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Use of an anti-tau ps422 antibody for the treatment of brain diseases

An antibody and phosphorylation technology, applied in the direction of antibodies, tumor rejection antigen precursors, antibody mimics/scaffolds, etc., can solve unclear problems

Active Publication Date: 2012-05-16
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to Pei, J.J. et al., Journal of Alzheimer's Disease 14 (2008) 385-392, the existing literature provides limited and unclear information on which of these loci are specific to the AD brain

Method used

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  • Use of an anti-tau ps422 antibody for the treatment of brain diseases
  • Use of an anti-tau ps422 antibody for the treatment of brain diseases
  • Use of an anti-tau ps422 antibody for the treatment of brain diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0233] Preparation and purification of antibodies

[0234] a) Antibody production

[0235] Mice use SEQ ID NO: 9 (Ser-Ile-Asp-Met-Val-Asp-Ser (PO 3 h 2 )-Pro-Gln-Leu-Ala-Thr-Leu-Ala-Asp), which corresponds to amino acids 416-430 of the longest human isoform of Tau. To allow direct coupling to KLH via thiols, a cysteine ​​was added to the Tau fragment at the N-terminus. Subsequent immunization schemes, fusion and cloning and screening for anti-TaupS422 specific antibodies are described in EP 1 876 185 .

[0236] b) Purified clones 2.10.3, 2.20.4 and 5.6.11

[0237] Cell-free hybridoma culture supernatant (250-300 ml) was loaded onto a 25 ml MEP Hypercell column (pall Biosciences) equilibrated with 50 mM TrisCl pH 8.0. After washing with equilibration buffer, the antibody was eluted with 30 mM sodium citrate, 100 mM NaCl pH 4.1. Antibody-containing fractions were pooled and then dialyzed overnight at 4° C. in Spectra-Por 6-8000 dialysis tubing against 5 liters of 10 mM Tri...

Embodiment 2

[0240] Preparation of Tau, Tau pS422, Aggregated Tau and Aggregated Tau pS422

[0241] Contains N-terminal (His) 6 -SUMO fusion-tagged Tau was expressed in Escherichia coli (E.coli) and passed through ion-exchange chromatography on HiTrapQ (GE Healthcare, Switzerland), followed by affinity affinity on Ni-NTA Sepharose (Qiagen, Switzerland) Chromatographic purification. The fusion tag was then cleaved by digestion with SUMO protease (Invitrogen, The Netherlands), followed by a second Ni-NTASepharose chromatography step to remove the fusion tag.

[0242] Tau-pSer422 was prepared by incubating Tau with ERK2 protein kinase. The molar ratio of ERK2:Tau (ca:1:10) was selected by adding 1 mM MgCl at 37 °C 2 and 2 mM ATP in 10 mM TrisCl pH 8.0 overnight to produce maximal phosphorylation at S422. This was then assumed to represent stoichiometric phosphorylation at position 422. Phosphorylation was checked by western blotting using an in-house monoclonal antibody specific for pS42...

Embodiment 3

[0245] Anti-Tau pS422 mAb is highly selective for Tau phosphorylated at S422

[0246] a) Peptide synthesis

[0247] Peptide synthesis was performed on an automated peptide synthesizer using Fmoc chemistry. In repeated cycles, the peptide sequence is assembled by sequential coupling of the corresponding Fmoc-amino acids. In each coupling step, the N-terminal Fmoc-group was removed by treating the resin with 20% piperidine in N-methylpyrrolidone. Couplings were performed using Fmoc-protected amino acids (1 mmol) activated by HBTU / HOBt (1 mmol each) and DIPEA (2 mmol) in DMF. After each coupling step, unreacted amino groups were capped by treatment (10 min shaking) with a mixture of acetic acid (0.5M), DIPEA (0.125M) and HOBt (0.015M) in NMP. Between steps, the resin was washed with N-methylpyrrolidone and DMF. Incorporation of sterically hindered amino acids is accomplished by automatic double coupling. For this purpose, the resin was treated twice with 1 mmol of the activa...

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PUM

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Abstract

An antibody binding to Tau, phosphorylated at serine 422 (pS422), characterized in specifically binding to phosphorylated Tau fragment of SEQ ID NO: 9 and to Tau pS422, but not binding to Tau and to phosphorylated MCAK fragment of SEQ ID NO: 17 is useful in the treatment of a Tauopathy.

Description

[0001] The present invention relates to the use of an antibody specifically binding to the phosphorylated Tau fragment (pS422) of SEQ ID NO: 9 for treating encephalopathy. Background of the invention [0002] Human Tau (microtubule-associated protein Tau (neurofibrillary tangle protein, paired helical filament-Tau (Paired helical filament-Tau), PHF-Tau) is a microtubule-associated protein of neurons mainly found in axons, and its Function is to promote tubulin polymerization and stabilize microtubules.Six isoforms (isoforms A, B, C, D, E, F, G, fetal-Tau) are found in the human brain, the longest isoform The Tau type contains 441 amino acids (isoform F, Uniprot P10636-8). Tau and its properties are also described by Reynolds, C.H. et al., J. Neurochem. 69 (1997) 191-198. [0003] Tau in its hyperphosphorylated form is a major component of paired helical filaments (PHFs), the structural unit of neurofibrillary damage in the Alzheimer's disease (AD) brain. Tau can be phosphoryl...

Claims

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Application Information

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IPC IPC(8): C07K16/18
CPCA61K38/1764C07K2317/92C07K2319/55C07K16/3053C07K2317/94C07K2316/96C07K14/4711C07K14/7051C07K16/18C07K2317/622C07K2319/40C07K14/4748A61K2039/505A61P25/00A61P25/14A61P25/28C12N15/11A61K39/395
Inventor 贝恩德·博尔曼乌尔里希·格普费特菲奥娜·格吕宁格尔瓦尔特·胡贝尔汉斯-威利·克雷尔瓦莱里娅·利夫可奥拉夫·蒙迪戈尔索尼亚·奥夫纳劳伦斯·奥兹曼迈克尔·施雷尔姆
Owner F HOFFMANN LA ROCHE & CO AG
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