DNA molecules encoding antibodies to tau and methods of making thereof

Abstract

Monoclonal antibodies to human tau aggregate, compositions comprising such tau antibodies, and methods of using such tau antibodies for the treatment of neurodegenerative diseases including Alzheimer's disease, Progressive Supranuclear Palsy and Pick's disease.

Claims

1. A DNA molecule comprising a polynucleotide sequence encoding a light chain (LC) of an antibody that binds human tau, the LC comprising a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO: 9 and comprising a polynucleotide sequence encoding a heavy chain (HC) of the antibody, the HC comprising a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO: 10.

2. The DNA molecule of claim 1, wherein the amino acid sequence of the LC is SEQ ID NO: 1.

3. The DNA molecule of claim 2, wherein the polynucleotide sequence encoding the LC is SEQ ID NO: 11.

4. The DNA molecule of claim 1, wherein the amino acid sequence of the HC is SEQ ID NO: 2.

5. The DNA molecule of claim 4, wherein the polynucleotide sequence encoding the HC is SEQ ID NO: 12.

6. The DNA molecule of claim 2, wherein the amino acid sequence of the HC is SEQ ID NO: 2.

7. The DNA molecule of claim 6, wherein the polynucleotide sequence encoding the LC is SEQ ID NO: 11 and the polynucleotide sequence encoding the HC is SEQ ID NO: 12.

8. A mammalian cell comprising a DNA molecule comprising a polynucleotide sequence encoding a LC of an antibody that binds human tau, the LC comprising a LCVR having the amino acid sequence of SEQ ID NO: 9 and a DNA molecule comprising a polynucleotide sequence encoding a HC of an antibody that binds human tau, the HC comprising a HCVR having the amino acid sequence of SEQ ID NO: 10, wherein the cell is capable of expressing the antibody.

9. The mammalian cell of claim 8, wherein the amino acid sequence of the LC is SEQ ID NO: 1 and the amino acid sequence of the HC is SEQ ID NO: 2, wherein the cell is capable of expressing the antibody.

10. The mammalian cell of claim 9, wherein the polynucleotide sequence encoding the LC having the amino acid sequence of SEQ ID NO: 1 is SEQ ID NO: 11 and the polynucleotide sequence encoding the HC having the amino acid sequence of SEQ ID NO: 2 is SEQ ID NO: 12.

11. A process for producing an antibody that binds human tau, the antibody comprising a LC comprising a LCVR having the amino acid sequence of SEQ ID NO: 9 and a HC comprising a HCVR having the amino acid sequence of SEQ ID NO: 10, the process comprising cultivating the mammalian cell of claim 8 under conditions such that the antibody is expressed, and recovering the expressed antibody.

12. The process of claim 11, wherein the amino acid sequence of the LC is SEQ ID NO: 1 and the amino acid sequence of the HC is SEQ ID NO: 2.

13. The process of claim 12, wherein the polynucleotide sequence encoding the LC having the amino acid sequence of SEQ ID NO: 1 is SEQ ID NO: 11 and the polynucleotide sequence encoding the HC having the amino acid sequence of SEQ ID NO: 2 is SEQ ID NO: 12.

Description

EXAMPLES

(1) Expression of Engineered Tau Antibody

(2) Engineered tau monoclonal antibodies of the present invention can be expressed and purified essentially as follows. A glutamine synthetase (GS) expression vector containing the DNA sequence of SEQ ID NO. 11 (encoding LC amino acid sequence of SEQ ID NO. 1) and the DNA sequence of SEQ ID NO. 12 (encoding HC amino acid sequence of SEQ ID NO. 2) is used to transfect a Chinese hamster ovary cell line (CHO) by electroporation. The expression vector encodes an SV Early (Simian Virus 40E) promoter and the gene for GS. Expression of GS allows for the biochemical synthesis of glutamine, an amino acid required by the CHO cells. Post-transfection, cells undergo bulk selection with 50 M L-methionine sulfoximine (MSX). The inhibition of GS by MSX is utilized to increase the stringency of selection. Cells with integration of the expression vector cDNA into transcriptionally active regions of the host cell genome can be selected against CHO wild type cells, which express an endogenous level of GS. Transfected pools are plated at low density to allow for close-to-clonal outgrowth of stable expressing cells. The masterwells are screened for antibody expression and then scaled up in serum-free, suspension cultures to be used for production. Clarified medium, into which the antibody has been secreted, is applied to a Protein A affinity column that has been equilibrated with a compatible buffer, such as phosphate buffered saline (pH 7.4). The column is washed with 1M NaCl to remove nonspecific binding components. The bound tau monoclonal antibody is eluted, for example, with sodium citrate at pH (approx.) 3.5 and fractions are neutralized with 1M Tris buffer. Tau monoclonal antibody fractions are detected, such as by SDS-PAGE or analytical size-exclusion, and then are pooled. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The tau monoclonal antibody of the present invention is concentrated and/or sterile filtered using common techniques. The purity of the tau monoclonal antibody after these chromatography steps is greater than 95%. The tau monoclonal antibody of the present invention may be immediately frozen at 70 C. or stored at 4 C. for several months.

(3) Binding Kinetics and Affinity

(4) Surface Plasmon Resonance (SPR) assay, measured with a BIACORE 2000 instrument (primed with HBS-EP+ running buffer (GE Healthcare, 10 mM Hepes pH7.4+150 mM NaCl+3 mM EDTA+0.05% surfactant P20) at 25 C.), is used to measure binding of exemplified tau monoclonal antibody of Example 1 to both human monomeric (e.g., native or non-aggregate) tau and human tau aggregates (both having the amino acid sequence as set forth in SEQ ID NO:13). Binding of humanized MC-1 antibody construct (having the framework combination: 5-51 heavy-chain, A27 light-chain) to human monomeric tau and human tau aggregate is measured in the same manner.

(5) Except as noted, all reagents and materials are from BIACORE AB (Upsala, Sweden). A CMS chip containing immobilized protein A (generated using standard NHS-EDC amine coupling) on all four flow cells (FC) is used to employ a capture methodology. Antibody samples are prepared at 0.5 g/mL by dilution into running buffer. Monomeric tau and fibril tau are prepared to concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.82, 3.91, 1.95, and 0 (blank) nM by dilution into running buffer. Each analysis cycle consists of: (1) capturing antibody samples on separate flow cells (FC2, FC3, and FC4); (2) injection of 250 L (300 sec) of either monomeric tau or tau fibril aggregate over respective FC at a rate of 50 t/min; (3) return to buffer flow for 20 mins. to monitor dissociation phase; (4) regeneration of chip surfaces with 25 L (30 sec) injection of glycine, pH1.5; (5) equilibration of chip surfaces with a 50 L (60 sec) injection of HBS-EP+.

(6) Data of binding to tau aggregate is processed using standard double-referencing and fit to a 1:1 binding model using Biacore 2000 Evaluation software, version 4.1, to determine the association rate (k.sub.on, M.sup.1 s.sup.1 units), dissociation rate (k.sub.off, s.sup.1 units), and R.sub.max (RU units). The equilibrium dissociation constant (K.sub.D) was calculated from the relationship K.sub.D=k.sub.off/k.sub.on, and is in molar units. Data of binding to monomeric tau cannot be determined accurately by SPR as described above due to rapid on- and off-rates. Therefore, K.sub.D for binding to monomeric tau is obtained by using a steady state binding fit model from plotting the concentration of antigen versus the response unit. Resulting binding data is provided in Table 2.

(7) TABLE-US-00002 TABLE 2 SPR binding data to both human monomeric and aggregate tau. k.sub.on k.sub.off K.sub.D* (M.sup.1s.sup.1 units) (M.sup.1s.sup.1 units) (nM) Exemplified Monomeric Not detectable Not detectable 235 Tau mAb of Tau Example 1 Tau Aggregate 4.59e4 <1e5 <0.22 Humanized Monomeric Not determined Not determined 550 MC-1 Ab Tau construct Tau Aggregate 5.75e4 1.02e4 1.77 *K.sub.D results are considered relative as the results are not normalized for influence of avidity.

(8) The results provided in Table 2 demonstrate tau monoclonal antibody of Example 1 does not possess measurable binding to monomeric tau such that an affinity value can be accurately determined by Biacore analysis (due to rapid on- and off-rates). Conversely, the results provided in Table 2 demonstrate tau monoclonal antibody of Example 1 possesses improved affinity to tau aggregate compared to humanized MC-1 antibody construct.

(9) Enzyme-Linked Immunosorbant Assay (ELISA) is used to determine relative binding affinity of the exemplified tau monoclonal antibody of Example 1 to aggregate tau fibrils from AD brain homogenates. AD brain homogenates are prepared from approx. 80 g of cortex from brain of AD patients. Briefly, buffer (TBS/1 mM PMSF/1 COMPLETE protease inhibitor cocktail (Roche, p/n. 11 697 498 001) and phosphatase inhibitor (ThermoFischer, p/n. 78428)) is added to the AD brain tissue at about 10 ml/1 g (tissue). Tissue is homogenized using a handheld Kinematica Polytron at speed 6-7. Tissue is then further homogenized using Parr Bomb (Parr Instrument, p/n. 4653) at 1500 psi of nitrogen for 30 mins. Homogenate is spun at 28,000 g (J14 Beckman rotor) for 30 min at 4 C. Supernatant is collected, pooled and run over a 4 cm high guard column of Sepharose 400 Superflow to remove larger debris, then run over 25 ml MC1-Affigel 10 column at a flow rate of 50-60 ml per hour, in order to purify MC1-binding tau fibrils. To maximize the recovery of purification, supernatants are recycled through MC-1 column over 18-20 hours at 4 C. Guard column is removed and MC1 column is washed with TBS with at least 40 column volumes. Bound tau aggregates are then eluted with 2 column volumes of 3M KSCN, collecting in approx. 1 ml fractions. Protein concentration in each eluted fraction is checked by microtiter plate Bradford assay. Fractions containing positive protein levels are pooled, concentrated to about 2 ml using Centricon (Millipore Ultracel-30K) at 4 C., and dialyzed using a Slide-A-Lyzer cassette (10K MWCO 3-12 ml, Pierce) overnight against 1 liter TBS. The concentration of tau within the tau fibrils purified from AD brain homogenate is measured by sandwich ELISA using DA-9 capture antibody and CP27 detection antibody.

(10) Purified tau fibrils (50 l) in PBS are coated on wells of 96-well plates (Coastar, p/n. 3690) at a concentration corresponding to 0.7 g/ml of total tau. Plates are incubated overnight at 4 C., then washed three times with 150 l of PBST (PBS containing 0.05% Tween-20), blocked in 100 l BB3 (ImmunoChemistry Technology, p/n. 643) at room temperature for at least 1 hr (usually 2 hrs). Following blocking, the blocking buffer is removed from the wells. Exemplified tau monoclonal antibody of Example 1 and a humanized MC-1 antibody construct (having the framework combination: 5-51 heavy-chain, A27 light-chain) are diluted in 0.25% casein buffer to 1000 nM stock, then diluted serially 23 times with two fold dilutions. 50 l of stock and serially diluted antibody (either exemplified tau monoclonal of Example 1 or humanized MC-1 antibody construct) are added to separate wells and incubated for 2 hours at room temperature, after which the plate is washed four times with 200 l PBST per well. 50 l of anti-human IgG-HRP antibodies (diluted at 1:4000 into 0.25% casein buffer) is added and incubated for 1 hour at room temperature, after which the plate is washed with 200 l PBST per well 4 times. 50 l of TMB/H2O2 is added and incubated at room temperature for about 10 minutes. Reaction is stopped by adding 50 l stop solution (2N H2SO4) and colorimetric signal is measured at 450 nm. Data is input into Prism 6 (GraphPad) program and EC.sub.50 values are generated using a nonlinear regression curve fit and sigmoidal dose response. Results are presented in Table 3.

(11) TABLE-US-00003 TABLE 3 EC.sub.50 Comparison of Binding to Purified AD Tau Fibrils EC.sub.50 Antibody Assayed (pM) exemplified tau mAb of Example 1 6.8 humanized MC-1 Ab construct 409.1

(12) As reflected in Table 3, exemplified tau monoclonal antibody of the present invention demonstrates a 60 fold improved affinity (as measured by EC.sub.50) to purified tau fibrils over humanized MC-1 antibody construct.

(13) Selectivity of tau monoclonal antibody of Example 1 to tau aggregates versus tau monomer is determined by direct ELISA. Following the ELISA procedure substantially as provided above, recombinant tau (rTau) is coated on 96-well plates at a concentration corresponding to either a high concentration (1 g/mL) or low concentration (15 ng/mL). High concentration of rTau, when coated on micro-well plates, aggregates, simulating binding to aggregated tau. Low concentration of rTau, when coated on micro-well plates, simulates binding to tau monomer. The plates, coated with high or low concentrations of rTau, respectively, are exposed to exemplified tau monoclonal antibody of Example 1 and binding of exemplified tau monoclonal antibody to the respective concentrations of rTau is measured substantially as described in the ELISA assay above. Results are provided in Table 4.

(14) TABLE-US-00004 TABLE 4 EC.sub.50 Comparison of Binding to High vs. Low Concentration of rTau EC.sub.50 rTau Monomer Concentration (pM) High (1 g/mL) 6.0 Low (15 ng/mL) 722.7

(15) As reflected in Table 4, exemplified tau monoclonal antibody of Example 1 demonstrates a 120 fold improved affinity (as measured by EC.sub.50) to aggregate tau over monomeric tau.

(16) Ex Vivo Target Engagement Studies

(17) Binding of exemplified tau monoclonal antibody of Example 1 to aggregated tau derived from human brains is determined through immunohistochemistry staining of formalin-fixed paraffin-embedded (FFPE) brain sections obtained from: a normal individual (displaying minimal tau aggregation); an AD patient (displaying severe tau aggregation and NFT formation, as well as amyloid plaque pathology); a PD patient (displaying severe tau aggregation). Staining is also performed on brain sections derived from a control wild type mouse that possess no human tau in order to determine background non-specific staining levels.

(18) FFPE sections are de-paraffinized and rehydrated. Thereafter, antigen retrieval (using the Lab Vision PT module system, Thermo Scientific) is performed on the sections which includes heating sections in citrate buffer (Thermo Scientific, p/n. TA-250-PM1) for 20 minutes at 100 C. then cooling the sections in dH2O. Sections are then exposed to the following seven incubation steps (at room temp.): (1) 10 min. in 0.03% H2O2; (2) 30 min. in 1:20 dilution of normal goat serum (Vector Labs., p/n. S-1000) diluted in PBST; (3) 60 min. in either exemplified tau monoclonal antibody of Example 1 or humanized MC-1 antibody construct (having the framework combination: 5-51 heavy-chain, A27 light-chain) (both the exemplified tau monoclonal antibody and humanized MC-1 antibody construct are normalized to 1 mg/ml, then diluted in PBST to a dilution of 1:4000 before incubation with sections); (4) 30 min. in rabbit anti-human IgG4 (raised against the Fc region of the exemplified antibody) at a concentration of 1.1 g/ml in PBST; (5) 30 min. in 1:200 dilution of biotinylated goat anti-rabbit IgG (Vector Labs., p/n. BA-1000) diluted in PBST: (6) 30 min. in avidin-biotin complex solution (Vector Labs., p/n. PK-7100); (7) 5 min. in 3,3-diaminobenzidine (Vector Labs., p/n. SK-4105). Sections are washed between each of the above 7 steps using PBST. Following the seven incubation steps above, sections are counterstained with haematoxylin, dehydrated and cover-slipped. For mouse control tissue sections the above protocol is modified in incubation step (3) by using a 1:8000 dilution (as opposed to a 1:4000 dilution) of both the exemplified tau monoclonal antibody and humanized MC-1 antibody construct; and by replacing incubation steps (4) and (5) with a single 30 min. 1:200 dilution of biotinylated goat anti-human IgG (Vector Labs. p/n. BA-3000) in PBST.

(19) Following procedures substantially as described above, an analysis of the binding of the exemplified tau monoclonal antibody of Example 1 to tau derived from human brains is performed. Results are provided in Table 5.

(20) TABLE-US-00005 TABLE 5 Semi-quantitative analysis of binding to aggregated tau in FFPE AD brain sections. Severity of aggregated tau detected as measured by semi quantitative scoring scheme (severe, +++; moderate, ++; mild, +; negative, ) WT Normal control control Alzheimer's Pick's (murine) (human) disease disease Exemplified Tau + +++ +++ mAb of Example 1 Humanized MC-1 + + Ab construct

(21) The results provided in Table 5 reflect that exemplified tau monoclonal antibody of Example 1 demonstrates significantly higher levels of staining to aggregated tau, from both AD and PD patients, in hippocampal brain sections as compared to humanized MC-1 antibody construct. The results provided in Table 5 also demonstrate that exemplified tau monoclonal antibody of Example 1 does not demonstrate higher non-specific binding than humanized MC-1 antibody construct (exemplified tau monoclonal antibody demonstrates binding to the minimal amount of aggregated tau in normal control human sections). Further, because AD and PD are characterized by distinct splicing variants of the gene encoding tau, these results support a conclusion that exemplified tau monoclonal antibody of Example 1 specifically binds the conformational epitope comprising amino acid residues 7-9 and 312-322 of human tau (residue numbering based on the exemplified human tau of SEQ ID NO. 13) common to tau aggregates of both AD and PD.

(22) In Vitro Neutralization of Tau Aggregate Propagation

(23) Homogenate brain preps from approx. 5 month old P301S mice are known, in the presence of native, non-aggregate tau, to induce aggregation of the native tau and to demonstrate a propagation-like effect of tau aggregation. Sarkosyl-insoluble homogenate preps of brain tissue from 4.5 to 5 month old P301S mice are sonicated and diluted with OPTI-MEM (GIBCO by Life Tech., p/n. 31985-062) to bring measured tau (per prep) to a final concentration of 0.77 g/ml. Each prep is incubated for 30 minutes at room temperature with one of exemplified tau monoclonal antibody of Example 1 (at concentrations: 21.00, 7.00, 2.33, 0.78, 0.26, 0.09, 0.03, and 0.01 g/ml) or humanized MC-1 antibody construct (at concentrations: 50.00, 16.67, 5.56, 1.85, 0.62, 0.21, 0.07, 0.02 and 0.01 g/ml).

(24) HEK293 cells (a human embryonic kidney cell line) are transfected by electroporation to inducibly express a mutant form of human tau (1N4L, which has a serine substituted for proline at residue 301 (P301S) (residue numbering based on the exemplified human tau of SEQ ID NO. 13)). (Falcon B., et al., J. Biol. Chem. 290:1049-1065, 2015). Stably transfected HEK293 cells are plated at a concentration of 110.sup.4 cells/well into the wells of a 96-well plate in complete medium (D-MEM medium (Invitrogen, p/n. 11965-092), 10% fetal bovine serum (Invitrogen, p/n. 16000), 1 pen. strep (Invitrogen, p/n. 15140-122), 5 g/ml Blasticin (Invitrogen, p/n. R210-01), 200 g/ml Zeocin (Invitrogen, p/n. R250-01)). Plates are incubated for three days at 37 C. Following incubation, 1 mg/ml tetracycline is added at a 1:1000 dilution per well (to a final concentration of 1 g tetracycline/ml medium) to induce expression of mutant tau. Plates are then incubated for 24 hours at 37 C. Following incubation, culture medium is removed and 50 l of homogenate prep with one of the respective concentrations of one of exemplified tau monoclonal antibody of Example 1 or humanized MC-1 antibody construct (prepared as described above) is added. Plates are incubated for three hours, after which homogenate prep is removed and 100 l complete medium with 1 g/ml tetracycline and the same respective concentration of either exemplified tau monoclonal antibody or humanized MC-1 antibody construct is added to each respective well. Plates are incubated for 24 hours at 37 C., after which medium is removed and 100 l complete medium and the same respective concentration of either exemplified tau monoclonal antibody or humanized MC-1 antibody construct is added to the respective wells. Plates are incubated for 48 hours at 37 C. Following incubation, cells are washed with 200 l DPBS and drained.

(25) Cells are resuspended in 50 l H buffer (TBS pH7.4 containing 2 mM EGTA, 5 mM EDTA, protease and phosphatase inhibitor (Thermo Scientific, p/n. 784420)) per well and bath-sonicated for 10 minutes. Total protein concentration is measured by BCA Protein Assay (Thermo Scientific, p/n. PI-23227). Tau aggregate levels are determined by sandwich ELISA. 96-well plates are coated with 50 l of 2 g/ml AT8 antibody at 4 C. overnight. Plates are washed three times with PBST, then blocked with 100 l of BB3 for 1 hour at room temperature. A standard curve is prepared using AD brain total extract by serially dilution in 0.25% casein buffer using two-fold dilutions from a starting concentration of 40 g/ml to a final concentration of 0.3125 g/ml. Cell lysates are diluted into 0.25% casein buffer to a total protein concentration of about 0.1 mg/ml. 50 ul of each standard sample dilution or of diluted cell samples are then added into separate wells of blocked plates and incubated at 4 C. overnight, after which plates are washed four times with PBST. Biotinylated CP27 antibody is diluted 1:2000 in 0.25% casein buffer and 50 l is then added to into wells containing samples. Plates are incubated at room temperature for 2 hours, after which plates are washed four times with PBST. Strepavidin-HRP (Invitrogen, p/n. SNN2004) is diluted 1:5000 in 0.25% casein buffer and 50 l is then added into each well and plates are incubated at room temperature for 1 hour. Following incubation, plates are washed 4 times with PBST and 50 l of a 1:1 mixture of H2O2 and TMB (Thermo Scientific, p/n. 34021) is added. Plates are incubated at room temperature for 10 min. and the reaction is stopped by adding 50 l of H2SO4. Colorimetric signal is measured at 450 nm or 650 nm. AT8-positive tau levels are normalized against total protein levels in each sample. The normalized values for each sample are further normalized against AT8-positive tau levels in control samples (not treated with antibody). Percentage inhibition of tau aggregate propagation in each sample is determined by subtracting the further normalized values from 100 and the percentage of inhibition value for each sample is input into Prism 6 Software program (GraphPad) applying nonlinear regression curve fit and sigmoidal dose response for generation of EC.sub.50 values. Results are provided in Table 6.

(26) TABLE-US-00006 TABLE 6 EC.sub.50 values representative of tau aggregate propagation inhibition. Exemplified Engineered Tau Humanized MC- Ab of Example 1 1 Ab Construct EC.sub.50 (representing inhibition of 16 476 AT8-Positive Tau Aggregate propagation (ng/mL))

(27) The results provided in Table 6 reflect that exemplified tau monoclonal antibody of Example 1 demonstrates an approximately 30 fold improvement in the inhibition of induced tau aggregate propagation.

(28) In Vivo Neutralization of Tau Aggregate Propagation

(29) Homogenate brain stem preps from approx. 5 month old P301S mice are known to, upon injection into hippocampus of normal 10 week old female P301S mice, induce aggregation of native, non-aggregate tau, demonstrating a propagation-like effect of tau aggregation. Homogenate preps of brain stem tissue from 4.5 to 5 month old P301S mice are prepared substantially the same as described above.

(30) Normal 10 week old female P301S mice are injected in the left hemisphere of the hippocampus with 5 l homogenate brain prep and either: 7.5 g exemplified tau monoclonal antibody of Example 1 (N=12); or 7.5 g of control human IgG4 antibody (N=11). Four weeks post-injection, the mice are sacrificed and the left and right hemispheres are collected, paraffin embedded, and 6 m serial sections are mounted on glass slides. Slides containing bregma (A-P=2.30) are de-paraffinized, embedded tissue is rehydrated, and antigen retrieval is performed by heating slide to 100 C. for 20 min. in citrate buffer. Slides are cooled in dH2O and incubated at room temperature according to the following steps: (a) 10 min. in (0.03%) H2O2; (b) 30 min. in a 1:20 dilution of normal goat serum; (c) 60 min. in a 1:8000 dilution of PG-5 antibody (diluted in PBST)(PG-5 antibody obtained from the lab of Dr. Peter Davies, Albert Einstein College of Medicine of Yeshiva University; PG-5 antibody specifically binds serine at residue 409 of tau when phosphorylated, residue numbering based on the exemplified human tau of SEQ ID NO. 13); (d) 30 min. in a 1:200 dilution of biotinylated goat anti-mouse IgG antibody (diluted in PBST); (e) 30 min. in avidin-biotin complex solution; and (f) 5 min. in 3,3-diaminobenzidine. PBST is used for washing between the respective steps. Following the 5 min. incubation in 3,3-diaminobenzidine, sections are counterstained with haematoxylin, then rehydrated and cover-slipped. Staining signal is measured by Scanscope AT Slide Scanner (Aperio) at 20 magnification. PG-5 immunoreactivity is quantified and expressed as a percentage using the positive pixel algorithm of Imagescope Software (v. 11.1.2.780, Aperio). Results are provided in Table 7.

(31) TABLE-US-00007 TABLE 7 Mean % PG-5 immunoreactivity in left and right hippocampus, respectively. (% PG-5 Immunoreactivity) Left Hippocampus Right Hippocampus Exemplified Tau 2.52 0.49 SEM 0.63 0.13 SEM mAb of Example 1 Control IgG4 Ab 6.38 0.93 SEM 1.88 0.31 SEM
The results provided in Table 7 demonstrate the exemplified tau monoclonal antibody of Example 1 reduces the level of tau aggregation in both the left and right hippocampus as compared to the control IgG4 antibody. As shown, the exemplified tau monoclonal antibody produces a 60.5% greater reduction in tau aggregation in the left hippocampus, and a 66.5% greater reduction in tau aggregation in the right hippocampus, respectively, compared to control IgG4 antibody. These results demonstrate the exemplified tau monoclonal antibody possesses neutralizing activity against propagation of tau aggregation.
In Vivo Efficacy Analysis in the Tg4510 Murine Model

(32) Transgenic Tg4510 mice express a mutant form of human tau (4R0N, which has a leucine substituted for proline at residue 301 (P301L), Ramsden M., et al., J. Neuroscience, 25: 10637-10647 (2005) and Santacruz K., et al., Science (2005); residue numbering based on the exemplified human tau of SEQ ID NO. 13). Tg4510 mice exhibit high levels of expression of the P301L mutant human tau in the hippocampus and neocortex regions, which demonstrates age-dependent tau aggregation progression.

(33) Tau antibodies of the present invention may induce an immunogenic response in Tg4510 mice. Therefore, in order to test therapeutic potential of the tau monoclonal antibodies of the present invention for chronic administration in a rodent model, a surrogate murine tau antibody is constructed targeting the same conformational epitope and reflecting similar levels of improved affinity relative to the exemplified tau monoclonal antibody of Example 1. The surrogate tau antibody has an affinity (EC.sub.50) to purified AD tau fibrils, measured by ELISA as described above (for exemplified tau monoclonal antibody of Example 1), to be 13.1 pM.

(34) Eight week old female Tg4510 mice are grouped into 3 separate groups. The first group (N=15) is injected with a control mouse IgG1 antibody (15 mg/kg) twice a week for 9 weeks. The second group (N=15) is injected twice a week for 9 weeks with recombinant MC-1 antibody (15 mg/kg) produced from mouse ascites injected with MC-1 hybridoma. The third group (N=15) is injected with surrogate murine tau antibody (15 mg/kg) twice a week for 9 weeks. Following the final administration, the mice are sacrificed and their brains collected. Portions of cortex and hippocampus sections are collected, paraffin embedded, and 6 m serial sections are mounted on glass slides for immunohistochemistry use.

(35) Remainder of cortex region of collected brains are homogenized by pulse sonication in a volume of H buffer 10 times greater than the cortex volume, spun at 21,000 g for 20 min. at 4 C. and an aliquot of supernatant from each cortex is collected and total protein levels are determined by BCA Protein Assay (Thermo Scientific, p/n. PI-23227) according to manufacturer's protocol. The remainder of the supernatant is spun at 100,000 g for 1 hour at 4 C., the supernatant discarded, and the insoluble pellet obtained is resuspended in H buffer (in a volume the volume of discarded supernatant). The resuspended pellet is sonicated and AT8-positive tau aggregate levels in each pellet are determined by ELISA using AT8 capture antibody and CP27 detection antibody substantially as described above. AT8-positive tau aggregate levels are normalized against total protein levels.

(36) Similarly, remainder of hippocampus from the collected brains are homogenized by pulse sonication in a volume of H buffer 10 times greater than the hippocampus volume, spun at 21,000 g for 20 min. at 4 C., and supernatant from each hippocampus is collected and total protein levels are determined. AT8-positive tau aggregate levels in supernatant are determined by ELISA using AT8 capture antibody and CP27 detection antibody substantially as described above. AT8-positive tau aggregate levels are normalized against total protein levels. Results are provided in Table 8.

(37) TABLE-US-00008 TABLE 8 Levels of AT8-positive tau aggregate in cortex and hippocampus brain homogenate measured via ELISA. AT8-Positive Tau Aggregate Level (g/mg) Cortex Hippocampus Surrogate murine 1416 195 SEM 386 71 SEM Tau Ab Control mIgG1 Ab 1872 198 SEM 591 66 SEM rMC-1 mIgG1 Ab 1703 138 SEM 510 62 SEM

(38) The results provided in Table 8 demonstrate the surrogate murine tau antibody reduced the levels of tau aggregate in both cortex and hippocampus by 24% and 35%, respectively, relative to the control mIgG1 treated mice. The results further show mice treated with recombinant murine MC-1 antibody did not show improved reduction in levels of tau aggregate over control mIgG1 treated mice.

(39) The level of tau aggregation in the cortex and hippocampus of the paraffin embedded sections prepared from collected brains is also measured by immunohistochemistry using PG-5 substantially as described above. Data is normalized by conversion to log.sub.10 values and results are summarized in Table 9.

(40) TABLE-US-00009 TABLE 9 Mean log.sub.10 value of % PG-5 immunoreactivity in cortex and hippocampus. Tau Aggregate (mean log.sub.10 value of % PG-5 Immunoreactivity) Cortex Hippocampus Surrogate murine 0.74 0.06 0.26 0.07 Tau Ab Control mIgG1 Ab 0.90 0.05 0.46 0.05 rMC-1 mIgG1 Ab 0.83 0.04 0.34 0.06

(41) The results provided in Table 9 demonstrate the surrogate murine tau antibody reduces the level of tau aggregate in both the cortex (by 18%) and hippocampus (by 43%) relative to control mIgG1 antibody whereas recombinant murine MC-1 antibody did not demonstrate noticeable reduction in the level of tau aggregate in either cortex or hippocampus relative to control mIgG1 antibody.

(42) Physical-Chemical Properties of Engineered Tau Monoclonal Antibody

(43) The exemplified tau monoclonal antibody of Example 1 demonstrates good solubility, chemical stability, and physical stability.

(44) Solubility:

(45) Sufficiently high solubility is desired to enable convenient dosing. For example, a 1 mg/kg dose administered by a 1.0 mL injection into a 100 kg patient will require solubility of 100 mg/ml. In addition, maintaining the antibody in monomeric state without high molecular weight (HMW) aggregation at high concentration is also desirable. Solubility of the exemplified tau monoclonal antibody of Example 1 is analyzed by concentrating 15 mg of the exemplified antibody with a 10 K molecular weight cut-off filter (Amicon U.C. filters, Millipore, catalog # UFC903024) to a volume of less than 100 l. The final concentration of the sample was measured by UV absorbance at A280 using a Nanodrop 2000 (Thermo Scientific).

(46) Following procedures substantially as described above, the exemplified tau monoclonal antibody of Example 1 displays a solubility of greater than: 140 mg/ml (at pH 6 in 10 mM citrate buffer); 177 mg/ml (at pH 6 in 10 mM citrate with 150 mM NaCl); and 170 mg/ml (at pH 7.4 in PBS buffer). In addition, only low levels of HMW (from 3 to 5.4%) are present at high concentration and no phase separation is observed.

(47) Chemical and Physical Stability:

(48) Chemical stability facilitates the development of drug formulations with sufficient shelf-life. Chemical stability of the exemplified tau monoclonal antibody of Example 1 is assessed by formulating the exemplified tau antibody to a concentration of 1 mg/ml in 10 mM citrate and buffered pH 4, 5, 6, or 7. Formulated samples are incubated for four weeks at 4 C., 25 C., or 40 C. in an accelerated degradation study. Changes in charge profile of the antibody, reflecting chemical changes, are assessed using capillary isoelectric focusing (cIEF) according to standard procedures.

(49) Following procedures substantially as described above, the exemplified tau monoclonal antibody of Example 1 demonstrates chemical stability results presented in Table 10.

(50) TABLE-US-00010 TABLE 10 Summary of change in % main peak over four weeks, relative to samples incubated at 4 C., measured by cIEF and % HMW aggregates measured by SEC. Change in Change in Change in % of main peak % HMW % HMW after 4 weeks aggregates aggregates (relative to 4 C.) (relative to 4 C.) (relative to 4 C.) pH 25 C. 25 C. 40 C. 4 8.43 0.1 49.8 5 4.13 0.1 1.1 6 3.95 0.2 0.3 7 3.69 0.2 0.9

(51) Results provided in Table 10 demonstrate that after 4 weeks storage at 40 C., the exemplified tau antibody of Example 1 has a percentage of main peak decrease of only 1.1 percentage points when formulated at pH5, and a decrease of only 0.3 percentage points when formulated at pH6 (a common pH used in antibody formulation). In addition, mass spectrometry analysis demonstrates only minimal degradation observed after 4 weeks storage at 40 C. (1.5% LCDR1 deamidation with less than 5% degradation in all CDR sequences), indicating that the exemplified tau monoclonal antibody of Example 1 has sufficient chemical stability to facilitate development of solution formulations with adequate shelf life.

(52) For purposes of comparison, chemical and physical stability of a humanized MC-1 antibody construct (having the framework combination: 5-51 heavy-chain, A27 light-chain) is performed by incubating the antibody for 2 weeks at 40 C. at pH8. The humanized MC-1 antibody construct showed significant chemical degradation including 12% deamidation of LCDR1, 5% deamidation and 10% isomerization in HCDR3 and 3% oxidation in HC framework.

(53) Binding affinity, following a four week accelerated degradation study of the exemplified tau monoclonal antibody of Example 1, is assessed by formulating the exemplified monoclonal antibody to a concentration of 1 mg/ml in 10 mM citrate and buffered pH 4 or 6. Formulated samples are incubated for four weeks at 4 C. or 40 C. in an accelerated degradation study. Following incubation, binding affinity of the exemplified tau monoclonal antibody of Example 1 to rTau (15 ng/ml) coated on 96-well plates is determined by direct ELISA following the ELISA procedure substantially as described above. Results of the above-described binding affinity study, performed in duplicate, are provided in Table 11.

(54) TABLE-US-00011 TABLE 11 EC.sub.50 comparison following an accelerated degradation study. Incubation EC.sub.50 EC.sub.50 Formulation Temp. (pM) (pM) pH ( C.) Study 1 Study 2 4 40 414 277 6 4 926 636 6 40 754 667

(55) Table 11 demonstrates the binding affinity of the exemplified tau monoclonal antibody of Example 1 to low concentrations of rTau remained similar for samples following a four week accelerated degradation, as compared to control samples incubated at 4 C.

(56) TABLE-US-00012 Sequences SEQIDNO:1-LCofexemplifiedtaumonoclonalantibody ofExample1 EIVLTQSPGTLSLSPGERATLSCRSSQSLVHSNQNTYLHWYQQKPGQAPRLLIYKVDNRF SGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCSQSTLVPLTFGGGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:2-HCofexemplifiedtaumonoclonalantibody ofExample1 EVQLVQSGAEVKKPGESLKISCKGSGYTFSNYWIEWVRQMPGKGLEWMGEILPGSDSIKY EKNFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARRGNYVDDWGQGTLVTVSSASTK GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLSLSLG SEQIDNO:3-LCDR1ofexemplifiedtaumonoclonalantibody ofExample1 RSSQSLVHSNQNTYLH SEQIDNO:4-LCDR2ofexemplifiedtaumonoclonalantibody ofExample1 YKVDNRFS SEQIDNO:5-LCDR3ofexemplifiedtaumonoclonalantibody ofExample1 SQSTLVPLT SEQIDNO:6-HCDR1ofexemplifiedtaumonoclonalantibody ofExample1 KGSGYTFSNYWIE SEQIDNO:7-HCDR2ofexemplifiedtaumonoclonalantibody ofExample1 EILPGSDSIKYEKNFKG SEQIDNO:8-HCDR3ofexemplifiedtaumonoclonalantibody ofExample1 ARRGNYVDD SEQIDNO:9-LCVRofexemplifiedtaumonoclonalantibody ofExample1 EIVLTQSPGTLSLSPGERATLSCRSSQSLVHSNQNTYLHWYQQKPGQAPRLLIYKVDNRF SGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCSQSTLVPLTFGGGTKVEIK SEQIDNO:10-HCVRofexemplifiedtaumonoclonalantibody ofExample1 EVQLVQSGAEVKKPGESLKISCKGSGYTFSNYWIEWVRQMPGKGLEWMGEILPGSDSIKY EKNFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARRGNYVDDWGQGTLVTVSS SEQIDNO:11-NucleotideSequenceEncodingthe ExemplifiedLC(SEQIDNO:1) gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccacc ctctcctgcagatctagtcagagccttgtacacagtaatcagaacacctatttacattgg taccagcagaaacctggccaggctcccaggctcctcatctataaagttgacaaccgattt tctggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatc agcagactggagcctgaagattttgcagtgtattactgttctcaaagtacactggttccg ctcacgttcggcggagggaccaaggtggagatcaaacggaccgtggctgcaccatctgtc ttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctg ctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaa tcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctc agcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaa gtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc SEQIDNO:12-NucleotideSequenceEncodingthe ExemplifiedHC(SEQIDNO:2) gaggtgcagctggtgcagtctggagcagaggtgaaaaagcccggggagtctctgaagatc tcctgtaagggttctggctacacattcagtaactactggatagagtgggtgcgccagatg cccgggaaaggcctggagtggatgggggagattttacctggaagtgatagtattaagtac gaaaagaatttcaagggccaggtcaccatctcagccgacaagtccatcagcaccgcctac ctgcagtggagcagcctgaaggcctcggacaccgccatgtattactgtgcgagaaggggg aactacgtggacgactggggccagggcaccctggtcaccgtctcctcagcttctaccaag ggcccatcggtcttcccgctagcgccctgctccaggagcacctccgagagcacagccgcc ctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggc gccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactcc ctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaagacctacacctgcaac gtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtccc ccatgcccaccctgcccagcacctgaggccgccgggggaccatcagtcttcctgttcccc ccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtg gacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtg cataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagc gtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctcc aacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccga gagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagc ctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggaaagcaat gggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttc ttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctca tgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtct ctgggt SEQIDNO:13-AminoAcidSequenceofHuman, Full-LengthTau MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPG SETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAG HVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPK TPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAK SRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHV PGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNI THVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMV DSPQLATLADEVSASLAKQGL