CN110028583B - Anti-Tau antibody and its application in the treatment of Alzheimer's disease and traumatic brain injury - Google Patents

Abstract

The object of the present invention is to provide an anti-Tau antibody and its application in the treatment of Alzheimer's disease and traumatic brain injury, the antibody can specifically bind to tau protein, especially to hyperphosphorylated tau protein; By administering the antibody, it can delay the expression of abnormally phosphorylated tau protein in a mouse model of Alzheimer's disease, and can prevent or treat tau protein-related neurodegeneration such as Alzheimer's disease and traumatic brain injury. effect of disease.

Description

Anti-Tau antibody and its application in the treatment of Alzheimer's disease and traumatic brain injury

technical field

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

Background technique

蛋白是一种微管相关蛋白，其可被分为四个区域，即N末端突出区、富含脯氨酸区、微管结合区、和C末端，Tau蛋白正常情况下与微管结合，但在被糖原合成酶激酶3β（glycogen synthase kinase-3β,GSK-3β）催化发生磷酸化后可以从微管脱落，引起微管稳定性下降或解聚；Tau蛋白磷酸化和去磷酸化之间的平衡是维持神经元微管稳定性、保证细胞完成线粒体等细胞器在轴浆中正常运输的前提。

The protein is a microtubule-associated protein, which can be divided into four regions, namely the N-terminal protruding region, the proline-rich region, the microtubule-binding region, and the C-terminus. Tau protein normally binds to microtubules, However, after being phosphorylated by glycogen synthase kinase-3β (glycogen synthase kinase-3β, GSK-3β), it can be detached from microtubules, resulting in decreased microtubule stability or depolymerization; Tau protein phosphorylation and dephosphorylation The balance between them is the premise to maintain the stability of neuronal microtubules and ensure that cells complete the normal transport of mitochondria and other organelles in the axoplasm.

Traumatic brain injury (TBI) is a persistent brain injury caused by accidental or non-accidental injury. Clinical manifestations include loss of consciousness, seizures, special postures, syncope, and hemiplegia. In the study of traumatic brain injury, it was found that hyperphosphorylation of tau protein can cause microtubule depolymerization, abnormal mitochondrial distribution and mitochondrial dysfunction, and eventually lead to neuronal degeneration or apoptosis; cis-phosphorylated tau protein is a neuron after brain injury. An early driver of degenerative disease that promotes tau pathology.

Alzheimer's disease (AD) mostly begins in old age, with insidious onset, slow and long-term irreversible course, and is a group of primary degenerative brain diseases with unknown etiology. The pathological features are senile plaques formed by Aβ deposition, neurofibrillary tangles formed by phosphorylated Tau protein deposition, and the loss of a large number of cholinergic neurons. At present, studies have shown that anti-tau protein antibodies can be used to treat or adjuvant Alzheimer's disease, which can play a role in alleviating disease progression.

Based on the above knowledge, the use of specific anti-tau protein antibodies to treat diseases associated with abnormal tau protein is considered to be a feasible measure.

SUMMARY OF THE INVENTION

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

Further, the present invention provides an encoding nucleotide, which can encode an anti-tau protein antibody.

Further, the present invention also provides a recombinant expression vector comprising the above-mentioned encoding nucleotides.

Furthermore, the present invention also provides a hybridoma cell capable of secreting anti-tau protein antibody.

The present invention also provides the use of the anti-tau protein antibody in the preparation of a medicament for treating hyperphosphorylated tau protein-related neurodegenerative diseases.

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

Further, the neurodegenerative disease is Alzheimer's disease, traumatic brain injury.

Further, the medicament also includes a pharmaceutically acceptable carrier.

The carrier is preferably an excipient and a stabilizer.

beneficial effect

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

The invention provides a new and effective way for the prevention and or treatment of neurodegenerative diseases related to hyperphosphorylated tau protein such as Alzheimer's disease and traumatic brain injury.

Description of drawings

Figure 1: Mouse serum titers after PHF-tau immunization.

Figure 2: Anti-tau antibody-specific western blot detection results, in which PHF-tau is phosphorylated tau protein, and tau is non-phosphorylated tau protein.

Figure 3: Affinity of antibody E2 to PHF-tau protein.

Figure 4: Therapeutic effect of monoclonal antibody E2 on Alzheimer's disease mice.

Detailed ways

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

It should be understood that terms or words used in the specification and claims should not be understood to have the meanings defined in the dictionary, but should be understood to have the meanings consistent with their meanings in the context of the present invention on the basis of the following principles Meaning: Concepts of terms may be appropriately defined by the inventor for the best description of the invention.

The experimental methods of unreceipted specific conditions in the following examples are usually in accordance with conventional conditions such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual, or in accordance with the conditions suggested by the manufacturer.

Example 1: Preparation of anti-tau protein antibody hybridomas

In order to obtain an anti-tau protein antibody that can specifically bind to tau protein diseased in neurological diseases such as traumatic brain injury and Alzheimer's disease, PHF-tau, which is closely related to the disease state, is selected as an antigen to prepare an anti-tau protein antibody.

The PHF-tau protein is prepared and purified by a known method, that is, selecting postmortem cortical tissue of Alzheimer's disease patients, using a tissue homogenizer to mix 5 mg of frontal cortex in 8-10 times the volume of cold buffer H at 1000 rpm. (10 nM Tris, 800 mM NaCl, 1 mM EGTA, and 10% sucrose, pH 7.4), and the homogenized material was centrifuged at 27000 g for 20 minutes in a high-speed centrifuge. Discard the pellet and adjust the supernatant to a final concentration of 1% (w/v) N-lauroylsarcosine and 1% (v/v) 2-mercaptoethanol and incubate at 37 °C for 2 h . The supernatant was then centrifuged in an ultracentrifuge at 108,000 g for 35 minutes at 20°C, the pellet was carefully washed in PBS and suspended in PBS, the supernatant was centrifuged a second time as described, and the final The pellets were dissolved to obtain purified PHF-tau protein, aliquoted and frozen at -80°C.

mouse immunization

Six 6-8-week-old female BALB/c mice were selected, marked with picric acid, and blood was collected from tails to prepare basal serum.

First Immunization:

The PHF-tau protein solution (0.4 mg/ml) was mixed with an equal volume of Freund's complete adjuvant, and the mixture was repeatedly pipetted and mixed until it was completely emulsified. The dose of 200 μl per mouse was injected intraperitoneally.

Boost your immunity:

Every 2 weeks after the initial immunization, the PHF-tau protein solution was mixed with an equal volume of incomplete Freund's adjuvant, and the mixture was repeatedly pipetted and mixed until completely emulsified, and injected intraperitoneally at a dose of 100 μl per mouse.

Shock Immunity:

After accumulative immunization for 8 times, booster immunization with PHF-tau protein was carried out by intrasplenic injection, and the dose was 25 μg/mouse. The method is: first anesthetize the mouse with ether, lay it on the right side of the table, expose the left flank, that is, part of the back, after disinfection with alcohol, cut the abdominal skin aseptically to expose the peritoneum, find the spleen through the peritoneum, and use 1ml The PHF-tau protein solution was slowly injected into the spleen by a syringe through the peritoneal extension shaft. After the injection, the incision was closed, and the spleen cells were taken for cell fusion three days later.

Hybridoma cell preparation:

Myeloma cells (SP2/0) were subcultured and expanded. When fusion, the cells were gently blown down from the bottle wall with a dropper, collected in a centrifuge tube, centrifuged at 1000 rpm for 5-10 min, discarded the supernatant, and added 30 ml of 1640 to culture. Then, the cells were resuspended in 10 ml of 1640 medium, mixed well, and the myeloma cell suspension was taken, and the cells were counted for later use.

The immunized BALB/c mice were taken, the eyeballs were removed for bloodletting, and the serum was separated as a positive control serum for antibody detection. The spleen of the mouse was removed by routine operations and the surrounding connective tissue was stripped, and the spleen was placed in an incomplete medium. In a dish, gently squeeze the spleen on a nylon mesh to obtain a single-cell suspension of splenocytes. The spleen cell suspension was harvested, centrifuged at 1000 rpm for 5-10 min, centrifuged and washed twice with incomplete 1640 medium, and then the cells were resuspended in 10 ml of incomplete medium and mixed.

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

Mix 1×10 ⁸ splenocytes and 2×10 ⁷ myeloma cells SP2/0 in a centrifuge tube, add 1640 medium to 30ml, mix well, centrifuge at 1000rpm for 5-10min, and aspirate the supernatant as much as possible. , tap the bottom of the fusion tube to loosen the precipitated cells, preheat in a water bath, add 1ml of PEG preheated to 37°C dropwise with a 1ml pipette within 1min, shake gently while adding, let stand for 1-2min, use 10ml Pipette slowly drop 20-30ml of incomplete 1640 medium preheated to 37°C within 2-3min to stop the reaction. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add 40 ml of 2 × HAT medium, gently pipette the pelleted cells to suspend and mix them, and inoculate them in a 96-well culture plate that has been added with feeder cells the day before. ml, then place the culture plate in a 37°C, 5% _CO2 incubator, and after 5 days, replace 1/2 medium with 2×HAT medium, and then replace 1/2 medium with 2×HAT medium every 3 days. 2 medium, replace HAT medium with HT medium after 14 days, and use common complete medium after 21 days.

Positive clone screening and subcloning

Observe the growth of hybridoma cells, and when they grow to more than 1/5 of the bottom area of the well, aspirate the culture supernatant and detect positive clones by direct ELISA method, that is, a 96-well plate coated with PHF-tau protein and horseradish peroxidase Conjugated rabbit anti-mouse IgG was used to screen positive clones, and the screened positive clones were subcloned by limiting dilution method.

Preparation and purification of monoclonal antibodies

Monoclonal antibodies were prepared from BALB/c mouse tail experimental animals using conventional monoclonal antibody preparation and purification methods.

The specificity of monoclonal antibodies was determined by Western blot.

The affinity constant was determined by ^PHF -tau protein competition binding antibody: the monoclonal antibody was first incubated with different concentrations of PHF-tau protein. The initial concentration of PHF-tau protein was diluted down from 100μg/ml to form 7 reaction systems. After incubation, the reaction solution was added to the ELISA plate coated with PHF-tau antigen, 100μl per well, and washed after incubation. Plate 3 times, add 100 μl HRP-labeled rabbit anti-mouse Erkang solution to each well, react at 37°C for 1 h, wash the plate 3 times, add TMB substrate for color development for 10 min, add stop solution, measure OD450 value to calculate the antigen binding of each reaction system Rate inferred affinity constant.

The results show:

When PHF-tau protein was injected intraperitoneally into immunized mice, the serum antibody titer could reach 1:4000 after the primary immunization, the antibody titer could reach 1:8000 after the third immunization, and the IgG antibody titer could reach 1:1 after the nine immunizations. 528,000, indicating that the mice immunized with PHF-tau protein obtained a good immune effect, and the results are shown in Figure 1.

After screening of positive clones, a total of 5 hybridoma cell lines that can stably secrete anti-PHF-tau protein antibodies were screened and named as A2, A4, B8, C6 and E2. The above 5 hybridoma cell lines were continuously subcultured. After 2 months, the antibodies were stably secreted, and the titers of the culture supernatants were all greater than 1:1000.

检测显示上述5株杂交瘤细胞株分泌的单克隆抗体中，E2、B8和A2具有较好的PHF-tau蛋白结合特异性，而C6和A4特异性结合PHF-tau蛋白的能力较差，结果见图2。

The detection showed that among the monoclonal antibodies secreted by the above 5 hybridoma cell lines, E2, B8 and A2 had better PHF-tau binding specificity, while C6 and A4 had poor specific binding ability to PHF-tau protein. See Figure 2.

The affinity constants of antibodies E2, B8 and A2 were determined, and the results showed that their affinity constants were between 2-8×10 ^-8 mol/l, and the affinity constant of monoclonal antibody E2 was 7.89×10 ^-8 mol/l (R2=0.9938), indicating that an excess of antigen is required to form a competitive inhibition of the antibody. The results are shown in Figure 3.

Example 2: Construction of Alzheimer's Disease Model

In order to study the effect of anti-tau protein antibody on hyperphosphorylated tau protein in physiological state, a mouse model of Alzheimer's disease was constructed.

小鼠用2%戊巴比妥钠（40mg/kg）腹腔注射麻醉后，固定于脑立体定位仪上，颅顶正中切开暴露至颅骨，根据小鼠颅脑定位立体图谱，对基底核定位，用牙科钻钻开枯骨，用1μl微型进样器注射针5min内缓慢注入Aβ_25-35和鹅膏蕈氨酸（IBO）（均购置于sigma公司）的混合液1μl，予以留针10min，注射完毕后以局部软组织封闭针孔，缝合皮肤，术后给予青霉素钠盐5万u肌肉注射，1次每天，连续注射3天。构建成功小鼠阿尔茨海默病模型。另外，空白组在定位下给予小鼠基底核一次性注射1μl灭菌生理盐水。

The mice were anesthetized by intraperitoneal injection of 2% pentobarbital sodium (40 mg/kg), and then fixed on a stereotaxic device. , drill the dead bone with a dental drill, and slowly inject 1 μl of the mixture of Aβ _25-35 and amantine (IBO) (both purchased from sigma) with a 1 μl micro-injector needle within 5 minutes, and retain the needle for 10 minutes. After completion, the needle hole was closed with local soft tissue, the skin was sutured, and intramuscular injection of penicillin sodium salt of 50,000 u was given after operation, once a day for 3 consecutive days. A successful mouse model of Alzheimer's disease was constructed. In addition, the blank group was given a one-time injection of 1 μl of sterile saline into the basal ganglia of mice under localization.

After the successful establishment of the mouse model, 3 mice were selected to measure and analyze the cumulative optical density and optical density of total tau protein and phosphorylated tau protein in the brain by immunohistochemistry.

The results showed that the cumulative optical densities of total tau protein and phosphorylated tau protein in the model group were 0.385±0.015, 0.339±0.018, respectively, and the cumulative optical densities of total tau protein and phosphorylated tau protein in the blank group were 0.363±0.011, 0.114±0.114± 0.023, that is, the cumulative optical density of total tau protein and phosphorylated tau protein in the model group was significantly higher than that in the blank group, indicating that the model was successfully constructed.

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

Based on the results of the preceding examples, monoclonal antibodies E2, B8 and A2 were selected to study their effect on reducing phosphorylated tau protein in Alzheimer's disease mice.

The Alzheimer's disease mouse model and the blank group mouse model were constructed as in Example 2, and 40 Alzheimer's disease model mice were selected as the experimental group and randomly divided into 4 groups of 10 mice, which were injected through the tail vein respectively. Administer normal saline, monoclonal antibodies E2, B8 and A2 (500 μg/mouse); select 10 normal mice and 10 blank group model mice to receive normal saline (500 μg/mouse) by tail vein injection, respectively. The rats had free access to food and water. The experimental group, the normal group and the blank group were given the medicine every other day after the first administration, 5 times after administration, 5 days apart, and then 6 times, the mice were sacrificed on the 50th day after the start of the experiment, and the brains were taken out and isolated hippocampal tissue.

The abnormal phosphorylation of tau protein in the hippocampus of mice was detected by western blot, that is, the total protein of the hippocampus was extracted and then the protein content was determined, and the quantitative protein was subjected to SDS-PAGE gel electrophoresis. , developing and fixing, using gel image analysis software to analyze the molecular weight and net optical density of the target band and determine the content of phosphorylated tau protein.

The results are shown in Table 1:

group normal blank group model group 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

Compared with the normal group, ^∞ P＜0.01; compared with the model group, ^※ P＜0.01

Namely, the results show that, relative to the monoclonal antibodies B2 and A2, the monoclonal antibody E2 is surprisingly effective in reducing hyperphosphorylated tau protein in Alzheimer's disease mice, which can be used for prevention and/or Treatment of neurodegenerative diseases related to hyperphosphorylation of tau protein such as Alzheimer's disease and traumatic brain injury.

Example 4: The effect of anti-tau antibody on the behavior of Alzheimer's disease mouse model

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 given monoclonal antibody E2 (500 μg/mouse), and the other group was given normal saline (500 μg/mouse). The morris water maze experiment was performed.

The water maze experiment is as follows: the water maze is a circular pool with a diameter of 100 cm and a height of 50 cm, the water depth is 30 cm, and the water temperature is kept at 26±1 °C. Called the first, second, third, and fourth quadrants, a circular transparent platform with a diameter of 9cm and a height of 29cm is placed in the middle of the first quadrant. Mice locomotion trajectories, the external reference in the maze remained unchanged during training.

Model mice and normal mice were administered with reference to Example 3, and the morris water maze test was started on the 5th day after the last administration. The mice were allowed to swim freely for 2 minutes for the first time on the first day, and the formal training was started for the second time on the first day. After that, the mice were trained twice a day for a total of 4 days. During the training, a water entry point was selected, and the mice were placed in the water facing the pool wall. Observe and record the route map, required time and swimming speed of the mouse to find and climb on the platform. If the mouse fails to find the platform within 120s, it needs to be led to the platform and let it stay on the platform for 20 seconds. Data collection And processing is done by Morris Maze Image Sub-East Surveillance Processing System.

The experimental results show that: the monoclonal antibody E2 experimental group found that the number of mice on the platform gradually increased with the test days, while the saline experimental group found that the number of mice on the platform did not show a trend of increasing with the test days. The results are shown in Figure 4 The above results show that the learning and memory ability of the mice in the monoclonal antibody E2 experimental group is improved compared with the saline experimental group, and the monoclonal antibody E2 can be used to prevent and or treat Alzheimer's disease, traumatic brain injury and other tau protein hyperphosphorylation related neurodegenerative diseases.

Example 5: The effect of anti-tau antibody on the behavior of Alzheimer's disease mouse model

The hybridoma E2 was handed over to Shanghai Sangong for sequencing of the monoclonal antibody. The sequencing results showed that the amino acid sequence of the light chain of the monoclonal antibody E2 was shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain was shown in SEQ ID NO: 2.

Alternatively, the monoclonal antibody E2 can be humanized by conventional antibody humanization technology in the art, so that it can be more suitable for preventing and or treating Alzheimer's disease and traumatic brain injury in humans. Neurodegenerative diseases associated with tau hyperphosphorylation.

The above is only the preferred embodiment of the present invention, it should be pointed out that: for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made, and these improvements and modifications are also It should be regarded as the protection scope of the present invention.

sequence listing

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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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