Patent ID: 12258386

SPECIFIC MODELS FOR CARRYING OUT THE PRESENT DISCLOSURE

The present disclosure discloses a humanized anti-Aβ monoclonal antibody and use thereof, and those skilled in the art can fulfill them by learning the contents of the present disclosure and appropriately improving the process parameters. In particular, it should be pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present disclosure. The methods and use of the present disclosure have been described through the preferred examples, and it is obvious that those skilled in the art can make changes or appropriate alternations and combinations to the methods and use described herein without departing from the content, spirit and scope of the present disclosure, so as to achieve and apply the technology of the present disclosure.

The humanized anti-Aβ monoclonal antibodies provided by the present disclosure and the raw materials and reagents used in the use were all commercially available.

The present disclosure is further illustrated in conjunction with the following examples:

Example 1: Preparation of Aβ Antigen and Positive Control Antibody

Preparation of Aβ Monomer and Polymer Mixture

Aβ1-42, Aβ1-16, and Aβ14-29polypeptides were synthesized by Ji'er Biochemical (Shanghai) Co., Ltd.

The amino acid sequence of Aβ1-42polypeptide was:(SEQ ID: 35)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA,the amino acid sequence of Aβ1-16polypeptide was:(SEQ ID: 36)DAEFRHDSGYEVHHQK,andthe amino acid sequence of Aβ14-29polypeptide was:(SEQ ID: 37)HQKLVFFAEDVGSNKGA.

Preparation method of Aβ1-42monomer (abbreviated as Aβ monomer): 1 ml of hexafluoro isopropanol (HFIP) was added to 1 mg of Aβ1-42polypeptide dry powder, subjected to vortex and shaking for 1 min, and sonicated in a water bath for 1-5 min until the dissolution was completed; placed in 37° C., 200 rpm shaking incubator and shaken for 1.5 hours; a vacuum rotary dryer was used to volatilize hexafluoro isopropanol; 192 μl of anhydrous dimethyl sulfoxide (DMSO) was added to the dried Aβ1-42polypeptide to dissolve the polypeptide, then added with 27 μl of 20×PBS solution, 54 μl of 2% SDS, 267 μl of ddH2O, mixed well, subpackaged in small amounts, stored in a refrigerator at −80° C., which was the Aβ1-42monomer; and the detection thereof was performed by SDS-PAGE and WB (hybridization detection was performed by using the positive antibody 066-P02 that specifically recognizes Aβ1-42) (seeFIG.1).

Preparation method of Aβ1-42polymer mixture (abbreviated as Aβ polymer mixture): 1 ml of hexafluoro isopropanol (HFIP) was added to 1 mg of Aβ1-42polypeptide dry powder, subjected to vortex and shaking for 1 min, sonicated in a water bath for 1-5 min until the dissolution was completed; placed in 37° C., 200 rpm shaking incubator and shaken for 1.5 h; a vacuum rotary dryer was used to volatilize hexafluoro isopropanol; 192 μl of DMSO was added to the dry Aβ1-42polypeptide to dissolve the polypeptide, then added with 27 μl of 20×PBS solution, 54 μl of 2% SDS, 267 μl of ddH2O, mixed well, and placed in a 37° C. water bath for 18-24 h; added with 1.62 ml of ddH2O, mixed well, and placed in a 37° C. water bath for 18-24 h; transferred into PBS by using 10 KDa ultrafiltration tube for buffer replacement, subpackaged in small amounts, stored in a refrigerator at −80° C., which was the Aβ1-42polymer mixture; and the detection thereof was performed by SDS-PAGE and WB (hybridization detection was performed using the positive antibody 066-P02 that specifically recognizes Aβ1-42) (seeFIG.1).

2. Construction of Positive Control Antibody Expression Vector

pGS003-hIgG1CH and pGS003-hIgKCL were separately selected as the expression vectors for constructing the heavy chain and the light chain of anti-human Aβ-positive antibodies (066-P01: Solanezumab, Eli lily; 066-P02: Aducanumab, Biogen); after the codon optimization of the amino acid sequences of the positive antibody variable regions, the positive antibody VH and VL genes were separately cloned into pGS003-hIgG1CH and pGS003-hIgKCL using restriction enzyme digestion method to obtain transient transfection expression vectors pGS003-066-P01VH-hIgG1CH, pGS003-066-P01VL-hIgKCL, pGS003-066-P02VH-hIgG1CH and pGS003-066-P02VL-hIgKCL of the heavy chain and the light chain of the positive antibody. The amino acid sequence of the heavy chain variable region of the positive antibody 066-P01 was as follows (SEQ ID: 38):

EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYSMSWVRQAPGKGLELVAQINSVGNSTYYPDTVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCASGDYWGQGTLVTVSS

The amino acid sequence of the light chain variable region of the positive antibody 066-P01 was as follows (SEQ ID: 39):

DVVMTQSPLSLPVTLGQPASISCRSSQSLIYSDGNAYLHWFLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGQGTKVEIK

The amino acid sequence of the heavy chain variable region of the positive antibody 066-P02 was as follows (SEQ ID: 40):

QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVIWFDGTKKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDVWGKGTTVTVSS

The amino acid sequence of the light chain variable region of the positive antibody 066-P02 was as follows (SEQ ID: 41):

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK
3. Expression by Transient TransfectionpGS003-066-P01VH-hIgG1CH and pGS003-066-P01VL-hIgKCL;pGS003-066-P02VH-hIgG1CH and pGS003-066-P02VL-hIgKCL were transiently expressed.

FreeStyle™ 293E cells were used for expression by transient transfection in Freestyle medium. Twenty-four hours before transfection, 30 ml of 293E cells were inoculated at 0.5×106cells/ml in a 125 ml conical flask, and cultured on a shaker at 130 rpm in a 37° C., 5% CO2incubator. During transfection, 60 μl of 293E Fectin was firstly taken and added to 1 ml of Opti-MEM, mixed well, and incubated at room temperature for 5 minutes; meanwhile, Total 30 μg plasmid DNA of transient transfection expression vectors (recombinant vectors) was dissolved in 1 ml of Opti-MEM. Then, the plasmid DNA and 293E Fectin were mixed thoroughly, with a total volume of 2 ml, incubated at room temperature for 15 minutes, and then all the mixture was added to the cell culture wells, mixed, and incubated on a shaker in a 37° C., 5% CO2incubator at 130 rpm for 7 days. The culture broth was centrifuged at a high speed and the supernatant was taken and subjected to vacuum filtration with a microporous membrane.

4. Purification of Protein

According to the operating method provided by the manufacturer, Protein A column (protein purification liquid chromatography system/AKTA Purifier 10, GE) and nickel column were used for purification to obtain purified positive antibodies 066-P01 and 066-P02. As shown inFIG.2.

Example 2: Preparation of Anti-Aβ Monoclonal Hybridoma

Immunization of BALB/c Mice

Aβ1-42polypeptide antigen and Freund's complete adjuvant were vortexed and mixed according to their doses, after emulsification was completed, first immunization was performed to 6-week-old BALB/c female mice. Each mouse was injected intraperitoneally with 200 μg of antigen, in total 3 groups of mice were immunized, 5 mice in each group. Two weeks after the first immunization, the mice were given second intraperitoneal immunization, in which Freund's incomplete adjuvant was used, while the dose of immune antigen was the same as the first immunization. After that, the mice were immunized intraperitoneally twice a month, and the adjuvant and antigen doses were the same as the second immunization.

After the first immunization, a small amount of blood was collected from mouse orbit and serum titer was tested every six weeks. After the serum titer reached 1:200000 or above by the indirect ELISA method, the mice used for fusion were subjected to booster immunization.

Preparation of Myeloma Cells for Fusion

Myeloma cells P3X63Ag8.653 used for fusion were resuscitated three weeks in advance, cultured in DMEM medium containing 1×8-azaguanine and 10% fetal bovine serum for two weeks, and cultured with DMEM containing 10% fetal bovine serum before one week of fusion, in which the density of P3X63Ag8.653 was maintained at 70% to 80% until the day of fusion.

Cell Fusion and HA Screening

Obtaining and preparation of spleen cells: 2 mice after booster immunization were taken, sacrificed after collection of immune serum, and soaked in 75% alcohol for 2-3 minutes. The skin and peritoneum on the abdomen side of the immunized mice were cut to expose spleen. The spleen was obtained by removing the surrounding tissues with scissor tip, ground with a grinding rod, and filtered through a cell sieve to prepare a single cell suspension. The supernatant was discarded after centrifugation.

Treatment before cell fusion: P3X63Ag8.653 in the culture flask was collected, centrifuged at 1000 rpm/5 min, then the supernatant was discarded, the cells were resuspended, and the live myeloma cells were counted. The spleen cell suspension was centrifuged to discard the supernatant, added with ACK lysate, incubated and centrifuged to discard supernatant to remove red blood cells, resuspended in DMED, and the viable spleen cells were counted.

Cell fusion: The cells were mixed at the ratio of spleen cells:P3X63Ag8.653=1:2, centrifuged at 2000 rpm/5 min to discard the supernatant, shaken to disperse cell pellets, added with 1 mg/ml Pronase at 400 μl/1×108spleen cells; after incubating for 15 seconds, 10 ml of fetal bovine serum was added to stop the reaction, electroporation solution (ECF) was supplemented to 50 ml, centrifuged at 2500 rpm for 5 minutes to discard the supernatant, resuspended with ECF and the viable cells were counted, and the spleen cell density was adjusted to 2×106/ml. The cell suspension with the well-adjusted density was added to an electrofusion tank, and an electroporator was run for cell fusion. After the fusion, the cell suspension was transferred from the fusion tank to ½ HA medium, allowed to stand for 3 hours and then cell plating was carried out.

HA medium selection: AT selection medium containing ½ HA, 1×penicillin-streptomycin, 20% fetal bovine serum and 80% DMEM medium was prepared. The mouse hybridoma cells were resuspended in the above ½ HA selection medium and mixed well. The cell suspension was added to a 96-well cell culture plate at 200 μl/well, 1×106spleen cells/plate, placed in a cell incubator and cultured at 37° C. After 1 week of culture, the ½ HA medium was used for the first renewing of the medium, and the culture was continued in 37° C. cell incubator. After 3 days of culture, the ½ HA medium was used for the second renewing of the medium.

Screening of Positive Cell Lines

Two weeks after the fusion, the cell supernatant was taken and subject to ELISA experiment to detect the binding of the cell supernatant to human Aβ1-42, and after the cells with positive ELISA result were screened out, the second ELISA experiment was retested. The cell supernatant with positive retested results was taken for subcloning and expansion culture.

Expansion Culture

The cell lines with positive ELISA test result were transferred from the 96 well-plate to a 24 well-plate and cultured, after the cells grew all over the plate, and they were transferred to a 25 cm2culture flask and cultured.

Subcloning by Limiting Dilution Method

The positive cell lines were mixed well by beating and pipetting, and a small amount thereof was pipetted to count the viable cells. About 200 cells were pipetted and added to 80 ml of complete medium and mixed well, and plated on 4 plates. In addition, about 400 cells were pipetted and added to 80 ml of complete medium and mixed well, and plated on 4 plates. In addition, about 1000 cells were pipetted and added to 20 ml of complete medium and mixed well, and plated on 1 plate. A total of 9 plates were plated at 3 different cell densities, respectively 0.5 cells/well, 1 cell/well, and 10 cells/well. The 96-well plates were plated in a 37° C., 5% CO2incubator for culture.

Clone Detection and Expansion Culture

The supernatants of the monoclonal cell wells were taken for ELISA to detect the binding of the cloned antibody to the full length of Aβ1-42as well as the N-terminal, C-terminal and middle peptide fragments of Aβ1-42, respectively.

Coating: Streptavidin was diluted with CBS (pH 9.6) to 1 μg/ml, added to 96-well microtiter plate, 50 μl per well, incubated overnight at 2-8° C.

Blocking: After washing the plate once with PBST, it was blocked with 1% BSA, 200 μl per well, and incubated for 1 hour at room temperature.

Antigen: After washing the plate three times with PBST, the biotinylated Aβ1-42, Aβ1-16, and Aβ14-29polypeptides were taken respectively, diluted with PBS (pH 7.2) to 1 μg/ml, and added to enzyme-labeled 96 well-plate, 50 μl per well, and incubated for 1 hour at room temperature.

Addition of primary antibody: After washing the plate three times with PBST, mouse candidate antibody was added, 50 μl/well, and incubated at room temperature for 2 hours.

Addition of secondary antibody: After washing the plate three times with PBST, anti-mouse IgG Fc-HRP antibody in 1:5000 diluent was added, 50 μl/well, and incubated for 1 hour at room temperature.

Color development: After washing the plate six times with PBST, TMB color development solution was added, 50 μl per well, and developed in the dark for 10 minutes at room temperature.

Stop: a stop solution was directly added to stop the reaction, 50 μl per well.

Detection: After stopping the reaction, the microtiter plate was immediately placed into a microplate reader, the OD value was measured at 450 nm, and the original data was stored.

Data processing: The raw data were input into the software SoftMax Pro 6.2.1 for data processing. See Table 1 for specific data. The results showed that the 12 murine candidate antibodies contained three different antigen binding epitopes, namely N-terminal (Aβ1-16), C-terminal (Aβ30-42), and middle (Aβ14-29) peptide fragments, in which, the antigen binding epitope of 066-4.26.14 was grouped into Aβ1-42C-terminal peptide fragment (because 066-4.26.14 could bind to the full length of Aβ1-42, but did not bind to Aβ1-16, Aβ14-29,it was deduced that it bound to Aβ30-42region).

The cell lines with positive ELISA result were transferred from the 96 well-plate to a 24 well-plate for culture, and the cells grew over the plate, they were transferred to a 25 cm2culture flask and cultured.

TABLE 1Grouping detection results of murine candidateantibody antigen binding epitopesAβ1-16Aβ14-29Full(N-terminal(middlelengthpeptidepeptideAntibody nameAβ1-42fragment)fragment)Epitope066-4.6.81.29440.05610.5115Middle peptidefragment066-4.17.281.45400.06901.1238Middle peptidefragment066-4.18.21.09251.22100.0848N-terminalpeptide fragment066-4.21.131.36470.0561.1457Middle peptidefragment066-4.22.11.25170.07731.0401Middle peptidefragment066-4.26.141.93120.06020.0674C-terminalpeptide fragment066-5.4.11.24830.05401.1566Middle peptidefragment066-6.1.11.37520.10010.1180C-terminalpeptide fragment066-6.1.31.36650.06180.0803C-terminalpeptide fragment066-6.2.11.30850.09750.1003C-terminalpeptide fragment066-6.7.21.47071.66080.3058N-terminalpeptide fragment066-7.17.21.08330.91970.0612N-terminalpeptide fragment
Identification of Subtypes

Goat anti-mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgM and IgGA were coated, 50 ng/100 μl/well, 4° C. overnight, blocked with BSA at room temperature, the cell supernatant to be tested was added, incubated at room temperature for 2 hours, added with enzyme-labeled secondary antibody goat anti-mouse IgG, κ, λ, after color development, stopping, and 450 nm reading, it was judged that the tested cell line was subtypes IgG1, κ or IgG2a, κ or IgG2b, κ. The results are shown in Table 2, in which for the antibody 066-4.26.14, its heavy chain constant region was murine IgG2a, and its light chain constant region was the constant region of the murine κ chain.

TABLE 2Detection results of mouse candidate subtypesAntibody nameSubtype066-4.6.8IgG1, Kappa066-4.17.28IgG1, Kappa066-4.18.2IgG2b, Kappa066-4.21.13IgG1, Kappa066-4.22.1IgG1, Kappa066-4.26.14IgG2a, Kappa066-5.4.1IgG1, Kappa066-6.1.1IgG2a, Kappa066-6.1.3IgG2a, Kappa066-6.2.1IgG2a, Kappa066-6.7.2IgG2a, Kappa066-7.17.2IgG1, Kappa
Cell Cryopreservation

Preparation of cryopreservation solution: 90% fetal bovine serum, 10% DMSO.

The cells in the culture flask were resuspended; after the cell counting, the cells were centrifuged at 1000 rpm/min for 5 min, the supernatant was discarded, and the suspension was beaten by pipetting with fetal bovine serum containing 10% DMSO, stored at 5×106cells/tube in a cryopreservation box at −80° C. overnight, and transferred into liquid nitrogen on the next day.

Preservation of Monoclonal Hybridoma Gene

Positive monoclonal cell lines were collected, added with TRizol to lyse the cells and extract RNA, which was reverse-transcribed into cDNA, and stored at −80° C.

Preparation of Antibodies by in Vitro Culture Method

The prepared hybridoma cell lines were resuscitated by a method as follows. The hybridoma cell lines were resuscitated in a DMEM medium containing 10% fetal bovine serum and 1% penicillin streptomycin, and cultured in a vial; after the cell confluence was about 90%, passage expansion was performed, the expansion was performed until the cell culture supernatant in total was about 200 ml, then the supernatant was collected, centrifuged and filtered for purification.

Example 3: Detection of Anti-Aβ Monoclonal Antibody in Inhibiting Aβ Polymerization

8.2% DMSO/DPBS solution (DMSO: sigma; DPBS: Hyclone) was used to dissolve Aβ dry powder to 1 mg/ml, the Aβ solution was diluted with DPBS to 33 μg/ml, the anti-Aβ monoclonal antibodies 066-4.6.8, 066-4.18.2, 066-4.22.1, 066-4.26.14, 066-5.4.1, 066-6.1.1, 066-6.1.3, 066-6.2.1, 066-6.7.2 were diluted to 450 μg/ml (IC100), and ThT (sigma) was diluted with ultrapure water to 20 μM. 50 μl of antibody diluent was taken and added to a 96-well black plate (corning), then added with 50 μl of Aβ diluent, finally added with 100 μl of ThT, incubated for 24 hours at room temperature in the dark, and the fluorescence intensity (Ex/Em=440/485) was detected with a multifunctional microplate reader. The abscissa represented different sample groups, and the ordinate represented relative fluorescence intensity. The results are shown inFIG.3. InFIG.3(A), when the relative fluorescence intensity of the IgG group was 1.0, the relative fluorescence intensity of the anti-Aβ monoclonal antibody 066-5.4.1 group was 0.59; inFIG.3(B), when the relative fluorescence intensity of the PBS group was 1.0, the relative fluorescence intensity of the anti-Aβ monoclonal antibody 066-4.22.1 group was 0.44, and the relative fluorescence intensity of the anti-Aβ monoclonal antibody 066-4.26.14 group was 0.46; it could be seen that the anti-Aβ monoclonal antibodies such as 066-4.22.1, 066-4.26.14 and 066-5.4.1 all could inhibit Aβ polymerization.

Example 4: Detection of Activity of Anti-Aβ Monoclonal Antibody in Promoting Macrophage Phagocytosis of Aβ

Mouse primary peritoneal macrophages that were in good condition after 3 days of adherent culture were digested with 0.25% trypsin and counted. The cell density was adjusted to 2×105/ml with DMEM medium (Gibco) containing 10% fetal bovine serum and the cells were inoculated on a 96-well cell culture plate, 100 μl/well; the anti-Aβ monoclonal antibodies 066-4.6.8, 066-4.17.28, 066-4.18.2, 066-4.21.13, 066-4.22.1, 066-4.26.14, 066-5.4.1, 066-6.1.1, 066-6.1.3, 066-6.2.1, 066-6.7.2, 066-7.17.2 were diluted with DMEM medium containing 1% fetal bovine serum to 20 μg/ml and used as working solutions, Aβ was diluted to 240 μg/ml, and ThT (sigma) was diluted to 20 μM with ultrapure water. The culture medium in the culture plate was discarded, 50 μl of antibody diluent was first added, then added with 50 μl of Aβ diluent, multiple wells were set; incubation was performed in a 37° C., 5% CO2incubator for 6 hours; 50 μl of supernatant was taken and add to a 96-well black plate, then added with 50 μl of ThT, and the fluorescence intensity (Ex/Em=440/485) was detected with a multifunctional microplate reader. The abscissa represented different sample groups, and the ordinate represented fluorescence intensity. The results are shown inFIG.4. Among them, the fluorescence intensity of the anti-Aβ monoclonal antibody 066-5.4.1 group was 650,000, and the fluorescence intensity of the anti-Aβ monoclonal antibody 066-7.17.2 group was 600,000. It could be seen that the anti-Aβ monoclonal antibodies 066-5.4.1, 066-7.17.2 had the activity of promoting the phagocytosis of Aβ by macrophages.

Example 5: Detection of Protective Activity of Anti-Aβ Monoclonal Antibody Against Cytotoxicity

Logarithmic growth phase SHSY5Y cells were digested with 0.25% trypsin, counted, adjusted with EMEM medium (ATCC) containing 10% fetal calf serum to a cell density of 3×104/ml, inoculated on a 96-well cell culture plate, 100 μl/well; the anti-Aβ monoclonal antibodies 066-4.6.8, 066-4.17.28, 066-4.18.2, 066-4.21.13, 066-4.22.1, 066-4.26.14, 066-5.4.1, 066-6.1.1, 066-6.1.3, 066-6.2.1, 066-6.7.2, 066-7.17.2 were diluted with EMEM medium containing 1% fetal bovine serum to 200 μg/ml (IC100), and used as working solution, Aβ was diluted to 240 μg/ml. The culture medium in the culture plate was discarded, 50 μl of antibody diluent was added, then added with 50 μl of AP diluent, multiple wells were set; incubation was performed in a 37° C., 5% CO2incubator for 48 hours; 50 μl of the supernatant was taken and added to a new 96 well-plate, then added with 50 μl of LDH assay buffer, reacted in the dark at room temperature for 30 minutes, added with 50 μl of stop solution, and the absorbance value was measured with a multifunctional microplate reader. The abscissa represented different sample groups, and the ordinate represented relative value of LDH release. The results are shown inFIG.5. Among them, when the relative value of LDH release of the Vehicle group was 1.0, the relative fluorescence intensity of the anti-Aβ monoclonal antibody 066-4.26.14 group was 1.2, the relative value of LDH release of the anti-Aβ monoclonal antibody 066-5.4.1 group was 1.37, the relative value of LDH release of the anti-Aβ monoclonal antibody 066-6.1.1 group was 1.43, the relative value of LDH release of the anti-Aβ monoclonal antibody 066-6.1.3 group was 1.35, the relative value of LDH release of the anti-Aβ monoclonal antibody 066-6.2.1 group was 1.34, the relative value of LDH release of the anti-Aβ monoclonal antibody 066-6.7.2 group was 1.44, the relative value of LDH release of the positive control antibody 066-P02 group was 1.26 (A) and 1.53 (B). It could be seen that the antibodies 066-4.26.14, 066-5.4.1, 066-6.1.1, 066-6.1.3, 066-6.2.1, 066-6.7.2, 066-7.17.2 all had protective effect against cytotoxicity, and the protective effect was equivalent to that of 066-P02.

Example 6: Morris Water Maze Experiment

1. Experimental Method and Steps:

Experimental animals 3×Tg mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and raised by the Experimental Animal Center of Medical College of Jilin University. The grouping situation was as follows:according to the different drugs to be injected, they were divided into antibody 066-4.26.14 treatment group, antibody 066-5.4.1 treatment group, antibody 066-7.17.2 treatment group, 3×Tg blank control group, wild-type PBS injection group, positive antibody 066-P02 control group, 8 animals in each group.

By referring to the literature method (Nabeshima, 2007), 6-month-old male 3×Tg mice (500 g/mouse) were intraperitoneally injected with monoclonal antibodies 066-4.26.14, 066-5.4.1, 066-7.17.2, once per week, continuously injected for 10 weeks, and Morris water maze test was performed 8 weeks after injection.

Morris Water Maze Test Steps:

(1) The specially designed water maze was mainly composed of a cylindrical pool and a movable platform. The pool had a height of 45 cm and a diameter of 100 cm, the platform had a diameter of 9 cm and an adjustable height from 15 to 40 cm, and digital camera was mounted above the pool and connected to a computer.(2) Clean water was filled into the pool in advance. The walls and bottom of the pool were all black. White pigment for food was added to the pool water to prevent mice from seeing the platform under the water surface. The water depth was 30 cm and the water surface was 1 cm higher than the platform.(3) The water temperature was controlled at 19±1° C., and except the quadrant where the platform was located, other quadrants on the pool were marked with points for entering water. On the sidewalls corresponding to each quadrant, markers of different shapes were adhered. The position of the platform was unchanged during the experiment.(4) Each test was carried out in a soundproof room, and the positions of laboratory objects such as the pool, light sources, and cages remained unchanged.(5) In the 8thweek, training was started on the 3rdday after the administration. The experiment lasted for 5 days (water maze-hidden platform test), 4 times a day. When the mouse entered the water, it faced the wall of the pool and was gently put into the water. Five training sessions (experiments) a day were conducted randomly in areas other than the quadrant where the platform was located, and the first two training sessions in the first two days of the experiment were performed as exercises. If the mouse found the platform within 60 seconds, it was allowed to stay on the platform for 15 seconds. If the mouse could not find the platform within 60 seconds (the latent period was recorded as 60 seconds), the experimenter would guide it to the platform and stay on the platform for 15 seconds. The average of four latent periods of the mouse was taken as the daily performance of the mouse.
2. Experimental Results (seeFIG.6):

On the second day of Hidden platform test, the time to find the hidden platform under the water (escape latent period) was significantly shortened for all antibody administration groups compared with the 3×Tg blank group, which was statistically significant. On day 3, the anti-Aβ monoclonal antibody 066-7.17.2 group took 18 s for escape latent period, the anti-Aβ monoclonal antibody 066-4.26.14 group took 27 s for escape latent period, the anti-Aβ monoclonal antibody 066-5.4.1 group took 30 s for escape latent period, and the blank control group took 39 s for escape latent period, in which the 066-7.17.2 and 066-4.26.14 administration groups had a significantly shorter escape latent period as compared with the 3×Tg blank group, and there was statistical significance. It could be seen that the anti-Aβ monoclonal antibodies 066-7.17.2 and 066-4.26.14 had a certain effect on improving the cognitive learning and memory ability in Alzheimer's dementia model mice.

Example 7: Monoclonal Antibody Gene Sequencing and Chimeric Antibody Preparation

1. Monoclonal Antibody Gene Sequencing

After immunization, fusion and monoclonalization, based on the experimental results of binding epitope, detection of inhibiting Aβ polymerization, detection of protective activity against cytotoxicity, Morris water maze, etc., the 066-4.26.14 monoclonal antibody cell line was selected for total RNA extraction which was reverse-transcribed into cDNA, and then the cDNA was used as a template for PCR amplification of the heavy chain variable region and light chain variable region of the antibody.

The TRIzol reagent kit (15596-026) of Invitrogen was used, and the total RNA was extracted from the 066-4.26.14 monoclonal antibody cell line according to its instructions. The results are shown inFIG.7.

The 5′RACE FULL kit (D315) of Takara was then used, the total RNA was reverse-transcribed into the first strand cDNA using the random primers in the kit, and then the PCR amplification of heavy chain was performed using the constant region primer mIgGR (5′-CTCAGGGAARTARCCYTTGAC-3′, SEQ ID NO: 42) and the RACE primer in the kit, and the PCR amplification of light chain was performed using the constant region primer mIgKR (5′-TCACTGCCATCAATCTTCCAC-3′, SEQ ID NO: 43) and the RACE primer in the kit. The results are shown inFIG.8.

The PCR fragments were recovered by the agarose gel recovery kit and subjected to TA cloning, and then single clones were picked up for colony PCR. The colony PCR primers were M13F (5′-TGTAAAACGACGGCCAGT-3′, SEQ ID NO: 44) and M13R (5′-CAGGAAACAGCTATGACC-3′, SEQ ID NO: 45). Part of the samples selected from the correct strains upon the identification were sent to Invitrogen for sequencing. It was finally determined that the nucleotide sequence of the heavy chain variable region was SEQ ID NO: 46, the nucleotide sequence of the light chain variable region was SEQ ID NO: 47, the amino acid sequence of the heavy chain variable region was SEQ ID NO: 48, and the amino acid sequence of the light chain variable region was SEQ ID NO: 49, see Table 3.

TABLE 3Specific sequences of heavy chain variable region andlight chain variable region of 066-4.26.14 antibodyNucleotide sequenceAmino acid sequenceHeavy chainLight chainHeavy chainLight chainvariablevariablevariablevariableAntibodyregionregionregionregion066-4.26.14SEQ IDSEQ IDSEQ IDSEQ IDNO: 46NO: 47NO: 48NO: 49

The nucleotide sequence of the heavy chain variable region of the 066-4.26.14 antibody was as follows (SEQ ID NO: 46):

GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGAAGTTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATCCATTAGTACTACTAGTAACACCTACTATCCAGACAGTGTGAAGGGCCGATTCACCACCTCCAGAGATAACGCCAGGAACATCGTGTACCTGCAAATGAGCAGTCTGAGGTCTGACGACACGGCCATGTATTACTGTGGAAGAGGCGTGATTACGAACCAGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

The nucleotide sequence of the light chain variable region of the 066-4.26.14 antibody was as follows (SEQ ID NO: 47):

GATATTGTGCTAACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGCGTCAGTCTTTCCTGCAGGGCCAGCCAAAGTATTAGCAACAACCTACACTGGTATCAGCAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAGTATGCTTCCCAGTCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACTCTCAGTGTCAACAATGTGGGGACTGAAGATTTTGGAATGTATTTCTGTCAACAGAGTAACAGCTGGCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

The amino acid sequence of the heavy chain variable region of the antibody 066-4.26.14 was as follows (SEQ ID NO: 48):

EVKLVESGGGLVKPGGSLKLSCAASGFTFRSYAMSWVRQTPEKRLEWVASISTTSNTYYPDSVKGRFTTSRDNARNIVYLQMSSLRSDDTAMYYCGRGVITNQAWFAYWGQGTLVTVSA

The amino acid sequence of the light chain variable region of the antibody 066-4.26.14 was as follows (SEQ ID NO: 49):

DIVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSVNNVGTEDFGMYFCQQSNSWPLTFGAGTKLELK
2. 066-4.26.14 Mouse-Human Chimeric Antibody Expression Vector Construction

The pGS003-hIgG1CH and pGS003-hIgKCL were selected as the expression vectors for constructing the heavy chain and the light chain of the anti-human Aβ mouse-human chimeric antibody, respectively. Using the synthesized 066-4.26.14 mouse antibody sequence as a template, the VH and the VL mouse antibody genes were PCR amplified and cloned into pGS003-hIgG1CH and pGS003-hIgKCL using restriction enzyme digestion and ligation methods to obtain the transient transfection expression vectors pGS003-066-4.26.14-chAbVH-hIgG1CH and pGS003-066-4.26.14-chAbVL-hIgKCL of the mouse-human chimeric antibody.

The amino acid sequence of the heavy chain of the 066-4.26.14 mouse-human chimeric antibody was as follows (SEQ ID NO: 50):

EVKLVESGGGLVKPGGSLKLSCAASGFTFRSYAMSWVRQTPEKRLEWVASISTTSNTYYPDSVKGRFTTSRDNARNIVYLQMSSLRSDDTAMYYCGRGVITNQAWFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

The amino acid sequence of the light chain of the 066-4.26.14 mouse-human chimeric antibody was as follows (SEQ ID NO: 51):

DIVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSVNNVGTEDFGMYFCQQSNSWPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
3. Expression by Transient Ttransfection

pGS003-066-4.26.14-chAbVH-hIgG1CH and pGS003-066-4.26.14-chAbVL-hIgKCL were subjected to transient expression.

FreeStyle™ 293E cells were used and subjected to transient transfection expression in Freestyle medium. Twenty-four hours before transfection, 30 ml of 293E cells were inoculated at 0.5×106cells/ml in a 125 ml conical flask, and cultured on a shaker at 130 rpm in a 37° C., 5% CO2incubator. During transfection, 60 μl of 293E Fectin was first taken and added to 1 ml of Opti-MEM, mixed well, and incubated at room temperature for 5 minutes; meanwhile, the total plasmid DNA of the recombinant vector in an amount of 30 μg was dissolved in 1 ml of Opti-MEM. Then, the plasmid DNA and 293E Fectin were mixed thoroughly, with a total volume of 2 ml, incubated at room temperature for 15 minutes, and then the whole mixture was added to the cell culture wells, mixed, and incubated in a 37° C., 5% CO2incubator on a shaker at 130 rpm for 7 days. The culture broth was centrifuged at a high speed and the supernatant was taken for vacuum filtration with a microporous membrane.

4. Purification of Protein

According to the operating method provided by the manufacturer, Protein A column (Protein Purification Liquid Chromatography System/AKTA Purifier 10, GE) and nickel column were used for purification to obtain the purified mouse-human chimeric antibody 066-4.26.14-chAb, as shown inFIG.9.

Example 8: Humanization of Antibodies

The mouse antibody 066-4.26.14 was selected for humanization. The humanization process comprised mainly human template search and reshaping.

The main goal of humanization was the FR sequence in the variable region. Using the amino acid sequences of the mouse antibody 066-4.26.14 VH and VL as templates, sequences alignment were performed on the NCBI website, and 5 humanized reference sequences were found, which were used as reference templates for the humanization of antibody FR regions to design the humanized sequences.

The specific sequences of the CDR regions are shown in Table 4, and the sequences of the humanized antibodies after reshaping are shown in Table 5.

TABLE 4Sequences of CDR regions of 066-4.26.14 antibodyAntibodyCDR1 sequenceCDR2 sequenceCDR3 sequence066-4.26.14SYAMSSISTTSNTYYPDSVKGGVITNQAWFAYH chain(SEQ ID NO: 1)(SEQ ID NO: 2)(SEQ ID NO: 3)066-4.26.14RASQSISNNLHYASQSISQQSNSWPLTL chain(SEQ ID NO: 4)(SEQ ID NO: 5)(SEQ ID NO: 6)

TABLE 5Humanized sequences of 066-4.26.14 antibodyHumanized sequence066-4.26.14H1EVQLVESGGGLVKPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVASISTTSNTYYPDSVKGRFTTSRDNAKNSLYLQMNSLRAEDTAVYYCGRGVITNQAWFAYWGQGTLVTVSS (SEQ ID NO: 15)066-4.26.14H2EVQLVESGGGLVKPGGSLRLSCAASGFTFRSYAMSWVRQTPEKRLEWVASISTTSNTYYPDSVKGRFTTSRDNAKNSVYLQMS SLRAEDTAVYYCGRGVITNQAWFAYWGQGTTVTVSS (SEQ ID NO: 16)066-4.26.14H3EVQLVQSGAEVKKPGESLKISCKGSGYSFRSYAMSWVRQMPGKGLEWVASISTTSNTYYPDSVKGRVTTSRDKSISTAYLQWSSLKASDTAMYYCGRGVITNQAWFAYVVGQGTLVTVSS (SEQ ID NO: 17)066-4.26.14H4EVQLVESGGGLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVASISTTSNTYYPDSVKGRFTTSRDNAKNSVYLQMSSLRDEDTAMYYCGRGVITNQAWFAYWGQGILVTVSS (SEQ ID NO: 18)066-4.26.14H5EVQLVESGGGLVQPGGSLRLSCVASGFTFRSYAMSWVRQAPGKGLEWVASISTTSNTYYPDSVKGRFTTSRDNSKNTVYLQMSSLRAEDTAVYYCGRGVITNQAWFAYWGQGTLVTVSS (SEQ ID NO: 19)066-4.26.14L1DIVLTQSPATLSVSPGERVTLSCRASQSISNNLHWYQQKSGQAPRLLIKYASQSISGIPSRFSGSGSGTDFTLTISSLQSEDFAVYFCQQSNSWPLTFGGGTQVEIK(SEQ ID NO: 20)066-4.26.14L2DIVLTQSPATLSVSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIKYASQSISGIPARFSGSGSGTDFTLTISSLQSEDFAVYFCQQSNSWPLTFGGGTKVEIK(SEQ ID NO: 21)066-4.26.14L3DIVLTQSPDFQSVTPKEKVTISCRASQSISNNLHWYQQKPDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLTINSLEAEDAAAYFCQQSNSWPLTFGPGTKVEIK(SEQ ID NO: 22)066-4.26.14L4EIVLTQSPGTLSLSPGERATLSCRASQSISNNLHWYQQKPGQAPRLLIKYASQSISGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSNSWPLTFGGGTKVEIK(SEQ ID NO: 23)066-4.26.14L5EIVLTQSPDFQSVTPKEKVTITCRASQSISNNLHWYQQKPDQSPKLLIKYASQSISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQSNSWPLTFGQGTKVEIK(SEQ ID NO: 24)

066-4.26.14H1(SEQ ID NO: 25)gaggtgcagctggtggaatcaggaggaggactggtgaagccaggcggatctctgagactgtcttgcgccgccagcggctttaccttcagatcttacgccatgtcttgggtccggcaggcaccaggaaaaggactggagtgggtggccagcatcagcaccaccagcaacacctactaccccgacagcgtgaagggcagattcaccaccagccgggacaacgccaagaacagcctgtacctgcagatgaacagcctgagggccgaggataccgccgtgtactattgcggacggggagtgatcaccaaccaggcttggttcgcctattgggggcagggaacactggtgaccgtgtctagc>066-4.26.14H2(SEQ ID NO: 26)gaggtgcagctggtggaatcaggaggaggactggtgaagccaggcggatctctgagactgtcttgcgccgccagcggctttaccttcagaagctacgccatgtcttgggtccggcagacaccagagaagagactggagtgggtggcctctatcagcaccaccagcaacacctactaccccgacagcgtgaagggcagattcaccaccagccgggacaacgccaagaacagcgtgtacctgcagatgagcagcctgagagccgaggacacagcagtgtactattgcggcaggggcgtgatcaccaaccaggcttggttcgcctattgggggcagggaacaaccgtgaccgtgtctagc>066-4.26.14H3(SEQ ID NO: 27)gaagtgcagctggtgcagagcggagcagaagtgaagaagcccggcgagtccctgaagatctcttgcaagggcagcggctacagcttcaggagctacgccatgtcttgggtccggcagatgccaggaaaaggactggagtgggtggcctctatcagcaccaccagcaacacctactaccccgacagcgtgaagggcagagtgacaaccagcagggacaagagcatcagcaccgcctacctgcagtggtctagcctgaaggccagcgataccgccatgtactattgcggccggggagtgatcaccaaccaggcttggttcgcctattgggggcagggaacactggtgaccgtgtctagc>066-4.26.14H4(SEQ ID NO: 28)gaggtgcagctggtggaatcaggaggaggactggtgcagccaggaggatctctgagactgtcttgcgccgccagcggctttaccttcagatcttacgccatgtcttgggtccggcaggcaccaggaaaaggactggagtgggtggccagcatcagcaccaccagcaacacctactaccccgacagcgtgaagggcagattcaccaccagccgggacaacgccaagaacagcgtgtacctgcagatgagcagcctgagggacgaggataccgccatgtactattgcggccggggagtgatcaccaaccaggcttggttcgcctattgggggcagggaatcctggtgaccgtgtctagc>066-4.26.14H5(SEQ ID NO: 29)gaggtgcagctggtggaatcaggaggaggactggtgcagccaggaggatctctgagactgtcttgcgtggccagcggcttcaccttcagatcttacgccatgtcttgggtccggcaggcaccaggaaaaggactggagtgggtggccagcatcagcaccaccagcaacacctactaccccgacagcgtgaagggcagattcaccaccagccgggacaacagcaagaacaccgtgtacctgcagatgagcagcctgagagccgaggacacagcagtgtactattgcggcaggggcgtgatcaccaaccaggcttggttcgcctattgggggcagggaacactggtgaccgtgtctagc>066-4.26.14L1(SEQ ID NO: 30)gacatcgtgctgacccagtctccagccacactgagcgtgtctccaggagagagagtgaccctgtcttgcagagccagccagagcatcagcaacaacctgcattggtaccagcagaagtccggccaggctcctaggctgctgatcaagtacgccagccagagcattagcggcatcccttctagattcagcggcagcggaagcggcacagatttcaccctgaccatcagcagcctgcagagcgaggacttcgccgtctacttctgccagcagagcaactcttggcccctgacctttggcggaggcacccaggtggagatcaag>066-4.26.14L2(SEQ ID NO: 31)gacatcgtgctgacccagtctccagccacactgagcgtgtctccaggagagagagccacactgtcttgcagagccagccagagcatcagcaacaacctgcattggtaccagcagaagccaggccaggctcctaggctgctgatcaagtacgcctctcagtctatcagcggcatcccagctagattcagcggcagcggaagcggcacagacttcaccctgaccatcagcagcctgcagagcgaggacttcgccgtctacttctgccagcagagcaactcttggcccctgacctttggcggaggcaccaaggtggagatcaag>066-4.26.14L3(SEQ ID NO: 32)gacatcgtgctgacccagagcccagacttccagtcagtgacccccaaggagaaggtcaccatcagctgcagagccagccagagcatcagcaacaacctgcattggtaccagcagaagcccgaccagagccccaagctgctgatcaagtacgccagccagtctatcagcggcatcccttctagattcagcggcagcggaagcggcacagatttcaccctgaccatcaacagcctggaggccgaagacgcagccgcctacttttgccagcagagcaactcttggcccctgacctttggccctggcaccaaggtggagatcaag>066-4.26.14L4(SEQ ID NO: 33)gagatcgtgctgacccagtctccaggcacactgtctctgagcccaggagagagagccacactgtcttgcagagccagccagagcatcagcaacaacctgcattggtaccagcagaagccaggccaggctcctaggctgctgatcaagtacgccagccagagcattagcggcatcccagatagattcagcggcagcggaagcggcacagatttcaccctgaccatcagcagactggagcccgaggacttcgccgtgtactattgccagcagagcaactcttggcccctgacctttggcggaggcaccaaggtggagatcaag>066-4.26.14L5(SEQ ID NO: 34)gagatcgtgctgacccagagcccagacttccagtcagtgacccccaaggagaaggtcaccatcacttgcagggccagccagagcatcagcaacaacctgcattggtaccagcagaagcccgaccagagccccaagctgctgatcaagtacgccagccagtctatcagcggagtgccttctagattcagcggcagcggaagcggcacagatttcaccctgaccatcaacagcctggaggcagaggacgcagccacctactattgccagcagagcaactcttggcccctgaccttcggacagggcaccaaggtggagatcaag

Example 9: Preparation of Anti-Aβ Humanized Full-Length Antibody

1. Construction of Expression Vectors for Transient Transfection of Full-Length Antibody

The pGS003-hIgG1CH and pGS003-hIgKCL were selected as the expression vectors for constructing the heavy chain and the light chain of the anti-Aβ humanized full-length antibody, respectively. Codon optimization was performed on the 066-4.26.14 humanized antibody sequence. After PCR amplification, the heavy chain was digested with HindIII and NheI, and the light chain was digested with HindIII and NarI, and then 5 VH and 5 VL antibody genes were cloned into pGS003-hIgG1CH and pGS003-hIgKCL, respectively, as shown in Table 6. After sequencing to identify the correct insertion of antibody gene, the recombinant expression vector was transformed intoE. coliTOP10F′, and a single colony was picked and inoculated in LB medium containing 100 μg/ml ampicillin, and cultured with shaking at 37° C. for 16 hours. The plasmids were extracted using endotoxin-free large-scale extraction kit of Zymo Research, and finally the plasmids were dissolved in 1 ml of ultrapure water, and the plasmids concentration and OD260/280were measured with a spectrophotometer. The plasmids DNA with OD260/280between 1.8 and 1.9 were of a relatively high purity.

TABLE 6List of expression vectors for transient transfectionof the heavy chain and the light chainName of Heavy chainName of Light chainexpression vectorexpression vectorH1L1H2L2H3L3H4L4H5L5
2. Transfection, Expression and Detection in Mammalian Cells 293E

The above 5 heavy chain expression vectors and 5 light chain expression vectors of 066-4.26.14 were combined in pairs (a total of 25 combinations), and then the transient transfection expression in 2 ml 293E system was evaluated, and the expression levels and ELISA values of the 25 combinations were evaluated. The results are shown in Table 7. Among them, 6 full-length antibodies were preferably selected, which were 066-4.26.14-H2L2, 066-4.26.14-H2L3, 066-4.26.14-H4L2, 066-4.26.14-H5L1, 066-4.26.14-H5L2, 066-4.26.14-H5L3, respectively.

TABLE 7Detection values of expression level and EC50 for smallsystem transient transfection expression of 066-4.26.14humanized full-length antibodies of 5 × 5 combinationCombination of heavyExpression levelEC50 value ofNo.chain and light chain(mg/L)Aβ42monomer1066-4.26.14H1L160—2066-4.26.14H1L261—3066-4.26.14H1L368.2—4066-4.26.14H1L432.1—5066-4.26.14H1L568.4—6066-4.26.14H2L189.70.021357066-4.26.14H2L286.60.012918066-4.26.14H2L351.80.015329066-4.26.14H2L47.72—10066-4.26.14H2L56.14—11066-4.26.14H3L15.42—12066-4.26.14H3L20—13066-4.26.14H3L36.78—14066-4.26.14H3L43.88—15066-4.26.14H3L53.68—16066-4.26.14H4L169.10.0494817066-4.26.14H4L271.20.0168518066-4.26.14H4L325.30.140619066-4.26.14H4L43.96—20066-4.26.14H4L50—21066-4.26.14H5L1118.50.00421822066-4.26.14H5L2113.20.0077223066-4.26.14H5L3103.30.0107524066-4.26.14H5L43.12—25066-4.26.14H5L50—Note:“—” in the table means no combination.

293E was used for transient transfection and expression of 6 candidate antibodies in Freestyle medium. Twenty-four hours before transfection, 300 ml of 293E cells were inoculated at 0.5×106cells/ml in a 1 L cell culture flask, and cultured in a 37° C., 5% CO2incubator with a shaker at 120 rpm. During transfection, 300 μl of 293 fectin was firstly taken and added to 5.7 ml of Opti-MEM, mixed well, and incubated at room temperature for 2 minutes; meanwhile, the expression plasmids for the heavy chain and the light chain in amount of 300 μg were diluted to 6 ml with Opti-MEM, respectively. The above-diluted transfection reagent and plasmid were mixed thoroughly, incubated at room temperature for 15 minutes, then the whole mixture was added to the cells, mixed well, and incubated in a 37° C., 5% CO2incubator with a shaker at 120 rpm for 7 days.

3. Purification and Detection of Antibodies

The cell culture medium was centrifuged at 2000 g for 20 min, the supernatant was collected, and the antibody expression level in the supernatant was detected by Octet. See Table 8.

TABLE 8Detection of expression level of 6 candidate antibodiesexpressed by transient transfection in 300 mlExpression levelHeavy chainLight chainof transientAntibody namesequencesequencetransfection (mg/L)066-4.26.14H2L2066-4.26.14H2066-4.26.14L2146066-4.26.14H2L3066-4.26.14H2066-4.26.14L356066-4.26.14H4L2066-4.26.14H4066-4.26.14L2101066-4.26.14H5L1066-4.26.14H5066-4.26.14L1164066-4.26.14H5L2066-4.26.14H5066-4.26.14L2128066-4.26.14H5L3066-4.26.14H5066-4.26.14L3135

The supernatant was filtered with a 0.22 μm filter, and then passed through a MabSelect SuRe affinity chromatography column (GE), eluted with 20 mM citrate-sodium citrate, pH 3.0, and the pH was adjusted to neutral with 1 M Tris base, and the solution was adjusted to an isotonic solution by adding with 10×PBS. The purified protein was detected by SDS-PAGE with 4-20% gradient gel (Nanjing Jinsirui Biotechnology Co., Ltd.). The results are shown inFIG.10below.

Example 10: Determination of EC50 Value of Humanized Candidate Antibody

Coating: The human Aβ42monomer was diluted with CBS (pH 9.4) to 1 μg/ml, added to 96-well microtiter plate, 50 μl per well, and incubated overnight at 2-8° C.

Blocking: After washing the plate three times with PBST, 3% BSA was used for blocking, 200 μl per well, and incubated for 1 hour at 25° C.

Sample processing: The humanized candidate antibody and chimeric antibody were taken respectively, subjected to 2-fold gradient dilution using 10 μg/ml as the starting concentration (20to 2−11), 50 μl/well, and incubated at 25° C. for 1 h.

Addition of antibody: After washing the plate four times with PBST, anti-human IgG (H+L)-HRP antibody in 1:5000 diluent was added, 50 μl/well, and incubated at 25° C. for 1 h.

Color development: After washing the plate four times, TMB color development solution was added, 50 μl per well, and developed in the dark for 3 minutes at room temperature.

Stop: The stop solution was directly added to stop the reaction, 50 μl per well.

Detection: After the reaction was stopped, the microtiter plate was immediately placed into a microplate reader to measure the OD value at 450 nm, and the original data were stored.

Data processing: The raw data were input into the software SoftMax Pro 6.2.1 for data processing. See Table 9 for the specific data. The results showed that the binding capability of the 6 humanized candidate antibodies to human AP was equivalent to that of the chimeric antibody.

TABLE 9EC50 values of 6 candidate antibodies binding to antigenAntibody nameEC50 value of Aβ42monomer066-4.26.14H2L20.0129066-4.26.14H2L30.0153066-4.26.14H4L20.0168066-4.26.14H5L10.0042066-4.26.14H5L20.0077066-4.26.14H5L30.0108066-4.26.14-chAb0.0255

Example 11: Determination of KD Value of Humanized Candidate Antibody

Biacore-T200 detection was performed, ProteinA chip was used to capture candidate antibodies or positive antibodies, different concentrations of human Aβ antigen were used to flow through the chip, and the fitting analysis was performed based on the collected data. The antigen sample was subjected to 2-fold gradient dilution using HBS-EP+ Buffer to obtain solutions with gradient concentrations of 400 nmol/L, 200 nmol/L, 100 nmol/L, 50 nmol/L, 25 nmol/L, 12.5 nmol/L, 6.25 nmol/L, 3.125 nmol/L, 1.56 nmol/L, 0.78 nmol/L, 0 nmol/L. The sample of 25 nmol/L was used for repeat concentration detection. The detection conditions were: capture time: 30 s; antigen binding time: 120 s; dissociation time: 900 s; flow rate: 30 μl/min. And the regeneration conditions were: 20 mM NaOH solution, flow rate: 30 μl/min. The specific experimental results are shown in Table 10. It could be seen from the experimental results that, compared with the mouse-human chimeric antibody, the KD value of the humanized antibody could be equivalent to that of the mouse antibody.

TABLE 10KD value detection of 6 candidate antibodiesAntibody nameKa (1/Ms)Kd (1/s)KD (M)066-4.26.14H2L21.92E+044.33E−042.25E−08066-4.26.14H2L31.83E+044.72E−042.58E−08066-4.26.14H4L21.28E+044.63E−043.63E−08066-4.26.14H5L11.89E+044.69E−042.48E−08066-4.26.14H5L21.34E+042.78E−042.08E−08066-4.26.14H5L31.42E+044.60E−043.25E−08066-4.26.14-chAb2.65E+043.33E−041.26E−08Note:E+04: ×104; E−04: ×10−4; E−08: ×10−08.

Example 12: Detection of Effect of Humanized Anti-Aβ Antibody Inhibiting Aβ Polymerization

8.2% DMSO/DPBS solution (DMSO: sigma; DPBS: Hyclone) was used to dissolve Aβ dry powder to 1 mg/ml, DPBS was used to dilute the Aβ solution to 33 μg/ml, and the humanized candidate antibody 066-4.26.14 was diluted to 450 μg/ml (IC100), and ThT (sigma) was diluted to 20 μM with ultrapure water. 50 μl of the candidate antibody diluent was taken and added to 96-well black plate (corning), then added with 50 μl of the Aβ diluent and finally added with 100 μl of ThT, incubated at room temperature for 24 hours in the dark, and measured with a mutifunctional microplate reader to determine fluorescence intensity (Ex/Em=440/485). The abscissa represented different sample groups, and the ordinate represented relative fluorescence intensity. The results are shown inFIG.11. The relative fluorescence intensity of hIgG was 1.00, the relative fluorescence intensity of the 066-4.26.14-mAb group was 0.70, the relative fluorescence intensity of the 066-4.26.14-chAb group was 0.71, the relative fluorescence intensity of the 066-4.26.14H2L2 group was 0.62, the relative fluorescence intensity of the 066-4.26.14H2L3 group was 0.67, the relative fluorescence intensity of the 066-4.26.14H4L2 group was 0.70, the relative fluorescence intensity of the 066-4.26.14H5L1 group was 0.68, the relative fluorescence intensity of the 066-4.26.14H5L2 group was 0.67, and the relative fluorescence intensity of the 066-4.26.14H5L3 group was 0.78. It could be seen that the humanized antibodies of 066-4.26.14 could inhibit Aβ polymerization.

Example 13: Detection of Protective Activity of Humanized Anti-Aβ Antibody Against Cytotoxicity

Logarithmic growth phase SHSY5Y cells were digested with 0.25% trypsin, counted, adjusted with EMEM medium (ATCC) containing 10% fetal calf serum to have a cell density of 3×104/ml, inoculated on a 96-well cell culture plate, 100 μl/well; the humanized candidate antibodies of 066-4.26.14 was diluted with EMEM medium containing 1% fetal bovine serum to 200 μg/ml (IC100) and used as working solutions, Aβ was diluted to 240 μg/ml. The culture medium in the culture plate was discarded, 50 μl of the candidate antibody diluent was firstly added, then added with 50 μl of the Aβ diluent, multiple wells were set; incubation was performed in a 37° C., 5% CO2incubator for 48 hours; 50 μl of the supernatant was taken and added to a new 96-well plate, then added with 50 μl of LDH assay buffer, reacted in the dark at room temperature for 30 minutes, added with 50 μl of stop solution, and measured with a multifunctional microplate reader to determine absorbance. The abscissa represented different sample groups, and the ordinate represented relative value of LDH release. The results are shown inFIG.12. The relative value of LDH release of hIgG was 1.00, the relative value of LDH release of the 066-P02 group was 1.26, the relative value of LDH release of the 066-4.26.14-mAb group was 1.20, the relative value of LDH release of the 066-4.26.14-chAb group was 1.32, the relative value of LDH release of the 066-4.26.14H2L2 group was 1.41, the relative value of LDH release of the 066-4.26.14H2L3 group was 1.42, the relative value of LDH release of the 066-4.26.14H4L2 group was 1.30, the relative value of LDH release of the 066-4.26.14H5L1 group was 1.37, the relative value of LDH release of the 066-4.26.14H5L2 group was 1.26, and the relative value of LDH release of the 066-4.26.14H5L3 group was 1.41. It could be seen that the humanized candidate antibodies of 066-4.26.14 all had protective effect against cytotoxicity, and the protective effect was equivalent to that of 066-P02, in which the 066-4.26.14H5L2 showed the best performance.

The humanized anti-Aβ monoclonal antibody and its use provided by the present disclosure have been introduced in detail above. The principle and implementation of the present disclosure are illustrated with specific examples, while the description of the above examples is only used to help understand the method and the core idea of the present disclosure. It should be pointed out that for those skilled in the art, without departing from the principle of the present disclosure, several improvements and modifications can be made to the present disclosure, and these improvements and modifications also fall within the protection scope of the claims of the present disclosure.