Anti-tau C3 antibody and its use

Abstract

Anti-tau C3 antibodies are described that are at least several orders of magnitude more specific for tau C3 than full-length tau (2N4R). Methods of using the anti-tau C3 antibodies are also described.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 829,774, filed April 5, 2019, which is incorporated herein by reference. [Background technology]

[0002] Tau protein is a microtubule-associated protein that is distributed mainly in axons and regulates the assembly, spatial organization, and behavior of microtubules (MTs) in neurons. Tau protein is encoded by a single gene located on chromosome 17.

[0003] There are six known isoforms of tau protein. These isoforms differ from each other by the presence or absence of a 29-amino acid or 58-amino acid insertion in the amino-terminal region of tau and by the addition or deletion of a tandem repeat (which can be repeated either three or four times) in the carboxyl-terminal region of tau, referred to as the microtubule-binding domain. The microtubule-binding domain region consists of an imperfect repeat of 31-32 amino acid residues. The longest tau protein isoform (2N4R) is 441 amino acids in length and contains four repeats (R1, R2, R3, and R4) and two insertions. The smallest tau isoform contains 352 amino acid residues with three tandem repeats (R1, R3, and R4) in the microtubule-binding domain and no amino-terminal insertion. The amino acid sequences corresponding to the isoforms of human tau protein are provided in SEQ ID NOs: 1-6.

[0004] SEQ ID NO: 1 is the longest tau isoform, htau40, which contains two N-terminal inserts and four microtubule-binding (2N4R) domains, as follows: MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 60 SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG 120 HVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPK 180 TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAK 240 SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINK KLDLSNVQSK CGSKDNIKHV 300 PGGGSVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI 360 THVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV 420 DSPQLATLAD EVSASLAKQG L 441

[0005] SEQ ID NO:2 contains two N-terminal inserts and three microtubule-binding domains (2N3R) as follows: MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 60 SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG 120 HVTQARMVSK SLDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPK 180 TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAK 240 SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIVYK PVDLSKVTSK CGSLGNIHHK 300 PGGGQVEVKS EKLDFKDRVQ SKIGSLDNIT HVPGGGNKKI ETHKLTFREN AKAKTDHGAE 360 IVYKSPVVSG DTSPAHLSNV SSTGSIDMVD SPQLATLADE VSASLAKQGL 410

[0006] SEQ ID NO:3 contains one N-terminal insert and four microtubule-binding domains (1N4R) as follows: MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTGDGSEEPG 60 SETSDAKSTP TAEAEEAGIG DTPSLEDEAA GHVTQARMVS KSLDGTGSDD KKAKGADGKT 120 LIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR 180 SRTPSLPTPP TREPKKVAVV RTPPKSPSSA KSRLQTAPVP MPDLKNVKSK IGSTENLKHQ 240 PGGGKVQIIN KKLDLSNVQS KCGSLDNILH VPGGGSVQIV YKPVDLSKVT SKCGSLGNIH 300 HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN ITHVPGGGNK KIETHKLTFR ENAKAKTDHG 360 AEIVYKSPVV SGDTSPRHLS NVSSTGSIDM VDSPQLATLA DEVSASLAKQ GL 412

[0007] SEQ ID NO:4 contains four microtubule-binding domains (ON4R) without an N-terminal insert as follows: MAEPRQEFEV MEDHAGTYGL GDRLDQGGYT MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA 60 AGHVTQARMV SKSKDGTGSD DKKAKGADGK TKIATPRGAA PPGQKGQANA TRIPAKTPPA 120 PKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTP PTREPKKVAV VATPPKSPSS 180 AKSRLQTAPV PMPDLKNVKS LIGSTENLKH QPGGGKVQII NKKLDLSNVQ SKCGSKDNIK 240 HVPGGGSVQI VYKPVDLSKV TSKCGSLGNI HHKPGGGQVE VKSEKLDFKD RVQSKIGSLD 300 NITHVPGGGN KKIETHKLTF RENAKALTDH GAEIVYKSPV VSGDTSPRHL SNVSSTGSID 360 MVDSPQLATL ADEVSASLAK QGL 383

[0008] SEQ ID NO:5 contains one N-terminal insert and three microtubule-binding domains (1N3R) as follows: MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 60 SETSDAKSTP TAEAEEAGIG DTPSLEDEAA GHVTQARMVS KSKDGTGSDD KKAKGADGKT 120 KIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR 180 SRTPSLPTPP TREPKKVAVV RTPPKSPSSA KSRLQTAPVP MPDLKNVKSK IGSTENLKHQ 240 PGGGKVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI 300 THVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV 360 DSPQLATLAD EVSASLAKQG L 381

[0009] SEQ ID NO:6 contains three microtubule-binding domains (ON3R) without an N-terminal insert as follows: MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA 60 AGHVTQARMV SKSKDGTGSD DKKAKGADGK TKIATPRGAA PPGQKGQANA TRIPAKTPPA 120 PKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTP PTREPKKVAV VRTPPKSPSS 180 AKSRLQTAPV PMPDLKNVKS KIGSTENLKH QPGGGKVQIV YKPVDLSKVT SKCGSLGNIH 240 HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN ITHVPGGGNK KIGTHKLTFR ENAKAKTDHG 300 AEIVYKSPVV SGDTSPRHLS NVSSTGSIDM VDSPQLATLA DEVSASLAKQ GL 352

[0010] Tau C3 is an extremely toxic, nucleating, pretangled, intracellularly and preferentially secreted C-terminally truncated tau fragment terminating at aspartate 421. Tau C3 is present at low abundance compared to full-length tau (FLT) (2N4R) but has been shown to exert disproportionately large pathological effects. Tau C3 may contribute, for example, to the seeding and propagation of pathological tau aggregates.

[0011] Pathological aggregation and propagation of tau in the brain are associated with over 20 neurodegenerative disorders, including, for example, Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), and frontotemporal lobar degeneration (FTLD) (collectively referred to as "tauopathies"). Summary of the Invention

[0012] It is an object of the present invention to provide chimeric antibodies that can be used in the diagnosis and treatment of neurodegenerative disorders associated with the pathological activity of tau C3 in the brain.

[0013] It is also an object of the present invention to provide humanized antibodies that can be used in the diagnosis and treatment of neurodegenerative disorders associated with the pathological activity of tau C3 in the brain.

[0014] It is an additional object of the present invention to provide human antibodies that can be used in the diagnosis and treatment of neurodegenerative disorders associated with the pathological activity of tau C3 in the brain.

[0015] It is a further object of the present invention to provide chimeric antibodies specific for the C-terminus of tau C3.

[0016] Specific for the C-terminus of tau C3, 1 × 10 -3 s -1 The following off-rates (K d It is a further object of the present invention to provide a chimeric antibody having

[0017] It is also an object of the present invention to provide a humanized antibody specific for the C-terminus of tau C3.

[0018] Specific for the C-terminus of tau C3, 1 × 10 -3 s -1 The following off-rates (K d It is also an object of the present invention to provide a humanized antibody having the following structure:

[0019] In furtherance of the above and other objects, the present invention relates to chimeric, humanized, and human antibodies specific for the C-terminus of tau C3 ("anti-tau C3 antibodies"). The anti-tau C3 antibodies are administered in a concentration of 1×10 -10 ~1×10 -12 Binding affinity (KD) of 1×10 for tau C3 -4 ~1×10 -8 For example, the anti-tau C3 antibody has a binding affinity (KD) for full-length tau ("FLT") (SEQ ID NO: 1) of approximately 5×10 -12 M ~ approx. 1.2×10 -10 M, about 1 x 10 -11 M ~ approx. 1×10 -10 M, about 1 x 10 -11 M ~ approx. 9×10 -11 M, about 1 x 10 -11 M ~ approx. 8×10 -11 M, about 1 x 10 -11 M ~ approx. 7×10 -11 M, about 1 x 10 -11 M ~ approx. 6×10 -11 M, about 1 x 10 -11 M ~ approx. 5×10 -11 M, or approximately 1 x 10 -11 M ~ approx. 4×10 -11 The binding affinity (KD) of M for tau C3 and 1 × 10 -4 ~1×10 -8The antibody may have a binding affinity (KD) for FLT of M. In a preferred embodiment, the antibody retains its binding ability after being subjected to temperatures of about 40°C to about 67°C for 10 minutes, and also retains its binding ability after incubation in serum (e.g., mouse) at 37°C for 21 days. The high potency of anti-tau C3 antibodies allows the antibody to target tau C3 without impairing the normal physiological function of FLT. In some embodiments, the specificity of the antibody allows it to target only the most toxic species of tau. This may potentially allow for a reduced therapeutically effective dose, for example, compared to antibodies that are not specific and do not distinguish between different species of tau. Anti-tau C3 antibodies, and antigen-binding fragments thereof, can be used in the diagnosis and treatment of neurodegenerative disorders associated with the pathological activity of tau C3 in the brain, including, for example, Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), frontotemporal lobar degeneration (FTLD), and the like. The anti-tau C3 antibody may have a water solubility of 50 mg / mL or more (e.g., about 50 mg / mL to about 200 mg / mL, about 55 mg / mL to about 180 mg / mL, about 55 mg / mL to about 170 mg / mL, about 55 mg / mL to about 150 mg / mL, about 55 mg / mL to about 140 mg / mL, about 55 mg / mL to about 130 mg / mL, or about 60 mg / mL to about 130 mg / mL).

[0020] The present invention further relates to chimeric, humanized, and human anti-tau C3 antibodies that have a higher binding affinity (KD) for tau C3 than murine anti-tau C3 antibodies. In some embodiments, the chimeric, humanized, and human anti-tau C3 antibodies have a binding affinity (KD) for tau C3 that is at least two-fold, three-fold, or four-fold higher than the binding affinity (KD) for tau C3 of the murine anti-tau C3 antibody. In some embodiments, the murine anti-tau C3 antibody has a binding affinity (KD) for tau C3 of about 4.9 x 10 -11 M, and chimeric, humanized, and human anti-tau C3 antibodies have a binding affinity (KD) for tau C3 of approximately 1 x 10-11 M ~ approx. 2.5×10 -11 Chimeric, humanized, and human anti-tau C3 antibodies have a binding affinity (KD) for tau C3 of, for example, about 1.1 x 10 -11 M, approx. 1.3 x 10 -11 M, approx. 1.5 x 10 -11 M, approx. 1.7 x 10 -11 M, approx. 1.9×10 -11 M, approx. 2.1 x 10 -11 M, or approximately 2.3 x 10 -11 The anti-tau C3 antibody may have a binding affinity (KD) for tau C3 of M. The anti-tau C3 antibody may have a water solubility of 50 mg / mL or more (e.g., about 50 mg / mL to about 200 mg / mL, about 55 mg / mL to about 180 mg / mL, about 55 mg / mL to about 170 mg / mL, about 55 mg / mL to about 150 mg / mL, about 55 mg / mL to about 140 mg / mL, about 55 mg / mL to about 130 mg / mL, or about 100 mg / mL to about 200 mg / mL, about 100 mg / mL to about 180 mg / mL, about 100 mg / mL to about 170 mg / mL, about 100 mg / mL to about 150 mg / mL, about 100 mg / mL to about 140 mg / mL, or about 100 mg / mL to about 130 mg / mL).

[0021] The present invention is -3 s -1 The following off-rates (K d ) with 1×10 -10 ~1×10 -12 and a binding affinity (KD) for tau C3 of 1×10 -4 ~1×10 -8 The present invention also relates to chimeric, humanized and human anti-tau C3 antibodies having a binding affinity (KD) for FLT of M.

[0022] The anti-tau C3 antibody, or antigen-binding fragment thereof, comprises (a) a heavy chain variable region comprising CDR1 represented by the sequence GFTFNTYA (SEQ ID NO: 7), CDR2 represented by IRSKSNNYAT (SEQ ID NO: 8), and CDR3 represented by VGGGDF (SEQ ID NO: 9), and (b) a light chain variable region comprising CDR1 represented by the sequence QEISVY (SEQ ID NO: 10), CDR2 represented by the sequence GAF (SEQ ID NO: 11), and CDR3 represented by the sequence LQYVRYPWT (SEQ ID NO: 12), and is administered in a concentration of 1 x 10 -10 ~1×10 -12 The binding affinity (KD) for tau C3 is 1×10 -3 The off-rate (K d ), 1×10 -4 ~1×10 -8 M, or has no detectable binding to FLT (SEQ ID NO: 1).

[0023] In certain embodiments, the anti-tau C3 antibody, or antigen-binding fragment thereof, comprises (a) a heavy chain variable region comprising a CDR1 homologous to the sequence GFTFNTYA (SEQ ID NO: 7), a CDR2 homologous to IRSKSNNYAT (SEQ ID NO: 8), and a CDR3 homologous to VGGGDF (SEQ ID NO: 9), and (b) a light chain variable region comprising a CDR1 homologous to the sequence QEISVY (SEQ ID NO: 10), a CDR2 homologous to the sequence GAF (SEQ ID NO: 11), and a CDR3 homologous to the sequence LQYVRYPWT (SEQ ID NO: 12), and is administered in a concentration of 1 x 10 -10 ~1×10 -12 The binding affinity (KD) for tau C3 is 1×10 -3 The off-rate (K d ), 1×10 -4 ~1×10 -8 M, or has no detectable binding to FLT (SEQ ID NO: 1).

[0024] In certain embodiments, the anti-tau C3 antibody, or antigen-binding fragment thereof, comprises (a) a heavy chain variable region comprising a CDR1 identical to the sequence GFTFNTYA (SEQ ID NO: 7), a CDR2 identical to the sequence IRSKSNNYAT (SEQ ID NO: 8), and a CDR3 identical to the sequence VGGGDF (SEQ ID NO: 9), and (b) a light chain variable region comprising a CDR1 identical to the sequence QEISVY (SEQ ID NO: 10), a CDR2 identical to the sequence GAF (SEQ ID NO: 11), and a CDR3 identical to the sequence LQYVRYPWT (SEQ ID NO: 12), and is administered in a concentration of 1 x 10 -10 ~1×10 -12 The binding affinity (KD) for tau C3 is 1×10 -3 The off-rate (K d ), 1×10 -4 ~1×10 -8 M, or has no detectable binding to FLT (SEQ ID NO: 1).

[0025] In certain embodiments, the anti-tau C3 antibody, or antigen-binding fragment thereof, comprises (a) a heavy chain variable region comprising a CDR1 of the sequence GFTFNTYA (SEQ ID NO: 7), a CDR2 of the sequence IRSKSNNYAT (SEQ ID NO: 8), and a CDR3 of the sequence VGGGDF (SEQ ID NO: 9), and (b) a light chain variable region comprising a CDR1 of the sequence QEISVY (SEQ ID NO: 10), a CDR2 of the sequence GAF (SEQ ID NO: 11), and a CDR3 of the sequence LQYVRYPWT (SEQ ID NO: 12), and is administered in a concentration of 1 x 10 -10 ~1×10 -12 The binding affinity (KD) for tau C3 is 1×10 -3 The off-rate (K d ), 1×10 -4 ~1×10 -8M, or has no detectable binding to SEQ ID NO: 1, and is used to treat Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), frontotemporal lobar degeneration (FTLD). The antibody may also be used to diagnose tauopathies, such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), or frontotemporal lobar degeneration (FTLD).

[0026] In one aspect, the present invention relates to a humanized antibody comprising: (a) a heavy chain variable region comprising CDR1 represented by SEQ ID NO:7, CDR2 represented by SEQ ID NO:8, and CDR3 represented by SEQ ID NO:9; and (b) a light chain variable region comprising CDR1 represented by SEQ ID NO:10, CDR2 represented by SEQ ID NO:11, and CDR3 represented by SEQ ID NO:12, wherein the antibody is administered in a concentration of 1×10 -10 ~1×10 -12 The binding affinity (KD) for tau C3 is 1×10 -3 s -1 Less than (e.g., 1×10 -4 ~1×10 -3 s -1 ) is the off-rate (K d ), 1×10 -4 ~1×10 -8 The present invention relates to an anti-tau C3 antibody having a binding affinity (KD) for FLT (SEQ ID NO: 1) of M or no detectable binding to FLT. Thus, the humanized antibody may comprise (a) a heavy chain variable region comprising CDR1 of SEQ ID NO: 7, CDR2 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 9, and (b) a light chain variable region comprising CDR1 of SEQ ID NO: 10, CDR2 of SEQ ID NO: 11, and CDR3 of SEQ ID NO: 12, and is capable of binding to FLT in a concentration of 1×10 -10 ~1×10 -12 The binding affinity (KD) for tau C3 is 1×10 -3 s -1 Less than (e.g., 1×10 -4 ~1×10-3 s -1 ) is the off-rate (K d ), 1×10 -4 ~1×10 -8 M, or has no detectable binding to FLT, and is used to treat tauopathies such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), and frontotemporal lobar degeneration (FTLD).

[0027] In one aspect, the invention relates to a humanized antibody comprising: (a) a heavy chain variable region comprising CDR1 identical to SEQ ID NO: 7, CDR2 identical to SEQ ID NO: 8, and CDR3 identical to SEQ ID NO: 9; and (b) a light chain variable region comprising CDR1 identical to SEQ ID NO: 7, CDR2 identical to SEQ ID NO: 11, and CDR3 identical to SEQ ID NO: 12; -10 ~1×10 -12 The binding affinity (KD) for tau C3 is 1×10 -3 s -1 Less than (e.g., 1×10 -4 ~1×10 -3 s -1 ) is the off-rate (K d ), 1×10 -4 ~1×10 -8 The present invention relates to an anti-tau C3 antibody having a binding affinity (KD) for FLT (SEQ ID NO: 1) of M or no detectable binding to FLT. Thus, the humanized antibody comprises (a) a heavy chain variable region having CDR1 of SEQ ID NO: 7, CDR2 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 9, and (b) a light chain variable region having CDR1 of SEQ ID NO: 10, CDR2 of SEQ ID NO: 11, and CDR3 of SEQ ID NO: 12, and is capable of binding to FLT in a concentration of 1×10 -10 ~1×10 -12 The binding affinity (KD) for tau C3 is 1×10 -3 s -1 Less than (e.g., 1×10 -4 ~1×10 -3 s -1 ) is the off-rate (Kd ), 1×10 -4 ~1×10 -8 M, or has no detectable binding to FLT, and is used to treat tauopathies such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), and frontotemporal lobar degeneration (FTLD).

[0028] In one aspect, the invention relates to a humanized antibody comprising: (a) a heavy chain variable region comprising CDR1 homologous to SEQ ID NO:7, CDR2 homologous to SEQ ID NO:8, and CDR3 homologous to SEQ ID NO:9; and (b) a light chain variable region comprising CDR1 homologous to SEQ ID NO:11, CDR2 homologous to SEQ ID NO:11, and CDR3 homologous to SEQ ID NO:12, wherein the antibody is administered in a concentration of 1×10 -10 ~1×10 -12 The binding affinity (KD) for tau C3 is 1×10 -3 s -1 Less than (e.g., 1×10 -4 ~1×10 -3 s -1 ) is the off-rate (K d ), 1×10 -4 ~1×10 -8 The present invention relates to an anti-tau C3 antibody having a binding affinity (KD) for FLT (SEQ ID NO: 1) of M or no detectable binding to FLT. Thus, the humanized antibody may comprise (a) a heavy chain variable region comprising CDR1 of SEQ ID NO: 7, CDR2 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 9, and (b) a light chain variable region comprising CDR1 of SEQ ID NO: 10, CDR2 of SEQ ID NO: 11, and CDR3 of SEQ ID NO: 12, and is capable of binding to FLT in a concentration of 1×10 -10 ~1×10 -12 The binding affinity (KD) for tau C3 is 1×10 -3 s -1 Less than (e.g., 1×10 -4 ~1×10 -3 s -1 ) is the off-rate (K d ), 1×10 -4 ~1×10-8 M, or has no detectable binding to FLT, and is used to treat tauopathies such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), and frontotemporal lobar degeneration (FTLD).

[0029] Humanized antibodies can be, for example, (a) a variable heavy chain comprising the sequence LVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQASGKGLEWVARIRSKS-NNYATYYAASVKGRFTISRDDSKSMAYLQMDSLKTEDTAVYYCVGGGDFWGQGTLVTVSS (SEQ ID NO: 13), or a sequence homologous to SEQ ID NO: 13; and (b) DIQMTQSPSSLSASVGDRVTITCRASQEISVYLGWFQQKPGKAPKRLIYGAFKLQSGVPSRFSGSRSGTEFTLTISSLQPEDFATYYCLQYVRYPWTFGGGTKVEIK (SEQ ID NO: 14) or a sequence homologous to SEQ ID NO: 14; DIQMTQSPSSLSASVGDRVTITCRASQEISVYLGWYQQKPGKAPKRLIYGAFTLQSGVPSRFSGSRSGTEYTLTISSLQPEDFATYYCLQYVRYPWTFGGGTKVEIK (SEQ ID NO: 15) or a sequence homologous to SEQ ID NO: 15, DIQMTQSPSSLSASVGDRVTITCRASQEISVYLGWYQQKPGKAPKRLIYGAFSLQSGVPSRFSGSRSGTEYTLTISSLQPEDFATYYCLQYVRYPWTFGGGTKVEIK (SEQ ID NO: 16) or a sequence homologous to SEQ ID NO: 16, DIQMTQSPSSLSASVGDRVTITCRASQEISVYLGWFQQKPGKAPKRLIYGAFKLQSGVPSRFSGSRSGTEYTLTISSLQPEDFATYYCLQYVRYPWTFGGGTKVEIK (SEQ ID NO: 17) or a sequence homologous to SEQ ID NO: 17, and a variable light chain comprising a sequence selected from the group consisting of: DIQMTQSPSSLSASVGDRVTITCRASQEISVYLSWFQQKPGKAIKRLIYGAFSLQSGVPSRFSGSRSGTEYTLTISSLQPEDFATYYCLQYVRYPWTFGGGTKVEIK (SEQ ID NO: 18), or a sequence homologous to SEQ ID NO: 18; 1×10 -10 ~9×10 -12 Binding affinity (KD) for tau C3 of 1 x 10 -4 ~1×10 -8 has a binding affinity (KD) for FLT (SEQ ID NO: 1) of M or has no detectable binding to FLT.

[0030] In certain embodiments, the humanized antibody comprises a variable heavy chain (V H ) polypeptide and the variable light chain (V) of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 18 L ) polypeptides.

[0031] In certain embodiments, the humanized antibody comprises: (a) a variable heavy chain (V) comprising a CDR1 represented by SEQ ID NO:7, a CDR2 represented by SEQ ID NO:8, and a CDR3 represented by SEQ ID NO:9; H ) polypeptide, comprising a variable heavy chain (V) having at least 70% sequence identity to SEQ ID NO: 13 H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptide having at least 70% sequence identity to SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. L ) polypeptides.

[0032] In certain embodiments, the humanized antibody has at least 75% sequence identity to SEQ ID NO: 13. V Hand a chain polypeptide having at least 75% sequence identity with SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. V L It includes a chain polypeptide.

[0033] In certain embodiments, the humanized antibody has at least 80% sequence identity to SEQ ID NO: 13. V H and a chain polypeptide having at least 80% sequence identity with SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. V L It includes a chain polypeptide.

[0034] In certain embodiments, the humanized antibody has at least 85% sequence identity to SEQ ID NO: 13. V H and a chain polypeptide having at least 85% sequence identity with SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. V L It includes a chain polypeptide.

[0035] In certain embodiments, the humanized antibody has at least 90% sequence identity to SEQ ID NO: 13. V H and a chain polypeptide having at least 90% sequence identity with SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. V L It includes a chain polypeptide.

[0036] In certain embodiments, the humanized antibody has at least 95% sequence identity to SEQ ID NO: 13. V H and a chain polypeptide having at least 95% sequence identity with SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. V L It includes a chain polypeptide.

[0037] In certain embodiments, the anti-tau C3 antibody comprises a variable heavy chain (V) comprising SEQ ID NO: 13. H ) polypeptide, and a variable light chain (V) comprising a sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18. L ) polypeptides.

[0038] In certain embodiments, the anti-tau C3 antibody comprises: (i) a variable heavy chain (V) comprising CDR1, CDR2, and CDR3; H ) polypeptide, wherein CDR1 is the polypeptide of SEQ ID NO: 7, CDR2 is the polypeptide of SEQ ID NO: 8, and CDR3 is the polypeptide of SEQ ID NO: 9; H ) polypeptide, and (ii) a variable light chain (V) comprising CDR1, CDR2, and CDR3. L ) polypeptide, wherein CDR1 is the polypeptide of SEQ ID NO: 10, CDR2 is the polypeptide of SEQ ID NO: 11, and CDR3 is the polypeptide of SEQ ID NO: 12. L ) polypeptides.

[0039] In certain embodiments, the anti-tau C3 antibody comprises: (i) a variable heavy chain (V H ) polypeptide, and (ii) a variable light chain (V L ) polypeptide, H ) polypeptide is the polypeptide of SEQ ID NO: 13, and the variable light chain (V L ) The polypeptide is the polypeptide of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.

[0040] The anti-tau C3 antibody also comprises (a) a heavy chain variable region comprising a CDR1 sequence represented by or homologous to SEQ ID NO:7, a CDR2 sequence represented by or homologous to SEQ ID NO:8, and a CDR3 sequence represented by or homologous to SEQ ID NO:9, and (b) a light chain variable region comprising a CDR1 sequence represented by or homologous to SEQ ID NO:10, a CDR2 sequence represented by or homologous to SEQ ID NO:11, and a CDR3 sequence represented by or homologous to SEQ ID NO:12, and is expressed in a concentration of 1×10 -10 ~1×10 -12 and a binding affinity (KD) for tau C3 of 1×10 -3- s -1 Less than (e.g., 1×10 -4 ~1×10 -3 s -1 ) is the off-rate (K d ), and 1 × 10 -4 ~1×10 -8 The antibody may be a chimeric antibody that has a binding affinity (KD) for FLT (SEQ ID NO: 1) that is M, or that has no detectable binding to FLT (SEQ ID NO: 1).

[0041] The present invention also provides a 1×10 antibody comprising (a) a heavy chain variable region comprising CDR1 represented by SEQ ID NO:7, CDR2 represented by SEQ ID NO:8, and CDR3 represented by SEQ ID NO:9, and (b) a light chain variable region comprising CDR1 represented by SEQ ID NO:10, CDR2 represented by SEQ ID NO:11, and CDR3 represented by SEQ ID NO:12. -10 ~1×10 -12 The binding affinity (KD) for tau C3 is 1×10 -3 s -1 Less than (e.g., 1×10 -4 ~1×10 -3 s -1 ) is the off-rate (K d ), 1×10 -4 ~1×10 -8The present invention relates to an antigen-binding fragment of an antibody that has a binding affinity (KD) for FLT (SEQ ID NO: 1) that is M, or has no detectable binding to FLT (SEQ ID NO: 1). The antigen-binding fragment of an antibody can be, for example, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or an scFv fragment.

[0042] The present invention also provides a method for producing a 1×10 antibody comprising: (a) a heavy chain variable region comprising a CDR1 that is homologous to SEQ ID NO: 7, a CDR2 that is homologous to SEQ ID NO: 8, and a CDR3 that is homologous to SEQ ID NO: 9; and (b) a light chain variable region comprising a CDR1 that is homologous to SEQ ID NO: 10, a CDR2 that is homologous to SEQ ID NO: 11, and a CDR3 that is homologous to SEQ ID NO: 12. -10 ~1×10 -12 The binding affinity (KD) for tau C3 is 1×10 -3 s -1 Less than (e.g., 1×10 -4 ~1×10 -3 s -1 ) is the off-rate (K d ), 1×10 -4 ~1×10 -8 The present invention relates to an antigen-binding fragment of an antibody that has a binding affinity (KD) for FLT (SEQ ID NO: 1) that is M, or has no detectable binding to FLT (SEQ ID NO: 1). The antigen-binding fragment of an antibody can be, for example, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or an scFv fragment.

[0043] The present invention also provides a method for producing a 1×10 antibody comprising: (a) a heavy chain variable region comprising a CDR1 identical to SEQ ID NO:7, a CDR2 identical to SEQ ID NO:8, and a CDR3 identical to SEQ ID NO:9; and (b) a light chain variable region comprising a CDR1 identical to SEQ ID NO:10, a CDR2 identical to SEQ ID NO:11, and a CDR3 identical to SEQ ID NO:12. -10 ~1×10 -12 The binding affinity (KD) for tau C3 is 1×10 -3 s -1 Less than (e.g., 1×10 -4 ~1×10 -3 s -1 ) is the off-rate (K d ), 1×10 -4~1×10 -8 The present invention relates to an antigen-binding fragment of an antibody that has a binding affinity (KD) for FLT (SEQ ID NO: 1) that is M, or has no detectable binding to FLT (SEQ ID NO: 1). The antigen-binding fragment of an antibody can be, for example, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or an scFv fragment.

[0044] The present invention also relates to methods for blocking pathological tau uptake, blocking pathological tau seeding activity, inhibiting pathological tau aggregation, and blocking the propagation of pathological tau, tau fibrils, and tau aggregates from one neuron to another, or from one part of the brain to another, comprising administering to a subject in need thereof an effective amount of an anti-tau C3 antibody. In some of these embodiments, the anti-tau C3 comprises (a) a variable heavy chain (V) comprising a CDR1 represented by SEQ ID NO:7, a CDR2 represented by SEQ ID NO:8, and a CDR3 represented by SEQ ID NO:9. H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0045] Upon administration, anti-tau C3 antibodies can block or reduce the propagation of pathological tau from one neuron to another or from one part of the brain to another, for example, by blocking or reducing tau C3 seeding activity, e.g., essentially blocking or reducing the cellular uptake of tau C3. This mechanism occurs extracellularly and does not require the anti-tau C3 antibody, which may be present inside the neuron. Anti-tau C3 antibodies can block or reduce the propagation of tau C3 tau, tau C3-containing fibrils, and tau C3-containing aggregates from one neuron to another and from one part of the brain to another. The aggregates can include heterogeneous populations of full-length tau (e.g., 2N4R), tau oligomers, and / or post-translationally modified tau (truncated or hyperphosphorylated). In addition to blocking the cellular uptake of tau C3 and fibrils containing tau C3, anti-tau C3 antibodies can also block or reduce pathological tau aggregation inside cells (e.g., neurons). Because the antibodies have substantially no affinity for full-length tau (e.g., 2N4R), the antibodies do not interfere with the normal, non-pathological functions of full-length tau. In some embodiments, the anti-tau C3 antibodies comprise (a) a variable heavy chain (V) comprising CDR1 represented by SEQ ID NO:7, CDR2 represented by SEQ ID NO:8, and CDR3 represented by SEQ ID NO:9. H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0046] Anti-tau C3 antibodies can also bind extracellular tau C3 and aggregates containing tau C3 released from cells, thereby reducing the propagation of tau C3-containing fibrils and aggregates. This prevents tau C3 and aggregates containing tau C3 from entering neighboring cells, reducing the propagation of tau aggregates from one neuron to another and from one part of the brain to another. Thus, anti-tau C3 antibodies can function as a means of preventing the entry of tau C3 or aggregates containing tau C3 into cells (e.g., neurons). In some embodiments, anti-tau C3 antibodies comprise (a) a variable heavy chain (V) comprising CDR1 represented by SEQ ID NO:7, CDR2 represented by SEQ ID NO:8, and CDR3 represented by SEQ ID NO:9. H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0047] Anti-tau C3 antibodies can also be used to delay and / or reduce the interneuronal propagation of tau aggregation. For example, anti-tau C3 antibodies can promote disaggregation of protein fibrils containing tau C3, block the intracellular conversion of monomeric tau C3 to fibrils and / or aggregates containing tau C3, and promote the intracellular degradation of fibrils and / or aggregates containing tau C3. In addition to tau C3, the fibrils and aggregates can contain heterogeneous populations of full-length tau (e.g., 2N4R), tau oligomers, and / or post-translationally modified tau (truncated or hyperphosphorylated). In some embodiments, the anti-tau C3 antibody comprises (a) a variable heavy chain (V) comprising CDR1 represented by SEQ ID NO:7, CDR2 represented by SEQ ID NO:8, and CDR3 represented by SEQ ID NO:9. H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0048] Anti-tau C3 antibodies can reduce brain atrophy in subjects with tauopathy. In some embodiments, the anti-tau C3 antibody comprises (a) a variable heavy chain (V) comprising CDR1 represented by SEQ ID NO:7, CDR2 represented by SEQ ID NO:8, and CDR3 represented by SEQ ID NO:9. H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0049] Anti-tau C3 antibodies can inhibit the formation of insoluble aggregates comprising heterogeneous populations of full-length tau (e.g., 2N4R), tau oligomers, and / or post-translationally modified tau (e.g., truncated or hyperphosphorylated). For example, this can reduce the amount of pathological tau (e.g., tau C3, tau C3-containing fibrils, and tau C3-containing aggregates) in the brain. In some embodiments, the anti-tau C3 antibody comprises (a) a variable heavy chain (V) comprising CDR1 represented by SEQ ID NO:7, CDR2 represented by SEQ ID NO:8, and CDR3 represented by SEQ ID NO:9. H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0050] In certain embodiments, the anti-tau C3 antibody inhibits the pathological aggregation of full-length tau (e.g., 2N4R). In some embodiments, the anti-tau C3 antibody comprises (a) a variable heavy chain (VH) comprising a CDR1 represented by SEQ ID NO:7, a CDR2 represented by SEQ ID NO:8, and a CDR3 represented by SEQ ID NO:9. H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0051] In certain embodiments, administration of an anti-tau C3 antibody can immunize a subject against the development of a tauopathy. In some embodiments, the anti-tau C3 antibody comprises: (a) a variable heavy chain (V) comprising CDR1 represented by SEQ ID NO:7, CDR2 represented by SEQ ID NO:8, and CDR3 represented by SEQ ID NO:9; H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0052] Administration of an anti-tau C3 antibody may reduce one or more symptoms of a tauopathy in a subject and / or reduce the progression of a tauopathy in a subject. For example, in certain embodiments, administration of an anti-tau C3 antibody may improve cognitive function and / or motor / sensorimotor function in a subject suffering from a tauopathy. In some embodiments, the anti-tau C3 antibody comprises (a) a variable heavy chain (V) comprising CDR1 represented by SEQ ID NO:7, CDR2 represented by SEQ ID NO:8, and CDR3 represented by SEQ ID NO:9. H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0053] The anti-tau C3 antibody of the present invention can be used to treat tauopathies in human subjects. For example, administration of the anti-tau C3 antibody to treat Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), and frontotemporal lobar degeneration (FTLD) is particularly contemplated. In some embodiments, the anti-tau C3 comprises (a) a variable heavy chain (V) comprising CDR1 represented by SEQ ID NO:7, CDR2 represented by SEQ ID NO:8, and CDR3 represented by SEQ ID NO:9; H) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0054] In certain embodiments, the present invention relates to a method for reducing the propagation of tau aggregates in the brain of a subject, comprising administering to the subject a therapeutically effective amount of an anti-tau C3 antibody, wherein the antibody binds to tau C3 but not to full-length tau. In some embodiments, the anti-tau C3 antibody comprises (a) a variable heavy chain (V) comprising CDR1 represented by SEQ ID NO:7, CDR2 represented by SEQ ID NO:8, and CDR3 represented by SEQ ID NO:9. H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0055] The present invention further relates to a method of treating a tauopathy in a subject, comprising administering a therapeutically effective amount of an anti-tau C3 antibody sufficient to block tau C3 seeding activity in the subject, wherein the anti-tau C3 antibody is a humanized antibody. In some embodiments, the humanized anti-tau C3 comprises (a) a variable heavy chain (VH) comprising CDR1 represented by SEQ ID NO:7, CDR2 represented by SEQ ID NO:8, and CDR3 represented by SEQ ID NO:9. H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0056] The present invention also relates to a method for treating a tauopathy in a subject, comprising administering to the subject a therapeutically effective amount of an anti-tau C3 antibody sufficient to block the reuptake of tau C3 by neurons, wherein the anti-tau C3 antibody is a chimeric antibody. In some embodiments, the anti-tau C3 comprises (a) a variable heavy chain (V) comprising CDR1 represented by SEQ ID NO:7, CDR2 represented by SEQ ID NO:8, and CDR3 represented by SEQ ID NO:9. H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0057] The present invention also provides a method of treating Alzheimer's disease in a subject, comprising administering to the subject a therapeutically effective amount of an anti-tau C3 antibody, wherein the anti-tau C3 antibody is at least 1×10 -10 ~1×10 -12 and has a binding affinity (KD) for tau C3 of 1×10 -3 s -1 The off-rate (K d ) and 1 × 10 -4 ~1×10 -8 In some of these embodiments, the anti-tau C3 is a humanized or chimeric antibody having a binding affinity (KD) for FLT that is M. In some of these embodiments, the anti-tau C3 comprises (a) a variable heavy chain (V) comprising CDR1 represented by SEQ ID NO:7, CDR2 represented by SEQ ID NO:8, and CDR3 represented by SEQ ID NO:9. H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0058] The present invention also provides a method of treating progressive supranuclear palsy (PSP) in a subject, comprising administering to the subject a therapeutically effective amount of an anti-tau C3 antibody, the anti-tau C3 antibody being at least 1×10 -10 ~1×10 -12and has a binding affinity (KD) for tau C3 of 1×10 -3 s -1 The off-rate (K d ) and 1 × 10 -4 ~1×10 -8 In some of these embodiments, the anti-tau C3 is a humanized or chimeric antibody having a binding affinity (KD) for FLT (SEQ ID NO: 1) that is M. In some of these embodiments, the anti-tau C3 comprises (a) a variable heavy chain (V) comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9. H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0059] The present invention also provides a method of treating frontotemporal dementia (FTD) in a subject, comprising administering to the subject a therapeutically effective amount of an anti-tau C3 antibody, wherein the anti-tau C3 antibody is at least 1×10 -10 ~1×10 -12 and has a binding affinity (KD) for tau C3 of 1×10 -3 s -1 The off-rate (K d ) and 1 × 10 -4 ~1×10 -8 In some of these embodiments, the anti-tau C3 is a humanized or chimeric antibody having a binding affinity (KD) for FLT (SEQ ID NO: 1) that is M. In some of these embodiments, the anti-tau C3 comprises (a) a variable heavy chain (V) comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9. H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0060] The present invention also provides a method of treating traumatic brain injury (TBI) in a subject, comprising administering to the subject a therapeutically effective amount of an anti-tau C3 antibody, the anti-tau C3 antibody being at least 1×10 -10 ~1×10 -12 and has a binding affinity (KD) for tau C3 of 1×10 -3 s -1 The off-rate (K d ) and 1 × 10 -4 ~1×10 -8 In some of these embodiments, the anti-tau C3 is a humanized or chimeric antibody having a binding affinity (KD) for FLT (SEQ ID NO: 1) that is M. In some of these embodiments, the anti-tau C3 comprises (a) a variable heavy chain (V) comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9. H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0061] The present invention also provides a method of treating Pick's disease (PiD) in a subject, comprising administering to the subject a therapeutically effective amount of an anti-tau C3 antibody, wherein the anti-tau C3 antibody is at least 1×10 -10 ~1×10 -12 and has a binding affinity (KD) for tau C3 of 1×10 -3 s -1 The off-rate (K d ) and 1 × 10 -4 ~1×10 -8 In some of these embodiments, the anti-tau C3 is a humanized or chimeric antibody having a binding affinity (KD) for FLT (SEQ ID NO: 1) that is M. In some of these embodiments, the anti-tau C3 comprises (a) a variable heavy chain (V) comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9. H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12.L ) polypeptides.

[0062] The present invention also provides a method of treating corticobasal degeneration (CBD) in a subject, comprising administering to the subject a therapeutically effective amount of an anti-tau C3 antibody, the anti-tau C3 antibody comprising at least 1×10 -10 ~1×10 -12 and has a binding affinity (KD) for tau C3 of 1×10 -3 s -1 The off-rate (K d ) and 1 × 10 -4 ~1×10 -8 In some of these embodiments, the anti-tau C3 is a humanized or chimeric antibody having a binding affinity (KD) for FLT (SEQ ID NO: 1) that is M. In some of these embodiments, the anti-tau C3 comprises (a) a variable heavy chain (V) comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9. H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0063] The present invention also provides a method of treating frontotemporal lobar degeneration (FTLD) in a subject, comprising administering to the subject a therapeutically effective amount of an anti-tau C3 antibody, the anti-tau C3 antibody being at least 1×10 -10 ~1×10 -12 and has a binding affinity (KD) for tau C3 of 1×10 -3 s -1 The off-rate (K d ) and 1 × 10 -4 ~1×10 -8 In some of these embodiments, the anti-tau C3 is a humanized or chimeric antibody having a binding affinity (KD) for FLT (SEQ ID NO: 1) that is M. In some of these embodiments, the anti-tau C3 comprises (a) a variable heavy chain (V) comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9. H) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptides.

[0064] The present invention also relates to therapeutic agents and compositions for blocking the cellular uptake of pathological tau, blocking tau seeding activity, blocking tau aggregation, and blocking the pathological propagation of tau, tau fibrils, tau aggregates, and fragments of any of the foregoing from one part of the brain to another, where this pathological propagation is induced or regulated by tau C3. The therapeutic agents and compositions include the anti-tau C3 antibodies described above and below. In addition to the anti-tau C3 antibody, the compositions of the present invention may contain one or more pharmaceutically acceptable excipients. The therapeutic agents or compositions may also be used for passive immunization from and treatment of tauopathies, such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), and frontotemporal lobar degeneration (FTLD). In certain embodiments, the composition may further comprise one or more agents that block tau-C3 production (e.g., a caspase inhibitor) or promote its removal (e.g., one or more small molecule tau-C3 aggregation inhibitors).

[0065] The present invention provides a composition comprising an anti-tau C3 antibody and one or more pharmaceutically acceptable excipients, wherein the anti-tau C3 antibody is present in an amount of 1×10 -10 ~1×10 -12 and has a binding affinity (KD) for tau C3 of 1×10 -3 s -1 The off-rate (K d ) and 1 × 10 -4 ~1×10 -8In some of these embodiments, the anti-tau C3 is a humanized or chimeric antibody having a binding affinity (KD) for FLT (SEQ ID NO: 1) that is M. In some of these embodiments, the anti-tau C3 comprises (a) a variable heavy chain (V) comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9. H ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. L ) polypeptide. The composition can be, for example, a liquid composition. The composition comprises an effective amount of an anti-tau C3 antibody for treating a tauopathy, including, for example, Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), frontotemporal lobar degeneration (FTLD), and the like. In certain preferred embodiments, the composition is stable (i.e., at least 90% of the anti-tau C3 antibodies in the composition retain their binding ability after storage of the composition at 37°C for 21 days). [Brief explanation of the drawings]

[0066] [Figure 1] Protein and DNA sequences of the MoTau01 kappa light chain variable region.

[0067] [Figure 2] Protein and DNA sequences of the MoTau01 kappa heavy chain variable region.

[0068] [Figure 3] pHuK LIC vector.

[0069] [Figure 4] pHuG4 LIC vector.

[0070] [Figure 5] Protein and DNA sequences of chimeric MoTau01 VK.

[0071] [Figure 6] Protein and DNA sequences of chimeric MoTau01 VH.

[0072] [Figure 7] Binding study of chimeric TauOl to tauC3 and FL tau using binding ELISA.

[0073] [Figure 8] Binding study of mouse TauO1 antibody and chimeric TauO1 antibody to Tau C3 and FL Tau using Octet.

[0074] [Figure 9] Protein and DNA sequences of TauO1 HA.

[0075] [Figure 10] Protein and DNA sequences of Tau01 HB.

[0076] [Figure 11] Protein and DNA sequences of TauO1 HC.

[0077] [Figure 12] Protein and DNA sequences of TauO1 KA.

[0078] [Figure 13] Protein and DNA sequences of Tau01 KB.

[0079] [Figure 14] Protein and DNA sequences of TauO1 KC.

[0080] [Figure 15] Binding of humanized and chimeric TauO1 to Tau C3: A and B versions.

[0081] [Figure 16]Binding ELISA of humanized TauO1 and chimeric TauO1 to Tau C3:HA-HL variants.

[0082] [Figure 17] Octet screening of humanized TauO1 antibodies against tau C3: HB, HC and HF with KA-KC.

[0083] [Figure 18] Binding ELISA of humanized Tau01 antibodies to tau C3:KA-KJ variants.

[0084] [Figure 19] Octet screening of humanized TauO1 antibodies against tau C3:HB with KA-KJ variants.

[0085] [Figure 20] Second round binding ELISA of humanized TauOl antibodies to tau C3: HM, HN and HO variants.

[0086] [Figure 21] Second round Octet screening of humanized TauO1 antibodies against tau C3: HM, HN and HO variants.

[0087] [Figure 22] Binding ELISA of HC and HM containing humanized antibodies against tau C3.

[0088] [Figure 23] Second round Octet screening of humanized Tau01 antibodies against Tau C3:HM variants.

[0089] [Figure 24] Second round of Octet screening of humanized Tau01 antibodies against tau C3 and FL tau: lead variants.

[0090] [Figure 25] Off-rate ranking of lead humanization candidates with Tau C3 using Biacore.

[0091] [Figure 26] Binding test for FL tau by Biacore.

[0092] [Figure 27] Thermostability of chimeric and humanized candidate antibodies.

[0093] [Figure 28] SEC-MALS aggregation analysis of purified MoTau01 HuG4K and Tau01 HCKB HuG4K antibodies.

[0094] [Figure 29] DLS analysis of purified Tau01 HMKN, HMKO, HMKP, HMKE, and HMKM HuG4K antibodies.

[0095] [Figure 30] Mass spectrometry of purified chimeric and humanized candidate antibodies.

[0096] [Figure 31] Biacore kinetics of humanized candidate antibodies.

[0097] [Figure 32] Thermal shift analysis of humanized candidate antibodies.

[0098] [Figure 33] Non-specific protein-protein interactions of humanized candidate antibodies (interaction chromatography).

[0099] [Figure 34] Purified humanized antibody candidates evaluated for solubility.

[0100] [Figure 35]Freeze / thaw and heat stress analysis of humanized candidate antibodies by circular dichroism.

[0101] [Figure 36] Capillary isoelectric focusing to determine the isoelectric point of humanized candidates.

[0102] [Figure 37] Serum stability assessment of humanized candidate antibodies.

[0103] definition As used herein, "antibody" refers to intact molecules (i.e., full-length antibodies (IgM, IgG, IgA, IgE)) and fragments thereof, including, for example, Fab, F(ab', and F V It is meant to include fragments thereof, or synthetic and biological derivatives thereof, of pIII or pVIII or other surface proteins expressed on the surface of filamentous phage or on the surface of bacteria that are capable of binding to antigens. Fab, F(ab'2 and F V The fragment is the F of the intact antibody. C The antibody lacks fragments and is cleared more rapidly from the circulation. In addition, it is largely free of non-specific tissue binding of the antibody. The antibody may be a monoclonal antibody. Recombinant antibodies are encompassed by the term "antibody." The term "antibody" includes chimeric and humanized antibodies. The antibody may also be fully human (e.g., derived from a transgenic mouse or phage).

[0104] As used herein, the term "humanized antibody" refers to an antibody in which the complementarity determining regions (CDRs) of a mouse or other non-human antibody are grafted onto a human antibody framework. A human antibody framework refers to a fully human antibody excluding the CDRs.

[0105] As used herein, the term "human antibody" refers to an antibody whose entire sequence is derived from a human gene repertoire (e.g., a transgenic mouse or phage).

[0106] As used herein, the term "homologous" means that a sequence is at least 80% identical to the homologous sequence, and that a polymeric peptide (e.g., an antibody) comprising one or more homologous sequences has substantially the same biological activity as the polymeric peptide comprising one or more homologous sequences. For example, a humanized antibody comprising (a) a heavy chain variable region comprising CDR1 represented by the sequence GFTFNTYA (SEQ ID NO:7), CDR2 represented by IRSKSNNYAT (SEQ ID NO:8), and CDR3 represented by VGGGDF (SEQ ID NO:9), and (b) a light chain variable region comprising CDR1 represented by the sequence QEISVY (SEQ ID NO:10), CDR2 represented by the sequence GAF (SEQ ID NO:11), and CDR3 represented by the sequence LQYVRYPWT (SEQ ID NO:12), and an antibody in which one or more CDR sequences are replaced by one or more homologous sequences, both exhibits a 1×10 -10 ~1×10 -12 The binding affinity (KD) for tau C3 is 1×10 -4 ~1×10 -8 have a binding affinity (KD) for FLT that is M, or have no detectable binding to FLT. By definition, homologous antibodies have substantially similar three-dimensional shapes.

[0107] As used herein, "CDR" means "complementarity determining region." CDRs may also be referred to as hypervariable regions. Unless otherwise specified, the CDR sequences disclosed herein are defined by the IMGT numbering system.

[0108] As used herein, "represented by a SEQ ID NO" in reference to a CDR sequence means that the sequence of the CDR is identical to or homologous to the recited SEQ ID NO.

[0109] As used herein, the term "chimeric antibody" refers to an antibody in which the entire variable region of a mouse or rat antibody is expressed along with human constant regions.

[0110] The term "murine anti-tau C3 antibody" as used herein refers to the "anti-tau C3 antibody" characterized in Nicholls, S.B., S.L. DeVos, C. Commins, C. Nobuhara, R.E. Bennett, D.L. Corjuc, E. Maury, et al. 2017. "Characterization of Tau C3 antibody and demonstration of its potential to block tau propagation." PLoS ONE 12(5):e0177914. doi:10.1371 / journal.pone.0177914.

[0111] As used herein, a "light chain" is the small polypeptide subunit of an antibody. A typical antibody contains two light chains and two heavy chains.

[0112] As used herein, a "heavy chain" is the large polypeptide subunit of an antibody. The heavy chain of an antibody contains a series of immunoglobulin domains, with at least one variable domain and at least one constant domain.

[0113] As used herein, the term "affinity" refers to the strength with which an antibody molecule binds to its epitope. Affinity is measured by surface plasmon resonance (SPR) using Biacore kinetics.

[0114] As used herein, the term "KD" refers to the equilibrium dissociation constant (KD=Kd / Ka, where Kd is the dissociation rate constant and Ka is the association rate constant).

[0115] As used herein, the term "immunodepletion" refers to the removal of proteins by the use of antibodies. The term "immunodepletion" is used interchangeably with the term "immunoprecipitation." This term refers to the ability of an antibody to precipitate or immunoprecipitate (IP) an antigen of interest from a sample (causing immunodepletion).

[0116] As used herein, the terms "therapeutically effective amount" and "effective amount" refer to an amount of a therapeutic agent (e.g., an anti-tau C3 antibody) or composition that causes a measurable clinical effect in a subject. The effective amount of a therapeutic agent is determined by the circumstances of the case, including the compound administered, the route of administration, the status of the condition being treated, and consideration of the subject and administration conditions, among other considerations. An "effective amount" generally includes about 0.0001 mg / kg to about 100 mg / kg, preferably 0.5 mg / kg to 20 mg / kg, of an anti-tau C3 antibody described herein. In certain embodiments, amounts of 1 mg / kg, 3 mg / kg, 4 mg / kg, 6 mg / kg, 8 mg / kg, or 10 mg / kg are used.

[0117] The term "pathological tau" encompasses tau C3, fibrils comprising tau C3, and aggregates comprising tau C3 (e.g., heterogeneous populations comprising full-length tau, tau oligomers, and / or post-translationally modified tau (truncated or hyperphosphorylated)). In addition to tau C3, pathological tau may include heterogeneous populations of full-length tau (e.g., 2N4R), tau oligomers, and / or post-translationally modified tau (truncated or hyperphosphorylated).

[0118] The term "tau C3" refers to a C-terminally truncated tau fragment ending at aspartate 421 of htau40 (SEQ ID NO: 1).

[0119] As used herein, "FLT" is an abbreviation for full-length tau (eg, htau40 (SEQ ID NO: 1)).

[0120] The term "treating" or "treatment" includes the attenuation, amelioration, or reversal of at least one symptom or symptomatic manifestation associated with a tauopathy.

[0121] The term "seeding" refers to extracellular activity that precedes the intracellular aggregation of tau C3, tau C3-containing fibrils, and / or tau C3-containing aggregates as part of a heterogeneous population of full-length tau (e.g., 2N4R), tau oligomers, and / or post-translationally modified tau (truncated or hyperphosphorylated).

[0122] The term "aggregation" refers to an activity that occurs intracellularly after cellular uptake of tau C3 and / or fibrils containing tau C3 and / or aggregates containing tau C3.

[0123] "ExpiCHO" is an abbreviation for Chinese Hamster Ovary (CHO High Density / Serum Free) cells.

[0124] "A" is an abbreviation for adenine.

[0125] "bp" is an abbreviation for base pair.

[0126] "℃" is the abbreviation for Celsius.

[0127] "C" is an abbreviation for cytosine.

[0128] "MEM" is an abbreviation for Minimal Essential Medium.

[0129] "DNA" is an abbreviation for deoxyribonucleic acid.

[0130] "ELISA" is an abbreviation for Enzyme linked immuno-adsorbent assay.

[0131] "EC50" is an abbreviation for the concentration of antibody that provides a 50% effective response.

[0132] "EC80" is an abbreviation for the concentration of antibody that provides 80% of the maximal response.

[0133] "ECD" is an abbreviation for extracellular domain.

[0134] "g" is an abbreviation for gram.

[0135] "G" is an abbreviation for guanine.

[0136] "HRP" is an abbreviation for horseradish peroxidase.

[0137] "IgG" is an abbreviation for immunoglobulin-G.

[0138] "K" is an abbreviation for G or T (IUPAC convention).

[0139] "LIC" is an abbreviation for Ligase independent cloning.

[0140] "min" is an abbreviation for minute.

[0141] "M" is an abbreviation for A or C (IUPAC convention).

[0142] "nm" is an abbreviation for nanometer.

[0143] "OD" is an abbreviation for optical density.

[0144] "PBS" is an abbreviation for phosphate buffered saline.

[0145] "PCR" is an abbreviation for polymerase chain reaction.

[0146] "R" is an abbreviation for A or G (IUPAC convention).

[0147] "RT" is an abbreviation for room temperature.

[0148] "s" is an abbreviation for seconds.

[0149] "S" is an abbreviation for C or G (IUPAC convention).

[0150] "T" is an abbreviation for thymine.

[0151] "TBS" is an abbreviation for Tris Buffered Saline.

[0152] "UV" is an abbreviation for ultraviolet light.

[0153] "V" is an abbreviation for A or C or G (IUPAC convention).

[0154] "VCI" is an abbreviation for Vernier, Canonical and Interface Residues.

[0155] "VH" is an abbreviation for immunoglobulin heavy chain variable region.

[0156] "VK" is an abbreviation for immunoglobulin kappa light chain variable region.

[0157] "W" is an abbreviation for A or T (IUPAC convention).

[0158] "Y" is an abbreviation for C or T (IUPAC convention). DETAILED DESCRIPTION OF THE INVENTION

[0159] Tau C3 is one of many high molecular weight species that are responsible for tau aggregation and seeding activity. In addition to tau C3, tau aggregates may contain heterogeneous populations of full-length (normal tau), tau oligomers, and / or post-translationally modified tau (truncated or hyperphosphorylated). Other neurodegenerative diseases besides sporadic AD also show elevated levels of tau C3.

[0160] Tau-C3 is neurotoxic and can cause microtubule dysfunction, and it may also be responsible for the propagation of tau fibrils from one part of the brain to another.

[0161] Anti-tau C3 antibody The anti-tau C3 antibodies of the present invention recognize the extracellular form of aggregated, pathological tau C3. The anti-tau C3 antibodies of the present invention can be, for example, chimeric, humanized, or human anti-tau C3 antibodies.

[0162] Anti-tau C3 antibodies exhibit very tight binding specificity for the target caspase-cleaved tau protein when tested against recombinant tau C3 protein. In certain embodiments, anti-tau C3 antibodies are effective in blocking seeding in biosensor assays and blocking entry of seeds into neurons that are responsible for inducing intracellular tau aggregation (i.e., effective in blocking entry of tau C3 and tau C3 fibrils into cells).

[0163] Anti-tau C3 antibodies generally have subnanomolar specificity for tau C3 and are at least 100-fold more specific for tau C3 than full-length tau (2N4R) (e.g., 100-fold or more specific for tau C3 than full-length tau). For example, an anti-tau C3 antibody may be 150-5000-fold more specific for tau C3 than full-length tau (2N4R). In certain embodiments, an anti-tau C3 antibody is 500-2500-fold more specific for tau C3 than full-length tau (2N4R). In certain embodiments, an anti-tau C3 antibody is 750-2000-fold more specific for tau C3 than full-length tau (2N4R). In certain embodiments, an anti-tau C3 antibody is 1000-1500-fold more specific for tau C3 than full-length tau (2N4R). In all of these embodiments, the anti-tau C3 antibody may have no detectable binding to full-length tau (2N4R).

[0164] In certain embodiments, the antibodies of the present invention are chimeric, humanized, and human anti-tau C3 antibodies that have a higher binding affinity (KD) for tau C3 than murine anti-tau C3 antibodies. In some embodiments, the chimeric, humanized, and human anti-tau C3 antibodies have a binding affinity (KD) for tau C3 that is at least 2-fold, 3-fold, or 4-fold higher than the binding affinity (KD) for tau C3 of the murine anti-tau C3 antibody. In some embodiments, the murine anti-tau C3 antibody has a binding affinity (KD) for tau C3 of about 4.9 x 10 -11 M, and chimeric, humanized, and human anti-tau C3 antibodies have a binding affinity (KD) for tau C3 of approximately 1 x 10 -11 M ~ approx. 2.5×10 -11 The chimeric anti-tau C3 antibody, humanized anti-tau C3 antibody and human anti-tau C3 antibody of the present invention have a binding affinity (KD) for tau C3 of, for example, about 1.1 × 10 -11 M, approx. 1.3 x 10 -11 M, approx. 1.5 x 10 -11 M, approx. 1.7 x 10 -11 M, approx. 1.9×10 -11 M, approx. 2.1 x 10 -11 M or approximately 2.3 x 10 -11 M. In some embodiments, the murine anti-tau C3 antibody may have a binding affinity (KD) for tau C3 of about 3.9 x 10 -11 M, and chimeric, humanized, and human anti-tau C3 antibodies have a binding affinity (KD) for tau C3 of approximately 1 x 10 -11 M ~ approx. 2.5×10 -11 The chimeric anti-tau C3 antibody, humanized anti-tau C3 antibody and human anti-tau C3 antibody of the present invention have a binding affinity (KD) for tau C3 of, for example, about 1.1 × 10 -11 M, approx. 1.3 x 10 -11 M, approx. 1.5 x 10 -11 M, approx. 1.7 x 10 -11 M, approx. 1.9×10 -11 M, approx. 2.1 x 10 -11 M or approximately 2.3 x 10 -11 The antibody may have a binding affinity (KD) for tau C3 of M.

[0165] In certain embodiments, the antibody is present in an amount of 1×10 -10 M~1×10 -11 It binds to tau C3 with an equilibrium constant KD of M and with 2N4R with an equilibrium constant KD of 1 x 10 -4 M~1×10 -8 In a preferred embodiment, the anti-tau C3 antibody is 1×10 -9 M~1×10 -12 Tau binds to C3 with an equilibrium constant KD of 1 x 10 M -8 M~9×10 -8 In some of these embodiments, the antibody binds to full-length (e.g., 2N4R) with an equilibrium constant KD of M or shows no detectable binding to 2N4R. In some of these embodiments, the antibody has a very slow off-rate from tau C3 (i.e., 1×10 -4 ~1×10 -3 s -1 off-rate) and has virtually no affinity for 2N4R (i.e., ka less than 100,000 1 / MS).

[0166] In certain embodiments, the anti-tau C3 antibody is a chimeric or humanized antibody that has a KD for tau C3 of about 5 pM to about 90 pM, about 10 pM to about 90 pM, about 10 pM to about 80 pM, about 10 pM to about 70 pM, about 10 pM to about 60 pM, about 10 pM to about 50 pM, about 10 to about 40 pM, or about 10 pM to about 35 pM.

[0167] In certain embodiments, the anti-tau C3 antibody has a KD of about 10 to about 90 pM and a RI of 2×10 -3 s -1 The antibody is a chimeric or humanized antibody that has a very slow off-rate from tau C3, as indicated by a Kd of less than 0. In other words, the antibody has a high degree of specificity for tau C3, a target protein that may be produced in disease states, and a slow off-rate, both of which are ideal for an antibody used in an immunization strategy.

[0168] Anti-tau C3 antibodies include, but are not limited to, monoclonal, chimeric, humanized, single chain, Fab fragments, and Fab expression libraries. Anti-tau C3 antibodies may be natural or recombinant, immobilized, free in solution, or displayed on various molecules or on the surface of bacteria, viruses, or other surfaces.

[0169] In certain embodiments, the anti-tau C3 antibody recognizes the sequence SSTGSIDMVD (SEQ ID NO: 23) at the C-terminus of tau C3, but does not recognize the same sequence when it is present internally in FLT.

[0170] Useful anti-tau C3 antibodies (e.g., humanized antibodies) according to the present invention can be administered to subjects who may be susceptible to or who are suffering from a tauopathy to block the seeding and / or aggregation of tau C3, thereby treating one or more symptoms of the tauopathy.

[0171] In yet another embodiment of the present invention, the anti-tau C3 antibodies of the present invention may be conjugated to a cytoprotective agent or an agent that promotes and / or enhances the antibody's ability to cross the blood-brain barrier ("BBB"). The cytoprotective agent may be an antioxidant (e.g., melatonin), and the agent that promotes or enhances the antibody's ability to cross the BBB is a hydrophobic substance that can cross the BBB and is generally recognized as being within the scope of (GRAS) by the U.S. Food and Drug Administration ("FDA"). The cytoprotective agent or the agent that promotes or enhances the antibody's ability to cross the BBB may be conjugated to the antibody directly or via a linker. The linker may include or be selected from the group consisting of a hydrazine linker, a disulfite linker, a thioether linker, and a peptide linker. In a specific embodiment, the antibody has an equilibrium constant KD for tau C3 that is 2 to 3 orders of magnitude higher than the antibody equilibrium constant KD for 2N4R, and the cytoprotective agent is melatonin.

[0172] How to use In one aspect, the invention provides an anti-tau C3 antibody for use in a living human suffering from or at risk of developing a tauopathy, such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), or frontotemporal lobar degeneration (FTLD).

[0173] Methods for blocking the propagation of pathological tau aggregation In one aspect, the invention relates to a method for blocking the propagation of pathological tau from one neuron to another or from one part of the brain to another.

[0174] In one aspect, the present invention relates to a method of blocking tau C3 seeding activity in the brain of a subject.

[0175] In an additional aspect, the invention relates to a method of reducing the propagation of pathological tau aggregates in the brain of a subject.

[0176] The present invention further relates to a method for reducing the propagation of aggregates comprising tau C3 in the brain of a subject.

[0177] The present invention further relates to a method for reducing the propagation of tau C3-containing fibrils in the brain of a subject.

[0178] In a further aspect, the present invention relates to a method for reducing intracellular aggregation of tau induced by cellular uptake of tau C3 and tau C3 fibrils.

[0179] In each aspect, the method comprises administering a therapeutically effective amount of an anti-tau C3 antibody to a human. The anti-tau C3 antibody is capable of uniquely recognizing the extracellular form of aggregated pathological tau without binding to physiological tau. In a preferred embodiment, an essential part of the epitope of the anti-tau C3 antibody is a carboxyl group that forms a neoepitope with the C-terminal residue of a peptide corresponding to the last 10 C-terminal residues of tau C3 (e.g., tau C3 or SEQ ID NO: 23). The anti-tau C3 antibody has an equilibrium constant KD for tau C3 that is 2 to 3 orders of magnitude higher than the antibody equilibrium constant KD for 2N4R, and one or more pharmaceutically acceptable excipients. The anti-tau C3 antibody has a 1×10 -10 M~1×10 -11 It binds to tau C3 with an equilibrium constant KD of M, whereas the equilibrium constant KD with full-length tau (e.g., 2N4R) is 1 × 10 -4 M~1×10 -8 In a preferred embodiment, the anti-tau C3 antibody is 1×10 -11 M~9×10 -11 Tau binds to C3 with an equilibrium constant KD of 1 x 10 M -8 M~9×10 -8 The anti-tau C3 antibody binds to 2N4R with an equilibrium constant KD of M or does not exhibit detectable binding to 2N4R. The anti-tau C3 antibody preferably has a very slow off-rate from tau C3 and has substantially no affinity for 2N4R (i.e., kA less than 100,000 1 / MS). The antibody may be selected from, for example, a humanized antibody, a chimeric antibody, or an immunological fragment of any of the foregoing. In a preferred embodiment, the antibody is an antibody selected from the humanized anti-tau C3 antibodies described herein.

[0180] The human may or may not have symptoms associated with tau aggregation prior to administration of a therapeutically effective amount of an anti-tau C3 antibody. In other words, the human may or may not experience symptoms associated with tau seeding and / or aggregation. One of skill in the art will understand that pathological tau seeding and aggregation likely begins before the diagnosis or onset of symptoms associated with tau aggregation. In some embodiments, the human has symptoms associated with tau seeding and / or aggregation. In other embodiments, the human does not have symptoms associated with tau seeding and / or aggregation. In yet other embodiments, the human has detectable tau pathology but does not have any other symptoms associated with tau symptoms and / or aggregation. Reducing the propagation of tau aggregation in the human brain by administering therapeutic agents and pharmaceutical compositions according to the present invention can reduce the onset and / or progression of symptoms associated with pathological tau seeding and / or aggregation.

[0181] Preventing, inhibiting or slowing the spread of pathological tau aggregation may therefore be used in the treatment of pathologies associated with the generation and propagation of tau aggregates. One definition of a condition associated with tau seeding and / or aggregation refers to any condition caused by the formation of tau aggregates that are composed in part of tau fibrils. Exemplary disorders with symptoms associated with tau aggregation include, but are not limited to, progressive supranuclear palsy, dementia pugilistica (chronic traumatic encephalopathy), frontotemporal dementia and chromosome 17-linked parkinsonism, Ritiko-Bodig disease (Parkinson-Dementia Complex of Guam), neurofibrillary tangle senile dementia, ganglioglioma and gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis complex, Hallervorden-Spatz syndrome, lipofuscinosis, Pick's disease, corticobasal degeneration, argyrophilic grain disease (AGD), frontotemporal lobar degeneration, Alzheimer's disease, and frontotemporal dementia. Methods for diagnosing these disorders are known in the art.

[0182] Exemplary disorders with symptoms associated with tau seeding or tau aggregation include, for example, impaired cognitive function, altered behavior, emotional dysregulation, epileptic seizures, and impaired nervous system structure or function. Impaired cognitive function includes, but is not limited to, difficulties with memory, attention, concentration, language, abstract thinking, creativity, executive function, planning, and body image. Altered behavior includes, but is not limited to, physical or verbal aggression, impulsivity, decreased inhibition, blunted affect, decreased arousal, personality changes, alcohol, tobacco, or drug abuse, and addiction-related behaviors. Emotional dysregulation includes, but is not limited to, depression, anxiety, mania, irritability, and emotional incontinence. Epileptic seizures include, but are not limited to, generalized tonic-convulsive seizures, complex partial seizures, and psychogenic non-epileptic seizures. Impaired nervous system structure or function includes, but is not limited to, hydrocephalus, parkinsonism, sleep disorders, psychiatric disorders, and balance and coordination disorders. This includes motor dysfunction such as monoparesis, hemiparesis, quadriparesis, ataxia, ballism, and tremor. It also includes sensory loss or dysfunction involving sensory functions such as smell, touch, taste, sight, and hearing. Furthermore, it includes autonomic nervous system dysfunction such as bowel dysfunction, bladder dysfunction, sexual dysfunction, blood pressure and temperature regulation dysfunction. Finally, it includes hormonal disorders that can result from dysfunction of the hypothalamus and pituitary gland, such as deficiencies and dysfunctions of growth hormone, thyroid-stimulating hormone, luteinizing hormone, follicle-stimulating hormone, gonadotropin-releasing hormone, prolactin, and numerous other hormones and modulators. Methods for detecting and assessing symptoms associated with tau aggregation are known in the art.

[0183] In some embodiments, the symptom associated with tau aggregation refers to dementia. Dementia is not a specific disease per se, but rather an overall term describing a broad range of symptoms associated with a decline in memory or other thinking skills severe enough to impair a person's ability to carry out daily activities. Dementia is also a common clinical symptom of many diseases associated with tau aggregation. Those skilled in the art will be familiar with the many methods available for diagnosing the severity of dementia. For example, multiple cognitive tests and screening questionnaires for dementia are known in the art, all with varying degrees of sensitivity and specificity. Non-limiting examples include the Mini-Mental State Examination (MMSE), the Abbreviated Mental Test May Score (AMTS), the Modified Mini-Mental State Examination (3MS), the Cognitive Abilities Screening Instrument (CASI), the Trail Making Test, the Clock Drawing Test, the Informant Questionnaire on Cognitive Decline in the Elderly, the General Practitioner Assessment of Cognition, the Clinical Dementia Scale (CDR), and the 8-Item Information Interview Differentiating Aging and Cognition (AD8).

[0184] In some embodiments, the severity of dementia symptoms is quantified using a clinical dementia scale. Using the clinical dementia scale, a score of 0 indicates no symptoms, a score of 0.5 indicates very mild symptoms, a score of 1 indicates mild symptoms, a score of 2 indicates moderate symptoms, and a score of 3 indicates severe symptoms. Therefore, any increase in a human's clinical dementia scale score indicates cognitive deterioration and an increase in dementia. Furthermore, a change in the clinical dementia scale from 0 to greater than 0 indicates the onset or onset of dementia.

[0185] In some embodiments, symptoms associated with tau seeding or tau aggregation refer to tau pathology or tauopathy. The terms "tau pathology" or "tauopathy" refer to pathological seeding of tau or aggregation of tau. In some embodiments, tau pathology refers to neurofibrillary tangles. In other embodiments, tau pathology refers to hyperphosphorylated tau. In yet other embodiments, tau pathology refers to high levels of tau aggregates detectable in blood, plasma, serum, CSF, or ISF, all of which are 2 to approximately 40 times higher than those detected in disease-free individuals.

[0186] Administration Administration of the anti-tau C3 antibodies described herein can be used as a therapy to treat or immunize against tauopathies.

[0187] Pharmaceutical-grade, preferably therapeutically effective, antibodies containing immunologically reactive fragments may be administered to humans. Administration is carried out using standard, effective techniques, including peripheral (i.e., not via administration into the central nervous system) or local to the central nervous system. Peripheral administration includes, but is not limited to, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, nasal, buccal, sublingual, or suppository administration. Local administration, including direct administration into the central nervous system (CNS), includes, but is not limited to, administration via the lumbar spine, intraventricular or intraparenchymal catheters, or the use of surgically implanted controlled-release formulations.

[0188] Those amenable to treatment include individuals at risk for disease but who do not exhibit symptoms, as well as those currently exhibiting symptoms. In the case of Alzheimer's disease, these essentially refer to individuals at risk of developing Alzheimer's disease. Therefore, the methods of the present invention can be administered prophylactically to the general population without the need to assess the subject's risk. Such prophylactic administration can begin, for example, at age 50 or older. The current methods are particularly useful for individuals without a known genetic risk for tauopathies (e.g., Alzheimer's disease). These individuals include those with relatives who have experienced the disease and those whose risk is measured by analysis of genetic or biochemical markers. For example, genetic markers of risk for Alzheimer's disease include mutations in the APP gene, particularly mutations at positions 717 and 670 and 671, known as the Hardy and Swedish mutations, respectively. Other markers of risk include mutations in the presenilin genes, PS1 and PS2, as well as ApoE4, family history, hypercholesterolemia, or atherosclerosis. Individuals currently suffering from Alzheimer's disease can be recognized by characteristic dementia due to the presence of the above risk factors. In addition, many diagnostic tests are available to identify individuals with AD. These diagnostic tests include imaging and / or measuring CSF tau and AJ342 levels. Elevated tau levels and decreased AJ342 levels indicate the presence of AD. Individuals suffering from Alzheimer's disease can also be diagnosed by the relevant criteria for Alzheimer's disease and related disorders.

[0189] In asymptomatic subjects, treatment can begin at any age (e.g., 10, 20, 30, 40, 50, or 60 years of age). However, treatment usually does not need to begin until the subject reaches 40, 50, 60, 70, 75, or 80 years of age. Treatment usually requires multiple doses over a period of time. Treatment can be monitored by assaying antibody or activated T cell or B cell responses to the therapeutic agent over time. If the response decreases, booster doses are suggested. In subjects with possible Down syndrome, treatment can begin before birth by administering a therapeutic agent to the mother or soon after birth.

[0190] In prophylactic applications, pharmaceutical compositions or drugs are administered to subjects susceptible to or at risk of a disease, in an amount sufficient to eliminate or reduce the risk, reduce the severity, or delay the onset of the disease, including the biochemical, histological, and / or behavioral symptoms of the disease, complications, and pathological endophenotypes manifested during the development of the disease. In therapeutic applications, compositions or drugs are administered to subjects suspected of or already suffering from these diseases, in an amount sufficient to cure or at least partially prevent the biochemical, histological, and / or behavioral symptoms of the disease, including complications and pathological endophenotypes manifested during the development of the disease. In some methods, administration of the drug reduces or eliminates mild cognitive impairment. An amount adequate to achieve therapeutic or prophylactic treatment is defined as a therapeutically effective dose or amount, or a prophylactically effective dose or amount. In both prophylactic and therapeutic regimes, drugs are typically administered in multiple doses until a sufficient immune response is achieved. Typically, the immune response is monitored and repeated dosing is administered if the immune response begins to wane.

[0191] Effective dosages of the compositions of the present invention for treating the above conditions vary depending on many different factors, including the means of administration, the target site, the physiological condition of the subject, other medications administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages must be tested on an individual basis in clinical trials and often titrated to optimize safety and efficacy. In certain embodiments, an additional advantage of the anti-tau C3 antibodies of the present invention may be that, for a comparable mass dosage, the anti-tau C3 dosage contains a higher molecular dose of the antibody, which is more effective at removing and / or "inactivating" tau C3 than compositions containing antibodies less specific for tau C3 than the anti-tau C3 antibodies of the present invention. Typically, the anti-tau C3 antibodies of the present invention are administered by intravenous infusion or subcutaneous injection. The amount of anti-tau C3 antibody for administration by intravenous infusion can vary from 0.5 mg to 10 mg per subject. Subcutaneous injection generally requires a higher dose to reach the brain in sufficient quantities. The antibody (eg, a whole IgG molecule) can be administered once a month.

[0192] In some methods, two or more antibodies (e.g., recombinant antibodies, monoclonal antibodies, chimeric antibodies, and / or humanized antibodies) with similar or different binding specificities are administered simultaneously, provided that the dosage of each antibody administered falls within the indicated range. In such a situation, two or more antibodies may both be directed, for example, to truncated tau. Alternatively, one or more antibodies may be directed, for example, to truncated tau, and one or more additional antibodies may be directed to amyloid-β (Aβ) peptides associated with Alzheimer's disease. Antibodies are typically administered multiple times. The interval between single doses may be hourly, daily, weekly, monthly, or yearly. In some methods, dosing is adjusted to obtain a plasma antibody concentration of 1-1000 μg / mL, and in some methods, this is 25-300 μg / mL. Alternatively, when less frequent administration is required, the antibody may be administered as a sustained-release formulation.

[0193] Dosage and frequency vary depending on the half-life of the antibody in the subject. Generally, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. Dosage and frequency of administration can vary depending on whether the treatment is preventative or therapeutic. In preventative applications, relatively low dosages are administered at relatively infrequent intervals over an extended period of time. Some subjects continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high dosages are sometimes required at relatively short intervals until the progression of the disease slows or terminates, preferably until the subject shows partial or complete remission of disease symptoms. Thereafter, the patient can be administered a preventative regimen.

[0194] The dosage of an anti-tau C3 antibody that blocks tau C3 seeding need not be the same as the dosage of an anti-tau C3 antibody that inhibits tau C3 aggregation, and in light of the information provided herein, the particular dosage can be determined by routine testing.

[0195] The efficacy of administration / treatment may be assessed by measuring the levels of pathological tau or phosphate-mediated tau in plasma and / or CSF. Based on this assessment, the dosage and / or frequency of administration may be adjusted accordingly.

[0196] In certain embodiments, effects on cognition may also be assessed.

[0197] Efficacy may also be assessed by the degree of brain atrophy, as measured by MRI.

[0198] Safety of dosing / treatment may be assessed by the number of adverse events (AEs) experienced by participants, serious AEs, abnormalities in clinical laboratory tests, vital signs, ECG, MRI, physical and neurological examinations, and cognitive deterioration. Based on this assessment, the dose and / or frequency of dosing may be adjusted accordingly.

[0199] The anti-tau C3 antibodies and immunogens may be administered intranasally, by subcutaneous injection, intramuscular injection, intravenous infusion, transdermally, bucally, etc., or as described in more detail below.

[0200] Pharmaceutical Composition Pharmaceutical compositions according to the present invention comprise an anti-tau C3 antibody, or a fragment thereof, described herein and one or more pharmaceutically acceptable excipients. The anti-tau C3 antibody is present in an amount of 1×10 -10 M~1×10 -11 It binds to tau C3 with an equilibrium constant KD of M, whereas the equilibrium constant KD with full-length tau (e.g., 2N4R) is 1 × 10 -4 M~1×10 -8 In a preferred embodiment, the anti-tau C3 antibody is 1×10 -11 M~9×10 -11 Tau binds to C3 with an equilibrium constant KD of 1 x 10 M -8 M~9×10 -8 The anti-tau C3 antibody preferably binds to 2N4R with an equilibrium constant KD of M or shows no detectable binding to 4R tau. The anti-tau C3 antibody preferably has a very slow off-rate from tau C3 (i.e., 1×10 -4 ~1×10 -3 s -1 ) and has substantially no affinity for 4R tau (i.e., ka less than 100,000 1 / MS). The antibody may be, for example, a humanized antibody, a chimeric antibody, or a human antibody (e.g., from a tg mouse).

[0201] Pharmaceutical compositions will be designed to be appropriate for the chosen mode of administration, and pharmaceutically acceptable excipients such as compatible dispersants, buffers, surfactants, preservatives, solubilizers, tonicity agents, stabilizers, etc. will be used as appropriate.

[0202] Peripheral, effective systemic delivery by intravenous or subcutaneous injection is the preferred method of administration to living subjects. Suitable vehicles for such injection are simple.

[0203] The concentration of the humanized antibody in the formulation to be administered is an effective amount, ranging from as low as about 0.1% by weight to about 95% or about 99.9% by weight. This range will be selected primarily based on fluid volume, viscosity, etc., depending on the particular mode of administration selected, if desired. In certain embodiments, the antibody may be about 15% to about 20% by weight of the composition.

[0204] Compositions for injection into a subject can be configured to contain 1 to 250 mL of sterile buffered water, phosphate-buffered saline, and about 1 to about 5,000 mg of any one or a combination of the anti-tau C3 antibodies of the present invention. The formulation can be sterile filtered after preparation or otherwise rendered microbiologically acceptable. A typical composition for intravenous infusion has a fluid volume of 1 to 250 mL, such as sterile Ringer's solution, which can have an anti-tau antibody concentration of 1 to 100 mg or more per mL. Therapeutics related to this discovery can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier before use. Lyophilization and reconstitution can vary the extent of antibody activity loss (e.g., with conventional immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies).

[0205] The dosage administered is an effective amount for the indicated purpose and may need to be adjusted to compensate. The pH of the formulation, which is generally of pharmaceutical grade quality, is selected to balance antibody stability (chemical and physical) and provide comfort to the subject upon administration. A pH of 4 to 8 is generally acceptable. Dosages vary from individual to individual based on the size, weight, and other physico-biological characteristics of the individual receiving the treatment.

[0206] In one aspect, a typical dosage contains about 0.1 mg to 10 mg of an anti-tau C3 antibody described herein. In certain embodiments, a typical dosage contains about 0.5 mg to 10 mg of an anti-tau C3 antibody. Dosages can range from about 0.55 mg / kg to about 10 mg / kg. With all IgG antibodies, the dosing frequency is typically monthly. In contrast, antibody fragments, given their short half-life, may need to be administered more frequently if needed to effectively treat symptoms.

[0207] The timing of administration of treatment for the disease itself and the duration of treatment are determined by the circumstances of the case. Treatment can be initiated after diagnosis of a disease associated with tau aggregation. Alternatively, treatment can be initiated after clinical confirmation of symptoms associated with tau aggregation. Furthermore, treatment can be initiated after detection of tau pathology. Treatment can be initiated immediately in a hospital or clinic, or after discharge from the hospital, or after confirmation in an outpatient clinic. The duration of treatment can range from a single dose administered at the appropriate time to a lifelong course of therapeutic treatment.

[0208] Although the foregoing methods, when appropriately adapted, will be the most convenient, suitable, and effective for administering proteins such as humanized antibodies, other effective techniques for administration, such as intracerebroventricular, transdermal, and oral administration, may also be used if the appropriate formulations are utilized therein.

[0209] Typical effective amounts or dosages can be determined and optimized using standard clinical techniques, and will depend on the mode of administration in light of the information provided herein and knowledge available in the art.

[0210] Example 1 (Sequencing of mouse MoTau01 antibody) Sequencing of MoTau01 antibody RNA preparation from hybridoma cells

[0211] Frozen pellets of mouse hybridoma cells (MoTau01) stored at -80°C were supplied by Genscript on behalf of Tau-Biologic and were processed using the Qiagen RNeasy kit and RNA was isolated according to the manufacturer's protocol.

[0212] First strand cDNA synthesis MoTau01 RNA (approximately 21 μg) was reverse transcribed and cDNA was produced using a first-strand cDNA synthesis kit from GE Life Sciences according to the manufacturer's protocol and purified as described in Example 5. Three independent cDNA products (round 1, round 2, and round 3) were generated in two rounds to detect and avoid reverse transcriptase-induced cDNA mutations.

[0213] cDNA sequencing MoTau01 cDNA was amplified by PCR as described in Example 5. Immunoglobulin cDNA was PCR amplified with MKC-spiked kappa light chain primers (Table 1) or MHC mix-spiked heavy chain primers (1-12 and 14) (Table 2) using Phusion Flash High-Fidelity PCR Master Mix. The MoTau01 VH PCR primer set failed to produce any product.

[0214] Therefore, based on the known sequences in the leader and tail regions, additional primers were designed to facilitate cloning of the VH domain from hybridoma cells. The additional primer sequences are listed in the MHV13 etc. primers table and in the additional primers section (Table 2).

[0215] [Table 1-1] [Table 1-2] Ambiguity code: W=A or T, Y=C or T, K=G or T

[0216] MKV indicates a primer that hybridizes to the leader sequence of the mouse kappa light chain variable region gene, and MKC indicates a primer that hybridizes to the mouse kappa constant region gene. The bold underlined portion indicates the M13 forward sequencing primer or M13 reverse sequencing primer. The wobble bases are defined in the definitions. [Table 2-1] [Table 2-2]

[0217] Ambiguity code: R=A or G, K=G or T, M=A or C.

[0218] MHV denotes a primer that hybridizes to the leader sequence of the mouse heavy chain variable region gene. MHCG denotes a primer that hybridizes to the mouse constant region gene. Bold underlined parts indicate M13 forward or reverse sequencing primers. Primer MHC mix consists of an equimolar mix of primers MHCG1, MHCG2a, MHCG2b, and MHCG3. The "wobble" bases are defined in the definitions.

[0219] The result of each PCR reaction was a single amplification product that was purified using a QIAquick PCR purification kit and sequenced (by Eurofins / GATC Genomics) in both directions using M13-forward and M13-reverse primers (Table 3) to obtain three independent sets of sequence information for each immunoglobulin chain. [Table 3]

[0220] VK and VH MoTau01 DNA sequences The consensus DNA sequences of the MoTau01 VK PCR product and the MoTau01 VH PCR product are shown in Figures 1 and 2, respectively. The resulting DNA sequences of the variable regions are identical to the sequences determined by Genscript. Germline analysis of the MoTau01 sequence confirmed that the kappa light chain is identical to the mouse VK1 IGKV9-124 * 01, and the heavy chain is mouse VH1 IGHV10-1 * Indicates that it is 02.

[0221] Example 2 (Generation of chimeric MoTau01 antibodies) Construction of chimeric MoTau01 expression vector The MoTau01 VH and VK genes were synthesized by Genscript. Using Genscript's proprietary software algorithms, the sequences for MoTau01 VH and MoTau01 VK were optimized and synthesized by silent mutagenesis using codons preferentially used by human cells.

[0222] Construction of chimeric expression vectors required cloning of the synthesized variable regions into IgG / kappa vectors (pHuK and pHuG4, Figures 3 and 4, respectively) using ligase-independent cloning (LIC). The vectors (pCMV-modified) were digested with BfuA1 (BspM1), and compatible overhangs were then generated using the 3'-5' exonuclease activity of T4 DNA polymerase (+dATP).

[0223] The antibody sequences (Figures 5 and 6) were generated by first amplifying the synthesized variable regions by PCR with a forward primer, which was a primer containing the 3' end of the leader sequence (most of the sequence is present in the vector), or a reverse primer, which was a primer containing the start of the constant region (IgG4 or kappa) followed by the start of the variable region (in each orientation) (Table 4).

[0224] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0225] Complementary overhangs were generated in the PCR product by T4 DNA polymerase + dTTP treatment (protocol provided in Example 5). The vector and insert were incubated at room temperature, transformed into chemically competent TOP10 bacteria, and plated on kanamycin plates. Multiple clones were isolated, and colonies were screened by PCR using primers such as HCMVi promoter forward and HuG4 LIC reverse for VH or HuK LIC reverse for VK (Table 3).

[0226] Clones generating PCR products of the correct size were selected, miniprepped using a QIAGEN kit, and sequenced using the same primers.

[0227] Generation of chimeric antibodies ExpiCHO suspension cells grown in ExpiCHO transfection medium and antibiotic were co-transfected with MoTau01_VH.pHuG4 and MoTau01_VK.pHuK (1 μg of DNA each) using ExpiFectamineCHO reagent. Cells were grown in 1 mL of growth medium for 7 days. MoTau01 HuG4k antibody concentrations up to 160 μg / mL (Table 14A) were measured in the conditioned medium by Octet quantification.

[0228] Tau C3 binding activity of chimeric antibodies Tau C3 and FL tau antigens were produced and purified by Genscript and supplied at concentrations of 2.54 mg / mL or 0.24 mg / mL, respectively. The binding of the chimeric antibodies to tau C3 and FL tau was assayed by binding ELISA. The chimeric antibodies were able to bind to tau C3 with an EC50 of 0.7 nM (Figure 7A), whereas no binding was observed to FL tau (Figure 7B). No nonspecific binding was observed to either antigen using the isotype, confirming the specificity of the observed binding. The binding of mouse Tau01 and chimeric Tau01 antibodies to purified tau C3 was assayed against a concentration series of 20 nM to 0.31 nM tau C3, as measured using Bio-Layer interferometry (OctetRed96, ForteBio, section 8.12) (Figures 8A and 8B). Both the murine and chimeric antibodies were able to bind to tau C3 in a concentration-dependent manner. The binding of the murine and chimeric antibodies was also tested against FL tau, but low or no binding was observed (Figures 8C and 8D), confirming that the antibody binding was specific to tau C3.

[0229] Example 3: Design of Tau01 humanized antibody variants Human VH and VK cDNA database A database of aligned human immunoglobulin sequences was compiled using human and mouse immunoglobulin protein sequences from the International Immunogenetics Database 2009 (Lefranc, 2015) and the Kabat Database Release 5 of Sequences of Proteins of Immunological Interest (last updated November 17, 1999) (Kabat et al. 1991). The database contains 10,406 VH and 2,894 VK sequences.

[0230] Molecular model of MoTau01 A humanized version of the MoTau01 antibody was designed using the MoTau01 VH and VK sequences. Homology models of the MoTau01 antibody variable regions were generated using the antibody prediction panel in Maestro 11.5. Ten loop models were generated using the selected human frameworks, which were then prepared using the one-step protein preparation wizard. Protein confidence reports were generated for all 10 models, and no major differences in model quality were identified. To capture different CDR orientations, all 10 models were used to determine the consensus of residues within 4 Å of the CDR loops.

[0231] Human Framework Selection Humanization requires the identification of suitable human V regions. The sequence analysis program Gibbs was used to search human VH and VK databases for the MoTau01 VH and VK protein sequences using various selection criteria. Using Maestro 11.5 (Schrodinger) software, FW residues (IMGT definition) within 4 Å of the CDR residues in the mouse Tau01 antibody structure were identified and designated "4 Å adjacent residues." The human VH sequence alignments with the highest identity to MoTau01 VH within 4 Å adjacent residues are shown in Table 5. Table 6 lists these envelope residues and VCI, as well as the number of residues in either the FW, VCI, or 4 Å adjacent residues that are identical to the mouse equivalent positions for the sequences in Table 5.

[0232] Humanized and incomplete sequences were excluded from the analysis. Sequence DQ840895.1 was selected as a candidate human heavy chain donor. This sequence scores highly for sequence identity and similarity. Its IGHV3-73 * There are only two somatic mutations from the 01VH germline, with changes in eight 4 Å adjacent residues and one VCI residue, which was the minimum number of changes obtainable (Table 8). [Table 5]

[0233] Similarly, sequence L33034 was selected as a candidate human kappa light chain donor. This sequence scores highly for sequence identity and similarity to Tau01 VK. Its IGKV1-17 * There is only one somatic mutation from the O1 germline, which has five potential 4 Å adjacent residue changes and one VCI residue change.

[0234] The sequences for the kappa light chain humanization strategy are shown in Table 12.

[0235] [Table 6]

[0236] Design of Tau01 humanized heavy chain variants Once a suitable human framework was identified, synthetic protein and DNA sequences could be designed. The initial design of a humanized version of TauO1 involved grafting CDR1, CDR2, and CDR3 from MoTauO1 VH onto the acceptor FW of DQ840895.1, creating the variant TauO1 HA. Next, in the humanized version of TauO1 HB, eight 4-Å adjacent residues at positions 1, 4, 35, 49, 58, 61, and 76-78 and one VCI residue were backmutated to their mouse equivalents, and sequences were constructed in silico, resulting in the following variants, designated TauO1 HD through TauO1 HL, mutated one at a time. Table 8 compares the mouse and humanized versions of the TauO1 VH protein sequences.

[0237] Design of Tau01 humanized light chain variants The framework from L33034 was used to design the DNA and protein for the humanized construct. The CDR1, CDR2, and CDR3 from TauO1 VK are shown, which are grafted into the acceptor FW of L33034 to generate the initial version of humanized TauO1. In TauO1 KA, which was backmutated to the mouse equivalent residues in variant TauO1 KB, there are five mismatched 4 Å adjacent residues at positions 34, 36, 44, 53, 69, and 71, and one VCI residue (Table 12).

[0238] These residues were mutated one at a time by constructing the sequence in silico and generating the following variants, designated Tau01 KD to Tau01 KI: Tau01 KG, in which the residue K backmutated to the mouse residue T was also mutated to the human germline residue S. This additional variant was designated Tau01 KJ.

[0239] Design of heavy and light chain C versions Following the design of the initial humanized variants, a homology model of Tau01 HAKA was constructed and evaluated. The latter model was superimposed on a model of the murine antibody. Each position identified for backmutation and the 3 Å residues surrounding those positions were highlighted and tested in the model. Based on this data, predictions were made about which residues would be most important to backmutate. These predictions were combined to form the HC version for the heavy chain (Table 8) and the KC version for the light chain (Table 12).

[0240] Example 4 (Generation and Characterization of Humanized Antibodies) Generation of Tau01 humanized antibodies The sequences for Tau01 HA / B / C and KA / B / C were codon-optimized using codons preferentially used by human cells and synthesized by Genscript. The KA / B / C and HA / B / C constructs were PCR-amplified, cloned into pHuK and pHuG4, respectively, using ligase-independent cloning, and used to transform TOP10 bacteria. Versions HA or KA were subsequently modified by PCR mutagenesis to obtain the other humanized variants annotated in Table 8 or Table 12, respectively, using the primers in Table 4.

[0241] Clones were sequenced and plasmid DNA was prepared using a QIAGEN Plasmid Miniprep kit or a Qiagen Plasmid Maxiprep kit. The expression construct sequences (HA, HB, HC, KA, KB, and KC) are shown in Figures 9-14.

[0242] Antibody expression ExpiCHO cells were transfected with expression plasmid preparations encoding (humanized or chimeric) VH and VK and cultured in serum-free medium for 7 days. The conditioned medium containing the secreted antibodies was then collected. The concentrations of IgG4κ antibodies in the ExpiCHO cell conditioned medium were measured by Octet and are shown in Tables 14A-C. Most antibodies were produced at good expression levels. [Table 7] [Table 8-1] [Table 8-2] [Table 9] *Expression of the transfection control, Hu1210 HuG1K, was also reduced (expected levels approximately 100 μg / mL).

[0243] Antigen binding by early versions of humanized antibodies The binding of the humanized variants to the provided tau C3 antigen was tested by the binding ELISA described in Example 5. The data shown in Figure 15 presents a binding ELISA for tau C3 using a humanized antibody consisting of HA / HB heavy chains combined with KA / KB light chains. The HAKA and HAKB humanized antibodies do not bind to tau C3. The MoTau01 HuG4K chimera binds to tau C3 with an EC50 value of 0.65 nM, and the HBKA and HBKB variants bind with similar EC50 values, with the HBKB version being the closest (0.78 nM).

[0244] Taking this data into consideration, further versions of the humanized heavy and light chains were expressed, each with a single backmutation (Tables 8 and 12). Figure 16 shows the results of the heavy chain single mutants and the HC version combined with KA-KC, tested for binding to tau C3 by ELISA. The data show that the heavy chain single mutants were unable to bind to tau C3 as well as the chimeric antibody. The HB and HC versions combined with KA-KC bound to tau C3 with the highest EC50 values, while HBKB and HBKC exhibited values ​​closest to the chimeric antibody (0.81 nM and 0.84 nM, respectively). These results were confirmed using a screening assay on an Octet instrument using a concentration of tau C3, as described in Section 8.12 (Figure 17). The Octet data are not optimal because a two-phase association and dissociation event may be observed, which is likely due to the nature of the tau C3 protein. However, the Octet data are sufficient for screening and ranking humanization candidates.

[0245] Because HB / HC was the best heavy chain version, we tested its binding to tau C3 in combination with all light chain versions (KA-KJ) by binding ELISA (Figure 18) and Octet screening assay (Figures 19A and 19B). While many of the light chain single mutants were able to retain binding to tau C3, light chain versions KE, KG, KI, and KJ ranked highest in both assays.

[0246] Second round humanized antibody design and generation Based on the suboptimal binding results for the initial humanized variants, second-round variants were designed by incorporating additional back mutations into the HC and KC versions (HM to HO and KL to KM, respectively) or by incorporating a combination of two back mutations in the KA version (KN to KP) (Tables 8 and 12). Mutagenesis, DNA preparation, expression, and quantification were performed. The resulting expression levels are shown in Tables 14D and 14E. Most antibodies were produced with good expression levels.

[0247] [Table 10] [Table 11]

[0248] Second-round antigen binding by humanized Tau01 antibodies To evaluate the heavy chain, the binding activity of antibody variants containing HM, HN, and HO combined with light chains KA, KB, KG, KI, and KJ was tested against tau C3 by ELISA (Figure 20) and Octet (Figure 21). The HM and HO variants ranked highest in both assays. The only difference between the HO and HM variants is the additional L-to-V backmutation at position 4, which is also present in the HN variant. Because the HN version did not bind as well as the HM version, the L-to-V backmutation was not deemed necessary. Therefore, HM was selected as the top heavy chain resulting from the second round of design.

[0249] The lead heavy chain HM was expressed with multiple light chains, KA-KC, selected single variants (KE, KG, KI, KJ) highlighted above, and the second-round designs KL, KN, KO, and KP. For comparison, these light chains were also expressed in combination with the earlier preferred heavy chain, HC. The binding of these humanized antibody variants to tau C3 was examined by binding ELISA (Figure 22). Overall, the HM variants bound with higher EC50s than the HC variants. Therefore, these variants were evaluated in the Octet screening assay (Figure 23). The antibody variants HMKE, HMKN, HMKO, and HMKP were ranked highest in both assays. The KM light chain in combination with the HC / HM was also screened against the leading antibody variants by Octet (Figure 24A). The HMKM version ranked very highly in the Octet screening assay, while HCKM ranked lowest. Binding of the humanized leads to FL-tau was also examined using Octet to determine how much selectivity remained (Figure 24B). The signal obtained with 500 nM FL-tau was very low (0.03-0.07 nM), which was in the range of nonspecific binding.

[0250] To further evaluate the binding data observed with Octet, off-rate ranking studies were performed on a Biacore200 instrument. Biacore offered even greater selectivity than Octet and facilitated immobilization of tau-C3 onto a CM5 chip via amine coupling, which stabilized the antigen. This generated higher-quality data with a stable baseline and better reproducible responses. Once tau-C3 was immobilized, a concentration (5 nM) of antibody was added, followed by dissociation and regeneration steps (Section 8.20). Off-rates were fitted using either a biphasic decay model (Figure 25B) or a monophasic decay model (Figure 25A) that fits only the second off-rate. Candidates were then ranked and compared based on the off-rates obtained using the fits. The data were consistent with the Octet results (although the ranking order differed). The lack of binding of FL tau to chimeric and humanized candidates, highlighting TauOl HMKM, HMKO, HMKP, HMKN, and HMKE as the humanized variants with the slowest off-rates, was also confirmed by Biacore by loading the antibodies onto a Protein G chip and adding 250 nM FL tau. Figure 26 shows that no binding was observed for either MoTauOl HuG4k or TauOlHCKB.

[0251] Thermal stability of humanized Tau01 candidate antibodies The purpose of this experiment was to test the thermal stability of chimeric and several humanized antibodies (Tau01, HCKG, HCKN, HMKE, HMKN, HMKO, and HMKP) when subjected to elevated temperatures ranging from 35°C to 95°C for 10 minutes, cooled to 4°C, and used in a binding ELISA at the EC80 concentration of each candidate (Section 8.15). All humanized candidate antibodies were more stable than the chimeric antibodies, retaining their ability to bind to tau C3 up to 67–68°C, after which their binding to tau C3 decreased (Figure 27). The humanized variants exhibit increased thermal stability compared to the chimeric antibody MoTau01 HuG4k, which only retained binding up to approximately 55°C. The variants containing the HM heavy chain retained binding at slightly higher temperatures than the HM heavy chain variants tested.

[0252] Selection of lead humanized Tau01 candidate antibodies Taking all these results together, the lead humanized antibody variants Tau01 HMKE, HMKM, HMKN, HMKO, and HMKP were selected for scale-up and purification using affinity and size-exclusion chromatography, as described in Section 8.16. The purified antibodies were further characterized in a series of biophysical assays. HCKB humanized candidates with weaker binding to tau C3 were scaled up for expression and purification to be used as comparative antibodies for ranking in the seeding assay. This allowed us to further examine the correlation between in vitro antibody affinity and potency in the cellular assay.

[0253] Aggregation of humanized Tau01 candidate antibodies As part of the post-purification QC process, antibody samples were subjected to SEC-MALS / DLS followed by mass spectrometry. To measure absolute molar mass and confirm aggregation, purified antibody samples were loaded onto a size-exclusion column in an HPLC system and analyzed by a multi-angle light scattering detector. The profiles of MoTau01 HuG4k and Tau01 HMKM showed no signs of aggregation and had an average molecular weight of approximately 147-148 kDa, which is within the expected range for IgG monomers in this analytical setup (Figure 28A). The profile of the Tau01 HCKB antibody has a broad peak that skews the data, resulting in a molecular weight of 179.6 kDa (Figure 28B). All three antibodies are monodisperse (Mw / Mn < 1.05) and show no signs of aggregation.

[0254] Dynamic light scattering is a complementary technique to static light scattering (SEC-MALS) for the detection of soluble aggregates and was used to QC the humanized variants Tau01 HMKN, HMKO, HMKP, and HMKE. For antibodies, the Z-average diameter, or hydrodynamic diameter, is expected to be approximately 10. The molecular weight dispersity (Pdl) should be less than 0.1 for the sample to be monodisperse. As shown in Figure 29, all antibody samples were monodisperse and contained one major population consistent with the size of the monoclonal antibody.

[0255] To confirm the correct molecular weight of the antibodies, mass spectrometry was performed on the intact and reduced antibody samples and is shown in Figures 30A-G. The molecular weights were consistent with the predicted molecular weight and confirmed amino acid sequence for all antibodies tested. No other trends were flagged. Overall, the purified chimeric and humanized antibodies passed QC.

[0256] Kinetic study of humanized Tau01 candidate antibody against tau C3 To measure the affinity of the binding interaction, a Biacore kinetic assay was developed, involving immobilization of tau-C3 on a CM5 chip by amine coupling and loading a concentration series of each antibody onto tau-C3. The chimeric antibody binds to tau-C3 with a KD of 57 pM (Figure 31A).

[0257] Figure 31B shows that Tau01 HCKB is the weakest binder to tau C3 (1.2 nM), followed by HMKM, which binds to tau C3 with an affinity of 110 pM (Figure 31G). As shown in Figures 31C-F, Tau01 HMKO, HMKN, HMKP, and HMKE have KD values ​​comparable to or closer to the chimeric antibody. However, when compared to the chimera, the off-rates are slightly faster for all humanized candidates (0.001-0.004 for humanized vs. 0.0007 for chimeric). Note that absolute values ​​should be compared with caution, as kA is at the instrument's limit. It can be concluded that antibodies HMKN, HMKP, and HMKE bind at picomolar concentrations and have the slowest off-rates of all humanized variants tested.

[0258] Determination of the melting temperature (Tm) of humanized Tau01 candidate antibodies A thermal shift assay was performed to measure the melting temperatures of the lead antibodies, Tau01 HMKE, HMKM, HMKN, HMKO, and HMKP. Samples were incubated with a fluorescent dye (Sypro Orange) in a qPCR thermal cycler for 71 cycles, increasing the temperature by 1°C per cycle (Section 8.21). The Tm of the humanized antibodies was calculated to be 68–69°C (Figure 32).

[0259] Non-specific protein-protein interaction (CIC) of humanized Tau01 candidate antibodies Interaction chromatography using bulk-purified human polyclonal IgG is a technique for monitoring nonspecific protein-protein interactions and can provide an indication of any solubility issues that may cause downstream manufacturing problems, as described in Example 5. An elevated retention index (k') indicates self-interacting propensity and poor solubility. Humanized TauOl HMKE, HMKM, HMKN, HMKO, HMKP candidate antibodies show retention indices less than 0.038, indicating poor propensity for nonspecific interactions and good solubility (Figure 33).

[0260] Solubility of humanized Tau01 candidate antibodies Humanized TauO1 HMKE, HMKM, HMKN, HMKO, and HMKP antibody candidates were concentrated using a solvent absorption concentrator (MWCO 7500 kDa), and concentrations were measured at regular intervals. TauO1 HMKP was concentrated to 123 mg / mL, and TauO1 HMKN, HMKM, and MoTauO1 HuG4K antibodies were concentrated to 87–88 mg / mL (Figure 34). TauO1 HMKO HuG4K was concentrated to 59 mg / mL without apparent precipitation, and TauO1 HMKE HuG4K was concentrated to 57 mg / mL. The data suggest that the antibodies are not prone to precipitation at concentrations up to 57 mg / mL. Freeze / thaw and heat stress analysis of a humanized Tau01 candidate antibody by circular dichroism

[0261] Circular dichroism (CD) is a spectroscopic technique that allows us to observe the global secondary structure of purified protein samples.

[0262] The freeze-thaw (FT) stress test involved subjecting samples of purified candidate antibodies to 10 cycles of 15 minutes at -80°C, followed by thawing for 15 minutes at room temperature. To perform the thermal stress test, samples of purified candidate antibodies were exposed to temperatures of a) 4°C, b) room temperature (RT), c) 37°C, and d) 50°C for 25 days.

[0263] The samples were then analyzed by circular dichroism to confirm whether the secondary structure was retained (Figure 35). All humanized variants tested passed our internal threshold. Overall, the data suggest that heat stress and freeze / thaw cycles did not affect the secondary structure of the humanized TauOl HMKE, HMKM, HMKN, HMKO, and HMKP candidate antibodies.

[0264] Isoelectric point analysis of humanized Tau01 candidate antibodies Capillary isoelectric focusing (cIEF) was used to perform pI analysis of the humanized antibody candidates. This technique allows for the separation of antibodies according to their isoelectric point (pI) using a pH gradient across a capillary. Figure 36 shows the chromatograms, and Table 15 lists one or more major pI isoforms (defined as greater than 10% of the peak area) for each antibody and the pI range for each antibody. The major isoelectric points for the humanized TauO1 HMKE, HMKM, HMKN, HMKO, and HMKP candidates are approximately 8.86 to 8.81.

[0265] Serum stability evaluation of humanized Tau01 candidate antibodies Purified samples of chimeric and humanized antibodies were incubated in mouse, human, and cynomolgus monkey serum for 21 days. Binding of the Tau01 HMKE, HMKM, HMKN, HMKO, and HMKP candidate antibodies incubated in three different serum samples was compared to antibodies incubated in PBS and a positive control sample at 4°C by binding ELISA against tau C3 (Figure 37). The Tau01 HMKE, HMKM, HMKN, HMKO, and HMKP candidate antibodies retained their binding ability after incubation in mouse, human, and cynomolgus monkey serum.

[0266] Summary of data on lead Tau01 HMKE, HMKN, HMKO, HMKP, and HMKM humanized candidates compared to MoTau01 HuG4k Table 16 summarizes the binding, kinetic affinity, and biophysical properties of the lead humanized TauO1 HMKE, HMKN, HMKO, HMKP, and HMKM antibody candidates compared to the chimeric antibody MoTauO1 HuG4k. All humanized candidates bind in the picomolar range, but among them, TauO1 HMKN, HMKO, and HMKP have the highest affinity and slowest off-rate and pass all of our biophysical assays. Considering all the data, TauO1 HMKP was selected as the lead humanization candidate because it had the highest affinity, slowest off-rate, and did not show any potential trends in the biophysical assays. TauO1 HMKN, TauO1 HMKO, and KMKE also have excellent properties, making them all good backup lead humanization candidates.

[0267] [Table 12]

[0268] conclusion The goal of this project is to humanize the MoTau01 antibody and ensure that the resulting antibody is capable of binding to tau C3 with comparable affinity when compared to the chimeric antibody. The MoTau01 antibody is designed and expressed as a humanized antibody without significant loss of binding affinity. The Tau01 HMKE, HMKM, HMKN, HMKO, and HMKP humanized antibodies demonstrate high affinity in the picomolar range in binding ELISA, Octet ranking, and kinetic studies using Biacore (Figure 31), and they also pass all of our biophysical assays.

[0269] The TauO1 HMKP antibody exhibits the best drug-like properties and excellent binding kinetics. Therefore, it was selected as a lead candidate (Table 16). In our opinion, the combination of excellent binding, expression, thermostability, affinity, and biophysical properties makes TauO1 HMKP a suitable candidate antibody for further development. TauO1 HMKN, HMKO, and HMKE also exhibit excellent properties and are very good backups for the humanized lead candidate.

[0270] Example 5 (protocol) The following protocols / procedures were used in Examples 1-4.

[0271] RNeasy Mini protocol for total RNA isolation (Qiagen) 1. Disrupt cells by adding Buffer RLT. For pelleted cells, thoroughly loosen the cell pellet by flicking the tube. Add Buffer RLT (600 μL) and proceed to step 2. Note: Incomplete loosening of the cell pellet may result in inefficient lysis and reduced yield. 2. Homogenize the cells by passing the lysate through an 18-20 gauge needle attached to an RNase-free syringe at least five times. 3. Add 1 volume of 70% ethanol to the homogenized lysate and mix thoroughly by pipetting. Do not centrifuge. The volume of lysate may be less than 600 μL due to losses during homogenization. 4. Transfer up to 700 μL of sample, including any precipitate that may have formed, to an RNeasy spin column placed in a 2 mL collection tube. Gently close the lid and centrifuge at ≥8000 x g for 15 seconds. Discard the flow-through. Reuse the centrifuge tube in step 5. 5. Add 700 μL of Buffer RW1 to the RNeasy column. Close the lid gently and centrifuge at ≥8000 x g for 15 seconds to wash the column membrane. Discard the flow-through. Reuse the centrifuge tube in step 6. 6. Add 500 μL of Buffer RPE to the RNeasy column. Close the lid gently and centrifuge at ≥8000 x g for 15 seconds to wash the column membrane. Discard the flow-through. Reuse the centrifuge tube in step 7. 6. Add another 500 μL of Buffer RPE to the RNeasy column. Close the lid gently and centrifuge at ≥8000 x g for 2 minutes to dry the RNeasy spin column membrane. 7. Place the RNeasy spin column in a new 2 mL collection tube and discard the old collection tube containing the flow-through. Gently close the lid and centrifuge at full speed for 1 minute. 8. To elute, transfer the RNeasy column to a new 1.5 mL collection tube. Add 30 μL of RNase-free water directly onto the RNeasy spin column membrane. Gently close the tube. Let stand for 1 minute, then centrifuge at ≥8000 x g for 1 minute.

[0272] Protocol for first-strand cDNA synthesis (GE Life Sciences) 1. Place RNA sample in a microfuge tube and add RNase-free water to bring the RNA to the appropriate volume (20 μL - 12x dilution, see Table A). 2. Heat the RNA solution to 65°C for 10 minutes, then cool on ice. Gently pipette the bulk first-strand cDNA reaction mix to obtain a uniform suspension. (BSA may precipitate in the mix upon storage; this precipitate will dissolve during incubation.) 3. Add 11 μL of the bulk first-strand cDNA reaction mix to a sterile 1.5 mL or 0.5 mL microcentrifuge tube. To this tube, add 1 μL of DTT solution, 1 μL (0.2 μg, 1:25 dilution) of NotI-d(T)18 primer, and heat-denatured RNA. Mix by pipetting up and down several times. 4. Incubate at 37°C for 1 hour and heat inactivate the transcriptase at 95°C for 5 minutes. [Table 13]

[0273] cDNA purification 1. A simple protocol designed to remove contaminating first-strand cDNA primers that may interfere with subsequent PCR reactions. Add 2.99 μL Buffer QG (from Qiagen Gel Extraction Kit, Cat. No: 28704) and 33 μL IPA. Mix and apply to a QiaQuick Gel Extraction column. Spin and discard the flow-through. 3. Wash the column once with 500 μL of Buffer QG. Discard the flow-through. 4. Wash the column once with 750 μL of Buffer PE. Discard the flow-through. 5. Spin the column to remove any residual alcohol and dry the column. 6. Elute the cDNA with 50 µL of distilled water preheated to 65 °C.

[0274] PCR cloning of mouse variable regions 1. Set up PCR reactions on the purified cDNA using the primers in Table 1 and Table 2. Use a different forward primer in each reaction (MHV1-12 and 14 and MKV1-11) as follows: [Table 14] 2. Cycle: [Table 15] 3. Electrophorese a 5 μL sample from each PCR reaction on a 2% (w / v) agarose gel to determine which leader primer produces a PCR product. Positive PCR clones are approximately 420-500 bp in size. 4. For positive clones, PCR purify the remaining sample using a QIAGEN PCR Purification kit, elute in 40 μL of nuclease-free water, and send to an external contractor (e.g., GATC) for sequencing of the PCR fragments using M13 forward and M13 reverse primers.

[0275] QIAquick PCR Purification Microcentrifuge and Vacuum Protocol (QIAGEN) 1. All centrifugation steps are performed in a conventional tabletop microcentrifuge at 17,900 x g (13,000 rpm). Add 2.5 volumes of Buffer PB to 1 volume of PCR reaction and mix. If the mixture is orange or purple, add 10 μL of 3 M sodium acetate (pH 5.0) and mix. The mixture will turn yellow. 3. Place the QIAquick column into the supplied 2 mL collection tube or into a vacuum manifold. 4. To bind the DNA, apply the sample to the QIAquick column and centrifuge for 30-60 seconds, or until the sample has passed through the column. Discard the flow-through and return the QIAquick column to the same tube. 5. To wash, add 0.75 mL Buffer PE to the QIAquick column and centrifuge or apply vacuum for 30-60 seconds. Discard the flow-through and return the QIAquick column to the same tube. 6. Centrifuge the column in a 2 mL collection tube (supplied) for 1 minute. 7. Place each QIAquick column in a clean 1.5 mL microcentrifuge tube. 8. To elute the DNA, add 40-50 μL of Buffer EB (10 mM Tris-Cl, pH 8.5) to the center of the QIAquick membrane and centrifuge the column for 1 minute.

[0276] Generation of mAb expression vectors by LIC Insert preparation 1. Use the sequence to generate LIC primers. 2. Perform LIC PCR on the codon-optimized, synthesized gene (Genscript) using LIC primers (Table 4). 3. Set up the PCR reaction as follows: [Table 16] NOTE: This step requires the use of a polymerase that generates blunt-ended PCR products. Other polymerases that produce T-overhangs are not suitable. 4. Cycle: [Table 17] 5. Run 5 μL of PCR product on a gel to confirm the correct size product (should be approximately 370 bp). 6. PCR purify the product using a QIAGEN PCR purification kit to remove nucleotides and primers. Elute in 40 μL of nuclease-free water. 7. T4 DNA polymerase treatment includes: PCR product 40 μL 4.5 μL of 10x NEB2 dTTP (100 mM) NEB 1.25 μL T4 DNA polymerase NEB 1 μL 8. Incubate at room temperature for 30 minutes, then inactivate the enzyme at 70°C for 20 minutes. Vector preparation 9. Digest the LIC vector (vector map shown in Figure 3 and Figure 4) using BfuAI by incubating the following at 50°C for 3 hours or overnight: 10 μL of 10x NEB buffer 3 1μL of 100x BSA BfuA1 5 μL LIC vector 5μg dH2O up to 100μL 10. After BfuAI digestion, add 2 μL of BamHI and incubate at 37°C for 2 hours. Run the digested vector on a 1x TAE gel containing 11.1% (w / v) agarose / 1x SYBR safe DNA stain. Two bands can be visualized - excise the higher MW band, extract it using a gel extraction kit, and elute it in 50 µL of EB. 12. T4 DNA polymerase processes the vector as follows: 10x NEB buffer 2 6μL 1.5 μL of 100 mM dATP T4 DNA Pol 1 μL BfuA1 digested vector 50 μL 13. Incubate at room temperature for 30 minutes, then inactivate the enzyme at 70°C for 20 minutes. Cloning 14. Mix 1 μL of insert with 0.5 μL of vector in a total of 10 μL of nuclease-free water for 20 minutes at room temperature. Always perform a single transformation of the vector. 15. Use the ligation mix to transform 25-50 µL of chemically competent Invitrogen TOP10 bacteria according to the manufacturer's instructions, spread onto a 90 mm LB agar plate containing kanamycin (50 µg / mL). Incubate overnight at 37°C. Selecting colonies from the transformation 16. Perform PCR verification using Phusion PCR Master Mix. [Table 18] [Table 19] Run each PCR reaction on a 17.2% agarose e-gel cassette and run for 15 min to determine the size of any PCR product bands on the gel. 18. Miniprep the constructs by growing overnight starter cultures using LB supplemented with kanamycin, and sequence DNA (using the same primers) from at least two separate positive clones of the variable genes to identify any possible errors due to the PCR reaction itself.

[0277] TOP10™ E. coli Transformation (Invitrogen Protocol) 1. Briefly centrifuge one or more vials containing one or more ligation reactions and place on ice. 2. For one or more ligations / transformations, thaw a 50 μL vial of One Shot cells on ice. 3. Pipette 1-2 μL of each ligation reaction directly into a vial of competent cells and mix by gently tapping. Do not mix by pipetting up and down. One or more remaining ligation mixtures can be stored at -20°C. 4. Incubate one or more vials on ice for 15-30 minutes. 5. Incubate in a 42°C water bath for exactly 30 seconds, then place on ice for 2 minutes. 6. Add 250 μL of pre-warmed SOC media to each vial. 7. Shake one or more vials in a shaking incubator at 37°C for exactly 1 hour at 225 rpm. 8. Spread 200 μL from each transformation vial onto individual labeled LB agar plates containing 500 μg / mL kanamycin. 9. Invert one or more plates and incubate at 37°C overnight.

[0278] Plasmid DNA miniprep isolation using QIAprep® (Qiagen protocol) 1. Resuspend the pelleted bacterial cells in 250 μL of Buffer P1 and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. 2. Add 250 µL of buffer P2 and gently invert the tube 4-6 times to mix. 3. Add 350 µL of Buffer N3 and immediately but gently invert the tube 4-6 times. The solution should become cloudy. 4. Centrifuge in a tabletop microcentrifuge at 13,000 rpm (approximately 17,900 x g) for 10 minutes. A small white pellet will form. 5. Apply the supernatant from step 4 to the QIAprep spin column by pipetting. 6. Centrifuge for 30-60 seconds. Discard the flow-through. 7. Wash the column by adding 0.5 mL of Buffer PB and centrifuging for 30-60 seconds. 8. Wash the column by adding 0.75 mL of Buffer PE and centrifuging for 30-60 seconds. 9. Discard the flow-through and centrifuge for a further 1 minute. 10. Place the QIAprep column in a clean 1.5 mL microcentrifuge tube. To elute the DNA, add 50 μL of nuclease-free water to the center of the QIAprep spin column, let it sit for 1 minute, and centrifuge for 1 minute.

[0279] ExpiCHO transfection in 1 mL transfection in 24-well plates (ExpiCHO™ Expression System kit - Invitrogen) 1. Approximately 4-6 x 10 cells 6 ExpiCHO cells are subcultured and expanded until a density of viable cells / mL is reached. 2. The day before transfection (day -1), the ExpiCHO culture was cultured at 3–4 × 10 6 The final density of viable cells is split and the cells are grown overnight. 3.6×10 6 Dilute cells to viable cells / mL. 4. Aliquot 0.9 mL of cells into each well of a 24-well plate to be used for transfection. 5. Prepare ExpiFectamine / DNA complex. 6. Dilute the plasmid DNA by adding 1 μL of DNA to each well to be transfected in OptiPro for a final volume of 50 μL (1 μg of plasmid DNA per mL of culture volume to be transfected). 7. For each well to be transfected, dilute 4 μL of ExpiFectamine CHO reagent in 46 μL of OptiPro medium (no incubation period necessary). 8. Add the diluted ExpiFectamine CHO to the diluted DNA and mix by gently pipetting 3-4 times (incubation time 1-5 minutes). 9. Add 100 μL of the complexation mixture to each well containing culture in a 24-well plate. 10. Cover the plate with a gas-permeable lid. 11. Incubate the 24-well plate in an incubator at 37° C. with 8% CO 2 on an orbital shaker (recommended shaking speed is 225 rpm for a shaker with an orbital throw of 19 mm). 12. 18-22 hours after transfection, add ExpiFectamine enhancer (6 μL of ExpiCHO enhancer) and ExpiCHO feed (190 μL of ExpiCHO feed). 13. Protein expression is usually complete and the supernatant is ready to be collected by 7-8 days after transfection.

[0280] IgG quantification by Octet 1. Prepare 100 μL of each concentration of standard curve and ExpiCHO supernatant as follows: a. HuG4K isotype standard at 500, 250, 125, 62.5, 31.25, 15.6, 7.81, and 3.9 μg / mL using ExpiCHO expression medium as the diluent. b. (Unknown) test sample 2. Pre-soak a protein G coated biosensor (Pall ForteBio) in 200 μL of ExpiCHO expression medium (for at least 10 minutes). Aliquot 3.45 μL of standard and test samples in duplicate into a 384-well slanted bottom plate, including a media-only control. Seal the plate and spin in a benchtop centrifuge (1000 rpm for 1 minute). 4. Remove the plate seal and insert the plate and pre-soaked sensors into the Octet. 5. Quantification is performed as follows: a. Regenerate the Protein G coated sensor in 10 mM glycine (pH 1.5) for 5 seconds and neutralize in ExpiCHO expression medium for 5 seconds. Repeat three times. b. Measure the standard or sample for 120 seconds. c. Repeat the regeneration and neutralization steps as above. 6. Import the data into the analysis software and fit the data to a dose response-5PL weighted fit to obtain the IgG concentration (μg / mL).

[0281] Tau C3 binding ELISA 1. Coat 94 / 384-well Maxisorp plates with 50 / 30 μL aliquots of tau-C3 in PBS at 1 μg / mL per well in each 96 / 384-well plate. Incubate overnight at 4°C. 2. Wash three times with PBS-T (0.1% Tween 20). 3. Block with 150 / 80 μL per well of PBS+5% BSA+0.1% Tween 20 in each 96 / 384 well plate. 4. Incubate for 1 hour at 37° C. Wash 3 times with PBS-T (0.1% Tween 20). 5. Add 50 / 30 μL of serially diluted primary antibody in PBS + 0.2% BSA + 0.1% Tween 20 to the assay plate (96 / 384 well plate, respectively). Use 3-fold serial dilutions starting at approximately 4 μg / mL. Repeat the incubation and washing steps (step 4). 6. Dilute anti-human kappa chain HRP (Sigma A7164-1mL) to 3 μL per 10 mL in PBS + 0.2% BSA + 0.1% Tween 20 and add 50 / 30 μL to each well of a 96 / 384 well plate respectively. Repeat the incubation and washing steps (step 4). 7. Add 75 / 20 μL of K-Blue substrate (Neogen) per well and incubate at room temperature for 5-10 minutes. The reaction is stopped by adding 8.50 / 10 μL of RED STOP solution (Neogen) to each well of the 96 / 384 well plate, respectively. 9. Read absorbance at 650 nm using a Pherastar Plus.

[0282] Screening and affinity determination of Tau01 variants with Octet 1. Immediately before use, the Protein G-coated sensor (Pall ForteBio) was placed in HBS-P + Incubate in buffer for 10 minutes. 2.1 μg / mL of antibody was applied to the Protein G sensor and HBS-P + Fill in for 600 seconds (immobilization level of 0.8-1 nm). 3. Install the sensor on the HBS-P + Equilibrate in PBS for 180 seconds. 4. For screening assays, the association step is carried out using 10 nM Tau C3 for 600 seconds. For kinetic assays, approximately 0.5 μg / mL of antibody is added to HBS-P + The mixture was filled in for 10 min, and an association step was performed for 10 min using tau-C3 at concentrations ranging from 20 nM to 0.31 nM. 5.HBS-P + A dissociation step is carried out in PBS for 600 seconds. 6. Regenerate the sensor with 10 mM glycine (pH 1.5–2.0) for 5–30 s and then resuspend the HBS-P + Neutralize by incubating in buffer for 30-60 seconds. Repeat three times.

[0283] QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene) 1. Prepare one or more reactions as shown below. a. 5 μL of 10x reaction buffer b. 0.12 μL (25 ng) of RHA or RKA template c. 1.3 μL (125 ng) of oligonucleotide mutagenic forward primer d. 1.3 μL (125 ng) of oligonucleotide mutagenic reverse primer e. 1 μL of dNTP mix f. 1.5 μL of QuikSolution reagent g. ddH2O to a final volume of 50 μL h. 1 μL QuikChange Lightning Enzyme 2. Cycle each reaction using the cycling parameters outlined in the table below. [Table 20] Add 3.2 μL of Dpn I restriction enzyme. 4. Gently and thoroughly mix each reaction, briefly microfuge, then immediately incubate at 37°C for 5 minutes to digest the parental dsDNA. 5. Transform 2 μL of Dpn I-treated DNA from each reaction into 45 μL (+ 2 μL β-ME) aliquots of XL10-Gold ultracompetent cells (see TOP10™ E. coli transformation). 6. Screen colonies using the Phusion method, miniprep, and sequence to confirm the exact mutation.

[0284] Qiagen HiSpeed ​​Maxiprep System Protocol 1. Select from a freshly streaked selection plate or a glycerol stock of the clone of interest and inoculate a starter culture of 2-5 mL of LB medium supplemented with kanamycin. Incubate at 37°C for approximately 8 hours with shaking at 250-300 rpm. 2. Dilute the starter culture 1 / 1000 and inoculate 150-250 mL of LB medium supplemented with kanamycin from the starter culture and incubate overnight (12-16 hours) at 37°C with shaking at 250-300 rpm. 3. Harvest the cells at 6,000 x g for 15 minutes. Discard the supernatant. 4. Thoroughly suspend the cell pellet in 10 mL of Buffer P1 by vortexing or pipetting. 5. Add 10 mL of Buffer P2. Mix by inverting vigorously 4-6 times. Incubate at room temperature for 5 minutes. 6. Add 10 mL of chilled Buffer P3. Mix by inverting vigorously 4-6 times. 7. Pour the lysate into the barrel of the QIAfilter cartridge. Incubate at room temperature for 10 minutes. 8. Equilibrate the HiSpeed ​​Maxi Tip by applying 10 mL of Buffer QBT and empty by gravity flow. 9. Using a QIAfilter, filter the lysate into an equilibrated HiSpeed ​​Maxi Tip. Apply the lysate to the resin by gravity flow. 10. Wash the HiSpeed ​​Maxi Tip with 60 mL of Buffer QC. 11. Elute the DNA with 15 mL of Buffer QF. 12. Precipitate the DNA by adding 10.5 mL of isopropanol to the eluted DNA. Mix and incubate at room temperature for 5 minutes. 13. Transfer the eluate / isopropanol mixture to a 30 mL syringe and filter through the QIAprecipitator module. 14. Wash the DNA with 2 mL of 70% ethanol in the QIAprecipitator. Dry the membrane by repeatedly pushing air through it. Using a 15.5 mL syringe, elute the DNA into 1 mL of nuclease-free water. Transfer the eluate to a syringe and elute again.

[0285] Comparison of thermal stability 1. Dilute fully humanized antibodies and chimeric controls to 1 μg / mL in PBS / 0.2% Tween and aliquot into PCR tubes in the appropriate volume for the EC80 concentration. Bring the volume up to 100 μL with the same buffer. 2. Heat each tube separately at temperatures between 30°C and 85°C at 5°C intervals for 10 minutes and cool to 4°C. The 3.1 μg / mL stock is frozen for 1 hour and then diluted to the EC80 concentration. 4. Perform the binding assay (section 8.11) against Tau-C3 in a 96-well plate using 100 μL of each antibody per well (assay each temperature in duplicate).

[0286] Biacore off-rate ranking and kinetic study of TauO1 humanized antibody Off-rate ranking 1. Amine-couple human tau C3 at 0.5 μg / mL in acetate buffer (pH 5) in one flow channel of a CM5 chip (GE Healthcare). Use HBS-EP+ as the running buffer and use the immobilization wizard to target approximately 15 RU. 2. Load antibody supernatant with 2.5 nM HBS-EP+ buffer at a flow rate of 30 μL / min for 300 seconds, followed by 600 seconds of dissociation and 30 seconds of regeneration with 3 M MgCl. Export raw data and fit data in GraphPad Prism using a monophasic or biphasic decay with buffer baseline subtraction. dynamics 1. Amine-couple human tau C3 at 0.5 μg / mL in acetate buffer (pH 5) in one flow channel in a CM5 chip using HBS-EP+ as the running buffer and using the immobilization wizard targeting approximately 15 RU. 2. Dilute each antibody to 5 nM and make a 2-fold dilution series down to 0.08 nM in HBS-EP+ buffer. Inject each concentration for 300 seconds at 30 μL / min, followed by a 600-second dissociation period and a 30-second regeneration period using 3 M MgCl2, with a 600-second stabilization period between cycles. Fit the data using a 1:1 global fit. Binding test FL Tau 1. Load 0.25 μg / mL of antibody in HBS-EP+ buffer onto a Protein G chip (GE Healthcare) at 10 μL / min for 30 seconds. Increase the flow rate to 30 μL / min and add 250 nM FL tau in HBS-EP+ buffer for 180 seconds. Regenerate with 10 mM glycine (pH 1.5) for 30 seconds. Purification of antibody candidates Equipment: GE Healthcare AKTAxpress™ purification system Software: UNICORN Column: HiTrap MabSelect SuRe, 1 mL; HiLoad 16 / 600 Superdex 200 pg Mobile phase: IgG elution buffer, Dulbecco's 1x PBS Sample preparation: filtration through 0.22 μm Injection volume: 200 mL of Expi293 conditioned medium in DPBS (1:1) Flow rate: Sample loading at 0.5 mL / min, gel filtration at 1.5 mL / min, elution at 1 mL / min

[0287] SEC-MALS 1.10 μL of each sample (1 mg / mL) was injected onto a SEC column (AdvanceBio SEC 300 Å, 4.6×150 mm, 2.7 μm, LC column, Agilent) and then detected by three in-line detectors: a.UV (Agilent 1260 Infinity HPLC system with thermostatted column compartment) b. Light scattering (Wyatt Technology DAWN HELEOS) c. Refractive index detector (Wyatt Technology Optilab TRex) 2. A constant flow rate of 0.4 mL / min was applied using a mobile phase of Gibco's PBS (ThermoFisher) containing 0.05% sodium azide. All experiments were performed at 25°C. 3. Data were analyzed with Wyatt Technology ASTRA software (version 6.1.2.83) and the refractive index increment (dn / dc) set to 0.185 (i.e., for protein analysis). 4. All samples were stored at 4°C before analysis by SEC-MALS.

[0288] Dynamic Light Scattering (DLS) 1. Prepare 50 μL samples at 1.3 mg / mL (in Dulbecco's PBS, Sigma D8537) and aliquot into 384-well polypropylene plates (Greiner bio-one). 2. Data was recorded on a Zetasizer APS (Malvern). All values ​​were recorded in triplicate and processed using the associated Zetasizer software (version 7.11). 3. Cumulant analysis was performed to obtain the average particle size (z-average) and the polydispersity index (PDI).

[0289] mass spectrometry Mass spectrometry analysis of purified chimeric and humanized candidate antibodies is shown in FIG.

[0290] Thermal Shift Comparison 1. Prepare samples directly into a 96-well white PCR plate in a final volume of 25 μL (final purified antibody concentration of 1-2 μM). 2. Make a 1:100 stock in Sypro Orange-PBS buffer, then add 1:10 to the final sample (e.g., 2.5 μL in 25 μL). 3. Load into qPCR instrument and use MxPro software, SYBR Green method (filter = FRROX, no reference dye). Thermal profile set to -1° ramp for 71 cycles. 4. Plot the results and determine the Tm.

[0291] Interaction Chromatography (CIC) 1. Samples were analyzed by two separate 20 μL injections (0.5 mg / mL): first onto 1 mL of NHS-activated resin (GE Healthcare) coupled with 30 mg of human polyclonal IgG (Sigma 14506), and then onto 1 mL of empty, coupled NHS-activated resin as a control column. 2. The mobile phase consisted of Dulbecco's PBS (Sigma D8537) containing 0.01% sodium azide (0.1 mL / min), and all experiments were performed at 25°C. 3. The eluted samples were detected by UV absorbance (Agilent 1260 Infinity HPLC system with a thermostatted column compartment) and the data was used to measure the sample peak retention times using Wyatt Technology ASTRA software (version 6.1.2.83). These were then used to calculate the retention factor k':

number

[0292] solubility Fill a Vivapore solvent absorption concentrator (7500 kDa MWCO) (VP0502 Satorius) with 3.5-5.0 mL of antibody solution at 1 mg / mL in PBS. 1. Monitor the antibody concentration every 10 minutes by sampling a small amount for measurement with a Nanodrop 2000 (ε=1.4) until the concentration reaches a dead volume of approximately 30-50 μL. 2. Plot the concentration values ​​(mg / mL) against the corresponding time points to generate a concentration profile.

[0293] circular dichroism Prepare 30 μL samples at 1.1 mg / mL (Dulbecco's PBS, Sigma D8537). The 2.1 mg / mL sample is diluted to 0.15 with 10 mM phosphate buffer. Readings were taken in 3.1 mM spectrosil cuvettes, using a DIT of 4 seconds and a scan rate of 20 nm / min with a step size of 1 nm. 4. The averaged blank spectrum was subtracted from the sample spectrum, and the spectrum was then converted to Δε. The spectrum was then zeroed for values ​​between 256 and 260 nm. Smoothing was performed with a Savitzky-Golay filter using a custom Excel function, sgFilter(), using a second-order polynomial with seven window sizes (-2, 3, 6, 7, 6, 3, -2). Spectra are shown with error bars, which represent the mean standard deviation at wavelengths of + / - 2 nm.

[0294] pI analysis using cIEF 1. Samples were concentrated to >5 mg / mL and desalted to a NaCl level of <50 mM. 10 μL was then diluted with 4.5 / 5.1 / 9.5 and 10 μL of HCl. pI 240 μL of pharmalyte / urea gel Master mix containing the marker was added. 2. The sample was mixed for at least 5 minutes, then 200 μL was added to a sample PCR vial. 3. The sample was loaded into the PA800 sample block along with the cIEF gel, catholyte, and anolyte, and the chemical mobilizer rinse buffer was loaded into the chemical buffer block. The PA800 was loaded with a neutral capillary and the default "Condition" method was run to prepare the capillary for sample analysis. 4. Each sample was run using a precise "separation" method that relies on the level of urea present in the sample. 5. Data were analyzed using 32Karat software. pI The markers are the sample peaks. pIA calibration curve is provided to quantify the values.

[0295] Antibody serum stability assessment 1. Prepare 600 μL of polished antibody at 0.4 mg / mL in PBS. 2. Use mouse serum (SCD-808), human serum (S-123), and cynomolgus monkey serum (S-118) from Seralab. Aliquot 150 μL of serum and PBS control into a round-bottom 96-well plate, and add 50 μL of 0.4 mg / mL antibody solution in PBS (final concentration of 100 μg / mL) in triplicate to each serum type in a tissue culture cabinet (BSL-2). Keep an aliquot at 4°C to use as a control. Serum incubation plate layout [Table 21] 3. Seal the plate and incubate at 37°C. 4. To avoid contamination, 20 μL samples are taken under sterile conditions (BSL-2) at specific intervals (e.g., on days 10 and 20). Freeze at -20°C until analysis. 5. Analyze the longest incubations first. Assay for antigen binding to Tau C3 by diluting the samples appropriately and generating an ELISA binding curve for each sample (3 * Use non-incubated antibody as a control (NI) and compare PBS / all serum per mAb samples on the same plate.

[0296] All references, publications and patent documents cited herein, and the text set forth in the Figures and Sequence Listing, are hereby incorporated by reference in their entirety for all purposes to the same extent as if each were individually indicated as such.

[0297] In the foregoing specification, the invention has been described with reference to certain exemplary embodiments and examples thereof. It will, however, be apparent that various modifications and changes can be made thereto without departing from the broader spirit and scope of the invention as set forth in the appended claims. The specification and drawings are therefore to be regarded in an illustrative manner and not in a restrictive sense.

[0298] The citation of any document herein is not an admission that such document is relevant prior art or is considered material to the patentability of any claim in this application. Any statement as to content or a date of a document is based on the information available to applicant at the time of filing and does not constitute an admission of correctness of such statement. [Sequence List Free Text]

[0299] Sequence Listing 1 <223> htau40 Sequence Listing 2 <223> Tau 2N3R Sequence Listing 3 <223> Tau 1N4R Sequence Listing 4 <223> Tau 0N4R Sequence Listing 5 <223> Tau 1N3R Sequence Listing 6 <223> Tau 0N3R Sequence Listing 7 <223> Heavy chain CDR1 Sequence Listing 8 <223> Heavy chain CDR2 Sequence Listing 9 <223> VGGGDF Sequence Listing 10 <223> Light chain CDR1 Sequence Listing 11 <223> Light chain CDR2 Sequence Listing 12 <223> LQYVRYPWT Sequence Listing 13 <223> Heavy chain HM Sequence Listing 14 <223> Light chain KE Sequence Listing 15 <223> Light chain KN Sequence Listing 16 <223> Light chain KO Sequence Listing 17 <223> Light chain KP Sequence Listing 18 <223> Light chain KM Sequence Listing 19 <223> Immunizing peptide Sequence Listing 20-104 <223> Primer Sequence list 105-121 <223> heavy chain Sequence list 122-138 <223> Light chain Sequence List 139-141 <223> Primer

Claims

1. An isolated anti-tau C3 antibody, said antibody being present in an amount of 1 x 10 －１０ ~1 x 10 －１２ has a binding affinity (KD) for tau C3 that is M, (a) a variable heavy chain (V) comprising a CDR1 represented by SEQ ID NO:7, a CDR2 represented by SEQ ID NO:8, and a CDR3 represented by SEQ ID NO:9; Ｈ ) polypeptide having a variable heavy chain (V) having at least 90% sequence identity to SEQ ID NO: 13 Ｈ ) polypeptides, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12; Ｌ ) polypeptide, comprising a variable light chain (V) having at least 90% sequence identity to SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:

18. Ｌ ) polypeptide, An anti-tau C3 antibody, wherein the antibody is a humanized antibody or a chimeric antibody.

2. An isolated anti-tau C3 antibody, said antibody being present in an amount of 1 x 10 －１０ ~1 x 10 －１２ Binding affinity (KD) for tau C3, which is M, and 1 x 10 －４ ~1 x 10 －８ has a binding affinity (KD) for full-length tau that is M, (a) a variable heavy chain (V) comprising a CDR1 represented by SEQ ID NO:7, a CDR2 represented by SEQ ID NO:8, and a CDR3 represented by SEQ ID NO:9; Ｈ ) polypeptide having a variable heavy chain (V) having at least 90% sequence identity to SEQ ID NO: 13 Ｈ ) polypeptides, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12; Ｌ ) polypeptide, comprising a variable light chain (V) having at least 90% sequence identity to SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:

18. Ｌ ) polypeptide, An anti-tau C3 antibody, wherein the antibody is a humanized antibody or a chimeric antibody.

3. Off-rate K for Tau C3 ｄ is 1 x 10 －４ ~1 x 10 －３ s －１ The anti-tau C3 antibody according to claim 1 or claim 2,

4. The variable heavy chain (V Ｈ ) polypeptide comprises SEQ ID NO: 13, Ｌ 3. The anti-tau C3 antibody of claim 1 or claim 2, wherein the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO:

18.

5. The variable heavy chain (V Ｈ ) polypeptide, wherein CDR1 is the polypeptide of SEQ ID NO: 7, CDR2 is the polypeptide of SEQ ID NO: 8, and CDR3 is the polypeptide of SEQ ID NO: 9; Ｌ 3. The anti-tau C3 antibody of claim 1 or claim 2, wherein in the polypeptide, CDR1 is the polypeptide of SEQ ID NO: 10, CDR2 is the polypeptide of SEQ ID NO: 11, and CDR3 is the polypeptide of SEQ ID NO:

12.

6. The variable heavy chain (V Ｈ ) polypeptide is the polypeptide of SEQ ID NO: 13, Ｌ 6. The anti-tau C3 antibody of claim 5, wherein the polypeptide is a polypeptide of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO:

18.

7. V having at least 95% sequence identity with SEQ ID NO: 13 H V chain polypeptides, and V having at least 95% sequence identity to SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18 L The anti-tau C3 antibody of claim 1 or claim 2, comprising a chain polypeptide.

8. An anti-tau C3 antibody described in claim 1 or claim 2, having a water solubility of 50 mg / mL to 200 mg / mL.

9. The anti-tau C3 antibody of claim 1 or claim 2, which is a humanized antibody.

10. The anti-tau C3 antibody according to claim 1 or claim 2, which is a chimeric antibody.

11. An anti-tau C3 antibody described in claim 1 or claim 2, which is a humanized antibody having a binding affinity (KD) for tau C3 of 10 pM to 40 pM.

12. An anti-tau C3 antibody described in claim 11, having a binding affinity (KD) for tau C3 of 10 pM to 35 pM.

13. A pharmaceutical composition comprising the anti-tau C3 antibody of claim 1 or the anti-tau C3 antibody of claim 2 for treating a tauopathy.

14. 14. The pharmaceutical composition of claim 13, wherein the tauopathy is selected from the group consisting of Alzheimer's disease, progressive supranuclear palsy, frontotemporal dementia, traumatic brain injury, Pick's disease, corticobasal degeneration, and frontotemporal lobar degeneration.

15. 15. The pharmaceutical composition of claim 14, wherein the tauopathy is Alzheimer's disease.

16. A pharmaceutical composition for treating a tauopathy, comprising: (i) an anti-tau C3 antibody according to claim 1 or an anti-tau C3 antibody according to claim 2; and (ii) one or more pharmaceutically acceptable excipients.

17. the anti-tau C3 antibody comprises (a) a variable heavy chain (V) comprising a CDR1 represented by SEQ ID NO:7, a CDR2 represented by SEQ ID NO:8, and a CDR3 represented by SEQ ID NO:9; Ｈ ) polypeptide, and (b) a variable light chain (V) comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO:

12. Ｌ 17. The pharmaceutical composition of claim 16, comprising a .

18. 18. The pharmaceutical composition of claim 17, wherein the anti-tau C3 antibody has a water solubility of 50 mg / ml to 200 mg / ml.

19. The anti-tau C3 antibody is 1×10 －１１ M~4 x 10 －１１ 19. The pharmaceutical composition of claim 18, which is a humanized antibody having a binding affinity (KD) for tau C3 that is M.

20. The pharmaceutical composition of claim 17, having a binding affinity (KD) for tau C3 of 10 pM to 40 pM.

21. The pharmaceutical composition of claim 13 , wherein the antibody is a humanized antibody.

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