HK1182722A - Methods and compositions for neural disease immunotherapy - Google Patents

Abstract

The invention provides antibodies to specific neural proteins and methods of using the same.

Description

Methods and compositions for immunotherapy of neurological diseases

RELATED APPLICATIONS

The benefit of U.S. provisional application No. 61/456,642 filed on 10/2010, U.S. provisional application No. 61/418,310 filed on 30/2010, U.S. provisional application No. 61/418,850 filed on 1/2010, and U.S. provisional patent application No. 61/426,425 filed on 22/2010, all of which are incorporated herein by reference in their entirety, is claimed.

Technical Field

The present invention relates generally to antibodies that are BACE1 antagonists, e.g., that inhibit or reduce BACE1 activity, and to compositions comprising the antibodies. Additional embodiments include methods for treating and diagnosing various neurological diseases or disorders, as well as methods of reducing APP and/or a β polypeptides in a patient.

Background

Amyloidosis is not a single disease entity, but a diverse group of progressive disease processes characterized by the extracellular tissue deposition of waxy, amyloid-like proteins called amyloid, which accumulate in one or more organs or body systems. As amyloid deposits accumulate, they begin to interfere with the normal function of organs or body systems. There are at least 15 different types of amyloidosis. The main forms are primary amyloidosis with no known antecedent, secondary amyloidosis followed by some other condition, and hereditary amyloidosis.

Many diseases of aging are based on or associated with amyloid-like proteins and are characterized in part by promoting the formation of extracellular deposits of pathogenic amyloid or amyloid-like substances, as well as the progression of the disease. Such diseases include, but are not limited to, neurological disorders such as Alzheimer's Disease (AD), Lewy body dementia (Lewy body dementia), Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (type Dutch); guam Parkinson-Dementia complex (Guam Parkinson-Dementia complex). Other diseases based on or associated with amyloid-like proteins are progressive supranuclear palsy (progressive supranuclear palsy), multiple sclerosis (multiple sclerosis), Creutzfeld Jacob disease, Parkinson's disease, HIV-related dementia (HIV-related dementias), ALS (amyotrophic lateral sclerosis), Adult Diabetes (Adult on set Diabetes), senile cardiac amyloidosis (sensor cardiac amyloidosis), endocrine tumors, and the like, including macular degeneration.

The polypeptide β -amyloid (a β) may play a central role in the pathogenesis of Alzheimer's Disease (AD). Vassar et al, J.Neurosci.29: 12787-12794(2009). Accumulation of a β polypeptides in the CNS leads to synaptic dysfunction, axonal degeneration and neuronal death. The brains of AD patients exhibit pathologies characteristic of prominent neuropathological pathologies, such as neurofibrillary disorders (NFTs), and senile plaques rich in amyloid. The major component of amyloid plaques is a β. These lesions are associated with a substantial loss of Central Nervous System (CNS) neuronal populations, and their progression is accompanied by clinical dementia associated with AD.

A β is a proteolytic product of a precursor protein (β amyloid precursor protein (β -APP or APP)). APP is a type I transmembrane protein that is cleaved by two proteases, β -and γ -secretases, in succession. The β -secretase enzyme, known as β -site amyloid precursor protein lyase 1(BACE1), first cleaves APP to expose the N-terminus of a β, thereby generating a membrane-bound fragment known as C99. Vassar et al, J.Neurosci,29: 12787-. Gamma-secretase can then cleave C99 to produce the mature a β polypeptide. The resulting a β has a heterologous C-terminus ranging from 38 amino acids to 43 amino acids in length. 42 amino acid form of A beta (A beta) ₄₂) Is a β in fibrinogen form and is overproduced in patients with down syndrome and is implicated in playing a role in the early onset of AD. Vassar et al, J.Neurosci.29: 12787-12794(2009). BACE1 thus becomes a therapeutic target as its inhibition would likely inhibit APP and a β production.

Indeed, BACE1 knockout mice (BACEl)^-/-) Brain a β is not produced, confirming that BACE1 is the primary, if not the only, enzyme responsible for the production of a β in the brain. Robrds et al, humanmol. genetics 10: 1317-1324(2001). In addition, BACE1 knockout mice in AD models do not form amyloid plaques; cognitive deficits and cholinergic dysfunction have also been rescued. Mccorlogue et al, j.biol.chem.282: 26326-26334 (2007); ohno et al, Neuron 41: 27-33 (2004); and Laird et al, j.neurosci.25: 11693-11709(2005). Furthermore, BACE1 hybrid knockout mice had reduced plaque formation, suggesting that complete inhibition of BACE1 activity is not necessary for plaque reduction. Mccorlogue et al, j.biol.chem.282: 26326-26334(2007).

Recently, APP has been shown to be a ligand for death receptor 6(DR6), which triggers caspase-dependent neuronal cell body death and axon reduction (axon pruning). Nikolaev et al, Nature 457: 981-989(2009). In addition, BACE1 compound inhibitors disrupt degeneration of axons and cell bodies. As above. These results point to a model in which APP can promote AD by binding to DR 6.

It would be advantageous to have a therapeutic inhibitor of BACE1 that is effective to reduce APP and a β production in patients with neurological diseases and disorders, such as AD. The invention provided herein relates to such inhibitors, including their use in various methods.

All references, including patent applications and publications, cited herein are hereby incorporated by reference in their entirety.

SUMMARY

The present invention provides BACE1 antagonist antibodies and methods of use thereof. In particular, the antibodies inhibit or reduce the activity of BACE 1.

In one embodiment, an isolated antibody that binds BACE1 is provided, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide. In particular, the antibodies bind to the active site of BACE1 or the exosite (exosite) of BACE 1.

In another embodiment, an isolated antibody that binds BACE1 is provided, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises at least one hypervariable region (HVR) sequence selected from the group consisting of: SEQ ID NO: 7-19, 22-26, 28-30, 35-47, 56-79 and 118- "122".

In another embodiment, an isolated antibody that binds BACE1 is provided, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises at least one sequence selected from the group consisting of seq id no: HVR-H1, HVR-H2 and HVR-H3, wherein HVR-H1 comprises the amino acid sequence GFX ₃₀FX₃₁X₃₂X₃₃X₃₄IH (SEQ ID NO: 45), wherein X₃₀N or T; x₃₁(ii) S, L or Y; x₃₂G or Y; x₃₃Y or S; and X₃₄A, G or S; HVR-H2 comprises amino acid sequence X₃₅X₃₆ISPX₃₇X₃₈GX₃₉TX₄₀YADSVKG (SEQ ID NO: 46), wherein X₃₅A or G; x₃₆W or S; x₃₇A or Y; x₃₈G or S; x₃₉(ii) S or Y; and X₄₀D or S; and HVR-H3 comprises amino acid sequence X₄₁PX₄₂X₄₃X₄₄X₄₅X₄₆X₄₇MDY (SEQ ID NO: 47), wherein X₄₁Q or G; x₄₂T or F; x₄₃H or S; x₄₄Y or P; x₄₅Y or W; x₄₆Y or V andwherein X₄₇Optionally comprising the sequence YAKGYKA (SEQ ID NO: 48). Alternatively, the antibody comprises a HVR-H1 sequence, the HVR-H1 sequence comprising the amino acid sequence GFTFX₁₃GYX₁₄IH (SEQ ID NO: 26), wherein X₁₃Or L and X₁₄A or G; or an amino acid sequence selected from the group consisting of: SEQ ID NO: 22; SEQ ID NO: 23; and SEQ ID NO: 28.

in another embodiment, an isolated antibody that binds BACE1 is provided, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises at least one sequence selected from the group consisting of seq id no: HVR-H1, HVR-H2 and HVR-H3, wherein HVR-H1 comprises the amino acid sequence GX₇₁X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇IH (SEQ ID NO: 120), wherein X₇₁F or Y; x₇₂F, N or T; x ₇₃F or Y; x₇₄L, Q, I, S or Y; x₇₅G or Y; x₇₆Y or S; and X₇₇A, G or S; HVR-H2 comprises amino acid sequence X₇₈X₇₉ISPX₈₀X₈₁GX₈₂X₈₃X₈₄YADSVKG (SEQ ID NO: 121), wherein X₇₈A or G; x₇₉W or S; x₈₀A, S, Q or Y; x₈₁G or S; x₈₂(ii) S, K, L or Y; x₈₃T or Y; and X₈₄D or S; and HVR-H3 comprises amino acid sequence X₈₅PX₈₆X₈₇X₈₈X₈₉X₉₀X₉₁MDY (SEQ ID NO: 122), where X₈₅Q or G; x₈₆T or F; x₈₇H, Y or S; x₈₈Y or P; x₈₉Y or W; x₉₀Y or V, and wherein X₉₁Optionally comprising the sequence YAKGYKA (SEQ ID NO: 48). Alternatively, the antibody comprises a HVR-H1 sequence, which HVR-H1 sequence comprises the amino acid sequence GX₅₃X₅₄X₅₅X₅₆GYGIH (SEQ ID NO: 68), wherein X₅₃F or Y; x₅₄T or F; x₅₅F or Y; x₅₆L, Q or I; orAn amino acid sequence selected from the group consisting of: SEQ ID NO: 71-73. Alternatively, the antibody comprises a HVR-H2 sequence, said HVR-H2 sequence comprising the amino acid sequence GWISPX₅₇X₅₈GX₅₉X₆₀DYASVKG (SEQ ID NO: 69), wherein X₅₇A, S or Q; x₅₈G or S; x₅₉S, K or L; x₆₀T or Y; or an amino acid sequence selected from the group consisting of: SEQ ID NO: 74-78. Alternatively, the antibody comprises a HVR-H3 sequence, which HVR-H3 sequence comprises the amino acid sequence GPFX ₆₁PWVMDY (SEQ ID NO: 70), wherein X₆₁= S or Y; or SEQ ID NO: 79.

In one embodiment, an isolated antibody that binds BACE1 is provided, wherein said antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises an HVR-H1 sequence comprising an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 22, SEQ ID NO: 23, SEQ id no: 28 and SEQ ID NO: 71-73.

In one embodiment, an isolated antibody that binds BACE1 is provided, wherein said antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises an HVR-H2 sequence comprising an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 24, SEQ ID NO: 29 and SEQ ID NO: 74-78.

In another embodiment, an isolated antibody that binds BACE1 is provided, wherein said antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises an HVR-H3 sequence comprising an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 25, SEQ ID NO: 30 and SEQ ID NO: 79.

in one embodiment, an isolated antibody that binds BACE1 is provided, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises HVR-H1, HVR-H2 and HVR-H3 sequences, said HVR-H1, HVR-H2 and HVR-H3 sequences corresponding to those described for clones YW412.8, yw412.8.31, yw412.8.30, yw412.8.2, yw412.8.29 and YW412.8.51 in fig. 1(B) or those described for clones Fab12, LC6, LC9 and LC10 in fig. 2(B) or those described in fig. 24A-C.

In one embodiment, an isolated antibody that binds BACE1 is provided, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises the amino acid sequence of SEQ ID NO: 22 or 23, the HVR-H1 sequence of SEQ ID NO: 24 and the HVR-H2 sequence of SEQ ID NO: 25, HVR-H3 sequence. In another embodiment, an isolated antibody that binds BACE1 is provided, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises the amino acid sequence of SEQ ID NO: 23, HVR-H1 sequence of SEQ ID NO: 24 and the HVR-H2 sequence of SEQ ID NO: 25, HVR-H3 sequence. In another embodiment, an isolated antibody that binds BACE1 is provided, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises the amino acid sequence of SEQ ID NO: 28, the HVR-H1 sequence of SEQ id no: 29, and the HVR-H2 sequence of SEQ ID NO: 30, HVR-H3 sequence.

In one embodiment, an isolated antibody that binds BACE1 is provided, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-73 selected from the group consisting of SEQ ID NOs: 74-78 and a HVR-H2 sequence selected from SEQ id nos: 79 HVR-H3.

In one embodiment, there is provided an isolated antibody that binds BACE1, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises a Variable Heavy (VH) chain having an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO: 20, 21, 27 and 80-98. In one aspect, the antibody comprises SEQ ID NO: 21, and VH chain amino acid sequence.

In another embodiment, there is provided an isolated antibody that binds BACE1, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises at least one sequence selected from the group consisting of seq id nos: HVR-L1, HVR-L2, and HVR-L3, wherein HVR-L1 comprises the amino acid sequence RASQX₁₇VX₁₈X₁₉X₂₀X₂₁A (SEQ ID NO: 42), wherein X₁₇(ii) S, D or V; x₁₈(ii) S or a; x₁₉(ii) S, T or N; x₂₀A or S; x₂₁V or L, HVR-L2 comprises the amino acid sequence X₂₂ASX₂₃LyS (SEQ ID NO: 43), wherein X₂₂(ii) S, W, Y or L; x₂₃F, S or W, and HVR-L3 comprises the amino acid sequence QQX₂₄X₂₅X₂₆X₂₇X₂₈X₂₉T (SEQ ID NO: 44), wherein X₂₄(ii) S, F, G, D or Y; x₂₅Y, P, S or a; x₂₆Y, T or N; x₂₇T, Y, D or S; x₂₈P or L; and X₂₉F, P or T.

In another embodiment, there is provided an isolated antibody that binds BACE1, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises at least one sequence selected from the group consisting of seq id nos: HVR-L1, HVR-L2, and HVR-L3, wherein HVR-L1 comprises the amino acid sequence RASQX₁₇VX₁₈X₁₉X₂₀X₂₁A (SEQ ID NO: 42), wherein X₁₇(ii) S, D or V; x₁₈(ii) S or a; x₁₉(ii) S, T or N; x₂₀A or S; x₂₁(vi) V or L, HVR-L2 comprises the amino acid sequence X₆₂ASX₆₃X₆₄YX₆₅(SEQ ID NO: 118) in which X₆₂S, W, Y, F or L; x ₆₃F, S, Y or W; x₆₄L or R; x₆₅(iii) S, P, R, K or W, and HVR-L3 comprises the amino acid sequence QQX₆₆X₆₇X₆₈X₆₉X₇₀X₇₁T (SEQ ID NO: 119), wherein X₆₆(ii) S, F, G, D or Y; x₆₇Y, P, S or a; x₆₈Y, T or N; x₆₉T, Y, D or S; x₇₀P, Q, S, K or L; and X₇₁= F, P or T.

In certain embodiments, an isolated antibody that binds BACE1 is provided, wherein said antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises an HVR-L1 sequence comprising the amino acid sequence RASQX₁VX₂X₃X₄X₅A (SEQ ID NO: 17), wherein X₁D or V; x₂(ii) S or a; x₃T or N; x₄(ii) S or a; x₅Or an amino acid sequence selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 35.

in one embodiment, an isolated antibody that binds BACE1 is provided, wherein said antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises an HVR-L2 sequence, said HVR-L2 sequence comprising amino acid sequence X₆ASFLYS (SEQ ID NO: 18), where X₆Or L or X₁₅ASX₁₆LYS (SEQ ID NO: 41), wherein X₁₅is-S, W or Y, and X₁₆(ii) S or W, or an amino acid sequence selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 36-39.

In one embodiment, an isolated antibody that binds BACE1 is provided, wherein said antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises an HVR-L3 sequence, said HVR-L3 sequence comprising the amino acid sequence QQX₇X₈X₉X₁₀X₁₁X₁₂T (SEQ ID NO: 19), wherein X₇(ii) S, F, G, D or Y; x₈Y, P, S, or a; x₉T or N; x₁₀T, Y, D or S; x₁₁P or L; x₁₂P or T, or an amino acid sequence selected from the group consisting of: SEQ ID NO: 11-16 and SEQ ID NO: 40.

in certain embodiments, an isolated antibody that binds BACE1 is provided, wherein said antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises an HVR-L1 sequence comprising the amino acid sequence RASQX₁VX₂X₃X₄X₅A (SEQ ID NO: 17), wherein X₁D or V; x₂(ii) S or a; x₃T or N; x₄(ii) S or a; x₅V or L, or selected from the group consisting of SEQ ID NO: 7.

In one embodiment, an isolated antibody that binds BACE1 is provided, wherein said antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises a HVR-L2 sequence, said HVR-L2 sequence comprising an amino groupSequence X₄₈ASX₄₉X₅₀YX₅₁(SEQ ID NO: 56), wherein X₄₈(ii) S or F; x₄₉F or Y; x₅₀L or R; x₅₁(ii) S, P, R, K or W, or an amino acid sequence selected from the group consisting of: SEQ ID NO: 58-64.

In one embodiment, an isolated antibody that binds BACE1 is provided, wherein said antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises an HVR-L3 sequence, said HVR-L3 sequence comprising the amino acid sequence QQFPPTYX₅₂PT (SEQ ID NO: 57), wherein X₅₂L, Q, S or K, or an amino acid sequence selected from the group consisting of: SEQ ID NO: 65-67.

In one embodiment, an isolated antibody that binds BACE1 is provided, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises HVR-L1, HVR-L2 and HVR-L3 sequences, said HVR-L1, HVR-L2 and HVR-L3 sequences corresponding to those described for clones YW412.8, yw412.8.31, yw412.8.30, yw412.8.2, yw412.8.29 and YW412.8.51 in fig. 1(a) or for clones Fab12, LC6, LC9 and LC10 in fig. 2(a) or for clones in fig. 23A-C.

In one embodiment, an isolated antibody that binds BACE1 is provided, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, HVR-L1 sequence; an HVR-L2 sequence selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 58-64; and an HVR-L3 sequence selected from the group consisting of: SEQ ID NO: 11-16 and 65-67. In another aspect, an isolated antibody that binds BACE1 is provided, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises the amino acid sequence of SEQ ID NO: 7, the HVR-L1 sequence of SEQ ID NO: 9 and the HVR-L2 sequence of SEQ id no: 12, HVR-L3 sequence.

In another embodiment, an isolated antibody that binds BACE1 is provided, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 35, HVR-L1 sequence.

In another embodiment, an isolated antibody that binds BACE1 is provided, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises the amino acid sequence of SEQ ID NO: 9-10, 36-39, or 58-64.

In another embodiment, an isolated antibody that binds BACE1 is provided, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises the amino acid sequence of SEQ ID NO: 11-16, 40 or 65-67 of HVR-L3.

In another embodiment, an isolated antibody that binds BACE1 is provided, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises a Variable Light (VL) chain sequence having an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO: 1-6, 31-34 and 99-117. In one aspect, the VL chain amino acid sequence is SEQ id no: 2.

in another embodiment, an isolated antibody that binds BACE1 is provided, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises the amino acid sequence of SEQ ID NO: 23, HVR-H1 sequence of SEQ ID NO: 24, the HVR-H2 sequence of SEQ ID NO: 25, HVR-H3 sequence of seq id NO: 7, HVR-L1 of SEQ ID NO: 9 HVR-L2 and SEQ ID NO: HVR-L3 of 12.

In one embodiment, an isolated antibody that binds BACE1 is provided, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide and comprises a heavy chain variable region comprising SEQ ID NO: 2 and a VL chain comprising the amino acid sequence of SEQ ID NO: 21, or a VH chain of the amino acid sequence of 21.

In another embodiment, an isolated antibody is provided that binds to an epitope comprising at least one amino acid residue of BACE1 selected from the group consisting of: SEQ ID NO: 314SER of 49; 316 GLU; 317 LYS; 327 GLN; 330 CYS; 331 TRP; 332 GLN; 335 THR; and 378 ASP. In certain embodiments, the antibody binds to an epitope of BACE1 comprising the amino acids: SEQ ID NO: 314SER of 49; 316 GLU; 317 LYS; 327 GLN; 330 CYS; 331 TRP; 332 GLN; 335 THR; and 378 ASP.

In other embodiments, the antibody binds to an epitope of BACE1 comprising at least one amino acid region of BACE1 selected from the group consisting of: SEQ ID NO: amino acids 315 of 49 and 318; SEQ ID NO: amino acids 331-335 of 49; SEQ ID NO: amino acids 370-381 of 49; and any combination thereof. In one embodiment, the antibody binds an epitope of BACE1 comprising the amino acid sequence of SEQ ID NO: amino acids 315, 331, 335 and 370, 381 of 49.

In another embodiment, the antibody binds to an epitope of BACE1, which upon binding results in a conformational change in the structure of the P6 and P7 sites of BACE 1. In another embodiment, the antibody binds a polypeptide comprising SEQ ID NO: the epitope of BACE1 at amino acids 218 and 231 of 49 thus assumes a random loop structure.

The antibodies of the invention can be in any number of forms. For example, the antibody of the present invention may be a human antibody, a humanized antibody or a chimeric antibody. In other aspects, an antibody of the invention is a full-length antibody or a fragment thereof (e.g., a fragment comprising an antigen-binding component). In other aspects of the invention, the antibody is a monoclonal antibody. In another aspect, the antibodies of the invention can be linked or conjugated to an agent or moiety, such as a cytotoxic agent, to produce an immunoconjugate.

In one embodiment, a pharmaceutical formulation is provided comprising an antibody of the invention and a pharmaceutically acceptable carrier. In additional embodiments, isolated nucleic acids encoding the antibodies of the invention are provided, as well as vectors comprising nucleic acids encoding the antibodies of the invention. In another aspect, there is provided a host cell comprising a nucleic acid encoding an antibody of the invention, and a method of producing an antibody of the invention, comprising culturing a host cell comprising a nucleic acid encoding an antibody of the invention under conditions suitable for production of the antibody.

In another embodiment, there is provided a method of treating a subject having a neurological disease or disorder, comprising administering to the subject an effective amount of an antibody of the invention.

In another embodiment, there is provided a method of reducing amyloid plaques, or inhibiting amyloid plaque formation, in a patient having, or at risk of contracting, a neurological disease or disorder, comprising administering to the individual an effective amount of an antibody of the invention.

In one embodiment, a method of reducing a β protein in a patient, the method comprising administering to the patient an effective amount of an antibody of the invention. In one aspect, the patient has a neurological disease or disorder, or is at risk of contracting a neurological disease or disorder.

In another embodiment, there is provided a method of inhibiting axonal degeneration in a patient comprising administering to the patient an effective amount of an antibody of the invention.

In another embodiment, a method of diagnosing a neurological disease or disorder in a patient, comprising contacting a biological sample isolated from said patient with an antibody of the invention under conditions suitable for binding of said antibody to a BACE1 polypeptide, and detecting whether a complex is formed between said antibody and said BACE1 polypeptide.

In one embodiment, a method of determining whether a patient is suitable for treatment with an anti-BACE 1 antibody, the method comprising contacting a biological sample isolated from said patient with an antibody of the invention under conditions suitable for binding of said antibody to a BACE1 polypeptide and detecting whether a complex is formed between said antibody and said BACE1 polypeptide, wherein the presence of a complex between said antibody and BACE1 indicates that the patient is suitable for treatment with an anti-BACE 1 antibody. In one aspect, the patient has a neurological disease or disorder, or is at risk of contracting a neurological disease or disorder.

In one aspect, can be used to diagnose a neurological disease or disorder; or for predicting responsiveness, a biological sample for determining the suitability of a patient for treatment with a BACE1 antibody includes, but is not limited to, a fluid such as serum, plasma, saliva, gastric secretions, mucus, cerebrospinal fluid, lymph fluid or the like, or a tissue or cell sample obtained from an organism such as neural tissue, brain tissue, heart tissue or vascular tissue.

In one aspect of the method of the invention, the patient is a mammal. In another aspect, the patient is a human. In another aspect, the neurological disease or disorder is selected from the group consisting of: alzheimer's Disease (AD), traumatic brain injury (traumatic brain injury), stroke, glaucoma, dementia, Muscular Dystrophy (MD), Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS), cystic fibrosis (cystic fibrosis), Angelman's syndrome, Liddle syndrome, Paget's disease, traumatic brain injury, Lewy body disease (Lewy body disease), post-polio syndrome (postpoliomyelitis syndrome), Charcot-Gray syndrome (Sheyard-Draeger syndrome), olivopontocerebellar atrophy (olivopontocerebellar atrophy), Parkinson's disease (Parkinson's disease), multiple sclerosis (multiple sclerosis), bovine spongiopathy (hypertrophic encephalopathy), bovine biliary degeneration (bovine biliary degeneration), Creutzfeldt-Jakob syndrome, kuru (kuru), Gerstmann-Straussler-Scheinker disease, Chronic wasting disease, fatal familial insomnia (facial fatigue syndrome), bulbar palsy (bulbar palsy), motor neuron disease (motor nerve disease), Kanner disease (Canavandiase), Huntington's disease, neuronal ceroid lipofuscinosis (neuronal cerbrosis), Alexander's disease, Tourette's syndrome, Mengkins twisting syndrome (Menkskin syndrome), Katsukura-ja syndrome (Korotkoff syndrome), Walkura-kura syndrome (Sporthia-scheinsis), Walkura-kura syndrome (Sporthia-Scheinker syndrome), Walkuru-kura-kuru (Sporthia-kura-kuru), Golay syndrome (Menksaki-kuru), Walkura-kuru (Sporthia-kura syndrome), Walkura-kura syndrome (Sporthia-kura syndrome), and pulsatile-long syndrome (Unverricht-Lundborg syndrome), dementia (including, but not limited to, Pick's disease, and spinocerebellar ataxia). In one aspect, the neurological disease or disorder is alzheimer's disease.

In one embodiment, a BACE1 epitope specifically recognized by an antibody or fragment thereof is provided, said epitope comprising at least one amino acid residue of BACE1 corresponding to an amino acid selected from the group consisting of: SEQ ID NO: 314SER of 49; 316 GLU; 317 LYS; 327 GLN; 330 CYS; 331 TRY; 332 GLN; 335 THR; and 378 ASP. In one aspect, the BACE1 epitope comprises a sequence corresponding to SEQ ID NO: 314SER of 49; 316 GLU; 317 LYS; 327 GLN; 330 CYS; 331 TRY; 332 GLN; 335 THR; and amino acids of 378 ASP.

In one embodiment, a BACE1 epitope specifically recognized by an antibody or fragment thereof, said epitope comprising at least one amino acid region of BACE1 selected from the group consisting of: SEQ ID NO: amino acids 315 of 49 and 318; SEQ ID NO: amino acids 331-335 of 49; SEQ ID NO: amino acids 370-381 of 49; and any combination thereof. In one aspect, the BACE1 epitope comprises SEQ ID NO: amino acids 315, 331, 335 and 370, 381 of 49.

Brief Description of Drawings

FIGS. 1A-1B show results obtained from first use in experimentsThe light and heavy chain amino acid sequences of clone YW412.8 and the affinity matured form of YW412.8 of the type of natural diversity phage display library are as described in example 1 (A). Figure 1A shows a light chain sequence alignment. Figure 1B shows a heavy chain sequence alignment. In FIGS. 1A and 1B, the HVR sequence of each clone consists of boxed regions Indicating, wherein the first box indicates HVR-L1(SEQ ID NOS: 7 and 8-FIG. 1A) or HVR-H1(SEQ ID NOS: 22 and 23-FIG. 1B), the second box indicates HVR-L2(SEQ ID NOS: 9 and 10-FIG. 1A) or HVR-H2(SEQ ID NOS: 24-FIG. 1B), and the third box indicates HVR-L3(SEQ ID NOS: 11-16-FIG. 1A) or HVR-H3(SEQ ID NOS: 25-FIG. 1B).

FIGS. 2A-2B show the light and heavy chain amino acid sequences of cloned Fab12 and affinity matured form of Fab12 obtained from a first experimental type of synthetic diversity phage display library, as described in example 1 (B). Figure 2A shows a light chain sequence alignment. Figure 2B shows a heavy chain sequence alignment. In FIGS. 2A and 2B, the HVR sequence of each clone is indicated by boxed regions, where the first box indicates HVR-L1(SEQ ID NO: 35-FIG. 2A) or HVR-H1(SEQ ID NO: 28-FIG. 2B), the second box indicates HVR-L2(SEQ ID NO: 36-39-FIG. 2A) or HVR-H2(SEQ ID NO: 29-FIG. 2B), and the third box indicates HVR-L3(SEQ ID NO: 40-FIG. 2A) or HVR-H3(SEQ ID NO: 30-FIG. 2B).

Fig. 3A and 3B show HVR or CDR sequences from light and heavy chain fabs isolated from synthetic diversity phage display libraries, as described in example 1 (B). Numbering is according to the nomenclature of Kabat et al. Fig. 3A discloses the amino acid sequence as set forth in SEQ ID NO: 133, as shown in SEQ id no: the "CDRL 2" sequence of 134, the "CDRL 3" sequence of SEQ ID NOS 135-144, 141 and 145 and 152, and the "CDRH 1" sequence of SEQ ID NOS 153-159, 158, 160 and 161, 159, 158, 162, 161 and 163 and 167 are each listed in order of occurrence. FIG. 3B discloses the "CDRH 2" sequences as set forth in SEQ ID NOS 168-177, 174, 171, 178-182, 177, and 183 and the "CDRH 3" sequence as set forth in SEQ ID NOS 184-202, respectively, all listed in order of occurrence.

FIG. 4 provides a graph showing the inhibition of BACE1 by different clones identified from naturally diverse and synthetic diverse phage display libraries. The clones were tested for inhibition of BACE1 in a homogeneous time-resolved fluorescence (HTRF) assay, as described in example 1 (a). All YW series antibodies were tested at a concentration of 500nM, except the YW434.6 antibody, which was tested at a concentration of 320 nM. Antibodies 12.IgG, 14.IgG lc6.IgG, lc9.IgG, lc10.IgG and lc11.IgG were tested at a concentration of 1 μ M.

FIG. 5 is a graph showing the activity of BACE1 in the presence of anti-BACE 1Fab identified from synthetic diverse phage display libraries in an HTRF assay, as described in example 1 (B). The line corresponds to 100% activity (0% inhibition) in the presence of BACE1 and substrate (PBS control), and 100% inhibition in the absence of BACE 1.

FIG. 6 shows the CDR or HVR sequences of an affinity matured anti-BACE 1Fab, as described in example 1 (B). Numbering is according to the nomenclature of Kabat et al. The competition ELISA ratio is the ratio of ELISA signals in the absence of 20nM BACE1 or the presence of 20nM BACE1 as competitor in a single-point (one-point) competition ELISA assay, as described in example 1 (B). FIG. 6 discloses the "CDRL 1" sequence as set forth in SEQ ID NOs 133, 133, 133, 133, 133 and 203; a "CDRL 2" sequence as set forth in SEQ ID NOs 134, 134 and 204-207; "CDRL 3" sequences as set forth in SEQ ID NOS 208-209, 145, 145 and 145-146; "CDRH 1" sequence as shown in SEQ ID NOs 157, 157, 158, 158, 158 and 162, "CDRH 2" sequence as shown in SEQ ID NOs 172, 172, 171, 171, 171 and 178; and the "CDRH 3" sequences, such as SEQ ID NO188, 188, 195, 195 and 195-196, are listed in order of occurrence, respectively.

FIGS. 7A-7C include graphs showing data from a competitive ELISA assay using affinity matured anti-BACE clones, as described in example 1 (B). Binding between Fab display phage and BACE1 immobilized on the plate competed with serial dilutions of BACE1 in solution. Fig. 7A, 7B and 7C show competition curves for the parent and corresponding affinity matured antibodies.

FIGS. 8A-8C show graphs showing the inhibition of BACE1 by anti-BACE 1Fab in HTRF enzymatic assays, as described in example 1 (B). The inhibitory activity of purified Fab on individual anti-BACE 1 clones was measured in HTRF enzyme assay. OM99-2(Catalog number 496000) Synthetic peptide inhibitors of BACE1, and were used as positive controls. FIGS. 8A, 8B and 8C are inhibition curves of the parent and corresponding affinity matured derivatives. In this assay, IC of OM99-2₅₀Is 11 nM.

Figure 9A provides a graph showing the effect of affinity matured yw412.8.31 anti-BACE 1 antibodies on the in vitro enzymatic activity of human recombinant BACE1 using either a long peptide substrate with enhanced sensitivity to BACE1 in the HTRF assay (left panel) or a short peptide substrate with enhanced sensitivity to BACE1 in the FRET assay (right panel), as described in example 2 (B). OM99-2( Cat # 496000), synthetic peptide inhibitors of BACE1, beta-secretase inhibitor IV (c)Catalog No. 565788), a small molecule inhibitor of BACE1 (BACE1SMI) and an IgG antibody that does not bind BACE1 were used as controls. FIGS. 9B-1 and 9B-2 also provide graphs showing the in vitro enzymatic activity of human recombinant BACE1 ectodomain, human recombinant BACE2 ectodomain, or cathepsin D ectodomain on short peptide substrates with enhanced sensitivity to BACE1 in the presence of YW412.8.31 or control IgG antibody, as described in example 2 (B).

FIG. 10 shows the results of experiments performed with different anti-BACE 1 antibodies (LC6, LC9, YW412.8, YW412.8.30, YW412.8.31 and YW412.8.51) during the processing of recombinant Amyloid Precursor Protein (APP) in 293-HEK cells, as described in example 2 (C). Does not bind BACE1The IgG antibody of (4) was used as a control.

FIGS. 11A-11D provide graphs showing the effect of the YW412.8.31 anti-BACE 1 antibody on the processing of recombinant or endogenous Amyloid Precursor Protein (APP) as described in example 2 (C). FIG. 11A shows results from experiments using 293-HEK cells stably expressing wild-type human APP. BACE1SMI is a small molecule BACE1 inhibitor that was used as a control (Compound 8E-Chartier et al, J.Med.chem.51: 3313-3317 (2008). FIG. 11B shows the results from experiments using E13.5 dorsal root ganglion neurons cultured from wild-type CD1 mice. additional experiments were performed using cultures of E16.5 cortical neurons (FIG. 11C) and E16.5 cultured hippocampal neurons (FIG. 11D) from wild-type CD1 mice.

Figures 12A-12C provide images of yw412.8.31 anti-BACE 1 antibody absorbed into primary mouse neurons, as described in example 2 (D). FIG. 12A shows internalization of YW412.8.31 anti-BACE 1 antibody into intracellular vesicles in neurons. Embryonic cortical neurons were incubated at 37 ℃ for the indicated time. Bound yw412.8.31 was detected on surface (non-permeabilized) or internal (permeabilized) cell fractions using α -human-Alexa 568. Most of the signal is internalized. By co-staining with the indicated markers for the vascular fraction, internalized yw412.8.31 was localized to the following subcellular fraction: early endosome (TfR); trans-golgi network (VAMP4) and lysosomes (LAMP 1). Scale bars 65 μm (top) and 20 μm (bottom). Fig. 12B shows: at two different temperatures and three different time points, anti-BACE 1 antibody was taken into E13.5 Dorsal Root Ganglion (DRG) neurons, as shown in the figure. Cells were permeabilized to allow labeling of intracellular BACE1 antibodies. In non-permeabilized cells, only the externally bound YW412.8.31 anti-BACE 1 antibody was labeled. FIG. 12(C) shows uptake of YW412.8.31 anti-BACE 1 antibody into E16.5 cortical neurons from mice expressing BACE1 or BACE1 knockout mice.

FIG. 13 provides a graphical representation of the ELISA results from example 3(A) with YW412.8 anti-BACE 1 antibody with itself, another anti-BACE 1 antibody (LC6), the active site BACE1 binding peptide (OM 99-2: (A))Catalog No. 496000)) and the exo-binding site BACE1 binding peptide (BMS1) (Kornacker et al, Biochemistry 44: 11567-11572(2005) -competitive binding of peptide 1).

FIG. 14 shows the preparation of Fab YW412.8.31 co-crystallized with the extracellular domain of human BACE1Different views of the structure, as described in example 3 (B). Fab binds to the BACE1 exo-binding site distal to the active site of secretase, which partially overlaps with another exo-binding site known to interact with certain peptides having BACE1 inhibitory properties.

FIG. 15 provides a partial magnified view of the interaction of Fab YW412.8.31 with the extracellular domain of human BACE 1. BACE1 is shown in surface representation, while Fab is shown as a ribbon. The dotted surface indicates the BACE1 epitope.

FIGS. 16A and 16B show the detection of BACE1 vs. A β in wild type mice_1-40Results of experiments on the contribution of levels. Detection of A β in BACE1+/+ mice_1-40Level comparison of A β in BACE 1-/-mice_1-40And (4) horizontal. Mice were dosed with a single dose of either control IgG antibody or anti-BACE yw412.8.31 antibody as described in example 4. FIG. 16A shows the detection of BACE1 vs. A β in mice _1-40The results of genetic studies of the resulting contributions. A β observed in BACE1 knockout mice (BACE1-/-)_1-40How a specific inhibitor of BACE1 levels alters a β in wild type mice_1-40The generation provides a control. FIG. 16B shows administration of control IgG or anti-BACE1YW412.8.31 (50mg/kg) at 24 or 48 hours post-administration versus A β in plasma and CNS (cortex)_1-40The resulting effect. A single dose of control IgG or anti-BACE 1 antibody (50mg/kg) was delivered by IV injection into C57B1/6 mice. After 24 or 48 hours, plasma and brain samples were collected for analysis of a β_1-40. Plasma Abeta_1-40Reduction of 35% (at 24 hours), cortical a β_1-40The reduction is 20 percent. Plotted values are mean (± SEM) × p < 0.01; p < 0.001.

FIGS. 17A-17B provide the results of the in vivo YW412.8.31 anti-BACE 1 antibody experiment described in example 4. FIG. 17A shows results in mice treated with YW412.8.31 anti-BACE 1 antibody at two different concentrations (compared to vehicle control treatment)Abeta observed in plasma and hippocampus_1-40Horizontal view. FIG. 17B is a graph of individual pharmacokinetics versus pharmacodynamic readings, indicating that for the YW412.8.31 anti-BACE 1 antibody, a PK/PD relationship is present in this mouse model.

Figures 18A and 18B show a comparison from experiments in which hAPP-transgenic mice were administered anti-BACE 1 antibody yw412.8.31 systemically (figure a, the same experiment described in figure 17A, redrawn for comparison) or by continuous ICV infusion (figure B). In FIG. 18A, animals received vehicle or anti-BACE 1 antibody (30 or 100mg/kg) by IP injection (3 doses of Q4D). At 2 hours after the last dose, plasma and brain samples were collected for analysis of a β _1-40And Abeta_1-42. anti-BACE 1 antibody, plasma A.beta.at 30 and 100mg/kg_1-40And Abeta_1-42Reduced to-30% control level. Hippocampus Abeta_1-40And Abeta_1-42Reduction (13-22%) of cortical A β by high dose of anti-BACE 1(100mg/kg)_1-40And Abeta_1-42Shows a trend of decrease (12-18%). In FIG. 18B, control IgG or anti-BACE 1 antibody was delivered by unilateral ICV infusion for 7 days. A β was observed at both doses_1-40And Abeta_1-42Consistent reduction of (d): in the cortex (15-23%), in the hippocampus (15-20%). Panel C shows the levels of anti-BACE 1 antibody in the brain following systemic versus ICV delivery. Plotted values are mean (± SEM) × p < 0.05; p < 0.001

FIGS. 19A and 19B show PK analysis of a single dose of YW412.8.31 anti-BACE 1(1 or 10mg/kg) delivered via IV injection to BALB/C mice (FIG. 19A). Serum PK was analyzed until 21 days post dose. Two separate PK assays were used: one assay detects all anti-BACE 1 (total mAb) in serum, while one assay detects only unbound anti-BACE 1 (free mAb) in serum. Single dose PK analysis in BACE1+/+, BACE1 +/-and BACE 1-/-mice confirmed the non-linearity observed in the initial study and suggested that the enhanced clearance was indeed target-mediated (figure 19B).

FIGS. 20A and 20B show PK analysis of cynomolgus monkeys dosed by IV delivery with control IgG or YW412.8.31 anti-BACE 1 antibody (30 mg/kg). Total anti-BACE 1 or control antibody concentrations in monkey sera (fig. 20A) and CSF samples (fig. 20B) were measured using monkey-adsorbed goat anti-human IgG polyclonal antibody (Bethyl, Montgomery, TX), as described in example 5.

FIGS. 21A-21D are the results of an experiment as described in example 5 in which cynomolgus monkeys were dosed IV with control IgG or the anti-BACE 1 antibody YW412.8.31. Data for individual animals are shown in hatched lines and group averages in solid lines. Plasma and CSF were sampled at 7 days, 2 days and prior to drug administration to set A β in each individual monkey_1-40Mean of baseline levels. Measurement of plasma Abeta at different times_1-40(FIG. 21A) and CSF Abeta_1-40(FIG. 21B). Abeta at baseline plasma (FIG. 21C) and CSF (FIG. 21D)_1-40Inter-animal variability was also shown.

Fig. 22A and 22B show a β production in wild type mice after systemic administration of yw412.8.31. FIG. 22A is a graph showing A β after a single dose of control IgG or YW412.8.31(100mg/kg) administered to C57B1/6J mice by IP injection_1-40And (4) generating. After 4 hours, plasma and brain samples were collected for analysis of a β_1-40. Plasma Abeta_1-4048% reduction, but in this example forebrain a β_1-40Is not reduced. FIG. 22B is a graph showing A β after administration of control IgG or YW412.8.31(30 or 100mg/kg) by 3 IP injections (4 days apart each)_1-40And (4) generating. Plasma and brain samples were collected for analysis of a β 4 hours after the final dose_1-40. Plasma Abeta_1-40Reduced by 50-53%, and forebrain Abeta _1-40There was no reduction when administered at 30mg/kg, but 42% reduction when administered at 100 mg/kg. Values plotted are mean (. + -. SEM). p < 0.0001

FIGS. 23A-23C show the light chain amino acid sequences of clone YW412.8.31 and the affinity matured form of YW412.8.31. FIGS. 23A-23C show a complete light chain sequence alignment. The HVR sequence of each clone was indicated by boxed regions, with the first box indicating HVR-L1(SEQ ID NO: 7-FIG. 23A), the second box indicating HVR-L2(SEQ ID NO: 9 and 58-64-FIG. 23B), and the third box indicating HVR-L3(SEQ ID NO: 12 and 66-67-FIG. 23C).

FIGS. 24A-24C show the heavy chain amino acid sequences of clone YW412.8.31 and the affinity matured form of YW412.8.31. FIGS. 24A-24C show a complete heavy chain sequence alignment. The HVR sequence of each clone was indicated by boxed regions, with the first box indicating HVR-H1(SEQ ID NOS: 24 and 71-73-FIG. 24A), the second box indicating HVR-H2(SEQ ID NOS: 24 and 74-78-FIG. 24B), and the third box indicating HVR-H3(SEQ ID NOS: 25 and 79-FIG. 24C).

FIGS. 25A and B show graphs showing inhibition of BACE1 by YW412.8.31 and affinity matured clones in the HTRF assay as described in example 6. Test clone YW412.8.31.3S; YW412.8.31.9S, respectively; YW412.8.31.25S, respectively; YW412.8.31.58S, respectively; YW412.8.31.53, respectively; YW412.8.31.69, respectively; YW412.8.31.77, respectively; YW412.8.31.81S and YW412.8.31.89S ability to inhibit protease activity of BACE 1.

Detailed description of the embodiments

Definition of

An "acceptor human framework" for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below. Acceptor human frameworks "derived from" human immunoglobulin frameworks or human consensus frameworks may comprise their same amino acid sequence, or they may comprise variations of the amino acid sequence. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the sequence of the VL acceptor human framework is identical to the VL human immunoglobulin framework sequence or human consensus framework sequence.

"affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise specified, "binding affinity" refers to a binding affinity that reflects a ratio between members of a binding pair (e.g., antibody and antigen) of 1: 1 intrinsic binding affinity of the interaction. The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) of the antibody, resulting in an increase in the affinity of the antibody for an antigen compared to a parent antibody without the alterations.

The terms "anti- β -secretase antibody", "anti-BACE 1 antibody", "antibody binding to β -secretase" and "antibody binding to BACE 1" refer to antibodies capable of binding BACE1 antibody with sufficient affinity such that the antibodies can be used as diagnostic and/or therapeutic agents in targeting BACE 1. In one embodiment, an anti-BACE 1 antibody binds to an unrelated, non-BACE 1 protein to a lesser extent than about 10% of the binding of said antibody to BACE1, as measured, for example, by Radioimmunoassay (RIA). In certain embodiments, an antibody that binds BACE1 has a dissociation constant (Kd) of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, less than or equal to 0.1nM, less than or equal to 0.01nM, or less than or equal to 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M). In certain embodiments, an anti-BACE 1 antibody binds to a BACE1 epitope that is conserved among BACE1 from different species and isoforms.

The term "antibody" is used herein in the broadest sense and includes different antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen binding activity.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab ', Fab ' -SH, F (ab ')₂(ii) a A diabody; linear reactanceA body; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of 50% or more of the reference antibody to its antigen in a competition assay, whereas a reference antibody blocks binding of 50% or more of the antibody to its antigen in a competition assay. An exemplary competition assay is provided herein.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remaining heavy and/or light chain is derived from a different source or species.

The "class" of antibodies refers to the type of constant domain or constant region that the heavy chain has. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG ₁，IgG₂，IgG₃，IgG₄，IgA₁And IgA₂. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to: radioisotope (e.g., At)²¹¹，I¹³¹，I¹²⁵，Y⁹⁰，Re¹⁸⁶，Re¹⁸⁸，Sm¹⁵³，Bi²¹²，P³²，pb²¹²And radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate), doxorubicin (adriamicin), vinca alkaloids (vinca alkaloids) (vincristine), vinblastine (vinblastine), etoposide (etoposide)), doxorubicin (doxorubicin), melphalan (melphalan), mitomycin (mitomycin) C, chlorambucil (chlorembucil), daunorubicin (daunorubicin), or other intercalating agents); a growth inhibitor; enzymes and fragments thereof such as nucleic acid hydrolases; (ii) an antibiotic; toxinsSuch as a small molecule toxin or an enzymatically active toxin of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various anti-tumor or anti-cancer agents disclosed below.

"Effector function" refers to those biological activities attributable to the Fc region of an antibody and which vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

An "effective amount" of an agent, e.g., a pharmaceutical formulation, is an amount effective to achieve the desired therapeutic or prophylactic result at the desired dosage and for the desired period of time.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pr0230 to the carbonyl end of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al, Sequences of proteins of immunological interest, 5th Ed. public Health Service, National Institutes of Health, Bethesda, Md, 1991.

"framework" or "FR" refers to variable domain residues other than the hypervariable region (HVR) residues. The FRs of a variable domain typically consist of four FR domains: FR1, FR2, FR3 and FR 4. Thus, HVR and FR sequences typically occur in the following sequences of VH (or VL): FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

The terms "full-length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid is introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primarily transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell.

"human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using a human antibody library or other human antibody coding sequence. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

By "human consensus framework" is meant a framework which, in the selection of human immunoglobulin VL or VH framework sequences, represents the most frequently occurring amino acid residues. Generally, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. In general, a subtype of this sequence is a subtype as in Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition, NIH Publication91-3242, Bethesda MD (1991), volumes 1-3. In one embodiment, for VL, this subtype is subtype K I as in Kabat et al (supra). In one embodiment, for the VH, this subtype is subtype III as in Kabat et al (supra).

A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized forms" of antibodies (e.g., non-human antibodies) refer to antibodies that have been humanized.

The term "hypervariable region" or "HVR", when used herein, refers to each region of an antibody variable domain whose sequence is hypervariable and/or forms structurally defined loops ("hypervariable loops"). Typically, a native four-chain antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically contain amino acid residues from hypervariable loops and/or "complementarity determining regions" (CDRs) that have the highest sequence variability and/or are involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2), and 96-101 (H3). (Chothia and Lesk, J.mol.biol.196: 901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at 24-34 of amino acid residues L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et al, Sequences of Proteins of immunological Interest, 5th Ed. public Health Service, National Institutes of Health, Bethesda, MD (1991)). In addition to the CDR1 in VH, the CDR typically comprises amino acid residues that form a hypervariable loop. CDRs also contain "specificity determining residues" or "SDRs," which are residues that are in contact with antigen. SDR is contained in a region of a CDR called an abbreviated (abbreviated) -CDR or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues L1 31-34, L2 50-55, L3 89-96, H1 31-35B, H2 50-58, and H3 95-102. (see Almagro and Fransson, front. biosci.13: 1619-1633 (2008)). Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al (supra).

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules (including but not limited to cytotoxic agents).

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al, j.chromatogr.b848: 79-87(2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

An "isolated nucleic acid encoding an anti-BACE 1 antibody" refers to one or more nucleic acid molecules encoding an antibody heavy and light chain (or fragments thereof), including such nucleic acid molecules in a single vector or in separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or occurring in the course of producing a monoclonal antibody preparation, such variants typically being present in minor amounts). Each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen, as compared to a polyclonal antibody preparation that typically includes different antibodies directed against different determinants (epitopes). Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be generated by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for making monoclonal antibodies are described herein.

By "naked antibody" is meant an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabeled. Naked antibodies may be present in pharmaceutical formulations.

"native antibody" refers to a naturally occurring immunoglobulin molecule with an altered structure. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains, which are bound by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH 3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also referred to as a variable light domain or light chain variable domain, followed by a Constant Light (CL) domain. The light chain of an antibody can be assigned to one of two types called kappa (K) and lambda (lambda) based on the amino acid sequence of its constant domain.

The term "package insert" is used to refer to instructions for use, typically included in commercial packaging for therapeutic products, that contain information about indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings concerning the use of such therapeutic products.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the reference polypeptide sequence, after the sequences are aligned (and gaps introduced, if necessary) to obtain the maximum percent sequence identity, and no conservative substitutions are considered as part of the sequence identity. Sequence alignments can be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to obtain maximum alignment over the full length of the sequences being compared. To this end, however,% amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The author of the ALIGN-2 sequence comparison computer program was Genentech, Inc and the source code had been submitted with the user document to the us copyright office (Washington d.c., 20559) with us copyright registration number TXU 510087. The ALIGN-2 program is publicly available through Genentech, Inc. (South San Francisco, Calif.) or may be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including numeral UNIXV4.0D. All sequence alignment parameters were set by the ALIGN-2 program and were unchanged.

In the case of ALIGN-2 as applied to amino acid sequence comparisons, the% amino acid sequence identity of a given amino acid sequence A relative to (to), with (with), or against (against) a given amino acid sequence B (or so to say that a given amino acid sequence A has or contains some% amino acid sequence identity relative to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the X/Y ratio

Wherein X is the number of amino acid residues scored as identical matches in the A and B alignments of the sequence alignment program using the program ALIGN-2, and wherein Y is the total number of amino acid residues in B. It will be understood that when the length of amino acid sequence a is not equal to that of amino acid sequence B, the% amino acid sequence identity of a relative to B will not equal the% amino acid sequence identity of B relative to a. Unless specifically stated otherwise, all% amino acid sequence identity values used herein are obtained as described in the preceding paragraph using the ALIGN-2 computer program.

The term "pharmaceutical formulation" refers to a formulation that is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional ingredients that have unacceptable toxicity to the subject to which the formulation is administered.

By "pharmaceutically acceptable carrier" is meant an ingredient in a pharmaceutical formulation that is distinct from the active ingredient and which is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

Unless otherwise indicated, the term "BACE 1" as used herein refers to any native β -secretase 1 (also known as β -amyloid precursor protein cleaving enzyme 1, membrane associated aspartic protease 2, memapsin2, aspartyl protease 2 or Asp2) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term includes "full-length" unprocessed BACE1 as well as any form of BACE1 that results from intracellular processing. The term also includes variants of naturally occurring BACE1, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary BACE1 polypeptide is shown in SEQ ID NO: 49 and the sequence of human BACE1, isoform a, as described in Vassar et al, Science 286: 735-741(1999), which is incorporated herein by reference in its entirety.

MAQALPWLLLWMGAGVLPAHGTQHGIRLPLRSGLGGAPLGLRLPRETDEEPEEPGRRGSFVEMVDNLRGKSGQGYYVEMTVGSPPQTLNILVDTGSSNFAVGAAPHPFLHRYYQRQLSSTYRDLRKGVYVPYTQGKWEGELGTDLVSIPHGPNVTVRANIAAITESDKFFINGSNWEGILGLAYAEIARPDDSLEPFFDSLVKQTHVPNLFSLQLCGAGFPLNQSEVLASVGGSMIIGGIDHSLYTGSLWYTPIRREWYYEVIWRVEINGQDLKMDCKEYNYDKSWDSGTTNLRLPKKVFEAAVKSIKAASSTEKFPDGFWLGEQLVCWQAGTTPWNIFPVISLYLMGEVTNQSFRITILPQQYLRPVEDVATSQDDCYKFAISQSSTGTVMGAVIMEGFYVVFDRARKRIGFAVSACHVHDEFRTAAVEGPFVTLDMEDCGYNIPQTDESTLMTIAYVMAAICALFMLPLCLMVCQWCCLRCLRQQHDDFADDISLLK(SEQ ID NO：49)

There are several other isoforms of human BACE1, including isoform B, C and D. See UniProtKB/Swiss-Prot Entry P56817, which is incorporated by reference herein in its entirety. Isoform B is shown in SEQ ID NO: 50 and differs from isoform A (SEQ ID NO: 49) in that it lacks amino acids 190-214 (i.e., lacks amino acids 190-214 of SEQ ID NO: 49). Isoform C is shown in SEQ ID NO: 51 and differs from isoform A (SEQ ID NO: 49) in that it lacks amino acids 146-189 (i.e., lacks amino acids 146-189 of SEQ ID NO: 49). Isoform D is shown in SEQ ID NO: 52 and differs from isoform A (SEQ ID NO: 49) in that it lacks amino acids 146-189 and 190-214 (i.e.lacks amino acids 146-189 and 190-214 of SEQ ID NO: 49).

As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual to be treated, and may be performed either for prophylaxis or in the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviation of symptoms, elimination of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of or slow the progression of disease.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain comprising four conserved Framework Regions (FRs) and three hypervariable regions (HVRs). (see, e.g., Kindt et al Kuby Immunology, 6^thed., w.h.freeman and co.91, page 2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains from antibodies that bind to a particular antigen can be used to isolate antibodies that bind the antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al, j.immunol.150: 880- & ltwbr & gt 887 & gt (1993); clarkson et al, Nature35 2：624-628(1991)。

The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

The term "neurological disorder" or "neurological disease" refers to or describes a disease or disorder in the central and/or peripheral nervous system of a mammal. Examples of neurological diseases include, but are not limited to, the following list of diseases and conditions. Neuropathic conditions are diseases or abnormalities of the nervous system characterized by inappropriate or uncontrolled nerve signaling or lack thereof, and include, but are not limited to, chronic pain (including nociceptive pain (pain caused by injury to body tissues, including cancer-related pain), neuropathic pain (pain caused by abnormalities in nerves, spinal cord, or brain), and psychiatric pain (complete or mostly related to psychological disorders), headache, migraine, neuropathy, and symptoms and syndromes often associated with such neuropathic conditions such as dizziness or nausea amyloid is a group of diseases and conditions associated with extracellular protein deposits in the CNS including, but not limited to, secondary amyloidosis, age-related amyloidosis, Alzheimer's Disease (AD), mild cognitive impairment (cognitive impairment, MCI), lewy body dementia, down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); guam Parkinson-dementia complex, cerebral amyloid angiopathy, huntington's disease, progressive supranuclear palsy, multiple sclerosis; creutzfeldt-jakob disease, parkinson's disease, transmissible spongiform encephalopathies, HIV-associated dementia, Amyotrophic Lateral Sclerosis (ALS), inclusion-body myositis (IBM), and ocular diseases involving β -amyloid deposition (i.e., macular degeneration, drusen-related optic neuropathy, and cataracts). Cancers of the CNS are characterized by abnormal proliferation of one or more CNS cells (i.e., nerve cells) and include, but are not limited to, glioma (glioma), glioblastoma multiforme (glioblastomas), meningioma (meningioma), astrocytoma (astrocytoma), acoustic neuroma (acoustic neuroma), chondroma (chloroglioma), oligodendroglioma, medulloblastoma, ganglioblastoma (ganglioglioma), ganglioglioma (ganlioma), Schwannoma, neurofibroma (neuroofibroma), neuroblastoma (neuroblastoma), and extradural, intramedullary or intradural tumors. An ocular disease or disorder is a disease or disorder of the eye, which for purposes herein is considered to be a CNS organ that is subject to the BBB. Eye diseases or conditions include, but are not limited to, conditions of the sclera, cornea, iris and ciliary body (i.e., scleritis), keratitis (keratitis), corneal ulcers (corneal ulcers), corneal abrasions (corneal abrasions), snow blindness (snowblinness), electro-optic ophthalmia (arc eye), Thygeson's superficial punctate keratopathy (Thygeson's punctate keratitis), corneal neovascularization (corneal), fucus ' dystrophy), keratoconus (keratoconus), keratoconjunctivitis sicca (keratoconjuctivitis sica), iritis (iritis) and uveitis (uveitis)), conditions of the cornea and retina (i.e., cataract), conditions of the choroid and retina (retinal detachment), retinopathy (retinopathy), retinopathy of prematurity (retinopathy), retinopathy (retinopathy of prematurity), age-related macular degeneration, macular degeneration (wet or dry), epiretinal membrane (epiretinal membrane), retinitis pigmentosa (retinitis pigmentosa) and macular edema (macular edema)), glaucoma (glaucoma), floaters (floats), disorders of the optic nerve and visual pathway (i.e., Leber's hereditary optic neuropathy (Leber's disease), and optic disc drusen), disorders of the optic nerve/binocular movement regulation/refraction (i.e., strabismus, oculomotor paralysis (opthalmopathy), progressive external ophthalmoplegia (opthalmopplegia), internal strabismus, external strabismus, hypermetropia, astigmatism, ametropia, presbyopia and ophthalmoplegia), visual impairment and amblyopia (uveromophytoia), congenital chromophoric dyschromia (congenital chromophoric), congenital chromophoric dyschromia), nyctalopia (nyctalopia), blindness (blindness), river blindness (river blindness) and microphthalmia/defect (micro-opthalmia/coloroma)), pinkeye, argyroson pupil (argyl Robertson pupil), keratomycosis (keratomycosis), dry eye and aniridia (aniridia). Viral or microbial infections of the CNS include, but are not limited to, infections caused by viruses (i.e., influenza, HIV, poliovirus, rubella), bacteria (i.e., Neisseria (Neisseria sp.), Streptococcus (Streptococcus sp.), Pseudomonas (Pseudomonas sp.), Proteus (Proteus sp.), escherichia coli (e.coli), staphylococcus aureus (s.aureus), Pneumococcus (pneucoccus sp.), Meningococcus (menigococcus sp.), Haemophilus (Haemophilus sp.), and Mycobacterium tuberculosis (Mycobacterium tuberculosis)) and other microorganisms such as fungi (i.e., yeast, Cryptococcus neoformans (cryptococci neoformans)), parasites) (i.e., toxoplasma (gonopsii)) or Proteus (amoebas)), which cause pathological encephalitis, including, chronic encephalitis, and chronic encephalitis, which may be acute or chronic encephalitis. Inflammation of the CNS is inflammation that results from injury to the CNS, which may be physical injury (i.e., due to accident, surgery, brain trauma, spinal cord injury, concussion (concussion)) or injury due to or associated with one or more other diseases or conditions of the CNS (i.e., abscess, cancer, viral or microbial infection). As used herein, ischemia of the CNS refers to a group of conditions related to abnormal blood flow or vascular behavior of the brain or its etiology, and includes, but is not limited to, focal cerebral ischemia (global cerebral ischemia), stroke (i.e., subarachnoid and intracerebral hemorrhage), and aneurysms. Neurodegenerative diseases are a group of diseases and disorders that are associated with neuronal cell loss of function or death in the CNS and include, but are not limited to, adrenoleukodystrophy (adrenalleukodystrophy), alexander disease, Alper's disease, amyotrophic lateral sclerosis, ataxia telangiectasia (ataxia telangiectasia), Batten disease (Batten disease), cockayne syndrome, corticobasal degeneration (corticobasal degeneration), degeneration caused by or associated with amyloidosis, Friedreich's ataxia, frontotemporal lobar degeneration (emporall lobar degeneration), kenne disease (Kennedy's disease), multiple system atrophy, multiple sclerosis, primary lateral sclerosis, progressive supranuclear palsy, spinal muscular atrophy, transverse myelitis, retney's disease, and Refsum spinal cord disorder. Seizure disorders and conditions of the CNS relate to inappropriate and/or abnormal electrical conduction in the CNS and include, but are not limited to, epilepsy (i.e., absence seizures (absentites), dystonic seizures (atonic seizures), benign motor seizures (benign rolandicepies), childhood absence (childhood absention), clonic seizures (sonic seizures), complex partial seizures (complex partial seizures), frontal epilepsy (frontal lobel epilepsy), febrile seizures (febrile seizures), infantile convulsions (infantile spasms), juvenile myoclonic epilepsy (juvenile myoclonic epilepsy), juvenile epilepsy (junctional mental epilepsy) (dominant seizure Syndrome), juvenile epilepsy (lateral seizure Syndrome), severe neuro epilepsy (lateral seizure Syndrome), neuro epilepsy (lateral Syndrome), neuro-grid Syndrome (len-Syndrome), neuro Syndrome-Syndrome (kalimes), neuro Syndrome (clavulan Syndrome), neuro Syndrome (throm Syndrome), progressive myoclonic seizures (progressive myoclonic seizures), psychiatric seizures (reflex seizures), Rasmussen's Syndrome, simple partial seizures (simple partial seizures), secondary generalized seizures (reflex seizures), temporal lobe seizures (temporal lobbies), clonic seizures (toxic partial seizures), tonic seizures (toxic partial seizures), psychomotor seizures (psychomotor seizures), limbic lobe seizures (1 immunologic epily), partial seizures (partial-ontoseireses), generalized seizures (systemic-episodics), status epilepticus (vegetative-persistent seizures), vegetative seizures (vegetative-episodics), vegetative-onset status epilepticus (vegetative-vegetative, focal seizures (focal fractures), laughing seizures (laser fractures), jiakson gorge (Jacksonian March), lava (laforadis), kinetic seizures (motor fractures), multifocal fractures (multifocal fractures), night seizures (noctual fractures), photosensitive seizures (photosensitive fractures), pseudo seizures (pseudo fractures), sensory seizures (sensory fractures), mini-seizures (subtle fractures), sylvan seizures (sylvan fractures), finger seizures (rodawal fractures), and visual reflex seizures (visual reflex fractures)). Behavioral disorders are disorders of the CNS characterized by abnormal behavior with respect to the afflicted subject and include, but are not limited to, sleep disorders (sleep disorders) (i.e., insomnia (insomnia), parasomnia (parasomnias), night terrors, circadian rhythm sleep disorders (circadian rhythm disorders), and narcolepsy (narcolepsy)), mood disorders (mood disorders) (i.e., depression (depression), suicidal depression (depression), anxiety (anxiety), chronic affective disorders (phobia), phobias (phobias), panic attacks (panic attacks), obsessive-compulsive disorders (obsessive-compulsive disorders), attention deficit hyperactivity disorder (attention deficit hyperactivity disorder), attention deficit hyperactivity disorder (attention deficit-deficit disorder) (attention-deficit disorder), attention-deficit disorder (attention-deficit disorder), post-stress syndrome (attention-deficit disorder) (attention-deficit-stress disorder), bipolar disorder), eating disorders (i.e., anorexia (anorexia) or bulimia (bulimia)), psychoses (psychoses), developmental behavior disorders (developmental behavial disorder) (i.e., autism (autism), Rett's syndrome (Rett's syndrome), Aspberger's syndrome (Aspberger's syndrome)), personality disorders, and psychiatric disorders (i.e., schizophrenia (schizophrenia), delusional disorder (delusional disorder), etc.). Lysosomal storage disorders (lysomal storage disorders) are metabolic disorders, in some cases associated with the CNS or having CNS-specific symptoms; such conditions include, but are not limited to, Tay-saxophone disease (Tay-Sachs disease), Gaucher's disease (Gaucher's disease), Fabry disease (Fabry disease), mucopolysaccharidosis (types I, II, III, IV, V, VI and VII), glycogenosis (glycogen storage disease), GMl gangliosidosis (GMl-gingliosis), metachromatic leukosis (metachromatic leukoencephalopathy), Farber disease (Farber's disease), Kannan leukodystrophy (Canavan's paradoxystosis), and neuronal ceroid lipofuscinosis (neuronal ceroid lipofuscinoses) types 1 and 2, Niemann-Pick disease (Niemann-Piase), Pompe (Pompe), and Kraberray's disease (Krabenzosis).

Compositions and methods

In one aspect, the invention is based, in part, on an antibody that binds BACE1 and reduces and/or inhibits BACE1 activity. In certain embodiments, antibodies are provided that bind to the active site or exo-binding site of BACE 1.

Exemplary anti-BACE 1 antibodies

In one aspect, the invention provides an anti-BACE 1 antibody, said anti-BACE 1 antibody comprising at least one, two, three, four, five or six HVRs selected from: (a) comprises the amino acid sequence of SEQ ID NO: 22, 23, 26, 28, 45, 68, 71, 72, 73, or 120 HVR-H1; (b) comprises the amino acid sequence of SEQ ID NO: 24, 29, 46, 69, 74, 75, 76, 77, 78, or 121; (c) comprises the amino acid sequence of SEQ ID NO: 25, 30, 47, 70, 79, or 122; (d) comprises the amino acid sequence of SEQ ID NO: 7, 8, 17, 35 or 42 HVR-L1; (e) comprises the amino acid sequence of SEQ ID NO: 9, 10, 18, 36-39, 41, 43, 56, 58, 59, 60, 61, 62, 63, 64, or 118; and (f) a polypeptide comprising the amino acid sequence of SEQ ID NO: 11-16, 19, 40, 44, 57, 65, 66, 67, or 119.

In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) comprises the amino acid sequence of SEQ ID NO: 22, 23, 26, 28, 45, 68, 71-73, or 120 HVR-H1; (b) comprises the amino acid sequence of SEQ ID NO: 24, 29, 46, 69, 74-78, or 121; and (c) a polypeptide comprising the amino acid sequence of SEQ ID NO: 25, 30, 47, 70, 79 or 122.

In one embodiment, the antibody comprises HVR-H1, which HVR-H1 comprises the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23 or SEQ ID NO: 28 or SEQ ID NO: 71 or SEQ ID NO: 72 or SEQ ID NO: 73. in another embodiment, the antibody comprises HVR-H2, which HVR-H2 comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 29 or SEQ ID NO: 74 or SEQ ID NO: 75 or SEQ ID NO: 76 or SEQ ID NO: 77 or SEQ ID NO: 78. in another embodiment, the antibody comprises HVR-H3, which HVR-H3 comprises the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 30 or SEQ ID NO: 79. in one embodiment, the antibody comprises HVR-H1, which HVR-H1 comprises the amino acid sequence of SEQ ID NO: 28. in another embodiment, the antibody comprises HVR-H2, which HVR-H2 comprises the amino acid sequence of SEQ ID NO: 29. in another embodiment, the antibody comprises HVR-H3, which HVR-H3 comprises the amino acid sequence of SEQ ID NO: 30.

in another embodiment, the antibody comprises (a) HVR-H1, which HVR-H1 comprises the amino acid sequence of SEQ ID NO: 22; (b) HVR-H2, said HVR-H2 comprising the amino acid sequence of SEQ ID NO: 24; and (c) HVR-H3, the HVR-H3 comprising the amino acid sequence of SEQ id no: 25, or the antibody comprises (a) HVR-H1, which HVR-H1 comprises the amino acid sequence of seq id NO: 23; (b) HVR-H2, said HVR-H2 comprising the amino acid sequence of SEQ ID NO: 24; and (c) HVR-H3, the HVR-H3 comprising the amino acid sequence of SEQ ID NO: 25. in another embodiment, the antibody comprises (a) HVR-H1, which HVR-H1 comprises the amino acid sequence of SEQ ID NO: 28; (b) HVR-H2, said HVR-H2 comprising the amino acid sequence of SEQ ID NO: 29; and (c) HVR-H3, the HVR-H3 comprising the amino acid sequence of SEQ ID NO: 30. in another embodiment, the antibody comprises (a) HVR-H1, which HVR-H1 comprises the amino acid sequence of SEQ ID NO: 23; (b) HVR-H2, said HVR-H2 comprising the amino acid sequence of SEQ ID NO: 74; and (c) HVR-H3, the HVR-H3 comprising the amino acid sequence of SEQ ID NO: 25, or the antibody comprises (a) HVR-H1, the HVR-H1 comprising the amino acid sequence of SEQ id no: 23; (b) HVR-H2, said HVR-H2 comprising the amino acid sequence of SEQ ID NO: 75; and (c) HVR-H3, the HVR-H3 comprising the amino acid sequence of SEQ ID NO: 25 or the antibody comprises (a) HVR-H1, which HVR-H1 comprises the amino acid sequence of SEQ ID NO: 71; (b) HVR-H2, said HVR-H2 comprising the amino acid sequence of SEQ ID NO: 24; and (c) HVR-H3, the HVR-H3 comprising the amino acid sequence of SEQ ID NO: 25, or the antibody comprises (a) HVR-H1, the HVR-H1 comprising the amino acid sequence of SEQ ID NO: 72; (b) HVR-H2, said HVR-H2 comprising the amino acid sequence of SEQ ID NO: 24; and (c) HVR-H3, the HVR-H3 comprising the amino acid sequence of SEQ ID NO: 25, or the antibody comprises (a) HVR-H1, the HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23; (b) HVR-H2, said HVR-H2 comprising the amino acid sequence of SEQ ID NO: 76; and (c) HVR-H3, the HVR-H3 comprising the amino acid sequence of SEQ id no: 25, or the antibody comprises (a) HVR-H1, which HVR-H1 comprises the amino acid sequence of seq id NO: 23; (b) HVR-H2, said HVR-H2 comprising the amino acid sequence of SEQ ID NO: 77; and (c) HVR-H3, the HVR-H3 comprising the amino acid sequence of SEQ ID NO: 79, or the antibody comprises (a) HVR-H1, the HVR-H1 comprising the amino acid sequence of SEQ ID NO: 73; (b) HVR-H2, said HVR-H2 comprising the amino acid sequence of SEQ ID NO: 78, a nitrogen source; and (c) HVR-H3, the HVR-H3 comprising the amino acid sequence of SEQ ID NO: 25.

In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1, the HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7, 8, 17, 35 and 42; (b) HVR-L2, the HVR-L2 comprising the amino acid sequence of SEQ ID NO: 9, 10, 18, 36-39, 41, 43, 56, 58-64, or 118; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11-16, 19, 40, 44, 57, 65-67, or 119.

In one embodiment, the antibody comprises HVR-L1, which HVR-L1 comprises the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. in another embodiment, the antibody comprises HVR-L2, which HVR-L2 comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 58 or SEQ ID NO: 59 or SEQ ID NO: 60 or SEQ ID NO: 61 or SEQ ID NO: 62 or SEQ ID NO: 63 or SEQ ID NO: 64. in another embodiment, the antibody comprises HVR-L3, which HVR-L3 comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15 or seq id NO: 16 or SEQ ID NO: 65 or SEQ ID NO: 66 or SEQ ID NO: 67. in another embodiment, the antibody comprises HVR-L1, which HVR-L1 comprises the amino acid sequence of seq id NO: 35. in another embodiment, the antibody comprises HVR-L2, comprising an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO: 36-39. In another embodiment, the antibody comprises HVR-L3, which HVR-L3 comprises the amino acid sequence of SEQ ID NO: 40.

In another embodiment, the antibody comprises (a) HVR-L1, wherein HVR-L1 comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2, said HVR-L2 comprising the amino acid sequence of SEQ ID NO: 9; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11, or the antibody comprises (a) HVR-L1, which HVR-L1 comprises the amino acid sequence of SEQ id no: 7; (b) HVR-L2, the HVR-L2 comprising the amino acid sequence of SEQ ID NO: 9; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12, or the antibody comprises (a) HVR-L1, which HVR-L1 comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2, the HVR-L2 comprising the amino acid sequence of SEQ ID NO: 9; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 13, or the antibody comprises (a) HVR-L1, which HVR-L1 comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2, the HVR-L2 comprising the amino acid sequence of SEQ ID NO: 9; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14, or the antibody comprises (a) HVR-L1, which HVR-L1 comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2, the HVR-L2 comprising amino acid sequence SEQ ID NO: 9; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16, or the antibody comprises (a) HVR-L1, which HVR-L1 comprises the amino acid sequence of SEQ ID NO: 8; (b) HVR-L2, the HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15. in another embodiment, the antibody comprises (a) HVR-L1, wherein HVR-L1 comprises the amino acid sequence of SEQ ID NO: 35; (b) HVR-L2, the HVR-L2 comprising the amino acid sequence of SEQ ID NO: 36; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40, or the antibody comprises (a) HVR-L1, which HVR-L1 comprises the amino acid sequence of SEQ ID NO: 35; (b) HVR-L2, the HVR-L2 comprising the amino acid sequence of SEQ ID NO: 37; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40, or the antibody comprises (a) HVR-L1, which HVR-L1 comprises the amino acid sequence of SEQ ID NO: 35; (b) HVR-L2, the HVR-L2 comprising the amino acid sequence of SEQ ID NO: 38; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40, or the antibody comprises (a) HVR-L1, which HVR-L1 comprises the amino acid sequence of SEQ ID NO: 35; (b) HVR-L2, said HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

In another embodiment, the antibody comprises (a) HVR-L1, wherein HVR-L1 comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2, said HVR-L2 comprising the amino acid sequence of SEQ ID NO: 58; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12, or the antibody comprises (a) HVR-L1, which HVR-L1 comprises the amino acid sequence of SEQ id no: 7; (b) HVR-L2, the HVR-L2 comprising the amino acid sequence of SEQ ID NO: 9; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 65, or the antibody comprises (a) HVR-L1, which HVR-L1 comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2, the HVR-L2 comprising the amino acid sequence of SEQ ID NO: 59; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12, or the antibody comprises (a) HVR-L1, which HVR-L1 comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2, the HVR-L2 comprising the amino acid sequence of SEQ ID NO: 9; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 66, or the antibody comprises (a) HVR-L1, which HVR-L1 comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2, the HVR-L2 comprising amino acid sequence SEQ ID NO: 9; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 67, or the antibody comprises (a) HVR-L1, which HVR-L1 comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2, the HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60, adding a solvent to the mixture; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 67, or the antibody comprises (a) HVR-L1, which HVR-L1 comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2, the HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 65, or the antibody comprises (a) HVR-L1, which HVR-L1 comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2, the HVR-L2 comprising the amino acid sequence of SEQ ID NO: 59; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 66, or the antibody comprises (a) HVR-L1, which HVR-L1 comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2, said HVR-L2 comprising the amino acid sequence of SEQ ID NO: 62, a first step of mixing; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 67, or the antibody comprises (a) HVR-L1, the HVR-L1 comprising the amino acid sequence of SEQ id no: 7; (b) HVR-L2, the HVR-L2 comprising the amino acid sequence of SEQ ID NO: 63; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12, or the antibody comprises (a) HVR-L1, which HVR-L1 comprises the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2, the HVR-L2 comprising the amino acid sequence of SEQ ID NO: 64; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 12.

In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1, said HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 22, 23, 26, 28, 45, 68, 71-73 or 120(ii) HVR-H2, said HVR-H2 comprising an amino acid sequence selected from: SEQ ID NO: 24, 29, 46, 69, 74-78 or 121 and (iii) an HVR-H3, said HVR-H3 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 25, 30, 47, 70, 79 or 122; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1, the HVR-L1 comprising an amino acid sequence selected from: SEQ ID NO: 7, 8, 17, 35, or 42, (ii) HVR-L2, the HVR-L2 comprising an amino acid sequence selected from: SEQ ID NO: 9, 10, 18, 36-39, 41, 43, 56, 58-64, or 118, and (c) HVR-L3, said HVR-L3 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 11-16, 19, 40, 44, 57, 65-67, or 119.

In another aspect, the invention provides an antibody comprising (a) HVR-H1, which HVR-H1 comprises the amino acid sequence of SEQ ID NO: 23; (b) HVR-H2, said HVR-H2 comprising the amino acid sequence of SEQ ID NO: 24; (c) HVR-H3, said HVR-H3 comprising the amino acid sequence of SEQ ID NO: 25; (d) HVR-L1, the HVR-L1 comprising amino acid sequence SEQ ID NO: 7; (e) HVR-L2, the HVR-L2 comprising the amino acid sequence of SEQ ID NO: 9; and (f) HVR-L3, said HVR-L3 comprising an amino acid sequence selected from: SEQ ID NO: 12.

In certain embodiments, the antibody comprises at least one sequence selected from the group consisting of: HVR-H1, HVR-H2, HVR-H3, wherein HVR-H1 comprises the amino acid sequence GFX₃₀FX₃₁X₃₂X₃₃X₃₄IH (SEQ ID NO: 45), wherein X₃₀N or T; x₃₁(ii) S, L or Y; x₃₂G or Y; x₃₃Y or S; and X₃₄A, G or S; wherein HVR-H2 comprises amino acid sequence X₃₅X₃₆ISPX₃₇X₃₈GX₃₉TX₄₀YADSVKG (SEQ ID NO: 46), wherein X₃₅A or G; x₃₆W or S; x₃₇A or Y; x₃₈G or S;X₃₉(ii) S or Y; and X₄₀D or S; and wherein HVR-H3 comprises sequence X₄₁PX₄₂X₄₃X₄₄X₄₅X₄₆X₄₇MDY (SEQ ID NO: 47), wherein X₄₁Q or G; x₄₂T or F; x₄₃H or S; x₄₄Y or P; x₄₅Y or W; x₄₆Y or V, and wherein X₄₇Optionally including the sequence YAKGYKA (SEQ ID NO: 48).

In certain embodiments, the antibody comprises at least one sequence selected from the group consisting of: HVR-H1, HVR-H2 and HVR-H3, wherein HVR-H1 comprises the amino acid sequence GX₇₁X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇IH (SEQ ID NO: 120), wherein X₇₁F or Y; x₇₂F, N or T; x₇₃F or Y; x₇₄L, Q, I, S or Y; x₇₅G or Y; x₇₆Y or S; and X₇₇A, G or S; HVR-H2 comprises amino acid sequence X₇₈X₇₉ISPX₈₀X₈₁GX₈₂X₈₃X₈₄YADSVKG (SEQ ID NO: 121), wherein X₇₈A or G; x₇₉W or S; x₈₀A, S, Q or Y; x₈₁G or S; x₈₂(ii) S, K, L or Y; x ₈₃T or Y; and X₈₄D or S; and HVR-H3 comprises amino acid sequence X₈₅PX₈₆X₈₇X₈₈X₈₉X₉₀X₉₁MDY (SEQ ID NO: 122), where X₈₅Q or G; x₈₆T or F; x₈₇H, Y or S; x₈₈Y or P; x₈₉Y or W; x₉₀Y or V, and wherein X₉₁Optionally comprising the sequence YAKGYKA (SEQ ID NO: 48).

In certain embodiments, the antibody comprises at least one sequence selected from the group consisting of: HVR-L1, HVR-L2, HVR-L3, wherein HVR-L1 comprises the amino acid sequence RASQX₁₇VX₁₈X₁₉X₂₀X₂₁A, (SEQ ID NO: 42) wherein X₁₇(ii) S, D or V; x₁₈(ii) S or a; x₁₉(ii) S, T or N; x₂₀A or S; x₂₁Wherein HVR-L2 comprises the amino acid sequence X₂₂ASX₂₃LYS (SEQ ID NO: 43), wherein X₂₂(ii) S, W, Y or L; x₂₃F, S or W, and wherein HVR-L3 comprises the amino acid sequence QQX₂₄X₂₅X₂₆X₂₇X₂₈X₂₉T (SEQ ID NO: 44), wherein X₂₄(ii) S, F, G, D or Y; x₂₅Y, P, S or a; x₂₆Y, T or N; x₂₇T, Y, D or S; x₂₈P or L; and X₂₉F, P or T.

In certain embodiments, the antibody comprises at least one sequence selected from the group consisting of seq id no: HVR-L1, HVR-L2, and HVR-L3, wherein HVR-L1 comprises the amino acid sequence RASQX₁₇VX₁₈X₁₉X₂₀X₂₁A (SEQ ID NO: 42), wherein X₁₇(ii) S, D or V; x₁₈(ii) S or a; x₁₉(ii) S, T or N; x₂₀A or S; x₂₁Wherein HVR-L2 comprises the amino acid sequence X ₆₂ASX₆₃X₆₄YX₆₅(SEQ ID NO: 118) in which X₆₂S, W, Y, F or L; x₆₃F, S, Y or W; x₆₄L or R; x₆₅(iii) S, P, R, K or W, and HVR-L3 comprises the amino acid sequence QQX₆₆X₆₇X₆₈X₆₉X₇₀X₇₁T (SEQ ID NO: 119), wherein X₆₆(ii) S, F, G, D or Y; x₆₇Y, P, S or a; x₆₈Y, T or N; x₆₉T, Y, D or S; x₇₀P, Q, S, K or L; and X₇₁= F, P or T.

In certain embodiments, the antibody comprises at least one sequence selected from the group consisting of: HVR-L1, HVR-L2, HVR-L3, wherein HVR-L1 comprises the amino acid sequence RASQX₁VX₂X₃X₄X₅A (SEQ ID NO: 17), wherein X₁D or V; x₂(ii) S or a; x₃T or N; x₄(ii) S or a; x₅Either the sum of V or L,wherein HVR-L2 comprises amino acid sequence X₆ASFLYS (SEQ ID NO: 18), where X₆(ii) S or L and wherein HVR-L3 comprises the amino acid sequence QQX₇X₈X₉X₁₀X₁₁X₁₂T (SEQ ID NO: 19), wherein X₇(ii) S, F, G, D or Y; x₈Y, P, S, or a; x₉T or N; x₁₀T, Y, D or S; x₁₁P or L; x₁₂P or T.

In certain embodiments, the antibody comprises at least one sequence selected from the group consisting of: HVR-L1, HVR-L2, HVR-L3, wherein HVR-L1 comprises the amino acid sequence RASQX₁VX₂X₃X₄X₅A (SEQ ID NO: 17), wherein X₁D or V; x₂(ii) S or a; x₃T or N; x ₄(ii) S or a; x₅Wherein HVR-L2 comprises the amino acid sequence X₄₈ASX₄₉X₅₀YX₅₁(SEQ ID NO: 56), wherein X₄₈(ii) S or F; x₄₉F or Y; x₅₀L or R; x₅₁(iii) S, P, R, K or W, wherein HVR-L3 comprises the amino acid sequence QQFPTYX₅₂PT (SEQ ID NO: 57), wherein X₅₂L, Q, S or K.

In certain embodiments, the antibody comprises at least one sequence selected from the group consisting of: HVR-H1, HVR-H2, HVR-H3, wherein HVR-H1 comprises the amino acid sequence GFTFX₁₃GYX₁₄IH (SEQ ID NO: 26), wherein X₁₃Or L and X₁₄A or G, wherein HVR-H2 comprises amino acid sequence GWISPAGGSTDYADSVKG (SEQ ID NO: 24), and wherein HVR-H3 comprises amino acid sequence GPFSPWVMDY (SEQ ID NO: 25).

In certain embodiments, the antibody comprises at least one sequence selected from the group consisting of: HVR-H1, HVR-H2, HVR-H3, wherein HVR-H1 comprises the amino acid sequence GX₅₃X₅₄X₅₅X₅₆GYGIH (SEQ ID NO: 68), wherein X₅₃F or Y; x₅₄T or F; x₅₅F or Y; x₅₆Wherein HVR-H2 isContaining the amino acid sequence GWISPX₅₇X₅₈GX₅₉X₆₀DYASVKG (SEQ ID NO: 69), wherein X₅₇A, S or Q; x₅₈G or S; x₅₉S, K or L; x₆₀T or Y and wherein the HVR-H3 sequence comprises the amino acid sequence GPFX₆₁PWVMDY (SEQ ID NO: 70), wherein X ₆₁S or Y.

In certain embodiments, the antibody comprises at least one sequence selected from the group consisting of: HVR-L1, HVR-L2, HVR-L3, wherein HVR-L1 comprises amino acid sequence RASQSVSSAVA (SEQ ID NO: 35), wherein HVR-L2 comprises amino acid sequence X₁₅ASX₁₆LYS (SEQ ID NO: 41), wherein X₁₅Or Y and X₁₆And wherein HVR-L3 comprises amino acid sequence QQYSYSPFT (SEQ ID NO: 40).

In certain embodiments, any one or more amino acids of an anti-BACE 1 antibody as provided above is substituted at the following HVR positions:

in HVR-H1(SEQ ID NO: 26): positions 5 and 8;

in HVR-L1(SEQ ID NO: 17): positions 5, 7, 8, 9 and 10;

in HVR-L2(SEQ ID NO: 18): position 1, or in HVR-L2(SEQ ID NO: 41), positions 1 and 4; and

in HVR-L3(SEQ ID NO: 19): positions 3, 4, 5, 6, 7 and 8.

In certain embodiments, the substitutions are conservative substitutions, as provided herein. In certain embodiments, any one or more of the following substitutions may be made in any combination:

in HVR-H1(SEQ ID NO: 26): serine or leucine at position 5 and alanine or glycine at position 8;

In HVR-L1(SEQ ID NO: 17): aspartic acid or valine at position 5; serine or alanine at position 7; threonine or asparagine at position 8; serine or alanine at position 9 and valine or leucine at position 10;

in HVR-L2(SEQ ID NO: 18): serine or leucine at position 1, or serine, tyrosine or tryptophan at position 1 or tyrosine, serine or tryptophan at position 4 of HVR-L2(SEQ ID NO: 41); and

in HVR-L3(SEQ ID NO: 19): serine, phenylalanine, glycine, aspartic acid or tyrosine at position 3; tyrosine or proline at position 4, serine, alanine, threonine or asparagine at position 5; tyrosine, threonine, aspartic acid or serine at position 6, aspartic acid, serine, proline or leucine at position 7 and proline or threonine at position 8.

In certain embodiments, the substitutions are conservative substitutions, as provided herein. In certain embodiments, any one or more of the following substitutions may be made in any combination:

in HVR-H1(SEQ ID NO: 26): S5L and A8G;

in HVR-L1(SEQ ID NO: 17): D5V; S7A; T8N; S9A and V10L;

In HVR-L2(SEQ ID NO: 18): S1L, or in HVR-L2(SEQ ID NO: 41), positions S1W or Y and S4W; and

in HVR-L3(SEQ ID NO: 19): position S3F, G, D or Y1; Y4P, S or A; T5N; T6Y, D or S; P7L and P8T.

In certain embodiments, any one or more amino acids of an anti-BACE 1 antibody as provided above is substituted at the following HVR positions:

in HVR-H1(SEQ ID NO: 120): positions 2, 3, 5, 6, 7 and 8;

in HVR-H2(SEQ ID NO: 121): positions 1, 2, 6, 7, 9, 10, and 11;

in HVR-H3(SEQ ID NO: 122): positions 1, 3, 4, 5, 6, 7, and 8

In HVR-L1(SEQ ID NO: 42): positions 5, 7, 8, 9 and 10;

in HVR-L2(SEQ ID NO: 118): positions 1, 4, 5 and 7; and

in HVR-L3(SEQ ID NO: 119): positions 3, 4, 5, 6, 7 and 8.

In certain embodiments, the substitutions are conservative substitutions, as provided herein.

Possible combinations of the above substitutions are as described above for SEQ ID NO: 42-47 and 118-122.

In any of the above embodiments, an anti-BACE 1 antibody is humanized. In one embodiment, an anti-BACE 1 antibody comprises an HVR as in any of the above embodiments, and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In another embodiment, an anti-BACE 1 antibody comprises an HVR as in any of the above embodiments, and further comprises a VH or VL comprising the amino acid sequence of SEQ ID NO: 1-6, 20, 21, 27, 31-34, 80-98 and 99-117 of FR1, FR2, FR3, or FR4 sequences.

In another aspect, an anti-BACE 1 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: SEQ ID NO: 20, 21, 27 and 80-98. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, while an anti-BACE 1 antibody comprising this sequence retains the ability to bind BACE1 and/or inhibit or attenuate BACE1 activity. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO: 20, 21, 27 and 80-98. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-BACE 1 antibody comprises SEQ ID NO: 20, 21, 27 or 80-98, including post-translational modifications of said sequences. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1, said HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22, 23, 26, 28, 45, 68, 71, 72, 73, or 120, (b) HVR-H2, said HVR-H2 comprising the amino acid sequence of SEQ ID NO: 24, 29, 46, 69, 74, 75, 76, 77, 78, or 121, and (c) HVR-H3, said HVR-H3 comprising the amino acid sequence of SEQ ID NO: 25, 30, 47, 70, 79 or 122.

In one aspect, the invention provides an anti-BACE 1 antibody comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1, said HVR-H1 comprising the amino acid sequence in FIGS. 1(B), 2(B) and 24 (A); (b) HVR-H2, said HVR-H2 comprising the amino acid sequence in FIGS. 1(B), 2(B) and 24 (B); (c) HVR-H3, said HVR-H3 comprising the amino acid sequence in FIGS. 1(B), 2(B) and 24 (C); (d) HVR-L1, said HVR-L1 comprises the amino acid sequence in FIGS. 1(A), 2(A) and 23 (A); (e) HVR-L2, said HVR-L2 comprises the amino acid sequence in FIGS. 1(A), 2(A) and 23 (B); and (f) HVR-L3, said HVR-L3 comprising the amino acid sequence in FIGS. 1(A) and 2(A) and 23 (C).

In another aspect, an anti-BACE 1 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: SEQ ID NO: 1-6, 31-34 and 99-117. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to a reference sequence comprises a substitution (e.g., a conservative substitution), insertion, or deletion, but an anti-BACE 1 antibody comprising the sequence retains the ability to bind BACE1 and/or inhibit or reduce BACE1 activity. In certain embodiments, in SEQ ID NO: 1-6, 31-34 and 99-117 in total 1 to 10 amino acids are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-BACE 1 antibody comprises SEQ ID NO: 1-6, 31-34, or 99-117, including post-translational modifications of the sequences. In a particular embodiment, the VL comprises one, two or three HVRs selected from: (a) HVR-L1, the HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7, 8, 17, 35 or 42; (b) HVR-L2, the HVR-L2 comprising the amino acid sequence of SEQ ID NO: 9, 10, 18, 36-39, 41, 43 and 56, 58-64 or 118; and (c) HVR-L3, said HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11-16, 19, 40, 44, 57, 65, 66, 67, or 119.

In another aspect, an anti-BACE 1 antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises a heavy chain variable region comprising SEQ ID NOs: 21 and SEQ ID NO: 2, including post-translational modifications of said sequences.

In another aspect, the invention provides antibodies that bind to the same epitope as an anti-BACE 1 antibody provided herein. For example, in certain embodiments, antibodies are provided that bind to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21, 27 and 80-98 and a VH sequence selected from seq id NOs: 1-6, 31-34 and 99-117, and an anti-BACE 1 antibody. In certain embodiments, antibodies are provided that bind to a polypeptide comprising an amino acid sequence set forth in SEQ ID NOs: 21 and SEQ ID NO: 2 VH and VL sequences are the same epitope as anti-BACE 1 antibodies.

In certain embodiments, antibodies are provided that bind an epitope within BACE1 comprising at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine amino acids corresponding to the amino acids set forth in SEQ id no: 49 amino acids 314SER, 316GLU, 317LYS, 318PHE, 319PRO, 327GLN, 328LEU, 329VAL, 330CYS, 331TRP, 332GLN, 333ALA, 335THR, 337PRO, 340ILE, 375THR, 378ASP, 380CYS, 426 PHE.

In certain embodiments, antibodies are provided that bind an epitope within BACE1 comprising at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine amino acids corresponding to the amino acids set forth in SEQ id no: amino acid 314SER of 49; 316 GLU; 317 LYS; 327 GLN; 330 CYS; 331 TRP; 332 GLN; 335 THR; and 378 ASP. In other embodiments, the conformational epitope comprises amino acids corresponding to: SEQ ID NO: 314SER of 49; 316 GLU; 317 LYS; 327 GLN; 330 CYS; 331 TRP; 332 GLN; 335 THR; and 378 ASP. It is understood that the amino acids identified in the BACE1 epitope correspond to the sequence of human BACE1 isoform a. However, such BACE1 conformational epitopes contain the corresponding amino acids included in other variants and isoforms of BACE1, and such epitopes may include amino acids other than the specified residues.

In certain embodiments, antibodies are provided that bind an epitope within BACE1 comprising at least one, at least two, or at least three amino acid regions of BACE1 that correspond to the amino acid sequences of SEQ ID NOs: amino acids 315 of 49 and 318; SEQ ID NO: amino acids 331-335 of 49; SEQ ID NO: amino acids 370-381 of 49; or any combination thereof. In one embodiment, the antibody binds an epitope of BACE1 comprising the amino acid sequence of SEQ ID NO: amino acids 315, 331, 335 and 370, 381 of 49.

In another embodiment, antibodies are provided that bind an epitope within BACE1 that results in: conformational changes at the P6 and/or P7 sites of BACE1 relative to BACE1 not bound to the antibody after binding (Turner et al Biochemistry 44: 105-112 (2005)). In another embodiment, antibodies are provided that bind to an epitope of BACE1 that induces the activity of BACE1 of seq id NO: amino acids 218-231 of 49 adopt a random loop structure. SEQ ID NO of BACE 1: 49 amino acid 218-231 is present in the substrate-binding complex in an alpha-helical structure.

In another embodiment, antibodies are provided that bind to a site within BACE1, as shown in figures 14 and 15 and in the crystal structures of BACE1 and the anti-BACE 1 antibody, yw412.8.31 (example 3 (B)).

In other embodiments, antibodies are provided that bind to an external binding site within BACE 1. In one embodiment, the exosite within BACE1 binds to a binding site within Kornacker et al, biochem.44: 11567-11573(2005) the identified external binding sites are identical external binding sites. In one embodiment, antibodies are provided that bind to a peptide found in Kornacker et al, biochem.44: 11567-11573(2005) (which reference is incorporated herein in its entirety by reference), peptides identified in (i.e., peptides 1, 2, 3, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, 4, 5, 6, 5-10, 5-9, Y5A, P6A, Y7A, F8A, I9A, P10A, and L11A) compete for binding to BACE 1.

In another embodiment, antibodies are provided that compete for binding (e.g., bind to the same epitope) as any anti-BACE 1 antibody described herein.

In another aspect of the invention, an anti-BACE 1 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, the anti-BACE 1 antibody is an antibody fragment, e.g., Fv, Fab, Fab ', scFv, diabody, or F (ab')₂And (3) fragment. In another embodiment, the antibody is a full length antibody, e.g., a complete IgG1 antibody or other antibody class or isotype as defined herein.

In another aspect, an anti-BACE 1 antibody according to any of the above embodiments may bind any of the features as described in portions 1-7 below, alone or in combination:

1. antibody affinity

In certain embodiments, an antibody provided herein has a dissociation constant (Kd) of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, less than or equal to 0.1nM, less than or equal to 0.01nM, or less than or equal to 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M)。

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab form of the antibody of interest and its antigen, as described by the following assay. Solution binding avidity of Fab for antigen by titration series in the presence of unlabeled antigen with minimum concentration of ¹²⁵I) Labeled antigen-balanced Fab, followed by capture of bound antigen with anti-Fab antibody-coated plates (see, e.g., Chen et al, j.mol.biol.293: 865-881(1999)). To determine the conditions of the assay, theThe well plates (Thermo Scientific) were coated overnight with 5. mu.g/ml of capture anti-Fab antibody (Cappel Labs) in 50mM sodium carbonate (pH9.6) and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for 2-5 hours at room temperature (about 23 ℃). In the non-adsorption plate (Nunc #269620), 100pM or 26pM [ alpha ], [ beta ]¹²⁵I]Mixing of antigen with serial dilutions of Fab of interest (consistent with the evaluation of anti-VEGF antibody, Fab-12, in Presta et al, Cancer research (Cancer Res). 57: 4593-4599 (1997)). Subsequently, the Fab of interest was incubated overnight; however incubation may be continued for a longer period (e.g. 65 hours) to ensure that equilibrium is reached. Subsequently, the mixture is transferred to a capture plate for incubation at room temperature (e.g., 1 hour). Next, the solution was removed and the plate was used with 0.1% polysorbate 20 in PBSAnd washing for 8 times. When the plates had dried, 150. mu.l/well of scintillator (MICROSCINT-20) was added^TM(ii) a Packard) and placing the plate in TOPCOUNT^TMCount on a gamma counter (Packard) for 10 minutes. The concentration of each Fab that provides less than or equal to 20% of the maximum binding is selected for use in a competitive binding assay.

According to another embodiment, Kd is determined by surface plasmon resonanceOrThe instrument (BIAcore, inc., Piscataway, NJ) measured at 25 ℃ using an immobilized antigen CM5 chip at-10 Response Units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxy-succinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5. mu.g/ml (. about.0.2. mu.M) with 10mM sodium acetate pH4.8 and then injected at a flow rate of 5. mu.l/min to obtain about 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, a solution containing 0.05% polysorbate 20 (TWEEN-20) was injected at 25 ℃ at a flow rate of about 25. mu.l/min^TM) Two-fold serial dilutions of Fab (0.78nM to 500nM) in surfactant pbs (pbst). Using a simple one-to-one Langmuir (Langmuir) binding model (Evaluation Software version3.2) calculation of the Association Rate (k) by Simultaneous fitting of Association and dissociation sensorgrams_on) And dissociation rate (k)_off). Equilibrium dissociation constant (Kd) in the ratio k _off/k_onAnd (4) calculating. See, e.g., Chen et al, j.mol.biol. (journal of molecular biology) 293: 865-881(1999). If according to the above surface plasmon resonance determination, the binding rate exceeds 10⁶M^-1s^-1The rate of binding can then be determined using fluorescence quenching techniques, i.e.according to a spectrometer such as an Aviv Instruments spectrophotometer or 8000 series SLM-AMINCO^TMMeasurement in a spectrophotometer (ThermoSpectronic) with a stirred cuvette (strained cuvette) in the presence of increasing concentrations of antigen, the increase or decrease in fluorescence emission intensity (295 nM for excitation; 340nM for emission; 16nM band pass) at 25 ℃ of 20nM anti-antigen antibody (Fab form) in PBS, pH7.2 was measuredLow.

2. Antibody fragments

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab ', Fab ' -SH, F (ab ')₂Fv, and scFv fragments, as well as other fragments described below. For a review of specific antibody fragments, see Hudson et al nat. med.9: 129-134(2003). For a review of scFv fragments, see, e.g., Pluckth ü n, the Pharmacology of monoclonal Antibodies (pharmacology of monoclonal Antibodies), Vol 113, edited by Rosenburg and Moore, (Springer-Verlag, New York), p.269-315 (1994); see also WO 93/16185; and U.S. patent nos. 5,571,894 and 5,587,458. For Fab and F (ab') containing rescue receptor (sample receptor) binding epitope residues and having increased in vivo half-life ₂See U.S. Pat. No. 5,869,046 for a discussion of fragments.

Diabodies are antibody fragments with two antigen binding sites, which may be bivalent or bispecific. See, for example, EP404,097; WO 1993/01161; hudson et al, nat. med.9: 129-134 (2003); and Hollinger et al, proc.natl.acad.sci.usa90: 6444-6448(1993). Triabodies and tetrabodies are also described in Hudson et al, nat. med.9: 129-134 (2003).

Single domain antibodies are antibody fragments that comprise all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B 1).

Antibody fragments can be prepared by different techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E.coli or phage), as described herein.

3. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. nos. 4,816,567; and Morrison et al, proc.natl.acad.sci.usa, 81: 6851 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class switch" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans, while maintaining the specificity and affinity of the parent non-human antibody. In general, humanized antibodies comprise one or more variable domains in which HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their preparation are described, for example, in Almagro and Fransson, front.biosci.13: 1619-: 323-329 (1988); queen et al, proc.natl.acad.sci.usa86: 10029-10033 (1989); U.S. Pat. nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; kashmiri et al, Methods (Methods) 36: 25-34(2005) (describing SDR (a-CDR) grafting) Padlan, mol. immunol. (molecular immunology) 28: 489-498(1991) (description "surface reforming"); dall' Acqua et al, Methods 36: 43-60(2005) (description "FR recombination (shuffling)"); and Osbourn et al, Methods 36: 61-68(2005) and Klimka et al, Br.J. cancer (J. cancer, UK), 83: 252-260(2000) (describing the "guided selection" method of FR recombination (shuffling)).

Human framework regions that may be used for humanization include, but are not limited to, framework regions selected using a "best fit" approach (see, e.g., Sims et al, J.Immunol. (J.Immunol.) 151: 2296 (1993); the framework regions of the human antibody common sequences derived from light or heavy chain variable regions of a particular subgroup (see, e.g., Carter et al, Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta et al, J.Immunol. (J.Immunol), 151: 2623 (1993); human mature (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, front.biosci.13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al, J.biol. chem. (J.Biol.Chem.) 272: 10678-10684(1997) and Rosok et al, J.biol. chem. (J.Biol.Chem.) 271: 22611-22618 (1996)).

4. Human antibodies

In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be made using various techniques known in the art. Human antibodies are generally described in van Dijk and van deWinkel, curr. 368-74(2001) and Lonberg, curr. opin. immunol. (current immunological view) 20: 450-459(2008).

Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or fully antibodies with human variable regions in response to an antigen challenge stimulus. These animals typically contain all or a portion of the human immunoglobulin locus, either in place of the endogenous immunoglobulin locus, or extrachromosomally or randomly integrated into the animal chromosome. In these transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For an overview of the method of obtaining human antibodies from transgenic animals, see Lonberg, nat. biotech, (natural biotechnology) 23: 1117-1125(2005). See also, for example, the description XENOMOUSE^TMU.S. Pat. nos. 6,075,181 and 6,150,584 of the art; description of the inventionU.S. patent No. 5,770,429 for technology; description of K-MU.S. Pat. No. 7,041,870 to Art, and descriptionU.S. patent application publication No. US2007/0061900 of the art. The human variable regions of the intact antibodies produced by these animals may be further modified, for example by combination with different human constant regions.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human fusion myeloma cell lines for the production of human monoclonal antibodies have been described. (see, e.g., Kozbor J.Immunol. (J. Immunol.), 133: 3001 (1984); Brodeur et al, monoclonal antibody Production Techniques and Applications, pp 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J.Immunol. (J. Immunol., 147: 86 (1991).) human antibodies produced by human B-cell hybridoma technology are also described in Li et al, Proc.Natl.Acad.Sci., USA, 103: 3557 and 3562 (2006). Other methods include, for example, U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26 (4): 265-268(2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) also available from Vollmers and Brandlein, Histology and Histopathology, 20 (3): 927-937(2005), and Vollmers and Brandlein, Methods and standards in Experimental and Clinical Pharmacology (Experimental and Clinical Methods and findings), 27 (3): 185-91 (2005).

Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from a human phage display library. These variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

5. Antibodies derived from libraries

Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies having the desired activity. For example, various methods are known in the art for generating phage display libraries and screening these libraries for antibodies with desired binding characteristics. These Methods are described, for example, in Hoogenboom et al, Methods in Molecular Biology 178: 1-37 (compiled by O' Brien et al, human Press, Totowa, NJ, 2001), and further compared to, for example, McCafferty et al, Nature 348: 552 and 554; clackso et al, Nature (Nature) 352: 624-628 (1991); marks et al, j.mol.biol. (journal of molecular biology) 222: 581-597 (1992); marks and Bradbury, Methods in Molecular Biology 248: 161-175(Lo eds., Press, Totowa, NJ, 2003); sidhu et al, j.mol.biol. (journal of molecular biology) 338 (2): 299-310 (2004); lee et al, j.mol.biol. (journal of molecular biology) 340 (5): 1073-1093 (2004); fellouse, proc.natl.acad.sci.usa101 (34): 12467-12472 (2004); and Lee et al, j.immunol.methods 284 (1-2): 119, and 132 (2004).

In some phage display methods, VH and VL gene banks (rettoire) are separately cloned by Polymerase Chain Reaction (PCR) and randomly recombined into phage libraries, which can then be used, for example, by Winter et al, ann. 433-455(1994) where antigen-binding phages were selected. The phage typically display antibody fragments in the form of single chain fv (scFv) fragments or Fab fragments. Libraries from immune sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, natural libraries can be cloned (e.g., from humans) to provide a single source of antibodies to multiple non-self antigens as well as self antigens without any immunization, such as Griffiths et al, EMBO J, 12: 725, 734 (1993). Finally, natural libraries can also be synthetically produced by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions and to effect in vitro rearrangement, such as Hoogenboom and Winter, j.mol.biol. (journal of molecular biology), 227: 381 and 388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and U.S. patent publication nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936 and 2009/0002360.

Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments herein.

6. Multispecific antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one binding specificity is for BACE1 and the other is for any other antigen. In certain embodiments, a bispecific antibody may bind to two different epitopes of BACE 1. Bispecific antibodies may also be used to localize cytotoxic agents to cells expressing BACE 1. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature (Nature) 305: 537 (1983); WO 93/08829; and Traunecker et al, EMBO J. 10: 3655(1991)), and "bump-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be made by: electrostatic guidance engineered for the preparation of antibody Fc-heterodimers (WO2009/089004a 1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al Science, 229: 81 (1985)); the use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al, J.Immunol. (J.Immunol.) (148 (5): 1547-1553 (1992)); bispecific antibody fragments were prepared using the "diabody" technique (see, e.g., Hollinger et al, Proc. Natl. Acad. Sci. USA, 90: 6444-; and the use of single chain fv (sFv) dimers (see, e.g., Gruber et al, J. Immunol. (J. Immunol.), 152: 5368 (1994); and as described, for example, by Tutt et al, j.immunol. (journal of immunology) 147: 60(1991) the trispecific antibody is prepared as described.

Also included herein are engineered antibodies having three or more functional antigen binding sites, including "Octopus antibodies" (see, e.g., US2006/0025576a 1).

Antibodies or fragments herein also include "dual action fabs or" DAFs "comprising an antigen binding site that binds to a BACEl and another different antigen (see, e.g., US 2008/0069820).

7. Antibody variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to increase the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications in the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

a) Substitution, insertion and deletion variants

In certain embodiments, antibody variants are provided having one or more amino acid substitutions. Relevant sites for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in table 1 under the heading "conservative substitutions". More substantial changes are provided under the heading of "exemplary substitutions" in table 1 and are described further below with respect to amino acid side chain species. Amino acid substitutions may be introduced into the relevant antibodies and screened for products having a desired activity, e.g., maintained/increased antigen binding, reduced immunogenicity, or increased ADCC or CDC.

TABLE 1

Amino acids can be grouped according to common side chain properties:

(1) hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilicity: cys, Ser, Thr, Asn, Gin;

(3) acidity: asp and Glu;

(4) alkalinity: his, Lys, Arg;

(5) residues that influence chain orientation: gly, Pro;

(6) aromaticity: trp, Tyr, Phe.

Non-conservative substitutions will require the exchange of a member of one of these classes for another.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). In general, certain biological properties of the resulting variants selected for further study are altered (e.g., improved) relative to the parent antibody (e.g., increased affinity, decreased immunogenicity) and/or certain biological properties of the parent antibody will be substantially maintained. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more HVR residues are mutated, and variant antibodies are presented on phage and screened for a particular biological activity (e.g., binding affinity).

Alterations (e.g., substitutions) may be made in HVRs, for example, to increase antibody affinity. These changes can be made at HVR "hot spots", i.e., residues encoded by codons that undergo high frequency mutations during somatic maturation (see, e.g., Chowdhury, Methods mol. biol. (Methods of molecular biology) 207: 179. 196 (2008)); and/or in SDRs (a-CDRs), wherein the resulting variant VH or VL is tested for binding affinity. Affinity maturation obtained by construction and re-selection from a second library has been described, for example, in Hoogenboom et al, Methods in Molecular Biology 178: 1-37 (O' Brien et al eds Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods, such as error-prone PCR (error-prone PCR), strand recombination (shuffling), or oligonucleotide-directed mutagenesis. A second library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves HVR targeting methods, in which several HVR residues are randomly selected (e.g., 4-6 residues at a time). HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. Specifically, CDR-H3 and CDR-L3 are commonly targeted.

In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made in HVRs. These changes may be outside of the HVR "hot spot" or SDR. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unaltered, or contains no more than one, two, or three amino acid substitutions.

Suitable methods for identifying residues or regions of an antibody that can be targeted for mutagenesis are referred to as "alanine scanning mutagenesis" such as Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether to affect the interaction of the antibody with the antigen. Additional substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. These contact residues and adjacent residues may be targeted or eliminated as replacement candidates. Variants can be screened to determine if they contain the desired property.

Amino acid sequence inserts include amino-terminal and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence inserts of single or multiple amino acid residues. Examples of terminal inserts include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusions of the N-or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

b) Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites of an antibody can be conveniently achieved by altering the amino acid sequence so as to create or remove one or more glycosylation sites.

If the antibody comprises an Fc region, the carbohydrate to which it is attached may be altered. Natural antibodies produced by mammalian cells typically comprise a branched chain bi-antennary oligosaccharide typically linked by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al, TIBTECH 15: 26-32(1997). Oligosaccharides may include a variety of saccharides, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention may be modified to produce antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a sugar structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in the antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose at Asn297 within the sugar chain relative to the sum of all sugar structures linked to Asn297 (e.g. complex, hybrid and high mannose type structures) measured according to MALDI-TOF mass spectrometry, e.g. as described in WO 2008/077546. Asn297 refers to the asparagine residue at about position 297 in the Fc region (Eu numbering of Fc region residues); however, due to minor sequence variations in the antibody, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300. These fucosylated variants may have an improved ADCC function. See, e.g., U.S. patent publication No. US2003/0157108(Presta, L.); US2004/0093621(Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosyl" or "fucose-deficient" antibody variants include US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; okazaki et al, j.mol.biol. (journal of molecular biology) 336: 1239-1249 (2004); yamane-ohniki et al, biotech.bioeng, (biotechnology and bioengineering) 87: 614(2004). Examples of cell lines capable of producing antibodies that are fucosylated include Lec13CHO cells deficient in protein fucosylation (Ripka et Al Arch. biochem. Biophys. 249: 533-545 (1986); U.S. patent application Nos. US2003/0157108Al, Presta, L; and WO2004/056312Al, Adams et Al, example 11, inter alia); and gene knockout cell lines, such as alpha-1, 6-fucosyltransferase genes, FUT8, gene knockout CHO cells (see, e.g., Yamane-Ohnuki et al, Biotech.Bioeng. (Biotechnology and bioengineering) 87: 614 (2004); Kanda, Y. et al, Biotechnol.Bioeng. (Biotechnology and bioengineering), 94 (4): 680-688 (2006); and WO 2003/085107).

Antibody variants having bisected oligosaccharides (biseculated oligosaccharides) are further provided, for example, wherein biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. These antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003/011878(Jean-Mairet et al); U.S. Pat. No. 6,602,684 (Umana et al); and US2005/0123546(Umana et al). Antibody variants having at least one galactose residue in an oligosaccharide linked to an Fc region are also provided. These antibody variants may have increased CDC function. Such antibody variants are described, for example, in WO1997/30087(Patel et al); WO1998/58964(Raju, S.); and WO1999/22764(Raju, S.).

c) Fc region variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to produce Fc region variants, in order to enhance, for example, the effectiveness of the antibody to treat a disease or disorder involving aberrant angiogenesis and/or vascular permeability or leakage. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In certain embodiments, the invention encompasses antibody variants with some, but not all, effector functions, which make them ideal candidates for applications in which the in vivo half-life of the antibody is important, but certain effector functions (e.g., complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays may be performed to confirm the reduction/failure of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks FcyR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Primary cells, NK cells, that mediate ADCC express FcyRIII only, whereas monocytes express Fc γ RI, Fc γ RII and FcyRIII. FcR expression on hematopoietic cells is described in ravatch and Kinet, annu.rev.immunol. (annual review in immunology) 9: 457-492(1991)Summarized in table 3 on page 464. Non-limiting examples of in vitro assays to assess ADCC activity of related molecules are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, i.e., proc.natl.acad.sci.usa 83: 7059-7063(1986)) and Hellstrom, i.e., proc.natl.acad.sci.usa 82: 1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al, J.Exp. Med. (J.Exp. Med.) 166: 1351-. Alternatively, non-radioactive assay methods can be employed (see, e.g., ACTI for flow cytometry) ^TMNonradioactive cytotoxicity assay (Celltechnology, Inc. mountain View, Calif.) andnon-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells suitable for use in these assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of the relevant molecule can be assessed in vivo, for example in the methods described in Clynes et al, proc.natl.acad.sci.usas 95: 652-. Clq binding assays may also be performed to demonstrate that antibodies are unable to bind Clq and therefore lack CDC activity. See, e.g., Clq and C3C binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al, J.Immunol. methods 202: 163 (1996); Cragg, M.S. et al, Blood 101: 1045-1052 (2003); and Cragg, M.S. and M.J.Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., Petkova, s.b. et al, intl.immunol. (international immunology) 18 (12): 1759-.

Antibodies with reduced effector function include those with substitutions in one or more of residues 238, 265, 269, 270, 297, 327 and 329 of the Fc region (U.S. patent 6,737,056). These Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

Certain antibody variants with increased or decreased binding to FcRs are described. (see, e.g., U.S. Pat. No. 6,737,056; WO2004/056312, and Shields et al, J.biol.chem. (J.Biol.Chem.) 9 (2): 6591-6604 (2001)).

In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, for example at positions 298, 333, and/or 334 (EU numbering of residues) of the Fc region.

In some embodiments, alterations are made in the Fc region that result in altered Clq binding and/or Complement Dependent Cytotoxicity (CDC) (i.e., by increasing or decreasing), for example as described in U.S. patent nos. 6, 194,551, WO99/51642 and Idusogie et al, j.immunol. (journal of immunology) 164: 4178 (2000).

Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn) responsible for the transfer of maternal IgG to the fetus are described in US2005/0014934A1(Hinto et al) (Guyer et al J.Immunol. (J.Immunol.) 117: 587(1976) and Kim et al J.Immunol. (J.Immunol.) 24: 249 (1994)). Those antibodies comprise an Fc region having one or more substitutions that increase binding of the Fc region to FcRn. These Fc variants include those having a substitution at one or more of residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434 of the Fc region (e.g., substitution of residue 434 of the Fc region) (U.S. patent No. 7,371,826).

For further examples of Fc region variants, see also Duncan and Winter, Nature 322: 738-40 (1988); U.S. Pat. nos. 5,648,260; U.S. Pat. nos. 5,624,821; and WO 94/29351.

d) Cysteine engineered antibody variants

In certain embodiments, it may be desirable to generate cysteine engineered antibodies, such as "thio mabs," in which one or more residues of the antibody are replaced with a cysteine residue. In particular embodiments, the substituted residue occurs at an accessible site of the antibody. By replacing those residues with cysteine, the reactive thiol group is accessible by localization at an antibody site and can be used to bind the antibody to other moieties, such as a drug moiety or linker-drug moiety, to produce an immunoconjugate as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: v205 of the light chain (Kabat numbering); a118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). Cysteine engineered antibodies can be produced as described, for example, in U.S. patent No. 7,521,541.

e) Antibody derivatives

In certain embodiments, the antibodies provided herein can be further modified to contain other non-protein moieties known and readily available in the art. Suitable antibody-derived moieties include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-di-poly-1-co-propylene glycol Alkane, poly-1, 3, 6-trioxaneAlkanes, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, it can be the same or different molecules. In general, for derivatizingThe number and/or type of polymers acted upon can be determined based on considerations including, but not limited to, the following: the specific properties or functions of the antibody to be improved, whether the antibody derivative is to be used in therapy under specified conditions, etc.

In another embodiment, conjugates of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al, Proc. Natl. Acad. Sci. USA 102: 11600-. The radiation can be of any wavelength, and includes, but is not limited to, wavelengths that do not damage normal cells but are capable of heating the non-protein portion to a temperature that can kill cells in the vicinity of the antibody-non-protein portion.

B. Recombinant methods and compositions

Antibodies can be produced using recombinant methods and compositions such as those described in U.S. Pat. No. 4,816,567. In one embodiment, isolated nucleic acids encoding anti-BACE 2 antibodies described herein are provided. Such nucleic acids may encode an amino acid sequence comprising a VL of an antibody and/or an amino acid sequence comprising a VH thereof (e.g., a light chain and/or a heavy chain of an antibody). In yet another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In yet another embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., is transformed with): (1) a vector comprising nucleic acids encoding an amino acid sequence comprising a VL of an antibody and an amino acid sequence comprising a VH of an antibody, or (2) a first vector comprising nucleic acids encoding an amino acid sequence comprising a VL of an antibody, and a second vector comprising nucleic acids encoding an amino acid sequence comprising a VH of an antibody. In one embodiment, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making an anti-BACE 2 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as provided above, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of anti-BACE 2 antibodies, nucleic acids encoding the antibodies (e.g., the antibodies described above) are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of an antibody).

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5,648,237,5,789,199, and 5,840,523. (see also Charlton, Methods in Molecular Biology, Vol.248 (B.K.C.Lo, eds., human a Press, Totowa, NJ, 2003), pp.245-. After expression, the antibody can be isolated from the bacterial cell paste in the soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains, whose glycosylation pathways have been "humanized" resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, nat. biotech. (natural biotechnology) 22: 1409-: 210-215(2006).

Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified which can be used in combination with insect cells, particularly for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells.

Plant cell cultures may also be used as hosts. See, for example, U.S. Pat. Nos. 5,959,177,6,040,498,6,420,548,7,125,978 and 6,417,429 (which describe PLANTIBODIIES that produce antibodies in transgenic plants^TMA technique).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered to be suitable for growth in suspension may be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (293 or 293 cells, as described, for example, in Graham et al, J.Gen Virol.) 36: 59 (1977); baby hamster kidney cells (BHK); mouse Sertoli (sertoli) cells (TM4 cells, as described, for example, in Mather, biol. reprod.23: 243-251 (1980)); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat (buffalo rat) hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as for example Mather et al, Annals n.y. acad.sci.383: 44-68 (1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, Proc. Natl.Acad.Sci.USA) 77: 4216 (1980); and myeloma cell lines such as Y0, NS0 and Sp 2/0. For a review of certain mammalian host cell lines suitable for antibody production see, e.g., Yazaki and Wu, Methods in Molecular Biology (Methods in Molecular Biology), Vol.248 (B.K.C.Lo, ed., human a Press, Totowa, NJ), pp.255-268 (2003).

C. Measurement of

anti-BACE 1 antibodies provided herein can be identified, screened for, or characterized for their physical/chemical properties and/or biological activity by various assays known in the art.

1. Binding assays and other assays

In one aspect, the antibodies of the invention are tested for antigen binding activity, e.g., by known methods such as ELISA, western blot, and the like.

In another aspect, a competition assay can be used to identify antibodies that compete for binding to BACE1 with any of the antibodies or Fabs described herein, e.g., YW412.8, yw412.8.31, yw412.8.30, yw412.8.2, yw412.8.29, yw412.8.51, Fab12, LC6, LC9, LC 10. In certain embodiments, such a competitive antibody binds to the same epitope (e.g., a linear or conformational epitope) bound by any of the antibodies or Fabs described herein (e.g., YW412.8, YW412.8.31, YW412.8.30, YW412.8.2, YW412.8.29, YW412.8.51, Fab12, LC6, LC9, LC 10). Detailed exemplary Methods for locating which Epitope an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," Methods in molecular biology volume 66 (human a Press, Totowa, NJ).

In an exemplary competition assay, immobilized BACE1 is incubated in a solution comprising a first labeled antibody that binds BACE1 (e.g., YW412.8, yw412.8.31, yw412.8.30, yw412.8.2, yw412.8.29, yw412.8.51, Fab12, LC6, LC9, LC10) as a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding BACE 1. The second antibody may be present in the hybridoma supernatant. As a control, immobilized BACE1 was incubated in a solution comprising a first labeled antibody but no second unlabeled antibody. After incubation under conditions that allow the first antibody to bind BACE1, excess unbound antibody is removed and the amount of label bound to immobilized BACE1 is measured. If the amount of label bound to immobilized BACE1 is significantly reduced in the test sample relative to the control sample, this indicates that the second antibody competes with the first antibody for binding to BACE 1. See Harlow and Lane (1988) Antibodies: laboratory Manual (antibodies: Laboratory Manual) ch.14(Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

2. Activity assay

In one aspect, assays are provided for identifying anti-BACE 1 antibodies that have biological activity. Biological activities may include, for example, inhibiting or reducing BACE1 aspartyl protease activity; or inhibiting or reducing BACE1 cleavage of APP; or inhibit or reduce a β production. Antibodies having such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, such biological activity of an antibody of the invention is determined. For example, BACE1 protease activity can be tested in homogeneous time-resolved fluorescence HTRF assays or Microfluidic Capillary Electrophoresis (MCE) assays (as detailed in examples 1 and 2 (B)) using synthetic substrate peptides.

Briefly, a homogeneous time-resolved fluorescence (HTRF) assay may be used to measure BACE1 aspartyl protease activity, using the amyloid precursor protein BACE1 cleavage site peptide. For example, a Bi27 Peptide (Biotin-KTEEISEVNLDAEFRHDSGYEVHHQKL (SEQ ID NO: 53), American Peptide Company)) was combined with an anti-BACE antibody in a BACE reaction buffer (50mM sodium acetate pH4.4 and 0.1% CHAPS) in 384-well plates (Proxiplate)^TMPerkin-Elmer) was used. The proteolytic reaction mixture was incubated at ambient temperature for 75 minutes and quenched by the addition of 5 μ L of an HTRF detection mixture containing 2nM streptavidin-D2 and 150nM anti-amyloid β antibody labeled with europium cryptate in detection buffer (200mM Tris ph8.0, 20mM edta, 0.1% BSA, and 0.8M KF). The final reaction mixture was incubated at ambient temperature for 60 minutes and an EnVision Multilabel Plate Reader was used ^TM(Perkin-Elmer) the TR-FRET signal was measured at an excitation wavelength of 320nm and emission wavelengths of 615 and 665 nm.

The MCE assay reaction can be carried out in a standard enzymatic reaction, starting with the addition of substrate to the enzyme and 4X compound containing human BACE1 (extracellular domain), amyloid precursor protein beta secretase active site peptide (FAM-KTEEISEVNLDAEFRWKK-CONH2(SEQ ID NO: 55)), 50mM NaOAc pH4.4 and 0.1% CHAPS. After incubation at ambient temperature for 60 minutes, each reaction was runThe product in (1) is separated from the substrate using a 12-suction (sipper) microfluidic chip in(both from Caliper Life Sciences). The separation of the product from the substrate was optimized by selecting the voltage and pressure using the manufacturer's optimization software. Substrate conversion was calculated from the electropherograms using HTS Well Analyzer software (Caliper Life Sciences).

Furthermore, BACE1 protease activity can be tested in vivo in cell lines expressing a BACE1 substrate, such as APP, or in transgenic mice expressing a BACE1 substrate, such as human APP, as described in examples 2(C) and 4.

In addition, BACE1 protease activity can be tested in animal models using anti-BACE 1 antibodies. For example, animal models of various neurological diseases and disorders, as well as related techniques for examining the pathological processes associated with these models, are readily available in the art. Animal models of various neurological diseases include non-recombinant and recombinant (transgenic) animals. Non-recombinant animal models include, for example, rodent, e.g., murine models. Such models can be established by introducing cells into syngeneic mice using standard techniques such as subcutaneous injection, tail vein injection, spleen transplantation, intraperitoneal transplantation, and transplantation under the renal capsule. In vivo models include models of stroke/cerebral ischemia, in vivo models of neurodegenerative diseases, such as mouse model of parkinson's disease; a mouse model of alzheimer's disease; a mouse model of amyotrophic lateral sclerosis (amyotrophic lateral sclerosis); mouse models of spinal muscular atrophy (spinal muscular atrophy); mouse/rat models of local (focal) and global (global) cerebral ischemia, e.g., models of common carotid artery occlusion or middle cerebral artery occlusion; or in whole embryo cultures ex vivo (ex vivo). As a non-limiting example, there are a number of mouse models of Alzheimer's disease known in the art ((see, e.g., Rakover et al, neurogene. Dis. (2007); 4 (5): 392 402; Mouri et al, FASEB J. (2007) Jul; 21 (9): 2135-48; Minkevicine et al, J. Pharmacol. exp. Ther. (2004) Nov; 311 (2): 677-82 and Yuede et al, BehavPharmacol. (2007) Sep; 18 (5-6): 347-63.) various such assays can be performed in known in vitro or in vivo assay modes, as known in the art and described in the literature.various such assays are also available from manufacturers such as Jackson in animal models, examples 4 and 5.

D. Immunoconjugates

The present invention also provides immunoconjugates comprising an anti-BACE 1 antibody herein conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof)) or a radioisotope.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs including, but not limited to, maytansinoids (see U.S. Pat. nos. 5,208,020, 5,416,064, and european patent EP0425235B 1); orlistatin (auristatin), such as monomethyl orlistatin drugs (monomethylauristatin) part DE and DF (MMAE and MMAF) (see U.S. Pat. nos. 5,635,483 and 5,780,588, and 7,498,298); dolastatin (dolastatin); calicheamicin (calicheamicin) or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al, Cancer Res. (Cancer Res.) 53: 3336-3342 (1993); and Lode et al, Cancer Res. (Cancer Res.) 58: 2925-2928 (1998)); anthracyclines such as daunomycin (daunomycin) or doxorubicin (see Kratz et al Current Med. chem. 13: 477-; methotrexate (methotrexate); vindesine (vindesine); taxanes (taxanes) such as docetaxel (docetaxel), paclitaxel (paclitaxel), larotaxel (larotaxel), docetaxel (tesetaxel) and docetaxel (ortataxel); trichothecene compounds (trichothecene); and CC 1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria (diphtheria) a chain, a non-binding active fragment of diphtheria toxin, exotoxin a chain (from Pseudomonas aeruginosa), ricin a chain, abrin a chain, modeccin a chain, alpha-sarcin (sarcin), aleurites fordii protein, dianthin protein (dianthin protein), phytolacca americana protein (PAPI, PAPII and PAP-S), Momordica charantia (mormocardia) inhibitor, curcin (curcin), crotin (crotin), saponaria officinalis (sapiensis), saponaria officinalis (PAPII) protein (PAPII and PAP-S), trichoderma viridin (trichoderma viride) inhibitor, curcin (curcin), trichoderma viridin (trichoderma), trichoderma viridin (trichoderma) inhibitor, trichoderma viridin (trichoderma viridin), trichoderma viridin (tricho, Phenomycin, enomycin and trichothecenes (trichothecenes).

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for making radioconjugates. Examples include At ²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、pb²¹²And radioactive isotopes of Lu. When a radioconjugate is used for detection, it may contain radioactive atoms for scintigraphic studies, for example tc99m or I123 or spin labels for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 and iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of the antibody with cytotoxic agents can be made using a variety of bifunctional protein coupling agents, for example, N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), imidoesters (such as dimethyladipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) hexanediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) is used. For example, a ricin immunotoxin may be identified as vietta et al, Science 238: 1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for binding radionucleotide to the antibody. See WO 94/11026. The linker may be a "cleavable linker" that facilitates the release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, Cancer Res. 52: 127-131 (1992); U.S. Pat. No. 5,208,020).

Immunoconjugates or ADCs herein expressly encompass, but are not limited to, such conjugates prepared with crosslinker agents including, but not limited to: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, thio-EMCS, thio-GMBS, thio-KMUS, thio-MBS, thio-SIAB, thio-SMCC, and thio-SMPB and SVSB (succinimidyl- (4-vinylsulfone) benzoate), crosslinker reagents are commercially available (e.g., from Pierce Biotechnology, inc., Rockford, il., u.s.a.).

E. Methods and compositions for diagnosis and detection

In certain embodiments, any of the anti-BACE 1 antibodies provided herein can be used to detect the presence of BACE1 in a biological sample. The term "detecting" as used herein includes quantitative or qualitative detection. In certain embodiments, the biological sample comprises a cell or tissue, such as serum, plasma, saliva, gastric secretions, mucus, cerebrospinal fluid, lymph fluid, neuronal tissue, brain tissue, cardiac tissue, or vascular tissue.

In one embodiment, anti-BACE 1 antibodies are provided for use in diagnostic or detection methods. In another aspect, methods are provided for detecting the presence of BACE1 in a biological sample. In certain embodiments, the methods comprise contacting a biological sample with an anti-BACE 1 antibody as described herein under conditions that allow binding of the anti-BACE 1 antibody to BACE1 and detecting whether a complex is formed between the anti-BACE 1 antibody and BACE 1. The method may be an in vitro or in vivo method. In one embodiment, an anti-BACE 1 antibody is used to select a subject suitable for treatment with an anti-BACE 1 antibody, e.g., wherein BACE1 is a biomarker for selecting patients.

Exemplary disorders that can be diagnosed using the antibodies of the invention include neurodegenerative diseases (including, but not limited to, Lewy body disease, post-polio syndrome, Chari-Delerger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatal substantia nigra degeneration, tauopathy (tauopathy) (including, but not limited to, Alzheimer's disease and supranuclear palsy), prion diseases (priondises) (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob disease, Kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), stroke, muscular dystrophy, multiple sclerosis, Amyotrophic Lateral Sclerosis (ALS), Angelman syndrome, Reed's syndrome, Paget's syndrome, traumatic brain injury, bulbar palsy, motor neuron disease, and nervous system degenerative (heterogenic) disorders (including, but not limited to, kanan's disease, huntington's disease, neuronal ceroid lipofuscinosis, alexander disease, tourette's syndrome, menkes kink syndrome, cockayne's syndrome, hallrewden-spatz syndrome, lafford's disease, rett syndrome, hepatolenticular degeneration, lesch-nyan syndrome, and pulsatile-long syndrome), dementia (including, but not limited to, pick's disease, and spinocerebellar ataxia).

In certain embodiments, labeled anti-BACE 1 antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and moieties that are detected indirectly, such as enzymes or ligands, for example, by enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes³²p，¹⁴C，¹²⁵I，³H, and¹³¹fluorophores such as rare earth chelates or luciferin and its derivatives, rhodamine and its derivatives, dansyl (dansyl), umbelliferone (umbelliferone), luciferase (luceriferase), for example, firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin (luciferin), 2, 3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, beta-galactosidase, glucoamylase, lytic enzymes, carbohydrate oxidases, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, plus enzymes that oxidize dye precursors using hydrogen peroxide such as HR, lactoperoxidase, or microperoxidase (microperoxidase), biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

F. Pharmaceutical preparation

Pharmaceutical formulations of anti-BACE 1 antibodies as described herein are prepared by mixing the antibody of the desired purity with one or more optional Pharmaceutical carriers (Remington's Pharmaceutical Sciences, 16 th edition, Osol, a. eds. (1980)), either as a lyophilized formulation or as an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; defendPreservatives (e.g., octadecyl dimethyl benzyl ammonium chloride, hexa hydroxy quaternary ammonium chloride, benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, e.g., methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other sugars including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers herein also include interstitial dispersing agents, such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, e.g., rHuPH20 (r: (r) ()) Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. patent publication nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more other glycosaminoglycanases, such as chondroitinase.

Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulation including histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient as required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. The active ingredients are suitably present in combination in an amount effective for the intended use.

The active ingredient may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These techniques are disclosed in Remington's pharmaceutical Sciences, 16 th edition, Osol, A. eds (1980).

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

Formulations intended for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filtration membranes.

G treatment methods and compositions

Any of the anti-BACE 1 antibodies provided herein may be used in methods of treatment.

In one aspect, anti-BACE 1 antibodies for use as a medicament are provided. In a further aspect, anti-BACE 1 antibodies are provided for use in treating a neurological disease or disorder (e.g., AD). In certain embodiments, anti-BACE 1 antibodies are provided for use in methods of treatment. In certain embodiments, the present invention provides an anti-BACE 1 antibody for use in a method of treating a subject having a neurological disease or disorder, comprising administering to the subject an effective amount of an anti-BACE 1 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In other embodiments, the invention provides anti-BACE 1 antibodies for use in reducing or inhibiting amyloid plaque formation in a patient at risk for or having a neurological disease or disorder (e.g., AD). In certain embodiments, the present invention provides an anti-BACE 1 antibody for use in a method of reducing or inhibiting a β production in a subject, the method comprising administering to the subject an effective anti-BACE 1 antibody. An "individual" according to any of the above embodiments is preferably a human. In a certain aspect, an anti-BACE antibody for use in a method of the invention reduces or inhibits BACE1 activity. For example, an anti-BACE 1 antibody reduces or inhibits the ability of BACE1 to cleave APP.

In another aspect, the invention provides the use of an anti-BACE 1 antibody in the manufacture or manufacture of a medicament. In one embodiment, the medicament is for treating a neurological disease or disorder. In another embodiment, the medicament is for use in a method of treating a neurological disease or disorder, the method comprising administering to a subject having a neurological disease or disorder an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In another embodiment, the medicament is for use in inhibiting BACE1 activity. In another embodiment, the medicament is for use in a method of inhibiting a β production or plaque formation in a subject, the method comprising administering to the subject an effective amount of the medicament to inhibit a β production or plaque formation. An "individual" according to any of the above embodiments may be a human.

In another aspect, the invention provides methods for treating alzheimer's disease. In one embodiment, the method comprises administering to a subject with AD an effective amount of an anti-BACE 1 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. An "individual" according to any of the above embodiments may be a human.

In another aspect, the invention provides a pharmaceutical formulation comprising any of the anti-BACE 1 antibodies provided herein, e.g., for use in any of the above methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the anti-BACE 1 antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-BACE 1 antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

In therapy, the antibodies of the invention may be used alone or in combination with other agents. For example, an antibody of the invention can be co-administered with at least one additional therapeutic agent.

Such combination therapies described above include combined administration (where two or more therapeutic agents are contained in the same or separate formulations), and separate administration, where administration of the antibody of the invention can occur before, simultaneously with, and/or after administration of additional therapeutic agents and/or adjuvants. The antibodies of the invention may also be used with radiation therapy.

The antibodies of the invention (and any additional therapeutic agent) may be administered by any suitable method, including parenteral, intrapulmonary and intranasal administration, and, if required for topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection, for example intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, a single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

Certain embodiments of the present invention provide antibodies or fragments thereof that cross the blood-brain barrier. Certain neurodegenerative diseases are associated with increased permeability of the blood-brain barrier, such that antibodies or active fragments thereof can be readily introduced into the brain. While the blood-brain barrier remains intact, there are several known methods for transporting molecules across the blood-brain barrier, including, but not limited to, physical methods, lipid-based methods, and receptor and channel-based methods.

Physical methods of transporting antibodies or fragments thereof across the blood-brain barrier include, but are not limited to, completely bypassing the blood-brain barrier, or by creating an opening in the blood-brain barrier. Bypassing methods include, but are not limited to, direct injection into the brain (see, e.g., Papanastassiou et al, Gene Therapy 9: 398-^TM，Guildford PharmaceuVertical). Methods of creating openings in a barrier include, but are not limited to, sonication (see, e.g., U.S. patent publication No. 2002/0038086), osmotic pressure (e.g., by application of hypertonic mannitol (Neuwelt, e.a.,Implication of the Blood-Brain Barrier and its Manipulation(blood brain Barrier and its manipulation) volume 1&2, Plenum Press, n.y. (1989))), permeabilization by, for example, bradykinin or a permeabilizer a-7 (see, e.g., U.S. Pat. nos. 5,112,596,5,268,164,5,506,206, and 5,686,416), and transfection of neurons crossing the blood-brain barrier with vectors containing genes encoding antibodies or fragments thereof (see, e.g., U.S. patent publication No. 2003/0083299).

Lipid-based methods of transporting antibodies or fragments thereof across the blood-brain barrier include, but are not limited to, encapsulating the antibodies or fragments thereof in liposomes that bind to antibody-binding fragments that bind to receptors on the vascular endothelium of the blood-brain barrier (see, e.g., U.S. patent application publication No. 20020025313), and encapsulating the antibodies or active fragments thereof in low-density lipoprotein particles (see, e.g., U.S. patent application publication No. 20040204354) or defatted lipoprotein E (see, e.g., U.S. patent application publication No. 20040131692).

The antibodies of the invention will be formulated, administered and administered in a manner consistent with good medical practice. Factors considered in this context include the particular condition to be treated, the particular mammal to be treated, the clinical status of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to medical practitioners. The antibody need not be, but is optionally, formulated with one or more agents currently used for the prevention or treatment of the condition in question. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dosage and using the route of administration as described herein, or at about 1-99% of the dosage described herein, or at any dosage and any route that is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (either alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient as a single treatment or over a series of treatments. Depending on the type and severity of the disease, about 1 μ g/kg to 15mg/kg (e.g., 0.1mg/kg to 10mg/kg) of antibody may be an initial candidate dose for administration to a patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dose may be in the range of about 1. mu.g/kg to 100mg/kg or more, depending on the factors mentioned above. For repeated administration for several days or longer, depending on the condition, the treatment will generally be continued until the desired suppression of disease symptoms occurs. An exemplary dose of the antibody should be in the range of about 0.05mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg, or 10mg/kg (or any combination thereof) may be administered to a patient. Such doses may be administered at intervals, e.g., weekly or every three weeks (e.g., such that the patient receives about 2 to about 20 or, e.g., about 6 doses of the antibody). An initial higher loading dose may be administered followed by one or more lower doses. However, other treatment regimens may be useful. The progress of the therapy can be readily monitored by conventional methods and assays.

It is to be understood that any of the above formulations or methods of treatment may be performed using the immunoconjugates of the invention in place of or in addition to anti-BACE 1 antibodies.

H. Article of manufacture

In another aspect of the invention, an article of manufacture is provided that comprises materials useful for the treatment, prevention and/or diagnosis of the above-mentioned conditions. The article of manufacture comprises a container and a label or package insert (package insert) on or with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be made of various materials such as glass or plastic. The container contains a composition that is effective for the treatment of the condition, either alone or in combination with another composition effective for the treatment, prevention and/or diagnosis of the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is for use in treating a particular condition. Further, the article of manufacture can comprise (a) a first container comprising a composition therein, wherein the composition comprises an antibody of the invention; and (b) a second container having a composition contained therein, wherein the composition comprises another cytotoxic or other therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. It may also include other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

It is understood that any of the above preparations may include an immunoconjugate of the invention in place of or in addition to the anti-BACE 1 antibody.

Examples

The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments may be implemented in accordance with the general description provided above.

Example 1: preparation and characterization of anti-BACE 1 antibodies

Antibodies that specifically bind BACE1 were prepared by the following method: panning (panning) was performed against the extracellular domain of human BACE1, SEQ ID NO: 49 (one with natural diversity (VH and VH/VL) and the other with diversity in specific CDR regions artificially restricted to a specific amino acid set (YSGX)).

A. Natural diversity library sorting and screening to identify anti-BACE-1 antibodies

Selection of phage-displayed anti-BACE 1 clones

Biotinylated human BACE-1 (1-457 of SEQ ID NO: 49) was used as an antigen for library sorting. The natural diversity phage library was sorted into five rounds against biotinylated BACE-1 pre-captured on alternating neutravidin/streptavidin plates. For the first round of sorting, NUNC96 well Maxisorp immunoplates were first coated with 10 μ g/mL neutravidin (Fisher Scientific, #21125) and blocked overnight with phage blocking buffer PBST (phosphate buffered saline (PBS) and 1% (w/v) Bovine Serum Albumin (BSA) and 0.05% (v/v) Tween 20). First, 10. mu.g/mL biotinylated BACE-1 was captured on an immunoplate within 30 minutes. Antibody phage library VH (see, e.g., Lee et al, J.Immunol. meth.284: 119-132(2004)) and VH/VL (see Liang et al, J.mol. biol.366: 815-829(2007)) pre-blocked with phage blocking buffer PBST were then added to the plates and incubated overnight at room temperature. The following day each plate was washed 10 times with PBT (PBS containing 0.05% Tween20) and bound phage were eluted with 1mL50mM HC1 and 500mM NaCl over 30 minutes and neutralized with 600 μ L of 1MTris base (ph 8.0). The recovered phage were amplified in E.coli XL-1Blue cells. During the subsequent selection round, the propagated phage library was first pre-absorbed 50ul in PBST/BSA MyOne^TMStreptavidin T1(Invitrogen, #65601) and in the chamberIncubate at room temperature for 30 minutes. Removing the neutravidin-bound phage particles from the phage pool and removingUnbound phage was then added to the BACE-1 antigen displayed on streptavidin plates and the incubation time was reduced to 2-3 hours. The stringency of the plate washes gradually increased.

After 5 rounds of panning, significant enrichment was observed. 96 clones were picked from the VH and VH/VL library partitions to determine if they specifically bound human BACE-1. The variable regions of these clones were PCR sequenced to identify unique sequence clones. 42 unique phage antibodies that bind human BACE-1 at least 5x above background were selected and transformed to full-length IgGs for evaluation in an in vitro cellular assay.

The clones of interest were transformed to IgG by the following method: v of Individual clones_LAnd V_HThe regions were cloned into LPG3 and LPG4 vectors and transiently expressed in mammalian CHO cells and purified using protein a column chromatography.

Selection of anti-BACE 1 inhibitory clones

BACE1 is an aspartyl protease that normally cleaves amyloid precursor protein at a site near the cell surface, adjacent to its transmembrane domain. Thus, the ability of the above-identified antibodies to modulate the proteolytic activity of BACE1 on a particular BACE1 substrate was evaluated in vitro using a Homogeneous Time Resolved Fluorescence (HTRF) assay.

HTRF measurements were performed as follows. Two microliters of 375nM Bi27 (biotin-KTEEISEVNLDAEFRHDSGYEVHHQKL (SEQ ID NO: 53), American peptide Company)), an amyloid precursor protein BACE1 cleavage site peptide with a substitution increasing the sensitivity to BACE1 cleavage, was preincubated with 3. mu.L of 125nM BACE1 with anti-BACE antibody in BACE reaction buffer (50mM sodium acetate pH4.4 and 0.1% CHAPS) in 384 well plates (Proxiplate)^TMPerkin-Elmer). Hydrolyzing the proteinThe reaction mixture was incubated at ambient temperature for 75 minutes and quenched by the addition of 5 μ L of an HTRF detection mixture containing 2nM streptavidin-D2 and 150nM of europium cryptate-labeled 6E10 anti-amyloid β antibody (Covance, Emoryville, CA) in detection buffer (200mM Tris ph8.0, 20mM EDTA, 0.1% BSA, and 0.8M KF). The final reaction mixture was incubated at ambient temperature for 60 minutes and an EnVisionMultilabel Plate Reader was used^TM(Perkin-Elmer) the TR-FRET signal was measured at an excitation wavelength of 320nm and emission wavelengths of 615 and 665 nm. Reactions lacking the BACE1 enzyme (0BACE) and reactions containing antibodies lacking anti-BACE 1 (PBS (100% BACE1 activity) were used as controls.

Of the 42 tested antibodies identified from the natural diversity library, the best BACE1 inhibitor, i.e. YW412.8, was selected for affinity maturation. See fig. 4.

anti-BACE 1 inhibits affinity maturation of clones

Libraries were constructed as follows to affinity-mature the YW412.8 antibody. Phagemid pW0703 (derived from phagemid pV0350-2b (Lee et al, J.mol.biol.340, 1073-1093(2004)) containing a stop codon (TAA) at all CDR-L3 positions and displaying a monovalent Fab on the surface of the M13 phage served as a library template for grafting the heavy chain variable domain (VH) of the clone of interest from the natural diversity library for affinity maturation. Hard (hard) and soft (soft) randomization strategies were used for affinity maturation. For hard randomization, a light chain library with selected positions of three light chain CDRs was randomized using amino acids designed to mimic natural human antibodies, and the designed DNA degeneracy was as described in Lee et al (j.mol.biol.340, 1073-. For soft randomization, residues at positions 91-94 and 96 of CDR-L3, 28-31 and 34-35 of CDR-H1, 50, 52 and 53-58 of CDR-H2, 95-99 and 100A of CDR-H3 were targeted; and two different combinations of CDR loops, L3/H1/H2 and L3/H3, were selected for randomization. To obtain soft randomization conditions, which introduce a mutation rate of about 50% at selected positions, mutagenized DNA was synthesized using a 70-10-10-10 mixture of bases favoring wild-type nucleotides (Gallop et al, J.Med.chem.37: 1233-1251 (1994)).

Selection of Fab with increased affinity was performed as follows. The affinity-enhanced phage library was subjected to a first round of plate sorting followed by four or five rounds of solution sorting. For the first round of plate sorting, the library was sorted against 10 μ g/ml biotinylated target (BACE1) captured by neutravidin coated plates (NUNC Maxisorp plates), with phage input of about 2OD/ml, in 1% BSA and 0.05% Tween20 for 2 hours at room temperature. After the first round of plate sorting, solution sorting was performed to increase the stringency of selection. For solution sorting, 1OD/ml phage propagated from the first round plate sorting were mixed with 100nM biotinylated target protein (the concentration is based on parental clonal phage IC)₅₀Values) were incubated in 100. mu.l buffer containing 1% superblock (Pierce Biotechnology) and 0.05% Tween20 for 30 minutes at room temperature. The mixture was further diluted 10x with 1% Superblock and 100 μ Ι/well was applied to neutravidin coated wells (5 μ g/ml) for 15 minutes at room temperature and gently shaken to bind the biotinylated target to the phage. The wells were washed ten times with PBS and 0.05% Tween 20. To determine background binding, control wells containing phage and non-biotinylated target were captured on neutravidin coated plates. Bound phage were eluted with 0.1N HCl for 20 minutes, neutralized with 1/10 volumes of 1m tris pH11, titrated, and propagated for the next round. Next, two more rounds of solution sorting were performed with increasing selection stringency. The first round was performed for binding rate selection by decreasing the biotinylated target protein concentration from 100nM to 5 nM. The second round was an off-rate selection by adding excess non-biotinylated target protein (100-fold more) at room temperature to compete away weaker binders. Also, phage input was reduced (0.1-0.5 OD/ml) to reduce background phage binding.

Colonies were picked from the fourth round of screening and grown overnight in 96-well plates (Falcon) at 37 ℃ in 150. mu.l/well 2YT medium containing 50. mu.g/ml carbenicillin and 1E10/ml KO7 phage. From the same plate, colonies of XL-1 infected parent phage were picked as controls. 96-well Nunc Maxisorp plates were plated with 100. mu.l/well neutravidin (2. mu.g/ml) in PBS overnight at 4 ℃ or 2 hours at room temperature. The plates were blocked with 65 μ l of 1% BSA for 30 min and 40 μ l of 1% Tween20 for an additional 30 min before the addition of biotinylated target protein (2 μ g/ml) and incubation at room temperature for 15 min.

Phage supernatants were plated in ELISA (enzyme-coupled immunoadsorption assay) buffer (PBS with 0.5% BSA, 0.05% Tween 20) with or without 10nM target protein 1: 10 dilutions, total volume 100 μ l, and incubation in F-plates (NUNC) at room temperature for at least 1 hour for single point competition assay. 75 μ l of the mixture with or without target protein (side byside) was transferred together to target protein captured by neutravidin coated plates. The plate was gently shaken for 15 minutes to allow unbound phage to be captured to neutravidin captured target protein. The plate was washed at least five times with PBS-0.05% Tween 20. Binding was quantified by adding horseradish peroxidase (HRP) conjugated anti-M13 antibody (1: 5000) to ELISA buffer and incubating for 30 minutes at room temperature. The plate was washed at least five times with PBS-0.05% Tween 20. Next, 100. mu.l/well of 1: 1 ratio of 3, 3 ', 5, 5' -Tetramethylbenzidine (TMB) peroxidase substrate to peroxidase solution B (H) ₂O₂) (Kirkegaard-Perry Laboratories (Gaithersburg, Md.)) was added to the wells and incubated for 5 minutes at room temperature. By adding 100. mu.l of 1M phosphoric acid (H) to each well₃PO₄) And allowed to incubate at room temperature for 5 minutes to terminate the reaction. The OD (optical density) of yellow in each well was determined at 450nm using a standard ELISA plate reader. OD reduction (%) was calculated by the following formula:

OD_450nmdecrease (%) - (OD of the well containing competitor_450nm) /(OD of wells without competitors)_450nm)]*100。

OD of well from parent phage_450nmReduction (%) (100%) comparison, such clones were picked for sequence analysis, for human and murine targetsTwo OD the clone has_450nmThe decrease (%) was less than 50%. Selection of unique clones for phage preparation to determine binding affinity for the target by comparison to parental clones (phage IC)₅₀). The clones with the greatest affinity enhancement were transformed into human IgG1 for antibody production and further analysis of binding kinetics by surface plasmon resonance using BIAcore and other in vitro or in vivo assays.

The sequences of the light and heavy chain HVR regions of YW412.8 selected from the natural diversity phage library are shown in fig. 1(a) and 1 (B). In addition, five antibodies obtained from affinity maturation of the YW412.8 antibody were also sequenced, and the light and heavy chain HVR sequences are also shown in fig. 1(a) and 1 (B). The consensus amino acid sequences for the light chain HVR regions that show variability in these antibodies are: HVR-L1: arg Ala Ser Gln X ₁Val X₂X₃X₄X₅Ala (SEQ ID NO: 17), wherein X₁Selected from aspartic acid and valine, X₂Selected from serine and alanine, X₃Selected from threonine and asparagine, X₄Is selected from alanine and serine, and X₅Selected from valine and leucine; HVR-L2: x₆Ala SerPhe Leu Tyr Ser (SEQ ID NO: 18), wherein X₆Selected from serine and leucine; and HVR-L3: gln Gln X₇X₈X₉X₁₀X₁₁X₁₂Thr (SEQ ID NO: 19), wherein X₇Selected from serine, phenylalanine, glycine, aspartic acid and tyrosine, X₈Selected from tyrosine, proline, serine and alanine, X₉Selected from threonine and asparagine, X₁₀Selected from threonine, tyrosine, aspartic acid and serine, X₁₁Is selected from proline and leucine, and X₁₂Selected from proline and threonine. In these antibodies, only the heavy chain hypervariable region H1 exhibited variability, and the consensus sequence for this region was: HVR-H1: gly Phe Thr Phe X₁₃Gly Tyr X₁₄Ile His (SEQ ID NO: 26), wherein X₁₃Is selected from serine and leucine, and X₁₄Selected from alanine and glycine.

B. Synthetic diversity library sorting and screening to identify anti-BACE-1 antibodies

A minimal synthetic antibody library with limited chemical diversity in the Complementarity Determining Regions (CDRs) has been constructed and demonstrated to be effective in obtaining high affinity antibody binders to a variety of proteins, as previously described in Fellouse, f.a. et al, j. mol.biol.373: 924, 940 (2007). Synthetic diversity libraries designated as YSGX libraries were used to search for inhibitory antibodies against BACE1 by solution sorting. Five rounds of combined panning were performed as described below.

The phagemid for Fab-phage display (pF1359) (library D in Fellouse, F.A. et al, J.mol.biol.373: 924-. The diversity of the library was about 2x10¹⁰。

For affinity maturation, all three CDRLs were randomized using immobilized CDRH for selected clones derived from primary sorting. Three types of oligonucleotides were used for randomization. Type I uses the degenerate codons TMC, which only encodes Tyr and Ser. Type II uses a conventional trimeric phosphoramidite mixture (trimer phosphoramidite mix) containing codons for Tyr, Ser, Gly and Trp in equimolar ratios. Type III uses a mixture of trimeric phosphoramidites encoding 10 amino acid residues in the following molar ratios: tyr (30%), Ser (15%), Gly (15%), Trp (10%), and Phe, Leu, His, Asp, Pro, Ala, 5% each. Mutations were introduced into the oligonucleotide for CDR-L3 to silence the KpnI site on the original template after mutation. For CDR-L1 length changes to 3 to 10 amino acids, for CDR-L2 length changes to 7 amino acids, and for CDR-L3 length changes to 2-10 amino acids. The oligonucleotides were suitably pooled together to form the final oligonucleotide set, i.e., all L1, L2, and L3 oligonucleotides of different lengths mixed within one type, and then all three types were mixed together as the oligonucleotide sets of CDR-L1, CDR-L2, and CDR-L3, respectively. Kunkel mutagenesis was used to replace all CDR-LC positions. After Kunkel mutagenesis (Kunkel, T.A., et al, Methods Enzymol.154: 367-382(1987)), the DNA was purified and treated with KpnI at 37 ℃ for 3 hours to digest the template DNA. The purified DNA was then subjected to electrophoresis for library construction.

Selection of phage-displayed anti-BACE 1 clones

Biotinylated human BACE-1 (1-457 of SEQ ID NO: 49) was used as the library-sorted antigen. For the first round of panning, 20 μ g of biotinylated BACE1 was combined with a 1ml library at 1X10¹³The pfu/ml concentration was incubated at 4 ℃ for 1.5 h. Blocked with blocking buffer (PBS, 0.5% (w/v) bovine serum albumin) before using 200. mu.l in 15 minMyOne streptavidin captures phage bound to the target. Bound phage were eluted with 0.1M HC1 and immediately neutralized with 1M Tris base. The eluted phage were amplified according to the standard protocol described previously (Sidhu, S.S. et al Methods enzymol.328: 333-363 (2000)). A second round was performed as the first round, using 10 μ g of biotinylated BACE1 incubated with 400 μ l of amplified phage. For all subsequent rounds, 2 μ g biotinylated BACE1 was incubated with 400 μ l of amplified phage. Within 15 minutes, phage bound to biotinylated BACE1 were captured using Maxisorp immunoplates (NUNC) that had been previously coated with Neutravidin or streptavidin (alternating between rounds) and blocked with blocking buffer.

After five rounds of selection, phage were prepared from individual clones grown in a 96-well format. And culture supernatants were diluted three-fold in Phosphate Buffered Saline (PBS), 0.5% (w/v) Bovine Serum Albumin (BSA) (Sigma-Aldrich, St Louis, Mo.), 0.1% (v/v) Tween20(Sigma-Aldrich) (PBT buffer) for phage dot ELISA. The diluted phage supernatant was incubated for 1 hour with biotinylated BACE1 immobilized on Neutravidin coated 384-well Maxisorp immunoplates (NUNC). The plates were plated with PBS, 0.05% (v/v) Tween20 Washed six times (PT buffer) and incubated with horseradish peroxidase/anti-M13 antibody conjugate (1: 5000 diluted in PBT buffer) (GE Healthcare) for 30 minutes. The plates were washed six times with PT buffer and two times with PBS using 3, 3 ', 5, 5' -tetramethylbenzidine/H₂O₂Peroxidase substrate (Kirkegaard-Perry Laboratories) for 15 minutes using 1.0MH₃PO₄Quench and read the absorbance spectrophotometrically at 450 nm.

Selection of anti-BACE 1 inhibitory clones

Panning of the YSGX library resulted in the identification of 18 unique clones that bound BACE 1. See fig. 3. The Fab proteins corresponding to these clones were purified as follows. A stop codon was introduced between the heavy chain and gene 3 encoding the Fab on the phagemid. The resulting phagemid was transformed into E.coli strain 3488. A single colony was cultured overnight at 37 ℃ in 30ml of LB medium supplemented with 50. mu.g/ml carbenicillin. The overnight culture (5ml) was inoculated into 500ml of complete c.r.a.p. medium supplemented with carbenicillin (50 μ g/ml) and grown at 30 ℃ for 24 h. Fab protein was purified by standard methods using protein a agarose beads.

Purified fabs were screened for inhibitory activity against BACE1 using HTRF enzyme activity assay, as described above. Fab2, 5, 8, 12, 14 and 19 were identified as inhibitors of BACE1, while Fab23 was identified as an activator. See fig. 5.

Fab2, 5, 8, 12, 14 and 19 were further characterized to determine its binding epitope. In a phage competition ELISA, a panel of purified fabs of all 6 antibodies was used to compete with individual Fab-displaying phage bound to plate-captured BACE1, as described below.

Individual colonies of selected clones (in XL1blue cells) were picked and grown for 2 hours at 37 ℃ in 1ml 2YT broth supplemented with 50. mu.g/ml carbenicillin, 10. mu.g/ml tetracycline and M13KO 7. Kanamycin (25. mu.g/ml) was added to the culture, which was continued for 6 hours. The culture was transferred to 30ml 2YT broth supplemented with 50. mu.g/ml carbenicillin and 25. mu.g/ml kanamycin and grown overnight at 37 ℃. The phage were collected and purified as described previously (Sidhu, S.S. et al Methods Enzymol.328: 333-363 (2000)). Purified Fab display phage were serially diluted in PBT buffer and tested for binding to BACE1 immobilized on the plate. Fixed phage concentrations that gave 80% saturation signal were selected for subsequent competition ELISA. The competition is performed by: immobilized, sub-saturated Fab display phages were incubated with serial dilutions of BACE1 for 1h and then transferred to BACE1 immobilized plates for 15 min to capture unbound phages. The plate was then washed 8 times and bound phage detected by anti-M13-HRP.

Purified Fab5 could compete with BACE1 for binding to phage displayed fabs 8 and 12 instead of Fab2, 14 and 19. Consistent with this data, purified Fab8 could compete with phage-displayed fabs 5 and 12. Taken together, these data indicate that Fab5, 8, and 12 bind to the same or overlapping epitopes on BACE 1. Based on the fact that any of these purified fabs can compete with any of the Fab 14-displaying phage and the Fab 19-displaying phage, the fabs 14 and 19 can also compete with each other. This suggests that these two antibodies bind the same or overlapping epitopes, which are different from the epitopes of Fab5, 8 and 12. In the phage ELISA assay, Fab2 display phage could not be competed away by any purified Fab protein (including Fab2 itself), suggesting that the binding between Fab2 and BACE1 is non-specific. Thus, Fab2 was excluded as a candidate for affinity maturation.

anti-BACE 1 inhibits affinity maturation of clones

To increase the binding affinity of the parental inhibitory antibodies obtained by the initial panning procedure, a new phage library was designed that randomized all three CDR-LCs of Fab5, 8, 12, 14 and 19. Based on their different epitopes, these five antibodies were divided into two subgroups-Fab 5, 8 and 12 as group 1, and Fab14 and 19 as group 2. Single stranded dna (ssdna) of individual clones was purified as a template for library construction. The ssDNA templates of group 1 were pooled together for affinity maturation library 1 (designated as LC-libl), while the ssDNA templates of group 2 were pooled together for library 2(LC-lib 2). Chemical diversity is limited within randomized CDRs based on the functional ability of natural amino acids for molecular recognition. See Birtalan, s. et al Mol biosystem.6: 1186-1194(2010). The minimal diversity (Tyr and Ser binary codons), the semi-minimal diversity (Tyr, Ser, Gly and Trp ternary codons) and the additional diversity where 10 amino acids are involved are mixed in order to obtain high avidity. As described above, the same set of oligonucleotide libraries was used to randomize all three CDR-LCs simultaneously to construct two affinity maturation libraries, LC-lib1 and LC-lib 2. For affinity maturation, all three CDR-LCs (complementarity determining region-light chains) were randomized with CDR-HC (complementarity determining region-heavy chain) fixation for selected clones derived from primary sorting.

Screening of the library for affinity maturation of the initially obtained antibody is performed analogously, as described above. Libraries were subjected to 3 rounds of sorting using biotinylated BACE1 in solution, which resulted in greater than 100-fold enrichment in binding. For round 1, 2 μ g of biotin-BACE 1 was incubated with the phage-displayed Fab library. For rounds 2 and 3, 20nM and 5nM biotinylated BACE1 were incubated with the amplified phage, respectively. Clones from each of the two libraries were screened in a single point competition ELISA (96) using 20nM of BACE1 in solution to compete with phage particles for binding to plate-immobilized BACE1, as described below.

Plates immobilized with BACE1 were prepared by using 384-well Maxisorp Immunoplate that was previously coated with 2. mu.g/ml Neutravidin at 4 ℃ overnight and blocked with blocking buffer to capture 2. mu.g/ml biotinylated BACE1 within 15 minutes. Culture supernatants from individual clones grown in 96-well format were diluted 20-fold in PBT buffer and incubated with (or without) 20nM BACE1 for 1 hour at room temperature. The mixture was transferred to plates immobilized with BACE1 and incubated for 15 minutes. The plate was washed six times with PT buffer and bound phage detected by anti-M13-HRP as described above. The ratio between the ELISA signal from a well in which BACE1 was not present in solution and the ELISA signal from a well in which BACE1 was present in solution indicates the affinity of the clone, with a higher ratio indicating a higher affinity.

Five clones from LC _ lib1 had a ratio > 4 between the ELISA signal from wells without BACE1 and the ELISA signal from wells with BACE1, while the ratio of one clone from LC _ lib2 was > 3. Two clones, designated LC4 and 11, were derived from Fab 5; three clones, LC6, LC9 and LC10, were derived from Fab12, whereas LC40 was derived from Fab14 (fig. 6).

To assess the affinity of these 6 clones, phage competition ELISAs were performed, as described above, and IC was determined₅₀Values (fig. 7). IC (integrated circuit)₅₀The values are determined by: the data was fitted to a four parameter logistic equation developed by Marquardt (Marquardt, d.w.siamj.appl.math.11: 431-.

TABLE 2

Parent strain

All LC clones actually showed increased affinity compared to their corresponding parents. Notably, the introduction of two Trp residues into CDR-L2 increased the affinity of Fab12 derivatives by more than 100-fold compared to the parent.

Fab proteins from 6 clones were purified and subjected to HTRF enzyme activity assay as described above. OM99-2(Cat 496000) which is a peptide inhibitor of BACE1, used as a control. For the antibody parental 5, Fab LC4 showed significantly improved inhibition, while LC11 lost inhibitory activity. To come The derivative LC40 from Fab14 also lost its inhibitory activity. Derivatives of Fab12, Fab LC6, LC9 and LC10 with increased affinity generally showed about 20-fold increase in their inhibitory activity (fig. 8). Based on this assay, Fab LC6 was the best inhibitor and showed almost 100% inhibition of enzyme activity, while the other fabs were partial inhibitors, with a degree of inhibition of about 60-70% (fig. 8). IC of the different Fab tested₅₀The values are shown in table 3 below. IC (integrated circuit)₅₀OM99-2 was 11nM in this assay.

TABLE 3

Fab ID	IC50(nM)
		Fab5*	130
LC4	480
		LC11	n.d.
Fab12*	n.d.
		LC9	140

Fab ID	IC50(nM)
		LC10	180
LC6	160
		Fab14*	740
LC40	n.d.
		Parent of Zhang

The sequences of the light and heavy chain HVR regions of Fab12 are shown in fig. 2(a) and 2 (B). The light and heavy chain HVR sequences of three antibodies generated by affinity maturation of Fab12 are also shown in fig. 2(a) and 2 (B). Of these antibodies, only light chain HVR-L2 showed variability: HVR-L2: x₁₅AlaSer X₁₆Leu Tyr Ser (SEQ ID NO: 41), where X₁₅X is selected from serine, tryptophan and tyrosine₁₆Selected from serine and tryptophan. Each of the three heavy chain HVR regions was identical in the four antibodies.

Fab was cloned as an IgG antibody for further use as follows. The variable domains of the light and heavy chains of selected fabs were cloned into pRK 5-based plasmids with human light or heavy chain (human IgGl) constant domains for transient IgG expression in 293T cells or Chinese Hamster Ovary (CHO) cells. IgG proteins were purified by standard methods using protein a agarose beads.

Example 2: further characterization of anti-BACE 1 antibody

Antibodies were identified by function and epitope bound on BACE1, as described above. The parent and affinity matured antibodies were further characterized using the following assays.

A. Binding kinetics

The binding kinetics of yw412.8.31 were evaluated. Briefly, the binding affinity of anti-BACE 1IgG was determined by Surface Plasmon Resonance (SPR) using BIAcore^TM3000 instruments. YW412.8.31 anti-BACE 1 human IgG was captured by mouse anti-human Fc antibody (GE Healthcare, cat # BR-1008-39) coated on a CM5 biosensor chip to obtain approximately 100 Response Units (RU). For kinetic measurements, two-fold serial dilutions (0.98nM to 125nM) of human BACE1ECD or murine BACE1ECD (amino acids 1-457) in PBT buffer (PBS containing 0.05% Tween 20) were injected at 25 ℃ at a flow rate of 30. mu.l/min. Using a simple one-to-one Langmuir binding model (BIAcore)^TMEvaluation software version 3.2) calculation of the Association Rate (k)_on) And dissociation rate (k)_off). Equilibrium dissociation constant (K)_D) Is calculated as the ratio k_off/k_on. The results of the binding of YW412.8.31 at pH7.0 are shown in Table 4.

TABLE 4-by BIAcore^TMMeasured binding kinetics values of anti-BACE 1 antibodies

Binding of YW412.8.31 to BACE1 was confirmed at pH7.0 and 5.0. This is important because BACE1 is most active at acidic pH, presumably in endocytic vesicles and/or trans-golgi networks.

B. In vitro inhibition assay

In addition, two activity assays were used: HTRF assay and Microfluidic Capillary Electrophoresis (MCE) assay, the ability of an antibody to modulate the proteolytic activity of BACE1 on a particular BACE substrate was evaluated in vitro using the extracellular domain of human recombinant BACE 1.

Affinity matured yw412.8.31 anti-BACE 1 antibodies were tested in HTRF assay as described in example 1. Synthetic peptide inhibitors of BACE1, OM99-2 (CCat No. 496000), small molecule inhibitors of BACE1 (β -secretase inhibitor IV,cat 5657688) and IgG antibodies that do not bind BACE1 were used as controls. See fig. 9 (panel a) (long peptide). In addition, a reaction using a short FRET peptide (Rh-EVNLDAEFK-quencher (SEQ ID NO: 54), Invitrogen) was also performed in the same manner as the HTRF reaction. The fluorescent product generated from the control reaction was measured as above, but at an excitation wavelength of 545nm and an emission wavelength of 585 nm. Using GraphPad Prism5^TM(LaJolla, CA) the data obtained were analyzed. See fig. 9 (panel a) (short peptide).

MCE assay reactions were performed in 384-well microplates at a final volume of 20. mu.L/well. A standard enzymatic reaction was initiated by adding 10. mu.L of 2X substrate to 5. mu.L of 4 Xenzyme and 5mL of 4 Xcompound containing 12nM human BACE1 extracellular domain, 1mM amyloid precursor protein beta secretase active site peptide (FAM-KTEEISEVNLDAEFRWKK-CONH) ₂(SEQ ID NO: 55)), 50mM NaOAc pH4.4 and 0.1% CHAPS. The same reaction conditions were used for the extracellular domain of human BACE2 enzyme (5nM) and the extracellular domain of cathepsin D (6nM,). After 60 minutes incubation at ambient temperature, the product and substrate in each reaction were separated and used in a 12-suction (sipper) microfluidic chip(both from Caliper Life Sciences). The separation of the product from the substrate was optimized by selecting the voltage and pressure using the manufacturer's optimization software. The isolation buffer contained 100mM HEPES pH7.2, 0.015% Brij-35, 0.1% coating reagent #3, 10mM EDTA and 5% DMSO. The separation conditions used a downstream voltage of-500V, an upstream voltage of-2250V and a screening pressure of-1.2 psi. Product and substrate fluorescence is excited at a wavelength of 488nm and detected at a wavelength of 530 nm. Substrate conversion was calculated from the electropherograms using HTS Well Analyzer software (Caliper Life Sciences).

The results from HTRF and MCE assays using yw412.8.31 antibody are shown in figure 9. IC of the antibody observed in Long peptide assays₅₀At 1.7nM, the maximum inhibition reached 77%. In addition, the YW412.8.31 antibody has IC in the short peptide assay ₅₀Was 17 nM. In addition, YW412.8.31 anti-BACE 1 antibody was expressed as an IC of 80pM in a microfluidic capillary electrophoresis assay₅₀Inhibits BACE1 activity, but not human BACE2 or cathepsin D, a lysosomal aspartyl protease. SPR analysis of yw412.8.31 antibody also demonstrated that the antibody did not bind BACE2, a protease most closely related to BACE 1. Together, these data indicate that the yw412.8.31 antibody is a potent and selective BACE1 antagonist. The antibody was further characterized to better understand its function.

Cell-based inhibition assay

To determine whether the observed in vitro inhibitory effect of anti-BACE 1 antibodies on APP processing was also present in the cellular environment, in vivo studies were performed. Evaluation of the inhibition of A.beta.by antibodies in 293-HEK cells stably expressing wild-type human amyloid precursor protein as follows_1-40The ability to generate. 293-APP (Ribose nucleic acid)^WTCells were cultured at 3X10⁴Cell/well density was seeded overnight in 96-well plates. 50 μ l of fresh medium (DMEM + 10% FBS) containing anti-BACE 1 antibody or control IgGl antibody was incubated with the cells for 24 hours at 37 ℃. A tricyclic small molecule BACE1 inhibitor (BACE1SMI) was also used as a control ((Compound 8e-Charrier, N. et al J.M)Chem.51: 3313-3317(2008)). Cell culture medium (cellular media) was collected and used with A.beta.according to the manufacturer's instructions _1-40Assay (CisBio) to determine A.beta._1-40Is present. Abeta (beta)_1-40Cell viability as determined using the CellTiter-GloLuminecent Cell ViabilityAssay (Promega) was normalized. The experiment was performed at least three times and each point in each experiment was repeated twice. Data were plotted using a four-parameter nonlinear regression curve fitting program (Kaleidagraph, Synergy Software).

Similar studies were also performed in dorsal root ganglia, cortical neurons and hippocampal neurons isolated from mice. Briefly, dissociated neuronal cultures were prepared from E13.5 Dorsal Root Ganglia (DRG), E16.5 cortical neurons and E16.5 hippocampal neurons. Neurons were cultured in vitro for five days. Fresh medium containing yw412.8.31 anti-BACE antibody or control IgGl was incubated with neurons for 24 hours. The medium was collected and used according to the manufacturer's instructionsRodent/Human (4G8) A beta 40Ultrasensitive kit (Ultrasensitive kit) for determining A beta₄₀Is present. Abeta (beta)₄₀Cell viability as determined using the CellTiter-Glo luminescennt Cell ViabilityAssay (Promega) was normalized. The experiment was performed at least three times and each point was repeated twice. Data were plotted using a four-parameter nonlinear regression curve fitting program (Kaleidagraph, Synergy Software).

non-BACE 1IgG antibody inhibitorsIn contrast, all anti-BACE 1 antibodies tested (LC6, LC9, YW412.8, yw412.8.30, yw412.8.31 and YW412.8.51) inhibited a β in 293 cells expressing APP_1-40And (4) generating. See fig. 10.

As shown in FIG. 11, the YW412.8.31 anti-BACE 1 antibody inhibits A β in APP-expressing 293 cells_1-40Production, to an extent similar to BACE1SMI control, IC₅₀17nM and a maximum reduction of-90%. Similar results were observed in DRG neurons, with yw412.8.31, a β at the highest concentration₄₀The production is reduced by about 50%, while IC₅₀It was 8.4 nM. YW412.8.31 anti-BACE 1 antibody also inhibits A β in cortex and hippocampal neurons₄₀Generation of, wherein IC₅₀2.3-2.6 nM. These findings indicate that anti-BACE 1 antibodies act on cells similar to those previously observed in vitro. Furthermore, yw412.8.31 antibody appears to show optimal efficacy in neurons of the CNS.

Intracellular localization of anti-BACE 1 antibodies

BACE1 is known to be expressed intracellularly, especially in the Golgi apparatus. To determine whether yw412.8.31 interacts with BACE1 in an intracellular environment, an internalization study was performed. One set of neuronal cultures was prepared from E13.5 Dorsal Root Ganglion (DRG) explants and a second set of neuronal cultures was prepared from E16.5 dissociated cortical neurons from BACE1+/+ or BACE 1-/-mice and cultured at 37 ℃ for 24 or 72 hours, respectively. Media containing 0.5 μ M YW412.8.31 anti-BACE 1 antibody was added to the culture over a period of 30 minutes to 2 hours and incubated at 4 ℃ or 37 ℃. After treatment, unbound antibody was washed out thoroughly with PBS. Cultures were fixed with 4% paraformaldehyde for 20 minutes at room temperature and selected samples were also permeabilized with 0.1% Triton X-100. Bound antibody was detected using a second Alexa 568-conjugated anti-human IgGl antibody (Molecular Probes) according to the manufacturer's instructions.

In high temperature samples, most of the antibody signal was found to be internalized. As can be seen in fig. 12(B), BACE1 can be detected intracellularly in DRG axons when the cells are permeabilized at 37 ℃ allowing detection of yw412.8.31 anti-BACE 1 antibodies with the second antibody. In contrast, very little BACE1 was detected on the cell surface when DRG was incubated at low temperature at 4 ℃ to prevent internalization, or when the cells were not permeabilized to allow intracellular detection of yw412.8.31. Internalization of antibodies into neurons is dependent on BACE1 binding, as it is only detectable in cortical neurons from BACE1+/+ animals, but not in neurons from BACE 1-/-animals (compare the middle and right panels of figure 12 (C)).

In addition, mouse cortical neurons were cultured in the presence of yw412.8.31 anti-BACE 1 antibody or control IgG for 10 minutes or 3 hours, after which the antibodies were detected by immunostaining. Neuronal cultures were prepared from E15.5 dissociated cortical neurons and 14DIV was cultured. Medium containing 1 μ M yw412.8.31 was added to the culture over 10 minutes to 3 hours and incubated at 37 ℃. Unbound antibody was washed out thoroughly after treatment with HBSS. Cultures were fixed with 2% paraformaldehyde for 10 minutes at room temperature and then permeabilized with 0.1% Triton X-100, or not only. Bound antibodies were detected using Alexa 568-conjugated anti-human IgGl secondary antibodies (molecular probes). Yw412.8.31 localization was analyzed in non-permeabilized cells to see how much binding is on the surface of the cells, and yw412.8.31 localization was analyzed in permeabilized cells to see how much antibody is internalized. Most of the antibody signals detected localized inside the cells, while almost no antibody staining was observed on the cell surface (fig. 12 (a)). Internalization was evident after only 10 minutes of yw412.8.31 treatment, indicating that the antibody was actively taken up by early endosomes. Most of the yw412.8.31 signal is dotted, indicating that it may be contained within a vesicle.

To better localize the subcellular fraction of the yw412.8.31 localization, we co-stained with markers of different vesicle fractions: early endosomes (transferrin receptor, TfR), Trans Golgi Network (TGN) (VAMP4), and lysosomes (LAMP 1). Cells were co-stained with anti-TfR (Novus, cat No. NB100-64979), anti-VAMP 4(Novus, cat No. NB300-533) or anti-LAMP 1(BDPharmingen, cat No. 553792). Yw412.8.31 immunoreactivity co-localized with markers of early endosomes and TGN, but not with markers of lysosomes (fig. 12 (a)). This pattern is consistent with antibodies localized to portions where BACE1 is active.

Example 3: anti-BACE 1 antibody binding site characterization

Further studies were performed to identify the binding site of specific anti-BACE 1 antibodies to human BACE 1. In one set of experiments, the binding of the antibodies to BACE1 (hbec 1) was assessed in the presence or absence of a known active site or exo-binding site BACE1 binding peptide to determine which antibodies showed competitive binding. In a second set of experiments, anti-BACE 1Fab was co-crystallized with the extracellular domain of human BACE1 to determine the three-dimensional binding site.

A. Competitive binding

As a method for indirectly determining the binding site of an anti-BACE 1 antibody of the invention on BACE1, a competitive ELISA was performed. Briefly, antibody YW412.8IgG (1. mu.g/ml) was coated onto NUNC96 well Maxisorp immunoplates, overnight at 4 ℃ and blocked with blocking buffer PBST (PBS and 1% BSA and 0.05% Tween20) for 1 hour at room temperature. Serial dilutions of anti-BACE 1 antibody YW412.8 or hbec 1 binding peptide were incubated with a predetermined amount of biotinylated hbec 1 and incubated for 60 minutes at room temperature. The serial dilutions were then added to YW412.8 coated plates and incubated for 30 minutes at room temperature. Subsequently, the plates were washed with washing buffer (PBS containing 0.05% T-20) and developed by adding streptavidin labeled with horseradish peroxidase (HRP) for 30 minutes at room temperature. The plates were then washed and developed using a Tetramethylbenzidine (TMB) substrate. HRP-conjugated streptavidin bound to captured biotinylated hbece 1 was measured at a wavelength of 630nm using standard techniques.

To determine the optimal concentration of biotinylated target protein for the above competition ELISA assay, NUNC96 well Maxisorp immunoplates were coated and blocked as described above. Serial dilutions of biotinylated target were incubated with antibody coated plates for 30 minutes at room temperature. The plates were then washed with PBST, followed by incubation with horseradish peroxidase-conjugated streptavidin for 30 minutes at room temperature. Detection of the binding signal is as described above. Data were plotted using a four-parameter nonlinear regression curve fitting program (Kaleidagraph, Synergy Software). The sub-saturation concentration of biotinylated hbec 1 was determined from curve fitting and applied to the above competition ELISA.

As expected, YW412.8 competed with itself for binding to hbec 1 (fig. 13). At YW412.8 and active site inhibitor peptide OM 99-2: (Directory number 496000) no competition was observed. Competition was observed between the LC6 and YW412.8 anti-BACE 1 antibodies and the known external binding site binding peptide BMS1 (peptide 1 from Kornacker et al, Biochemistry 44: 11567-11572 (2005)). Taken together, these results indicate that YW412.8 binds at a binding site outside of BACE1 that is different from the active site of BACE1 for APP cleavage. The shape of the curves in fig. 13 suggests that YW412.8, LC6, and BMS1 may have overlapping binding sites on BACE 1.

B. Crystal structure

To better understand the interaction of YW412.8 antibody with BACE1, yw412.8.31fab was co-crystallized with the extracellular domain of human recombinant BACE1 extracellular domain.

Protein expression and purification

Protein expression and purification of BACE1 (amino acids 57-453 of SEQ ID NO: 49). DNA tagged with a C-terminal His6 (SEQ ID NO: 210) was synthesized by Blue Heron, cloned into pET29a (+) vector (Novagen) and transformed into BL21(DE3) cells (Invitrogen). Expression was performed at 37 ℃ for 4 hours and induced with 1mM isopropyl beta-D-1-thiogalactoside (IPTG). Cells were lysed using a microfluidizer (microfluidizer) and inclusion bodies (which contained BACE1) were isolated and washed twice with TE (10mM Tris pH8.0 and 1mM ethylenediaminetetraacetic acid (EDTA)) buffer. Protein solubilization was performed using 7.5M urea, 100mM MAMPSO pH10.8 and 100mM β -mercaptoethanol (BME) at room temperature for 2 hours, followed by centrifugation at 12,000rpm for 30 minutes. The supernatant was then diluted with 7.5M urea, 100mM AMMPSO pH10.8To obtain an OD of about 1.5-2.0₂₈₀. Protein refolding was performed by: solubilized BACE1 was first solubilized as follows 1: 20 are diluted in cold water and the sample is then gently stirred at 4 ℃ for 3 weeks to allow refolding to occur. Purification of refolded BACE1 involved 3 column chromatography steps. First, BACE1 was loaded onto a 50ml Q sepharose Fast Flow (GE Healthcare) column pre-equilibrated with 20mM Tris pH8.0 and 0.4M urea and eluted with a salt gradient from 0-0.5M NaCl. The peak fractions were collected, diluted 5-fold with 20mM Tris pH8.0 buffer, and applied to a SourceTM15Q column (GE Healthcare). A gradient of 0-0.3M NaCl was used to elute BACE 1. The fractions containing BACE1 protein were collected, concentrated and concentrated in Superdex ^TMFurther purification on S75 column (GE Healthcare) in 25mM Hepes pH7.5, 150mM NaCl.

YW412.8.31Fab was expressed in E.coli and cell bodies were thawed in PBS, 25mM EDTA and 1mM PMSF. The mixture was homogenized, passed twice through a microfluidizer, and centrifuged at 12,000rpm for 60 minutes. The supernatant was then applied to a protein G column at 5 ml/min. The column was washed with PBS to baseline, and the protein was eluted with 0.58% acetic acid. The YW412.8.31Fab containing fraction was collected and loaded onto a SP-Sepharose column equilibrated with 20mM MES, pH5.5, and the Fab was eluted with a salt gradient from 0 to 0.25M NaCl. In Superdex^TMFab was further purified on S75 column in 25mM Hepes pH7.5 and 150mM NaCl.

Crystallization of

Purified BACE1 protein (amino acids 57 to 453 of SEQ ID NO: 49) was mixed with purified YW412.8.31Fab in a 1: molar ratio (excess Fab) of 1.5. The complex was incubated on ice for 1 hour and purified on a S20026/60 gel filtration column (GE Healthcare) to separate it from excess Fab. The complex was then concentrated to 15 mg/ml. Crystallization was performed by sitting drop vapor diffusion, in which 1 μ l of BACE1/Fab complex solution was mixed with 1 μ l of a well solution containing 20% PEG4000, 0.1M Tris pH8.5 and 0.2M sodium acetate. The crystallized droplets were then incubated at 19 ℃. Crystals appeared after 4 days and continued to grow for more than 2 days. The crystals were then collected and snap frozen in a cryoprotectant solution containing mother liquor and 20% glycerol.

Data collection and texture determination

Diffraction data was collected using a monochromatic X-ray beam (12658.4eV) in a Stanford Synchrotron radiation exposure (SSRL) beam 7-1. The X-ray detection device is an ADSC quantum-315 CCD detector, which is placed 430mm from the crystal. The rotation method was applied to a single crystal for collecting a complete data set, with a 0.5 ° swing per frame and a total wedge (wedge) size of 180 °. The data is then indexed, integrated, and the program used(HLK Research, Inc.) scaling (scaled).

The structure was analyzed by the Molecular Replacement (MR) method using the program Phaser (Read, R.J., Acta Crystal.D57: 1373-1382 (2000)). Matthews coefficient calculations indicate that each asymmetric unit consists of one BACE1/Fab complex and 48% solvent. Thus, MR calculations were performed to search for a group containing three subunits including the N-and C-domains of Fab, as well as the extracellular domain of BACE 1. The N-and C-terminal Fab domains were searched separately to allow flexible corner angles (elbow). The search model for Fab subunits was derived from the crystal structure of the HGFA/Fab complex (PDB code: 2R0L, Wu, Y. et al Proc. Natl. Acad. Sci. USA 104: 19784-19789 (2007)). Search model for BACE1 from the published BACE1 structure, the PDB of the BACE1 structure encodes: 1FKN (Hong, L. et al Science 290: 150-. Significant conformational changes occurred at the BACE1/Fab interface. Using the program COOT (Crystallographic Object-OrientationToolkit) (Emsley) &Cowtan, acta.cryst.d60: 2126-2132(2004)) for manual reconstruction. The most used programs REFMAC5(Murshudov, G.N. et al, Acta Crystal.D53: 240-Large similarity target function was structurally refined to obtain a final R factor of 0.221 and an R of 0.274_free. Statistical data for structure refinement are shown in table 5.

TABLE 5 statistics of crystallographic data

¹Rsym＝∑|I_hi-I_h|/∑I_hiIn which I_hiIs the intensity of the scaled I-th symmetric correlation observation of the reflection h, and I_hAre average values.

²The value in parentheses is the highest resolution shellThe value of (c).

³Rcryst＝∑_h|F_oh-F_ch|/∑_hF_ohIn which F is_ohAnd F_chIs the observed and calculated structure factor amplitude of the reflection h.

⁴The value of Rfree was calculated for 5% of the randomly selected reflections not included in the refinement.

The crystals diffract and are inAnd refining the structure by resolution. The overall structure of BACE1 in complexes is very similar to its free form (Hong et al, Science 290: 150-153(2000)), which can be inRMSD was aligned at the C α carbon position of 96% (373/385) residues. YW412.8.31Fab is covered on the surface of BACE1 molecule And does not bind in the vicinity of the active site. The epitope comprises structural elements indicated by Hong et al (Science 290: 150-153(2000)) as loop C (amino acid 315-318 of full length BACE 1), D (amino acid 331-335 of full length BACE 1), and F (amino acid 370-381 of full length BACE 1), which are compactly positioned and in three-dimensional space. In addition, the portion of BACE1 at and near the yw412.8.31 binding site assumed a conformational change and resulted in a shape complementation score of 0.71, consistent with strong binding. Antibody-induced conformational changes are thought to promote allosteric inhibition of secretase activity.

Fab binds to an external binding site distant from the active site of BACE1 of amyloid precursor protein, which partially overlaps with an external binding site previously identified as the binding site for a set of BACE1 binding peptides (Kornecker et al, Biochemistry 44: 11567-, become random loops in the antibody complex, which adversely affects APP proteolytic cleavage, perhaps by preventing APP from entering the BACE1 catalytic cleft in a catalytic manner. This structural epitope includes the amino acid residue of BACE1 that comprises one or more atoms located within 4 angstroms of any portion of the yw412.8.31fab in the crystal structure. In table 6 below, the Fab light chain residues belong to the L chain and the Fab heavy chain residues belong to the H chain. The residue numbering of BACE1 amino acid is based on the full length sequence of BACE1 (SEQ ID NO: 49). Residue numbering of Fab amino acids is based on the Kabat numbering scheme (Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

TABLE 6-residues located in the YW412.8.31-BACE1 binding interface

BACE1 residue	Fab residues
		314SER	H26GLY
316GLU	H27PHE
		317LYS	H28THR
318PHE	H30LEU
		319PRO	H31GLY
327GLN	H32TYR
		328LEU	H53ALA
329VAL	H58ASP
		330CYS	H94ARG
331TRP	H96PRO
		332GLN	H97PHE
333ALA	H98SER
		335THR	H99PRO
337PRO	H100TRP
		340ILE
375THR	L49TYR
		378ASP	L53PHE
380CYS	L55TYR
		426PHE	L56SER
	L94TYR

The detailed atomic interactions are in the form of van der Waals contacts (van der Waalscreentact) which are polar interactions. Polar interactions include hydrogen bonds and salt bridges. Table 7 below includes a list of pairwise polar interactions between BACE1 and yw412.8.31fab. Fab light chain residues belong to the L chain, while Fab heavy chain residues belong to the H chain. The residue numbering of BACE1 amino acid is based on the full length sequence of BACE1 (SEQ ID NO: 49). Residue numbering of Fab amino acids is based on the Kabat numbering scheme.

TABLE 7 Pair-polar interactions between BACE1 and YW412.8.31Fab

BACE1 residue- -Fab residue
	314SER---H98SER
317LYS---H58ASP
	327GLN---H53ALA
330CYS---H31GLY
	331TRP---H98SER
331TRP---H32TYR
	332GLN---H32TYR
378ASP---H32TYR
	316GLU---L94TYR
332GLN---L55TYR
	335THR---L49TYR

As shown below, the amino acid composition in BACE1 epitope of yw412.8.31 antibody was poorly conserved in the corresponding region in BACE2 and cathepsin D. This amino acid difference in the epitope of the yw412.8.31 antibody is consistent with the following observations: this antibody is highly selective against BACE 1. The numbering is based on the full-length sequence of BACE1 (SEQ ID NO: 49). The sequences of BACE2 and cathepsin D are aligned to BACE1 based on their respective crystal structures. YW412.8.31BACE1 are boxed.

Example 4: in vivo characterization-mouse

The effect of yw412.8.31 was evaluated in vivo. To determine the maximum Abeta obtainable by a BACE 1-specific inhibitor_1-40Reduction, examination of A.beta.in plasma and forebrain of BACE1 vs. BACE 1-/-mice (compared to BACE1+/+ control)_1-40The resulting contribution. In BACE 1-/-mice, plasma A β_1-40The signal is reduced by 45%, while the brain A beta_1-40The signal decreased by 80% (fig. 16, panel a). These results suggest that BACE1 is indeed the major β -secretase in the forebrain, but that in the periphery BACE1 is responsible for only part of A β_1-40Produced, the remainder being from another beta-secretase.

The ability of the anti-BACE 1 antibody yw412.8.31 to modulate amyloidogenic processing in hAPP transgenic and wild type mice was evaluated under understanding the contribution of BACE1 to a β production.

hAPP transgenic mice

Briefly, 30mg/kg or 100mg/kg of YW412.8.31 antibody orMice expressing human APP at 5 months of age were treated with vehicle for a total of three doses (i.e., on days 1, 5, and 9). Animals were euthanized two hours after the final dose. Serum, plasma and brain were collected and processed. Plasma, cortex and hippocampus were analyzed for soluble A β using an amyloid β (A β) ELISA test kit according to The manufacturer's instructions (The Genetics Company) _1-40And Abeta_1-42And (4) horizontal. Pharmacokinetic analyses were performed on serum and brain homogenates.

The results show that: plasma Abeta at 30mg/kg and 100mg/kg YW412.8.31 antibody dose levels_1-40And Abeta_1-42The levels were all reduced to about 30% of the control levels (fig. 17(a), top panel, and fig. 18, panel a). However, the yw412.8.31 antibody, a β, at a dose level of 100mg/kg compared to that observed in wild-type mice discussed below_1-40And Abeta_1-42Levels in the brain were only reduced by 15-22% (fig. 17(a), bottom, and fig. 18, panel a). The concentration of the yw412.8.31 antibody in the brain of the treated animals increased in a dose-dependent manner, with the concentration of the antibody observed in 30mg/kg treated animals being 4.8 ± 3.6nM in the brain and the concentration of the antibody observed in 100mg/kg treated animals being 14.0 ± 9.3nM in the brain, confirming that the higher dose of antibody administered intraperitoneally did switch to the higher dose of antibody observed in the brain. Individual pharmacokinetic mapping to pharmacodynamic readings suggested a PK/PD relationship for this antibody in this model (fig. 17 (B)).

A similar experiment was also performed in which yw412.8.31 anti-BACE 1 antibody was delivered systemically or directly into hAPP transgenic mouse brains by continuous ICV infusion. For ICV delivery, antibodies were delivered continuously via a unilaterally implanted Alzet osmotic mini-pump (model 2001) for 7 days. The amount of yw412.8.31 antibody delivered was 0.041 mg/day (low dose) or 0.41 mg/day (high dose); control IgG at 0.33 mg/day was delivered to the control group. At euthanasia, plasma, cortex and hippocampus were collected and analyzed for soluble a β by elisa (the Genetics company) according to the manufacturer's instructions _1-40And Abeta_1-42The level of (c).

Table 8 below shows the concentration of yw412.8.31 antibody in the brain of mice dosed at 30mg/kg or 100mg/kg (by systemic delivery) or 0.041 mg/day and 0.41 mg/day (by ICV delivery).

TABLE 8

However, although the levels of antibody in the brain were high after infusion, the a β reduction was moderate at 15-23% and similar to that observed with systemic delivery (fig. 18, panel B). This observation suggests that while high dose systemic injection may be able to reduce a β levels in hAPP transgenic mice, the reduction is moderate. The reduced efficacy in hAPP transgenic mice is thought to be a result of animal models, as high concentrations in the brain, comparable to the concentration in serum after systemic delivery, did not further reduce a β production. Furthermore, the reduction in brain in hAPP transgenic mice was moderate compared to that observed in wild type mice and described below. Thus, transgenic hAPP mice may not be ideal for studying the in vivo effect of anti-BACE 1. Also from a disease perspective, wild-type mice are a more appropriate model for antibody efficacy, as an overwhelming majority of alzheimer patient populations harbor wild-type APP alleles.

Wild type mouse

The ability of the anti-BACE 1 antibody yw412.8.31 to modulate amyloid forming processing was also evaluated in wild type mice. Briefly, the experiment was performed as described above. A single dose of control IgG antibody or yw412.8.31 anti-BACE 1 antibody (50mg/kg) was delivered systemically to wild type mice by Intravenous (IV) injection. After 24 or 48 hours, plasma and brain samples were collected and analyzed for A β_1-40And (4) horizontal. Total mouse Abeta in plasma and brain was determined using a sandwich ELISA following a procedure similar to that described below for measuring total anti-BACE 1 antibody concentration_1-40The concentration of (c). In brief introductionThen, will be to Abeta_1-40The C-terminal specific rabbit polyclonal antibody (Millipore, Bedford, MA) was coated on a plate, and biotinylated anti-mouse a β monoclonal antibody M3.2(Covance, Dedham, MA) was used for detection. The assay has a lower limit of quantitation of 1.96Pg/ml in plasma and 39.1Pg/g in brain. As shown in FIGS. 16 and B, plasma Abeta_1-40Reduction of 35% and cortical a β_1-40The reduction is 20%.

Additional experiments with wild-type C5781/6J mice were performed in which 100mg/kg of YW412.8.31 or control IgG was administered systemically. Determination of A β in plasma and forebrain of treated animals four hours after a single Intraperitoneal (IP) injection _1-40And (4) horizontal. Blood was collected from the animals and plasma was isolated by cardiac puncture. After PBS perfusion, brains were harvested and the forebrain from one hemisphere (hemibrain) was prepared in PK buffer (1% NP-40 in PBS containing Roche complete protease inhibitor) while the forebrain from the other hemisphere was homogenized in 5M GuHCL, 50mM Tris pH8.0 and further diluted in casein blocking buffer (0.25% casein/0.05% sodium azide, 20. mu.g/ml aprotinin/5 mM EDTA, pH 8.0/10. mu.g/ml leupeptin in PBS) for A.beta._1-40And (6) analyzing.

As shown in FIG. 22, panel A, a 100mg/kg dose was able to convert plasma A β_1-40Reduce control levels by-50%, and resemble BACE1 knock-out levels described previously. However, no a β in the forebrain was detected 4 hours after dosing_1-40A change in (c). This early time point may be too close to administration of yw412.8.31 to see an effect in the brain. A longer period of time after administration may be required to observe reduced a β in the brain, particularly because a reduction in a β is observed in wild type mice treated at a lower dose (50mg/kg) at 24 hours, as described above. The concentration of YW412.8.31 in serum was very high, 1040. + -. 140. mu.g/mL (6.9. + -. 0.9. mu.M) at 4 hours after administration. The YW412.8.31 concentration in the brain is much lower, at 0.7 + -0.4 μ g/g (4.7 + -2.7 nM), which represents a 0.07% concentration in serum, very close to the predicted 0.1% steady state penetration of the antibody into the CNS (Reiber and Felgenerhauer, C) lin, chim, acta, 163: 319-328(1987). Importantly, the concentration of antibody obtained in brain (4.7 ± 2.7nM) approximates the cellular IC previously observed₅₀(FIG. 11). Thus, anti-BACE 1 antibodies are highly effective in vivo, e.g., by plasma A β_1-40Reduction to levels observed in BACE1 knockout mice. However, by 4 hours post-administration to mice, a single systemic dose did not result in brain loss, most likely because this time point was too early to observe any effect.

Additional experiments were performed to determine the effect of elevated brain antibody levels by repeated dosing. Yw412.8.31 antibody or control IgG was administered by intraperitoneal injection at 30 or 100mg/kg every 4 days for a total of 3 doses. In this study, a β in plasma and forebrain of treated animals was measured 4 hours after the last dose_1-40And (4) horizontal. Again, plasma A β was observed after multiple dosing at 30 and 100mg/kg_1-40The level was reduced by-50% (FIG. 22, panel B). Significantly, forebrain a β was observed at high doses of anti-BACE 1_1-40A 42% reduction, although no reduction was observed at low doses. After administration of 30 and 100mg/kg every 4 days, respectively, the YW412.8.31 antibody concentrations in serum were 480. + -. 210 and 1500. + -. 440. mu.g/mL, while the concentrations in brain were 0.9. + -. 0.6. mu.g/g (5.9. + -. 4.3nM) and 3.0. + -. 1.6. mu.g/g (20. + -. 10 nM). Thus, as predicted, higher antibody levels in the brain result in a dramatic reduction in a β levels. Notably, there was no difference in peripheral Α β levels at the 30mg/kg dose compared to the 100mg/kg dose, suggesting that maximum peripheral inhibition was obtained at 30mg/kg and therefore, simply lowering peripheral Α β levels was not sufficient to lower brain levels.

In addition, PK data were obtained after administration of yw412.8.31 anti-BACE 1 antibody in wild type and BACE1 knockout mice. See fig. 19. A single dose of anti-BACE 1(1 or 10mg/kg) was delivered to BALB/C mice via IV injection. Serum PK was analyzed until 21 days post dose.

The total anti-BACE 1 antibody concentration in mouse serum and brain samples was measured as follows. Antibody concentrations in mouse serum and brain samples were measured using enzyme-linked immunosorbent assay (ELISA).NUNC 384-well Maxisorp immunoplates (Neptune, NJ) were coated with F (ab') of donkey anti-human IgG, Fc fragment-specific polyclonal antibody (Jackson ImmunoResearch, West Grove, Pa.)₂Fragments, overnight at 4 ℃. The next day, the plates were blocked with Phosphate Buffered Saline (PBS) containing 0.5% Bovine Serum Albumin (BSA) for 1 hour at room temperature. Each antibody (control IgG and anti-BACE 1) was used as a standard to quantify the respective antibody concentrations. After washing the plates with PBS containing 0.05% Tween20 using a microplate washer (Bio-Tek Instruments, inc., Winooski, VT), standards and samples diluted in PBS containing 0.5% BSA, 0.35M NaCl, 0.25% CHAPS, 5mM EDTA, 0.05% Tween20 and 15ppm Proclin were incubated on the plates for 2 hours at room temperature with gentle stirring. F (ab') conjugated with horseradish peroxidase ₂Goat anti-human IgG, Fc specific polyclonal antibody (Jackson ImmunoResearch) detects bound antibody. Finally, the plates were developed using the substrate 3, 3 ', 5, 5' -Tetramethylbenzidine (TMB) (KPL, inc., Gaithersburg, MD). The absorbance was measured at a wavelength of 450nm (reference 630nm) on a Multiskan Ascent detector (Thermo Scientific, Hudson, NH). Concentrations were determined from standard curves using a four parameter non-linear regression procedure. The lower limit of quantitation (LLOQ) value of this assay was 3.12ng/ml in serum and 15.6ng/g in brain.

Free yw412.8.31 antibody concentration in mice was detected following a similar procedure as described above using BACE1ECD as coating (coat) and anti-human IgG, Fc specific antibody for detection (Jackson ImmunoResearch). The LLOQ value of the free anti-BACE 1 mouse ELISA was 0.626ng/ml in serum and 3.13ng/g in brain.

Two separate PK assays were used: one assay was used to detect all yw412.8.31 (total mAb) in serum, while one assay was used to detect only unbound yw412.8.31 (free mAb) in serum. The observed PK kinetics are non-linear, and the difference between total and free mAb values for samples where yw412.8.31 concentration < 10 μ g/mL suggests target-mediated clearance. See fig. 19, fig. a. Furthermore, the difference between total and unbound mAb indicates that some yw412.8.31 in serum is likely to bind soluble BACE 1. Single dose PK analyses in BACE1+/+, BACE1 +/-and BACE 1-/-mice confirmed the non-linearity observed in the initial study and indicate that the enhanced clearance is indeed target-mediated. BACE 1-/-mice showed linear PK. See fig. 19 (fig. B).

Example 5: in vivo characterization-monkey

Cynomolgus monkeys were dosed with control IgG or yw412.8.31 anti-BACE 1 antibody (30mg/kg) by IV delivery. Plasma and CSF were sampled up to 7 days prior to dosing to establish an average baseline Α β in each individual animal_1-40Level, then sampled at different times after dosing. Total anti-BACE 1 or control antibody concentrations in monkey serum and CSF samples were measured using monkey-adsorbed goat anti-human IgG polyclonal antibody (Bethyl, Montgomery, TX), both as coating and for detection (figure 20). BACE1ECD as a coating and monkey-adsorbed goat anti-human IgG antibody (Bethyl) for detection were used to determine the free anti-BACE 1 antibody concentration in monkeys. In serum or CSF, the LLOQ values determined for both total and free anti-BACE 1 monkeys were 6.25 ng/ml. PK was as expected for IgGl administered in monkeys and showed predicted exposure (exposure).

Also determined are Abeta of plasma and CSF from cynomolgus monkeys tested_1-40And (4) horizontal. Briefly, total cynomolgus monkey A β in plasma was determined using MSD MA6000 human (6E10) A β kit (catalog No. K111BVE-2, Meso Scale diagnostics) according to the manufacturer's instructions_1-40The concentration of (c). Will be for A beta_1-40The C-terminal specific capture antibody of (a) was pre-coated on the plate, and the Sulfo-Tag anti- Α β monoclonal antibody 6E10 was used for detection. The lower limit of quantitation of this assay in plasma was 49.4 pg/ml. Determination of total cynomolgus monkey Abeta in CSF Using Sandwich ELISA _1-40The concentration of (c). Will be for A beta_1-40The C-terminal specific rabbit polyclonal antibody of (cat. No. AB5737, Millipore, Bedford, MA) was coated on a plate, and biotinylated anti- Α β monoclonal antibody 6E10 (cat. No. SIG-39340,covance, Dedham, MA) was used for the detection. The lower limit of quantification of this assay in CSF is 15.6 pg/ml.

As shown in fig. 21 (panel a), plasma a β was present in all individuals_1-40Levels were reduced by-50% of baseline. The 50% maximal plasma Α β reduction was sustained throughout the 7 day observation period. The serum concentration-time profile (profile) of yw412.8.31 anti-BACE 1 antibody appeared similar to that observed for the control IgG antibody, suggesting that the kinetics were similar to those of the typical IgGl administered in the linear range (fig. 20, panel a). At the first sample collection 15 minutes after dosing, a peak serum antibody concentration of 800 μ g/mL was observed and dropped to 232 μ g/mL by 7 days after dosing. Notably, serum concentrations of yw412.8.31 exceeded cellular IC at all time points measured post-dose₅₀(-2.5 nM, see FIG. 11).

CSF Aβ_1-40Levels, as shown in figure 21 (panel B), although variable, showed a 50% reduction at 1 and 3 days post-dose, followed by a trend toward return to baseline a β at day 7 post-dose. The variability of baseline plasma and CSF levels is shown in figure 21 (figures C and D). In animals, baseline plasma levels were fairly consistent, while CSF Abeta _1-40The level may vary. Thus, all A β will be_1-40Measurements were normalized to baseline for each individual monkey.

These data show that a single dose of yw412.8.31 significantly reduced plasma and CSF Α β levels in monkeys. In CSF, a concentration of YW412.8.31 of 0.2-0.3. mu.g/ml was observed over this time, which shifted to 2nM (FIG. 20, panel B). From this data, it was concluded that the brain concentration of yw412.8.31 is within a similar range. Comparing PK and PD data, these results show: the exposure of the drug to plasma was sufficient to maximally inhibit A β production over a period of 7 days, while at the tested dose levels (30mg/kg), the drug concentration in CSF approached the cellular IC₅₀And transiently decrease a β levels in the brain. In summary, these data provide strong evidence that systemic administration of anti-BACE 1 can reduce BACE1 activity in the brain, such as by CSF Abeta in non-human primatesAnd (4) measuring and determining.

Example 6: affinity maturation of the YW412.8.31 antibody

The yw412.8.31 antibody undergoes affinity maturation as directed by the structural data provided by the crystal structure described previously. Antibody residues that are contacted with BACE1 are mutated to enhance the avidity of the yw412.8.31 antibody. The affinity matured clone prepared by this strategy was named yw412.8.31xs. Yw412.8.31 affinity matured clones were also prepared by soft randomization of all CDR targeting as described previously and named yw412.8.31x. The heavy and light chain variable sequences of clones that bind BACE1 are depicted in fig. 23(a) - (C) and 24(a) - (C).

Clones that bound BACE1 were tested for BACE1 protease inhibition in cell-based HTRF assays, as previously described in example 2C. The results of this assay are shown in fig. 25A and 25B. FIG. 25B shows A β from primary cortical neurons treated with different affinity matured anti-BACE 1 antibodies at the indicated concentrations for 24 hours_1-40Results were generated (pg/mi). Several antibodies tested inhibited BACE1 at levels similar to those observed with yw412.8.31.

＊＊＊＊＊＊＊＊＊

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and example should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims (73)

1. An isolated antibody or fragment thereof that binds BACE1, wherein the antibody reduces or inhibits the activity of a BACE1 polypeptide.

2. The antibody of claim 1, wherein said antibody binds to the active site of BACE 1.

3. The antibody of claim 1, wherein said antibody binds the exosite of BACE 1.

4. The antibody of claim 1, comprising at least one hypervariable region (HVR) sequence selected from the group consisting of: SEQ ID NO: 7-19, 22-26, 28-30, 35-47, 56-79 and 118- "122".

5. The antibody of claim 1, comprising at least one sequence selected from the group consisting of: HVR-H1, HVR-H2 and HVR-H3, wherein HVR-H1 comprises the amino acid sequence GFX₃₀FX₃₁X₃₂X₃₃X₃₄IH (SEQ ID NO: 45), wherein X₃₀N or T; x₃₁(ii) S, L or Y; x₃₂G or Y; x₃₃Y or S; and X₃₄A, G or S; HVR-H2 comprises amino acid sequence X₃₅X₃₆ISPX₃₇X₃₈GX₃₉TX₄₀YADSVKG (SEQ ID NO: 46), wherein X₃₅A or G; x₃₆W or S; x₃₇A or Y; x₃₈G or S; x₃₉(ii) S or Y; and X₄₀D or S; and HVR-H3 comprises amino acid sequence X₄₁PX₄₂X₄₃X₄₄X₄₅X₄₆X₄₇MDY (SEQ ID NO: 47), wherein X₄₁Q or G; x₄₂T or F; x₄₃H or S; x₄₄Y or P; x₄₅Y or W; x₄₆Y or V and wherein X₄₇Optionally comprising the sequence YAKGYKA (SEQ ID NO: 48).

6. The antibody of claim 1, comprising at least one sequence selected from the group consisting of: HVR-H1, HVR-H2 and HVR-H3, wherein HVR-H1 comprises the amino acid sequence GX₇₁X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇IH (SEQ ID NO: 120), wherein X₇₁F or Y; x₇₂F, N or T; x₇₃F or Y; x₇₄L, Q, I, S or Y; x₇₅G or Y; x₇₆Y or S; and X₇₇A, G or S; HVR-H2 comprises amino acid sequence X₇₈X₇₉ISPX₈₀X₈₁GX₈₂X₈₃X₈₄YADSVKG (SEQ ID NO: 121), wherein X₇₈A or G; x₇₉W or S; x₈₀A, S, Q or Y; x₈₁G or S; x₈₂(ii) S, K, L or Y; x₈₃T or Y; and X₈₄D or S; and HVR-H3 comprises amino acid sequence X₈₅PX₈₆X₈₇X₈₈X₈₉X₉₀X₉₁MDY (SEQ ID NO: 122), where X₈₅Q or G; x₈₆T or F; x₈₇H, Y or S; x₈₈Y or P; x₈₉Y or W; x₉₀Y or V and wherein X₉₁Optionally comprising the sequence YAKGYKA (SEQ ID NO: 48).

7. The antibody of claim 1, comprising at least one sequence selected from the group consisting of: HVR-H1, HVR-H2 and HVR-H3, wherein HVR-H1 comprises the amino acid sequence GX₅₃X₅₄X₅₅X₅₆GYGIH (SEQ ID NO: 68), wherein X₅₃F or Y; x₅₄T or F; x₅₅F or Y; x₅₆(ii) L, Q or I, HVR-H2 comprises the amino acid sequence GWISPX₅₇X₅₈GX₅₉X₆₀DYASVKG (SEQ ID NO: 69), wherein X₅₇A, S or Q; x ₅₈G or S; x₅₉S, K or L; x₆₀Wherein the HVR-H3 sequence comprises the amino acid sequence GPFX₆₁PWVMDY (SEQ ID NO: 70), wherein X₆₁S or Y or SEQ ID NO: 79.

8. The antibody of claim 5, comprising an HVR-H1 sequence comprising the amino acid sequence GFTFX, and the HVR-H1 sequence₁₃GYX₁₄IH (SEQ ID NO: 26), wherein X₁₃Or L and X₁₄A or G.

9. The antibody of claim 6, comprising an HVR-H1 sequence, which HVR-H1 sequence comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 28 and SEQ ID NO: 71-73.

10. The antibody of claim 6, comprising an HVR-H2 sequence comprising an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 24, SEQ ID NO: 29 and SEQ ID NO: 74-78.

11. The antibody of claim 6, comprising an HVR-H3 sequence comprising an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 25; SEQ ID NO: 30 and SEQ ID NO: 79.

12. the antibody of claim 6, comprising HVR-H1, HVR-H2, and HVR-H3 sequences, the HVR-H1, HVR-H2, and HVR-H3 sequences corresponding to those shown for clones YW412.8, yw412.8.31, yw412.8.30, yw412.8.2, yw412.8.29, and YW412.8.51 in figure 1(B) or the clones in figures 24(a) - (C).

13. The antibody of claim 6, comprising HVR-H1, HVR-H2, and HVR-H3 sequences, the HVR-H1, HVR-H2, and HVR-H3 sequences corresponding to those shown for clones Fab12, LC6, LC9, and LC10 in figure 2 (B).

14. The antibody of claim 6, comprising the amino acid sequence of SEQ ID NO: 22 or 23, the HVR-H1 sequence of SEQ ID NO: 24 and the HVR-H2 sequence of SEQ ID NO: 25, HVR-H3 sequence.

15. The antibody of claim 14, wherein the HVR-H1 sequence is SEQ ID NO: 23.

16. the antibody of claim 6, comprising the amino acid sequence of SEQ ID NO: 28, the HVR-H1 sequence of SEQ ID NO: 29, and the HVR-H2 sequence of SEQ ID NO: 30, HVR-H3 sequence.

17. The antibody of claim 6, comprising a VH chain having an amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 20, 21, 27 and 80-98.

18. The antibody of claim 16, wherein the VH chain amino acid sequence is SEQ id no: 21.

19. the antibody of claim 1, comprising at least one sequence selected from the group consisting of seq id no: HVR-L1, HVR-L2, and HVR-L3, wherein HVR-L1 comprises the amino acid sequence RASQX₁₇VX₁₈X₁₉X₂₀X₂₁A (SEQ ID NO: 42), wherein X₁₇(ii) S, D or V; x₁₈(ii) S or a; x ₁₉(ii) S, T or N; x₂₀A or S; x₂₁(vi) V or L, HVR-L2 comprises the amino acid sequence X₂₂ASX₂₃LYS (SEQ ID NO: 43), wherein X₂₂(ii) S, W, Y or L; x₂₃F, S or W, and HVR-L3 comprises the amino acid sequence QQX₂₄X₂₅X₂₆X₂₇X₂₈X₂₉T (SEQ ID NO: 44), wherein X₂₄(ii) S, F, G, D or Y; x₂₅Y, P, S or a; x₂₆Y, T or N; x₂₇T, Y, D or S; x₂₈P or L; and X₂₉F, P or T.

20. The antibody of claim 1, comprising at least one sequence selected from the group consisting of seq id no: HVR-L1, HVR-L2, and HVR-L3, wherein HVR-L1 comprises the amino acid sequence RASQX₁₇VX₁₈X₁₉X₂₀X₂₁A (SEQ ID NO: 42), wherein X₁₇(ii) S, D or V; x₁₈(ii) S or a; x₁₉(ii) S, T or N; x₂₀A or S; x₂₁(vi) V or L, HVR-L2 comprises the amino acid sequence X₆₂ASX₆₃X₆₄YX₆₅(SEQ ID NO: 118) in which X₆₂S, W, Y, F or L; x₆₃＝F，S，Y or W; x₆₄L or R; x₆₅(iii) S, P, R, K or W, and HVR-L3 comprises the amino acid sequence QQX₆₆X₆₇X₆₈X₆₉X₇₀X₇₁T (SEQ ID NO: 119), wherein X₆₆(ii) S, F, G, D or Y; x₆₇Y, P, S or a; x₆₈Y, T or N; x₆₉T, Y, D or S; x₇₀P, Q, S, K or L; and X₇₁F, P or T.

21. The antibody of claim 1, comprising at least one sequence selected from the group consisting of: HVR-L1, HVR-L2, and HVR-L3, wherein HVR-L1 comprises the amino acid sequence RASQX ₁VX₂X₃X₄X₅A (SEQ ID NO: 17), wherein X₁D or V; x₂(ii) S or a; x₃T or N; x₄(ii) S or a; x₅(vi) V or L, HVR-L2 comprises the amino acid sequence X₄₈ASX₄₉X₅₀YX₅₁(SEQ ID NO: 56), wherein X₄₈(ii) S or F; x₄₉F or Y; x₅₀L or R; x₅₁(iii) S, P, R, K or W, and HVR-L3 comprises the amino acid sequence QQFPTYX₅₂PT (SEQ ID NO: 57), wherein X₅₂L, Q, S or K.

22. The antibody of claim 17, comprising an HVR-L1 sequence comprising the amino acid sequence RASQX₁VX₂X₃X₄X₅A (SEQ ID NO: 17), wherein X₁D or V; x₂(ii) S or a; x₃T or N; x₄(ii) S or a; x₅V or L.

23. The antibody of claim 19, comprising a HVR-L2 sequence, the HVR-L2 sequence comprising the amino acid sequence X₆ASFLYS (SEQ ID NO: 18) or X₁₅ASX₁₆LYS (SEQ ID NO: 41), wherein X₆(ii) S or L; x₁₅(ii) S, W or Y; and X₁₆S or W。

24. The antibody of claim 19, comprising a HVR-L3 sequence, the HVR-L3 sequence comprising the amino acid sequence qqqx₇X₈X₉X₁₀X₁₁X₁₂T (SEQ ID NO: 19), wherein X₇(ii) S, F, G, D or Y; x₈Y, P, S, or a; x₉T or N; x₁₀T, Y, D or S; x₁₁P or L; x₁₂P or T.

25. The antibody of claim 19, comprising an HVR-L1 sequence comprising an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 35.

26. The antibody of claim 20, comprising an HVR-L2 sequence comprising an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 36-39 and SEQ ID NO: 58-64.

27. The antibody of claim 20, comprising an HVR-L3 sequence comprising an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 11-16, seq id NO: 40 and SEQ ID NO: 65-67.

28. The antibody of claim 20, which comprises HVR-L1, HVR-L2, and HVR-L3 sequences, which HVR-L1, HVR-L2, and HVR-L3 sequences correspond to those shown for clones YW412.8, yw412.8.31, yw412.8.30, yw412.8.2, yw412.8.29, and YW412.8.51 in figure 1(a) and clones in figures 23(a) - (C).

29. The antibody of claim 20, which comprises HVR-L1, HVR-L2, and HVR-L3 sequences, which HVR-L1, HVR-L2, and HVR-L3 sequences correspond to those shown for clones Fab12, LC6, LC9, and LC10 in figure 2 (a).

30. The antibody of claim 20, comprising the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, HVR-L1 sequence; SEQ ID NO: 9 or SEQ ID NO: 10, HVR-L2 sequence; and is selected from the group consisting of SEQ ID NO: 11-16.

31. The antibody of claim 30, wherein the HVR-L1 sequence is SEQ ID NO: 7, the HVR-L2 sequence is SEQ ID NO: 9, and the HVR-L3 sequence is SEQ ID NO: 12.

32. the antibody of claim 20, comprising the amino acid sequence of SEQ ID NO: 35, the HVR-L1 sequence; selected from the group consisting of SEQ ID NO: 36-39 and the HVR-L2 sequence of the group consisting of SEQ ID NO: 40, HVR-L3 sequence.

33. The antibody of claim 20, comprising a VL chain sequence having an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 1-6, 31-34 and 99-117.

34. The antibody of claim 33, wherein the VL chain amino acid sequence is SEQ ID NO: 2.

35. the antibody of claim 15, further comprising a heavy chain variable region comprising SEQ ID NO: 7, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9 and HVR-L2 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L3.

36. The antibody of claim 34, further comprising a VH chain comprising the amino acid sequence of SEQ ID NO: 21.

37. An isolated antibody or fragment thereof that binds an epitope comprising at least one amino acid residue of BACE1 selected from the group consisting of: SEQ ID NO: 314SER of 49; 316 GLU; 317 LYS; 327 GLN; 330 CYS; 331 TRP; 332 GLN; 335 THR; and 378 ASP.

38. The antibody of claim 37, wherein the epitope comprises SEQ ID NO: 314SER of 49; 316 GLU; 317 LYS; 327 GLN; 330 CYS; 331 TRP; 332 GLN; 335 THR; and 378 ASP.

39. An isolated antibody or fragment thereof that binds an epitope comprising at least one amino acid region of BACE1 selected from the group consisting of: SEQ ID NO: amino acids 315 of 49 and 318; SEQ ID NO: amino acids 331-335 of 49; SEQ ID NO: amino acids 370-381 of 49; and any combination thereof.

40. The antibody of claim 39, wherein the epitope comprises SEQ ID NO: amino acids 315, 331, 335 and 370, 381 of 49.

41. An isolated antibody or fragment thereof that binds to an epitope of BACE1 and which upon binding results in a conformational change in the structure of the P6 and P7 sites of BACE 1.

42. An isolated antibody or fragment thereof that binds to an epitope of BACE1 and that upon binding induces the amino acid sequence of SEQ ID NO: amino acids 218-231 of 49 adopt a random loop structure.

43. The antibody of claims 37-42, wherein said antibody reduces or inhibits the activity of BACE 1.

44. The antibody of any one of claims 1-43, which is a monoclonal antibody.

45. The antibody of any one of claims 1-44, which is a human, humanized, or chimeric antibody.

46. The antibody of any one of claims 1-45, which is an antibody fragment.

47. The antibody of any one of claims 1-45, which is a full length IgG1 antibody.

48. An isolated nucleic acid encoding the antibody of any one of claims 1-47.

49. A host cell comprising the nucleic acid of claim 48.

50. A method of producing an antibody, the method comprising culturing the host cell of claim 49 to produce the antibody.

51. An immunoconjugate comprising the antibody of any one of claims 1-47 and a cytotoxic agent.

52. A pharmaceutical formulation comprising the antibody of any one of claims 1-47 and a pharmaceutically acceptable carrier.

53. A method of treating a subject having a neurological disease or disorder, said method comprising administering to said subject an effective amount of the antibody of any one of claims 1-47.

54. A method of reducing amyloid plaques in a patient having or at risk of contracting a neurological disease or disorder, comprising administering to the individual an effective amount of the antibody of any one of claims 1-47.

55. A method of inhibiting amyloid plaque formation in a patient having or at risk of developing a neurological disease or disorder, comprising administering to the individual an effective amount of the antibody of any one of claims 1-47.

56. The method of any one of claims 53-55, wherein the neurological disease or disorder is selected from the group consisting of: alzheimer's Disease (AD), traumatic brain injury, stroke, glaucoma, dementia, Muscular Dystrophy (MD), Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS), cystic fibrosis, Agilerman syndrome, Leidel syndrome, Paget's disease, traumatic brain injury, Lewy body disease, post-polio syndrome, Charpy-Derager's syndrome, Olive pontine cerebellar atrophy, Parkinson's disease, multiple system atrophy, striatal substantia nigra degeneration, supranuclear palsy, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, Kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, fatal familial insomnia, bulbar palsy, motor neuron disease, Carnanvan disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexandria, Tourette syndrome, Mengkins knot syndrome, kekahn syndrome, Hallervorden-Spatz syndrome, Lafula's disease, Rett syndrome, hepatolenticular degeneration, Leishin-Nehn syndrome, and Pulsatilla-Long syndrome, pick's disease, and spinocerebellar ataxia.

57. The method of claim 56, wherein the neurological disease or disorder is selected from the group consisting of: alzheimer's disease, stroke, traumatic brain injury and glaucoma.

58. A method of reducing amyloid- β (A β) protein in a patient, the method comprising administering to the patient an effective amount of the antibody of any one of claims 1-47.

59. The method of claim 58, wherein the patient has, or is at risk of contracting, a neurological disease or disorder.

60. The method of claim 59, wherein the neurological disease or disorder is selected from the group consisting of: alzheimer's disease, stroke, traumatic brain injury and glaucoma.

61. A method of diagnosing a neurological disease or disorder in a patient, said method comprising contacting a biological sample isolated from said patient with an antibody of any one of claims 1-47 under conditions suitable for binding of said antibody to a BACE1 polypeptide, and detecting whether a complex is formed between said antibody and said BACE1 polypeptide.

62. A method of determining whether a patient is suitable for treatment with an anti-BACE 1 antibody, the method comprising contacting a biological sample isolated from said patient with an antibody according to any one of claims 1-47 under conditions suitable for binding of said antibody to a BACE1 polypeptide and detecting whether a complex is formed between said antibody and said BACE1 polypeptide, wherein the presence of a complex between said antibody and BACE1 indicates that the patient is suitable for treatment with an anti-BACE 1 antibody.

63. The method of claim 61 or 62, wherein the biological sample is selected from the group consisting of: serum, plasma, saliva, gastric secretions, mucus, cerebrospinal fluid, lymph, neural tissue, brain tissue, cardiac tissue or vascular tissue.

64. The antibody of any one of claims 1-47 for use as a medicament.

65. The antibody of any one of claims 1-47 for use in treating a neurological disease selected from the group consisting of: alzheimer's Disease (AD), traumatic brain injury, stroke, glaucoma, dementia, Muscular Dystrophy (MD), Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS), cystic fibrosis, Agilerman syndrome, Leidel syndrome, Paget's disease, traumatic brain injury, Lewy body disease, post-polio syndrome, Charpy-Derager's syndrome, Olive pontine cerebellar atrophy, Parkinson's disease, multiple system atrophy, striatal substantia nigra degeneration, supranuclear palsy, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, Kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, fatal familial insomnia, bulbar palsy, motor neuron disease, Carnanvan disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexandria, Tourette syndrome, Mengkins knot syndrome, kekahn syndrome, Hallervorden-Spatz syndrome, Lafula's disease, Rett syndrome, hepatolenticular degeneration, Leishin-Nehn syndrome, and Pulsatilla-Long syndrome, pick's disease, and spinocerebellar ataxia.

66. The antibody of any one of claims 1-47 for use in reducing and/or inhibiting amyloid- β (A β) protein production.

67. Use of the antibody of any one of claims 1-47 in the manufacture of a medicament.

68. The use of claim 67, wherein the medicament is for treating a neurological disease selected from the group consisting of: alzheimer's Disease (AD), traumatic brain injury, stroke, glaucoma, dementia, Muscular Dystrophy (MD), Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS), cystic fibrosis, Agilerman syndrome, Leidel syndrome, Paget's disease, traumatic brain injury, Lewy body disease, post-polio syndrome, Charpy-Derager's syndrome, Olive pontine cerebellar atrophy, Parkinson's disease, multiple system atrophy, striatal substantia nigra degeneration, supranuclear palsy, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, Kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, fatal familial insomnia, bulbar palsy, motor neuron disease, Carnanvan disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexandria, Tourette syndrome, Mengkins knot syndrome, kekahn syndrome, Hallervorden-Spatz syndrome, Lafula's disease, Rett syndrome, hepatolenticular degeneration, Leishin-Nehn syndrome, and Pulsatilla-Long syndrome, pick's disease, and spinocerebellar ataxia.

69. The use of claim 67, wherein said medicament is for reducing and/or inhibiting amyloid- β (A β) protein production.

70. A BACE1 epitope specifically recognized by an antibody or fragment thereof, said BACE1 epitope comprising at least one amino acid residue of BACE1 corresponding to an amino acid selected from the group consisting of: SEQ ID NO: 314SER of 49; 316 GLU; 317 LYS; 327 GLN; 330 CYS; 331 TRP; 332 GLN; 335 THR; and 378 ASP.

71. The BACE1 epitope of claim 70 wherein the epitope comprises a sequence corresponding to seq id NO: 314SER of 49; 316 GLU; 317 LYS; 327 GLN; 330 CYS; 331 TRP; 332 GLN; 335 THR; and amino acids of 378 ASP.

72. A BACE1 epitope specifically recognized by an antibody or fragment thereof, said BACE1 epitope comprising at least one region of amino acids of BACE1 selected from the group consisting of: SEQ ID NO: amino acids 315 of 49 and 318; SEQ ID NO: amino acids 331-335 of 40; SEQ ID NO: amino acids 370-381 of 49; and any combination thereof.

73. The BACE1 epitope of claim 72 wherein the epitope comprises the amino acid sequence of SEQ id no: amino acids 315, 331, 335 and 370, 381 of 49.