阅读说明：本技术 抗PHF-tau抗体及其用途 (anti-PHF-tau antibodies and uses thereof ) 是由 M.默肯 T.马利亚 M.博格斯 K.范科伦 于 2018-03-16 设计创作，主要内容包括：本发明描述了单克隆抗PHF-tau抗体及其抗原结合片段。还描述了编码抗体的核酸、包含抗体的组合物、产生抗体以及使用抗体治疗或预防病症诸如tau蛋白病变的方法。(Monoclonal anti-PHF-tau antibodies and antigen binding fragments thereof are described. Also described are nucleic acids encoding the antibodies, compositions comprising the antibodies, methods of producing the antibodies, and methods of using the antibodies to treat or prevent disorders such as tauopathies.)

1. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to phosphorylated tau protein at a phosphorylated epitope comprising:

(a) phosphorylated T212 and phosphorylated T217 of the tau protein, and the phosphorylated epitope has the amino acid sequence of SEQ ID NO: 48 or in the amino acid sequence of SEQ ID NO: 48;

(b) phosphorylated T217 of the tau protein, and the phosphorylated epitope has the amino acid sequence of SEQ ID NO: 52 or in the amino acid sequence of SEQ ID NO: 52; or

(c) Phosphorylated T212 of the tau protein, and the phosphorylated epitope has the amino acid sequence of SEQ ID NO: 54 or in the amino acid sequence of SEQ ID NO: 54, or a pharmaceutically acceptable salt thereof.

2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the monoclonal antibody comprises the amino acid sequence of SEQ ID NO: 1. HCDR1 of any one of 4, 7, 10, 71, 80; SEQ ID NO: 2.5, 8, 11, 72, 81, HCDR 2; SEQ ID NO: 3. 6, 9, 12, 73, HCDR 3; SEQ ID NO: 13. LCDR1 of any one of 16, 19, 22, 70; SEQ ID NO: 14. LCDR2 of any one of 17, 20, 23; SEQ ID NO: 15. LCDR3 of any one of 18, 21, 24.

3. The isolated monoclonal antibody or antigen-binding fragment of claim 2, comprising:

(a) have the amino acid sequences of SEQ ID NO: 7. 8 and 9 and immunoglobulin heavy chain complementarity determining regions (HCDR) HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 19. 20 and 21, the immunoglobulin light chain complementarity determining regions (LCDR) LCDR1, LCDR2 and LCDR 3;

(b) have the amino acid sequences of SEQ ID NO: 1. 2 and 3 and HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of seq id NOs: 13. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 14 and 15;

(c) have the amino acid sequences of SEQ ID NO: 4.5 and 6 and HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of seq id NOs: 16. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 17 and 18;

(d) have the amino acid sequences of SEQ ID NO: 10. 11 and 12 and HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 22. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 23 and 24;

(e) have the amino acid sequences of SEQ ID NO: 80. 81 and 9 and HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 70. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 20 and 21;

(f) have the amino acid sequences of SEQ ID NO: 71. 72, 73 and HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 70. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 20 and 21;

(g) have the amino acid sequences of SEQ ID NO: 71. 72 and 73 and HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 19. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 20 and 21;

(h) has the sequence shown in SEQ ID NO: 26 of a polypeptide sequence V_HHCDR1, HCDR2 and HCDR3 of the regions and a nucleic acid molecule having the sequence of SEQ ID NO: 31 of the polypeptide sequence V_LLCDR1, LCDR2, and LCDR3 of the region;

(i) has the sequence shown in SEQ ID NO: 28 of the polypeptide sequence V_HHCDR1, HCDR2 and HCDR3 of the regions and a nucleic acid molecule having the sequence of SEQ ID NO: 34 of the polypeptide sequence V_LLCDR1, LCDR2, and LCDR3 of the region;

(j) has the sequence shown in SEQ ID NO: 26 of a polypeptide sequence V_HHCDR1, HCDR2 and HCDR3 of the regions and a nucleic acid molecule having the sequence of SEQ ID NO: 34 of the polypeptide sequence V_LLCDR1, LCDR2, and LCDR3 of the region; or

(k) Has the sequence shown in SEQ ID NO: 28 of the polypeptide sequence V_HHCDR1, HCDR2 and HCDR3 of the regions and a nucleic acid molecule having the sequence of SEQ ID NO: 31 of the polypeptide sequence V_LLCDR1, LCDR2, and LCDR3 of the region;

wherein the antibody or antigen binding fragment thereof binds PHF-tau.

4. The isolated monoclonal antibody or antigen-binding fragment of claim 3, comprising a light chain variable region having a sequence identical to SEQ ID NO: 26. 27, 28, and 29, and a heavy chain variable region having a polypeptide sequence at least 95% identical to any one of seq id NOs: 31. 32, 33, and 34, or a light chain variable region of a polypeptide sequence that is at least 95% identical to the polypeptide sequence.

5. The isolated monoclonal antibody or antigen-binding fragment of claim 4, comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 26. 27, 28 and 29, and a heavy chain variable region having the polypeptide sequence of any one of SEQ ID NOs: 31. 32, 33 and 34, or a light chain variable region of the polypeptide sequence of any one of seq id no.

6. The isolated monoclonal antibody or antigen-binding fragment of claim 3, comprising a heavy chain variable region having a sequence identical to a sequence comprising SEQ ID NO: 45. v of the heavy chain of the polypeptide sequence of any one of 74, 76 and 78_HA heavy chain variable region having a polypeptide sequence of at least 95% identity to SEQ ID NO: 31. 75, 77 and 79, and a polypeptide sequence having at least 95% identity to the VL of the light chain.

7. The isolated monoclonal antibody or antigen-binding fragment of claim 6, comprising a light chain variable region having a sequence comprising SEQ id no: 45. v of the heavy chain of the polypeptide sequence of any one of 74, 76 and 78_HAnd a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 31. 75, 77 and79 of the light chain of any one of the above.

8. The isolated monoclonal antibody or antigen-binding fragment of claim 3, comprising

(a) Has the sequence shown in SEQ ID NO: 26 of a polypeptide sequence V_HAnd a polypeptide having the sequence of SEQ ID NO: 31, VL of the polypeptide sequence of;

(b) has the sequence shown in SEQ ID NO: 28 of the polypeptide sequence V_HAnd a polypeptide having the sequence of SEQ ID NO: 34, VL of the polypeptide sequence of;

(c) has the sequence shown in SEQ ID NO: 26 of a polypeptide sequence V_HAnd a polypeptide having the sequence of SEQ ID NO: 34, VL of the polypeptide sequence of;

(d) has the sequence shown in SEQ ID NO: 28 of the polypeptide sequence V_HAnd a polypeptide having the sequence of SEQ ID NO: 31, VL of the polypeptide sequence of;

(e) has the sequence shown in SEQ ID NO: 27 of the polypeptide sequence V_HAnd a polypeptide having the sequence of SEQ ID NO: 31, VL of the polypeptide sequence of;

(f) SEQ ID NO: 74 of the heavy chain V_HAnd SEQ ID NO: v of light chain of 75_L；

(g) SEQ ID NO: v of the heavy chain of 76_HAnd SEQ ID NO: 77V of the light chain_L(ii) a Or

(h) SEQ ID NO: v of heavy chain of 78_HAnd SEQ ID NO: 79V of light chain_L。

9. The isolated monoclonal antibody or antigen-binding fragment of claim 8, comprising a light chain variable region having a sequence identical to SEQ id no: 45. 74, 76, and 78, and a heavy chain having a polypeptide sequence at least 95% identical to any one of SEQ id nos: 46. a light chain of a polypeptide sequence that is at least 95% identical to any one of 75, 77 and 79.

10. The isolated monoclonal antibody or antigen-binding fragment of claim 9, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 45. 74, 76, and 78, and a light chain having the polypeptide sequence of any one of SEQ ID NOs: 46. 75, 77 and 79.

11. The isolated monoclonal antibody or antigen-binding fragment of claim 10, comprising:

(a) SEQ ID NO: 45 and SEQ ID NO: 46 with a light chain;

(b) SEQ ID NO: 74 and SEQ ID NO: 75 a light chain;

(c) SEQ ID NO: 76 and the heavy chain of SEQ ID NO: 77; or

(d) SEQ ID NO: 78 and SEQ ID NO: 79 light chain.

12. The isolated monoclonal antibody or antigen-binding fragment of any one of claims 1 to 11, comprising a human heavy chain IgG1 constant region and a human light chain kappa constant region.

13. An isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 12.

14. A vector comprising the isolated nucleic acid of claim 13.

15. A host cell comprising the nucleic acid of claim 14.

16. A pharmaceutical composition comprising an isolated monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

17. A method of reducing pathological tau aggregation or tauopathy propagation in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition according to claim 16.

18. A method of treating a tauopathy in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of claim 16.

19. The method of claim 18, wherein the tauopathy is selected from the group consisting of familial alzheimer's disease, sporadic alzheimer's disease, frontotemporal dementia associated with chromosome 17 and parkinson's syndrome (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, pick's disease, progressive subcortical hyperplasia, tangle-dementia only, diffuse neurofibrillary tangle with calcification, silvery particle dementia, amyotrophic lateral sclerosis parkinson's syndrome-dementia complex, down's syndrome, gerstmann-straussler-scheinker disease, halleword-schutz disease, inclusion body myositis, creutzfeldt-jakob disease, multiple system atrophy, niemann-pick type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-guam motor neuronopathy with neurotangles, neurofibrillary, and combinations thereof, Postencephalitic parkinsonism, chronic traumatic encephalopathy, and dementia pugilistica (boxing disease).

20. A method of producing the monoclonal antibody or antigen-binding fragment of any one of claims 1 to 12, comprising culturing a cell comprising a nucleic acid encoding the antibody or antigen-binding fragment under conditions in which the antibody or antigen-binding fragment is produced, and recovering the antibody or antigen-binding fragment from the cell or cell culture.

21. A method of detecting the presence of PHF-tau in a biological sample from a subject, the method comprising contacting the biological sample with the antibody or antigen-binding fragment of any one of claims 1 to 12, and detecting binding of the antibody or antigen-binding fragment to PHF-tau in the sample from the subject.

22. The method of claim 21, wherein the biological sample is a blood, serum, plasma, interstitial fluid, or cerebrospinal fluid sample.

Technical Field

The present invention relates to anti-PHF-tau antibodies, nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors, and compositions comprising the antibodies. Also provided are methods of making the antibodies, methods of using the antibodies to treat disorders including tauopathies, and methods of using the antibodies to diagnose diseases such as tauopathies.

Background

Alzheimer's Disease (AD) is a degenerative brain disorder characterized by progressive loss of memory, cognition, reasoning, judgment and emotional stability that gradually leads to extreme mental decline and ultimately death. AD is a very common cause of progressive mental disorders (dementia) in the elderly, and is considered the fourth most common medical cause of death in the united states. AD has been observed in ethnic groups worldwide and represents a major public health problem both currently and in the future.

The brain of individuals with AD exhibits characteristic lesions known as senile (or amyloid) plaques, amyloid angiopathy (deposition of amyloid in blood vessels), and neurofibrillary tangles. A large number of these lesions, particularly amyloid plaques and neurofibrillary tangles of paired helical filaments, are commonly found in several regions of the human brain important for memory and cognitive function in patients with AD.

Current AD treatment regimens include only therapies approved for the treatment of cognitive symptoms in patients with dementia. There is no approved therapy to alter or slow the progression of AD. Potential disease modulators include humanized anti-a of EliLilly for patients with mild AD_βMonoclonal solivacizumab, and small molecule BACE inhibitor Verubecestat of Merck for patients with mild to moderate AD. These therapies, and most other potential disease modulators that may be marketed in the next decade, target a_β(the major component of amyloid plaques, i.e., one of the two "hallmark" pathological signs of AD).

Neurofibrillary tangles, the second hallmark pathological feature of AD, are composed mainly of aggregates of hyperphosphorylated tau protein. the main physiological function of tau is microtubule polymerization and stabilization. Binding of tau to microtubules occurs through ionic interactions between the positive charges of the microtubule binding domain of tau and the negative charges on the microtubule network frame (Butner and Kirschner, J Cell biol.115 (3): 717-30, 1991). tau protein contains 85 possible phosphorylation sites, and phosphorylation at many of these sites interferes with the major function of tau. Tau bound to the axonal microtubule network is in a hypophosphorylated state, whereas aggregated tau in AD is hyperphosphorylated, providing a unique epitope distinct from the physiologically active pool of tau.

the hypothesis of propagation and spread of tauopathies (tauopathies) has been described and is based on the Braak stage of development of tauopathies in the human brain and the spread of tauopathies following tau aggregate injection in preclinical tau models (Frost et al, J Biol chem.284: 12845-52, 2009; Clavaguera et al, Nat Cell biol.11: 909-13, 2009).

Development of therapeutic agents to prevent or clear tau aggregation has been of interest for many years, and Drug candidates, including anti-aggregation compounds and kinase inhibitors, have entered clinical testing (Brunden et al, Nat Rev Drug Discov.8: 783-93, 2009). Several studies have been published showing beneficial therapeutic effects of both active and passive tau immunity in transgenic mouse models (Chai et al, J biol chem.286: 34457-67, 2011; Boutajango et al, J neurochem.118: 658-67, 2011; Boutaiangout et al, J neurosci.30: 16559-66, 2010; Asuni et al, JNeurosci.27: 9115-29, 2007). The activity of both phospho-and non-phospho-targeted antibodies has been reported (Schroeder et al, J Neurohimmune Pharmacol.11 (1): 9-25, 2016).

Despite advances in the art, there remains a need for effective therapeutic agents that prevent tau aggregation and the development of tauopathies to treat tauopathies such as AD and other neurodegenerative diseases.

Disclosure of Invention

The present invention fills this need by providing anti-helical filament (PHF) -tau antibodies or antigen-binding fragments thereof that have high binding affinity for paired-helical filament (PHF) -tau and are selective for phosphorylated tau. The antibodies of the invention were generated by Human Framework Adaptation (HFA) of mouse PHF-tau specific antibodies. It is believed that the selectivity of the antibody for phosphorylated tau allows efficacy against pathogenic tau without interfering with normal tau function. The invention also provides nucleic acids encoding the antibodies, compositions comprising the antibodies, and methods of making and using the antibodies. The anti-PHF-tau antibody or antigen-binding fragment thereof of the invention inhibits tau seeds as measured by a cellular assay using tau seeds derived from HEK cell lysate or spinal cord lysate of mutant tau transgenic mice. In addition, chimeric antibodies with the variable regions of the anti-PHF-tau antibody of the invention and a mouse Ig constant region (such as the mouse IgG2a constant region) blocked seeding activity in an in vivo mutant tau transgenic mouse model.

The development of tauopathies in the AD brain follows a distinct and characteristic spreading pattern. Preclinical models have shown that extracellular phosphotau seeds can induce tauopathies in neurons (Clavaguera et al, PNAS 110 (23): 9535-40, 2013). Thus, it is believed that tauopathies can spread from one brain region to the next in a prion-like manner. This spreading process will involve the externalisation of tau seeds, which can be taken up by nearby neurons and induce further tauopathies. Without being bound by theory, it is believed that the anti-PHF-tau antibodies or antigen binding fragments thereof of the present invention prevent tau aggregation or the spread of tauopathies in the brain by interacting with phosphotau seeds.

In one general aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof that binds PHF-tau. In a specific embodiment, the antibody is a humanized monoclonal antibody.

According to a particular aspect, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to phosphorylated tau protein at a phosphorylated epitope of the proline-rich domain of the tau protein. In a more specific aspect, the phosphorylated epitope comprises phosphorylated T212 and/or phosphorylated T217 of the tau protein, and the phosphorylated epitope has the amino acid sequence of SEQ ID NO: 48. 52 and 54 or in the amino acid sequence of SEQ ID NO: 48. 52 and 54, or a pharmaceutically acceptable salt thereof. In some embodiments, the antibodies of the invention bind to a phosphorylated epitope comprising phosphorylated T212 and phosphorylated T217 of tau protein.

According to a particular aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising:

(1) have the amino acid sequences of SEQ ID NO: 4.5 and 6 and immunoglobulin heavy chain complementarity determining regions (HCDR) HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 16. 17 and 18, an immunoglobulin light chain complementarity determining region (LCDR) LCDR1, LCDR2, and LCDR 3;

(2) have the amino acid sequences of SEQ ID NO: 1. 2 and 3 and HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 13. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 14 and 15;

(3) have the amino acid sequences of SEQ ID NO: 7. 8 and 9 and HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 19. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 20 and 21;

(4) have the amino acid sequences of SEQ ID NO: 10. 11 and 12 and HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 22. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 23 and 24;

(5) have the amino acid sequences of SEQ ID NO: 80. 81 and 9 and HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 70. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 20 and 21;

(6) have the amino acid sequences of SEQ ID NO: 71. 72, 73 and HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 70. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 20 and 21;

(7) have the amino acid sequences of SEQ ID NO: 71. 72 and 73 and HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 19. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 20 and 21;

(8) has the sequence shown in SEQ ID NO: 26 of a polypeptide sequence V_HHCDR1, HCDR2 and HCDR3 of the regions and a nucleic acid molecule having the sequence of SEQ id no: 31 of the polypeptide sequence V_LLCDR1, LCDR2, and LCDR3 of the region;

(9) has the sequence shown in SEQ ID NO: 28 of the polypeptide sequence V_HHCDR1, HCDR2 and HCDR3 of the regions and a nucleic acid molecule having the sequence of SEQ id no: 34 of the polypeptide sequence V_LLCDR1, LCDR2, and LCDR3 of the region;

(10) has the sequence shown in SEQ ID NO: 26 of a polypeptide sequence V_HHCDR1, HCDR2, and HCDR3 of the region and the region havingSEQ ID NO: 34 of the polypeptide sequence V_LLCDR1, LCDR2, and LCDR3 of the region; or

(11) Has the sequence shown in SEQ ID NO: 28 of the polypeptide sequence V_HHCDR1, HCDR2 and HCDR3 of the regions and a nucleic acid molecule having seq id NO: 31 of the polypeptide sequence V_LLCDR1, LCDR2, and LCDR3 of the region;

wherein the antibody or antigen binding fragment thereof binds PHF-tau, preferably human PHF-tau.

In a more specific aspect, the framework regions in the heavy chain variable region domain and the light chain variable region domain comprise the amino acid sequences of a human immunoglobulin.

According to another particular aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region having an amino acid sequence identical to SEQ ID NO: 26. 27, 28 and 29 or SEQ ID NO: 74. v of any heavy chain of either of 76 and 78_HA heavy chain variable region of a polypeptide sequence having a region of at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identity to the sequence of SEQ id no: 31. 32, 33 and 34 or SEQ ID NO: 75. v of any one of the light chains of 77 and 79_LA light chain variable region of a polypeptide sequence whose regions are at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98% identical, and most preferably 100% identical.

According to another particular aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region having an amino acid sequence identical to SEQ ID NO: 45. 74, 76 and 78, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical; and has a sequence identical to SEQ id no: 46. 75, 77 and 79, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical.

According to another particular aspect, the isolated monoclonal antibody or antigen-binding fragment thereof of the invention further comprises a constant region, such as a human or mouse heavy chain IgG constant region, and a human or mouse antibody light chain kappa or lambda constant region.

In another general aspect, the present invention relates to an isolated nucleic acid encoding a monoclonal antibody or antigen binding fragment thereof of the present invention.

In another general aspect, the present invention relates to a vector comprising an isolated nucleic acid encoding a monoclonal antibody of the invention, or an antigen binding fragment thereof.

In another general aspect, the present invention relates to a host cell comprising an isolated nucleic acid encoding a monoclonal antibody or antigen binding fragment thereof of the present invention.

In another general aspect, the present invention relates to a pharmaceutical composition comprising an isolated monoclonal antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.

In another general aspect, the present invention relates to a method of reducing pathological tau aggregation or tauopathy propagation in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the invention.

In another general aspect, the present invention relates to a method of treating a tauopathy in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the present invention. tauopathies include, but are not limited to, one or more selected from the group consisting of: familial Alzheimer's disease, sporadic Alzheimer's disease, frontotemporal dementia associated with chromosome 17 and Parkinson's syndrome (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, pick's disease, progressive subcortical hyperplasia, tangle dementia only, diffuse neurofibrillary tangle with calcification, silvery granular dementia, amyotrophic lateral sclerosis Parkinson's syndrome-dementia complex, Down's syndrome, Gerstmann-Straussler-Scheinz disease, Hallervorden-Spatz disease, Inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-pick disease type C, prion protein amyloid cerebrovascular disease, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guillain-Motor neuropathy with neurofibrillary tangles, post-encephalitic Parkin syndrome, chronic traumatic encephalopathy, chronic inflammatory disease, chronic inflammatory bowel disease, and dementia pugilistica (boxing disease).

In another general aspect, the invention features a method of producing a monoclonal antibody or antigen-binding fragment thereof of the invention, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof under conditions in which the monoclonal antibody or antigen-binding fragment thereof is produced, and recovering the monoclonal antibody or antigen-binding fragment thereof from the cell or cell culture.

In another general aspect, the present invention relates to a method of producing a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof of the present invention, comprising combining the monoclonal antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

In another general aspect, the present invention relates to a method of detecting the presence of phosphorylated PHF-tau in a subject or a method of diagnosing a tauopathy in a subject by detecting the presence of PHF-tau in a subject using a monoclonal antibody or antigen binding fragment thereof of the invention.

Other aspects, features and advantages of the present invention will be apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments, and the appended claims.

Drawings

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It is to be understood that the invention is not limited to the precise embodiments shown in the drawings.

Figure 1 shows binding of recombinantly expressed PT3 ("R3788") and hybridoma expressed PT3 ("hyb") to PHF-tau and soluble tau.

FIG. 2 shows Western blot analysis of mouse anti-tau monoclonal antibodies after SDS-PAGE of recombinant normal human tau ("NT") and sarcosyl insoluble PHF-tau ("PT").

Figures 3A-3E show immunohistochemical analysis of PT3 on AD hippocampal tissue, which was positive against amyloid 4G 8. The monoclonal antibodies used were (A) PT1, (B) PT2, (C) PT3, (D) AT8 and (E) HT 7.

Figures 4A-4E show immunohistochemical analysis of PT3 on control hippocampal tissue, which was negative for anti-amyloid 4G 8. The monoclonal antibodies used were (A) PT1, (B) PT2, (C) PT3, (D) AT8 and (E) HT 7.

FIGS. 5A-5B show phospho-tau specific staining patterns of PT3 in (A) tau knockout or (B) wild type mouse brain.

FIGS. 6A-6B show non-phospho-tau specific staining patterns of tau-1 in (A) tau knockout or (B) wild type mouse brain.

Fig. 7 shows the crystal structure of the PT3Fab + PT212/PT217-tau peptide complex, with PT3Fab shown in a space-filling representation (light grey) and tau peptide shown in a rod representation (black).

FIG. 8 shows the crystal structure of the PT3Fab + pT212/pT217-tau peptide complex, with PT3 shown as a band (light grey) and its paratope residues shown as a rod, and the tau peptide shown as a rod (black).

Fig. 9 shows a graph of the interaction of the PT3Fab + PT212/PT217-tau peptide structure, where peptide residues are shown in black boxes with white text, VH residues are shown in dark grey, VL residues are shown in light grey, and where dashed lines indicate hydrogen bonds and solid lines indicate van der waals contacts.

Figure 10 shows the sequences of HFA PT3 heavy and light chain variable regions in which the HFA variant is aligned with PT3 mouse parental V regions (VH10 and VL7), the parental CDRs transferred to human FRs are underlined, and the residue numbering is sequential.

Fig. 11 shows the crystal structure of the B324+ pT212/pT217-tau peptide complex, with B324 shown in a space-filling representation (light grey) and tau peptide shown in a rod representation (black).

Fig. 12 shows the crystal structure of the B324+ pT212/pT217tau peptide complex, with B324 shown as a band (light grey) and its paratope residues shown as a rod representation, and the tau peptide shown as a rod representation (black), noting that D92(L) and E93(L) do not have electron densities with respect to C γ and side chain carboxylate atoms.

Fig. 13 shows an interaction diagram of the B324+ pT212/pT217tau peptide structure, where peptide residues are shown in black boxes with white text, VH residues are shown in dark grey, VL residues are shown in light grey, and where dashed lines indicate hydrogen bonds and solid lines indicate van der waals contacts.

Fig. 14 shows a schematic diagram of a FRET biosensor cell model.

Figure 15 shows PT3 induced inhibition of K18 aggregates seeded with HEK cell homogenates containing GFP-tauP301L aggregates as determined using BRET assay.

Figure 16 shows PT 3-induced inhibition of K18 aggregates seeded with TgP301S spinal cord homogenate as determined using FRET assay.

Figure 17 shows the results of a mouse TgP301S spinal cord extract immunodepletion assay, where the data were from 2 independent experiments.

Figure 18 shows the results of a human AD brain extract immunodepletion assay, where the data was from 2 experiments (with the exception of HT7 and AT8, n-1). PT3 inhibited tau seeding as determined using FRET assay.

Figures 19A-19G show schematic diagrams of injection models in transgenic mice expressing mutant human P301L tau. IHC images show representative AT8 staining of injected hemispheres from mice injected with (a-B) 3 months after injection of control extract (C-D) 1 month after injection and (E-F) 3 months after injection of ePHF-tau from AD brain. (G) The histogram shows representative biochemical data for mice treated with increased amounts of ePHF.

Figure 20 shows the effect of peripheral administration of (IP) PT3 followed by seeding with PHF-tau from AD brain on tau aggregation in transgenic mice expressing mutant human P301L tau.

Figure 21 shows the effect on tau aggregation of co-injection of reduced dose of PT3 in transgenic mice expressing mutant human P301L tau, followed by seeding with PHF-tau from AD brain.

Figures 22A-22C show the effect on tau aggregation by co-injection, i.e. IP peripheral administration, of PT3 isoform in transgenic mice expressing mutant human P301L tau, followed by seeding with PHF-tau derived from AD brain. Mice treated according to (a) showed effects in (B) injected hemisphere and (C) non-injected hemisphere.

Figures 23A-23B show the level of aggregated tau in brain homogenates from PSP patients compared to the level of brain homogenates from AD patients. The monoclonal antibodies used were (A) AT8 and (B) PT 3.

FIGS. 24A-24J show staining with (A-C) AT8 or (D-F) PT3 antibodies on cryosections of brain tissue from (A, D) AD patients or (B, C, E, F) PSP patients, showing staining in the infected anatomical areas in PSP. (G-J) control shows no staining.

FIGS. 25A-2525H show SPR binding sensorgrams for affinity matured mAb and its Fab to PHF-tau. The solid line (grey) indicates the kinetic fit using the bivalent binding model (mAb) or the 1: 1 Langmuir model (Fab). (A) B296 mAb (B) B711mAb (C) B809 mAb (d) B333mAb (E) B324 Fab of B296 (f) B330 Fab of B711 (g) B332Fab of B809 (H) B331 Fab of B333.

FIGS. 26A-26B show the binding of PT3-HFA and affinity matured variants to pT212/pT217 peptide in direct ELISA experiments using (A) mAb or (B) Fab.

Detailed Description

Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles and the like which has been included in this specification is intended to provide a context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Otherwise, certain terms used herein have the meanings described in the specification. All patents, published patent applications, and publications cited herein are hereby incorporated by reference as if fully set forth herein. It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Unless otherwise indicated, any numerical value, such as concentrations or concentration ranges described herein, are understood to be modified in all instances by the term "about. Accordingly, numerical values typically include the stated values ± 10%. For example, a concentration of 1mg/mL includes 0.9mg/mL to 1.1 mg/mL. Also, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, unless the context clearly indicates otherwise, a numerical range used explicitly includes all possible subranges, all individual numerical values within the range, including integers within such range and fractions within the range.

As used herein, the term "isolated" means that a biological component (such as a nucleic acid, peptide, or protein) has been substantially separated, or purified from other biological components of the organism in which the component naturally occurs (i.e., other chromosomal and extra-chromosomal DNA and RNA, and proteins). Thus, nucleic acids, peptides and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. "isolated" nucleic acids, peptides and proteins can be part of a composition and still be isolated if such a composition is not part of the environment of the nucleic acid, peptide or protein itself. The term also includes nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

As used herein, the term "antibody" or "immunoglobulin" is used broadly and includes immunoglobulins or antibody molecules (including polyclonal antibodies), monoclonal antibodies (including murine, human-adapted, humanized and chimeric monoclonal antibodies and antibody fragments).

Generally, an antibody is a protein or peptide chain that exhibits binding specificity for a particular antigen. Antibody structures are well known. Depending on the heavy chain constant domain amino acid sequence, immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM. IgA and IgG are further sub-classified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG 4. Thus, the antibodies of the invention can be any of the five main classes or corresponding subclasses. Preferably, the antibody of the invention is IgG1, IgG2, IgG3 or IgG 4. Antibodies of the invention include those having a variation in their Fc region such that they have altered properties compared to the wild-type Fc region, including but not limited to increased half-life, reduced or increased ADCC or CDC, and silent Fc effector function. The light chain of an antibody of any vertebrate species can be assigned to one of two completely different types, κ and λ, based on the amino acid sequence of its constant domain. Thus, an antibody of the invention may contain a kappa or lambda light chain constant domain. According to a particular embodiment, the antibody of the invention comprises heavy and/or light chain constant regions from a mouse antibody or a human antibody.

In addition to the heavy and light chain constant domains, antibodies also contain light and heavy chain variable regions. The immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by an "antigen binding site". Antigen binding sites are defined by various terms and numbering schemes as follows:

(i) kabat: "complementarity determining regions" or "CDRs" are based on sequence variability (Wu and Kabat, J Exp Med.132: 21l-50, 1970). Generally, the antigen binding site has three CDRs per variable region (e.g., HCDR1, HCDR2, and HCDR3 in the heavy chain variable region (VH), and LCDR1, LCDR2, and LCDR3 in the light chain variable region (VL);

(ii) chothia: the terms "hypervariable region", "HVR" or "HV" refer to regions of an antibody variable domain which are structurally hypervariable as defined by Chothia and Lesk (Chothia and Lesk, J Mol biol.196: 901-17, 1987). Generally, the antigen binding site has three hypervariable regions in each VH (H1, H2, H3) and VL (L1, L2, L3). The numbering system and annotation of the CDRs and HV have been revised by abinandan and Martin (abinandan and Martin, Mol immunol.45: 3832-9, 2008);

(iii) IMGT: another definition of the regions forming the antigen binding site has been proposed by Lefranc (Lefranc et al, Dev Comp Immunol.27: 55-77, 2003) based on a comparison of the V domains of immunoglobulins and T cell receptors. The Interrnonal ImmunoGeneTiCs (IMGT) database provides standardized numbering and definitions of these regions. The correspondence between CDR, HV and IMGT depictions (delication) is described in Lefranc et al, 2003 (supra);

(iv) AbM: the trade-off between Kabat and Chothia numbering schemes is the AbM numbering convention described by Martin (Martin ACR (2010) Antibody Engineering, compiled by Kontermann R, Dubel S (Springer-Verlag, Berlin), Vol.2, p.33-51).

(v) Antigen binding sites may also be described based on the "specificity determining residues used" (SDRU) (Almagro, Mol recognit.17: 132-43, 2004), where SDR refers to the amino acid residues of an immunoglobulin that are directly involved in antigen contact.

The exact definition of the antigen binding site can be determined by a variety of divisions as described above, so the exact framework sequence depends on the definition of the antigen binding site Framework Regions (FRs) are more highly conserved portions of the variable domains of the native heavy and light chains each comprise four FRs (FR 1, FR2, FR3 and FR4, respectively), which typically take on a β -sheet configuration, linked by three hypervariable loops, the hypervariable loops in each chain are tightly bound together by FRs and the hypervariable loops of the other chain, and contribute to the formation of the antigen binding site of the antibody.

The term "antigen-binding fragment" as used herein refers to antibody fragments such as, for example, diabodies, fabs, Fab ', F (ab') 2, Fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv)₂Bispecific dsFv (dsFv-dsFv'), disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scF)v), single domain antibodies (sdab), scFv dimers (diabodies), multispecific antibodies formed from a portion of an antibody comprising one or more CDRs, camelized single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not comprise the entire antibody structure. The antigen binding fragment is capable of binding to the same antigen as the parent antibody or the antigen to which the parent antibody fragment binds. According to a specific embodiment, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment of a heavy chain constant region. According to other specific embodiments, the antigen binding fragment comprises Fab and F (ab').

As used herein, the term "humanized antibody" refers to a non-human antibody that has been modified to increase sequence homology to a human antibody such that the antigen-binding properties of the antibody are retained, but its antigenicity in humans is reduced.

As used herein, the term "epitope" refers to a site on an antigen to which an immunoglobulin, antibody, or antigen-binding fragment thereof specifically binds. Epitopes can be formed both from contiguous amino acids or from non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. Epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods of determining the spatial conformation of an epitope include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., epipope Mapping Protocols in Methods in Molecular Biology, vol 66, g.e. morris editors (1996).

As used herein, the term "tau" or "tau protein" refers to an abundant central and peripheral nervous system protein with multiple isoforms. In the human Central Nervous System (CNS), there are six major tau isoforms ranging in size from 352 to 441 amino acids in length due to alternative splicing (Hanger et al Trends Mol Med.15: 112-9, 2009). The isoforms differ from each other by regulatory content of 0-2N-terminal inserts and 3 or 4 serially arranged microtubule binding repeats, and are designated 0N3R (SEQ ID NO: 64), 1N3R (SEQ ID NO: 65), 2N3R (SEQ ID NO: 66), 0N4R (SEQ ID NO: 67), 1N4R (SEQ ID NO: 68) and 2N4R (SEQ ID NO: 69). As used herein, the term "control tau" refers to seq id NO: 69, which lacks phosphorylation and other post-translational modifications. As used herein, the term "tau" includes proteins comprising mutations, e.g., point mutations, fragments, insertions, deletions, and splice variants, of full-length wild-type tau. The term "tau" also encompasses post-translational modifications of the tau amino acid sequence. Post-translational modifications include, but are not limited to, phosphorylation.

tau binds to microtubules and regulates the transport of cargo through cells, a process that can be regulated by tau phosphorylation. In AD and related disorders, abnormal phosphorylation of tau is prevalent and is thought to precede and/or trigger aggregation of tau into fibrils known as Paired Helical Filaments (PHFs). The major component of PHF is hyperphosphorylated tau. As used herein, the term "paired helical filament-tau" or "PHF-tau" refers to tau aggregates in paired helical filaments. The two major regions in the PHF structure are evident in the electron microscope, viz, the villiated shell and core filaments; the villous outer shell is sensitive to proteolysis and is located outside the filament, and the protease resistant core of the filament forms the backbone of the PHF (Wischik et al, Proc Natl Acadsi USA.85: 4884-8, 1988).

As used herein, "isolated humanized antibody that binds PHF-tau" or "isolated humanized anti-PHF-tau antibody" is intended to refer to a humanized anti-PHF-tau antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated humanized anti-PHF-tau antibody is substantially free of antibodies that specifically bind antigens other than PHF-tau). However, an isolated humanized anti-PHF-tau antibody may be cross-reactive with other relevant antigens, e.g. from other species (such as PHF-tau species homologues).

As used herein, the term "specifically binds" or "specifically binds" means that the anti-PHF-tau antibody of the invention is present at about 1X 10^-6M or less (e.g., about 1X 10)^-7M or less, about 1X 10^-8M or less, about 1X 10^-9M or less, about 1X 10^-10M or less, about 1X 10^-11M or less, about 1X 10^-12M or less, or about 1X 10^-13M or less) of a dissociation constant (K)_D) Ability to bind to a predetermined target. KD is obtained from the ratio of KD to Ka (i.e. KD/Ka) and is expressed as molar concentration (M). In accordance with the present disclosure, the KD value of an antibody can be determined using methods in the art. For example, the KD value of an anti-PHF-tau antibody can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g.System, Proteon instrument (BioRad), KinExA instrument (Sapidyne), ELISA or competitive binding assays known to those skilled in the art. In general, K for an anti-PHF-tau antibody to bind to a predetermined target (i.e., PHF-tau)_DK to nonspecific target_DAt least ten times smaller as measured by surface plasmon resonance using, for example, a ProteOn instrument (BioRad). However, anti-PHF-tau antibodies that specifically bind to PHF-tau may be cross-reactive with other related targets, e.g. with the same predetermined target from other species (homologues).

As used herein, the term "polynucleotide," synonymously referred to as a "nucleic acid molecule," "nucleotide," or "nucleic acid," refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single-and double-stranded RNA, and RNA that is a mixture of single-and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single-and double-stranded regions. Furthermore, "polynucleotide" refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, as well as DNA or RNA having backbones modified for stability or other reasons. "modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA; thus, "polynucleotide" includes chemically, enzymatically, or metabolically modified forms of polynucleotides that normally occur naturally, as well as chemical forms of DNA and RNA that are unique to viruses and cells. "Polynucleotide" also includes relatively short nucleic acid strands, often referred to as oligonucleotides.

As used herein, the term "vector" is a replicon in which another nucleic acid segment may be operably inserted to cause replication or expression of the segment.

The term "host cell" as used herein refers to a cell comprising a nucleic acid molecule of the invention. The "host cell" may be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In one embodiment, a "host cell" is a cell transfected with a nucleic acid molecule of the invention. In another embodiment, a "host cell" is a progeny or potential progeny of such a transfected cell. Progeny of a cell may or may not be identical to the parent cell, e.g., due to mutations or environmental influences that may occur in the progeny or due to integration of the nucleic acid molecule into the host cell genome.

As used herein, the term "expression" refers to the biosynthesis of a gene product. The term encompasses gene to RNA transcription. The term also encompasses translation of RNA into one or more polypeptides, and also encompasses all naturally occurring post-transcriptional and post-translational modifications. The expressed humanized antibody or antigen-binding fragment thereof that binds PHF-tau may be within the cytoplasm of the host cell, in an extracellular environment such as the growth medium of a cell culture, or anchored to the cell membrane.

As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid-containing vesicle, microsphere, liposome encapsulation, or other material known in the art for use in pharmaceutical formulations. It will be appreciated that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the effect of, or the biological activity of, the composition according to the present invention. According to the present disclosure, any pharmaceutically acceptable carrier suitable for use in antibody pharmaceutical compositions may be used in the present invention, according to a specific embodiment.

As used herein, the term "subject" refers to an animal, and preferably to a mammal. According to a particular embodiment, the subject is a mammal, including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, rabbit, guinea pig or mouse) or a primate (e.g., a monkey, chimpanzee or human). In particular embodiments, the subject is a human.

As used herein, the term "therapeutically effective amount" refers to the amount of an active ingredient or component that elicits a desired biological or pharmaceutical response in a subject. The therapeutically effective amount can be determined empirically in a routine manner for the intended purpose. For example, in vitro assays may optionally be employed to help determine optimal dosage ranges. Selection of a particular effective dose can be determined by one of skill in the art (e.g., via clinical trials) based on consideration of several factors, including the disease to be treated or prevented, the symptoms involved, the weight of the patient, the immune status of the patient, and other factors known to those of skill. The precise dose to be employed in the formulation will also depend on the route of administration and the severity of the disease and should be decided according to the judgment of the physician and the circumstances of each patient. Effective doses can be derived from dose response curves derived from in vitro or animal model test systems.

As used herein, the terms "treatment" and "treatment" are intended to refer to the improvement or reversal of at least one measurable physical parameter associated with a tauopathy, which is not necessarily identifiable in a subject, but which is identifiable in the subject. The terms "treat" and "treating" may also refer to causing regression, preventing progression, or at least delaying progression of a disease, disorder, or condition. In a particular embodiment, "treating" and "treatment" refer to reducing, preventing the development or onset of, or shortening the duration of one or more symptoms associated with a tauopathy. In a particular embodiment, "treating" and "treatment" refer to preventing the recurrence of a disease, disorder, or condition. In a particular embodiment, "treating" and "treatment" refer to an increase in survival of a subject having a disease, disorder, or condition. In a particular embodiment, "treating" and "treatment" refer to the elimination of a disease, disorder, or condition in a subject.

As used herein, "tauopathy" encompasses any neurodegenerative disease involving the pathological aggregation of tau within the brain. In addition to familial and sporadic AD, other exemplary tauopathies are frontotemporal dementia and parkinsonism associated with chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, pick's disease, progressive subcortical hyperplasia, tangle dementia only, diffuse neurofibrillary tangle with calcification, silvery granular dementia, amyotrophic lateral sclerosis parkinsonism-dementia complex, down's syndrome, gerstmann-shaggar's disease, halloweden-stutz disease, inclusion body myositis, creutzfeldt-jakob disease, multiple system atrophy, niemann-pick type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-guaranular motor neuronopathy with neurofibrillary tangles, postencephalitic parkinsonism, and chronic traumatic encephalopathy such as dementia (Morris) (Morris et al, neuron, 70: 410-26, 2011).

As used herein, the term "combination" in the context of administering two or more therapies to a subject refers to the use of more than one therapy. The use of the term "in combination" does not limit the order in which the therapies are administered to a subject. For example, a first therapy (e.g., a composition described herein) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the second therapy is administered to the subject.

anti-PHF-tau antibodies

In one general aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof that binds PHF-tau. Such anti-PHF-tau antibodies may have the property of binding to a phosphorylated epitope on PHF-tau or to a non-phosphorylated epitope on PHF-tau. anti-PHF-tau antibodies are useful as therapeutic agents, as well as research or diagnostic reagents to detect PHF-tau in biological samples, such as tissues or cells.

According to a particular aspect, the invention relates to an isolated humanized antibody or antigen-binding fragment thereof that binds to phosphorylated tau protein at an epitope of the proline-rich domain of tau protein. In a more specific aspect, the invention relates to an isolated humanized antibody or antigen-binding fragment thereof that binds to phosphorylated tau protein at an epitope comprising phosphorylated T212 and/or T217 residues. In a more specific aspect, the invention relates to a polypeptide that binds to SEQ ID NO: 48. 52 and 54, or an antigen binding fragment thereof. In an even more particular aspect, the invention relates to a polypeptide that binds to SEQ ID NO: 48 or an antigen binding fragment thereof. The antibody of the invention may be a humanized antibody.

Table 1 the heavy and light chain variable regions of 5 humanized mabs binding to phosphotau are shown by SEQ ID NOs. The heavy and light chain sequences of humanized mAb B296 are also shown. The mAb was affinity matured (see table 3).

Table 2 shows the antigen binding site residues (i.e., CDR regions) of exemplary antibodies of the invention as defined according to Chothia, ABM, Kabat, and IMGT numbering schemes. The amino acid sequence of an exemplary heavy chain variable region is as set forth in SEQ ID NO: 26-29, and the amino acid sequence of the exemplary light chain variable region is set forth in SEQ ID NO: 31-34.

Table 3 shows the sequences of affinity matured monoclonal antibodies generated by B296 (i.e., B333, B711, and B809). Variable region sequences are underlined in the heavy and light chain sequences. Bold amino acids in the CDRs of the affinity matured monoclonal antibody indicate substitutions compared to the B296 CDR sequences. CDR sequences were determined by Kabat numbering scheme.

Table 1: humanized phosphotau mAb

mAb	VH	VL	Heavy chain	Light chain
					B235	26	31
B252	28	34
					B280	26	34
B282	28	31
					B296	27	31	45	46

Table 2: CDR sequences of the VH (VH10) and VL (VL7) domains of humanized anti-PHF-tau antibody B296

Table 3: affinity matured B296

Humanized antibodies have variable region framework residues substantially from a human antibody (referred to as the acceptor antibody) and complementarity determining regions substantially from a non-human antibody (i.e., the mouse antibody) (referred to as the donor immunoglobulin). See Queen et al, proc.natl.acad.sci.usa.86: 10029-10033, 1989, WO 90/07861, US5693762, US5693761, US5585089, US5530101 and US 5225539. One or more constant regions, if present, are also substantially or entirely derived from a human immunoglobulin. The human variable regions are typically selected from human antibodies whose framework sequences exhibit a high degree of sequence identity with the murine variable region domains from which the CDRs are derived. The heavy and light chain variable region framework residues may be derived from the same or different human antibody sequences. The human antibody sequences can be naturally occurring human antibody sequences, or can be consensus sequences of several human antibodies. See WO 92/22653. Specific amino acids of the human variable region framework residues are selected for substitution based on their possible effect on CDR conformation and/or antigen binding. Such possible effects are studied by modeling, examining the characteristics of amino acids at specific positions, or experimentally observing the effects of specific amino acid substitutions or mutagenesis.

For example, when an amino acid differs between murine variable region framework residues and selected human variable region framework residues, the amino acid: (1) direct non-covalent binding of antigen, (2) adjacent to the CDR regions, (3) additional interaction with the CDR regions (e.g., within about 6 angstroms of the CDR regions), or (4) participation in the VL-VH interface, human framework amino acids should typically be replaced with equivalent framework amino acids of a mouse antibody.

Other candidates for substitution are acceptor human framework amino acids, which are less common for human immunoglobulins at this position. These amino acids may be substituted with amino acids from equivalent positions of a mouse donor antibody or more typically equivalent positions of a human immunoglobulin. Other candidates for substitution are acceptor human framework amino acids, which are less common for human immunoglobulins at this position. The variable region framework of a humanized immunoglobulin typically shows at least 85% sequence identity to, or shares such sequence with, a human variable region framework sequence.

Antibody humanization can be accomplished using well-known methods such as Specificity Determining Residue Resurfacing (SDRR) (US2010/0261620), resurfacing (Padlan et al, mol. Immunol.28: 489-98, 1991), super-humanization (WO 04/006955), and human cross-talk optimization (US 7657380). Human framework sequences useful for human transplantation or humanization can be selected from relevant databases by those skilled in the art. The selected framework can also be modified to retain or enhance binding affinity by techniques such as those disclosed by Queen et al, 1989 (supra). According to specific embodiments, methods for humanizing anti-PHF-tau antibodies from a mouse parent antibody include those described in example 4 below.

The antibodies of the invention can be produced by a variety of techniques, such as the hybridoma method (Kohler and Milstein, Nature.256: 495-7, 1975). Chimeric monoclonal antibodies comprising variable light and heavy chain regions derived from a donor antibody (typically murine) in combination with constant light and heavy chain regions derived from an acceptor antibody (typically another mammalian species such as human) can be prepared by the methods disclosed in US 4816567. CDR-grafted monoclonal antibodies with CDRs derived from a non-human donor immunoglobulin (typically a murine) and portions thereof derived from one or more human immunoglobulins derived from the remaining immunoglobulins of the molecule can be prepared by techniques known to those skilled in the art, such as the techniques disclosed in US 5225539. Fully human monoclonal antibodies lacking any non-human sequence can be prepared from human immunoglobulin transgenic mice by techniques mentioned in the following references (Lonberg et al, Nature.368: 856-9, 1994; Fishwild et al, Nat Biotechnol.14: 845-51, 1996; Mendez et al, Nat Genet.15: 146-56, 1997). Human monoclonal antibodies can also be prepared and optimized from phage display libraries (Knappik et al, J Mol biol. 296: 57-86, 2000; Krebs et al, J Immunol methods. 254: 67-84, 2001; Shi et al, J Mol biol. 397: 385-96, 2010).

The monoclonal antibody of the present invention includes an antibody having the amino acid sequence of SEQ ID NO: 1. HCDR1 of any one of 4, 7, 10, 71, 80; SEQ ID NO: 2.5, 8, 11, 72, 81, HCDR 2; SEQ ID NO: 3. 6, 9, 12, 73, HCDR 3; SEQ ID NO: 13. LCDR1 of any one of 16, 19, 22, 70; SEQ ID NO: 14. LCDR2 of any one of 17, 20, 23; SEQ ID NO: 15. LCDR3 of any one of 18, 21, 24. Monoclonal antibodies also encompassed by the invention have CDR sequences at least 90%, more preferably at least 95%, more preferably at least 98%, more preferably at least 99% identical to: SEQ ID NO: 1. HCDR1 of any one of 4, 7, 10, 71, 80; SEQ ID NO: 2.5, 8, 11, 72, 81, HCDR 2; SEQ ID NO: 3. 6, 9, 12, 73, HCDR 3; SEQ ID NO: 13. LCDR1 of any one of 16, 19, 22, 70; SEQ ID NO: 14. LCDR2 of any one of 17, 20, 23; SEQ ID NO: 15. LCDR3 of any one of 18, 21, 24.

According to a particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising:

(1) have the amino acid sequences of SEQ ID NO: 4.5 and 6, HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of seq id NOs: 16. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 17 and 18;

(2) have the amino acid sequences of SEQ ID NO: 1. 2 and 3 and HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 13. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 14 and 15;

(3) have the amino acid sequences of SEQ ID NO: 7. 8 and 9 and HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 19. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 20 and 21;

(4) have the amino acid sequences of SEQ ID NO: 10. 11 and 12 and HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 22. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 23 and 24;

(5) have the amino acid sequences of SEQ ID NO: 80. HCDR1 of the polypeptide sequences of 81 and 9,

HCDR2 and HCDR3 and a nucleic acid molecule having SEQ ID NOs: 70. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 20 and 21;

(6) have the amino acid sequences of SEQ ID NO: 71. 72, 73 and HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NOs: 70. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 20 and 21;

(7) have the amino acid sequences of SEQ ID NO: 71. 72 and 73, HCDR1,

HCDR2 and HCDR3 and a nucleic acid molecule having SEQ ID NOs: 19. LCDR1, LCDR2 and LCDR3 of the polypeptide sequences of 20 and 21;

(8) has the sequence shown in SEQ ID NO: 26 of a polypeptide sequence V_HHCDR1, HCDR2 and HCDR3 of the regions and a nucleic acid molecule having the sequence of SEQ id no: 31, a polypeptide sequence ofV of_LLCDR1, LCDR2, and LCDR3 of the region;

(9) has the sequence shown in SEQ ID NO: 28 of the polypeptide sequence V_HHCDR1, HCDR2 and HCDR3 of the regions and a nucleic acid molecule having the sequence of SEQ id no: 34 of the polypeptide sequence V_LLCDR1, LCDR2, and LCDR3 of the region;

(10) has the sequence shown in SEQ ID NO: 26 of a polypeptide sequence V_HHCDR1, HCDR2 and HCDR3 of the regions and a nucleic acid molecule having seq id NO: 34 of the polypeptide sequence V_LLCDR1, LCDR2, and LCDR3 of the region; or

(11) Has the sequence shown in SEQ ID NO: 28 of the polypeptide sequence V_HHCDR1, HCDR2 and HCDR3 of the regions and a nucleic acid molecule having seq id NO: 31 of the polypeptide sequence V_LLCDR1, LCDR2, and LCDR3 of the region;

wherein the antibody or antigen-binding fragment thereof binds PHF-tau, preferably human PHF-tau, and wherein the framework regions in the heavy chain variable region domain and the light chain variable region domain comprise the amino acid sequences of a human immunoglobulin.

According to another particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region having a sequence identical to SEQ ID NO: 26. 27, 28 or 29, or a heavy chain variable region having a polypeptide sequence which is at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical to SEQ ID NO: 31. 32, 33 or 34, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical.

According to another particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region having a sequence identical to SEQ ID NO: 74. 76 and 78, or a heavy chain variable region having a polypeptide sequence at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical to the variable region in the heavy chain of any one of SEQ ID NOs: 75. 77 and 79, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical in the variable region of the light chain.

According to another particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region having a sequence identical to SEQ ID NO: 26, and a heavy chain variable region having a polypeptide sequence at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical to SEQ ID NO: 31, a light chain variable region of a polypeptide sequence that is at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical.

According to another particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region having a sequence identical to SEQ ID NO: 28, and a heavy chain variable region having a polypeptide sequence at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical to SEQ ID NO: 34, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identity.

According to another particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region having a sequence identical to SEQ ID NO: 26, and a heavy chain variable region having a polypeptide sequence at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical to SEQ ID NO: 34, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identity.

According to another particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region having a sequence identical to SEQ ID NO: 28, and a heavy chain variable region having a polypeptide sequence at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical to SEQ ID NO: 31, a light chain variable region of a polypeptide sequence that is at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical.

According to another particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region having a sequence identical to SEQ ID NO: 27, and a heavy chain variable region having a polypeptide sequence at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical to SEQ ID NO: 31, a light chain variable region of a polypeptide sequence that is at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical.

According to another particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region having a sequence identical to SEQ ID NO: 74, and a heavy chain variable region having a polypeptide sequence at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical to the variable region in the heavy chain of SEQ ID NO: 75, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical in the variable region of the light chain.

According to another particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region having a sequence identical to SEQ ID NO: 76, and a heavy chain variable region having a polypeptide sequence at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical to the variable region in the heavy chain of SEQ ID NO: 77, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical.

According to another particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region having a sequence identical to SEQ ID NO: 78, a heavy chain variable region of a polypeptide sequence having at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identity to the variable region in the heavy chain of SEQ ID NO: 79, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identity to the variable region of the light chain.

According to another particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region having a sequence identical to SEQ ID NO: 45, and a heavy chain having a polypeptide sequence at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical to seq id NO: 46, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identity. According to another particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 45 and a light chain having the polypeptide sequence of SEQ ID NO: 46, or a light chain of the polypeptide sequence of seq id no.

According to another particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region having a sequence identical to SEQ ID NO: 74, and a heavy chain having a polypeptide sequence at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical to seq id NO: 75, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical. According to another particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 74 and a light chain having the polypeptide sequence of SEQ ID NO: 75, or a light chain of the polypeptide sequence of seq id no.

According to another particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region having a sequence identical to SEQ ID NO: 76, a heavy chain having a polypeptide sequence at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical to seq id NO: 77, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identity. According to another particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 76 and a light chain having the polypeptide sequence of SEQ ID NO: 77.

According to another particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region having a sequence identical to SEQ ID NO: 78, and a heavy chain having a polypeptide sequence at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identical to seq id NO: 79, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably 100% identity. According to another particular aspect, the present invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 78 and a light chain having the polypeptide sequence of SEQ ID NO: 79.

According to another particular aspect, the invention relates to an isolated humanized antibody or antigen binding fragment thereof comprising a human heavy chain IgG1 constant region and a human light chain kappa constant region.

According to another toolIn one aspect, the invention relates to an isolated humanized antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment is present at 5x 10^-9Dissociation constant (K) of M or less_D) Preferably 1X 10^-9M or less, or 1X 10^-10K of M or less_DBinding to human PHF-tau, wherein K_DBy surface plasmon resonance analysis, such as by measurement using a Biacore or ProteOn system.

The functional activity of humanized antibodies and antigen-binding fragments thereof that bind PHF-tau can be characterized by methods known in the art and as described herein. Methods for characterizing antibodies and antigen-binding fragments thereof that bind PHF-tau include, but are not limited to, affinity and specificity assays, including Biacore, ELISA, and FACS analysis; immunohistochemical analysis; in vitro cell assays and in vivo injection assays to determine the efficacy of antibodies to inhibit tau seeding; a cytotoxicity assay to detect the presence of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities of the antibody; and the like. According to particular embodiments, methods for characterizing antibodies and antigen-binding fragments thereof that bind PHF-tau include those described in examples 5, 6, 8, and 9 below. An exemplary mouse parent antibody for a humanized antibody that binds PHF-tau but not control tau is antibody PT3, which has the amino acid sequence of SEQ ID NO: 25 and SEQ ID NO: 30 (see, e.g., U.S. patent 9,371,376, which is incorporated by reference in its entirety).

Several well-known methods can be employed to determine the binding epitope of an antibody of the invention. For example, when the structures of two individual components are known, a silicon wafer protein-protein docking can be performed to identify compatible interaction sites. Hydrogen-deuterium (H/D) exchange can be performed with the antigen and antibody complex to localize the region on the antigen to which the antibody binds. Segmental and point mutagenesis of an antigen can be used to locate amino acids important for antibody binding. The co-crystal structure of the antibody-antigen complex is used to identify residues that contribute to the epitope and paratope. According to particular embodiments, methods for determining the binding epitope of an antibody of the invention include those described in examples 2, 3 and 7 below.

An exemplary bispecific antibody can bind to two different epitopes on PHF-tau, or can bind to PHF-tau and β amyloid (A β). Another exemplary bispecific antibody can bind to PHF-tau and endogenous blood brain barrier transcytosis receptors, such as insulin receptor, metastasis receptor, insulin-like growth factor-1 receptor, and lipoprotein receptor.

The immune effector properties of the antibodies of the invention can be enhanced or silenced by Fc modification by techniques known to those skilled in the art. For example, Fc effector functions such as C1q binding, Complement Dependent Cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors; BCR), and the like, can be provided and/or controlled by modifying residues in the Fc that contribute to these activities. Pharmacokinetic properties may also be enhanced by mutating residues in the Fc domain that prolong the half-life of the antibody (Strohl, Curr Opin Biotechnol.20: 685-91, 2009).

In addition, the antibodies of the invention can be post-translationally modified by processes such as glycosylation, isomerization, deglycosylation, or non-naturally occurring covalent modifications such as the addition of polyethylene glycol moieties and lipidation. Such modifications can be made in vivo or in vitro. For example, the antibodies of the invention may be conjugated to polyethylene glycol (pegylated) to improve their pharmacokinetic properties. Conjugation can be performed by techniques known to those skilled in the art. Conjugation of therapeutic antibodies to PEG has been shown to enhance pharmacodynamics without interfering with function (Knight et al, Plateleles.15: 409-18, 2004; Leong et al, cytokine.16: 106-19, 2001; Yang et al, Protein Eng.16: 761-70, 2003).

In another general aspect, the present invention relates to an isolated polynucleotide encoding a monoclonal antibody of the invention, or an antigen-binding fragment thereof. One skilled in the art will appreciate that the coding sequence of a protein can be altered (e.g., substituted, deleted, inserted, etc.) without altering the amino acid sequence of the protein. Thus, one skilled in the art will appreciate that the nucleic acid sequence encoding the humanized antibody or antigen-binding fragment thereof of the present invention can be altered without altering the amino acid sequence of the protein. Exemplary isolated polynucleotides encode a polypeptide comprising the amino acid sequence set forth in SEQ ID NOs: 4.5 and 6, or a polypeptide comprising the immunoglobulin heavy chain CDRs CDR HCDR1, HCDR2 and HCDR3, or a polypeptide comprising the amino acid sequence set forth in SEQ ID NOs: 16. 17 and 18, LCDR1, LCDR2, and LCDR 3. Other exemplary isolated polynucleotides are those encoding the antibody variable regions of the invention having the amino acid sequence of SEQ ID NO: 36-39 or 41-44. Other polynucleotides encoding the antibodies of the invention in view of the degeneracy of the genetic code or codon preference in a given expression system are also within the scope of the invention. The isolated nucleic acids of the invention can be prepared using well known recombinant or synthetic techniques. DNA encoding the monoclonal antibody is readily isolated and sequenced using methods known in the art. In the case of hybridoma production, such cells may be used as a source of such DNA. Alternatively, display techniques in which the coding sequences and translation products are related, such as phage or ribosome display libraries, can be used.

In another general aspect, the present invention relates to a vector comprising an isolated polynucleotide encoding a monoclonal antibody of the invention, or an antigen-binding fragment thereof. Any vector known to those of skill in the art may be used in light of this disclosure, such as a plasmid, cosmid, phage vector, or viral vector. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any elements that establish the conventional function of an expression vector, such as a promoter, ribosome binding elements, terminator, enhancer, selection marker and origin of replication. The promoter may be a constitutive, inducible or repressible promoter. A variety of expression vectors capable of delivering a nucleic acid to a cell are known in the art and are useful herein for producing an antibody or antigen-binding fragment thereof in a cell. Conventional cloning techniques or artificial gene synthesis may be used to generate recombinant expression vectors according to embodiments of the present invention.

In another general aspect, the present invention relates to a host cell comprising an isolated polynucleotide encoding a monoclonal antibody of the invention, or an antigen-binding fragment thereof. In view of this disclosure, any host cell known to those skilled in the art can be used to recombinantly express an antibody or antigen-binding fragment thereof of the present invention. Such host cells may be eukaryotic cells, bacterial cells, plant cells, or archaeal cells. Exemplary eukaryotic cells can be of mammalian, insect, avian, or other animal origin. Mammalian eukaryotic cells include immortalized cell lines such as hybridoma or myeloma cell lines such as SP2/0 (American type culture Collection (ATCC), Manassas, Va., CRL-1581), NS0 (European cell culture Collection (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646), and Ag653(ATCC CRL-1580) murine cell lines. An exemplary human myeloma cell line is U266(ATTC CRL-TIB-196). Other useful cell lines include cell lines derived from Chinese Hamster Ovary (CHO) cells, such as CHO-K1 SV (Lonza biologics), CHO-K1 (ATCCRL-61, Invitrogen) or DG 44.

In another general aspect, the invention features a method of producing a monoclonal antibody or antigen-binding fragment thereof of the invention, comprising culturing a cell comprising a polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof under conditions in which the monoclonal antibody or antigen-binding fragment thereof of the invention is produced, and recovering the antibody or antigen-binding fragment thereof from the cell or cell culture (e.g., from the supernatant). The expressed antibody or antigen-binding fragment thereof can be harvested from the cells and purified according to conventional techniques known in the art.

Pharmaceutical compositions and methods of treatment

The anti-PHF-tau antibodies of the invention or fragments thereof of the invention are useful for treating, alleviating or preventing symptoms in a patient suffering from a neurodegenerative disease involving pathological aggregation of tau within the brain, or tauopathy such as in a patient suffering from AD.

Thus, in another general aspect, the present invention relates to a pharmaceutical composition comprising an isolated monoclonal antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.

In another general aspect, the present invention relates to a method of treating or alleviating a symptom of a disease, disorder or condition, such as a tauopathy, in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the present invention.

In another general aspect, the present invention relates to a method of reducing pathological tau aggregation or tauopathy propagation in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the invention.

According to an embodiment of the invention, the pharmaceutical composition comprises a therapeutically effective amount of a monoclonal anti-PHF-tau antibody or an antigen binding fragment thereof. As used herein, with reference to a humanized anti-PHF-tau antibody or antigen-binding fragment thereof, a therapeutically effective amount means that the amount of a monoclonal anti-PHF-tau antibody or antigen-binding fragment thereof results in the treatment of a disease, disorder or condition; preventing or slowing the progression of a disease, disorder, or condition; or alleviating or completely alleviating symptoms associated with an immune disease, disorder or condition.

According to particular embodiments, a therapeutically effective amount refers to a therapeutic amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or ameliorating the severity of the disease, disorder or condition being treated or the symptoms associated therewith; (ii) reducing the duration of the disease, disorder or condition being treated or symptoms associated therewith; (iii) preventing the development of the disease, disorder or condition being treated or symptoms associated therewith; (iv) causing regression of the disease, disorder or condition being treated or symptoms associated therewith; (v) preventing the development or onset of the disease, disorder or condition being treated or symptoms associated therewith; (vi) preventing the recurrence of the disease, disorder or condition being treated or symptoms associated therewith; (vii) reducing hospitalization of the subject with the treated disease, disorder or condition or symptoms associated therewith; (viii) reducing the hospitalization time of a subject having the treated disease, disorder or condition or symptoms associated therewith; (ix) increasing survival of a subject having the treated disease, disorder, or condition or symptoms associated therewith; (xi) Inhibiting or reducing the treated disease, disorder or condition or symptoms associated therewith in a subject; and/or (xii) enhances or improves the prophylactic or therapeutic effect of the other therapy.

According to a particular embodiment, the disease, disorder or condition treated is a tauopathy. According to more specific embodiments, the disease, disorder or condition treated includes, but is not limited to, familial alzheimer's disease, sporadic alzheimer's disease, frontotemporal dementia associated with chromosome 17 and parkinsonism (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, pick's disease, progressive subcortical gliosis, tangle dementia only, diffuse neurofibrillary tangle with calcification, silvery particle dementia, amyotrophic lateral sclerosis parkinsonism-dementia complex, down's syndrome, gerstmann-straussler-scherzehnson disease, hallrewarden-schutzfeldt-jakob disease, inclusion body myositis, creutzfeldt-jakob disease, multiple system atrophy, niemann-pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-kandao motor neuronopathy with neurofibrillary tangles, neuro-synephrosis, sporadic dementia, and/or neurodegenerative diseases, Postencephalitic parkinsonism, chronic traumatic encephalopathy, or dementia pugilistica (boxing disease).

tauopathy-related behavioral phenotypes include, but are not limited to, cognitive impairment, early personality changes and loss of control, apathy, anhedonia, mutism, apraxia, persistent speech, stereotyped actions/behaviors, hypersexuality, confusion, unplanned or organized sequential tasks, selfishness/passivity, antisocial characteristics, lack of transposition thinking, hesitancy, grammatical speech with frequent semantic errors but relatively well-preserved understanding, impaired understanding and word-selection disorders, slow progressive gait instability, desynchrony, immobility, frequent falls, non-levodopa-responsive axial stiffness, nuclear gaze paralysis, square wave twitching, slow vertical saccades, pseudobulbar paralysis, limb apraxia, dystonia, cortical sensation loss, and tremor.

Treatment-compliant patients include, but are not limited to, asymptomatic individuals at risk for AD or other tauopathies, as well as patients who are currently showing symptoms.treatment-compliant patients include individuals with known genetic risk for AD such as family history of AD or the presence of genetic risk factors in the genome.an exemplary risk factor is a mutation in Amyloid Precursor Protein (APP), particularly mutations at position 717 and positions 670 and 671 (haddi and swedish type mutations, respectively). other risk factors are mutations in the presenilin genes, PS1 and PS2, and ApoE4, hypercholesterolemia, or family history of atherosclerosis.

The anti-PHF-tau antibodies of the invention are suitable as both therapeutic and prophylactic agents for the treatment or prevention of neurodegenerative diseases involving pathological aggregation of tau, such as AD or other tauopathies. In asymptomatic patients, treatment may begin at any age (e.g., at about 10, 15, 20, 25, 30 years of age). Typically, however, treatment does not need to be initiated until the patient reaches about the age of 40, 50, 60 or 70. Treatment typically requires multiple doses over a period of time. Treatment can be monitored by measuring antibody, or activated T cell or B cell responses to the therapeutic over time. If the response drops, a booster dose may be indicated.

In prophylactic applications, the pharmaceutical composition or agent is administered to a patient susceptible to or otherwise at risk for AD in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, including biochemical, histological, and/or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes present during the course of disease progression. In therapeutic applications, the composition or agent is administered to a patient suspected of having or having such a disease in an amount sufficient to reduce, prevent or delay any of the symptoms (biochemical, histological, and/or behavioral) of the disease. Administration of the therapeutic agent can reduce or eliminate mild cognitive impairment in patients who have not yet developed the pathology characteristic of alzheimer's disease.

A therapeutically effective amount or dose may vary depending on various factors, such as the disease, disorder, or condition to be treated, the mode of administration, the site of interest, the physiological state of the subject (including, for example, age, weight, health), whether the subject is human or animal, other drugs administered, and whether prophylactic or therapeutic treatment is employed. Therapeutic doses are optimally titrated to optimize safety and efficacy.

The antibodies of the invention can be prepared as pharmaceutical compositions containing a therapeutically effective amount of the antibody as the active ingredient in a pharmaceutically acceptable carrier. The carrier can be a liquid, such as water and oils, including those derived from petroleum, animal, vegetable, or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional known sterilization techniques, such as filtration. The composition may contain pharmaceutically acceptable auxiliary substances as necessary to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizers, thickening agents, lubricants, and coloring agents, and the like. The concentration of the antibody of the invention in such pharmaceutical formulations can vary widely, i.e., from less than about 0.5%, typically at or at least about 1% to as much as 15% or 20% by weight, and will be selected based primarily on the desired dosage, fluid volume, viscosity, etc., depending on the particular mode of administration selected.

The mode of administration for therapeutic use of the antibodies of the invention may be any suitable route of delivery of the agent to the host. For example, the compositions described herein can be formulated to be suitable for parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, or intracranial administration, or they can be administered into the cerebrospinal fluid of the brain or spine.

Treatment may be administered on a single dose schedule or as a multiple dose schedule in which the initial course of treatment may use 1-10 discrete doses followed by other doses administered at subsequent intervals required to maintain and/or enhance response, for example at 1-4 months for the second dose and, if desired, one or more subsequent doses several months later. Examples of suitable therapy sessions include: (i)0, 1 month, and 6 months, (ii)0, 7 days, and 1 month, (iii)0 and 1 month, (iv)0 and 6 months, or other treatment courses sufficient to elicit a desired response expected to reduce the symptoms or severity of the disease.

The antibodies of the invention can be lyophilized for storage and reconstituted in a suitable vehicle prior to use. This technique has been shown to be effective for antibody and other protein formulations, and lyophilization and reconstitution techniques known in the art can be employed.

In accordance with a particular embodiment, the compositions used in the treatment of tauopathies can be used in combination with other agents effective in the treatment of the associated neurodegenerative diseases PHF-tau and A β pathologies may act synergistically with the antibodies of the present invention may be administered in combination with agents that reduce or prevent β amyloid (A β) deposition in the case of AD.

In another general aspect, the present invention relates to a method of producing a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof of the present invention, comprising combining the monoclonal antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

Diagnostic method and kit

The monoclonal anti-PHF-tau antibodies of the invention are useful in methods of diagnosing AD or other tauopathies in a subject.

Thus, in another general aspect, the present invention relates to methods of detecting the presence of PHF-tau in a subject and methods of diagnosing tauopathy in a subject by using the monoclonal antibodies or antigen-binding fragments thereof of the invention to detect the presence of PHF-tau in a subject.

Phosphorylated tau can be detected in a biological sample from a subject (e.g., a blood, serum, plasma, interstitial fluid, or cerebrospinal fluid sample) by contacting the biological sample with a diagnostic antibody reagent and detecting binding of the diagnostic antibody reagent to phosphorylated tau in the sample from the subject. Assays for performing detection include well-known methods such as ELISA, immunohistochemistry, western blotting, or in vivo imaging. An exemplary diagnostic antibody is antibody PT3 of the invention.

Diagnostic antibodies or similar agents may be administered by intravenous injection into a patient, or by direct injection into the brain by any suitable route that delivers the agent to the host. The dosage of the antibody should be within the same range as the therapeutic method. Typically, the antibody is labeled, although in some methods, the primary antibody having affinity for phosphorylated tau is unlabeled, and a second labeled antibody is used to bind to the primary antibody. The choice of label depends on the detection method. For example, fluorescent labels are suitable for optical detection. The use of paramagnetic markers is suitable for tomographic detection without surgical intervention. Radiolabels can also be detected using PET or SPECT.

Diagnosis is performed by comparing the number, size and/or intensity of labeled PHF-tau, tau aggregates and/or neurofibrillary tangles in a sample from or in a subject with corresponding baseline values. The baseline value may represent the average level in a population of healthy individuals. The baseline value may also represent a previous level determined in the same subject.

The diagnostic methods described above can also be used to monitor the response of a subject to treatment by detecting the presence of phosphorylated tau in the subject prior to, during, or after treatment. A decrease from the baseline value signals a positive response to the treatment. Values may also increase transiently in biological fluids as pathological tau is cleared from the brain.

The invention also relates to a kit for carrying out the above diagnostic and monitoring method. Typically, such kits contain a diagnostic reagent such as an antibody of the invention and optionally a detectable label. The diagnostic antibody itself may contain a detectable label (e.g., a fluorescent molecule, biotin, etc.) that can be detected directly or can be detected via a secondary reaction (e.g., reaction with streptavidin). Alternatively, a second reagent comprising a detectable label may be used, wherein the second reagent has binding specificity for the primary antibody. In a diagnostic kit suitable for measuring PHF-tau in a biological sample, the antibodies of the kit may be provided pre-bound to a solid phase, such as to a well of a microtiter dish.

The contents of all cited references in this patent application, including references, issued patents, issued patent applications, and co-pending patent applications, are hereby expressly incorporated by reference.