CN110028583B - anti-Tau antibody and application thereof in treating Alzheimer disease and traumatic brain injury - Google Patents

Abstract

The object of the present invention is to provide anti-Tau antibodies capable of specifically binding Tau proteins, in particular of specifically binding hyperphosphorylated Tau proteins, and their use in the treatment of alzheimer's disease, traumatic brain injury; administration of the antibody can delay the expression of abnormally phosphorylated tau in a mouse model of Alzheimer's disease, and can have an effect of preventing or treating tau-related neurodegenerative diseases such as Alzheimer's disease and traumatic brain injury.

Description

anti-Tau antibody and application thereof in treating Alzheimer disease and traumatic brain injury

Technical Field

The invention belongs to the field of antibodies, and particularly relates to an anti-Tau antibody and application thereof in treating Alzheimer disease and traumatic brain injury.

Background

The protein is a microtubule-associated protein which can be divided into four regions, namely an N-terminal overhang region, a proline-rich region, a microtubule-binding region, and a C-terminal region, and Tau protein normally binds to microtubules but can be detached from microtubules to cause decrease or depolymerization of microtubules after phosphorylation catalyzed by glycogen synthase kinase 3 β (glycosynthase kinase-3 β -3 β), and the balance between phosphorylation and dephosphorylation of Tau protein is to maintain neuronal microtubule stabilitySex, and guarantee the cell to finish the premise that organelles such as mitochondria and the like are normally transported in the axial fluid.

Traumatic Brain Injury (TBI) is a persistent brain injury caused by either accidental or non-accidental injury. The clinical manifestations include loss of consciousness, seizures, special postures, syncope, hemiplegia, etc. In the research of traumatic brain injury, tau protein hyperphosphorylation can cause microtubule depolymerization, abnormal mitochondrial distribution and mitochondrial dysfunction, and finally neuronal degeneration or apoptosis is caused; cis-phosphorylated tau is an early driver of neurodegenerative disease following brain injury, which promotes tauopathy.

Alzheimer Disease (AD) mostly develops in the elderly, develops secretly, has a slow and irreversible course, is a group of primary degenerative diseases of the brain with unclear etiology at present, and has the basic pathological characteristics of senile plaques formed by deposition of Abeta, neurofibrillary tangles formed by deposition of phosphorylated Tau protein, loss of a large number of cholinergic neurons and the like. Research shows that the anti-tau antibody can be used for treating or assisting in treating the Alzheimer disease and can play a role in relieving the disease progression.

Based on the above recognition, the treatment of tau abnormality-related diseases with specific anti-tau antibodies is considered to be a viable approach.

Disclosure of Invention

The invention aims to provide an anti-tau antibody, which can specifically bind phosphorylated tau, and the light chain amino acid sequence of the antibody is shown in SEQ ID NO: 1, and the heavy chain amino acid sequence is shown as SEQ ID NO: 2, respectively.

Further, the present invention provides an encoding nucleotide encoding an anti-tau antibody.

Still further, the present invention provides a recombinant expression vector comprising the above-described encoding nucleotide.

Still further, the present invention also provides a hybridoma cell capable of secreting an anti-tau antibody.

The invention also provides the use of an anti-tau antibody in the preparation of a medicament for the treatment of a hyperphosphorylated tau-associated neurodegenerative disease.

Further, the light chain amino acid sequence of the anti-tau antibody is as set forth in SEQ ID NO: 1, and the heavy chain amino acid sequence is shown as SEQ ID NO: 2, respectively.

Further, the neurodegenerative disease is alzheimer disease, traumatic brain injury.

Still further, the medicament further comprises a pharmaceutically acceptable carrier.

The carrier is preferably excipient and stabilizer.

Advantageous effects

The anti-tau antibody of the present invention can specifically bind to phosphorylated tau, and can significantly improve the symptoms of neurodegenerative diseases related to hyperphosphorylated tau, such as alzheimer's disease.

The invention provides a new effective way for preventing and/or treating hyperphosphorylation tau protein related neurodegenerative diseases such as Alzheimer disease, traumatic brain injury and the like.

Drawings

FIG. 1: serum titers of mice after PHF-tau immunization.

FIG. 2: and (3) detecting the anti-tau antibody specificity western blot, wherein PHF-tau is phosphorylated tau protein, and tau is non-phosphorylated tau protein.

FIG. 3: affinity of antibody E2 for PHF-tau protein.

FIG. 4: therapeutic effect of monoclonal antibody E2 on alzheimer's disease mice.

Detailed Description

The present invention is described in more detail below to facilitate an understanding of the present invention.

It should be understood that the terms or words used in the specification and claims should not be construed as having meanings defined in dictionaries, but should be interpreted as having meanings that are consistent with their meanings in the context of the present invention on the basis of the following principles: the concept of terms may be defined appropriately by the inventors for the best explanation of the invention.

The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, such as those described in Sambrook et al, molecular cloning, in laboratory manuals, or according to conditions recommended by the manufacturer.

Example 1: preparation of anti-tau antibody hybridoma

In order to obtain an anti-tau antibody capable of specifically binding to tau which is a pathological change in a nervous system disease such as traumatic brain injury and Alzheimer's disease, PHF-tau which is closely related to a disease state is selected as an antigen to prepare an anti-tau antibody.

The PHF-tau protein is purified by a known method, i.e.selecting dead cortical tissue from Alzheimer's patients, homogenizing 5mg of frontal cortex in 8-10 volumes of cold buffer H (10 nM Tris, 800mM NaCl, 1mM EGTA and 10% sucrose, pH 7.4) using a tissue homogenizer at 1000rpm, and centrifuging the homogenized material at 27000g for 20 minutes in a high speed centrifuge. The pellet was discarded and the supernatant adjusted to a final concentration of 1% (W/V) N-lauroylsarcosine and 1% (V/V) 2-mercaptoethanol and incubated at 37 ℃ for 2 hours. The supernatant was then centrifuged at 108000g in a super speed centrifuge at 20 ℃ for 35 minutes, the pellet was carefully washed in PBS and suspended in PBS, the supernatant was centrifuged a second time as described, and the final pellet was solubilized to obtain purified PHF-tau protein, aliquoted and frozen at-80 ℃.

Immunization of mice

6 female BALB/c mice in 6-8 weeks are taken, the picric acid is used for staining and marking, and blood is taken after tail breaking to prepare the basic serum.

First immunization:

PHF-tau protein solution (0.4 mg/ml) was mixed with equal volume of Freund's complete adjuvant, repeatedly whipped and mixed until complete emulsification, and injected intraperitoneally at a dose of 200. mu.l per mouse.

And (3) boosting immunity:

after 2 weeks of initial immunization, PHF-tau protein solution was mixed with an equal volume of Freund's incomplete adjuvant, repeatedly whipped and mixed until complete emulsification, and injected intraperitoneally at a dose of 100. mu.l per mouse.

Impact immunization:

after 8 cumulative immunizations, a booster immunization with PHF-tau was performed at a dose of 25. mu.g/mouse by intrasplenic injection. The method comprises the following steps: the mice were anesthetized with ether, placed on the right side of the table, the left flank, i.e., part of the dorsal part, was exposed, sterilized with alcohol, the abdominal skin was cut open to expose the peritoneum, the spleen was found through the peritoneum, a PHF-tau protein solution was slowly injected into the spleen through a needle inserted through the peritoneal membrane extension shaft with a 1ml syringe, the incision was closed after the injection was completed, and the spleen cells were taken three days later for cell fusion.

Preparation of hybridoma cells:

passage expanding culture of myeloma cells (SP2/0), during fusion, gently blowing the cells from the bottle wall by using a dropper, collecting the cells in a centrifuge tube, centrifuging at 1000rpm for 5-10min, discarding the supernatant, adding 30ml of 1640 culture medium, centrifuging and washing once by the same method, then resuspending the cells in 10ml of 1640 culture medium, mixing uniformly, taking myeloma cell suspension, and counting the cells for later use.

Taking immunized BALB/c mice, removing eyeballs for bloodletting, separating serum to serve as positive control serum during antibody detection, taking spleens of the mice through conventional operation, removing surrounding connective tissues, placing the spleens in a flat dish containing incomplete culture medium, and lightly pressing the spleens on a nylon net to obtain a splenocyte single cell suspension. Harvesting spleen cell suspension, centrifuging at 1000rpm for 5-10min, centrifuging and washing with incomplete 1640 culture medium for 2 times, then re-suspending the cells in 10ml incomplete culture medium, mixing well, and taking the suspension for standby after living cell counting.

Feeder cells were prepared and cultured 1 day before cell fusion.

1 × 10⁸Spleen cells and 2 × 10⁷Mixing myeloma cells SP2/0 in a centrifuge tube, adding 1640 culture medium to 30ml, mixing, centrifuging at 1000rpm for 5-10min, sucking the supernatant to the greatest extent, tapping the bottom of the fusion tube to loosen and homogenize the precipitated cells, preheating in water bath, adding 1ml PEG preheated to 37 ℃ in 1min with a 1ml suction tube, gently shaking while adding, standing for 1-2min, and slowly adding 20-30m in 2-3min with a 10ml suction tubel incomplete 1640 medium preheated to 37 ℃ until reaction is stopped, centrifuging at 1000rpm for 5min, discarding supernatant, adding 40ml 2 × HAT medium, gently sucking precipitated cells, suspending and mixing, inoculating to 96-well culture plate containing feeder cells in the previous day, culturing, placing the culture plate at 37 ℃ and 5% CO for 0.1ml per well, and culturing₂Culturing in incubator, changing out 2 × HAT culture medium to 1/2 culture medium after 5 days, changing out 2 × HAT culture medium to 1/2 culture medium every 3 days, changing out HT culture medium after 14 days, and using normal complete culture medium after 21 days.

Positive clone screening and subcloning

Observing the growth condition of the hybridoma cells, sucking out culture supernatant when the hybridoma cells grow to a bottom area of a hole of more than 1/5, detecting positive clones by a direct ELISA method, namely screening the positive clones by a PHF-tau protein coated 96-well plate and horseradish peroxidase conjugated rabbit anti-mouse IgG, and subcloning the screened positive clones by a limiting dilution method.

Preparation and purification of monoclonal antibodies

The monoclonal antibody is prepared and obtained by BALB/c mouse tail experimental animals by using a conventional monoclonal antibody preparation and purification method.

The specificity of the monoclonal antibody was determined by using Western blot.

Determination of affinity constant by competitive binding of PHF-tau antibody monoclonal antibody was first incubated with different concentrations of PHF-tau protein, initial concentration of monoclonal antibody in reaction system was 400ng/ml (2.6 × 10)^-12mol/l), diluting the initial concentration of the PHF-tau protein from 100 mu g/ml to the lower multiple to form 7 reaction systems, adding the reaction solution into an ELISA plate coated with the PHF-tau antigen after incubation, washing the plate for 3 times after incubation, adding 100 mu l of HRP-labeled rabbit anti-rat kan solution into each hole, reacting for 1h at 37 ℃, washing the plate for 3 times, adding TMB substrate for developing for 10min, adding a stop solution, measuring OD450 values, and calculating the antigen binding rate of each reaction system to calculate the affinity constant.

The results show that:

the PHF-tau protein is injected into an immune mouse in an abdominal cavity, the serum antibody titer after primary immunization can reach 1:4000, the antibody titer after three times of immunization can reach 1:8000, and the IgG antibody titer after nine times of immunization can reach 1:528000, which shows that the PHF-tau protein immune mouse obtains good immune effect, and the result is shown in figure 1.

After positive clone screening, 5 hybridoma cell strains capable of stably secreting anti-PHF-tau protein antibodies are obtained through co-screening and are respectively named as A2, A4, B8, C6 and E2, the 5 hybridoma cell strains can stably secrete the antibodies after continuous subculture for 2 months, and the titer of culture supernatants is greater than 1: 1000.

The detection shows that E2, B8 and A2 in the monoclonal antibodies secreted by the 5 hybridoma cell lines have better PHF-tau protein binding specificity, and C6 and A4 have poorer PHF-tau protein binding capacity, and the results are shown in figure 2.

The affinity constant determination of the antibodies E2, B8 and A2 shows that the affinity constant is 2-8 × 10^-8Between mol/l, wherein the affinity constant of the monoclonal antibody E2 is 7.89 × 10^-8mol/l (R2=0.9938), indicating that an antigen excess is required to form competitive inhibition of the antibody, the results are shown in figure 3.

Example 2: construction of Alzheimer's disease model

To investigate the effect of anti-tau antibodies on hyperphosphorylated tau under physiological conditions, a mouse model of alzheimer's disease was constructed.

The mouse is anesthetized by intraperitoneal injection with 2% sodium pentobarbital (40 mg/kg), fixed on a brain stereotaxic apparatus, incised and exposed to the skull in the middle of the vertex of the cranium, positioning the basal nucleus according to the stereogram for positioning the skull of the mouse, drilling the dead bone by a dental drill, and slowly injecting A β into the dead bone by a 1-microliter miniature injector needle within 5min_25-35Mixing with 1 μ l of amanitic acid (IBO) (purchased from Sigma), holding for 10min, sealing the needle hole with local soft tissue, suturing skin, and administering after operationPenicillin sodium salt is injected into muscle of 5 thousands of u, 1 time each day, and the injection is continuously carried out for 3 days. And (3) successfully constructing a mouse Alzheimer disease model. In addition, the blank group was given a one-time injection of 1 μ l of sterilized physiological saline to the basal nuclei of the mice under localization.

After the mouse model is successfully constructed, 3 mice are respectively selected to carry out determination and analysis on the cumulative optical density and the optical density of total tau protein and phosphorylated tau protein in the brain through immunohistochemistry.

The results show that: the cumulative optical densities of the total tau protein and the phosphorylated tau protein in the model group are respectively 0.385 +/-0.015 and 0.339 +/-0.018, and the cumulative optical densities of the total tau protein and the phosphorylated tau protein in the blank group are respectively 0.363 +/-0.011 and 0.114 +/-0.023, namely the cumulative optical densities of the total tau protein and the phosphorylated tau protein in the model group are obviously higher than those of the blank group, which indicates that the model is successfully constructed.

Example 3: effect of anti-tau antibodies on phosphorylated tau protein in Alzheimer's disease mice

Based on the results of the previous examples, monoclonal antibodies E2, B8 and a2 were selected to investigate their effect on reducing phosphorylated tau protein in alzheimer's disease mice.

An alzheimer disease mouse model and a blank group mouse model were constructed as in example 2, 40 alzheimer disease model mice were selected as an experimental group, randomly divided into 4 groups of 10 mice each, and administered with physiological saline, monoclonal antibodies E2, B8 and a2 (500 μ g/mouse) by tail vein injection, respectively; 10 normal mice and 10 mice of the blank group model were selected and given physiological saline (500. mu.g/mouse) by tail vein injection, respectively, and the experimental mice were freely fed and drunk water. The experimental, normal and blank groups were administered every other day after the first administration, 5 times 5 days after administration, 6 times again, mice were sacrificed 50 days after the start of the experiment, brains were removed and hippocampal tissues were isolated.

Abnormal phosphorylation of tau protein in mouse hippocampal tissue is detected by western blot, namely protein content determination is carried out after total protein of hippocampal tissue is extracted, SDS-PAGE gel electrophoresis is carried out on quantitative protein, immunoreaction, chemiluminescence, development and fixation are carried out after the electrophoresis is finished and membrane transfer is carried out, and molecular weight and net light density value of a target band are analyzed by utilizing gel image analysis software and the content of phosphorylated tau protein is determined.

The results are shown in table 1:

group of

Is normal

Blank group

Model set

E2

B8

A2

Phosphorylated tau protein content

38.52±8.16

39.98±6.53

102.32±10.94∞

55.49±4.46※

89.91±10.49

92.78±11.93

In comparison with the normal group,^∞p is less than 0.01; in comparison to the set of models,^※P＜0.01

that is, the results show that, compared with the monoclonal antibodies B2 and a2, the monoclonal antibody E2 has an unexpectedly good effect of reducing hyperphosphorylation of tau protein in alzheimer mice, and can be used for preventing and/or treating neurodegenerative diseases related to hyperphosphorylation of tau protein, such as alzheimer disease and traumatic brain injury.

Example 4: effect of anti-tau antibodies on the behavioural Studies of mouse models of Alzheimer's disease

A mouse model of Alzheimer's disease was constructed as in example 2, and 20 model mice were selected and divided into 2 groups, one group was administered with monoclonal antibody E2 (500. mu.g/mouse), and one group was administered with physiological saline (500. mu.g/mouse), and a morris water maze experiment was performed.

The water maze experiment was: the water maze is a circular water pool with the diameter of 100cm and the height of 50cm, the water depth is 30cm, the water temperature is kept at 26 +/-1 ℃, four water inlet points of south, east, west and north are marked on the wall of the water pool, the water pool is divided into four quadrants which are respectively called as a first quadrant, a second quadrant, a third quadrant and a fourth quadrant, a circular transparent platform with the diameter of 9cm and the height of 29cm is placed in the center of the first quadrant, the top of the platform is 1cm lower than the water surface, a camera connected with a computer is arranged above the maze, the motion trail of a mouse is synchronously recorded, and a reference object outside the maze is kept unchanged during training.

Model and normal mice were dosed according to example 3, and the morris water maze experiment was started 5 days after the completion of the last dose. The method comprises the following steps of enabling a mouse to swim freely for 2min in the 1 st time of the first day, starting formal training in the 2 nd time of the first day, then training for 2 times every day for 4 days, selecting a water inlet point during training, putting the mouse into water facing to the pool wall, observing and recording a route map of the mouse finding and climbing a platform, required time and swimming speed, leading the mouse to the platform if the mouse cannot find the platform within 120s, and enabling the mouse to stay on the platform for 20 seconds, wherein data collection and processing are completed by a Morris maze image son-east monitoring processing system.

The experimental results show that: the monoclonal antibody E2 experimental group finds that the number of mice of the platform is gradually increased along with the number of test days, while the physiological saline experimental group finds that the number of mice of the platform does not show the trend of increasing along with the number of test days, the result is shown in figure 4, the results show that the learning and memory capacity of the mice of the monoclonal antibody E2 experimental group is improved compared with that of the physiological saline experimental group, and the monoclonal antibody E2 can be used for preventing and or treating neurodegenerative diseases related to tau protein hyperphosphorylation, such as Alzheimer disease, traumatic brain injury and the like.

Example 5: effect of anti-tau antibodies on the behavioural Studies of mouse models of Alzheimer's disease

The sequencing result of the monoclonal antibody by the hybridoma E2 handed over from Shanghai to Shanghai works shows that the light chain amino acid sequence of the monoclonal antibody E2 is shown as SEQ ID NO: 1, and the heavy chain amino acid sequence is shown as SEQ ID NO: 2, respectively.

Alternatively, monoclonal antibody E2 can be humanized by antibody humanization techniques conventional in the art, so that it can be more suitably used for preventing and/or treating human neurodegenerative diseases related to tau hyperphosphorylation, such as Alzheimer's disease, traumatic brain injury, etc.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Sequence listing

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<120> anti-Tau antibody and use thereof in treating alzheimer's disease, traumatic brain injury

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Claims (8)

1. An anti-tau antibody, or an antigen-binding portion thereof, characterized by: the antibody is capable of specifically binding phosphorylated tau protein, and the light chain amino acid sequence of the antibody is as set forth in SEQ ID NO: 1, the heavy chain amino acid sequence is shown as SEQ ID NO: 2, respectively.

2. An encoding nucleotide, characterized in that: the coding nucleotides encode the antibody or antigen-binding portion thereof of claim 1.

3. A recombinant expression vector characterized by: the vector comprises the coding nucleotide of claim 2.

4. A hybridoma cell, characterized by: the hybridoma cells comprising the recombinant expression vector of claim 3.

5. Use of an anti-tau antibody for the manufacture of a medicament for the treatment of a neurodegenerative disease associated with hyperphosphorylated tau, characterized in that: the light chain amino acid sequence of the anti-tau antibody is shown as SEQ ID NO: 1, and the heavy chain amino acid sequence is shown as SEQ ID NO: 2, respectively.

6. Use according to claim 5, characterized in that: the neurodegenerative disease is Alzheimer disease and traumatic brain injury.

7. Use according to claim 5, characterized in that: the medicament also comprises a pharmaceutically acceptable carrier.

8. Use according to claim 7, characterized in that: the carrier is excipient and stabilizer.

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