NOs:7 and 11, respectively. Other antibodies having such a binding specificity can be produced by immunizing mice with tau or a portion thereof including, consisting essentially of or consisting of the desired epitope (e.g. 199-213 and/or 262-276 of SEQ ID NO:3, corresponding to residues 257-271 and/or 320-334, respectively, of SEQ ID NO: l; or e.g., 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: l, any combination of 2, 3 or all 4 thereof) and screening resulting antibodies for binding to tau optionally in competition with an antibody having the variable regions of mouse 3D6 (IgGl kappa).
Fragments of tau including the desired epitope can be linked to a carrier that helps elicit an antibody response to the fragment and/or be combined with an adjuvant the helps elicit such a response. Such antibodies can be screened for differential binding to tau or a fragment thereof compared with mutants of specified residues. Screening against such mutants more precisely defines the binding specificity to allow identification of antibodies whose binding is inhibited by mutagenesis of particular residues and which are likely to share the functional properties of other exemplified antibodies. The mutations can be systematic replacement substitution with alanine (or serine if an alanine is present already) one residue at a time, or more broadly spaced intervals, throughout the target or throughout a section thereof in which an epitope is known to reside. If the same set of mutations significantly reduces the binding of two antibodies, the two antibodies bind the same epitope.
[0330] Antibodies having the binding specificity of a selected murine antibody (e.g., 3D6) can also be produced using a variant of the phage display method. See Winter, WO 92/20791. This method is particularly suitable for producing human antibodies. In this method, either the heavy or light chain variable region of the selected murine antibody is used as a starting material. If, for example, a light chain variable region is selected as the starting material, a phage library is constructed in which members display the same light chain variable region (i.e., the murine starting material) and a different heavy chain variable region. The heavy chain variable regions can for example be obtained from a library of rearranged human heavy chain variable regions. A phage showing strong specific binding for tau or a fragment thereof (e.g, at least 108 and preferably at least 109 M 1) is selected. The heavy chain variable region from this phage then serves as a starting material for constructing a further phage library. In this library, each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) and a different light chain variable region. The light chain variable regions can be obtained for example from a library of rearranged human variable light chain regions. Again, phage showing strong specific binding for tau or a fragment thereof are selected. The resulting antibodies usually have the same or similar epitope specificity as the murine starting material. [0331] Kabat/Chothia Composite CDRs of the heavy chain of 3D6 are designated SEQ ID NOs:8, 9, and 10, respectively, and Kabat CDRs of the light chain of 3D6 are designated SEQ ID NOs: 12, 13, and 14, respectively.
[0332] Table 2 indicates the 3D6 CDRs as defined by Kabat, Chothia, Composite of Chothia and Kabat (also referred to herein as“Kabat/Chothia Composite”), AbM, and Contact.
[0333] Table 2: 3D6 CDRs as defined by Kabat, Chothia, Composite of Chothia and Kabat,
AbM, and Contact
Figure imgf000055_0001
[0334] Other antibodies can be obtained by mutagenesis of cDNA encoding the heavy and light chains of an exemplary antibody, such as 3D6. Monoclonal antibodies that are at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to 3D6 in amino acid sequence of the mature heavy and/or light chain variable regions and maintain its functional properties, and/or which differ from the respective antibody by a small number of functionally inconsequential amino acid substitutions ( e.g ., conservative substitutions), deletions, or insertions are also included in the invention. Monoclonal antibodies having at least one or all six CDR(s) as defined by any conventional definition, but preferably Kabat, that are 90%, 95%, 99% or 100% identical to corresponding CDRs of 3D6 are also included. [0335] The invention also provides antibodies having some or all ( e.g ., 3, 4, 5, and 6) CDRs entirely or substantially from 3D6. Such antibodies can include a heavy chain variable region that has at least two, and usually all three, CDRs entirely or substantially from the heavy chain variable region of 3D6 and/or a light chain variable region having at least two, and usually all three, CDRs entirely or substantially from the light chain variable region of3D6. The antibodies can include both heavy and light chains. A CDR is substantially from a corresponding 3D6 CDR when it contains no more than 4, 3, 2, or 1 substitutions, insertions, or deletions, except that CDR-H2 (when defined by Rabat) can have no more than 6, 5, 4, 3, 2, or 1 substitutions, insertions, or deletions. Such antibodies can have at least 70%, 80%, 90%, 95%, 96%, 97%,
98%, or 99% identity to 3D6 in the amino acid sequence of the mature heavy and/or light chain variable regions and maintain their functional properties, and/or differ from 3D6 by a small number of functionally inconsequential amino acid substitutions (e.g., conservative
substitutions), deletions, or insertions.
[0336] Some antibodies identified by such assays can bind to monomeric, misfolded, aggregated, phosphorylated, or unphosphorylated forms of tau or otherwise. Likewise, some antibodies are immunoreactive on non-pathological and pathological forms and conformations of tau.
B. Non-Human Antibodies
[0337] The production of other non-human antibodies, e.g, murine, guinea pig, primate, rabbit or rat, against tau or a fragment thereof (e.g., amino acid residues 199-213 or 262-276 of SEQ ID NO:3, corresponding to amino acid residues 257-271 or 320-334, respectively, of SEQ ID NO: l; or amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: l) can be accomplished by, for example, immunizing the animal with tau or a fragment thereof . See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Such an immunogen can be obtained from a natural source, by peptide synthesis, or by recombinant expression. Optionally, the immunogen can be administered fused or otherwise complexed with a carrier protein. Optionally, the immunogen can be administered with an adjuvant. Several types of adjuvant can be used as described below. Complete Freund’s adjuvant followed by incomplete adjuvant is preferred for immunization of laboratory animals. Rabbits or guinea pigs are typically used for making polyclonal antibodies. Mice are typically used for making monoclonal antibodies. Antibodies are screened for specific binding to tau or an epitope within tau (e.g., an epitope comprising one or more of amino acid residues 199-213 or 262-276 of SEQ ID NO:3; corresponding to amino acid residues 257-271 or 320-334, respectively, of SEQ ID NO: 1 or an epitope comprising one or more of amino acid residues 259- 268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: 1). Such screening can be accomplished by determining binding of an antibody to a collection of tau variants, such as tau variants containing amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or 320-334, respectively, of SEQ ID NO: 1) or tau variants containing amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: l, or mutations within these residues, and determining which tau variants bind to the antibody. Binding can be assessed, for example, by Western blot, FACS or ELISA.
C. Humanized Antibodies
[0338] A humanized antibody is a genetically engineered antibody in which CDRs from a non human“donor” antibody are grafted into human“acceptor” antibody sequences (see, e.g.,
Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205; and Foote, US 6,881,557). The acceptor antibody sequences can be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Thus, a humanized antibody is an antibody having at least three, four, five or all CDRs entirely or substantially from a donor antibody and variable region framework sequences and constant regions, if present, entirely or substantially from human antibody sequences. Similarly a humanized heavy chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody heavy chain, and a heavy chain variable region framework sequence and heavy chain constant region, if present, substantially from human heavy chain variable region framework and constant region sequences. Similarly a humanized light chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody light chain, and a light chain variable region framework sequence and light chain constant region, if present, substantially from human light chain variable region framework and constant region sequences. Other than nanobodies and dAbs, a humanized antibody comprises a humanized heavy chain and a humanized light chain.
A CDR in a humanized antibody is substantially from a corresponding CDR in a non-human antibody when at least 85%, 90%, 95% or 100% of corresponding residues (as defined by any conventional definition but preferably defined by Rabat) are identical between the respective CDRs. The variable region framework sequences of an antibody chain or the constant region of an antibody chain are substantially from a human variable region framework sequence or human constant region respectively when at least 85%, 90%, 95% or 100% of corresponding residues defined by Kabat are identical. To be classified as humanized under the 2014 World Health Organization (WHO) International non-proprietary names (INN) definition of humanized antibodies, an antibody must have at least 85% identity to human germline antibody sequences (i.e., prior to somatic hypermutation). Mixed antibodies are antibodies for which one antibody chain (e.g., heavy chain) meets the threshold but the other chain (e.g., light chain) does not meet the threshold. An antibody is classified as chimeric if neither chain meets the threshold, even though the variable framework regions for both chains were substantially human with some murine backmutations. See, Jones et al. (2016) The INNs and outs of antibody nonproprietary names, mAbs 8: 1, 1-9, DOI: 10.1080/19420862.2015.1114320. See also“WHO-INN:
International nonproprietary names (INN) for biological and biotechnological substances (a review)” (Internet) 2014. Available from: http://www.
who.int/medicines/services/inn/BioRev2014.pdf), incorporated herein by reference. For the avoidance of doubt, the term“humanized” as used herein is not intended to be limited to the 2014 WHO INN definition of humanized antibodies. Some of the humanized antibodies provided herein have at least 85% sequence identity to human germline sequences and some of the humanized antibodies provided herein have less than 85% sequence identity to human germline sequences. Some of the heavy chains of the humanized antibodies provided herein have from about 60% to 100% sequence identity to human germ line sequences, such as, for example, in the range of about 60% to 69%, 70% to 79%, 80% to 84%, or 85% to 89%. Some heavy chains fall below the 2014 WHO INN definition and have, for example, about 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, or 82%, 83%, or 84% sequence identity to human germ line sequences, while other heavy chains meet the 2014 WHO INN definition and have about 85%, 86%, 87%, 88%, 89% or greater sequence identity to human germ line sequences. Some of the light chains of the humanized antibodies provided herein have from about 60% to 100% sequence identity to human germ line sequences, such as, for example, in the range of about 80% to 84% or 85% to 89%. Some light chains fall below the 2014 WHO INN definition and have, for example, about 81%, 82%, 83% or 84% sequence identity to human germ line sequences, while other light chains meet the 2014 WHO INN definition and have about 85%, 86%, 87%, 88%, 89% or greater sequence identity to human germ line sequences. Some humanized antibodies provided herein that are "chimeric" under the 2014 WHO INN definition have heavy chains with less than 85% identity to human germ line sequences paired with light chains having less than 85% identity to human germ line sequences. Some humanized antibodies provided herein are "mixed" under the 2014 WHO INN definition, for example, having a heavy chain with at least 85% sequence identity to human germ line sequences paired with a light chain having less than 85% sequence identity to human germ line sequences, or vice versa. Some humanized antibodies provided herein meet the 2014 WHO INN definition of "humanized" and have a heavy chain with at least 85% sequence identity to human germ line sequences paired with a light chain having at least 85% sequence identity to human germ line sequences. Additional humanized antibodies of the invention meet the 2014 WHO INN definition of“mixed.”
[0339] Although humanized antibodies often incorporate all six CDRs (defined by any conventional definition but preferably as defined by Kabat) from a mouse antibody, they can also be made with less than all CDRs ( e.g ., at least 3, 4, or 5 CDRs) from a mouse antibody (e.g, Pascalis et a/., J. Immunol. 169:3076, 2002; Vajdos et al., J. of Mol. Biol., 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36: 1079-1091, 1999; Tamura et al, J. Immunol., 164: 1432-1441, 2000).
[0340] In some antibodies only part of the CDRs, namely the subset of CDR residues required for binding, termed the SDRs, are needed to retain binding in a humanized antibody. CDR residues not contacting antigen and not in the SDRs can be identified based on previous studies (for example residues H60-H65 in CDR H2 are often not required), from regions of Kabat CDRs lying outside Chothia hypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987), by molecular modeling and/or empirically, or as described in Gonzales et al., Mol. Immunol. 41 : 863, 2004. In such humanized antibodies at positions in which one or more donor CDR residues is absent or in which an entire donor CDR is omitted, the amino acid occupying the position can be an amino acid occupying the corresponding position (by Kabat numbering) in the acceptor antibody sequence. The number of such substitutions of acceptor for donor amino acids in the CDRs to include reflects a balance of competing considerations. Such substitutions are potentially advantageous in decreasing the number of mouse amino acids in a humanized antibody and consequently decreasing potential immunogenicity and/or for meeting the WHO INN definition of“humanized”. However, substitutions can also cause changes of affinity, and significant reductions in affinity are preferably avoided. Positions for substitution within CDRs and amino acids to substitute can also be selected empirically.
[0341] The human acceptor antibody sequences can optionally be selected from among the many known human antibody sequences to provide a high degree of sequence identity ( e.g ., 65- 85% identity) between a human acceptor sequence variable region frameworks and
corresponding variable region frameworks of a donor antibody chain.
[0342] Some humanized and chimeric antibodies have the same (within experimental error) or improved functional properties, e.g., binding affinity for human tau, inhibition of tau
internalization into neurons as described in the examples as a murine antibody from which they were derived. For example, some humanized and chimeric antibodies have a binding affinity within a factor of 3, 2 or 1 of the murine antibody from which they were derived or an affinity indistinguishable within experimental error. Some humanized and chimeric antibodies inhibit tau internalization into neurons as described in the examples within a factor of 3, 2 or 1 of the murine antibody from which they were derived or inhibit the same within experimental error as the mouse antibody from which they were derived. Some humanized antibodies exhibit reduced immunogenicity, increased affinity, increased thermostability and/or improved expression relative to previously described humanized forms of the 3D6 antibody (see WO 2017/191560) hu3D6VHvlbAl l/L2-DIM4 demonstrated improved affinity, as evidenced by on-rate, off-rate, and Kd numbers, over parental hu3D6VHvlbAl 1/ hu3D6VLv2. hu3D6VHvlbAl 1/L2-DIM4 demonstrated higher thermostability and titer over parental hu3D6VHvlbAl 1/ hu3D6VLv2.
[0343] An example of an acceptor sequence for the heavy chain is the human mature heavy chain variable region of humanized 48G7 Fab with PDB accession code 2RCS-VH_huFrwk (SEQ ID NO:75). The variable domains of 3D6 and 48G7 Fab also share identical lengths for the CDR-H1, H2 loops. An example of an acceptor sequence for the heavy chain is the human mature heavy chain variable region IMGT# IGHVl-69-2*01 (SEQ ID NO:25). IMGT# IGHV1- 69-2*01 (SEQ ID NO:25) shares the canonical form of mouse 3D6 heavy chain CDR-H1 and H2. IMGT# IGHVl-69-2*01 (SEQ ID NO:25) belongs to human heavy chain subgroup 1. An example of an acceptor sequence for the light chain is the human mature light chain variable region with PDB accession code human antibody ARX71335 VL (SEQ ID NO:82). The variable light domain of 3D6 and ARX71335 antibody also share identical lengths for the CDR- LI, L2 and L3 loops. An example of an acceptor sequence for the light chain is the human mature light chain variable region with IMGT#IGKV2-30*02 (SEQ ID NO:27). IMGT#IGKV2- 30*02 (SEQ ID NO:27) has the same canonical classes for CDR-L1, CDR-L2 and L3 as mouse 3D6. IMGT#IGKV2-30*02 (SEQ ID NO:27) belongs to human kappa subgroup 2.
[0344] If more than one human acceptor antibody sequence is selected, a composite or hybrid of those acceptors can be used, and the amino acids used at different positions in the humanized light chain and heavy chain variable regions can be taken from any of the human acceptor antibody sequences used. For example, the human mature heavy chain variable regions of IMGT# IGHV1 -69-2*01 (SEQ ID NO:25) and PDB accession code # 2RCS-VH_huFrwk (SEQ ID NO: 75) were used as acceptor sequences for humanization of the 3D6 mature heavy chain variable region. An example of a positions in which these two acceptors differ is position HI 7 (T or S). Humanized versions of the 3D6 heavy chain variable region can include either amino acid at this position. For example, the human mature light chain variable regions IMGT# IGKV2-30*02 (SEQ ID NO:27) and PDB code # ARX71335-VL_huFrwk (SEQ ID NO:82) were used as acceptor sequences for humanization of the 3D6 mature light chain variable region. An example of a position in which these two acceptors differ is position LI 00 (Q or A).
Humanized versions of the3D6 light chain variable region can include either amino acid at this position.
[0345] Certain amino acids from the human variable region framework residues can be selected for substitution based on their possible influence on CDR conformation and/or binding to antigen. Investigation of such possible influences is by modeling, examination of the characteristics of the amino acids at particular locations, or empirical observation of the effects of substitution or mutagenesis of particular amino acids.
[0346] For example, when an amino acid differs between a murine variable region framework residue and a selected human variable region framework residue, the human framework amino acid can be substituted by the equivalent framework amino acid from the mouse antibody when it is reasonably expected that the amino acid:
(1) noncovalently binds antigen directly;
(2) is adjacent to a CDR region or within a CDR as defined by Chothia but not Rabat; (3) otherwise interacts with a CDR region ( e.g ., is within about 6 A of a CDR region),
( e.g ., identified by modeling the light or heavy chain on the solved structure of a homologous known immunoglobulin chain); or
(4) is a residue participating in the VL-VH interface.
[0347] In an embodiment, humanized sequences are generated using a two-stage PCR protocol that allows introduction of multiple mutations, deletions, and insertions using QuikChange site- directed mutagenesis [Wang, W. and Malcolm, B.A. (1999) BioTechniques 26:680-682)].
[0348] Framework residues from classes (1) through (3) as defined by Queen, US 5,530,101, are sometimes alternately referred to as canonical and vernier residues. Framework residues that help define the conformation of a CDR loop are sometimes referred to as canonical residues (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Thornton & Martin, J. Mol. Biol. 263:800- 815 (1996)). Framework residues that support antigen-binding loop conformations and play a role in fine-tuning the fit of an antibody to antigen are sometimes referred to as vernier residues (Foote & Winter, J. Mol. Biol 224:487-499 (1992)).
[0349] Other framework residues that are candidates for substitution are residues creating a potential glycosylation site. Still other candidates for substitution are acceptor human framework amino acids that are unusual for a human immunoglobulin at that position. These amino acids can be substituted with amino acids from the equivalent position of the mouse donor antibody or from the equivalent positions of more typical human immunoglobulins.
[0350] Other framework residues that are candidates for substitution are N-terminal glutamine residues (Q) that may be replaced with glutamic acid (E) to minimize potential for pyroglutamate conversion [ Y. Diana Liu, et ak, 2011, J. Biol. Chem., 286: 11211-11217] Glutamic acid (E) conversion to pyroglutamate (pE) occurs more slowly than from glutamine (Q). Because of the loss of a primary amine in the glutamine to pE conversion, antibodies become more acidic. Incomplete conversion produces heterogeneity in the antibody that can be observed as multiple peaks using charge-based analytical methods. Heterogeneity differences may indicate a lack of process control.
[0351] Exemplary humanized antibodies are humanized forms of the mouse 3D6, designated Hu3D6. [0352] The mouse antibody 3D6 comprises mature heavy and light chain variable regions having amino acid sequences comprising SEQ ID NO:7 and SEQ ID NO: 11, respectively. The invention provides humanized forms of the murine 3D6 antibody including 10 exemplified humanized heavy chain mature variable regions (hu3D6VHvbl (SEQ ID NO:76), hu3D6VHvb2 (SEQ ID NO: 77), hu3D6VHvb3 (SEQ ID NO: 78), hu3D6VHvb4 (SEQ ID NO: 79),
hu3D6VHvb5 (SEQ ID NO:80), hu3D6VHvb6 (SEQ ID NO:90), hu3D6VHvb7 (SEQ ID NO:91), hu3D6VHvlbAl l D60E (h3D6VHvb8, SEQ ID NO: 146), hu3D6VHvlbAl 1 L82cV (SEQ ID NO: 147), and hu3D6VHvlbAl 1 D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9, SEQ ID NO: 148)) and 56 exemplified humanized light chain mature variable regions
(hu3D6VLvbl (SEQ ID NO:83), hu3D6VLvb2 (SEQ ID NO:84), hu3D6VLvb3 (SEQ ID NO:85), hu3D6VLv2 L54D (SEQ ID NO:93), hu3D6VLv2 L54G (SEQ ID NO:94),
hu3D6VLv2 L54N (SEQ ID NO:95), hu3D6VLv2 L54E (SEQ ID NO:96), hu3D6VLv2 L50E (SEQ ID NO:97), hu3D6VLv2 L54Q (SEQ ID NO:98), hu3D6VLv2 L50D (SEQ ID NO:99), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 I48G (SEQ ID NO: 104), hu3D6VLv2 I48D (SEQ ID NO: 105), hu3D6VLv2 L47G (SEQ ID NO: 106), hu3D6VLv2 Y49E (SEQ ID NO: 107), hu3D6VLv2 L54V (SEQ ID NO: 108), hu3D6VLv2 L54S (SEQ ID NO: 109), hu3D6VLv2 S52G (SEQ ID NO: 110), hu3D6VLv2 L47N (SEQ ID NO: 111), hu3D6VLv2 L47D (SEQ ID NO: 112), hu3D6VLv2 L47E (SEQ ID NO: 113), hu3D6VLv2 L47P (SEQ ID NO: 114), hu3D6VLv2 L47T (SEQ ID NO: 115), hu3D6VLv2 L47S (SEQ ID NO: 116), hu3D6VLv2 L47A (SEQ ID NO: 117), hu3D6VLv2 L50V (SEQ ID NO: 118), hu3D6VLv2 L37Q L50G L54R (SEQ ID NO: 119), hu3D6VLv2 L37Q L50G L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q S52G L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q S52G L54R (SEQ ID NO: 122), hu3D6VLv2 L37Q S52G L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q S52G L54D (SEQ ID NO: 124), hu3D6VLv2 L37Q L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q L50D L54G (SEQ ID NO: 132), hu3D6VLv2
L37Q L50D L54R (SEQ ID NO: 133), hu3D6VLv2 L37Q L50E L54G (SEQ ID NO: 134), hu3D6VLv2 L37Q L50E L54R (SEQ ID NO: 135), hu3D6VLv2 L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2 L37Q L50G L54G G100Q (SEQ ID NO: 137), hu3D6VLv2 L37Q_S52G_L54R_G100Q (SEQ ID NO: 138), hu3D6VLv2 L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), hu3D6VLv2 L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2
L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), and hu3D6VLv2 G100Q (SEQ ID
NO: 144)).
[0353] Figures 2 and 3 show alignments of the heavy chain variable region and light chain variable region, respectively, of murine 3D6 and various humanized antibodies. Figures 9A and 9B show alignment of the heavy chain variable region of the murine 3D6 with the heavy chain variable region of various humanized antibodies. Figures 10 A, 10B, IOC, and 10D show alignment of the light chain variable region of hu3D6VLv2 with the light chain variable region of various humanized antibodies.
[0354] For reasons such as possible influence on CDR conformation and/or binding to antigen, mediating interaction between heavy and light chains, interaction with the constant region, being a site for desired or undesired post-translational modification, being an unusual residue for its position in a human variable region sequence and therefore potentially immunogenic, getting aggregation potential, and other reasons, the following 35 variable region framework positions were considered as candidates for substitutions in the 56 exemplified human mature light chain variable regions and the 10 exemplified human mature heavy chain variable regions, as further specified in the examples: L7 (T7S), L10 (T10S), L15 (I15L), L17 (Q17E), L37 (L37Q), L45 (K45R), L47 (L47G, L47N, L47D, L47E, L47P, L47T, L47S, or L47A) , L48 (I48G or I48D), L49 (Y49E), L83 (L83V), L86 (H86Y), L100 (A100Q), L106 (L106I), HI (Q1E), H5 (Q5V),
HI 1 (LI IV), H17 (S17T), H20 (L20I), H23 (T23K), H38 (K38R), H42 (E42G), H43 (Q43K), H66 (K66R), H67 (A67V), H75 (S75T), H76 (N76D), H80 (L80M), H81 (Q81E), H82c
(L82cV), H83 (T83R), H91 (Y91F), H93 (A93S), H94 (S94T), H108 (T108L), and H109 (LI 09V). The following 9 variable region CDR positions were considered as candidates for substitutions in the 56 exemplified human mature light chain variable regions and 10 exemplified human mature heavy chain variable regions, as further specified in the examples: L24 (K24R), L50 (L50E, L50D, L50G, or L50V), L52 (S52G), L54 (L54D, L54G, L54N, L54E, L54Q, L54K, L54R, L54T, L54V, or L54S), H28 (N28T), H54 (N54D), H56 (D56E), H58 (V58I), and H60 (D60E). In some humanized 3D6 antibodies, Rabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 87. In some humanized 3D6 antibodies, Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 149. In some humanized 3D6 antibodies, Kabat-Chothia Composite CDR-H1 has an amino acid sequence comprising SEQ ID NO:86, and Kabat CDR- H2 has an amino acid sequence comprising SEQ ID NO:87. In some humanized 3D6 antibodies, Kabat-Chothia Composite CDR-H1 has an amino acid sequence comprising SEQ ID NO:86 and Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO:88. In some humanized 3D6 antibodies, Kabat-Chothia Composite CDR-H1 has an amino acid sequence comprising SEQ ID NO:86 and Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO:92. In some humanized 3D6 antibodies, Kabat CDR-L1 has an amino acid sequence comprising SEQ ID NO:89. In some humanized 3D6 antibodies, Kabat CDR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 150-175.
[0355] Here, as elsewhere, the first-mentioned residue is the residue of a humanized antibody formed by grafting Kabat CDRs or a composite Chothia-Kabat CDR in the case of CDR-H1 into a human acceptor framework, and the second-mentioned residue is a residue being considered for replacing such residue. Thus, within variable region frameworks, the first mentioned residue is human, and within CDRs, the first mentioned residue is mouse.
[0356] Exemplified antibodies include any permutations or combinations of the exemplified mature heavy and light chain variable regions VHvbl/VLvbl, VHvbl/VLvb2, VHvbl/VLvb3, VHvb2/VLvbl, VHvb2/VLvb2, VHvb2/VLvb3, VHvb3/VLvbl, VHvb3/VLvb2,
VHvb3/VLvb3, VHvb4/VLvbl, VHvb4/VLvb2, VHvb4/VLvb3, VHvb5/VLvbl,
VHvb5/VLvb2, VHvb5/VLvb3, VHvb6/VLvbl, VHvb6/VLvb2, VHvb6/VLvb3,
VHvb7/VLvbl, VHvb7/VLvb2, VHvb7/VLvb3. Exemplified antibodies include any
permutations or combinations of the exemplified mature heavy chain variable regions hu3D6VHvbl (SEQ ID NO: 76), hu3D6VHvb2 (SEQ ID NO: 77), hu3D6VHvb3 (SEQ ID NO:78), hu3D6VHvb4 (SEQ ID NO:79), hu3D6Hvb5 (SEQ ID NO:80), hu3D6VHvb6 (SEQ ID NO:90), hu3D6VHvb7 (SEQ ID NO:91), hu3D6VHvb7 (SEQ ID NO:91), hu3D6VHvlbAl 1 D60E (h3D6VHvb8, SEQ ID NO: 146), hu3D6VHvlbAl 1 L82cV (SEQ ID NO: 147), and hu3D6VHvlbAl 1 D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9, SEQ ID NO: 148) with any of the humanized 3D6VL light chain variable regions hu3D6VLvbl (SEQ ID NO:83), hu3D6VLvb2 (SEQ ID NO:84), hu3D6VLvb3 (SEQ ID NO:85), hu3D6VLv2 L54D (SEQ ID NO:93), hu3D6VLv2 L54G (SEQ ID NO:94), hu3D6VLv2 L54N (SEQ ID NO:95), hu3D6VLv2 L54E (SEQ ID NO:96), hu3D6VLv2 L50E (SEQ ID NO:97), hu3D6VLv2 L54Q (SEQ ID NO:98), hu3D6VLv2 L50D (SEQ ID NO:99), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 I48G (SEQ ID NO: 104), hu3D6VLv2 I48D (SEQ ID NO: 105), hu3D6VLv2 L47G (SEQ ID NO: 106), hu3D6VLv2 Y49E (SEQ ID NO: 107), hu3D6VLv2 L54V (SEQ ID NO: 108), hu3D6VLv2 L54S (SEQ ID NO: 109), hu3D6VLv2 S52G (SEQ ID NO: 110), hu3D6VLv2 L47N (SEQ ID NO: 111), hu3D6VLv2 L47D (SEQ ID NO: 112), hu3D6VLv2 L47E (SEQ ID NO: 113), hu3D6VLv2 L47P (SEQ ID NO: 114), hu3D6VLv2 L47T (SEQ ID NO: 115), hu3D6VLv2 L47S (SEQ ID NO: 116), hu3D6VLv2 L47A (SEQ ID NO: 117), hu3D6VLv2 L50V (SEQ ID NO: 118), hu3D6VLv2 L37Q L50G L54R (SEQ ID NO: 119), hu3D6VLv2 L37Q L50G L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q S52G L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q S52G L54R (SEQ ID NO: 122), hu3D6VLv2 L37Q S52G L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q S52G L54D (SEQ ID NO: 124), hu3D6VLv2
L37Q L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q L50D L54G (SEQ ID NO: 132), hu3D6VLv2 L37Q L50D L54R (SEQ ID NO: 133), hu3D6VLv2 L37Q L50E L54G (SEQ ID NO: 134), hu3D6VLv2 L37Q L50E L54R (SEQ ID NO: 135), hu3D6VLv2 L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2
L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2 L37Q_S52G_L54R_G100Q (SEQ ID NO: 138), hu3D6VLv2 L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), hu3D6VLv2
L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), and hu3D6VLv2 G100Q (SEQ ID NO: 144).
[0357] Exemplified antibodies include any permutations or combinations of the exemplified mature heavy chain variable regions hu3D6VHvb l (SEQ ID NO:76), hu3D6VHvb2 (SEQ ID NO:77), hu3D6VHvb3 (SEQ ID NO:78), hu3D6VHvb4 (SEQ ID NO:79), hu3D6Hvb5 (SEQ ID NO: 80), hu3D6VHvb6 (SEQ ID NO: 90), hu3D6VHvb7 (SEQ ID NO:91), hu3D6VHvb7 (SEQ ID NO:91), hu3D6VHvlbAl l D60E (h3D6VHvb8, SEQ ID NO: 146), hu3D6VHvlbAl 1 L82cV (SEQ ID NO: 147), and hu3D6VHvlbAl 1 D60E_L80M_Q81E_L82cV T83R (h3D6VHvb9, SEQ ID NO: 148) with any of the humanized 3D6VL light chain variable regions hu3D6VLvl (SEQ ID NO:20), hu3D6VLv2 (SEQ ID NO:21), hu3D6VLv3 (SEQ ID NO:22), and
hu3D6VLv4 (SEQ ID NO:22). Exemplified antibodies include any permutations or
combinations of the exemplified mature light chain variable regions hu3D6VLvbl (SEQ ID NO:83), hu3D6VLvb2 (SEQ ID NO:84), hu3D6VLvb3 (SEQ ID NO:85), hu3D6VLv2 L54D (SEQ ID NO:93), hu3D6VLv2 L54G (SEQ ID NO:94), hu3D6VLv2 L54N (SEQ ID NO:95), hu3D6VLv2 L54E (SEQ ID NO:96), hu3D6VLv2 L50E (SEQ ID NO:97), hu3D6VLv2 L54Q (SEQ ID NO:98), hu3D6VLv2 L50D (SEQ ID NO:99), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 I48G (SEQ ID NO: 104), hu3D6VLv2 I48D (SEQ ID NO: 105), hu3D6VLv2 L47G (SEQ ID NO: 106), hu3D6VLv2 Y49E (SEQ ID NO: 107), hu3D6VLv2 L54V (SEQ ID NO: 108), hu3D6VLv2 L54S (SEQ ID NO: 109), hu3D6VLv2 S52G (SEQ ID NO: 110), hu3D6VLv2 L47N (SEQ ID NO: 111), hu3D6VLv2 L47D (SEQ ID NO: 112), hu3D6VLv2 L47E (SEQ ID NO: 113), hu3D6VLv2 L47P (SEQ ID NO: 114), hu3D6VLv2 L47T (SEQ ID NO: 115), hu3D6VLv2 L47S (SEQ ID NO: 116), hu3D6VLv2 L47A (SEQ ID NO: 117), hu3D6VLv2 L50V (SEQ ID NO: 118), hu3D6VLv2 L37Q L50G L54R (SEQ ID NO: 119), hu3D6VLv2 L37Q L50G L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q S52G L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q S52G L54R (SEQ ID NO: 122), hu3D6VLv2 L37Q S52G L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q S52G L54D (SEQ ID NO: 124), hu3D6VLv2
L37Q L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q L50D L54G (SEQ ID NO: 132), hu3D6VLv2 L37Q L50D L54R (SEQ ID NO: 133), hu3D6VLv2 L37Q L50E L54G (SEQ ID NO: 134), hu3D6VLv2 L37Q L50E L54R (SEQ ID NO: 135), hu3D6VLv2 L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2
L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2 L37Q_S52G_L54R_G100Q (SEQ ID NO: 138), hu3D6VLv2 L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), hu3D6VLv2
L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), and hu3D6VLv2 G100Q (SEQ ID NO: 144) with any of the humanized 3D6 heavy chain variable regions hu3D6VHvl (SEQ ID NO: 15); hu3D6VHv2 (SEQ ID NO: 16); hu3D6VHvlb (SEQ ID NO: 17); hu3D6VHvlbAl 1 (SEQ ID NO: 18); hu3D6VHv5 (SEQ ID NO: 19); hu3D6VHvlbAl 1B6G2 (SEQ ID NO:46); hu3D6VHvlbAl 1B6H3 (SEQ ID NO:47); hu3D6VHvlc (SEQ ID NO:48); hu3D6VHvld (SEQ ID NO:49); hu3D6VHvle (SEQ ID NO:50); hu3D6VHvlf (SEQ ID NO:51); hu3D6VHv3 (SEQ ID NO:52); hu3D6VHv3b (SEQ ID NO:53); hu3D6VHv3c (SEQ ID NO:54); hu3D6VHv4 (SEQ ID NO:55); hu3D6VHv4b (SEQ ID NO:56); and hu3D6VHv4c (SEQ ID NO:57).
[0358] The invention provides an antibody in which humanized heavy chain variable region hu3D6VHvlbAl 1, also known as h3D6Hu5, (SEQ ID NO: 18) is combined with humanized light chain variable region hu3D6VLv2 L37Q S52G L54R (L2-DIM4, SEQ ID NO: 122). The invention provides an antibody in which humanized heavy chain variable region
hu3D6VHvlbAl 1, also known as h3D6Hu5, (SEQ ID NO: 18) is combined with humanized light chain variable region hu3D6VLv2 L37Q S52G L54T (L2-DIM5, SEQ ID NO: 123). The invention provides an antibody in which humanized heavy chain variable region h3D6VHvb8 (SEQ ID NO: 146) is combined with humanized light chain variable region hu3D6VLv2
L37Q S52G L54R (L2-DIM4, SEQ ID NO: 122). The invention provides an antibody in which humanized heavy chain variable region hu3D6VHvlbAl 1, also known as h3D6Hu5, (SEQ ID NO: 18) is combined with humanized light chain variable region hu3D6VLv2
L37Q S52G L54G (L2-DIM3, SEQ ID NO: 121). The invention provides an antibody in which humanized heavy chain variable region hu3D6VHvlbAl 1, also known as h3D6Hu5, (SEQ ID NO: 18) is combined with humanized light chain variable region hu3D6VLv2 S52G (L2-DIM9, SEQ ID NO: 110). The invention provides an antibody in which humanized heavy chain variable region h3D6VHvb8 (SEQ ID NO: 146) is combined with humanized light chain variable region hu3D6VLv2 L54G (L2-DIM7, SEQ ID NO:94). The invention provides an antibody in which humanized heavy chain variable region hu3D6VHvlbAl 1, also known as h3D6Hu5, (SEQ ID NO: 18) is combined with humanized light chain variable region hu3D6VLv2 L50G (L2-DIM22, SEQ ID NO: 103).
[0359] The invention provides an antibody in which any one of the exemplified humanized heavy chain variable regions is combined with a human heavy chain constant region. An exemplary human heavy chain constant region is provided as SEQ ID NO: 176 (IgGl : allotype Glml7,l). For example, SEQ ID NO: 178 sets forth the amino acid sequence of a mature heavy chain of a 3D6 humanized variant (hu3D6VHvlbAl 1 IgGl Glml7 allotype). For example,
SEQ ID NO: 180 sets forth the amino acid sequence of a heavy chain of a 3D6 humanized variant (hu3D6VHvlbAl 1 IgGl Glml7 allotype) bovine alpha-lactalbumin signal peptide at the N- terminus. The invention provides an antibody in which any one of the exemplified humanized light chain variable regions is combined with a light chain constant region. An exemplary light chain constant region is provided as SEQ ID NO: 177 (kappa). For example, SEQ ID NO: 179 sets forth the amino acid sequence of a mature light chain of a 3D6 humanized variant
(hu3D6VLv2 variant L37Q S52G L54K, L2-DIM4 kappa). For example, SEQ ID NO: 181 sets forth the amino acid sequence of a light chain of a 3D6 humanized variant (hu3D6VLv2 variant L37Q__S52G__L54R, L2-DIM4 kappa) with bovine alpha-lactalbumin signal peptide at the N- terminus.
[0360] The invention provides variants of the 3D6 humanized antibody in which the humanized mature heavy chain variable region shows at least 90%, 95%, 96%, 97%, 98%, or 99% identity to hu3D6VHvbl (SEQ ID NO:76), hu3D6VHvb2 (SEQ ID NO:77), hu3D6VHvb3 (SEQ ID NO:78), hu3D6VHvb4 (SEQ ID NO:79), hu3D6Hvb5 (SEQ ID NO:80), hu3D6VHvb6 (SEQ ID NO:90), hu3D6VHvb7 (SEQ ID NO:91), hu3D6VHvb7 (SEQ ID NO:91),
hu3D6VHvlbAl l D60E (h3D6VHvb8, SEQ ID NO: 146), hu3D6VHvlbAl 1 L82cV (SEQ ID NO: 147), or hu3D6VHvlbAl 1 D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9, SEQ ID NO: 148) and the humanized mature light chain variable region shows at least 90%, 95%, 96%, 97%, 98%, or 99% identity to hu3D6VLvbl (SEQ ID NO:83), hu3D6VLvb2 (SEQ ID NO:84), hu3D6VLvb3 (SEQ ID NO:85), hu3D6VLv2 L54D (SEQ ID NO:93), hu3D6VLv2 L54G (SEQ ID NO: 94), hu3D6VLv2 L54N (SEQ ID NO: 95), hu3D6VLv2 L54E (SEQ ID NO: 96), hu3D6VLv2 L50E (SEQ ID NO:97), hu3D6VLv2 L54Q (SEQ ID NO:98), hu3D6VLv2 L50D (SEQ ID NO:99), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 I48G (SEQ ID NO: 104), hu3D6VLv2 I48D (SEQ ID NO: 105), hu3D6VLv2 L47G (SEQ ID NO: 106), hu3D6VLv2 Y49E (SEQ ID NO: 107), hu3D6VLv2 L54V (SEQ ID NO: 108), hu3D6VLv2 L54S (SEQ ID NO: 109), hu3D6VLv2 S52G (SEQ ID NO: 110), hu3D6VLv2 L47N (SEQ ID NO: 111), hu3D6VLv2 L47D (SEQ ID NO: 112), hu3D6VLv2 L47E (SEQ ID NO: 113), hu3D6VLv2 L47P (SEQ ID NO: 114), hu3D6VLv2 L47T (SEQ ID NO: 115), hu3D6VLv2 L47S (SEQ ID NO: 116), hu3D6VLv2 L47A (SEQ ID NO: 117), hu3D6VLv2 L50V (SEQ ID NO: 118), hu3D6VLv2 L37Q L50G L54R (SEQ ID NO: 1 19), hu3D6VLv2 L37Q L50G L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q S52G L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q S52G L54R (SEQ ID NO: 122), hu3D6VLv2 L37Q S52G L54T (SEQ ID NO: 123), hu3D6VLv2
L37Q S52G L54D (SEQ ID NO: 124), hu3D6VLv2 L37Q L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q L50D L54G (SEQ ID
NO: 132), hu3D6VLv2 L37Q L50D L54R (SEQ ID NO: 133), hu3D6VLv2 L37Q L50E L54G (SEQ ID NO: 134), hu3D6VLv2 L37Q L50E L54R (SEQ ID NO: 135), hu3D6VLv2
L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2 L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2 L37Q_S52G_L54R_G100Q (SEQ ID NO: 138), hu3D6VLv2
L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), hu3D6VLv2 L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2
L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), or hu3D6VLv2 G100Q (SEQ ID NO: 144). In some such antibodies at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, or all 44 of the backmutations or other mutations in SEQ ID
NOs:76-80, SEQ ID NOs:90-91, SEQ ID NOs: 146-148, SEQ ID NOs:83-85, and SEQ ID
NOs:93-145 are retained.
[0361] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by the amino acid as specified: H93 is occupied by S and H94 is occupied by T. In some humanized 3D6 antibodies, positions H93 and H94 are occupied by S and T, respectively.
[0362] In some humanized 3D6 antibodies, position H91 in the VH region is occupied by F.
[0363] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by the amino acid as specified: HI is occupied by E, H5 is occupied by V,
HI 1 is occupied by V, H20 is occupied I, H23 is occupied by K, H38 is occupied by R, H42 is occupied by G, H43 is occupied by K, H66 is occupied by R, H75 is occupied by T, H76 is occupied by D, H81 is occupied by E, HI 08 is occupied by L, HI 09 is occupied by V. In some humanized 3D6 antibodies, positions HI, H5, HI 1, H20, H23, H38, H42, H43, H66, H75, H76, H81, HI 08, and HI 09 in the VH region are occupied by E, V, V, I, K, R, G, K, R, T, D, E, L, and V, respectively.
[0364] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by the amino acid as specified: HI 7 is occupied by T, H80 is occupied by M, H83 is occupied by R. In some humanized 3D6 antibodies, positions HI 7, H80, and H83 in the VH region are occupied by T, M, and R, respectively.
[0365] In some humanized 3D6 antibodies, position H58 in the VH region is occupied by I.
[0366] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by the amino acid as specified: H28 is occupied by T, H67 is occupied by V. In some humanized 3D6 antibodies, positions H28 and H67 in the VH region are occupied by T and V, respectively.
[0367] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by the amino acid as specified: H54 is occupied by D, H56 is occupied by E. In some humanized 3D6 antibodies, positions H54 and H56 in the VH region are occupied by D and E, respectively.
[0368] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by the amino acid as specified: HI is occupied by Q or E, H5 is occupied by Q or V, HI 1 is occupied by L or V , HI 7 is occupied by S or T, H20 is occupied by L or I, H23 is occupied by T or K, H28 is occupied by N or T, H38 is occupied by K or R, H42 is occupied by E or G, H43 is occupied by Q or K, H54 is occupied by N or D, H56 is occupied by D or E, H58 is occupied by V or I, H66 is occupied by K or R, H67 is occupied by A or V, H75 is occupied by S or T, H76 is occupied by N or D, H80 is occupied by L or M, H81 is occupied by Q or E, H83 is occupied by T or R , H91 is occupied by F or Y, H93 is occupied by S, H94 is occupied by T, HI 08 is occupied by T or L, HI 09 is occupied by L or V.
[0369] In some humanized 3D6 antibodies, positions H91, H93, and H94 in the VH region are occupied by F, S, and T, respectively, as in huVHvbl . In some humanized 3D6 antibodies, positions HI, H5, Hl l, H20, H23, H38, H42, H43, H66, H75, H76, H81, H91, H93, H94, H108, and HI 09 in the VH region are occupied by E, V, V, I, K, R, G, K, R, T, D, E, F, S, T,L, and V, respectively, as in huVHvb2. In some humanized 3D6 antibodies, positions HI, H5, Hl l, HI 7, H20, H23, H38, H42, H43, H58, H66, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T,I, K, R, G, K, I, R, T, D, M, E, R, S, T, L, and V, respectively, as in huVHvb3. In some humanized 3D6 antibodies, positions HI, H5, HI 1, HI 7, H20, H23, H28, H38, H42, H43, H58, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108, and HI 09 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, I, R, V, T, D, M, E, R, S, T, L, and V, respectively, as in huVHvb4. In some humanized 3D6 antibodies, positions HI,
H5, HI 1, H17, H20, H23, H28, H38, H42, H43, H54, H56, H58, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, I, R, V, T, D, M, E, R, S, T, L, and V, respectively, as in huVHvb5. In some humanized 3D6 antibodies, positions HI, H5, HI 1, H17, H20, H23, H28, H38, H42, H43, H54, H56, H66, H67, H75, H76, H80, H81, H83, H91, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, R, V, T, D, M, E, R, F, S, T, L, and V, respectively, as in huVHvb6. In some humanized 3D6 antibodies, positions HI, H5, HI 1, HI 7, H20, H23, H28, H38, H42, H43, H54, H56, H66, H67, H75, H76, H80, H81, H83, H93, H94,
HI 08, and HI 09 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, R, V, T, D, M, E, R, S, T, L, and V, respectively, as in huVHvb7.
[0370] In some humanized 3D6 antibodies, position H60 is occupied by E, as in
hu3D6VHvlbAl 1 D60E (h3D6VHvb8). In some humanized 3D6 antibodies, position H82C is occupied by V, as in hu3D6VHvlbAl 1 L82cV. In some humanized 3D6 antibodies, positions H60, H80, H81, H82c, and H83 are occupied by E, M, E, V, and R, as in hu3D6VHvlbAl 1 D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9).
[0371] The heavy chain variable region of any of the above-referenced antibodies can be modified to further reduce immunogenicity. For example, in some of the humanized antibodies position H80 is occupied by M and/or position H82c is occupied by V.
[0372] In some humanized 3D6 antibodies, at least one of the following positions in the VL region is occupied by the amino acid as specified: L7 is occupied by S, L10 is occupied by S,
L15 is occupied by L, L83 is occupied by V, L86 is occupied by Y, and LI 06 is occupied by I.
In some humanized 3D6 antibodies, positions L7, L10, L15, L83, L86, and L106 are occupied by S, S, L, V, Y, and Y, respectively.
[0373] In some humanized 3D6 antibodies, at least one of the following positions in the VL region is occupied by the amino acid as specified: L7 is T or S, L10 is T or S, L15 is I or L, L17 is Q or E, L24 is K or R, L37 is L or Q, L45 is K or R, L83 is L or V, L86 is H or Y, L100 is A or Q, L106 is L or I. [0374] In some humanized 3D6 antibodies, positions L7, L10, L15, L83, L86, and L106 in the VL region are occupied by S, S, L, V, Y, and I, respectively, as in huVLvb2. In some humanized 3D6 antibodies, positions L7, L10, L15, L17, L24, L37, L45, L83, L86, L100, and LI 06 in the VL region are occupied by S, S, L, E, R, Q, R, V, Y, Q, and I, respectively, as in huVLvb3.
[0375] The light chain variable region of any of the above referenced antibodies can be modified to further reduce immunogenicity. For example, in some of the humanized antibodies position L47 is occupied by G, N, D, E, P, T, S or A; position L48 is occupied by G or D;
position L49 is occupied by E; position L50 is occupied by E, D, G, or V; position L52 is occupied by G; and/or position L54 is occupied by D, G, N, E, Q, K, R, T, V or S. The heavy chain variable region of any of the above-referenced antibodies can be modified to further reduce immunogenicity. For example, in some of the humanized antibodies position H80 is occupied by M and/or position H82c is occupied by V.
[0376] In some humanized 3D6 antibodies, position L54 is occupied by D, as in hu3D6VLv2 L54D. In some humanized 3D6 antibodies, position L54 is occupied by G, as in hu3D6VLv2 L54G. In some humanized 3D6 antibodies, position L54 is occupied by N, as in hu3D6VLv2 L54N, In some humanized 3D6 antibodies, position L54 is occupied by E, as in hu3D6VLv2 L54E. In some humanized 3D6 antibodies, position L50 is occupied by E, as in hu3D6VLv2 L50E. In some humanized 3D6 antibodies, position L54 is occupied by Q, as in hu3D6VLv2 L54Q. In some humanized 3D6 antibodies, position L50 is occupied by D, as in hu3D6VLv2 L50D. In some humanized 3D6 antibodies, position L54 is occupied by K, as in hu3D6VLv2 L54K. In some humanized 3D6 antibodies, position L54 is occupied by R, as in hu3D6VLv2 L54R. In some humanized 3D6 antibodies, position L54 is occupied by T, as in hu3D6VLv2 L54T. In some humanized 3D6 antibodies, position L50 is occupied by G, as in hu3D6VLv2 L50G. In some humanized 3D6 antibodies, position L48 is occupied by G, as in hu3D6VLv2 I48G. In some humanized 3D6 antibodies, position L48 is occupied by D, as in hu3D6VLv2 I48D. In some humanized 3D6 antibodies, position L47 is occupied by G, as in hu3D6VLv2 L47G. In some humanized 3D6 antibodies, position L49 is occupied by E, as in hu3D6VLv2 Y49E. In some humanized 3D6 antibodies, position L54 is occupied by V, as in hu3D6VLv2 L54V. In some humanized 3D6 antibodies, position L54 is occupied by S, as in hu3D6VLv2 L54S. In some humanized 3D6 antibodies, position L52 is occupied by G, as in hu3D6VLv2 S52G. In some humanized 3D6 antibodies, position L47 is occupied by N, as in hu3D6VLv2 L47N. In some humanized 3D6 antibodies, position L47 is occupied by D, as in hu3D6VLv2 L47D. In some humanized 3D6 antibodies, position L47 is occupied by E, as in hu3D6VLv2 L47E. In some humanized 3D6 antibodies, position L47 is occupied by P, as in hu3D6VLv2 L47P. In some humanized 3D6 antibodies, position L47 is occupied by T, as in hu3D6VLv2 L47T. In some humanized 3D6 antibodies, position L47 is occupied by S, as in hu3D6VLv2 L47S. In some humanized 3D6 antibodies, position L47 is occupied by A, as in hu3D6VLv2 L47A. In some humanized 3D6 antibodies, position L50 is occupied by V, as in hu3D6VLv2 L50V.
[0377] In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, G, and R, respectively, as in hu3D6VLv2 L37Q_L50G_L54R. In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, G, and G, respectively, as in hu3D6VLv2 L37Q_L50G_L54G. In some humanized 3D6 antibodies, positions L37, L52, and L54 are occupied by Q, G, and G, respectively, as in hu3D6VLv2 L37Q S52G L54G. In some humanized 3D6 antibodies, positions L37, L52, and L54 are occupied by Q, G, and R, respectively, as in hu3D6VLv2 L37Q S52G L54R. In some humanized 3D6 antibodies, positions L37, L52, and L54 are occupied by Q, G, and T, respectively, as in hu3D6VLv2 L37Q S52G L54T. In some humanized 3D6 antibodies, positions L37, L52, and L54 are occupied by Q, G, and D, respectively, as in hu3D6VLv2 L37Q S52G L54D.
[0378] In some humanized 3D6 antibodies, positions L37 and L54 are occupied Q and R, respectively, as in hu3D6VLv2 L37Q L54R. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and G, respectively, as in hu3D6VLv2 L37Q L54G. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and D, respectively, as in hu3D6VLv2 L37Q L54D. In some humanized 3D6 antibodies, positions L37 and L50 are occupied by Q and G, respectively, as in hu3D6VLv2 L37Q L50G. In some humanized 3D6 antibodies, positions L37 and L50 are occupied by Q and D, respectively, as in hu3D6VLv2 L37Q L50D. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and T, respectively, as in hu3D6VLv2 L37Q L54T. In some humanized 3D6 antibodies, positions L37 and L52 are occupied by Q and G, respectively, as in hu3D6VLv2 L37Q S52G. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and E, respectively, as in hu3D6VLv2 L37Q L54E. [0379] In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, D, and G, respectively, as in hu3D6VLv2 L37Q L50D L54G. In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, D, and R, respectively, as in hu3D6VLv2 L37Q L50D L54R. In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, E, and G, respectively, as in hu3D6VLv2 L37Q L50E L54G. In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, E, and R, respectively, as in hu3D6VLv2 L37Q_L50E_L54R.
[0380] In some humanized 3D6 antibodies, positions L37, L50, L54, and L100 are occupied by Q, G, R, and Q, respectively, as in hu3D6VLv2 L37Q_L50G_L54R_G100Q. In some humanized 3D6 antibodies, positions L37, L50, L54, and L100 are occupied by Q, G, G, and Q, respectively, as in hu3D6VLv2 L37Q_L50G_L54G_G100Q. In some humanized 3D6 antibodies, positions L37, L52, L54, and LI 00 are occupied by Q, G, R, and Q, respectively, as in hu3D6VLv2 L37Q_S52G_L54R_G100Q. In some humanized 3D6 antibodies, positions L37, L52, L54, and LI 00 are occupied by Q, G, D, and Q, respectively, as in hu3D6VLv2
L37Q_S52G_L54D_G100Q. In some humanized 3D6 antibodies, positions L37, L50, L54, and LI 00 are occupied by Q, D, G, and Q, respectively, as in hu3D6VLv2
L37Q_L50D_L54G_G100Q. In some humanized 3D6 antibodies, positions L37, L50, L54, and LI 00 are occupied by Q, D, R, and Q, respectively, as in hu3D6VLv2
L37Q_L50D_L54R_G100Q. In some humanized 3D6 antibodies, positions L37, L50, L54, and LI 00 are occupied by Q, V, D, and Q, respectively, as in hu3D6VLv2
L37Q_L50V_L54D_G100Q.
[0381] In some humanized 3D6 antibodies, position L37 is occupied by Q, as in hu3D6VLv2 L37Q. In some humanized 3D6 antibodies, position LI 00 is occupied by Q as in hu3D6VLv2 G100Q.
[0382] Some humanized 3D6 antibodies comprise a mature heavy chain variable region comprising CDRs HI, H2 and H3 comprising SEQ ID NOs:8, 9, and 10, respectively except that position H28 can be occupied by N or T, H54 can be occupied by N or D, H56 can be occupied by D or E, position H58 occupied by V or I, and position H60 can be occupied by D or E, and a mature light chain variable region comprising CDRs LI, L2 and L3 comprising SEQ ID NOs.:
12, 13, and 14 respectively, except that position L24 can be occupied by K or R, position L50 can be occupied by L, E. D, G, or V, position L52 can be occupied by S or G, and position L54 can be occupied by L, D, G, N, E, Q, K, R, T, V, or S, wherein at least one of the following positions is occupied by the amino acid as specified: HI is occupied by Q, H5 is occupied by Q, HI 1 is occupied by L, H20 is occupied by L, H23 is occupied by T, H38 is occupied by K, H75 is occupied by S, H56 is occupied by E, H58 is occupied by I, H60 is occupied by E, H82c is occupied by V, L10 is occupied by T, L17 is occupied by E, L24 is occupied by R, L37 is occupied by Q, L47 is occupied by G, N, D, E, P, T, S, or A, L48 is occupied by G or D, L49 is occupied by E, L50 is occupied by E, D, G, or V, L52 is occupied by G, L54 is occupied by D,
G, N. E, Q, K, R, T, V, or S, L83 is occupied by L, L86 is occupied by H, LI 00 is occupied by Q, L106 is occupied by L.
[0383] Some humanized 3D6 antibodies comprise three light chain CDRs and three heavy chain CDRs of monoclonal antibody 3D6, wherein 3D6 is a mouse antibody characterized by a heavy chain variable region having an amino acid sequence comprising SEQ ID NO:7 and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 11, except that position H27 can be occupied by F or Y, position H28 can be occupied by N or T, position H29 can be occupied by I or F, position H30 can be occupied by K or T, position H51 can be occupied by I or V, position H54 can be occupied by N or D, position H60 can be occupied by D, A, or E, position H61 can be occupied by P or E, position HI 02 can be occupied by F or Y, position L50 can be occupied by L, E. D, G, or V, position L52 can be occupied by S or G, and position L54 can be occupied by L, D, G, N, E, Q, K, R, T, V, or S, wherein at least one of the following positions is occupied by the amino acid as specified: L37 is occupied by Q, L47 is occupied by G, N, D, E, P, T, S, or A, L48 is occupied by G or D, L49 is occupied by E, L50 is occupied by E, D, G, or V, L52 is occupied by G, L54 is occupied by D, G, N. E, Q, K, R, T, V, or S, LI 00 is occupied by Q, H60 is occupied by E, H82c is occupied by V.
[0384] In some humanized 3D6 antibodies, the variable heavy chain has > 85% identity to human sequence. In some humanized 3D6 antibodies, the variable light chain has > 85% identity to human sequence. In some humanized 3D6 antibodies, each of the variable heavy chain and variable light chain has > 85% identity to human germline sequence. In some humanized 3D6 antibodies, the three heavy chain CDRs are as defined by Kabat/Chothia Composite (SEQ ID NOs:8, 9, and 10) and the three light chain CDRs are as defined by
Kabat/Chothia Composite (SEQ ID NOs: 12, 13, and 14); provided that position H28 is occupied by N or T, position H54 is occupied by N or D, position H56 is occupied by D or E, position H58 is occupied by V or I, position H60 is occupied by D or E, position L24 is occupied by K or R, position L50 is occupied by L, E, D, G, or V, position L52 is occupied by S or G, and position L54 is occupied by L, D, G, N, E, Q, K, R, T, V, or S. In some humanized 3D6 antibodies, Kabat/Chothia Composite CDR-H1 has an amino acid sequence comprising SEQ ID NO:86. In some humanized 3D6 antibodies, Rabat CDR-H2 has an amino acid sequence comprising SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:92, or SEQ ID NO: 149. In some humanized 3D6 antibodies, Rabat CDR-L1 has an amino acid sequence comprising SEQ ID NO:89. In some humanized 3D6 antibodies, Rabat CDR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 150-175.
[0385] The CDR regions of such humanized antibodies can be identical or substantially identical to the CDR regions of 3D6, The CDR regions can be defined by any conventional definition (e.g., Chothia, or composite of Chothia and Rabat) but are preferably as defined by Rabat.
[0386] Variable regions framework positions are in accordance with Rabat numbering unless otherwise stated. Other such variants typically differ from the sequences of the exemplified Hu3D6 heavy and light chains by a small number (e.g., typically no more than 1, 2, 3, 5, 10, or 15) of replacements, deletions or insertions. Such differences are usually in the framework but can also occur in the CDRs.,
[0387] A possibility for additional variation in humanized 3D6 variants is additional backmutations in the variable region frameworks. Many of the framework residues not in contact with the CDRs in the humanized mAb can accommodate substitutions of amino acids from the corresponding positions of the donor mouse mAb or other mouse or human antibodies, and even many potential CDR-contact residues are also amenable to substitution. Even amino acids within the CDRs may be altered, for example, with residues found at the corresponding position of the human acceptor sequence used to supply variable region frameworks. In addition, alternate human acceptor sequences can be used, for example, for the heavy and/or light chain.
If different acceptor sequences are used, one or more of the backmutations recommended above may not be performed because the corresponding donor and acceptor residues are already the same without backmutations. [0388] Preferably, replacements or backmutations in humanized 3D6 variants (whether or not conservative) have no substantial effect on the binding affinity or potency of the humanized mAb, that is, its ability to bind to tau.
[0389] The humanized 3D6 antibodies are further characterized by their ability to bind both phosphorylated and unphosphorylated tau and misfolded/aggregated forms of tau.
D. Chimeric and Veneered Antibodies
[0390] The invention further provides chimeric and veneered forms of non-human antibodies, particularly the 3D6 antibodies of the examples.
[0391] A chimeric antibody is an antibody in which the mature variable regions of light and heavy chains of a non-human antibody ( e.g ., a mouse) are combined with human light and heavy chain constant regions. Such antibodies substantially or entirely retain the binding specificity of the mouse antibody, and are about two-thirds human sequence. In an embodiment, a chimeric 3D6 antibody has a heavy chain amino acid sequence of SEQ ID NO: 72 and a light chain amino acid sequence of SEQ ID NO: 73.
[0392] A veneered antibody is a type of humanized antibody that retains some and usually all of the CDRs and some of the non-human variable region framework residues of a non-human antibody but replaces other variable region framework residues that may contribute to B- or T- cell epitopes, for example exposed residues (Padlan, Mol. Immunol. 28:489, 1991) with residues from the corresponding positions of a human antibody sequence. The result is an antibody in which the CDRs are entirely or substantially from a non-human antibody and the variable region frameworks of the non-human antibody are made more human-like by the substitutions.
Veneered forms of the 3D6 antibody are included in the invention.
E. Human Antibodies
[0393] Human antibodies against tau or a fragment thereof (e.g., amino acid residues 199-213 and/or 262-276 of SEQ ID NO:3, corresponding to amino acid residues 257-271 and/or 320-334, respectively, of SEQ ID NO: l or amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: 1 or any combination of 2, 3 or all 4 thereof) are provided by a variety of techniques described below. Some human antibodies are selected by competitive binding experiments, by the phage display method of Winter, above, or otherwise, to have the same epitope specificity as a particular mouse antibody, such as one of the mouse monoclonal antibodies described in the examples. Human antibodies can also be screened for a particular epitope specificity by using only a fragment of tau, such as a tau fragment containing only amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257- 271 or 320-334, respectively, of SEQ ID NO: l) or containing only amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: 1, as the target antigen, and/or by screening antibodies against a collection of tau variants, such as tau variants containing various mutations within amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or 320-334, respectively, of SEQ ID NO: 1), or within amino acid residues 259- 268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: l.
[0394] Methods for producing human antibodies include the trioma method of Oestberg et al ., Hybridoma 2:361-367 (1983); Oestberg, U.S. Patent No. 4,634,664; and Engleman et al., US Patent 4,634,666, use of transgenic mice including human immunoglobulin genes (see, e.g., Lonberg et al, W093/12227 (1993); US 5,877,397; US 5,874,299; US 5,814,318; US 5,789,650; US 5,770,429; US 5,661,016; US 5,633,425; US 5,625,126; US 5,569,825; US 5,545,806;
Neuberger, Nat. Biotechnol. 14:826 (1996); and Kucherlapati, WO 91/10741 (1991)) phage display methods (see, e.g., Dower et al., WO 91/17271; McCafferty et al., WO 92/01047; US 5,877,218; US 5,871,907; US 5,858,657; US 5,837,242; US 5,733,743; and US 5,565,332); and methods described in WO 2008/081008 (e.g., immortalizing memory B cells isolated from humans, e.g., with EBV, screening for desired properties, and cloning and expressing
recombinant forms).
F. Selection of Constant Region
[0395] The heavy and light chain variable regions of chimeric, veneered or humanized antibodies can be linked to at least a portion of a human constant region. The choice of constant region depends, in part, whether antibody-dependent cell-mediated cytotoxicity, antibody dependent cellular phagocytosis and/or complement dependent cytotoxicity are desired. For example, human isotypes IgGl and IgG3 have complement-dependent cytotoxicity and human isotypes IgG2 and IgG4 do not. Human IgGl and IgG3 also induce stronger cell mediated effector functions than human IgG2 and IgG4. Light chain constant regions can be lambda or kappa. Numbering conventions for constant regions include EU numbering (Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969)), Kabat numbering (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1991, IMGT unique numbering (Lefranc M.-P. et al., IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains, Dev. Comp. Immunol., 29, 185- 203 (2005), and IMGT exon numbering (Lefranc, supra).
[0396] One or several amino acids at the amino or carboxy terminus of the light and/or heavy chain, such as the C-terminal lysine of the heavy chain, may be missing or derivatized in a proportion or all of the molecules. Substitutions can be made in the constant regions to reduce or increase effector function such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., US Patent No. 5,624,821; Tso et al., US Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). Exemplary substitutions include a Gin at position 250 and/or a Leu at position 428 (EU numbering is used in this paragraph for the constant region) for increasing the half-life of an antibody. Substitution at any or all of positions 234, 235, 236 and/or 237 reduce affinity for Fey receptors, particularly FcyRI receptor (see, e.g., US
6.624.821). An alanine substitution at positions 234, 235, and 237 of human IgGl can be used for reducing effector functions. Some antibodies have alanine substitution at positions 234, 235 and 237 of human IgGl for reducing effector functions. Optionally, positions 234, 236 and/or 237 in human IgG2 are substituted with alanine and position 235 with glutamine (see, e.g., US
5.624.821) . In some antibodies, a mutation at one or more of positions 241, 264, 265, 270, 296, 297, 322, 329, and 331 by EU numbering of human IgGl is used. In some antibodies, a mutation at one or more of positions 318, 320, and 322 by EU numbering of human IgGl is used. In some antibodies, positions 234 and/or 235 are substituted with alanine and/or position 329 is substituted with glycine. In some antibodies, positions 234 and 235 are substituted with alanine. In some antibodies, the isotype is human IgG2 or IgG4.
[0397] Antibodies can be expressed as tetramers containing two light and two heavy chains, as separate heavy chains, light chains, as Fab, Fab', F(ab')2, and Fv, or as single chain antibodies in which heavy and light chain mature variable domains are linked through a spacer.
[0398] Human constant regions show allotypic variation and isoallotypic variation between different individuals, that is, the constant regions can differ in different individuals at one or more polymorphic positions. Isoallotypes differ from allotypes in that sera recognizing an isoallotype bind to a non-polymorphic region of a one or more other isotypes. Thus, for example, another heavy chain constant region is of IgGl Glm3 with or without the C-terminal lysine. Reference to a human constant region includes a constant region with any natural allotype or any permutation of residues occupying positions in natural allotypes. An exemplary heavy chain constant region is SEQ ID NO: 176, with or without the C-terminal lysine, and an exemplary light chain constant region is SEQ ID NO: 177.
(7. Expression of Recombinant Antibodies
[0399] A number of methods are known for producing chimeric and humanized antibodies using an antibody-expressing cell line ( e.g ., hybridoma). For example, the immunoglobulin variable regions of antibodies can be cloned and sequenced using well known methods. In one method, the heavy chain variable VH region is cloned by RT-PCR using mRNA prepared from hybridoma cells. Consensus primers are employed to the VH region leader peptide
encompassing the translation initiation codon as the 5' primer and a g2b constant regions specific 3' primer. Exemplary primers are described in U.S. patent publication US 2005/0009150 by Schenk et al. (hereinafter“Schenk”). The sequences from multiple, independently derived clones can be compared to ensure no changes are introduced during amplification. The sequence of the VH region can also be determined or confirmed by sequencing a VH fragment obtained by 5' RACE RT-PCR methodology and the 3' g2b specific primer.
[0400] The light chain variable VL region can be cloned in an analogous manner. In one approach, a consensus primer set is designed for amplification of VL regions using a 5’ primer designed to hybridize to the VL region encompassing the translation initiation codon and a 3' primer specific for the Ck region downstream of the V-J joining region. In a second approach, 5'RACE RT-PCR methodology is employed to clone a VL encoding cDNA. Exemplary primers are described in Schenk, supra. The cloned sequences are then combined with sequences encoding human (or other non-human species) constant regions.
[0401] In one approach, the heavy and light chain variable regions are re-engineered to encode splice donor sequences downstream of the respective VDJ or VJ junctions and are cloned into a mammalian expression vector, such as pCMV-hyl for the heavy chain and pCMV-Mcl for the light chain. These vectors encode human gΐ and Ck constant regions as exonic fragments downstream of the inserted variable region cassette. Following sequence verification, the heavy chain and light chain expression vectors can be co-transfected into CHO cells to produce chimeric antibodies. Conditioned media is collected 48 hours post-transfection and assayed by western blot analysis for antibody production or ELISA for antigen binding. The chimeric antibodies are humanized as described above. [0402] Chimeric, veneered, humanized, and human antibodies are typically produced by recombinant expression. Recombinant polynucleotide constructs typically include an expression control sequence operably linked to the coding sequences of antibody chains, including naturally associated or heterologous expression control elements, such as a promoter. The expression control sequences can be promoter systems in vectors capable of transforming or transfecting eukaryotic or prokaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences and the collection and purification of the crossreacting antibodies.
[0403] These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, e.g ., ampicillin resistance or hygromycin resistance, to permit detection of those cells transformed with the desired DNA sequences.
[0404] E. coli is one prokaryotic host useful for expressing antibodies, particularly antibody fragments. Microbes, such as yeast, are also useful for expression. Saccharomyces is a yeast host with suitable vectors having expression control sequences, an origin of replication, termination sequences, and the like as desired. Typical promoters include 3 -phosphogly cerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.
[0405] Mammalian cells can be used for expressing nucleotide segments encoding
immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH
Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed, and include CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas including Sp2/0 and NS0. The cells can be nonhuman. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al ., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Expression control sequences can include promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. See Co et al., J.
Immunol. 148: 1149 (1992). [0406] Alternatively, antibody coding sequences can be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g ., U.S. Pat. No. 5,741,957; U.S. Pat. No. 5,304,489; and U.S. Pat. No. 5,849,992). Suitable transgenes include coding sequences for light and/or heavy chains operably linked with a promoter and enhancer from a mammary gland specific gene, such as casein or beta lactoglobulin.
[0407] The vectors containing the DNA segments of interest can be transferred into the host cell by methods depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics, or viral-based transfection can be used for other cellular hosts. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. For production of transgenic animals, transgenes can be microinjected into fertilized oocytes or can be incorporated into the genome of embryonic stem cells or induced pluripotent stem cells (iPSCs), and the nuclei of such cells transferred into enucleated oocytes.
[0408] Having introduced vector(s) encoding antibody heavy and light chains into cell culture, cell pools can be screened for growth productivity and product quality in serum -free media. Top-producing cell pools can then be subjected of FACS-based single-cell cloning to generate monoclonal lines. Specific productivities above 50 pg or 100 pg per cell per day, which correspond to product titers of greater than 7.5 g/L culture, can be used. Antibodies produced by single cell clones can also be tested for turbidity, filtration properties, PAGE, IEF, UV scan, HP- SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and binding assay, such as ELISA or Biacore. A selected clone can then be banked in multiple vials and stored frozen for subsequent use.
[0409] Once expressed, antibodies can be purified according to standard procedures of the art, including protein A capture, HPLC purification, column chromatography, gel electrophoresis and the like (see generally , Scopes, Protein Purification (Springer-Verlag, NY, 1982)).
[0410] Methodology for commercial production of antibodies can be employed, including codon optimization, selection of promoters, selection of transcription elements, selection of terminators, serum-free single cell cloning, cell banking, use of selection markers for
amplification of copy number, CHO terminator, or improvement of protein titers (see, e.g., US 5,786,464; US 6,114,148; US 6,063,598; US 7,569,339; W02004/050884; W02008/012142; W02008/012142; W02005/019442; W02008/107388; W02009/027471; and US 5,888,809).
IV. Active Immunogens
[0411] An agent used for active immunization serves to induce in a patient the same types of antibody described in connection with passive immunization above. Agents used for active immunization can be the same types of immunogens used for generating monoclonal antibodies in laboratory animals, e.g ., a peptide of 3-15 or 3-12 or 5-12, or 5-8 contiguous amino acids from a region of tau corresponding to residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to residues 257-271 or 320-334, respectively, of SEQ ID NO: l), such as, for example, a peptide including residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to residues 257-271 or 320-334, respectively, of SEQ ID NO: 1) or from a region of tau corresponding to residues 259- 268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: l, such as for example, a peptide including residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: l. For inducing antibodies binding to the same or overlapping epitope as 3D6, the epitope specificity of these antibodies can be mapped (e.g, by testing binding to a series of overlapping peptides spanning tau). A fragment of tau consisting of or including or overlapping the epitope can then be used as an immunogen. Such fragments are typically used in unphosphorylated form.
[0412] The heterologous carrier and adjuvant, if used may be the same as used for generating monoclonal antibody, but may also be selected for better pharmaceutical suitability for use in humans. Suitable carriers include serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, or a toxoid from other pathogenic bacteria, such as diphtheria (e.g, CRM 197), E. coli, cholera, or H. pylori, or an attenuated toxin derivative. T cell epitopes are also suitable carrier molecules. Some conjugates can be formed by linking agents of the invention to an immunostimulatory polymer molecule (e.g, tripalmitoyl- S-glycerine cysteine (Par Cys), mannan (a mannose polymer), or glucan (a b 1— >2 polymer)), cytokines (e.g, IL-1, IL-1 alpha and b peptides, IL-2, g-INF, IL-10, GM-CSF), and chemokines (e.g, MIPl-a and b, and RANTES). Immunogens may be linked to the carriers with or without spacers amino acids (e.g, gly-gly). Additional carriers include virus-like particles. Virus-like particles (VLPs), also called pseudovirions or virus-derived particles, represent subunit structures composed of multiple copies of a viral capsid and/or envelope protein capable of self-assembly into VLPs of defined spherical symmetry in vivo. (Powilleit, et ak, (2007) PLoS ONE 2(5):e415.) Alternatively, peptide immunogens can be linked to at least one artificial T-cell epitope capable of binding a large proportion of MHC Class II molecules., such as the pan DR epitope ("PADRE"). PADRE is described in US 5,736,142, WO 95/07707, and Alexander J et al, Immunity, 1 :751-761 (1994). Active immunogens can be presented in multimeric form in which multiple copies of an immunogen and/or its carrier are presented as a single covalent molecule.
[0413] Fragments are often administered with pharmaceutically acceptable adjuvants. The adjuvant increases the titer of induced antibodies and/or the binding affinity of induced antibodies relative to the situation if the peptide were used alone. A variety of adjuvants can be used in combination with an immunogenic fragment of tau to elicit an immune response.
Preferred adjuvants augment the intrinsic response to an immunogen without causing
conformational changes in the immunogen that affect the qualitative form of the response.
Preferred adjuvants include aluminum salts, such as aluminum hydroxide and aluminum phosphate, 3 De-O-acylated monophosphoryl lipid A (MPL™) ( see GB 2220211 (RIBI
ImmunoChem Research Inc., Hamilton, Montana, now part of Corixa). Stimulon™ QS-21 is a triterpene glycoside or saponin isolated from the bark of the Quillaja Saponaria Molina tree found in South America (see Kensil et al. , in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); US 5,057,540), (Aquila
BioPharmaceuticals, Framingham, MA; now Antigenics, Inc., New York, NY). Other adjuvants are oil in water emulsions (such as squalene or peanut oil), optionally in combination with immune stimulants, such as monophosphoryl lipid A (see Stoute et al. , N. Engl. J. Med. 336, 86- 91 (1997)), pluronic polymers, and killed mycobacteria. Ribi adjuvants are oil-in-water emulsions. Ribi contains a metabolizable oil (squalene) emulsified with saline containing Tween 80. Ribi also contains refined mycobacterial products which act as immunostimulants and bacterial monophosphoryl lipid A. Another adjuvant is CpG (WO 98/40100). Adjuvants can be administered as a component of a therapeutic composition with an active agent or can be administered separately, before, concurrently with, or after administration of the therapeutic agent.
[0414] Analogs of natural fragments of tau that induce antibodies against tau can also be used. For example, one or more or all L-amino acids can be substituted with D amino acids in such peptides. Also the order of amino acids can be reversed (retro peptide). Optionally a peptide includes all D-amino acids in reverse order (retro-inverso peptide). Peptides and other compounds that do not necessarily have a significant amino acid sequence similarity with tau peptides but nevertheless serve as mimetics of tau peptides and induce a similar immune response. Anti- idiotypic antibodies against monoclonal antibodies to tau as described above can also be used. Such anti-id antibodies mimic the antigen and generate an immune response to it (see Essential Immunology, Roit ed., Blackwell Scientific Publications, Palo Alto, CA 6th ed., p. 181).
[0415] Peptides (and optionally a carrier fused to the peptide) can also be administered in the form of a nucleic acid encoding the peptide and expressed in situ in a patient. A nucleic acid segment encoding an immunogen is typically linked to regulatory elements, such as a promoter and enhancer that allow expression of the DNA segment in the intended target cells of a patient. For expression in blood cells, as is desirable for induction of an immune response, promoter and enhancer elements from light or heavy chain immunoglobulin genes or the CMV major intermediate early promoter and enhancer are suitable to direct expression. The linked regulatory elements and coding sequences are often cloned into a vector. Antibodies can also be administered in the form of nucleic acids encoding the antibody heavy and/or light chains. If both heavy and light chains are present, the chains are preferably linked as a single chain antibody. Antibodies for passive administration can also be prepared e.g ., by affinity chromatography from sera of patients treated with peptide immunogens.
[0416] The DNA can be delivered in naked form (z.e., without colloidal or encapsulating materials). Alternatively a number of viral vector systems can be used including retroviral systems (see, e.g. , Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993));
adenoviral vectors (see, e.g. , Bett et al, J. Virol. 67, 591 1 (1993)) including retrovirus derived vectors such MMLV, HIV-1, and ALV; adeno-associated virus vectors (see, e.g. , Zhou et al., J. Exp. Med. 179, 1867 (1994)), lentiviral vectors such as those based on HIV or FIV gag sequences, viral vectors from the pox family including vaccinia virus and the avian pox viruses, viral vectors from the alpha virus genus such as those derived from Sindbis and Semliki Forest Viruses (see, e.g. , Dubensky et al., J. Virol. 70, 508-519 (1996)), Venezuelan equine encephalitis virus (see US 5,643,576) and rhabdoviruses, such as vesicular stomatitis virus (see WO
96/34625)and papillomaviruses (Ohe et al., Human Gene Therapy 6, 325-333 (1995); Woo et al, WO 94/12629 and Xiao & Brandsma, Nucleic Acids. Res. 24, 2630-2622 (1996)). [0417] DNA encoding an immunogen, or encoding the antibody heavy and/or light chains, or a vector containing the same, can be packaged into liposomes. Suitable lipids and related analogs are described by US 5,208,036, US 5,264,618, US 5,279,833, and US 5,283,185. Vectors and DNA encoding an immunogen or encoding the antibody heavy and/or light chains, can also be adsorbed to or associated with particulate carriers, examples of which include polymethyl methacrylate polymers and polylactides and poly(lactide-co-glycolides), (see, e.g ., McGee et al., J. Micro Encap. 1996).
[0418] Vectors or segments therefrom encoding the antibody heavy and/or light chains can be incorporated in cells ex vivo, for example to cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy ) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the transgenes (see, e.g., WO 2017/091512). Exemplary patient-derived cells include patient derived induced pluripotent stem cells (iPSCs) or other types of stem cells (embryonic, hematopoietic, neural, or mesenchymal).
[0419] A vector or segment therefrom encoding the antibody heavy and/or light chains can be introduced into any region of interest in cells ex vivo, such as an albumin gene or other safe harbor gene. Cells incorporating the vector can be implanted with or without prior
differentiation. Cells can be implanted into a specific tissue, such as a secretory tissue or a location of pathology, or systemically, such as by infusion into the blood. For example, cells can be implanted into a secretory tissue of a patient, such as the liver, optionally with prior differentiation to cells present in that tissue, such as hepatocytes in the case of a liver. Expression of the antibody in the liver results in secretion of the antibody to the blood.
H. Antibody Screening Assays
[0420] Antibodies can be initially screened for the intended binding specificity as described above. Active immunogens can likewise be screened for capacity to induce antibodies with such binding specificity. In this case, an active immunogen is used to immunize a laboratory animal and the resulting sera tested for the appropriate binding specificity.
[0421] Antibodies having the desired binding specificity can then be tested in cellular and animal models. The cells used for such screening are preferentially neuronal cells. A cellular model of tau pathology has been reported in which neuroblastoma cells are transfected with a four-repeat domain of tau, optionally with a mutation associated with tau pathology ( e.g ., delta K280, see Khlistunova, Current Alzheimer Research 4, 544-546 (2007)). In another model, tau is induced in the neuroblastoma N2a cell line by the addition of doxycyclin. The cell models enable one to study the toxicity of tau to cells in the soluble or aggregated state, the appearance of tau aggregates after switching on tau gene expression, the dissolution of tau aggregates after switching the gene expression off again, and the efficiency of antibodies in inhibiting formation of tau aggregates or disaggregating them.
[0422] Antibodies or active immunogens can also be screened in transgenic animal models of diseases associated with tau. Such transgenic animals can include a tau transgene (e.g., any of the human isoforms) and optionally a human APP transgene among others, such as a kinase that phosphorylates tau, ApoE, presenilin or alpha synuclein. Such transgenic animals are disposed to develop at least one sign or symptom of a disease associated with tau.
[0423] An exemplary transgenic animal is the K3 line of mice (Itner et al., Proc. Natl. Acad. Sci. USA 105(41): 15997-6002 (2008)). These mice have a human tau transgene with a K 369 I mutation (the mutation is associated with Pick’s disease) and a Thy 1.2 promoter. This model shows a rapid course of neurodegeneration, motor deficit and degeneration of afferent fibers and cerebellar granule cells. Another exemplary animal is the JNPL3 line of mice. These mice have a human tau transgene with a P301L mutation (the mutation is associated with frontotemporal dementia) and a Thy 1.2 promoter (Taconic, Germantown, N.Y., Lewis, et al., Nat Genet.
25:402-405 (2000)). These mice have a more gradual course of neurodegeneration. The mice develop neurofibrillary tangles in several brain regions and spinal cord, which is hereby incorporated by reference in its entirety). This is an excellent model to study the consequences of tangle development and for screening therapy that may inhibit the generation of these aggregates. Another advantage of these animals is the relatively early onset of pathology. In the homozygous line, behavioral abnormalities associated with tau pathology can be observed at least as early as 3 months, but the animals remain relatively healthy at least until 8 months of age. In other words, at 8 months, the animals ambulate, feed themselves, and can perform the behavioral tasks sufficiently well to allow the treatment effect to be monitored. Active immunization of these mice for 6-13 months with - Al wl KLH-PHF-1 generated titers of about 1,000 and showed fewer neurofibrillary tangles, less pSer422, and reduced weight loss relative to untreated control ice. [0424] The activity of antibodies or active agents can be assessed by various criteria including reduction in amount of total tau or phosphorylated tau, reduction in other pathological characteristics, such as amyloid deposits of Ab, and inhibition or delay or behavioral deficits. Active immunogens can also be tested for induction of antibodies in the sera. Both passive and active immunogens can be tested for passage of antibodies across the blood brain barrier into the brain of a transgenic animal. Antibodies or fragments inducing an antibody can also be tested in non-human primates that naturally or through induction develop symptoms of diseases characterized by tau. Tests on an antibody or active agent are usually performed in conjunction with a control in which a parallel experiment is conduct except that the antibody or active agent is absent ( e.g ., replaced by vehicle). Reduction, delay or inhibition of signs or symptoms disease attributable to an antibody or active agent under test can then be assessed relative to the control. V. Patients Amenable to Treatment
[0425] The presence of neurofibrillary tangles has been found in several diseases including Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia,
frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal
degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), and progressive supranuclear palsy (PSP). The present regimes can also be used in treatment or prophylaxis of any of these diseases. Because of the widespread association between
neurological diseases and conditions and tau, the present regimes can be used in treatment or prophylaxis of any subject showing elevated levels of tau or phosphorylated tau (e.g., in the CSF) compared with a mean value in individuals without neurological disease. The present regimes can also be used in treatment or prophylaxis of neurological disease in individuals having a mutation in tau associated with neurological disease. The present methods are particularly suitable for treatment or prophylaxis of Alzheimer’s disease, and especially in patients.
[0426] Patients amenable to treatment include individuals at risk of disease but not showing symptoms, as well as patients presently showing symptoms. Patients at risk of disease include those having a known genetic risk of disease. Such individuals include those having relatives who have experienced this disease, and those whose risk is determined by analysis of genetic or biochemical markers. Genetic markers of risk include mutations in tau, such as those discussed above, as well as mutations in other genes associated with neurological disease. For example, the ApoE4 allele in heterozygous and even more so in homozygous form is associated with risk of Alzheimer’s disease. Other markers of risk of Alzheimer’s disease include mutations in the APP gene, particularly mutations at position 717 and positions 670 and 671 referred to as the Hardy and Swedish mutations respectively, mutations in the presenilin genes, PS1 and PS2, a family history of AD, hypercholesterolemia or atherosclerosis. Individuals presently suffering from Alzheimer’s disease can be recognized by PET imaging, from characteristic dementia, as well as the presence of risk factors described above. In addition, a number of diagnostic tests are available for identifying individuals who have AD. These include measurement of CSF tau or phospho-tau and Ab42 levels. Elevated tau or phospho-tau and decreased Ab42 levels signify the presence of AD. Some mutations associated with Parkinson’s disease. Ala30Pro or Ala53, or mutations in other genes associated with Parkinson's disease such as leucine-rich repeat kinase, PARK8. Individuals can also be diagnosed with any of the neurological diseases mentioned above by the criteria of the DSM IV TR.
[0427] In asymptomatic patients, treatment can begin at any age ( e.g ., 10, 20, 30). Usually, however, it is not necessary to begin treatment until a patient reaches 40, 50, 60 or 70 years of age. Treatment typically entails multiple dosages over a period of time. Treatment can be monitored by assaying antibody levels over time. If the response falls, a booster dosage is indicated. In the case of potential Down’s syndrome patients, treatment can begin antenatally by administering therapeutic agent to the mother or shortly after birth.
I. Nucleic Acids
[0428] The invention further provides nucleic acids encoding any of the heavy and light chains described above (e.g., SEQ ID NO:7, SEQ ID NO: l l, SEQ ID NOs: 76-80, SEQ ID NOs:90-91, SEQ ID NOs: 146-148, SEQ ID NOs:83-85, SEQ ID NOs:93-145, and SEQ ID NOs: 178-181). An exemplary nucleic acid encoding a heavy chain of the invention is SEQ ID NO: 182, and an exemplary nucleic acid encoding a light chain of the invention is SEQ ID NO: 183. Optionally, such nucleic acids further encode a signal peptide and can be expressed with the signal peptide linked to the variable region. Coding sequences of nucleic acids can be operably linked with regulatory sequences to ensure expression of the coding sequences, such as a promoter, enhancer, ribosome binding site, transcription termination signal, and the like. The regulatory sequences can include a promoter, for example, a prokaryotic promoter or a eukaryotic promoter. The nucleic acids encoding heavy or light chains can be codon-optimized for expression in a host cell. The nucleic acids encoding heavy and light chains can encode a selectable gene. The nucleic acids encoding heavy and light chains can occur in isolated form or can be cloned into one or more vectors. The nucleic acids can be synthesized by, for example, solid state synthesis or PCR of overlapping oligonucleotides. Nucleic acids encoding heavy and light chains can be joined as one contiguous nucleic acid, e.g., within an expression vector, or can be separate, e.g., each cloned into its own expression vector.
J. Conjugated Antibodies
[0429] Conjugated antibodies that specifically bind to antigens, such as tau, are useful in detecting the presence of tau; monitoring and evaluating the efficacy of therapeutic agents being used to treat patients diagnosed with Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP); inhibiting or reducing aggregation of tau; inhibiting or reducing tau fibril formation; reducing or clearing tau deposits; stabilizing non-toxic
conformations of tau; or treating or effecting prophylaxis of Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann- Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies,
Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP) in a patient. For example, such antibodies can be conjugated with other therapeutic moieties, other proteins, other antibodies, and/or detectable labels. See WO 03/057838; US 8,455,622. Such therapeutic moieties can be any agent that can be used to treat, combat, ameliorate, prevent, or improve an unwanted condition or disease in a patient, such as Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).
[0430] Conjugated therapeutic moieties can include cytotoxic agents, cytostatic agents, neurotrophic agents, neuroprotective agents, radiotherapeutic agents, immunomodulators, or any biologically active agents that facilitate or enhance the activity of the antibody. A cytotoxic agent can be any agent that is toxic to a cell. A cytostatic agent can be any agent that inhibits cell proliferation. A neurotrophic agent can be any agent, including chemical or proteinaceous agents, that promotes neuron maintenance, growth, or differentiation. A neuroprotective agent can be agent, including chemical or proteinaceous agents, that protects neurons from acute insult or degenerative processes. An immunomodulator can be any agent that stimulates or inhibits the development or maintenance of an immunologic response. A radiotherapeutic agent can be any molecule or compound that emits radiation. If such therapeutic moieties are coupled to a tau- specific antibody, such as the antibodies described herein, the coupled therapeutic moieties will have a specific affinity for tau-related disease-affected cells over normal cells. Consequently, administration of the conjugated antibodies directly targets cancer cells with minimal damage to surrounding normal, healthy tissue. This can be particularly useful for therapeutic moieties that are too toxic to be administered on their own. In addition, smaller quantities of the therapeutic moieties can be used.
[0431] Some such antibodies can be modified to act as immunotoxins. See, e.g ., U.S. Patent No. 5,194,594. For example, ricin, a cellular toxin derived from plants, can be coupled to antibodies by using the bifunctional reagents S-acetylmercaptosuccinic anhydride for the antibody and succinimidyl 3-(2-pyridyldithio)propionate for ricin. See Pietersz et al, Cancer Res. 48(16):4469-4476 (1998). The coupling results in loss of B-chain binding activity of ricin, while impairing neither the toxic potential of the A-chain of ricin nor the activity of the antibody. Similarly, saporin, an inhibitor of ribosomal assembly, can be coupled to antibodies via a disulfide bond between chemically inserted sulfhydryl groups. See Polito el al ., Leukemia 18: 1215-1222 (2004).
[0432] Some such antibodies can be linked to radioisotopes. Examples of radioisotopes include, for example, yttrium90 (90Y), indium111 (l l lln), 131I, "mTc, radiosilver-111, radiosilver- 199, and Bismuth213. Linkage of radioisotopes to antibodies may be performed with conventional bifunction chelates. For radiosilver- 111 and radiosilver- 199 linkage, sulfur-based linkers may be used. See Hazra et al., Cell Biophys. 24-25:1-7 (1994). Linkage of silver radioisotopes may involve reducing the immunoglobulin with ascorbic acid. For radioisotopes such as 11 lln and 90Y, ibritumomab tiuxetan can be used and will react with such isotopes to form 11 lln-ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol ., 48 Suppl LS91-S95 (2001).
[0433] Some such antibodies can be linked to other therapeutic moieties. Such therapeutic moieties can be, for example, cytotoxic, cytostatic, neurotrophic, or neuroprotective. For example, antibodies can be conjugated with toxic chemotherapeutic drugs such as maytansine, geldanamycin, tubulin inhibitors such as tubulin binding agents ( e.g ., auristatins), or minor groove binding agents such as calicheamicin. Other representative therapeutic moieties include agents known to be useful for treatment, management, or amelioration of Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann- Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).
[0434] Antibodies can also be coupled with other proteins. For example, antibodies can be coupled with Fynomers. Fynomers are small binding proteins (e.g., 7 kDa) derived from the human Fyn SH3 domain. They can be stable and soluble, and they can lack cysteine residues and disulfide bonds. Fynomers can be engineered to bind to target molecules with the same affinity and specificity as antibodies. They are suitable for creating multi-specific fusion proteins based on antibodies. For example, Fynomers can be fused to N-terminal and/or C- terminal ends of antibodies to create bi- and tri-specific FynomAbs with different architectures. Fynomers can be selected using Fynomer libraries through screening technologies using FACS, Biacore, and cell-based assays that allow efficient selection of Fynomers with optimal properties. Examples of Fynomers are disclosed in Grabulovski et al, ./. Biol. Chem. 282:3196-3204 (2007); Bertschinger et al., Protein Eng. Des. Sel. 20:57-68 (2007); Schlatter et al., MAhs. 4:497-508 (2011); Banner et al., Acta. Crystallogr. D. Biol. Crystallogr. 69(Pt6): 1124-1137 (2013); and Brack et al, Mol. Cancer Ther. 13:2030-2039 (2014).
[0435] The antibodies disclosed herein can also be coupled or conjugated to one or more other antibodies ( e.g ., to form antibody heteroconjugates). Such other antibodies can bind to different epitopes within tau or can bind to a different target antigen.
[0436] Antibodies can also be coupled with a detectable label. Such antibodies can be used, for example, for diagnosing Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP), and/or for assessing efficacy of treatment. Such antibodies are particularly useful for performing such determinations in subjects having or being susceptible to Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age- related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP), or in appropriate biological samples obtained from such subjects. Representative detectable labels that may be coupled or linked to an antibody include various enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such streptavidin/biotin and avidin/biotin; fluorescent materials, such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as luminol; bioluminescent materials, such as luciferase, luciferin, and aequorin; radioactive materials, such as radiosilver-1 1 1, radiosilver- 199, Bismuth213, iodine (131I, 125I, 123I, 121I,), carbon (14C), sulfur (5S), tritium (¾), indium (115In, 113In, 112In, U 1ln,), technetium (99Tc), thallium (201Ti), gallium (68Ga, 67 Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97RU, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, and 117Tin; positron emitting metals using various positron emission tomographies; nonradioactive paramagnetic metal ions; and molecules that are radiolabelled or conjugated to specific radioisotopes.
[0437] Linkage of radioisotopes to antibodies may be performed with conventional bifunction chelates. For radiosilver- 1 1 1 and radiosilver- 199 linkage, sulfur-based linkers may be used. See Hazra et al., Cell Biophys. 24-25 : 1 -7 (1994). Linkage of silver radioisotopes may involve reducing the immunoglobulin with ascorbic acid. For radioisotopes such as 1 1 lln and 90Y, ibritumomab tiuxetan can be used and will react with such isotopes to form 1 1 lln-ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother.
Pharmacol., 48 Suppl L S91-S95 (2001).
[0438] Therapeutic moieties, other proteins, other antibodies, and/or detectable labels may be coupled or conjugated, directly or indirectly through an intermediate ( e.g ., a linker), to an antibody of the invention. See e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475- 506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of
Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985); and Thorpe et al., Immunol. Rev., 62: 1 19-58 (1982). Suitable linkers include, for example, cleavable and non- cleavable linkers. Different linkers that release the coupled therapeutic moieties, proteins, antibodies, and/or detectable labels under acidic or reducing conditions, on exposure to specific proteases, or under other defined conditions can be employed.
VI. Pharmaceutical Compositions and Methods of Use
[0439] In prophylactic applications, an antibody or agent for inducing an antibody or a pharmaceutical composition the same is administered to a patient susceptible to, or otherwise at risk of a disease ( e.g ., Alzheimer’s disease) in regime (dose, frequency and route of
administration) effective to reduce the risk, lessen the severity, or delay the onset of at least one sign or symptom of the disease. In particular, the regime is preferably effective to inhibit or delay tau or phospho-tau and paired filaments formed from it in the brain, and/or inhibit or delay its toxic effects and/or inhibit/or delay development of behavioral deficits. In therapeutic applications, an antibody or agent to induce an antibody is administered to a patient suspected of, or already suffering from a disease (e.g., Alzheimer’s disease) in a regime (dose, frequency and route of administration) effective to ameliorate or at least inhibit further deterioration of at least one sign or symptom of the disease. In particular, the regime is preferably effective to reduce or at least inhibit further increase of levels of tau, phosphor-tau, or paired filaments formed from it , associated toxi cities and/or behavioral deficits.
[0440] A regime is considered therapeutically or prophylactically effective if an individual treated patient achieves an outcome more favorable than the mean outcome in a control population of comparable patients not treated by methods of the invention, or if a more favorable outcome is demonstrated in treated patients versus control patients in a controlled clinical trial (e.g, a phase II, phase II/III or phase III trial) at the p < 0.05 or 0.01 or even 0.001 level.
[0441] Effective doses of vary depending on many different factors, such as means of administration, target site, physiological state of the patient, whether the patient is an ApoE carrier, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
[0442] Exemplary dosage ranges for antibodies are from about 0.01 to 60 mg/kg, or from about 0.1 to 3 mg/kg or 0.15-2 mg/kg or 0.15-1.5 mg/kg, of patient body weight. Antibody can be administered such doses daily, on alternative days, weekly, fortnightly, monthly, quarterly, or according to any other schedule determined by empirical analysis. An exemplary treatment entails administration in multiple dosages over a prolonged period, for example, of at least six months. Additional exemplary treatment regimes entail administration once per every two weeks or once a month or once every 3 to 6 months.
[0443] The amount of an agent for active administration varies from 0.1-500 pg per patient and more usually from 1-100 or 1-10 pg per injection for human administration. The timing of injections can vary significantly from once a day, to once a year, to once a decade. A typical regimen consists of an immunization followed by booster injections at time intervals, such as 6 week intervals or two months. Another regimen consists of an immunization followed by booster injections 1, 2 and 12 months later. Another regimen entails an injection every two months for life. Alternatively, booster injections can be on an irregular basis as indicated by monitoring of immune response.
[0444] Antibodies or agents for inducing antibodies are preferably administered via a peripheral route (i.e., one in which an administered or induced antibody crosses the blood brain barrier to reach an intended site in the brain. Routes of administration include topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, intranasal, intraocular, or intramuscular. Preferred routes for administration of antibodies are intravenous and subcutaneous. Preferred routes for active immunization are subcutaneous and intramuscular. This type of injection is most typically performed in the arm or leg muscles. In some methods, agents are injected directly into a particular tissue where deposits have accumulated, for example intracranial injection.
[0445] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration).
Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of
administration chosen. For injection, antibodies can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline or acetate buffer (to reduce discomfort at the site of injection). The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Alternatively antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g, sterile pyrogen-free water, before use. [0446] The present regimes can be administered in combination with another agent effective in treatment or prophylaxis of the disease being treated. For example, in the case of Alzheimer’s disease, the present regimes can be combined with immunotherapy against Ab
(WO/2000/072880), cholinesterase inhibitors or memantine or in the case of Parkinson’s disease immunotherapy against alpha synuclein WO/2008/103472, Levodopa, dopamine agonists, COMT inhibitors, MAO-B inhibitors, Amantadine, or anticholinergic agents.
[0447] Antibodies are administered in an effective regime meaning a dosage, route of administration and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration, and/or ameliorates at least one sign or symptom of a disorder being treated. If a patient is already suffering from a disorder, the regime can be referred to as a therapeutically effective regime. If the patient is at elevated risk of the disorder relative to the general population but is not yet experiencing symptoms, the regime can be referred to as a prophylactically effective regime. In some instances, therapeutic or prophylactic efficacy can be observed in an individual patient relative to historical controls or past experience in the same patient. In other instances, therapeutic or prophylactic efficacy can be demonstrated in a preclinical or clinical trial in a population of treated patients relative to a control population of untreated patients.
[0448] Exemplary dosages for an antibody are 0.1-60 mg/kg (e.g., 0.5, 3, 10, 30, or 60 mg/kg), or 0.5-5 mg/kg body weight (e.g., 0.5, 1, 2, 3, 4 or 5 mg/kg) or 10-4000 mg or 10-1500 mg as a fixed dosage. The dosage depends on the condition of the patient and response to prior treatment, if any, whether the treatment is prophylactic or therapeutic and whether the disorder is acute or chronic, among other factors.
[0449] Administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Some antibodies can be administered into the systemic circulation by intravenous or subcutaneous administration. Intravenous administration can be, for example, by infusion over a period such as 30-90 min.
[0450] The frequency of administration depends on the half-life of the antibody in the circulation, the condition of the patient and the route of administration among other factors. The frequency can be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the patient’s condition or progression of the disorder being treated. An exemplary frequency for intravenous administration is between weekly and quarterly over a continuous cause of treatment, although more or less frequent dosing is also possible. For subcutaneous administration, an exemplary dosing frequency is daily to monthly, although more or less frequent dosing is also possible.
[0451] The number of dosages administered depends on whether the disorder is acute or chronic and the response of the disorder to the treatment. For acute disorders or acute exacerbations of a chronic disorder, between 1 and 10 doses are often sufficient. Sometimes a single bolus dose, optionally in divided form, is sufficient for an acute disorder or acute exacerbation of a chronic disorder. Treatment can be repeated for recurrence of an acute disorder or acute exacerbation. For chronic disorders, an antibody can be administered at regular intervals, e.g ., weekly, fortnightly, monthly, quarterly, every six months for at least 1, 5 or 10 years, or the life of the patient.
A. Diagnostics and Monitoring Methods
In Vivo Imaging, Diagnostic Methods, and Optimizing Immunotherapy
[0452] The invention provides methods of in vivo imaging tau protein deposits (e.g, neurofibrillary tangles and tau inclusions) in a patient. The methods work by administering a reagent, such as antibody that binds tau (e.g, a mouse, humanized, chimeric or veneered 3D6 antibody), to the patient and then detecting the agent after it has bound. Antibodies binding to an epitope of tau within amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or 320-334, respectively, of SEQ ID NO: 1) or within amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: l, are preferred. In some methods, the antibody binds to an epitope within amino acid residues 199-213 of SEQ ID NO: 3 (corresponding to amino acid residues 257-271 of SEQ ID NO: 1) , or within amino acids 262- 276 of SEQ ID NO:3 (corresponding to amino acid residues 320-334 of SEQ ID NO: 1). In some methods, the antibody binds to an epitope within amino acid residues 259-268 of SEQ ID NO: 1, within amino acids 290-299 of SEQ ID NO: l, within amino acids 321-330 of SEQ ID NOl, or within amin acids 353-362 of SEQ ID NO: 1. A clearing response to the administered antibodies can be avoided or reduced by using antibody fragments lacking a full-length constant region, such as Fabs. In some methods, the same antibody can serve as both a treatment and diagnostic reagent.
[0453] Diagnostic reagents can be administered by intravenous injection into the body of the patient, or directly into the brain by intracranial injection or by drilling a hole through the skull. The dosage of reagent should be within the same ranges as for treatment methods. Typically, the reagent is labeled, although in some methods, the primary reagent with affinity for tau is unlabeled and a secondary labeling agent is used to bind to the primary reagent. The choice of label depends on the means of detection. For example, a fluorescent label is suitable for optical detection. Use of paramagnetic labels is suitable for tomographic detection without surgical intervention. Radioactive labels can also be detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).
[0454] The methods of in vivo imaging of tau protein deposits are useful to diagnose or confirm diagnosis of a tauopathy, such as Alzheimer's disease, frontotemporal lobar
degeneration, progressive supranuclear palsy and Pick’s disease, or susceptibility to such a disease. For example, the methods can be used on a patient presenting with symptoms of dementia. If the patient has abnormal neurofibrillary tangles, then the patient is likely suffering from Alzheimer's disease. Alternatively, if the patient has abnormal tau inclusions, then depending on the location of the inclusions, the patient may be suffering from frontotemporal lobar degeneration. The methods can also be used on asymptomatic patients. Presence of abnormal tau protein deposits indicates susceptibility to future symptomatic disease. The methods are also useful for monitoring disease progression and/or response to treatment in patients who have been previously diagnosed with a tau-related disease.
[0455] Diagnosis can be performed by comparing the number, size, and/or intensity of labeled loci, to corresponding baseline values. The base line values can represent the mean levels in a population of undiseased individuals. Baseline values can also represent previous levels determined in the same patient. For example, baseline values can be determined in a patient before beginning tau immunotherapy treatment, and measured values thereafter compared with the baseline values. A decrease in values relative to baseline signals a positive response to treatment.
[0456] In some patients, diagnosis of a tauopathy may be aided by performing a PET scan. A PET scan can be performed using, for example, a conventional PET imager and auxiliary equipment. The scan typically includes one or more regions of the brain known in general to be associated with tau protein deposits and one or more regions in which few if any deposits are generally present to serve as controls. [0457] The signal detected in a PET scan can be represented as a multidimensional image.
The multidimensional image can be in two dimensions representing a cross-section through the brain, in three dimensions, representing the three dimensional brain, or in four dimensions representing changes in the three dimensional brain over time. A color scale can be used with different colors indicating different amounts of label and, inferentially, tau protein deposit detected. The results of the scan can also be presented numerically, with numbers relating to the amount of label detected and consequently amount of tau protein deposits. The label present in a region of the brain known to be associated with deposits for a particular tauopathy ( e.g .,
Alzheimer's disease) can be compared with the label present in a region known not to be associated with deposits to provide a ratio indicative of the extent of deposits within the former region. For the same radiolabeled ligand, such ratios provide a comparable measure of tau protein deposits and changes thereof between different patients.
[0458] In some methods, a PET scan is performed concurrent with or in the same patient visit as an MRI or CAT scan. An MRI or CAT scan provides more anatomical detail of the brain than a PET scan. However, the image from a PET scan can be superimposed on an MRI or CAT scan image more precisely indicating the location of PET ligand and inferentially tau deposits relative to anatomical structures in the brain. Some machines can perform both PET scanning and MRI or CAT scanning without the patient changing positions between the scans facilitating superimposition of images.
[0459] Suitable PET ligands include radiolabeled antibodies of the invention (e.g., a mouse, humanized, chimeric or veneered 3D6 antibody). The radioisotope used can be, for example,
C11, N13, O15, F18, or I123. The interval between administering the PET ligand and performing the scan can depend on the PET ligand and particularly its rate of uptake and clearing into the brain, and the half- life of its radiolabel.
[0460] PET scans can also be performed as a prophylactic measure in asymptomatic patients or in patients who have symptoms of mild cognitive impairment but have not yet been diagnosed with a tauopathy but are at elevated risk of developing a tauopathy. For asymptomatic patients, scans are particularly useful for individuals considered at elevated risk of tauopathy because of a family history, genetic or biochemical risk factors, or mature age. Prophylactic scans can commence for example, at a patient age between 45 and 75 years. In some patients, a first scan is performed at age 50 years. [0461] Prophylactic scans can be performed at intervals of for example, between six months and ten years, preferably between 1-5 years. In some patients, prophylactic scans are performed annually. If a PET scan performed as a prophylactic measure indicates abnormally high levels of tau protein deposits, immunotherapy can be commenced and subsequent PET scans performed as in patients diagnosed with a tauopathy. If a PET scanned performed as a prophylactic measure indicates levels of tau protein deposits within normal levels, further PET scans can performed at intervals of between six months and 10 years, and preferably 1-5 years, as before, or in response to appearance of signs and symptoms of a tauopathy or mild cognitive impairment. By combining prophylactic scans with administration of tau-directed immunotherapy if and when an above normal level of tau protein deposits is detected, levels of tau protein deposits can be reduced to, or closer to, normal levels, or at least inhibited from increasing further, and the patient can remain free of the tauopathy for a longer period than if not receiving prophylactic scans and tau-directed immunotherapy ( e.g ., at least 5, 10, 15 or 20 years, or for the rest of the patient's life).
[0462] Normal levels of tau protein deposits can be determined by the amount of
neurofibrillary tangles or tau inclusions in the brains of a representative sample of individuals in the general population who have not been diagnosed with a particular tauopathy (e.g.,
Alzheimer's disease) and are not considered at elevated risk of developing such disease (e.g, a representative sample of disease-free individuals under 50 years of age). Alternatively, a normal level can be recognized in an individual patient if the PET signal according to the present methods in a region of the brain in which tau protein deposits are known to develop is not different (within the accuracy of measurement) from the signal from a region of the brain in which it is known that such deposits do not normally develop. An elevated level in an individual can be recognized by comparison to the normal levels (e.g, outside mean and variance of a standard deviation) or simply from an elevated signal beyond experimental error in a region of the brain associated with tau protein deposits compared with a region not known to be associated with deposits. For purposes of comparing the levels of tau protein deposits in an individual and population, the tau protein deposits should preferably be determined in the same region(s) of the brain, these regions including at least one region in which tau protein deposits associated with a particular tauopathy (e.g, Alzheimer's disease) are known to form. A patient having an elevated level of tau protein deposits is a candidate for commencing immunotherapy. [0463] After commencing immunotherapy, a decrease in the level of tau protein deposits can be first seen as an indication that the treatment is having the desired effect. The observed decrease can be, for example, in the range of 1-100%, 1-50%, or 1-25% of the baseline value. Such effects can be measured in one or more regions of the brain in which deposits are known to form or can be measured from an average of such regions. The total effect of treatment can be approximated by adding the percentage reduction relative to baseline to the increase in tau protein deposits that would otherwise occur in an average untreated patient.
[0464] Maintenance of tau protein deposits at an approximately constant level or even a small increase in tau protein deposits can also be an indication of response to treatment albeit a suboptimal response. Such responses can be compared with a time course of levels of tau protein deposits in patients with a particular tauopathy ( e.g ., Alzheimer's disease) that did not receive treatment, to determine whether the immunotherapy is having an effect in inhibiting further increases of tau protein deposits.
[0465] Monitoring of changes in tau protein deposits allows adjustment of the immunotherapy or other treatment regime in response to the treatment. PET monitoring provides an indication of the nature and extent of response to treatment. Then a determination can be made whether to adjust treatment and if desired treatment can be adjusted in response to the PET monitoring.
PET monitoring thus allows for tau-directed immunotherapy or other treatment regime to be adjusted before other biomarkers, MRI or cognitive measures have detectably responded. A significant change means that comparison of the value of a parameter after treatment relative to basement provides some evidence that treatment has or has not resulted in a beneficial effect. In some instances, a change of values of a parameter in a patient itself provides evidence that treatment has or has not resulted in a beneficial effect. In other instances, the change of values, if any, in a patient, is compared with the change of values, if any, in a representative control population of patients not undergoing immunotherapy. A difference in response in a particular patient from the normal response in the control patient (e.g., mean plus variance of a standard deviation) can also provide evidence that an immunotherapy regime is or is not achieving a beneficial effect in a patient.
[0466] In some patients, monitoring indicates a detectable decline in tau protein deposits but that the level of tau protein deposits remains above normal. In such patients, if there are no unacceptable side effects, the treatment regime can be continued as is or even increased in frequency of administration and/or dose if not already at the maximum recommended dose.
[0467] If the monitoring indicates levels of tau protein deposits in a patient have already been reduced to normal, or near-normal, levels of tau protein deposits, the immunotherapy regime can be adjusted from one of induction (i.e., that reduces the level of tau protein deposits) to one of maintenance ( i.e . , that maintains tau protein deposits at an approximately constant level). Such a regime can be affected by reducing the dose and or frequency of administering
immunotherapy.
[0468] In other patients, monitoring can indicate that immunotherapy is having some beneficial effect but a suboptimal effect. An optimal effect can be defined as a percentage reduction in the level of tau protein deposits within the top half or quartile of the change in tau protein deposits (measured or calculated over the whole brain or representative region(s) thereof in which tau protein deposits are known to form) experienced by a representative sample of tauopathy patients undergoing immunotherapy at a given time point after commencing therapy.
A patient experiencing a smaller decline or a patient whose tau protein deposits remains constant or even increases, but to a lesser extent than expected in the absence of immunotherapy ( e.g ., as inferred from a control group of patients not administered immunotherapy) can be classified as experiencing a positive but suboptimal response. Such patients can optionally be subject to an adjustment of regime in which the dose and or frequency of administration of an agent is increased.
[0469] In some patients, tau protein deposits may increase in similar or greater fashion to tau deposits in patients not receiving immunotherapy. If such increases persist over a period of time, such as 18 months or 2 years, even after any increase in the frequency or dose of agents, immunotherapy can if desired be discontinued in favor of other treatments.
[0470] The foregoing description of diagnosing, monitoring, and adjusting treatment for tauopathies has been largely focused on using PET scans. However, any other technique for visualizing and/or measuring tau protein deposits that is amenable to the use of tau antibodies of the invention (e.g., a mouse, humanized, chimeric or veneered 3D6 antibody) can be used in place of PET scans to perform such methods.
[0471] Also provided are methods of detecting an immune response against tau in a patient suffering from or susceptible to diseases associated with tau. The methods can be used to monitor a course of therapeutic and prophylactic treatment with the agents provided herein. The antibody profile following passive immunization typically shows an immediate peak in antibody concentration followed by an exponential decay. Without a further dose, the decay approaches pretreatment levels within a period of days to months depending on the half-life of the antibody administered. For example, the half-life of some human antibodies is of the order of 20 days.
[0472] In some methods, a baseline measurement of antibody to tau in the subject is made before administration, a second measurement is made soon thereafter to determine the peak antibody level, and one or more further measurements are made at intervals to monitor decay of antibody levels. When the level of antibody has declined to baseline or a predetermined percentage of the peak less baseline (e.g, 50%, 25% or 10%), administration of a further dose of antibody is administered. In some methods, peak or subsequent measured levels less background are compared with reference levels previously determined to constitute a beneficial prophylactic or therapeutic treatment regime in other subjects. If the measured antibody level is significantly less than a reference level (e.g, less than the mean minus one or, preferably, two standard deviations of the reference value in a population of subjects benefiting from treatment) administration of an additional dose of antibody is indicated.
[0473] Also provided are methods of detecting tau in a subject, for example, by measuring tau in a sample from a subject or by in vivo imaging of tau in a subject. Such methods are useful to diagnose or confirm diagnosis of diseases associated with tau, or susceptibility thereto. The methods can also be used on asymptomatic subjects. The presence of tau indicates susceptibility to future symptomatic disease. The methods are also useful for monitoring disease progression and/or response to treatment in subjects who have been previously diagnosed with Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).
[0474] Biological samples obtained from a subject having, suspected of having, or at risk of having Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia,
frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal
degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP) can be contacted with the antibodies disclosed herein to assess the presence of tau. For example, levels of tau in such subjects may be compared to those present in healthy subjects. Alternatively, levels of tau in such subjects receiving treatment for the disease may be compared to those of subjects who have not been treated for Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). Some such tests involve a biopsy of tissue obtained from such subjects. ELISA assays may also be useful methods, for example, for assessing tau in fluid samples.
VII. Kits
[0475] The invention further provides kits ( e.g ., containers) comprising an antibody disclosed herein and related materials, such as instructions for use (e.g., package insert). The instructions for use may contain, for example, instructions for administration of the antibody and optionally one or more additional agents. The containers of antibody may be unit doses, bulk packages (e.g, multi-dose packages), or sub-unit doses.
[0476] Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage,
administration, contraindications and/or warnings concerning the use of such therapeutic products
[0477] Kits can also include a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It can also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
VIII. Other Applications
[0478] The antibodies can be used for detecting tau, or fragments thereof, in the context of clinical diagnosis or treatment or in research. For example, the antibodies can be used to detect the presence of tau in a biological sample as an indication that the biological sample comprises tau deposits. Binding of the antibodies to the biological sample can be compared to binding of the antibodies to a control sample. The control sample and the biological sample can comprise cells of the same tissue origin. Control samples and biological samples can be obtained from the same individual or different individuals and on the same occasion or on different occasions. If desired, multiple biological samples and multiple control samples are evaluated on multiple occasions to protect against random variation independent of the differences between the samples. A direct comparison can then be made between the biological sample(s) and the control sample(s) to determine whether antibody binding (z.e., the presence of tau) to the biological sample(s) is increased, decreased, or the same relative to antibody binding to the control sample(s). Increased binding of the antibody to the biological sample(s) relative to the control sample(s) indicates the presence of tau in the biological sample(s). In some instances, the increased antibody binding is statistically significant. Optionally, antibody binding to the biological sample is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, or 100-fold higher than antibody binding to the control sample.
[0479] In addition, the antibodies can be used to detect the presence of the tau in a biological sample to monitor and evaluate the efficacy of a therapeutic agent being used to treat a patient diagnosed with Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). A biological sample from a patient diagnosed with Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia,
frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal
degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP) is evaluated to establish a baseline for the binding of the antibodies to the sample (i.e., a baseline for the presence of the tau in the sample) before commencing therapy with the therapeutic agent. In some instances, multiple biological samples from the patient are evaluated on multiple occasions to establish both a baseline and measure of random variation independent of treatment. A therapeutic agent is then administered in a regime. The regime may include multiple administrations of the agent over a period of time. Optionally, binding of the antibodies (i.e., presence of tau) is evaluated on multiple occasions in multiple biological samples from the patient, both to establish a measure of random variation and to show a trend in response to immunotherapy. The various assessments of antibody binding to the biological samples are then compared. If only two assessments are made, a direct comparison can be made between the two assessments to determine whether antibody binding (i.e., presence of tau) has increased, decreased, or remained the same between the two assessments. If more than two measurements are made, the measurements can be analyzed as a time course starting before treatment with the therapeutic agent and proceeding through the course of therapy. In patients for whom antibody binding to biological samples has decreased (i.e., the presence of tau), it can be concluded that the therapeutic agent was effective in treating the Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP) in the patient. The decrease in antibody binding can be statistically significant. Optionally, binding decreases by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Assessment of antibody binding can be made in conjunction with assessing other signs and symptoms of Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).
[0480] The antibodies can also be used as research reagents for laboratory research in detecting tau, or fragments thereof. In such uses, antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes, and can be provided in the form of kit with all the necessary reagents to perform the detection assay. The antibodies can also be used to purify tau, or binding partners of tau, e.g. , by affinity chromatography.
[0481] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. EXAMPLES
[0482] Example 1. Identification of tau Monoclonal Antibodies
[0483] Monoclonal antibodies against tau were generated as follows. Immunizations were performed with either recombinant N-terminally His-tagged 383 a.a. human tau (4R0N), containing a P301S mutation [immunogen A] or recombinant 383 a.a. human tau (4R0N), containing a P301S mutation, lacking an N-terminal His-tag [immunogen B] Immunogens were emulsified in RIBI adjuvant.
[0484] Five week old female Balb/c mice were intraperitoneally immunized with 25 pg of immunogen A on day 0, and 10pg of immunogen A each on days 7, 14, 21, 27, 34, 48, 55, and 62. Mice were immunized with 10pg of immunogen B on days 76 and 90. On days 43 and 98, mice were bled and titered against immunogen A; on day 101 the animals with highest titers were boosted with a terminal immunization of 50pg immunogen B, which was delivered ½ intraperitoneally and ½ intravenously. Fused hybridomas were screened via ELISA against both immunogens, and positives with the highest signal were epitope mapped (see Example 2). 3D6 reacted to peptides corresponding to amino acid residues 199-213 of SEQ ID NO: 3 and to amino acid residues. 262-276 of SEQ ID NO:3 (following numbering of the longest CNS isoform of tau, this corresponds to amino acid residues 257-271 of SEQ ID NO:l and to amino acid residues 320-334 of SEQ ID NO: 1).
[0485] Example 2. Epitope mapping of antibody 3D6
[0486] A range of overlapping biotinylated peptides spanning the entire 383aa 4R0N human tau protein were used for mapping the murine 3D6 antibody. Additional peptides were used to model potential post-translational modifications of the C- and N-terminal ends of the protein.
[0487] Biotinylated peptides were bound to separate wells of a streptavidin-coated ELISA plate. The plate was blocked and treated with murine 3D6, followed by incubation with a horseradish peroxidase-conjugated anti-mouse antibody. After thorough washing, OPD was applied to the plate and allowed to develop. The plate was read at 450nm absorbance.
Background subtraction was performed with absorbance values from wells containing no primary antibody, and a threshold for positive binding was set to 0.2 absorbance units.
Positive binding was detected for the peptide spanning amino acid residues 199-213 (of SEQ ID NO:3), and amino acid residues 262-276 (of SEQ ID NO:3). Using the numbering of the full- length 4R2N human tau protein (441 amino acids) these peptides correspond to amino acid residues 257-271 (of SEQ ID NO: 1) and 320-334 (of SEQ ID NO: 1). (Figure 1)
[0488] Example 3. Design of Humanized 3D6 Antibodies
[0489] The starting point or donor antibody for humanization was the mouse antibody 3D6.
The heavy chain variable amino acid sequence of mature m3D6 is provided as SEQ ID NO:7.
The light chain variable amino acid sequence of mature m3D6 is provided as SEQ ID NO: 11.
The heavy chain Kabat/Chothia Composite CDR1, CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOs:8-10, respectively. The light chain Rabat CDR1, CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOsl2-14 respectively. Rabat numbering is used throughout.
[0490] The variable kappa (Vk) of 3D6 belongs to mouse Rabat subgroup 2 which corresponds to human Rabat subgroup 2 and the variable heavy (Vh) to mouse Rabat subgroup 2c which corresponds to human Rabat subgroup 1 [Rabat E. A., et ah, (1991), Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242] 16 residue Chothia CDR- L1 belongs to canonical class 4, 7 residue Chothia CDR-L2 to class 1, 9 residue Chothia CDR- L3 to class 1 in Vk [Martin A.C, and Thornton J.M. (1996) J. Mol. Biol. 263:800-15. [Martin & Thornton, 1996] 10 residue Chothia CDR-H1 belongs to class 1, 17 residue Chothia CDR-H2 to class 2 [Martin & Thornton, 1996]]. CDR-H3 has no canonical classes. A search was made over the protein sequences in the PDB database [Deshpande N, et ak, (2005) Nucleic Acids Res. 33: D233-7.] to find structures which would provide a rough structural model of 3D6. To build up a Fv model of 3D6, a .structure of murine anti-pyroglutamate-Abeta antibody Fab c#24 (pdb code 5MYX) [Piechotta, A. et ak, 2017, J Biol Chem. 292: 12713-12724] with a resolution of 1.4 A was used. It retained the same canonical structure for the loops as 3D6.
[0491] The frameworks of 3D6 VH share a high degree of sequence similarity with the corresponding regions of humanized 48G7 Fab PDB: 2RCS, designed by Wedemayer, G. J., et ak (1997; Science 276: 1665-1669). The variable domains of 3D6 and 48G7 fab also share identical lengths for the CDR-H1, H2 loops. Similarly, the frameworks of 3D6 VL share a high degree of sequence similarity with the corresponding regions of human antibody ARX71335 VL, cloned by Dafferner, A. J., et ak (2017; . Direct Submission). The variable light domain of 3D6 and ARX71335 antibody also share identical lengths for the CDR-L1, L2 and L3 loops.
Accordingly, the framework regions of 48G7 VH (2RCS-VH) and ARX71335 VL were chosen as the acceptor sequences for the CDRs of 3D6. A model of the 3D6 CDRs grafted onto the respective human frame-works for VH and VL was built and used as a guidance for further backmutations.
[0492] Heavy and light chain variant sequences resulting from antibody humanization process were further aligned to human germ line sequences using IMGT Domain GapAlign tool to assess the humanness of the heavy and light chain as outlined by WHO INN committee guidelines. (WHO-INN: International nonproprietary names (INN) for biological and biotechnological substances (a review) (Internet) 2014. Available from: http://www.
who.int/medicines/services/inn/BioRev2014.pdf) Residues were changed to align with corresponding human germ line sequence, where possible, to enhance humanness and to reduce potential immunogenicity. For humanized VLvb2 and VLvb3 variants, mutations were introduced to render the sequences more similar to human germ line gene IGKV2-30*02 (SEQ ID NO:27) For humanized VHvb2, VHvb3, VHvb4, VHvb5, VHvb6, and VHvb6 variants, mutations were introduced to render the sequences more similar to human germ line gene IGHV1 -69-2*01 (SEQ ID NO:25)
[0493] Versions of hu3D6-VH and hu3D6-VL were designed to enable assessment of various framework residues for their contributions to antigen binding, thermostability, and
immunogenicity, and for optimization of glycosylation, aggregation, N-term heterogeneity, thermostability, surface exposed charged patches, surface exposed charge patches, deamination, and proteinase susceptibility. The positions considered for mutation include those that:
- define the canonical CDR conformations (summarized in Martin, A.C.R. (2010) Protein sequence and structure analysis of antibody variable domains. In: Kontermann R and Diibel S (eds). Antibody Engineering. Heidelberg, Germany: Springer International Publishing AG.),
- are within the Vernier zone (Foote J and Winter G. (1992) Antibody framework residues affecting the conformation of the hypervariable loops. JMol Biol. 224(2):487-99.),
- localize to the VH/VL domain interface (summarized in Leger OJP and Saldanha J.
(2000) Preparation of recombinant antibodies from immune rodent spleens and the design of their humanisation by CDR grafting. In: Shepherd P and Dean C (eds). Monoclonal Antibodies: a Practical Approach. Oxford, UK: Oxford University Press.),
- are susceptible to post-translational modifications, such as glycosylation or
pyroglutamination,
- are occupied by residues that are predicted to clash with CDRs, according to the model of 3D6 CDRs grafted onto VH and VL frameworks, or - are occupied by residues that are rare among sequenced human antibodies, where either the parental mouse 3D6 residue or some other residue is much more prevalent within human antibody repertoire.
[0494] Alignments of the murine 3D6 and various humanized antibodies are shown for the light chain variable regions (Table 4 and Figure 3), and heavy chain variable regions (Table 3 and Figure 2).
[0495] 7 humanized heavy chain variable region variants and 3 humanized light chain variable region variants were constructed containing different permutations of substitutions:
hu3D6VHvbl, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6Hvb5, hu3D6VHvb6, or hu3D6VHvb7 (SEQ ID NOs: 76-80 and 90-91, respectively); and hu3D6VLvbl, hu3D6VLvb2, or hu3D6VLvb3 (SEQ ID NOs:83-85, respectively) (Tables 3 and 4). The exemplary humanized Vk and Vh designs, with backmutations and other mutations based on selected human frameworks, are shown in Tables 3 and 4, respectively. The bolded areas in Tables 3 and 4 indicate the CDRs as defined by Kabat/Chothia Composite. SEQ ID NOs:76-80 and SEQ ID NOs:90-91 contain backmutations and other mutations as shown in Table 5. The amino acids at positions in hu3D6VHvbl, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7 are listed in Table 6. The amino acids at positions in hu3D6VLvbl, hu3D6VLvb2, and hu3D6VLvb3 are listed in Table 7.
[04961 Table 3
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
00
Figure imgf000120_0001
[0497] Table 4
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
[0498] Table 5: VH, VL Backmutations and Other Mutations for Humanized 3D6
Figure imgf000126_0001
[0499] Table 6: Kabat Numbering of Framework (or CDR) Residues (based on Kabat/Chothia Composite CDRs) for Backmutations and Other Mutations in Heavy Chains of Humanized 3D6 Antibodies
Figure imgf000127_0001
[0500] Table 7: Kabat Numbering of Framework Residues (based on Kabat/Chothia Composite CDRs) for Backmutations and Other Mutations in Light Chains of Humanized
3D6 Antibodies
Figure imgf000128_0001
[0501] Positions at which canonical, vernier, or interface residues differ between mouse and human acceptor sequences are candidates for substitution. Examples of canonical/CDR interacting residues include Rabat residues H54 and H94 in Table 3. Examples of vernier residues include Rabat residues H28, H67, H93, and H94 in Table 3. Examples of
interface/packing (VH+VL) residues include Rabat residues H91 and H93 in Table 3.
[0502] The rationales for selection of the positions indicated in Table 3 in the heavy chain variable region as candidates for substitution are as follows.
[0503] heavy chain variable regions
[0504] hu3D6VHvbl
- consists of the CDR-H1, H2, and H3 loops of 3D6-VH grafted onto the framework of 48G7-VH (RCS-VH), with backmutations at positions H91 (Y91F), H93 (A93S), and H94 (S94T).
[0505] hu3D6VHvb2
- reverts all framework substitutions at positions that are key for defining the Chothia canonical classes, are part of the Vernier zone, or localize to the VH/VL domain interface or contribute to structural stability. 3D6-VH_vb2 incorporates backmutations or substitutions Q1E, Q5V, LI IV, L20I, T23K, K38R, E42G, Q43K, K66R, S75T, N76D, Q81E,Y91F, A93S, S94T T108L, and LI 09 V, to enable assessment of these positions’ contributions to antigen-binding affinity and immunogenicity.
[0506] hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7 consists of further substitutions and either add to antibody stability and/or for optimization of glycosylation, aggregation, N-term heterogeneity, thermostability, surface exposed charged patches, surface exposed charge patches, deamination, and proteinase susceptibility.
[0507] Q1E: is a stability enhancing mutation to mitigate pyroglutamate formation potential (Liu, supra.)
[0508] Q5V: is a frequency -based and germ line-aligning mutation. Val is most frequent in human sequences at this position. Val is in human germ line gene IMGT# IGHV1 -69-2*01 (SEQ (ID NO: 25) at this position.
[0509] LI IV: is a germ line-aligning mutation. Val is in human germ line gene IMGT# IGHV1- 69-2*01 (SEQ (ID NO: 25) at this position. [0510] S17T: is a germ line-aligning mutation. Thr is in human germ line gene IMGT# IGHV1- 69-2*01 (SEQ (ID NO: 25) at this position.
[0511] L20I: is a germ line-aligning mutation. He is in human germ line gene IMGT# IGHV1- 69-2*01 (SEQ (ID NO: 25) at this position.
[0512] T23K: is a frequency -based and germ line-aligning mutation. Lys is more frequent at this position. Lys is in human germ line gene IMGT# IGHVl-69-2*01 (SEQ (ID NO: 25) at this position.
[0513] N28T: This is a CDR-H1 residue substitution to Thr.
[0514] K38R: is a frequency -based back-mutation. Arg is most frequent at this position. Arg at this position is predicted to make two H-bonds with Glu 46 in addition to one H-bond each with Asp86 and Tyr 90 in heavy chain; therefore, Arg substitution may enhance stability over Lys at this position.
[0515] E42G: is a frequency -based and germ line-aligning mutation. Gly is in human germ line gene IMGT# IGHVl-69-2*01 (SEQ (ID NO: 25) at this position. Gly is most frequent at this position. Gly substitution is predicted not to affect stability.
[0516] Q43K: Lys side chain at this position is predicted to make H-bond with G42 besides main chain making H-bonds with Gin 39 and Arg 40, thereby Lys substitution may enhance stability over Q at this position.
[0517] N54D and D56E are substitutions of CDR residues and are predicted to be non-antigen contact positions as per homology model. N54D and D56E substitutions are predicted to stabilize antibody structure.
[0518] V58I: is a substitution of a CDR-H2 residue. Germline gene IMGT# IGHVl-69-2*01 (SEQ (ID NO: 25) has He at this position. This residue is predicted not to contact antigen.
[0519] K66R: Arg at this position is predicted to make H-bonds with Ser 82a and Thr 83 in addition to making H-bond and salt-bridge with Asp 86. [0520] A67V: is a substitution of a Vernier zone residue. Germ line gene IMGT# IGHV1-69- 2*01 (SEQ (ID NO: 25) has Val at this position.
[0521] S75T: Ser at this position is predicted to make H-bond with Asp 72 and Tyr 76. Thr at this position is predicted to also make these contacts but being surface exposed residue Thr may enhance antibody stability.
[0522] N76D: Asp is a germ line-aligning mutation. Asp is in human germ line gene IMGT# IGHV1 -69-2*01 (SEQ (ID NO: 25) at this position.
[0523] L80M: Met is a germ line-aligning mutation. Met is in human germ line gene IMGT# IGHV1 -69-2*01 (SEQ (ID NO: 25) at this position.
[0524] Q81E: Glu is predicted to make H-bond plus salt-bridge with K19 hence Glu at this position make enhance antibody stability.
[0525] T83R to enhance thermostability and to increase humanness. Arg is a germ-line aligning mutation. Arg is in human germ line gene IMGT# IGHVl-69-2*01 (SEQ (ID NO: 25) at this position. Arg is most frequent at this position.
[0526] F91Y: is mutation of an interface residue, and is a frequency-based back-mutation. Tyr is typically at this position, and may enhance antibody stability.
[0527] A93S: is a back-mutation of a Vernier zone and interface zone residue.
[0528] S94T: is a back-mutation of a Chothia defined canonical structural residue and vernier residue.
[0529] T108L: Leu is a germ line-aligning mutation. Leu is in human germ line gene IMGT# IGHVl-69-2*01 (SEQ (ID NO: 25) at this position. Leu at this position is predicted to make the antibody less immunogenic and to have no impact on antibody stability.
[0530] L109V: is a frequency -based mutation. Val is most frequent at this position.
[0531] The rationales for selection of the positions indicated in Table 4 in the light chain variable region as candidates for substitution are as follows. [0532] kappa light chain variable regions
[0533] hu3D6VLvbl
- consists of the CDR-L1, L2, and L3 loops of 3D6-VL grafted onto the framework of ARX71335 VL.
[0534] hu3D6VLvb2 and hu3D6VLvb3
- reverts all framework substitutions at positions that are key for defining the Chothia canonical classes, are part of the Vernier zone, or locate to the VH/VL domain interface. Hu3D6- VLvb2 & Hu3D6-VLvb3 also include substitutions that contribute to structural stability hu3D6- VL_vb2 incorporates backmutations T7S, I15L, L83V, H86Y and L106I, to enable assessment of these positions’ contributions to antigen-binding affinity and immunogenicity.
Hu3D6-VL_vb3 all substitutions mentioned for vb2 along with additional changes at Q17E, K24R, L37Q, K45R, and LI 061
[0535] T7S: is a germ line-aligning mutation. Ser is in human germ line gene IGKV2-30*02 (SEQ (ID NO: 27) at this position.
[0536] T10S: is a frequency-based and germ line-aligning mutation. Ser is frequent at this position. Ser is in human germ line gene IGKV2-30*02 (SEQ (ID NO: 27) at this position.
[0537] II 5L: is a germline-aligning mutation. Leu is in human germ line gene IGKV2-30*02 (SEQ (ID NO: 27) at this position.
[0538] Q17E: Glu at this position is predicted to make H-bond with T14 and salt-bridge with Lys 107, both light chain residues, and to enhance antibody stability.
[0539] K24R: is a mutation of a CDR residue. Both Lys and Arg are predicted to make H-bond and salt-bridge with Asp 70 in the light chain. Arg is predicted to fit better in the conformation. Arg is also a germ line-aligning mutation. Arg is in human germ line gene IGKV2-30*02 (SEQ (ID NO: 27) at this position.
[0540] L37Q: This is predicted to be a deep buried residue, Leu is not predicted to interact with surrounding residues, whereas, Gin is predicted to make H-bonds with Q38 and Asp 82 in the light chain. Gin is also a germ line-aligning mutation. Gin is in human germ line gene IGKV2- 30*02 (SEQ (ID NO: 27) at this position. [0541] K45R: Although, Lys is predicted to make H-bonds with S56 and Gly 57; Arg’s predicted interaction with neighboring residues is much more extensive as it is predicted to form salt-bridges with D55, H-bond with Arg46 and double H-bonds with S56. Arg is also a germ line-aligning mutation. Arg is in human germ line gene IGKV2-30*02 (SEQ (ID NO: 27) at this position.
[0542] L83V: This is a frequency -based mutation of a residue predicted to be surface exposed. Val is also a germ line-aligning mutation. Val is in human germ line gene IGKV2-30*02 (SEQ (ID NO: 27) at this position.
[0543] H86Y: Murine 3D6 VL has Tyr at this position. Tyr is also the most frequent residue at this position.
[0544] A100Q: Ala is rare at this position. Ala is predicted to be surface-exposed residue and is not predicted to interact surrounding residues. Gin is most frequent at this position and is also a germline-aligning mutation. Gin is in human germ line gene IGKV2-30*02 (SEQ (ID NO: 27) at this position. Gin is predicted to make H-bond with Ser 7 stabilizing intra-chain.
[0545] L106I: is a frequency-based and germ line-aligning mutation. He is most frequent at this position. lie is in human germ line gene IGKV2-30*02 (SEQ (ID NO: 27) at this position.
[0546] The designs based on these human frameworks were: heavy chain variable regions
>hu3D6VHvbl (SEQ ID NO: 76)
QVQLQQSGAELVKPGASVKLSCTASGFNIKDYYLHWVKQRPEQGLEWIGWIDPENGDT VYDPKFQGKATIT ADTS SNTAYLQLS SLTSEDTAVYFC STLDFW GQGTTLTV S S
>hu3D6VHvb2 (SEQ ID NO: 77)
EVQLVQSGAEVVKPGASVKISCKASGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDT V YDPKF Q GRATIT ADT S TDT A YLEL S SLT SEDT A V YF C S TLDF W GQ GTL VT V S S
>hu3D6VHvb3 (SEQ ID NO: 78) EVQLVQSGAEVVKPGATVKISCKASGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDTI YDPKFQGRATIT ADT STDT AYMELS SLRSEDT AVYY C STLDFW GQGTL VT V S S hu3D6VHvb4 (SEQ ID NO: 79)
EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPENGDTI YDPKFQGRVTIT ADT STDT AYMELS SLRSEDT AVYY C STLDFW GQGTL VTV S S
>hu3D6VHvb5 (SEQ ID NO:80)
EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGETI YDPKFQGRVTIT ADT STDT AYMELS SLRSEDT AVYY C STLDFW GQGTL VTV S S
>hu3D6VHvb6 (SEQ ID NO: 90)
EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGET VYDPKF Q GRVTIT ADT S TDT A YMEL S SLRSEDT A VYF C S TLDF W GQ GTL VT V S S
>hu3D6VHvb7 (SEQ ID NO:91)
EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGET VYDPKF Q GRVTIT ADT S TDT A YMEL S SLRSEDT AVYY C S TLDF W GQGTL VTV S S kayya light chain variable regions hu3D6VLvbl (SEQ ID NO:83)
DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLD SGVPDRF SGSGSGTDFTLKISRVEAEDLGVHY CWQGTHFP YTF GAGTKLELK
>hu3D6VLvb2 (SEQ ID NO: 84)
DVVMTQSPLSLSVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLD S GVPDRF S GS GS GTDF TLKISRVE AED V G V Y Y CWQGTHFP YTF G AGTKLEIK
>hu3D6VLvb3 (SEQ ID NO:85) D VVMT Q SPL SL S VTLGEP ASISCRS SQ SLLD SDGKT YLNWLQQRPGQ SPRRLIYL V SKLD S GVPDRF S GS GS GTDF TLKISRVE AED V G V Y Y C WQGTHFP YTF GQGTKLEIK
[0547] Humanized sequences are generated using a two-stage PCR protocol that allows introduction of multiple mutations, deletions, and insertions using QuikChange site-directed mutagenesis [Wang, W. and Malcolm, B.A. (1999) BioTechniques 26:680-682).
[0548] Example 4. Mouse monoclonal antibodies bind tau in ELISA assays
[0549] Methods: Indirect ELISA: 96-well polystyrene plates were coated with capture antibodies anti-6xHis (Figure 4A) or polyclonal anti-tau (Dako #A0024, Figure 4B) suspended in lxPBS for 2 hr at RT or 16 hr at 4°C. Coating was removed, and plates were blocked for 1 hr with 1%BSA in lxPBS, followed by incubation with human recombinant tau, either with (Figure 5A) or without (Figure 4B) a polyhistidine tag at the N-terminus of the protein. After washing, plates were incubated with indicated antibodies, washed, and incubated with HRP-conjugated goat anti-mouse secondary antibody. Plates were developed with TMB, and A450 was measured with a plate reader.
[0550] Sandwich ELISA: 96-well polystyrene plates were coated with anti-mouse antibodies in lxPBS for 2 hr at RT or 16 hr at 4°C. Coating was removed, and plates were blocked for 1 hr with 1%BSA in lxPBS. The plate was next incubated with the Indicated antibodies at identical concentrations, diluted in 0.1% BSA in lxPBS. Plates were successively treated with human tau, polyclonal rabbit anti-tau (Dako #A0024), and HRP-conjugated goat anti-rabbit antibody, all diluted in 0.1%BSA in PBS with washes occurring between each step. Streptavidin-HRP was added, plates were developed with TMB, and A450 was measured with a plate reader. See Figure 4C.
[0551] Results: A panel of hybridoma-produced antibodies were assayed for binding to tau via a number of different ELISA formats. Detection of tau was confirmed using an indirect format, using tau protein immobilized by its N-terminally fused polyhistidine tag (Figure 4A). Binding to the native, untagged protein was also confirmed (Figure 4B). To assess the solution affinity of the various antibodies, a sandwich ELISA format was used in which tested hybridoma antibodies were used as capture reagents (Figure 4C). [0552] Example 5. Affinity of mouse monoclonal antibodies to tau
[0553] Methods: SPR analysis was performed using a Biacore T200 to determine the binding kinetics of murine antibodies to recombinant human tau. To prepare a sensor surface, anti-mouse antibody (GE Life Sciences) was immobilized on sensor chip CM5 via amine coupling, and antibody was captured at a level to ensure maximum binding of 50 RU. Various concentrations of recombinant tau ranging from 10-0.14 nM were passed over the captured ligand at a flow rate of 50 pL/min in running buffer (HBS + 0.05% P-20, 1 mg/mL BSA), for 180 sec association and 900 sec dissociation. Data were double-referenced to both an irrelevant sensor not containing antibody ligand, and 0 nM analyte concentration to account for the dissociation of ligand from the capture moiety. Data was then analyzed using a global 1 : 1 fit.
[0554] Results: Multiple murine antibodies were selected based on their performance in a battery of ELISA assays, and their binding affinities were assessed via SPR. Antibodies were tested in parallel sets, and their binding association and dissociation rates were compared to select the highest binder to recombinant human tau. The highest binding affinity was observed with antibody clone 3D6. Binding affinities are shown in Figure 5.
[0555] Example 6. Mouse monoclonal antibodies prevent binding of human tau to the surface of immortalized neuronal cells
[0556] Methods: Inhibition_of Tau Binding to B103 Neuroblastoma Cells with anti-Tau Monoclonal Antibodies
1. Resuspend B103 cells in PBS at 5 x 105 cells/mL. Plate 50pL of cell suspension per well in a MSD High Bind plate. This results in 25K cells/well. Cover the plate and allow cells to attach at 37°C, 5% CO2, for 2 hrs.
2. Following cell attachment, remove PBS from wells by inverting plate and gently tapping to remove excess buffer. Add 50pL of 3% MSD Blocker A in PBS or other suitable blocking buffer to each well and incubate plate at RT for 1 hr without shaking.
3. During the plate blocking step co-incubate Tau and anti-Tau antibodies as follows: a. Start with anti-Tau antibody at 2 mg/mL and serial dilute in PBS, 1 :2, for 7 additional dilutions. b. Dilute Tau to 20 nM in PBS. The Tau concentration will be constant in each well. c. Mix the Tau and anti-Tau antibody, 1 : 1, for a final Tau concentration of 10 nM and a starting concentration of anti-Tau of 1 mg/mL. d. Incubate the mixture for approximately 1 hr at RT with shaking (600rpm).
4. After plate blocking, step 2, remove blocking buffer from wells by inverting plate and gently tapping and wash plate 2x with PBS using a multichannel pipette. Ensure excess buffer is completely removed. Cool the plated cells to 4°C prior to adding the Tau: anti-Tau complexes.
5. Add 50pL of cooled complex, step 3, to the plated cells and incubate on ice for 30 minutes.
6. Wash plate 2x with chilled PBS as previously described.
7. Add 50pL per well of the 16B5.SULFO-TAG for detection of cell surface bound Tau. Incubate for 30 minutes on ice.
8. Wash plate 2x with chilled PBS again as previously described.
9. Add 150 pL per well of IX Read Buffer T Without Surfactant (diluted in EhO) and read immediately on the MSD SECTOR™ 600 instrument. Avoid introducing bubbles when adding read buffer.
10. Report the MSD signals vs. concentration of anti-Tau.
[0557] Antibodies tested were anti -tau antibodies 3D6, 16G7, 3H9, 4C5, and 5G8, and isotype control.
[0558] Results: [0559] Decreasing SulfoTag anti-tau signal occurring with increasing test antibody indicates functional blocking of the binding of tau to neuronal cell surfaces. No blocking was observed with isotype control, 16G7, or 3H9. Increasing amounts of functional blocking activity were observed with 4C5, 5G8, and 3D6. 3D6 demonstrated the deepest blocking activity of the antibodies tested. See Figure 6.
[0560] Example 7. Disaggregation activity
[0561] Methods: Aggregation of recombinant tau— Purified recombinant tau with an N- terminal 6xHis tag was combined with equimolar amounts of low-molecular weight heparin in lxPBS (pH 7.4), and incubated at 37°C for 96 hr on a nutator. Aggregation of the sample was confirmed by binding to Thioflavin T.
Incubation with antibodies— Antibodies were incubated with aggregated, recombinant tau at the indicated molar ratios incubated at 37°C for 96 hr without rotation or nutation. At the end of the experiment, aggregation was measured by incubating samples with 25 mM Thioflavin T, and measuring emitted fluorescence (450/482 ex/em). Signals were background subtracted to buffer samples.
[0562] Results: As shown in Figure 7, 3D6 preferentially disassembles intact tau fibrils.
Varying molar ratios of 3D6 (triangles), isotype control (circles) and 16G7 (squares) were incubated with amyloid-containing tau fibrils for 96 hours. At the end of this period, the extent of aggregation was assessed by binding to Thioflavin T. 3D6 preferentially decreases the Thioflavin T signal present in the sample, compared to both an isotype control antibody as well as to 16G7, an anti-tau antibody that binds to a different region of tau.
[0563] Example 8. 3D6 and 5G8 immunocapture tau from human disease tissue.
[0564] Methods: High-salt soluble protein fractions were prepared to 1 mg/ml. For each immunoprecipitation, 200 pg of sample was used. 10pg of the indicated antibody (either an isotype control, 3D6, or anti-tau antibody 5G8) was added to the high-salt sample preparations, and incubated for 2 hr. Protein G magnetic beads were then added to the mixtures, and incubated for a further hour to capture antibody/antigen complexes. Samples were thoroughly washed with lxPBS, and beads were boiled in reducing/denaturing sample buffer to release captured proteins. Resulting samples were resolved by SDS-PAGE and Western blotting was performed using a polyclonal anti-tau antibody (Dako, #A0024).
[0565] Results: As shown in Figure 8, 3D6 and 5G8 immunoprecipitated tau from Alzheimer disease tissue. High-salt soluble fractions were immunoprecipitated with the indicated antibody, and detected with a polyclonal anti-tau antibody directed towards a separate region of the tau molecule from the binding sites for 3D6 and tau antibody A. 3D6 robustly captured tau from this fraction. The input (high-salt soluble sample) is shown at right.
[0566] Example 9. Immunohistochemistry immunoreactivity of 3D6,
[0567] Frontotemporal cortices were obtained from patients without neurodegenerative disease or with Alzheimer disease, which was confirmed upon post-mortem assessment.
Immunohistochemistry was performed on lightly acetone-fixed, lOum slide-mounted
cryosections. All staining steps were performed using a Leica BOND Rx autostainer, using Leica consumables. Either murine or a human form of 3D6 was incubated with tissue sections followed by addition of species-appropriate secondary antibodies conjugated to an HRP polymer. To prevent non-specific binding of endogenous immunoglobulin when using humanized antibodies on human tissue, the antibodies were non-covalently labeled with a biotin-conjugated anti-human monovalent Fab fragment in vitro before incubation on tissue. Tissue labeled with the primary antibody -biotin Fab fragment complex was further amplified using an avi din-biotin amplification system (Vector Laboratories, Burlingame, CA). The staining was visualized with a DAB chromogen, which produced a brown deposit. Negative control consisted of performing the entire immunohistochemical procedure on adjacent sections with an IgG isotype control antibody.
[0568] Antibodies tested were murine CD6, chimeric 3D6 (which contained VH and VL from the murine antibody with human constant regions, heavy chain SEQ ID NO: 72 and light chain SEQ ID NO:73), and humanized variant hu3D6VHv5/hu3D6VLv2.
[0569] Staining performed with murine, chimeric, and humanized forms of 3D6 were qualitatively compared and assessed for the strength and intensity of staining, as well as localization of immunoreactivity. Intensity of staining was similar for chimeric and humanized forms of 3D6, and displayed similar localization patterns compared with the murine form of the antibody. Tau was detected in neurofibrillary tangles, fibrils, neuropil threads, and in degenerating axons. There was also notable somal staining detected.
[0570] Example 10. Affinity of humanized variants towards tau
[0571] Methods ;Indirect ELISA 96-well polystyrene plates are coated with human recombinant tau suspended in lxPBS for 2h at RT or 16h at 4°C. Coating is removed, and plates are blocked for lh with 1%BSA in lxPBS. Humanized variant antibodies at 1 pg/mL in 0.1% BSA in lxPBS are added to plates for 1 hour followed by washing, and HRP-conjugated goat anti-human antibody are added. Plates are developed with TMB, and A450 is measured with a plate reader.
[0572] Sandwich ELISA 96-well polystyrene plates are coated with anti-human antibodies in lxPBS for 2 hr at RT or 16 hr at 4°C. Coating is removed, and plates are blocked for 1 hr with 1%BSA in lxPBS. Humanized variant antibodies at varying concentrations diluted in 0.1% BSA in lxPBS are added to plates for 1 hour followed by washing, and biotinylated recombinant human tau diluted in 0.1% BSA in lxPBS is added. After washing, Streptavidin-HRP is added, plates are developed with TMB, and A450 is measured with a plate reader.
[0573] SPR analysis is performed using a Biacore T200 to determine the binding kinetics of h3D6-VHv5-L2 to recombinant human tau. To prepare a sensor surface, anti-human antibody (GE Life Sciences) is immobilized on sensor chip CM5 via amine coupling, and humanized variant antibody is captured at a level to ensure maximum binding of 50 RU. Various concentrations of recombinant tau ranging from 10-0.14 nM are passed over the captured ligand at a flow rate of 50 pL/min in running buffer (HBS + 0.05% P-20, 1 mg/mL BSA), for 180s association and 900s dissociation. Data are double-referenced to both an irrelevant sensor not containing antibody ligand, and 0 nM analyte concentration to account for the dissociation of ligand from the capture moiety. Data is then analyzed using a global 1 : 1 fit.
[0574] Example 11 Immunogenicity of hu3D6VLv2 light chain variable region
[0575] The amino acid sequences of the hu3D6VLv2 light chain variable region (SEQ ID NO:21) of were analyzed using iedb.org Deimmunization Tool (Dhanda et al, Immunology. 2018 Jan; 153(1): 118-132). Table 9 shows the peptides that may be selected for deimmunization of hu3D6VLv2, i.e., suggesting areas where further substitutions could be made to reduce potential immunogenicity.
[0576] Table 9. Results of immunogenicity analysis identifying potential peptides for deimmunization
Figure imgf000141_0001
[0577] Based on the results of the analysis shown in Table 9, variants of hu3D6VLv2 light chain variable region were designed, targeting the amino acid residues bolded in Table 10. Each variant incorporates one of the following amino acid substitutions, as shown in Table 11.
[05781 Table 10. Amino acid residues in hu3D6VLv2 targeted for substitution.
Figure imgf000142_0001
[0579] Table 11. Amino acid substitutions in hu3D6VLv2 designed to reduce immunogenicity
Figure imgf000143_0001
[0580] Additional variants of hu3D6VLv2 light chain variable region were designed, as shown in Table 12, which incorporate substitutions at one or two of the amino acid residues highlighted in Table 10. Some variants in Table 12 also incorporate the substitution L37Q (SEQ ID
NOs: 119-135, and 145), or both the substitution L37Q and the substitution G100Q (SEQ ID NOs: 136-142).
[0581] The rationales for selection of the positions L37Q and G100Q as indicated in Table 12 and Table 13 in the light chain variable region as candidates for substitution are as follows. [0582] L37Q is a mutation to increase humanness of the sequence. Gin is a germline-aligning mutation. Gin is in human germline gene IGKV2-30*02 (SEQ (ID NO: 27) at this position.
[0583] G100Q is a mutation to increase humanness of the sequence. Gin is a germline- aligning mutation. Gin is in human germline gene IGKV2-30*02 (SEQ (ID NO: 27) at this position.
[0584] Table 12: Amino acid substitutions in hu3D6VLv2 designed to reduce
immunogenicity
Figure imgf000144_0001
Figure imgf000145_0001
[0585] Additional variants of hu3D6VLv2 light chain variable region were designed to incorporate L37Q (SEQ ID NO: 143) or to incorporate G100Q (SEQ ID NO: 144), as shown in Table 13.
[0586] Table 13: Additional Amino acid substitutions in hu3D6VLv2
Figure imgf000145_0002
[0587] Figures 10A, 10B, IOC, and 10D show alignments of the light chain variable region of humanized versions of the 3D6 antibody: hu3D6VLv2 (SEQ ID NO:21), hu3D6VLv2 L37Q (SEQ ID NO: 143), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 S52G (SEQ ID
NO: 110), hu3D6VLv2 L54G (SEQ ID NO:94), hu3D6VLv2 L54D (SEQ ID NO:93), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L37Q L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q L50G L54R (SEQ ID NO: 119), hu3D6VLv2
L37Q L50G L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q L50D L54R (SEQ ID NO: 133), hu3D6VLv2 L37Q L50D L54G (SEQ ID NO: 132), hu3D6VLv2 L37Q S52G L54D (SEQ ID NO: 124), hu3D6VLv2 L37Q S52G L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q S52G L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q S52G L54R (SEQ ID NO: 122), hu3D6VLv2
L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2 L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2
L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), and hu3D6VLv2 L37Q_S52G_L54R_G100Q (SEQ ID NO: 138).
[0588] Example 12 Immunogenicity of hu3D6VHvlbAl 1 (heavy chain variable region)
[0589] Additional heavy chain variable region variants of hu3D6VHvlbAl 1 (also known as h3D6Hu5) (SEQ ID NO: 18) were designed, as shown in Table 14.
[0590] Figures 9A and 9B show alignments of the heavy chain variable region of murine 3D6 (SEQ ID NO:7) and of humanized versions of the 3D6 antibody (hu3D6VHvbl, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, hu3D6VHvb7, hu3D6VHvlbAl 1, h3D6VHvb8, and h3D6VHvb9) with human germline heavy chain variable region sequence IGHVl-69-2*01 (SEQ ID NO:25) and with human acceptor heavy chain variable region sequence 2RCS VH hFrwk (SEQ ID NO:75). hu3D6VHvbl is SEQ ID NO:76, hu3D6VHvb2 is SEQ ID NO:77, hu3D6VHvb3 is SEQ ID NO:78, hu3D6VHvb4 is SEQ ID NO:79,
hu3D6VHvb5 is SEQ ID NO:80, hu3D6VHvb6 is SEQ ID NO:90, hu3D6VHvb7 is SEQ ID NO:91, hu3D6VHvlbAl l is SEQ ID NO: 18, h3D6VHvb8 is SEQ ID NO: 146, and h3D6VHvb9 is SEQ ID NO: 148. [0591] Table 14: Additional Amino acid substitutions in hu3D6VHvlbAll (also known as h3D6Hu5)
Figure imgf000147_0001
[0592] The rationales for selection of the positions indicated in Table 14 in the heavy chain variable region as candidates for substitution are as follows.
[0593] D60E: to reduce potential proteolysis. Asp-Pro motifs are known to be potential proteolytic cleavage sites. There is an Asp-Pro motif in hu3D6VHvlbAl 1 (h3D6Hu5) VH at Kabat position 60-61. Based upon homology model, Glu substitution at position 60 is considered as conformation conserving. Therefore, D60E substitution is made to reduce potential proteolysis.
[0594] Q81E, to enhance thermostability and to increase humanness. Glu is predicted to make
H-bond plus salt-bridge with K19 hence Glu at this position make enhance antibody stability.
Glu is a germ-line aligning mutation. Glu is in human germline gene IMGT# IGHV1 -69-2*01 (SEQ (ID NO: 25) at this position.
[0595] L82cV to reduce immunogenicity. In silico analysis of humanized hu3D6VHvlbAl 1 (h3D6Hu5) immunogenicity using IEDB (immune epitope database) immunogenicity analysis tool predicted a potential immunogenic peptide that included L82c. L82cV substitution is designed to reduce immunogenicity. Valine substitution at this position conserves loop conformation.
[0596] L80M, to reduce immunogenicity; and to increase humanness. Met is a germline aligning mutation. Met is in human germ line gene IMGT# IGHVl-69-2*01 (SEQ (ID NO: 25) at this position. [0597] T83R to enhance thermostability and to increase humanness. Arg is a germline aligning mutation. Arg is in human germ line gene IMGT# IGHVl-69-2*01 (SEQ (ID NO: 25) at this position. Arg is most frequent at this position.
[0598] Example 13 Analysis of Humanized 3D6 Variants
[0599] Humanized 3D6 variants with predicted deimmunizing substitutions were analyzed for several characteristics, including target binding affinity, activity in cell-based assays,
thermostability, expression characteristics, and number of substitutions. In all cases, results were compared with the parental sequence, hu3D6VHvlbAl 1/ hu3D6VLv2 to determine if any loss of activity or stability was noted.
[0600] Target binding analysis was performed using a Biacore T200 to compare the binding affinity of humanized 3D6 variants to recombinant human 4R0N tau. Anti -Human Fc antibody was immobilized on sensor chip CM3 via amine coupling, and humanized 3D6 variants were captured to equivalent levels. Various concentrations of recombinant 4R0N human tau (ranging from 0.02 nM to 12.5 nM) were passed over the captured ligand at 50 pL/min in running buffer (HBS + 0.05% P-20, 1 mg/mL BSA) for 180 seconds association/420 seconds dissociation as a single cycle. Data was blank subtracted to both an irrelevant sensor not containing antibody and 0 nM analyte concentration. Analysis was performed using a global 1 : 1 fit with Biacore
Evaluation software.
[0601] Affinity determination revealed a number of deimmunized variants that retained the affinity of the parental hu3D6VHvlbAl 1/ hu3D6VLv2, which was determined by comparison of the KD of each antibody to that of parental hu3D6VHvlbAl 1/ hu3D6VLv2. Antibodies determined to have KD comparable within 4-fold of hu3D6VHvlbAl 1/ hu3D6VLv2 (Table 15) include hu3D6VHvlbAl 1/L2-DIM4, hu3D6VHvlbAl 1/L2-DIM9, hu3D6VHvlbAl 1/L2- DIM5, hu3D6VHvlbAl 1/L2-DIM3, h3D6VHvb8/L2-DIM4, hu3D6VHvlbAl 1/L2-DIM7, h3D6VHvb8/L2-DIM7, h3D6VHvb8/L2-DIM8, hu3D6VHvlbAl 1/L2-DIM12,
hu3D6VHvlbAl 1/L2-DIM21, and hu3D6VHvlbAl 1/L2-DIM22. hu3D6VHvlbAl 1/L2-DIM4 demonstrated improved affinity, as evidenced by on-rate, off-rate, and Kd numbers, over parental hu3D6VHvlbAl 1/ hu3D6VLv2. [0602] Table 15. Affinity of 3D6 Humanized Variants
Figure imgf000149_0001
[0603] In addition, as a secondary characteristic, thermostability and titer were analyzed for all deimmunized variants. Thermostability and titer levels were compared for antibodies that were comparable to hu3D6VHvlbAl 1/ hu3D6VLv2 based on affinity measurements, and antibodies in Table 16 are listed in order based on variance from Tm of hu3D6VHvlbAl 1/ hu3D6VLv2.
[0604] Thermostability values were determined using differential scanning calorimetry (DSC). All DSC scans were performed using a VP-Capillary DSC system (Malvern). All samples were prepared to 0.5 mg/mL inlxPBS, and referenced to lxPBS. Approximately 0.5 mL of protein solution and buffer were introduced into the sample and reference cell. Calorimetric scan rates were carried out at scan rates of 60°C/hour, from 25°C to 110°C under constant pressure.
Analysis was performed using origin software. Reported values are the temperature at which the maximal heat capacity of the Fab peak is recorded.
[0605] Titer was determined as follows. After expression in 293 suspension cells, antibodies were purified using Protein A chromatography utilizing standard methods. Following
purification, the antibodies were exchanged into lxPBS and protein concentrations were determined by absorbance at 280nm. Titers were calculated by dividing the final yield of purified protein by the starting volume of the expression culture, and reported in milligrams per liter. hu3D6VHvlbAl 1/L2-DIM4 demonstrated higher thermostability and titer over parental hu3D6VHvlbAl 1/ hu3D6VLv2. Thermostability and transient expression levels (titer) are both important considerations for drug development.
[0606] Table 16. Thermostability and Titer of 3D6 Humanized Variants
Figure imgf000150_0001
[0607] Based on analysis of the above-listed findings, the neutralization activity of seven antibodies in Table 16 were selected for further analysis in a cell based model of tau
internalization: Substitutions in the heavy and light chains relative to parental hu3D6VHvlbAl 1/ hu3D6VLv2 are noted. [0608] An internalization assay employing fluorescence activated cell sorting (FACS) was performed to evaluate the ability of various antibodies to block neuronal internalization of tau. Antibodies that block internalization will likely block transmission of tau.
[0609] Soluble tau aggregates were generated by incubation of recombinant full length tau with equimolar amounts of low molecular weight heparin for 3d at 37°C. After incubation, insoluble and soluble tau was separated by centrifugation at 1 O,OOOc# for 15 minutes. The supernatant was then resolved by preparative size exclusion chromatography, and aggregate peaks (greater than 100 kDa) were collected and concentrated. To measure internalization, the soluble aggregate fraction was labeled with pHrodo Red succinimidyl ester, which fluoresces when internalized into the endolysosomal pathway.
[0610] pHrodo-labeled 4R0N human tau P301L soluble oligomer (1.5 pg/ml final concentration) was preincubated with anti-tau antibodies (dose titration: 80 pg/ml starting concentration followed by 4-fold serial dilutions) for 30 min at room temperature in cell culture media. Tau/antibody mixture was then added to B 103 neuroblastoma cell lines at 500,000 cells/ml final concentration and incubated for 3-4 hrs at 37°C in a tissue culture incubator (5% CO2). Cells were then washed 3x with culture media, followed by 10 minutes culture media incubation, and washed 2x with FACS buffer (1% FBS in PBS). Cells were resuspended in 100 pi FACS buffer and Texas Red mean fluorescence intensity measured by FACS LSR II. Texas red fluorescence from pHrodo is activated by low pH associated with endolysosomal compartments upon internalization. Because FACS detects cells and pHrodo only fluoresces upon internalization, only tau internalized by the cells will be detected. The lower the mean fluorescence intensity, the lower the amount of internalized tau, which suggests a higher blocking activity of the antibody tested.
[0611] With the exception of hu3D6VHvlbAl 1/L2-DIM22, all tested variants displayed a high degree of inhibitory activity in the model of tau internalization at an equivalent concentration (Figure 11 and Table 17). [0612] Table 17. Humanized 3D6 Variants Tested in Cell-Based Assay of Tau Internalization
Figure imgf000152_0001
[0613] Example 14 Refinement of Epitope Mapping of 3 D6
[0614] Initial peptide mapping indicated that the epitope for 3D6 is in the microtubule binding repeat region (MTBR, Figure 1). Furthermore, peptide mapping indicated that 3D6 binds to multiple sites within the MTBR, suggesting a repeated and discontinuous epitope.
[0615] As the initial peptide map used for epitope studies utilized overlapping peptides overlaps that did not contain intact microtubule binding repeats, additional peptide mapping was carried out with the individual intact microtubule repeats. Biotinylated peptides corresponding to the amino acids for MTBR repeat 1-4 were added to a streptavi din-coated ELISA plate, washed, and blocked in 1% BSA/PBS. Varying amounts of 3D6 (ranging from 50 ug/ml-10 ng/ml) were then incubated on the plate. After washing, goat anti-mouse HRP was added to the ELISA plate, washed, and developed using OPD. Absorbance was detected at 490 nm, and binding curves were fitted using a 4-parameter log curve to determine EC50 values (Table 18). 3D6 bound MTBRs 1-3 relatively equivalently, with EC50 values of 72.5, 35.5 and 25.1 ng/ml respectively. However, binding to MTBR 4 was approximately 60-170 times lower, with an apparent EC50 of 4309 ng/ml. Sequence variations in the repeated epitope in MTBR 4 from MTBRs 1, 2, 3 and 4 may explain the weaker binding to MTBR 4.
[0616] Table 18
Figure imgf000153_0001
[0617] In order to more closely elucidate the specific amino acids responsible for 3D6 binding, substitution mapping via microarray was performed using the peptide NVKSKIGSTENLKHQPG (SEQ ID NO: 184), which is the sequence for residues 255-271 using the numbering of the full length tau isoform. The assay exchanged each of the amino acids in the peptide sequence with all 20 amino acids, to determine with a high degree of accuracy the core epitope for 3D6. The microarray was printed with all peptides in triplicate, along with HA control peptides to ensure assay performance. To perform the assay, the array was incubated with 3D6 (lOug/ml) in blocking buffer, washed with PBS-T, followed by incubation with a goat anti-mouse secondary antibody conjugated to DyLight-680. In addition, the microarray was incubated with anti-HA antibody 12CA5 directly conjugated to DyLight-800. Microarrays were scanned using a LiCor scanner, and intensities of triplicates were averaged.
[0618] Substitution effects for each residue were calculated by determining the ratio of 3D6 binding lost when the native residue was mutated to the sum of all other 19 amino acids. Results are plotted in Figure 12A. Loss of binding greater than 50% (dashed line) signifies an important residue for 3D6 binding. Binding of the probe peptide (residues 255-271 of the 441aa isoform) with 3D6 revealed an epitope substitution pattern with a conserved core motif KIGSTENLKH (SEQ ID NO: 188), framed by variable N- and C-terminal stretches NVKS (SEQ ID NO: 189) and QPG (SEQ ID NO: 190), both of which are not important for binding. Within this core epitope, residues that do not permit substitution are Lys5, Asn11, and His14; in addition, position 13 demonstrates reduced substitution tolerance for residues other than Lys and His. In addition to these important residues, Ser8 only allows limited substitution. This results in a discontinuous epitope of KXXSXXNX(K/H)H (SEQ ID NO: 191) as shown in Figure 12B. Pertinent sections of the tau microtubule binding repeats are presented in alignment, with important 3D6 binding residues highlighted. When compared to the microtubule repeats in tau, the important residues are present in all repeats, with the exception of the penultimate position in the core epitope (K/H, indicated by L). In this position, repeat 4 contains a threonine, which may be responsible for the lower 3D6 ELISA binding. The core epitope is defined at KIGSTENLKH (SEQ ID NO: 188, residues 259-268 of SEQ ID NO: 1, in MBTR repeat 1), KCGSKDNIKH (SEQ ID NO: 192, residues 290-299 of SEQ ID NO: 1, in MBTR repeat 2), KCGSLGNIHH (SEQ ID NO: 193, residues 321-330 of SEQ ID NO: 1, in MBTR repeat 3), with lesser binding at KIGSLDNITH (SEQ ID NO: 194, residues 353-362 of SEQ ID NO: 1, in MBTR repeat 4). [0619] Example 15 Mouse 3D6 binds patient samples from Alzheimer’s disease patients, immunohistochemistry experiments
[0620] Autopsy blocks of fresh frozen human brain samples (~ 0.5 g) were embedded in OCT, and cut using a cryostat to generate 10 pm sections. Sections were placed into a solution of glucose oxidase and beta D-glucose, in the presence of sodium azide, to block endogenous peroxidase. Once tissue sections were prepared, they were stained with 3D6 at the indicated concentrations using an anti-mouse polymer and DAB-based detection Kit (Leica BOND refine detection kit) as per the manufacturer's instructions. Results are shown in Figure 13.
[0621] In AD tissue, 3D6 binds hallmark pathological features of tau, including neurofibrillary tangles as well as dystrophic neurites. In addition, 3D6 binds soluble intracellular tau; this is evident by the staining present in normal healthy control tissue.
[0622] Example 16 Mouse 3D6 binds soluble tau aggregates, cross-linking high-mass MALDI Mass Spectrometry experiments
[0623] In order to determine if 3D6 bound a single or multiple tau subunits, crosslinking high- mass MALDI mass spectrometry was utilized to assess the binding stoichiometry. Cross-linking allow the direct analysis of non-covalent interaction. By mixing a protein sample containing non-covalent interactions with a cross-linking mixture (Bich, C et al. Anal. Chem., 2010, 82 (1), pp 172-179), it is possible to specifically detect non-covalent complex with high-sensitivity. In this assay, the interaction of 3D6 with soluble tau aggregates were tested. Soluble tau aggregates were generated by incubation of recombinant full length tau with equimolar amounts of low molecular weight heparin for 3d at 37°C. After incubation, insoluble and soluble tau was separated by centrifugation at 10,000xg- for 15 minutes. The supernatant was then resolved by preparative size exclusion chromatography, and aggregate peaks (greater than 100 kDa) were collected and concentrated.
[0624] 3D6 was mixed with various ratios of soluble tau aggregate, and detected by high mass
MALDI mass spectrometry. In addition, control experiments were performed of individual components to allow for correct labeling of detected peaks. Analysis was performed in linear positive mode with the HM4 interaction module (CovalX) with a standard nitrogen laser, focusing on the mass range of 0-1500 kDa. [0625] Results are shown in Figure 14. In the control (uncrosslinked) results, both tau and 3D6 are detected as individual entities of 40.93 and 146.938 kDa, respectively. However, when crosslinked, the antibody peak for 3D6 disappears, whereas two new peaks appear representing 3D6 interacting with one and two antigens (at 194.788 and 228.963, respectively. As detected in the overlay inset, the 3D6 peak at 146.938 kDa is notably absent from the crosslinked samples. These data suggest that 3D6 very strongly binds soluble aggregates of tau, and carries this out either through binding of one or two complexes per antibody.
[0626] Example 17 Mouse 3D6 interrupts tau seeding in an in vivo disease model of Alzheimer’s disease pathology
[0627] The ability of 3D6 to interrupt tau seeding were investigated in an in vivo disease model of the propagation of AD pathology. Pathological seeds were generated using brainstem homogenates from end-stage hTauP301L mice, or wild type controls. To induce pathology, seeds were unilaterally injected in the hippocampus of 3.5 month old mice, well before the onset of the progression of natural pathology. In order to test the ability of antibodies to impact seeding of tau, antibodies were preincubated before injection. After one month, the amount of seeding in the ipsilateral side of the mice was assessed by AT8 immunoreactivity; the number of neurons per section within the hippocampus was determined.
[0628] Results are shown in Figure 15. Compared to isotype control, preincubation of tau seeds with 3D6 was able to significantly decrease the amount of seeding detected in mice after one month, suggesting the interruption of tau internalization proximally to the injection site and suggesting that 3D6 can impair tau uptake and propagation.
[0629] Example 18 Mouse 3D6 and humanized variants interrupt tau interaction with heparin in vitro
[0630] Several studies indicate that tau internalization into cells is mediated via initial interaction of tau with surface-associated heparin sulfate proteoglycans (HSPGs), and that this interaction is an essential initial step leading to tau uptake, seeding, and propagation (Holmes et ak, PNAS, 110(33), 2013; Katsinelos et al., Cell Rep, 23, 2018). Therefore, a favorable property of a therapeutic anti-tau antibody would be the ability to block the interaction between tau and heparin that would be present on the surface of cells. [0631] To test the ability of murine and humanized 3D6 variants to interrupt tau interaction with heparin, ELISA plates coated with low molecular weight heparin were first blocked with 2% BSA in PBS for 2h at room temperature. Plates were washed with TBST. After blocking, plates were incubated with biotinylated recombinant tau along with indicated concentrations of unlabeled antibody in 1% BS A/PBS for one hour at room temperature. Plates were again washed, and then incubated with streptavidin-HRP in 0.1% BSA/PBS for 45min. Plates were then washed and developed with OPD, and absorbance was measured at 490nm. Inhibition curves were analyzed using a 4-parameter log fit.
[0632] Results are shown in Figure 16 and Table 19. Murine, as well as both humanized forms of 3D6 tested in the assay displayed the ability to block the tau/heparin interaction, indicating that the 3D6 epitope is important for this interaction, and that antibodies are able to inhibit the interaction. This suggests the ability to interrupt tau uptake into cells. Though all antibodies tested displayed inhibitory activity, the variant hu3D6VHvlbAl 1/L2-DIM4 retained activity closer to the murine form of the antibody (IC50=422.7 pM, compared to 409.2 pM for murine).
In contrast, hu3D6VHvlbAl l/hu3D6VLv2 demonstrated lower inhibitory activity (IC50=564.6 pM), compared to hu3D6VHvlbAl 1/L2-DIM4, indicating hu3D6VHvlbAl 1/L2-DIM4 has a higher degree of retention of murine antibody properties and suggesting an improvement of inhibitory activity of hu3D6VHvlbAl 1/L2-DIM4 due to the amino acid variants in the CDR of the light chain.
[0633] Table 19
Figure imgf000157_0001
[0634] Example 19 3D6 humanized variants bind fibrillar forms of tau
[0635] Recombinant tau fibrils were generated by either incubation with equimolar amounts of low molecular weight heparin with either 4-repeat tau (“fibrillar tau”) or seeding growth of 3- repeat and 4-repeat tau (“mixed fibrils”). Tau was incubated for 3-5d at 37°C. To isolate fibrils, prepared samples were ultracentrifuged at 100, 000 x# for 30 minutes. Pellets were resuspended in their original volume before storage at -80°C before use. [0636] ELISA plates were coated with preformed fibrils (overnight at 4°C in 50 mM carbonate-bicarbonate buffer, pH 9.6), washed, and blocked with 1% BSA in PBS. After blocking, plates were incubated with the indicated concentrations of biotinylated primary antibody in 0.1% BSA/PBS for lh at room temperature. Plates were washed, and then incubated with streptavidin-HRP in 0.1% BSA/PBS for 45min. Plates were then washed and developed with OPD, and absorbance was measured at 490nm. Binding curves were analyzed using a 4- parameter log fit.
[0637] Results are shown in Figure 17 and Table 20. Both humanized forms of 3D6 tested in the assay displayed strong binding to fibrillar forms of tau, whether fibrils contained 4R tau only or both 3R and 4R tau. The variant hu3D6VHvlbAl 1/L2-DIM4 displayed a preference for binding to both species (EC50=191.8 pM) compared to hu3D6VHvlbAl l/hu3D6VLv2
(EC50=236.7 pM), indicating an improvement in binding capacity _of hu3D6VHvlbAl l/L2-DIM4 due to the amino acid variants in the CDR of the light chain.
[0638] Table 20
Figure imgf000158_0001
[0639] Example 20 Exemplary CDRs
[0640] Exemplary CDRs of antibodies of the invention are in Table 21.
[0641] Table 21: Exemplary CDRs
Figure imgf000159_0001
Figure imgf000160_0001
Figure imgf000161_0001
Listing of Sequences
[0642] P10636-8 (SEQ ID NO: 1)
MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSE
EPGSET SD AK S TPT AED VT APL VDEGAPGKQ A A AQPHTEIPEGTT AEE AGIGDTP SLEDE
AAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAK
TPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSP
SSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKD
NIKHVPGGGS VQIVYKP VDL SK VT SKCGSLGNIHHKPGGGQ VEVKSEKLDFKDRVQ SKI
GSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSST
GSIDMVDSPQLATLADEVSASLAKQGL
[0643] P10636-7 (SEQ ID NO:2)
MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSE EPGSET SD AK S TPT AE AEE AGIGDTP SLEDE A AGH VT Q ARM V SK SKDGT GSDDKK AKG ADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPG TPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTEN LKHQPGGGK V QIINKKLDL SNVQ SKCGSKDNIKHVPGGGS VQIVYKP VDLSKVT SKCGS LGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAK AKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL
[0644] P10636-6 (4RON human tau) (SEQ ID NO:3)
MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSLE DEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIP AKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPK SP S S AK SRLQT AP VPMPDLKNVK SKIGS TENLKHQPGGGK VQIINKKLDL SN V Q SKC GS KDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQ VEVKSEKLDFKDRVQ SKIGSLDNITHVPGGGNKKIETHKLTFREN AK AKTDHGAEI V YK SP V V S GDT SPRHL SN V S STGSIDMVD SPQL ATL ADEV S ASL AKQGL
[0645] P10636-5 (SEQ ID NO:4)
MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSE EPGSET SD AK S TPT AED VT APL VDEGAPGKQ A A AQPHTEIPEGTT AEE AGIGDTP SLEDE AAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAK TPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSP SSARSRLQTAPVPMPDLRNVRSRIGSTENLRHQPGGGRVQIVYRPVDLSRVTSRCGSLG NIHHRPGGGQVEVRSERLDFRDRVQSRIGSLDNITHVPGGGNRRIETHRLTFRENARAR TDHGAEIVYRSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLARQGL
[0646] PI 0636-4 (SEQ ID NO:5)
MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSE EPGSET SD AR S TPT AE AEE AGIGDTP SLEDE A AGH VT Q ARM V SK SKDGT GSDDKK AKG ADGRTRIATPRGAAPPGQRGQANATRIPARTPPAPRTPPSSGEPPRSGDRSGYSSPGSPG TPGSRSRTPSLPTPPTREPRRVAVVRTPPRSPSSARSRLQTAPVPMPDLRNVRSRIGSTEN LKHQPGGGK V QI V YKP VDL SK VT SKC GSLGNIHHKPGGGQ VE VK SEKLDFKDRV Q SKI GSLDNITHVPGGGNRRIETHRLTFRENARARTDHGAEIVYRSPVVSGDTSPRHLSNVSST GSIDMVDSPQLATLADEVSASLARQGL
[0647] PI 0636-2 (SEQ ID NO: 6)
MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSLE
DEAAGHVTQARMVSRSRDGTGSDDRRARGADGRTRIATPRGAAPPGQRGQANATRIP
ARTPPAPRTPPSSGEPPRSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPRRVAVVRTPPR
SPSSARSRLQTAPVPMPDLRNVRSRIGSTENLRHQPGGGRVQIVYRPVDLSRVTSRCGS
LGNIHHRPGGGQVEVRSERLDFRDRVQSRIGSLDNITHVPGGGNRRIETHRLTFRENAR
ARTDHGAEIVYRSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLARQGL
[0648] SEQ ID NO:7; Murine 3D6 VH amino acid sequence:
EVQLQQSGADLVRPGALVKLSCKASGFNIKDYYLHWVRQRPEQGLEWIGWIDPENGDT VYDPKFQGKATITADTS SNTAYLQLGSLTSEDTAVYFC STLDFW GQGTTLTV S S
[0649] SEQ ID NO:8; Kabat/Chothia HCDR1 :
GFNIKDYYLH
[0650] SEQ ID NO: 9; Rabat HCDR2:
WIDPENGDT VYDPKF QG
[0651] SEQ ID NO: 10; Rabat HCDR3 :
LDF
[0652] SEQ ID NO: 1 1 ; Murine 3D6 VL amino acid sequence:
DVVMTQTPLTLSVTIGQPASISCRSSQSLLDSDGRTYLNWLLQRPGQSPRRLIYLVSRLD S GVPDRF TGS GS GTDF TLRISRVEAEDLGV Y Y C WQGTHFP YTF GGGTRLEIR
[0653] SEQ ID NO: 12; Murine Rabat LCDR1 : KSSQSLLDSDGKTYLN
[0654] SEQ ID NO: 13; Murine Rabat LCDR2:
LVSKLDS
[0655] SEQ ID NO: 14; Murine Rabat LCDR3 :
WQGTHFPYT
[0656] SEQ ID NO: 15; hu3D6VHvl :
EVQLVQSGAEVVRPGALVRVSCRASGFNIRDYYLHWVRQAPEQGLEWIGWIDPENGDT
V YDPRF Q GR ATIT ADT S TNT A YLQL S SLT SEDT A V YF C S TLDF W GQGTL VT V S S
[0657] SEQ ID NO: 16; hu3D6VHv2:
EVQLVQSGAEVRRPGASVRVSCRVSGFNIRDYYLHWVRQAPEQGLEWMGWIDPENGD T VYDPRF QGR VTIT ADT S TNT A YMEL S SLT SEDT AVY Y C S TLDF W GQGTL VTV S S
[0658] SEQ ID NO: 17; hu3D6VHvlb:
EVQLVQSGAEVVRPGALVRISCRASGFNIRDYYLHWVRQRPEQGLEWIGWIDPENGDT VYDPRF Q GR ATIT ADT S TNT A YLQLGSLT SEDT A V YF C S TLDF W GQGTL VTV S S
[0659] SEQ ID NO: 18; hu3D6VHvlbAl l :
EVQLVQSGAEVVRPGATVRISCRASGFNIRDYYLHWVRQRPGQGLEWIGWIDPENGDT VYDPRF Q GRATIT ADT S TDT A YLQLGSLT SEDT A V YF C S TLDF W GQ GTL VT V S S
[0660] SEQ ID NO: 19; hu3D6VHv5:
EVQLVQSGAEVVRPGATVRISCRASGFTIRDYYLHWVRQRPGQGLEWIGWIDPEDGDT
V Y APRF Q GRATIT ADT S TDT A YLQLGSLT SEDT A V YF C S TLDF W GQ GTL VTV S S
[0661] SEQ ID NO:20; hu3D6VLvl :
DVVMTQSPLSLSVTLGQPASISCRSSQSLLDSDGRTYLNWLLQRPGQSPRRLIYLVSRLD S GVPDRF S GS GS GTDF TLRISRVE AED V G V Y Y C WQGTHFP YTF GGGTRLEIR
[0662] SEQ ID NO:21; hu3D6VLv2:
D VVMT Q SPL SLP VTLGQP ASISCRS SQ SLLD SDGRT YLNWLLQRPGQ SPRRLIYL V SRLD S GVPDRF S GS GS GTDF TLRISRVE AED V G V Y Y C WQGTHFP YTF GGGTRLEIR
[0663] SEQ ID NO:22; hu3D6VLv3 :
D VVMT Q SPL SLP VTLGQP ASISCRS SQ SLLD SDGRT YLNWLLQRPGQ SPRRLIYL V SRLD S GVP SRF S GS GS GTDF TLRISRVEAED V GV Y Y C WQGTHFP YTF GGGTRLEIR
[0664] SEQ ID NO:23; hu3D6VLv4: DIVMTQTPLSLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPKRLIYLVSKLDS GVPDRF S GS GS GTDF TLKISRVE AED V G V Y Y C WQGTHFP YTF GGGTKLEIK
[0665] SEQ ID NO:24 ; heavy chain variable acceptor Acc.# BAC01986.1
QVQLQQSGAEVKKPGSSVKVSCKASGGTFGSYAISWVRQAPGQGLEWMGRIIPILGIAT Y AQKF QGRVTIT ADK S T S T A YMDL S SLRSEDT A V Y Y C ARGKGEFEGMD VW GQ GTT VT
vss
[0666] SEQ ID NO:25 ; heavy chain variable acceptor Acc.# IMGT# IGHVl-69-2*01
EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDG
ETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCAT
[0667] SEQ ID NO:26 ; heavy chain variable acceptor Acc.# IMGT#IGKJ 1 *01
QHW GQGTL VT V S S
[0668] SEQ ID NO:27; light chain variable acceptor Acc. # IMGT#IGKV2-30*02 Acc. # IMGT#IGKV2-30*02
D VVMT Q SPL SLP VTLGQP ASISCRS SQ SLVHSDGNT YLNWFQQRPGQ SPRRLIYK V SNRD SGVPDRF SGSGSGTDFTLKISRVEAED VGVYY CMQGTHWP
[0669] SEQ ID NO:28 ; light chain variable acceptor Acc. # IMGT#IGKJ2*01
YTF GQGTKLEIK
[0670] SEQ ID NO:29; Light chain variable acceptor Acc. # AAZ09048.1
D VVMT Q SPL SLT VTLGQP ASISCRS SQ SLVY SDGNT YLNWF QQRPGQ SPRRLIYRV SHW D S GVPDRF S GS GS GTDF TLKISRVE AED V G V Y Y CMQGT YWPLTF GQGTKLEIK
[0671] SEQ ID NO:30 ; Murine 3D6 VH nucleic acid sequence:
GAGGTTCAGCTGCAGCAGTCTGGGGCTGACCTTGTGAGGCCAGGGGCCTTAGTCAA
GTTGTCCTGCAAAGCTTCTGGCTTCAACATTAAAGACTACTATTTGCACTGGGTGAG
GC AGAGGC C T G A AC AGGGC C T GG AGT GG ATT GG AT GG ATT GAT C C T GAG A AT GGT G
ATACTGTATATGACCCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACATCC
TCCAATACAGCCTACCTGCAGCTCGGCAGCCTGACATCTGAGGACACTGCCGTCTAT
TTCTGTTCTACCCTTGACTTCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA
[0672] SEQ ID NO:31 ; Murine 3D6 VL nucleic acid sequence: GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCC
TCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTG
AATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCT
AAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTC
ACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCA
AGGTACACATTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGT
[0673] SEQ ID NO:32; Murine CDR-H1 Rabat
DYYLH
[0674] SEQ ID NO:33; Murine CDR-H1 Chothia
GFNIKDY
[0675] SEQ ID NO:34; Murine CDR-H2 Chothia
DPENGD
[0676] SEQ ID NO : 35 ; Murine CDR-H2 AbM
WIDPENGDTV
[0677] SEQ ID NO: 36; Murine CDR-L1 Contact
KTYLNWL
[0678] SEQ ID NO: 37; Murine CDR-L2 Contact
RLIYLVSKLD
[0679] SEQ ID NO:38; Murine CDR-L3 Contact
WQGTHFPY
[0680] SEQ ID NO:39; Murine CDR-H1 Contact
KDYYLH
[0681] SEQ ID NO:40; Murine CDR-H2 Contact
WIGWIDPENGDTV
[0682] SEQ ID NO:41; Murine CDR-H3 Contact
STLD
[0683] SEQ ID NO:42; Alternate Kabat-Chothia CDR-H1
GFTIKDYYLH
[0684] SEQ ID NO:43; Alternate Rabat CDR-H2
WIDPEDGDTVYAPRFQG [0685] SEQ ID NO:44; consensus VH amino acid sequence from Figure 2 of
PCT/IB2017/052544
EVQLVQSGAEVVXPGALVKISCKASGFNIKDYYLHWVRQRPEQGLEWIGWIDPENGDT VYDPKF Q GX ATIT ADT S TNT A YLQLGSLT SEDT A V YF C S TLDF W GQGTL VT V S S
[0686] SEQ ID NO:45; consensus VL amino acid sequence of Figure 3 of
PCT/IB2017/052544
DVVMTQSPLSLSVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLD S GVPDRF S GS GS GTDF TLKISRVE AED V G V Y Y C WQGTHFP YTF GGGTKLEIKR
[0687] SEQ ID NO:46; hu3D6VHvlbAl 1B6G2:
EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWVDPEDGDT VY APKFQGRATIT ADT STDT AYLELGSLTSEDT AVYF C STLDFW GQGTL VTV S S
[0688] SEQ ID NO:47; hu3D6VHvlbAl 1B6H3 :
EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGDT VY APKFQGRATIT ADT STDT AYLELGSLTSEDT AVYF C STLDFW GQGTL VTV S S
[0689] SEQ ID NO:48; hu3D6VHvlc:
EVQLVQSGAEVKRPGALVKISCKASGFNFKDYYLHWVRQRPEQGLEWMGWIDPENGD TVYDEKFQGRVTITADTSTNTAYLQLGSLTSEDTAVYFCSTLDFW GQGTL VTVSS
[0690] SEQ ID NO:49; hu3D6VHvld:
EVQLVQSGAEVKRPGALVKISCKASGYTFTDYYLHWVRQRPEQGLEWMGWVDPEDGD TVYAEKFQGRVTITADTSTNTAYLQLGSLTSEDTAVYFCSTLDFW GQGTL VTVSS
[0691] SEQ ID NO:50; hu3D6VHvle:
EVQLVQSGADVvkPGALVKISCKASGFTIKDYYLHWVRQRPEQGLEWIGWIDPENGDTV Y AEKF QGRVTIT ADT STNT AYLeLGSLT SEDT AVYF C STLDFW GQGTTLT V S S
[0692] SEQ ID NO:51; hu3D6VHvlf:
EVQLVQSGADVVKPGALVKISCKASGFTIKDYYLHWVRQRPGQGLEWIGWVDPEDGD TVYAEKFQGRVTITADTSTDTAYMELGSLTSEDTAVYFCSTLDYWGQGTTLTVSS
[0693] SEQ ID NO: 52; hu3D6VHv3 :
EVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYLHWVRQAPGKGLEWMGWIDPENGD T VYDPKF QGRVTITADTSTDTAYMELS SLRSEDT AVYYCSTLDFWGQGTLVT V S S
[0694] SEQ ID NO:53; hu3D6VHv3b: E VQL V Q S GAE VKKPGAL VKIS CK V S GYNFKD YYLHW VRQAPGKGLEWMGWIDPEN G DTVYDEKFQGRVTITADTSTNTAYMELGSLRSEDTAVYYCSTLDFWGQGTLVTVSS
[0695] SEQ ID NO:54; hu3D6VHv3c:
EVQLVQSGAE VKKPGAL VKISCKVSGYTFTDYYLHWVRQAPGKGLEWMGWVDPEDG DTVYAEKFQGRVTITADTSTNTAYMELGSLRSEDTAVYYCSTLDFWGQGTLVTVSS
[0696] SEQ ID NO: 55; hu3D6VHv4:
EVQLVQSGAEVVKPGATVKISCKVSGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDT VYDPKF Q GK ATIT ADT S TNT A YLELGSLT SEDT A V Y Y C S TLDF W GQGTL VT V S S
[0697] SEQ ID NO:56; hu3D6VHv4b:
E VQL V Q S GAE VVKPGAL VKIS CKV S GYNFKD YYLHW VRQRPGKGLEWMGWIDPEN GD TVYDEKFQGRVTITADTSTDTAYLELGSLTSEDTAVYYCSTLDFWGQGTLVTVSS
[0698] SEQ ID NO:57; hu3D6VHv4c:
EVQLVQSGAEVVKPGALVKISCKVSGYTFTDYYLHWVRQRPGKGLEWMGWVDPEDG DTVYAEKFQGRVTITADTSTDTAYLELGSLTSEDTAVYYCSTLDFWGQGTLVTVSS
[0699] SEQ ID NO:58; Alternate Kabat-Chothia CDR-H1 (as in hu3D6VHlc).
GFNFKDYYLH
[0700] SEQ ID NO:59; Alternate Kabat-Chothia CDR-H1, (as in hu3D6VHvld,
hu3D6VHv3c, and hu3D6VHv4c).
GYTFTDYYLH
[0701] SEQ ID NO:60; Alternate Kabat-Chothia CDR-H1 (as in hu3D6VHv3b and hu3D6VHv4b)
GYNFKD YYLH
[0702] SEQ ID NO:61; Alternate Rabat CDR-H2 (as in hu3D6VHvlbAl 1B6G2).
WVDPEDGDTVYAPKFQG
[0703] SEQ ID NO: 62, Alternate Rabat CDR-H2 (as in hu3D6VHvlc, hu3D6VHv3b, AND hu3D6VHv4b.
WIDPENGDT VYDEKF QG
[0704] SEQ ID NO: 63; Alternate Rabat CDR-H2 as in hu3D6VHvld, hu3D6VHvlf, hu3D6VHv3c, and hu3D6VHv4c).
WVDPEDGDT VY AEKF QG
[0705] SEQ ID NO:64; Alternate Rabat CDR-H2 (as in hu3D6VHvle). WIDPENGDTVYAEKFQG
[0706] SEQ ID NO: 65; Alternate Rabat CDR-H3 (as in hu3D6VHvlf)
LDY
SEQ ID NO:66; heavy chain variable region of the mouse 6A10 antibody.
EVQLQQSGAELVRSGASVKLSCTASGLNIKDYYIHWVKQRPEQGLEWIGWIDPENDDTE YAPKFQGRATLTTDTSSNTAYLQLSSLTSEDTAVYYCTPLDYWGQGTSVTVSS
[0707] SEQ ID NO:67; Kabat/Chothia composite CDR-H1 of the mouse 6A10 antibody. GLNIKDYYIH
[0708] SEQ ID NO:68; Rabat CDR-H2 of the mouse 6A10 antibody.
WIDPENDDTE Y APRF QG
[0709] SEQ ID NO: 69; Rabat CDR-H3 of the mouse 6A10 antibody
LDY
[0710] SEQ ID NO:70; Mus VH structure template (PDB# 1 CR9 H)
RVRLQQSGAELVRSGASVRLSCTASGFNIRDYYIQWVRQRPEQGLEWIGWIDPENGNSEYAPRF QGRATMTADTLSNTAYLQLSSLTSEDTAVYYCNADLHDYWGQGTTLTVSS
[0711] SEQ ID NO:71 ; consensus VH amino acid sequence from Figures 4A and 4B of PCT/IB2017/052544
EVQLVQSGAEVVRPGALVRISCRASGFNIRDYYLHWVRQRPGQGLEWIGWIDPENGDT V YDPRF Q GRVTIT ADT S TNT A YLELGSLT SEDT A V YF C S TLDF W GQGTL VT V S S
[0712] SEQ ID NO: 72; heavy chain of chimeric 3D6 antibody
EVQLQQSGADLVRPGALVRLSCRASGFNIRDYYLHWVRQRPEQGLEWIGWIDPENGDT VYDPRFQGRATITADTSSNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTTLTVSSASTRG P S VFPL AP S SR S T S GGT A ALGOL VRD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S S VVTVP S S SLGTQT YICNVNHRP SNTRVDRRVEPRSCDRTHT CPPCP APELLGGP S VFL FPPRPRDTLMISRTPEVTC VVVD V SHEDPEVRFNWYVDGVEVHNARTRPREEQYNST Y RVVSVLTVLHQDWLNGREYRCRVSNRALPAPIERTISRARGQPREPQVYTLPPSREEMT RN Q V SLTCL VRGF YP SDI A VEWE SN GQPENN YRTTPP VLD SDGSFFL Y SRLT VDR SRW Q QGNVFSCSVMHEALHNHYTQRSLSLSPGR
[0713] SEQ ID NO: 73; light chain of chimeric 3D6 antibody
DVVMTQTPLTLSVTIGQPASISCRSSQSLLDSDGRTYLNWLLQRPGQSPRRLIYLVSRLD S GVPDRF TGS GS GTDF TLRISRVE AEDLGV Y Y C WQGTHFP YTF GGGTRLEIRRT V A AP S VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SL S S TLTL SK AD YEKHK V Y ACE VTHQ GL S SP VTK SFNRGEC
[0714] SEQ ID NO:74; amino acid sequence of heavy chain variable structural model Acc 5MYX- VH_m St
EVQLQQSGAELVRPGSSVKISCKASGYIFNNYWINWVKQRPGQGLEWIGQIYPGDGDTN
YNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAREGYIVYWGQGTLVTVSA
[0715] SEQ ID NO:75; amino acid sequence of heavy chain variable acceptor Acc.# 2RCS- VH huFrwk
QVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNT KYDPKFQGKATIT ADTS SNTAYLQLS SLTSEDTAVYYC AS YYGIYWGQGTTLT VS S
[0716] SEQ ID NO:76; amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvbl
QVQLQQSGAELVKPGASVKLSCTASGFNIKDYYLHWVKQRPEQGLEWIGWIDPENGDT VYDPKFQGKATIT ADTS SNTAYLQLS SLTSEDTAVYFC STLDFW GQGTTLTV S S
[0717] SEQ ID NO:77; amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb2
EVQLVQSGAEVVKPGASVKISCKASGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDT VYDPKF Q GRATIT ADT S TDT A YLEL S SLT SEDT A VYF C S TLDF W GQ GTL VT V S S
[0718] SEQ ID NO:78; amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb3
EVQLVQSGAEVVKPGATVKISCKASGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDTI YDPKFQGRATIT ADT STDT AYMELS SLRSEDT AVYY C STLDFW GQGTL VT V S S
[0719] SEQ ID NO:79; amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb4
EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPENGDTI YDPKFQGRVTIT ADT STDT AYMELS SLRSEDT AVYY C STLDFW GQGTL VTV S S
[0720] SEQ ID NO: 80; amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb5
EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGETI YDPKFQGRVTIT ADT STDT AYMELS SLRSEDT AVYY C STLDFW GQGTL VTV S S [0721] SEQ ID NO:81; amino acid sequence of light chain variable structural model Acc.# 5MYX- VL_m St
DVVLTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYVVSKLDS
GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPFTFGSGTKLEIK
[0722] SEQ ID NO: 82; amino acid sequence of light chain variable acceptor Acc.#
ARX71335 - VL_huF rwk
DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYLVSKLD S GVPDRF S GS GS GTDF TLKISRVE AEDLGVH Y CEQGTHFPLTF GAGTKLELK
[0723] SEQ ID NO: 83; amino acid sequence of light chain variable region of the humanized 3D6 antibody hu3D6VLvbl
DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLD SGVPDRF SGSGSGTDFTLKISRVEAEDLGVHY CWQGTHFP YTF GAGTKLELK
[0724] SEQ ID NO: 84; amino acid sequence of light chain variable region of the humanized 3D6 antibody hu3D6VLvb2
DVVMTQSPLSLSVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLD S GVPDRF S GS GS GTDF TLKISRVE AEDV G V Y Y CWQGTHFP YTF G AGTKLEIK
[0725] SEQ ID NO:85; amino acid sequence of light chain variable region of the humanized 3D6 antibody hu3D6VLvb3
D VVMT Q SPL SL S VTLGEP ASISCRS SQ SLLD SDGKT YLNWLQQRPGQ SPRRLIYL V SKLD S GVPDRF S GS GS GTDF TLKISRVE AEDV G V Y Y CWQGTHFP YTF GQGTKLEIK
[0726] SEQ ID NO:86; amino acid sequence of an alternate Kabat-Chothia Composite CDR- H1 of a humanized 3D6 antibody (as in hu3D6VHvb4 and hu3D6VHvb5)
GFTIKDYYLH
[0727] SEQ ID NO: 87; amino acid sequence of an alternate Rabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvb3 and hu3D6VHvb4)
WIDPENGDTIYDPKF QG
[0728] SEQ ID NO:88; amino acid sequence of an alternate Rabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvb5)
WIDPEDGETI YDPRF QG
[0729] SEQ ID NO:89; amino acid sequence of an alternate Rabat CDR-L1 of a humanized 3D6 antibody (as in hu3D6VLvb3) RSSQSLLDSDGKTYLN
[0730] SEQ ID NO:90; amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb6
EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGET VYDPKF Q GRVTIT ADT S TDT A YMEL S SLRSEDT A VYF C S TLDF W GQ GTL VT V S S
[0731] SEQ ID NO:91; amino acid sequence of heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb7
EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGET VYDPKF Q GRVTIT ADT S TDT A YMEL S SLRSEDT A VYY C S TLDF W GQGTL VT V S S
[0732] SEQ ID NO: 92; amino acid sequence of an alternate Rabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvb6 and hu3D6VHvb7)
WIDPEDGET VYDPKF QG
[0733] SEQ ID NO:93; light chain variable region of a hu3D6VLv2 variant L54D, also known as L2-DIM21
DWMTQS PLSLPVTLGQPAS I SCKS SQSLLDSDGKTYLNWLLQRPGQS PRRL I YLVSKDDSGVP DRFSGSGSGTDFTLKI SRVEAEDVGVYYCWQGTHFPYT FGGGTKLE IK
[0734] SEQ ID NO:94; light chain variable region of a hu3D6VLv2 variant L54G, also known as L2-DIM7
DWMTQS PLSLPVTLGQPAS I S CKS SQSLLDSDGKT YLNWLLQRPGQS PRRL I YLVSKGDSGVP DRFSGSGSGTDFTLKI SRVEAEDVGVYYCWQGTHFPYT FGGGTKLE IK
[0735] SEQ ID NO: 95; light chain variable region of a hu3D6VLv2 variant L45N
DWMTQS PLSLPVTLGQPAS I S CKS SQSLLDSDGKT YLNWLLQRPGQS PRRL I YLVSKNDSGVP DRFSGSGSGTDFTLKI SRVEAEDVGVYYCWQGTHFPYT FGGGTKLE IK
[0736] SEQ ID NO: 96; light chain variable region of a hu3D6VLv2 variant L54E
DWMTQS PLSLPVTLGQPAS I S CKS SQSLLDSDGKT YLNWLLQRPGQS PRRL I YLVSKEDSGVP DRFSGSGSGTDFTLKI SRVEAEDVGVYYCWQGTHFPYT FGGGTKLE IK
[0737] SEQ ID NO:97; light chain variable region of a hu3D6VLv2 variant L50E
DWMTQS PLSLPVTLGQPAS I S CKS SQSLLDSDGKT YLNWLLQRPGQS PRRL I YEVSKLDSGVP DRFSGSGSGTDFTLKI SRVEAEDVGVYYCWQGTHFPYT FGGGTKLE IK
[0738] SEQ ID NO:98; light chain variable region of a hu3D6VLv2 variant L54Q DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKQDSGVP
DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0739] SEQ ID NO:99; light chain variable region of a hu3D6VLv2 variant L50D
DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYDVSKLDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0740] SEQ ID NO: 100; light chain variable region of a hu3D6VLv2 variant L54K
DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKKDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0741] SEQ ID NO: 101; light chain variable region of a hu3D6VLv2 variant L54R
DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKRDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0742] SEQ ID NO: 102; light chain variable region of a hu3D6VLv2 variant L54T
DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKTDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0743] SEQ ID NO: 103; light chain variable region of a hu3D6VLv2 variant L50G, also known as L2-DIM22
DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYGVSKLDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0744] SEQ ID NO: 104; light chain variable region of a hu3D6VLv2 variant I48G
DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLGYLVSKLDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0745] SEQ ID NO: 105; light chain variable region of a hu3D6VLv2 variant I48D
DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLDYLVSKLDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0746] SEQ ID NO: 106; light chain variable region of a hu3D6VLv2 variant L47G
DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRGIYLVSKLDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0747] SEQ ID NO: 107; light chain variable region of a hu3D6VLv2 variant Y49E
DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIELVSKLDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0748] SEQ ID NO: 108; light chain variable region of a hu3D6VLv2 variant L54V DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKVDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0749] SEQ ID NO: 109; light chain variable region of a hu3D6VLv2 variant L54S
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKSDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0750] SEQ ID NO: 110; light chain variable region of a hu3D6VLv2 variant S52G, also known as L2-DIM9
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVGKLDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0751] SEQ ID NO: 111; light chain variable region of a hu3D6VLv2 variant L47N
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRNIYLVSKLDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0752] SEQ ID NO: 112; light chain variable region of a hu3D6VLv2 variant L47D
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRDIYLVSKLDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0753] SEQ ID NO: 113; light chain variable region of a hu3D6VLv2 variant L47E
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRREIYLVSKLDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0754] SEQ ID NO: 114; light chain variable region of a hu3D6VLv2 variant L47P
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRPIYLVSKLDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0755] SEQ ID NO: 115; light chain variable region of a hu3D6VLv2 variant L47T
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRTIYLVSKLDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0756] SEQ ID NO: 116; light chain variable region of a hu3D6VLv2 variant L47S
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRSIYLVSKLDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0757] SEQ ID NO: 117; light chain variable region of a hu3D6VLv2 variant L47A
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRAIYLVSKLDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0758] SEQ ID NO: 118, light chain variable region of a hu3D6VLv2 variant L50V DWMTQSPLSLPVTLGQPAS ISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLI YWSKLDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0759] SEQ ID NO: 119; light chain variable region of a hu3D6VLv2 variant
L 37 Q_L 50 G_L 54R, also known as L2-DIM1
DWMTQSPLSLPVTLGQPAS TSCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLTYGVSKGDSGVP
DRFSGSGSGTDFTLKIS R VE AE D VG V Y YCWQGTHFPYTFGGGTKLETK
[0760] SEQ ID NO: 120; light chain variable region of a hu3D6VLv2 variant
L 37 Q_L 50 G_L 54 G, also known as L2-DIM2
DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYEVSKGDSGVP
DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0761] SEQ ID NO: 121; light chain variable region of a hu3D6VLv2 variant
L37Q_S52G_L54G, also known as L2-DIM3
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLN LQQRPGQSPRRLIYLVGKGDSGVP D R F S G S G S G T D F T L K I S R VE AE D VG V Y YCWQGTHFPYTFGGGTKLETK
[0762] SEQ ID NO: 122; light chain variable region of a hu3D6VLv2 variant
L37Q S52G L54R, also known as L2-DIM4
DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVGKRDSGVP D R F S G S G S G T D F T L K I S R YE AE D V G V Y Y C W Q G T H F P Y T F G G G T K L E I K
[0763] SEQ ID NO: 123; light chain variable region of a hu3D6VLv2 variant
L37Q S52G L54T, also known as L2-DIM5
DWMTQSPLSLPVTLGQPAS T SCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLI YLVGKTDSGVP
DRFSGSGSGTDFTLKIS R VE AE D VG V Y YCWQGTHFPYTFGGGTKLETK
[0764] SEQ ID NO: 124; light chain variable region of a hu3D6VLv2 variant
L37Q_S52G_L54D, also known as L2-DIM6
DWMTQSPLSLPVTLGQPAS I SCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVGKDDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0765] SEQ ID NO: 125; light chain variable region of a hu3D6VLv2 variant L37Q_L54R DWMTQSPLSLPVTLGQPASISCKSSGSLLDSDGKTYLNWLQQRPGGSPRRLIYLVSKGDSGVP
DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0766] SEQ ID NO: 126; light chain variable region of a hu3D6VLv2 variant L37QJL54G DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVSKGDSGVP
DRFSGSGSGTDFTLKIS R VE AE D V G V Y Y C WQGTHFPYTFGGGTKLEI K
[0767] SEQ ID NO: 127; light chain variable region of a hu3D6VLv2 variant L37Q L54D, also known as L2-DIM12
DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVSKDDSGVP
DRFSGSGSGTDFTLKIS R VE AE D V G V Y Y C WQGTHFPYTFGGGTKLEI K
[0768] SEQ K) NO: 128; light chain variable region of a hu3D6VLv2 variant L37Q L50G, also known as L2-DIM13
DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYGVSKLDSGVP D R F S G S G S G T D F T L K I S R YE AE D V G V Y Y C W Q G T H F P Y T F G G G T K L E I K
[0769] SEQ ID NO: 129; light chain variable region of a hu3D6VLv2 variant L37Q_L50D, also known as L2-DIMI4
DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYDVSKLDSGVP
DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0770] SEQ ID NO: 130; light chain variable region of a hu3D6VLv2 variant L37QJL54T DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLN LQQRPGQSPRRLIYDVSKLDSGVP
DRFSGSGSGTDFTLKIS R VE AE D VG V Y YCWQGTHFPYTFGGGTKLETK
[0771] SEQ ID NO: 131; light chain variable region of a hu3D6VLv2 variant L37Q S52G DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVGKLDSGVP
DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0772] SEQ ID NO: 132; light chain variable region of a hu3D6VLv2 variant
L37Q L50D L54G, also known as L2-DIM17
DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYDVSKGDSGVP
DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK [0773] SEQ ID NO: 133; light chain variable region of a hu3D6VLv2 variant.
L37Q L50D L54R, also known as L2-DIM18
DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYDVSKRDSGVP
DRFSGSGSGTDFTLKISRVEAEDVGVYYC QGTHFPYTFGGGTKLEIK
[0774] SEQ ID NO: 134; light chain variable region of a hu3D6VLv2 variant.
L37Q L50E L54G, also known as L2-DIMI9
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLN LQQRPGQSPRRLIYEVSKGDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLETK
[0775] SEQ ID NO: 135; light chain variable region of a hu3D6VLv2 variant
L37Q_L50E_L54R, also known as L2-DIM20
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLN LQQRPGQSPRRLIYEVSKRDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLETK
[0776] SEQ ID NO: 136; light chain variable region of a hu3D6VLv2 variant
L37Q_L50G_L54R_G100Q
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLN LQQRPGQSPRRLIYGVSKRDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLETK
[0777] SEQ ID NO: 137; light chain variable region of a hu3D6VLv2 variant
L 37 Q_L 50 G_L 54 G_G 100 Q
DVVMTQSPLSLPVTLGQPAS TSCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLTYGVSKGDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK
[0778] SEQ ID NO: 138; light chain variable region of a hu3D6VLv2 variant
L37Q_S52G_L54R_G100Q
DVVMTQSPLSLPVTLGQPAS TSCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLTYLVGKRDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK
[0779] SEQ ID NO: 139; light chain variable region of a hu3D6VLv2 variant
L37Q_S52G_L54D_G1G0Q
DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVGKDDSGVP
DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK [0780] SEQ ID NO: 140; light chain variable region of a hu3D6VLv2 variant
1. 7 Q L 5 QD L 54 G G 100Q
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYDVSKGDSGVP
DRFSGSGSGTDFTLKISRVEAEDVGVYYC QGTHFPYTFGQGTKLEIK
[0781] SEQ ID NO: 141; light chain variable region of a hu3D6VLv2 variant
L 37 Q L 5 QD L 54R G 100 Q
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLN LQQRPGQSPRRLIYDVSKRDSGVP
DRFSGSGSGTDFTLKI SRVEAEDVGVYYCWQGTHFPYTFGQGTKLETK
[0782] SEQ ID NO: 142; light chain variable region of a hu3D6VLv2 variant
L37 Q__L 50 V_L 54D_G 1 Q0Q
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLN LQQRPGQSPRRLIYVVSKDDSGVP
DRFSGSGSGTDFTLKI SRVEAEDVGVYYCWQGTHFPYTFGQGTKLETK
[0783] SEQ ID NO: 143; light chain variable region of a hu3D6VLv2 variant L37Q, also known as L2-DIM8
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLN LQQRPGQSPRRLIYLVSKLDSGVP
DRFSGSGSGTDFTLKI SRVEAEDVGVYYCWQGTHFPYTFGGGTKLETK
[0784] SEQ ID NO: 144 light chain variable region of a hu3D6VLv2 variant G10QQ
DVVMTQSPLSLPVTLGQPAS ISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKLDSGVP
DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK
[0785] SEQ ID NO: 145 light chain variable region of a hu3D6VLv2 variant L37Q_L54E DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVSKEDSGVP
DRFSGSGSGTDFTLKI SRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK
[0786] SEQ ID NO: 146; heavy chain variable region of a hu3D6VHvlbAl 1 variant D6QE, also known as h3D6VHvb8
EVQLVQSGAEV^/KPGATVKI SCKASGFNIKDYYLHWVRQRPGQGLEWIGWTDPENGDTVYEPKF QGRAT I TADTSTDTAYLQLGSLTSEDTAVY”FCSTLDFWGQGTLVTVSS
[0787] SEQ K) NO: 147 heavy chain variable region of a hu3D6VHvIbAl 1 variant L82cV EVQLVQSGAEWKPGATVKISCKASGFNIKDYYLHWVRQRPGQGLEWIGWIDPENGDTVYDPKF QGRAT I TADΪSTDΪAYLQLGSVTSEDTAVYFCSTLDFWGQGTLVTVSS
[0788] SEQ ID NO: 148; heavy chain variable region of a hu3D6VHvlbAl 1 variant
D60E L80M Q81E L82cV T83R, also known as h3D6VHvb9
EVQLVQSGAEWKPGATVKISCKASGFNIKDYYLHWVRQRPGQGLEWIGWIDPENGDTVYEPKF
QGRATITADTSTDTAYMELGSVRSEDTAVYECSTLDFWGQGTLVTVSS
[0789] SEQ ID NO: 149; amino acid sequence of an alternate Kabat CDR-H2 of a humanized 3D6 antibody (as in h3D6VHvb8 and in h3D6VHvb9)
WIDPENGDT VYEPKF QG
[0790] SEQ ID NO: 150; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54D and in hu3D6VLv2 L37Q L54D):
LVSKDDS
[0791] SEQ ID NO: 151; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54G and in hu3D6VLv2 L37Q L54G):
LVSKGDS
[0792] SEQ ID NO: 152; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54N):
LVSKNDS
[0793] SEQ ID NO: 153; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54E and in hu3D6VLv2 L37Q L54E):
LVSKEDS
[0794] SEQ ID NO: 154; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L50E):
EVSKLDS
[0795] SEQ ID NO: 155; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54Q):
LVSKQDS
[0796] SEQ ID NO: 156; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L50D and in hu3D6VLv2 L37Q L50D):
DVSKLDS [0797] SEQ ID NO: 157; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54K):
LVSKKDS
[0798] SEQ ID NO: 158; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54R and in hu3D6VLv2 L37Q L54R):
LVSKRDS
[0799] SEQ ID NO: 159; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54T and in hu3D6VLv2 L37Q L54T):
LVSKTDS
[0800] SEQ ID NO: 160; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L50G and in hu3D6VLv2 L37Q L50G):
GVSKLDS
[0801] SEQ ID NO: 161; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54V):
LVSKVDS
[0802] SEQ ID NO: 162; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54S):
LVSKSDS
[0803] SEQ ID NO: 163; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 S52G and in hu3D6VLv2 L37Q S52G):
LVGKLDS
[0804] SEQ ID NO: 164; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L50V):
VVSKLDS
[0805] SEQ ID NO: 165; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q_L50G_L54R and hu3D6VLv2
L37Q_L50G_L54R_G100Q):
GVSKRDS
[0806] SEQ ID NO: 166; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q_L50G_L54G and in hu3D6VLv2
L37Q_L50G_L54G_G100Q): GVSKGDS
[0807] SEQ ID NO: 167; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q S52G L54G):
LVGKGDS
[0808] SEQ ID NO: 168; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q S52G L54R and in hu3D6VLv2
L37Q_S52G_L54R_G100Q):
LVGKRDS
[0809] SEQ ID NO: 169; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q S52G L54T):
LVGKTDS
[0810] SEQ ID NO: 170; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q S52G L54D and in hu3D6VLv2
L37Q_S52G_L54D_G100Q):
LVGKDDS
[0811] SEQ ID NO: 171; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q L50D L54G and in hu3D6VLv2
L37Q_L50D_L54G_G100Q):
DVSKGDS
[0812] SEQ ID NO: 172; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q L50D L54R and in hu3D6VLv2
L37Q_L50D_L54R_G100Q):
DVSKRDS
[0813] SEQ ID NO: 173; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q L50E L54G):
EVSKGDS
[0814] SEQ ID NO: 174; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q_L50E_L54R):
EVSKRDS
[0815] SEQ ID NO: 175; amino acid sequence of an alternate Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L37Q_L50V_L54D_G100Q): VVSKDDS
[0816] SEQ ID NO: 176; amino acid sequence of a heavy chain constant region (IgGl : allotype Glml7,l):
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGK
[0817] SEQ ID NO: 177; amino acid sequence of a light chain constant region (kappa):
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
[0818] SEQ ID NO: 178; amino acid sequence of a mature heavy chain of a 3D6 humanized variant (hu3D6VHvlbAl 1 IgGl Glml7 allotype)
EVQLVQSGAEWKPGATVKISCKASGFNIKDYYLHWVRQRPGQGLEWIGWIDPENGDTVYDPKF
QGRATITADTSTDTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC
VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSN
KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0819] SEQ ID NO: 179; amino acid sequence of a mature light chain of a 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4 kappa)
DWMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVGKRDSGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIKRTVAAPSVFIFPPSDE QLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE
KHKVYACEVTHQGLSSPVTKSFNRGEC
[0820] SEQ ID NO: 180; amino acid sequence of a heavy chain of a 3D6 humanized variant (hu3D6VHvlbAl 1 IgGl Glml7 allotype) with bovine alpha-lactalbumin signal peptide at the N-terminus
MMSFVSLLLVGILFHATQAEVQLVQSGAEWKPGATVKISCKASGFNIKDYYLHWVRQRPGQGL
EWIGWIDPENGDTVYDPKFQGRATITADTSTDTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPGK
[0821] SEQ ID NO: 181; amino acid sequence of a light chain of a 3D6 humanized variant (hu3D6VLv2 variant L37Q__S52G_L54R, L2-DIM4 kappa) with bovine alpha-lactalbumin signal peptide at the N- terminus .
MMSFVSLLLVGILFHATQADVVMTQSPLSLPVTLGQPAS ISCKSSQSLLDSDGKTYLNWLQQRP GQSPRRLIYLVGKRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKL EIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
[0822] SEQ ID NO: 182; nucleotide sequence encoding a heavy chain of a 3D6 humanized variant (hu3D6VHvlbAl 1 IgGl Glml7 allotype) with bovine alpha-lactalbumin signal peptide at the N-terminus
ATGATGTCCTTTGTCTCTCTGCTCCTGGTTGGCATCCTATTCCATGCCACCCAGGCCGAGGTGC AGCTGGTGCAGTCTGGGGCAGAGGTTGTGAAGCCAGGGGCCACAGTCAAGATCTCCTGTAAGGC TTCTGGCTTCAACATTAAAGACTACTATCTGCACTGGGTGCGGCAGAGGCCTGGACAGGGCCTG GAGTGGATTGGATGGATTGATCCTGAGAATGGTGATACTGTGTATGACCCGAAGTTCCAGGGCA GGGCCACTATAACAGCAGACACATCCACCGACACAGCCTACCTGCAGCTCGGCAGCCTGACATC TGAGGACACTGCCGTCTATTTCTGTTCTACCCTGGACTTCTGGGGCCAAGGCACCCTTGTCACA GTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCTAGCAAGAGCACCT CTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTC GTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGA CTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTTGAGCCCAAATCTTGTGA CAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTC TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGG TGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA TAATGCCAAGACAAAGCCGAGAGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC TCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATTCCAAACTCACCGTGGA CAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC CACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAATGATGAGATCTCGAG
[0823] SEQ ID NO: 183; nucleotide sequence encoding a light chain of a 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4 kappa) with bovine alpha-lactalbumin signal peptide at the N-terminus
ATGATGTCCTTTGTCTCTCTGCTCCTGGTTGGCATCCTATTCCATGCCACCCAGGCCGATGTTG TGATGACCCAGTCTCCACTCTCTTTGCCCGTTACCCTTGGACAACCTGCCTCCATCTCTTGCAA GTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGCAACAGAGGCCA GGCCAGTCTCCACGGCGCCTAATCTATCTGGTGGGCAAACGGGACTCTGGAGTCCCTGACAGGT TCAGTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGT GGGAGTTTATTATTGCTGGCAAGGCACACATTTTCCGTACACGTTCGGAGGGGGGACCAAGCTG GAAATAAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGCTTA AGTCCGGAACTGCTAGCGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACA GTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC AAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACA AAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAG GGGAGAGTGTTAGTGAGATCTCGAG
[0824] SEQ ID NO: 184; amino acid sequence of a region of tau microtubule binding repeat 1 (amino acid residues 255-271 of SEQ ID NO: l)
NVKSKIGSTENLKHQPG
[0825] SEQ ID NO: 185; amino acid sequence of of a region of tau microtubule binding repeat 2 (amino acid residues 286-302 of SEQ ID NO: 1)
NVQSKCGSKDNIKHVPG [0826] SEQ ID NO: 186; amino acid sequence of of a region of tau microtubule binding repeat 3 (amino acid residues 317-333 of SEQ ID NO: l)
KVTSKCGSLGNIHHKPG
[0827] SEQ ID NO: 187; amino acid sequence of of a region of tau microtubule binding repeat 4 (amino acid residues 349-365 of SEQ ID NO: l)
RV Q SKIGSLDNITH VPG
[0828] SEQ ID NO: 188; amino acid sequence of a core motif of tau bound by 3D6 KIGSTENLKH
[0829] SEQ ID NO: 189; amino acid sequence of tau sequence N-terminal to a core motif of tau bound by 3D6
NVKS
[0830] SEQ ID NO: 190; amino acid sequence of tau sequence C-terminal to core motif of tau bound by 3D6
QPG
[0831] SEQ ID NO: 191; amino acid sequence of epitope of 3D6
KXXSXXNX (K/H) H
[0832] SEQ ID NO: 192; amino acid sequence of a core motif of tau bound by 3D6
KCGSKDNIKH
[0833] SEQ ID NO: 193; amino acid sequence of a core motif of tau bound by 3D6
KCGSLGNIHH
[0834] SEQ ID NO: 194; amino acid sequence of a core motif of tau bound by 3D6
KIGSLDNITH

Claims

WHAT IS CLAIMED IS:
1. An isolated antibody specifically binding to human tau, comprising: a mature heavy chain variable region comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9 or SEQ ID NO: 149, and CDR-H3 comprising SEQ ID NO: 10, wherein the heavy chain variable region is at least 90% identical to SEQ ID NO: 18; and a mature light chain variable region comprising CDR-L1 comprising SEQ ID NO: 12, CDR-L2 comprising SEQ ID NO: 150, 151, 153, 156, 158, 159, 160, 163, 165, 166, 167, 168, 169, 170, 171, 172, 173 or 174, and CDR-L3 comprising SEQ ID NO: 14, wherein the light chain variable region is at least 90% identical to SEQ ID NO: 122.
2. The antibody of claim 1, wherein at least one of positions H12, H13, H17, H24, H40, H43, H48, H66, H67, H76, H80, H81, and H91 can be occupied by V, K, T, A, R, Q,
I, R, A, D, L, Q, and F, respectively, and at least one of positions L2, L12, L15, L37, L39, L45, L60 and LI 00 can be occupied by V, P, L, Q, R, R, D and Q respectively.
3. The antibody of claim 1, wherein CDR-L2 comprises SEQ ID NO: 150, 151, 163, 167, 168, or 169.
4. The antibody of claim 3, wherein the heavy chain variable region comprises SEQ ID NO: 18 and the light chain variable region comprises SEQ ID NO: 110, 121, 122 or 123.
5. The antibody of claim 4, wherein the light chain variable region comprises
SEQ ID NO: 110.
6 The antibody of claim 4, wherein the light chain variable region comprises
SEQ ID NO: 121.
7. The antibody of claim 4, wherein the light chain variable region comprises
SEQ ID NO: 122.
8 The antibody of claim 4, wherein the light chain variable region comprises
SEQ ID NO: 123.
9. The antibody of claim 3, wherein the heavy chain variable region comprises SEQ ID NO: 146 and the light chain variable region comprises SEQ ID NO:94 or SEQ ID NO: 122.
10. The antibody of claim 9, wherein the light chain variable region comprises SEQ ID NO:94.
11. The antibody of claim 9, wherein the light chain variable region comprises SEQ ID NO: 122.
12. The antibody of claim 3, wherein the heavy chain variable region comprises SEQ ID NO: 18 or 146 and the light chain variable region comprises SEQ ID NO: 122.
13. The antibody of any one of claims 1-12 wherein the antibody is a chimeric, veneered, or humanized antibody.
14. The antibody of any one of claims 1-13 that is an intact antibody.
15. The antibody of any one of claims 1-13 that is a binding fragment.
16. The antibody of claim 15, wherein the binding fragment is a single-chain antibody, Fab, or Fab’2 fragment.
17. The antibody of any one of claims 1-13 that is a Fab fragment, or single chain Fv.
18. The antibody of any one of the preceding claims, wherein the isotype is human IgGl .
19. The humanized antibody of any one of claims 1-14 and 18 wherein the mature light chain variable region is fused to a light chain constant region and the mature heavy chain variable region is fused to a heavy chain constant region.
20. The humanized antibody of claim 19, wherein the heavy chain constant region is a mutant form of a natural human heavy chain constant region which has reduced binding to a Fey receptor relative to the natural human heavy chain constant region.
21. The humanized antibody of claim 19 or claim 20, wherein the heavy chain constant region is of IgGl isotype.
22. The humanized antibody of claim 21, wherein the heavy chain constant region has an amino acid sequence of SEQ ID NO: 176.
23. The humanized antibody of claim 19, wherein the mature heavy chain variable region fused to a heavy chain constant region has an amino acid sequence of SEQ ID NO: 178.
24. The humanized antibody of claim 19, further comprising a signal peptide fused to the mature heavy and/or light chain variable region.
25. The humanized antibody of claim 23, wherein the heavy chain has an amino acid sequence of SEQ ID NO: 180.
26. The humanized antibody of claim 19, wherein the light chain constant region has an amino acid sequence of SEQ ID NO: 177.
27. The humanized antibody of claim 19, wherein the mature light chain variable region is fused to a light chain constant region has an amino acid sequence of SEQ ID NO: 179.
28. The humanized antibody of claim 27, wherein the light chain has an amino acid sequence of SEQ ID NO: 181.
29. The humanized antibody of claim 23, wherein the heavy chain has an amino acid sequence of SEQ ID NO: 178 and the light chain has an amino acid sequence of SEQ ID NO: 179.
30. The humanized antibody of claim 25, wherein the heavy chain has an amino acid sequence of SEQ ID NO: 180 and the light chain has an amino acid sequence of SEQ ID NO: 181.
31. The antibody of any one of the preceding claims having at least one mutation in the constant region.
32. The antibody of claim 31, wherein the mutation reduces complement fixation or activation by the constant region.
33. The antibody of claim 32 having a mutation at one or more of positions 241, 264, 265, 270, 296, 297, 318, 320, 322, 329 and 331 by EU numbering.
34. The antibody of claim 33 having alanine at positions 318, 320 and 322.
35. The antibody of any one of claims 1-20 wherein the isotype is of human IgG2 or IgG4 isotype.
36. The antibody of any one of claims 1-35, wherein the antibody is at least
95% w/w pure.
37. The antibody of any preceding claim, wherein the antibody is conjugated to a therapeutic, cytotoxic, cytostatic, neurotrophic, or neuroprotective agent.
38. A pharmaceutical composition comprising an antibody as defined in any of claims 1-37 and a pharmaceutically-acceptable carrier.
39. A nucleic acid encoding the heavy chain and/or light chain of an antibody as described in any one of claims 1-38.
40. The nucleic acid of claim 39, wherein the heavy chain is encoded by a sequence comprising SEQ ID NO: 182 and the light chain is encoded by a sequence comprising SEQ ID NO: 183.
41. A method of producing a humanized antibody, the method comprising:
(a) culturing cells transformed with the nucleic acid of claim 40, so that the cells secrete the antibody; and
(b) purifying the antibody from cell culture media.
42. A method of producing a cell line producing a humanized, chimeric, or veneered antibody, the method comprising:
(a) introducing a vector encoding the nucleic acid of claim 40 and a selectable marker into cells;
(b) propagating the cells under conditions to select for cells having increased copy number of the vector;
(c) isolating single cells from the selected cells; and
(d) banking cells cloned from a single cell selected based on yield of antibody.
43. A method of producing a humanized, chimeric, or veneered antibody, the method comprising:
(a) culturing cells transformed with nucleic acids encoding the heavy and light chains of the antibody of claim 1, so that the cells secrete the antibody; and
(b) purifying the antibody from cell culture media.
44. A method of producing a cell line producing a humanized, chimeric, or veneered antibody, the method comprising:
(a) introducing a vector encoding heavy and light chains of the antibody of claim 1 and a selectable marker into cells;
(b) propagating the cells under conditions to select for cells having increased copy number of the vector;
(c) isolating single cells from the selected cells; and
(d) banking cells cloned from a single cell selected based on yield of antibody.
45. A method of inhibiting or reducing aggregation of tau in a subject having or at risk of developing a tau-mediated amyloidosis, comprising administering to the subject an effective regime of the antibody of any one of claims 1-38, thereby inhibiting or reducing aggregation of tau in the subject.
46. The method of claim 45, wherein the antibody is a humanized version of
3D6.
47. A method of treating or effecting prophylaxis of a tau-related disease in a subject, comprising administering an effective regime of an antibody as defined by any one of claims 1-38 and thereby treating or effecting prophylaxis of the disease.
48. A method of claim 47 wherein the tau-related disease is Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).
49. A method of claim 47 wherein the tau-related disease is Alzheimer’s disease.
50. The method of claim 49 wherein the patient is an ApoE4 carrier.
51. A method of reducing aberrant transmission of tau comprising administering an effective regime of an antibody as defined in any of claims 1-38 and thereby reducing transmission of tau.
52. A method of inducing phagocytosis of tau comprising administering an effective regime of an antibody as defined in any of claims 1-38 and thereby inducing
phagocytosis of tau.
53. A method of inhibiting tau aggregation or deposition comprising administering an effective regime of an antibody as defined in any of claims 1-38 thereby inhibiting tau aggregation or deposition.
54. A method of inhibiting formation of tau tangles comprising administering an effective regime of an antibody as defined in any of claims 1-38.
55. A method of detecting tau protein deposits in a subject having or at risk of a disease associated with tau aggregation or deposition, comprising administering to a subject an antibody defined by any one of claims 1-38, and detecting the antibody bound to tau in the subject.
56. A method of claim 55, wherein the disease associated with tau aggregation or deposition is Alzheimer’s disease, Down’s syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick’s disease, type C Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).
57. A method of claim 55, wherein the antibody is administered by
intravenous injection into the body of the subject.
58. A method of claim 55, wherein the antibody is administered directly to the brain of the subject by intracranial injection or by drilling a hole through the skull of the subject.
59. A method of claim 55, wherein the antibody is labeled.
60. A method of claim 59, wherein the antibody is labeled with a fluorescent label, a paramagnetic label, or a radioactive label.
61. A method of claim 60, wherein the radioactive label is detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).
62. A method of measuring efficacy of treatment in a subject being treated for a disease associated with tau aggregation or deposition, comprising (a) measuring a first level of tau protein deposits in the subject prior to treatment by administering to a subject an antibody defined by any one of claims 1-38, and detecting a first amount of the antibody bound to tau in the subject,
(b) administering the treatment to the subject,
(c) measuring a second level of tau protein deposits in the subject after treatment by administering to a subject the antibody, and detecting the antibody bound to tau in the subject, wherein a decrease in the level of tau protein deposits indicates a positive response to treatment.
63. A method of measuring efficacy of treatment in a subject being treated for a disease associated with tau aggregation or deposition, comprising
(a) measuring a first level of tau protein deposits in the subject prior to treatment by administering to a subject an antibody defined by any one of claims 1-38, and detecting a first amount of antibody bound to tau in the subject,
(b) administering the treatment to the subject,
(c) measuring a second level of tau protein deposits in the subject after treatment by administering to a subject the antibody, and detecting a second amount of antibody bound to tau in the subject, wherein no change in the level of tau protein deposits or a small increase in tau protein deposits indicates a positive response to treatment.
64 A method of producing an antibody that specifically binds to human tau at an epitope within a motif of the formula KXXSXXNX(K/H)H (SEQ ID NO: 191) or
KIGSLDNITH (SEQ ID NO: 194) comprising immunizing an animal with human tau or a fragment thereof to produce antibodies and screening for an antibody of the produced antibodies that specifically binds within the motif.
65. A method of producing an antibody that specifically binds to a peptide consisting of residues KXXSXXNX(K/H)H (SEQ ID NO: 191) or KIGSLDNITH (SEQ ID NO: 194) comprising immunizing an animal with human tau or a fragment thereof to produce antibodies and screening for an antibody of the produced antibodies that specifically binds to the peptide.
66. A method of producing an antibody that specifically binds to an epitope comprising KXXSXXNX(K/H)H (SEQ ID NO: 191) comprising immunizing an animal with tau or a fragment thereof, and screening for antibodies that specifically bind to the epitope.
67. The method of any one of claims 64-66, wherein the animal is immunized with 383 amino acid human tau (4R0N).
68. The method of claim 67, wherein the human tau contains a P301 S mutation.
69. The method of claim 68, wherein the human tau is recombinant N- terminally His-tagged.
70. The method of claim 66, wherein the screening determines specific binding between the antibodies and one or more peptides of up to 15 amino acids comprising KIGSTENLKH (SEQ ID NO: 188), KC GSKDNIKH (SEQ ID NO: 192), KCGSLGNTHH (SEQ ID NO: 193) respectively or any other consensus motif represented by KXXSXXNX(K/H)H (SEQ ID NO: 191).
71. The method of claim 70, wherein the one or more peptides comprise KIGSTENLKH (SEQ ID NO: 188) or KCGSKDNIKH (SEQ ID NO: 192) or KCGSLGNTHH (SEQ ID NO: 193) respectively.
72. The method of claim 66, wherein the animal is immunized with a tau fragment of up to 15 amino acids comprising KXXSXXNX(K/H)H (SEQ ID NO: 191), linked to a carrier.
73. The method of claim 72, wherein the peptide is KIGSTENLKH (SEQ ID NO: 188) or KCGSKDNIKH (SEQ ID NO: 192) or KCGSLGNIHH (SEQ ID NO: 193).
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10906964B2 (en) 2016-05-02 2021-02-02 Prothena Biosciences Limited Antibodies recognizing tau
US10961302B2 (en) 2019-03-03 2021-03-30 Prothena Biosciences Limited Antibodies recognizing tau
EP3877410A4 (en) * 2018-11-08 2022-08-10 Prothena Biosciences Limited Antibodies recognizing tau
WO2022174026A1 (en) * 2021-02-14 2022-08-18 Prothena Biosciences Limited Methods of using antibodies recognizing tau
US11492393B2 (en) 2016-05-02 2022-11-08 Prothena Biosciences Limited Tau immunotherapy
US11584791B2 (en) 2016-05-02 2023-02-21 Prothena Biosciences Limited Antibodies recognizing tau
US11643457B2 (en) 2013-03-13 2023-05-09 Prothena Biosciences Limited Tau immunotherapy
WO2023092004A1 (en) 2021-11-17 2023-05-25 Voyager Therapeutics, Inc. Compositions and methods for the treatment of tau-related disorders
US11958896B2 (en) 2017-05-02 2024-04-16 Prothena Biosciences Limited Antibodies recognizing tau

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9321841B2 (en) * 2011-06-01 2016-04-26 Antitope Limited Humanised anti-CD52 antibodies
WO2017191560A1 (en) * 2016-05-02 2017-11-09 Prothena Biosciences Limited Antibodies recognizing tau

Family Cites Families (162)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CH652145A5 (en) 1982-01-22 1985-10-31 Sandoz Ag METHOD FOR IN VITRO PRODUCTION OF HYBRID OMEN WHAT human monoclonal antibodies GENERATE.
US4634666A (en) 1984-01-06 1987-01-06 The Board Of Trustees Of The Leland Stanford Junior University Human-murine hybridoma fusion partner
US5208036A (en) 1985-01-07 1993-05-04 Syntex (U.S.A.) Inc. N-(ω, (ω-1)-dialkyloxy)- and N-(ω, (ω-1)-dialkenyloxy)-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
EP0832981A1 (en) 1987-02-17 1998-04-01 Pharming B.V. DNA sequences to target proteins to the mammary gland for efficient secretion
WO1988007089A1 (en) 1987-03-18 1988-09-22 Medical Research Council Altered antibodies
US5057540A (en) 1987-05-29 1991-10-15 Cambridge Biotech Corporation Saponin adjuvant
US4912094B1 (en) 1988-06-29 1994-02-15 Ribi Immunochem Research Inc. Modified lipopolysaccharides and process of preparation
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5633076A (en) 1989-12-01 1997-05-27 Pharming Bv Method of producing a transgenic bovine or transgenic bovine embryo
US5859205A (en) 1989-12-21 1999-01-12 Celltech Limited Humanised antibodies
JP3068180B2 (en) 1990-01-12 2000-07-24 アブジェニックス インコーポレイテッド Generation of heterologous antibodies
US5279833A (en) 1990-04-04 1994-01-18 Yale University Liposomal transfection of nucleic acids into animal cells
US5264618A (en) 1990-04-19 1993-11-23 Vical, Inc. Cationic lipids for intracellular delivery of biologically active molecules
US5427908A (en) 1990-05-01 1995-06-27 Affymax Technologies N.V. Recombinant library screening methods
AU665190B2 (en) 1990-07-10 1995-12-21 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
GB9015198D0 (en) 1990-07-10 1990-08-29 Brien Caroline J O Binding substance
US5789650A (en) 1990-08-29 1998-08-04 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5877397A (en) 1990-08-29 1999-03-02 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5814318A (en) 1990-08-29 1998-09-29 Genpharm International Inc. Transgenic non-human animals for producing heterologous antibodies
US5874299A (en) 1990-08-29 1999-02-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
ATE158021T1 (en) 1990-08-29 1997-09-15 Genpharm Int PRODUCTION AND USE OF NON-HUMAN TRANSGENT ANIMALS FOR THE PRODUCTION OF HETEROLOGUE ANTIBODIES
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5194594A (en) 1990-09-07 1993-03-16 Techniclone, Inc. Modified antibodies
US5858657A (en) 1992-05-15 1999-01-12 Medical Research Council Methods for producing members of specific binding pairs
DE69230142T2 (en) 1991-05-15 2000-03-09 Cambridge Antibody Technology Ltd. METHOD FOR PRODUCING SPECIFIC BINDING PAIRS
WO1992022653A1 (en) 1991-06-14 1992-12-23 Genentech, Inc. Method for making humanized antibodies
US5283185A (en) 1991-08-28 1994-02-01 University Of Tennessee Research Corporation Method for delivering nucleic acids into cells
ES2136092T3 (en) 1991-09-23 1999-11-16 Medical Res Council PROCEDURES FOR THE PRODUCTION OF HUMANIZED ANTIBODIES.
JPH07503132A (en) 1991-12-17 1995-04-06 ジェンファーム　インターナショナル，インコーポレイティド Transgenic non-human animals capable of producing xenoantibodies
US5733743A (en) 1992-03-24 1998-03-31 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
US5674703A (en) 1992-12-02 1997-10-07 Woo; Savio L. C. Episomal vector systems and related methods
WO1994013804A1 (en) 1992-12-04 1994-06-23 Medical Research Council Multivalent and multispecific binding proteins, their manufacture and use
DE69318420T2 (en) 1992-12-14 1999-01-28 Naamloze Vennootschap Innogenetics S.A., Gent MONOCLONAL ANTIBODIES AGAINST THE MICROTUBULUS ASSOCIATED TAUPROTEIN, HYBRIDOMES THAT SECRETORE THESE ANTIBODIES, ANTIGENT DETECTION THROUGH THESE MONOCLONAL ANTIBODIES AND THE USE THEREOF
DE69435171D1 (en) 1993-09-14 2009-01-08 Pharmexa Inc PAN DR BINDEPROTEINS FOR INCREASING THE IMMUNE RESPONSE
US5827690A (en) 1993-12-20 1998-10-27 Genzyme Transgenics Corporatiion Transgenic production of antibodies in milk
US5877218A (en) 1994-01-10 1999-03-02 Teva Pharmaceutical Industries, Ltd. Compositions containing and methods of using 1-aminoindan and derivatives thereof and process for preparing optically active 1-aminoindan derivatives
US5505947A (en) 1994-05-27 1996-04-09 The University Of North Carolina At Chapel Hill Attenuating mutations in Venezuelan Equine Encephalitis virus
US5786464C1 (en) 1994-09-19 2012-04-24 Gen Hospital Corp Overexpression of mammalian and viral proteins
US7427392B1 (en) 1994-11-14 2008-09-23 Elan Pharmaceuticals, Inc. Methods for aiding in the diagnosis of alzheimer's disease by measuring amyloid-β peptide (x-≧41) and tau
US7153510B1 (en) 1995-05-04 2006-12-26 Yale University Recombinant vesiculoviruses and their uses
DE19539493A1 (en) 1995-10-24 1997-04-30 Thomae Gmbh Dr K Strong homologous promoter from hamster
US5834597A (en) 1996-05-20 1998-11-10 Protein Design Labs, Inc. Mutated nonactivating IgG2 domains and anti CD3 antibodies incorporating the same
US6114148C1 (en) 1996-09-20 2012-05-01 Gen Hospital Corp High level expression of proteins
AUPO390396A0 (en) 1996-11-29 1996-12-19 Csl Limited Novel promiscuous T helper cell epitopes
AU753688B2 (en) 1997-03-10 2002-10-24 Ottawa Civic Loeb Research Institute Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
US5888809A (en) 1997-05-01 1999-03-30 Icos Corporation Hamster EF-1α transcriptional regulatory DNA
US7964192B1 (en) 1997-12-02 2011-06-21 Janssen Alzheimer Immunotherapy Prevention and treatment of amyloidgenic disease
KR100444778B1 (en) 1999-02-05 2004-08-18 삼성전자주식회사 Image texture describing method and apparatus
TWI255272B (en) 2000-12-06 2006-05-21 Guriq Basi Humanized antibodies that recognize beta amyloid peptide
AT500379B8 (en) 2001-02-02 2009-08-15 Axon Neuroscience TAU PROTEINS
JP2005538706A (en) 2001-07-12 2005-12-22 ジェファーソン フーテ， Super humanized antibody
EP1470146B8 (en) 2001-12-28 2007-09-12 Amgen Fremont Inc. Antibodies against the muc18 antigen
WO2004016655A1 (en) 2002-08-14 2004-02-26 Mitsubishi Chemical Corporation ANTIBODY SPECIFIC TO CENTRAL τ-PROTEIN
MXPA05005481A (en) 2002-11-29 2005-07-25 Boehringer Ingelheim Pharma Novel neomycin phosphotransferase genes and method for the selection of high-producing recombinant cells.
US7847146B2 (en) 2003-03-28 2010-12-07 Baylor College Of Medicine Model for neurodegenerative disorders
DE10338531A1 (en) 2003-08-19 2005-04-07 Boehringer Ingelheim Pharma Gmbh & Co. Kg Method for recloning production cells
US20050132424A1 (en) 2003-10-21 2005-06-16 Envivo Pharmaceuticals, Inc. Transgenic flies expressing Abeta42-Dutch
US7910100B2 (en) 2004-10-01 2011-03-22 Max-Planck-Gesellschaft zur Forderung der Wissen Antibodies directed to the mammalian EAG1 ion channel protein
KR101235658B1 (en) 2004-11-10 2013-02-21 베링거 잉겔하임 파르마 게엠베하 운트 코 카게 Use of flow-cytometric analysis to optimize cell banking strategies for CHO cells
AU2005318171B2 (en) 2004-12-20 2011-09-29 Crucell Holland B.V. Binding molecules capable of neutralizing West Nile virus and uses thereof
US7855276B2 (en) 2004-12-30 2010-12-21 The United States Of America As Represented By The Department Of Health And Human Services Framework residue substituted humanized COL-1 antibodies and their use
US8012936B2 (en) 2006-03-29 2011-09-06 New York University Tau fragments for immunotherapy
AU2007261584A1 (en) 2006-06-16 2007-12-27 Envivo Pharmaceutical Inc. Transgenic flies expressing tau and amyloid precursor fragment
EP2047252B1 (en) 2006-06-22 2013-01-23 Walter and Eliza Hall Institute of Medical Research Structure of the insulin receptor ectodomain
US20080124760A1 (en) 2006-07-26 2008-05-29 Barbara Enenkel Regulatory Nucleic Acid Elements
WO2008022153A2 (en) 2006-08-14 2008-02-21 The Regents Of The University Of California Inhibitors of pde4 and methods of use
PT2099823E (en) 2006-12-01 2014-12-22 Seattle Genetics Inc Variant target binding agents and uses thereof
EP2097097B1 (en) 2006-12-01 2018-05-30 E. R. Squibb & Sons, L.L.C. Antibodies, in particular human antibodies, that bind cd22 and uses thereof
HUE033325T2 (en) 2007-01-05 2017-11-28 Univ Zuerich Method of providing disease-specific binding molecules and targets
DK3067066T3 (en) 2007-02-23 2019-05-20 Prothena Biosciences Ltd PREVENTION AND TREATMENT OF SYNUCLEINOPATIC AND AMYLOIDOGENIC DISEASE
TWI419902B (en) 2007-03-02 2013-12-21 百靈佳殷格翰製藥公司 Improvement of protein production
EP2031064A1 (en) 2007-08-29 2009-03-04 Boehringer Ingelheim Pharma GmbH & Co. KG Method for increasing protein titres
JP2009056790A (en) 2007-09-03 2009-03-19 Takao Otogawa Pocket for file
SG187477A1 (en) 2007-09-26 2013-02-28 U3 Pharma Gmbh Heparin-binding epidermal growth factor-like growth factor antigen binding proteins
EP2220122A2 (en) 2007-11-13 2010-08-25 Teva Biopharmaceuticals USA, Inc. Humanized antibodies against tl1a
JP2010014691A (en) 2008-06-20 2010-01-21 Igaku Seibutsugaku Kenkyusho:Kk Methods, kits, reagents, and devices for detecting mesothelin and/or megakaryocyte enhancer in ascites
SG193216A1 (en) 2008-08-18 2013-09-30 Amgen Fremont Inc Antibodies to ccr2
US9605054B2 (en) 2009-02-23 2017-03-28 The Board Of Trustees Of The University Of Illinois Composition and method for treating a tauopathy
RU2016142225A (en) 2009-05-13 2018-12-18 Джензим Корпорейшн IMMUNOGLOBULINS TO HUMAN CD52
WO2010144711A2 (en) 2009-06-10 2010-12-16 New York University Immunological targeting of pathological tau proteins
US20110045534A1 (en) 2009-08-20 2011-02-24 Cell Signaling Technology, Inc. Nucleic Acid Cassette For Producing Recombinant Antibodies
AU2010286501B2 (en) 2009-08-28 2015-06-11 The Board Of Regents Of The University Of Texas System Antibodies that bind Tau oligomers
WO2011053565A2 (en) 2009-10-29 2011-05-05 Biomedical Sciences Research Centre "Alexander Fleming" Compositions and methods for detecting a tauopathy
EP2512600A2 (en) 2009-12-18 2012-10-24 Amgen Inc. Wise binding agents and epitopes
JP5697044B2 (en) 2010-01-08 2015-04-08 国立大学法人京都大学 Tauopathy treatment vaccine
WO2011106297A2 (en) 2010-02-23 2011-09-01 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
MX360403B (en) 2010-04-15 2018-10-31 Abbvie Inc Amyloid-beta binding proteins.
EP2560681A4 (en) 2010-04-22 2013-09-25 Janssen Alzheimer Immunotherap Use of tau to monitor immunotherapy
ITRM20100320A1 (en) 2010-06-11 2011-12-11 Consiglio Nazionale Ricerche METHOD FOR DIAGNOSTICS AND TREATMENT OF TAUPATIES
US20120023911A1 (en) 2010-07-28 2012-02-02 Gm Global Technology Operations, Inc. Detection of exhaust particulate filter substrate failure
JP2014503178A (en) 2010-10-11 2014-02-13 バイオジェン アイデック インターナショナル ニューロサイエンス ゲーエムベーハー Human anti-tau antibody
US8741298B2 (en) 2011-02-07 2014-06-03 Neotope Biosciences Limited APOE immunotherapy
EP2701743A4 (en) 2011-04-27 2015-08-19 Univ Northwestern Antibodies selective for pathological tau dimers and prefibrillar pathological tau oligomers and their uses in treatment, diagnosis and monitoring of tauopathies
GB201111361D0 (en) 2011-07-04 2011-08-17 Nordic Bioscience As Biochemical markers for neurodegenerative conditions
GB201112056D0 (en) 2011-07-14 2011-08-31 Univ Leuven Kath Antibodies
EP2748194A4 (en) 2011-08-22 2015-01-28 Cangene Corp Clostridium difficile antibodies
PT2758433T (en) 2011-09-19 2018-01-19 Axon Neuroscience Se Protein-based therapy and diagnosis of tau-mediated pathology in alzheimer's disease
TW201817745A (en) 2011-09-30 2018-05-16 日商中外製藥股份有限公司 Therapeutic antigen-binding molecule with a FcRn-binding domain that promotes antigen clearance
CA2850686C (en) 2011-10-07 2020-09-08 Ac Immune S.A. Phosphospecific antibodies recognising tau
SG10201702396QA (en) 2012-03-28 2017-04-27 Sanofi Sa Antibodies to bradykinin b1 receptor ligands
MX356800B (en) 2012-04-05 2018-06-13 Ac Immune Sa Humanized tau antibody.
US9428864B2 (en) 2012-04-18 2016-08-30 E I Du Pont De Nemours And Company Multilayered sheet
DE102012211455A1 (en) 2012-07-02 2014-01-02 Wobben Properties Gmbh Handling device for handling a rotor blade for making a rotor blade of a wind turbine
SG11201408626YA (en) 2012-07-03 2015-03-30 Univ Washington Antibodies to tau
JP6290212B2 (en) 2012-08-16 2018-03-07 アイピエリアン，インコーポレイティド How to treat tauopathy
US20140056901A1 (en) 2012-08-21 2014-02-27 The Institute For Molecular Medicine Anti-tau antibodies and compositions for and methods of making and using in treatment, diagnosis and monitoring of tauopathies
EP2906596B1 (en) 2012-10-12 2023-12-27 Arizona Board of Regents on behalf of Arizona State University Antibody based reagents that specifically recognize toxic oligomeric forms of tau
US9200068B2 (en) 2012-12-18 2015-12-01 Regents Of The University Of Minnesota Compositions and methods related to tauopathy
US8980270B2 (en) 2013-01-18 2015-03-17 Ipierian, Inc. Methods of treating a tauopathy
US9364191B2 (en) 2013-02-11 2016-06-14 University Of Rochester Method and apparatus of spectral differential phase-contrast cone-beam CT and hybrid cone-beam CT
EP2970453B1 (en) 2013-03-13 2019-12-04 Prothena Biosciences Limited Tau immunotherapy
CN105283196B (en) 2013-03-15 2018-10-12 贝丝以色列女执事医疗中心 For generate and using conformation-specific antibody method and composition
WO2014152773A1 (en) 2013-03-15 2014-09-25 The Trustees Of The University Of Pennsylvania Blood biomarkers that predict persistent cognitive dysfunction after concussion
AU2014308751B2 (en) 2013-08-22 2020-03-05 Acceleron Pharma, Inc. TGF-beta receptor type II variants and uses thereof
EA038994B1 (en) 2013-11-27 2021-11-18 Айпириэн, Инк. Methods of treating a tauopathy
CN106030310B (en) 2013-12-13 2019-01-04 通用医疗公司 Soluble high-molecular amount (HMW) TAU type and its application
DK3736292T3 (en) 2013-12-17 2024-07-22 Genentech Inc Anti-CD3 Antibodies and Methods of Use
US9629801B2 (en) 2014-01-10 2017-04-25 Wisconsin Alumni Research Foundation Blood-brain barrier targeting antibodies
US20150253341A1 (en) 2014-02-10 2015-09-10 Merck Sharp & Dohme Corp. Quantification of tau in biological samples by immunoaffinity enrichment and mass spectrometry
SG11201610459XA (en) 2014-06-26 2017-01-27 Janssen Vaccines & Prevention Bv Antibodies and antigen-binding fragments that specifically bind to microtubule-associated protein tau
TWI734975B (en) 2014-06-27 2021-08-01 美商Ｃ２Ｎ醫療診斷有限責任公司 Humanized anti-tau antibodies
JP2016051255A (en) 2014-08-29 2016-04-11 凸版印刷株式会社 Touch panel, and touch type information input image display device using the same
RS61431B1 (en) * 2014-11-19 2021-03-31 Axon Neuroscience Se Humanized tau antibodies in alzheimer's disease
CA2874083C (en) 2014-12-05 2024-01-02 Universite Laval Tdp-43-binding polypeptides useful for the treatment of neurodegenerative diseases
EP3261670A4 (en) 2015-02-24 2018-08-01 Rpeptide, LLC Anti-tau antibodies
RU2732122C2 (en) 2015-06-05 2020-09-11 Дженентек, Инк. Anti-tau protein antibodies and methods of using said antibodies
US20200030445A1 (en) 2015-06-12 2020-01-30 C2N Diagnostics, Llc Stable formulations of humanized anti-tau antibody
TN2017000543A1 (en) 2015-07-06 2019-04-12 Ucb Biopharma Sprl Tau-binding antibodies
JO3711B1 (en) 2015-07-13 2021-01-31 H Lundbeck As Antibodies specific for hyperphosphorylated tau and methods of use thereof
WO2017013599A1 (en) 2015-07-21 2017-01-26 Bioarctic Neuroscience Ab Method for treatment of traumatic brain injury targeting aggregated peptides
US20190330335A1 (en) 2015-10-06 2019-10-31 Alector Llc Anti-trem2 antibodies and methods of use thereof
EP3380622A4 (en) 2015-11-23 2019-08-07 Sangamo Therapeutics, Inc. Methods and compositions for engineering immunity
CU24538B1 (en) 2016-05-02 2021-08-06 Prothena Biosciences Ltd MONOCLONAL ANTIBODIES COMPETING TO JOIN HUMAN TAU WITH THE 16G7 ANTIBODY
WO2017191559A1 (en) 2016-05-02 2017-11-09 Prothena Biosciences Limited Tau immunotherapy
WO2018085653A1 (en) 2016-11-04 2018-05-11 The Regents Of The University Of California Compositions targeting 3-repeat tau for the treatment of neurodegenerative disorders, and methods for making and using them
MX2019006334A (en) 2016-12-07 2019-08-01 Genentech Inc Anti-tau antibodies and methods of use.
CA3045294A1 (en) 2016-12-07 2018-06-14 Genentech, Inc. Anti-tau antibodies and methods of use
MX2019009817A (en) 2017-02-17 2019-11-21 Denali Therapeutics Inc Anti-tau antibodies and methods of use thereof.
WO2018156250A1 (en) 2017-02-21 2018-08-30 Remd Biotherapeutics, Inc. Cancer treatment using antibodies that bind cytotoxic t-lymphocyte antigen-4 (ctla-4)
JOP20180021A1 (en) 2017-03-16 2019-01-30 Janssen Biotech Inc Anti-phf-tau antibodies and uses thereof
JP7109467B2 (en) 2017-03-28 2022-07-29 ヤンセン ファッシンズ アンド プリベンション ベーフェー Binding molecules that specifically bind to tau
CN110494446A (en) 2017-03-28 2019-11-22 基因泰克公司 The method for treating neurodegenerative disease
IL270375B1 (en) * 2017-05-02 2024-08-01 Prothena Biosciences Ltd Antibodies recognizing tau
AU2017418317A1 (en) 2017-06-16 2019-12-05 Bristol-Myers Squibb Company Compositions and methods for treating tauopathies
WO2019077500A1 (en) 2017-10-16 2019-04-25 Eisai R&D Management Co., Ltd. Anti-tau antibodies and uses thereof
WO2019094595A2 (en) 2017-11-09 2019-05-16 Pinteon Therapeutics Inc. Methods and compositions for the generation and use of humanized conformation-specific phosphorylated tau antibodies
SG11202004904XA (en) 2017-12-04 2020-06-29 Janssen Vaccines & Prevention Bv Binding molecules that specifically bind to tau
EP3768706A1 (en) 2018-03-23 2021-01-27 AbbVie Deutschland GmbH & Co. KG Stable aqueous anti-tau antibody formulations
SG11202008098TA (en) 2018-03-28 2020-10-29 Axon Neuroscience Se Antibody-based methods of detecting and treating alzheimer's disease
EP3784274A1 (en) 2018-04-27 2021-03-03 Fondazione Ebri Rita Levi-Montalcini Antibody directed against a tau-derived neurotoxic peptide and uses thereof
PE20211777A1 (en) 2018-11-08 2021-09-08 Prothena Biosciences Ltd ANTIBODIES THAT RECOGNIZE TAU
WO2020096608A1 (en) 2018-11-08 2020-05-14 Prothena Biosciences Limited Antibodies recognizing tau
EP3883602A4 (en) 2018-11-19 2023-04-12 The Board Of Regents Of The University Of Texas System Tau peptide antigens and antibodies binding thereto for the treatment of tauopathies
KR20210125037A (en) 2019-02-08 2021-10-15 프로테나 바이오사이언시즈 리미티드 Tau Recognizing Antibodies
CU20210073A7 (en) 2019-03-03 2022-04-07 Prothena Biosciences Ltd ANTIBODIES THAT BIND WITHIN THE CDRS-DEFINED MICROTUBULE-BINDING REGION OF TAU
JP2022526334A (en) 2019-03-25 2022-05-24 アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル Methods of Treatment of Tauopathy Disorders by Targeting New Tau Species
AU2020313751A1 (en) 2019-07-15 2022-02-10 Adel, Inc. Anti-tau antibody and use of same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9321841B2 (en) * 2011-06-01 2016-04-26 Antitope Limited Humanised anti-CD52 antibodies
WO2017191560A1 (en) * 2016-05-02 2017-11-09 Prothena Biosciences Limited Antibodies recognizing tau

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3935083A4 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11643457B2 (en) 2013-03-13 2023-05-09 Prothena Biosciences Limited Tau immunotherapy
US10906964B2 (en) 2016-05-02 2021-02-02 Prothena Biosciences Limited Antibodies recognizing tau
US11492393B2 (en) 2016-05-02 2022-11-08 Prothena Biosciences Limited Tau immunotherapy
US11584791B2 (en) 2016-05-02 2023-02-21 Prothena Biosciences Limited Antibodies recognizing tau
US11958896B2 (en) 2017-05-02 2024-04-16 Prothena Biosciences Limited Antibodies recognizing tau
EP3877410A4 (en) * 2018-11-08 2022-08-10 Prothena Biosciences Limited Antibodies recognizing tau
US10961302B2 (en) 2019-03-03 2021-03-30 Prothena Biosciences Limited Antibodies recognizing tau
US11926659B2 (en) 2019-03-03 2024-03-12 Prothena Biosciences Limited Antibodies recognizing tau
WO2022174026A1 (en) * 2021-02-14 2022-08-18 Prothena Biosciences Limited Methods of using antibodies recognizing tau
US20220267426A1 (en) * 2021-02-14 2022-08-25 Prothena Biosciences Limited Methods of using antibodies recognizing tau
WO2023092004A1 (en) 2021-11-17 2023-05-25 Voyager Therapeutics, Inc. Compositions and methods for the treatment of tau-related disorders

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