HUMANIZED ANTI-IL-4Ra SINGLE DOMAIN ANTIBODY AND APPLICATION THEREOF

Abstract

Provided is a humanized anti-IL-4Rα single domain antibody. The antibody has complementarity determining regions and humanized and modified framework regions.

Claims

1. A humanized anti-IL-4Rα single domain antibody, comprising complementarity determining regions comprising CDR1, CDR2 and CDR3 and framework regions comprising FR1, FR2, FR3 and FR4; wherein CDR1 has an amino acid sequence as set forth in SEQ ID NO. 3, CDR2 has an amino acid sequence as set forth in SEQ ID NO. 12, and CDR3 has an amino acid sequence as set forth in SEQ ID NO. 18; and FR1 has an amino acid sequence as set forth in SEQ ID NO. 2, FR2 has an amino acid sequence selected from any one of SEQ ID NO. 4-11, FR3 has an amino acid sequence selected from any one of SEQ ID NO. 13-17, and FR4 has an amino acid sequence as set forth in SEQ ID NO. 20 or 21.

2. The humanized anti-IL-4Rα single domain antibody according to claim 1, wherein the amino acid sequence of FR2 is as set forth in SEQ ID NO. 11, the amino acid sequence of FR3 is as set forth in SEQ ID NO. 15, and the amino acid sequence of FR4 is as set forth in SEQ ID NO. 21.

3. A fusion protein comprising the anti-IL-4Rα single domain antibody according to claim 1.

4. A bispecific antibody comprising the anti-IL-4Rα single domain antibody according to claim 1.

5. A method for treating a disease in a subject, comprising administrating the anti-IL-4Rα single domain antibody according to claim 1 to the subject, wherein the disease is selected from asthma, allergic dermatitis, eczema, arthritis, herpes, chronic primary urticaria, scleroderma, hypertrophic cicatrix, chronic obstructive pulmonary disease, atopic dermatitis, idiopathic pulmonary fibrosis, Kawasaki disease, sickle cell disease, Graves' disease, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, and nephropathy.

6. (canceled)

7. A drug for treating a disease, comprising the anti-IL-4Rα single domain antibody according to claim 1, and a pharmaceutically acceptable excipient.

8. An isolated nucleic acid molecule encoding the anti-IL-4Rα single domain antibody according to claim 1.

9. A vector or recombinant cell containing the nucleic acid molecule according to claim 8.

10. A method for preparing the anti-IL-4Rα single domain antibody according to claim 1, comprising: culturing a recombinant cell, and isolating and purifying the anti-IL-4Rα single domain antibody from the culture product, the recombinant cell containing a recombinant expression vector comprising the nucleic acid molecule encoding the anti-IL-4Rα single domain antibody.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] To describe the technical solutions of the embodiments of the present disclosure more clearly, the following briefly describes the accompanying drawings for describing the embodiments. It should be understood that, the following accompanying drawings show only some embodiments of the present disclosure, which cannot be considered as limitation on the scope. A person of ordinary skill in the art may still derive other accompanying drawings from such accompanying drawings without creative efforts.

[0044] FIG. 1 is a schematic diagram of IL4/IL-13 and IL-5 signaling pathways;

[0045] FIG. 2 shows a sequence alignment result of some clones after humanization of an anti-IL-4Rα monoclonal antibody 4E9;

[0046] FIG. 3 shows assay results of different humanized 4E9 antibody clones neutralizing IL-4-induced TF-1 proliferation;

[0047] FIG. 4 shows assay results of humanized 4E9 antibody clones neutralizing IL-13-induced TF-1 proliferation;

[0048] FIG. 5 shows assay results (ELISA) of an IL-4Rα/IL-5 bispecific antibody 4E9V10-2B3V2 blocking binding of IL-4Rα to IL-4;

[0049] FIG. 6 shows assay results (ELISA) of an IL-4Rα/IL-5 bispecific antibody 4E9V10-2B3V2 blocking binding of IL-5R to IL-5;

[0050] FIG. 7 shows assay results of an IL-4Rα/IL-5 bispecific antibody 4E9V10-2B3V2 neutralizing IL-4-induced TF-1 proliferation;

[0051] FIG. 8 shows assay results of an IL-4Rα/IL-5 bispecific antibody 4E9V10-2B3V2 neutralizing IL-13-induced TF-1 proliferation; and

[0052] FIG. 9 shows assay results of an IL-4Rα/IL-5 bispecific antibody 4E9V10-2B3V2 neutralizing IL-5-induced TF-1 proliferation.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0053] To make the objective, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be described clearly and completely below. If specific conditions are not indicated in the embodiments, it is carried out in accordance with the conventional conditions or conditions suggested by manufacturers. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

[0054] The features and performance of the present disclosure will be further described in detail below in conjunction with the examples.

Example 1

[0055] Humanized Modification

[0056] Humanized modification was carried out on the basis of an anti-IL-4Rα single domain antibody 4E9 (named 4E9-V0) of SEQ ID NO. 22.

[0057] The sequences of the modified humanized single domain antibodies (named 4E9 V1-V14, respectively) are shown in Table 1, and alignment results of partial humanized antibody sequences are shown in FIG. 2.

TABLE-US-00001 TABLE 1 Sequence identifier (SEQ ID NO.) corresponding to each region of each humanized single domain antibody FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 4E9-V0 1 3 4 12 13 18 20 4E9-V1 2 3 5 12 14 18 21 4E9-V2 2 3 6 12 14 18 21 4E9-V3 2 3 6 12 15 18 21 4E9-V4 1 3 4 12 13 19 20 4E9-V5 2 3 5 12 14 19 21 4E9-V6 2 3 7 12 15 18 21 4E9-V7 2 3 8 12 16 18 21 4E9-V8 2 3 9 12 16 18 21 4E9-V9 2 3 10 12 15 18 21 4E9-V10 2 3 11 12 15 18 21 4E9-V11 2 3 8 12 15 18 21 4E9-V12 2 3 7 12 16 18 21 4E9-V13 2 3 7 12 17 18 21 4E9-V14 2 3 7 12 14 18 21

Example 2

[0058] Assay for Humanized Single Domain Antibodies Neutralizing IL-4-Induced or IL-13-Induced TF1 Cell Proliferation

[0059] (1) TF-1 cells passaged 3-4 times after recovery were plated in a 96-well plate at 10000 cells/well.

[0060] (2) Different Tabs and the humanized single domain antibodies in Table 1 were formulated into 10 μg/mL solutions, and subjected to 5-fold gradient dilution.

[0061] (3) The Tab antibodies (obtained referring to a method disclosed in the Chinese invention patent application no. CN202010576200.7 titled with “Anti-IL-4Rα single domain antibody as well as application and drug”) and the humanized single domain antibodies both subjected to gradient dilution were mixed with IL-4 or IL13 of EC80 concentration (obtained referring to the method disclosed in the Chinese invention patent application no. CN202010576200.7 titled with “Anti-IL-4Rα single domain antibody as well as application and drug”) at 1:1 to prepare a mixed solution. EC80 is defined as follows: EC80, namely the concentration for 80% of maximal effect (EC80), refers to a concentration that can cause 80% of the maximal effect.

[0062] (4) The mixed solution in the previous step was added into cell culture wells in an equal volume to the cell culture solution.

[0063] (5) After incubation for 72 h, the cell viability was detected with a luminescence cell viability assay kit.

[0064] (6) The EC50 concentrations of different humanized single domain antibodies for neutralizing IL-4-induced or IL13-induced TF-1 cell proliferation was calculated according to the assay results. EC50, namely the concentration for 50% of maximal effect (EC50), refers to the concentration that can cause 50% of the maximal effect. The results are shown in Table 2, Table 3, FIG. 3 and FIG. 4.

TABLE-US-00002 TABLE 2 Assay results of humanized anti-IL-4Rα single domain antibodies neutralizing IL-4-induced TF-1 proliferation Antibody name 4E9-V1 4E9-V2 4E9-V3 4E9-V4 4E9-V5 4E9-V6 4E9-V7 EC50 56898284406318 52.08 13.84 2.917e+20 0.7460 0.7070 185.0 (nM) Antibody name 4E9-V8 4E9-V9 4E9-V10 4E9-V11 4E9-V12 4E9-V13 4E9-V14 EC50 2.768 0.7519 0.2708 1880000 2.031 4.521 18630000 (nM)

TABLE-US-00003 TABLE 3 Assay results of humanized anti-IL-4Rα single domain antibodies neutralizing IL-13-induced TF-1 proliferation 4E9-V6 4E9-V8 4E9-V10 4E9-V12 4E9-V13 EC50 0.7595 3.107 0.1798 2.111 4.204 (nM)

[0065] The results show that among all humanized antibodies, the 4E9-V10 antibody has the strongest cell proliferation neutralizing ability and has an EC50 of 0.27 nM, 4E9-V1 has the worst effect, and 4E9-V6, 4E9-V8, 4E9-V12 and 4E9-V13 have certain neutralizing effects.

Example 3

[0066] Assay for Thermal Stability of Humanized Single Domain Antibodies by Differential Scanning

[0067] Assay Method:

[0068] (1) To an 8-tube strip or a 96-well PCR plate, 45 μL of a solution of 0.1 mg/mL aforementioned humanized single domain antibodies was added, followed by 5 μL of a 100×Sypro orange dye. The dye was at a final concentration of 5×. 3 replicates were made for each sample, with 1×PBS as a blank.

[0069] (2) A Melt curve assay was carried out, with a reporter group of ROX and a quencher of None, according to a heating program of 25° C. for 5 min and a scanning range of 25° C.-95° C., at a heating rate of 1% (about 1° C./min).

[0070] (3) The temperature corresponding to the maximum value of the first derivative of the melting curve was taken as the denaturation temperature (Tm value) of the protein.

[0071] The assay results are as shown in Table 4.

TABLE-US-00004 TABLE 4 Sample name Tm value (° C.) 4E9-V13 65.1 4E9-V12 65 4E9-V10 64.6 4E9-V6 64.5 4E9-V7 64.3 4E9-V14 64.2 4E9-V11 64.1 4E9-V4 64.1 4E9-V9 64 4E9-V5 63.8 4E9-V8 63.5 4E9-V1 63.3 4E9-V2 63.2 4E9-V3 N/A

[0072] The results show that among all the humanized single domain antibodies, 4E9-V13 has the highest thermal stability, 4E9-V3 has the lowest undetectable thermal stability, and 4E9-V10 has the third highest thermal stability among all the antibodies.

Example 4

[0073] Determination of Affinity of Humanized Single Domain Antibodies

[0074] Preparation of an SD buffer: an appropriate amount of bovine serum albumin and Tween 20 were dissolved in 1×PBS (pH 7.4), so that the mass (or volume) fractions of the bovine serum albumin and the Tween 20 were 0.1% and 0.02% respectively. The IL-4Rα binding molecules (the aforementioned partially humanized single domain antibodies) were formulated with the SD buffer to a concentration of 10 μg/mL.

[0075] Preparation of an antigen working solution: an antigen was firstly formulated to 200 nM with the SD buffer, and then subjected to 2-fold gradient dilution. A total of 5 concentration gradients were set, in addition to the SD buffer as a blank control.

[0076] Preparation of a regenerating solution: an appropriate amount of a 0.1 M glycine stock solution was diluted 10 times in deionized water and mixed well to obtain the regenerating solution.

[0077] Assay steps: Octet 96 and Data Acquisition software in the supporting computer were run. The bottom and sides of an acquisition probe were cleaned using a lens tissue with an appropriate amount of 75% ethanol, and the instrument was preheated for 15 min or more. Sensor pre-wetting: before the assay, the Sensor was soaked in the SD buffer for 10 min or more for later use. Then, the machine procedure was set according to: baseline.fwdarw.antibody.fwdarw.baseline.fwdarw.antigen binding.fwdarw.antigen dissociation.fwdarw.sensor regeneration for assay operation. The assay results are shown in Table 5.

TABLE-US-00005 TABLE 5 Antibody name KD(M) KD Error Ka(1/Ms) Ka Error Kd(1/s) Kd Error X.sup.2 R.sup.2 4E9-V10 3.08E−09 3.98E−11 1.73E+05 9.99E+02 5.34E−04 6.18E−06 0.236 0.9946 4E9-V13 3.41E−09 4.03E−11 1.66E+05 9.02E+02 5.65E−04 5.92E−06 0.1934 0.9956 4E9-V12 9.30E−09 1.55E−11 2.88E+04 1.20E+03 2.68E−04 4.33E−06 0.1415 0.9957 4E9-V6 3.05E−09 3.71E−11 1.66E+05 8.51E+02 5.06E−04 5.58E−06 0.2055 0.9963 KD: Affinity constant, in moles (M). Ka: Association rate constant, in the reciprocal of molar time (1/Ms). Kd: Dissociation rate constant, in the reciprocal of time. R.sup.2: Degree of fitting, that is, the degree of fitting between a measured curve and a fitted curve. The closer R.sup.2 is to 1, the closer a fitted value is to the measured value, and in this system, R.sup.2 should be at least greater than 0.95. X.sup.2: Statistical parameter performance of the values measured by the system, which should be less than 3, and the smaller it is, the more credible the measured value.

[0078] Other error values are the error values of their corresponding parameters, which should be an order of magnitude (10-fold) smaller than the corresponding parameters or less.

[0079] The results show that 4E9-V6 and 4E9-V10 have the lowest KD values, and the highest antigen affinity.

[0080] Based on the above results as well as the results of antibody function assay in cells and the results of physicochemical analysis, 4E9-V10 is the humanized single domain antibody with the best comprehensive effect among all the humanized sequences, which was unexpected by the inventors, and 4E9-V10 was used for assays in subsequent examples.

Example 5

[0081] Construction of Eukaryotic Expression Vector of Fc Fusion Antibody of Anti-IL-4Rα/IL-5 Protein Bispecific Single Domain Antibody

[0082] (1) The gene sequence (positions 1-345 of SEQ ID NO. 23) of the codon-optimized humanized anti-IL-4Rα single domain antibody (4E9V10) or the gene sequence (positions 1069-1434 of SEQ ID NO. 23) of the humanized anti-IL-5 single domain antibody (named 2B3V2) were synthesized and inserted into a vector RJK-V4-3 (obtained referring to the method disclosed in the Chinese invention patent application no. CN202010576200.7 titled with “Anti-IL-4Rα single domain antibody as well as application and drug”) respectively by means of sequence synthesis.

[0083] (2) The constructed recombinant eukaryotic expression vector was transformed into DH5a Escherichia coli, and cultured for plasmid maxiprep extraction and removal of endotoxin.

[0084] (3) The extracted plasmids were sequenced and identified.

[0085] (4) The identified anti-IL-4Rα antibody sequence was subcloned into the eukaryotic expression vector containing the anti-IL-S antibody sequence: specifically, the anti-IL-4Rα antibody sequence was cut from the eukaryotic expression vector where it was located by using restriction endonucleases Xba I and BamH I, and ligated with the eukaryotic expression vector containing the anti-IL-S antibody sequence with the same restriction endonuclease sticky end. The ligated vector was subjected to transformation, sequencing and identification; the clones confirmed by sequencing were subjected to plasmid maxiprep extraction for removal of endotoxin; the extracted plasmids were then sequenced and identified; and confirmed recombinant vectors were prepared for subsequent transfection and expression in eukaryotic cell. The eukaryotic expression vector of the anti-IL-4Rα/IL-5 protein bispecific single domain antibody was obtained. The anti-IL-4Rα/IL-5 protein bispecific single domain antibody (having an amino acid sequence shown in SEQ ID NO. 24, and a gene sequence shown in SEQ ID NO. 23) was named 4E9V10-2B3V2, and includes three moieties: 4E9V10-Fc fragment-2B3V2, where 4E9V10 is at the amino terminus (positions 1-115 in SEQ ID NO. 24), the Fc fragment is at positions 116-356 in SEQ ID NO. 24, and 2B3V2 is at the carboxy terminus (positions 357-478 in SEQ ID NO. 24).

Example 6

[0086] The anti-IL-4Rα/IL-5 protein bispecific single domain antibody was expressed in a suspension ExpiCHO-S cell by an assay method referring to the Chinese invention patent application no. CN202010576200.7 titled with “Anti-IL-4Rα single domain antibody as well as application and drug”.

Example 7

[0087] The anti-IL-4Rα/IL-5 protein bispecific single domain antibody was expressed in a suspension 293F cell by an assay method referring to the Chinese invention patent application no. CN202010576200.7 titled with “Anti-IL-4Rα single domain antibody as well as application and drug”.

Example 8

[0088] The anti-IL-4Rα/IL-5 protein bispecific single domain antibody was purified by an assay method referring to the Chinese invention patent application no. CN202010576200.7 titled with “Anti-IL-4Rα single domain antibody as well as application and drug”.

Example 9

[0089] Blocking Assay of Receptor-Ligand Binding Using Bispecific Single Domain Antibody

[0090] (1) A receptor protein (IL-4Rα or IL-5R) was diluted with a protein diluent to 1 μg/mL, and coated overnight at 4° C.

[0091] (2) The plate was washed and blocked with 5% skim milk at 37° C.

[0092] (3) A biotin-conjugated ligand protein (IL-4 or IL-5) was diluted to 2 times the EC80 concentration, and the antibody was diluted to 2 times the initial concentration, and 5-fold gradient dilution was carried out. The ligand protein, and the diluted antibody (dupilumab, 4E9V10-2B3V2, 4E9-V0 or 2B3 (SEQ ID NO. 25)) and hIgG respectively, were transferred at 1:1 into a new dispensing plate and mixed well.

[0093] (4) The plate was washed, and the diluted ligand protein/antibody mixture was transferred into an ELISA plate in duplicate and incubated at 37° C.

[0094] (5) The plate was washed, and diluted Streptavidin[HRP] was added and incubated at 37° C.

[0095] (6) The plate was washed, single-component TMB was added for color development at room temperature in the dark.

[0096] (7) A stopping solution was added, and the values of samples in different wells were immediately read at the wavelength of 450 nM with a microplate reader and recorded as OD450. EC50 was calculated by plotting. 4E9-V0, non-humanized anti-IL-S single domain antibody 2B3, dupilumab, reslizumab, and hIgG were used as controls, and the results are shown in Tables 6 and 7 and FIGS. 5 and 6, respectively.

TABLE-US-00006 TABLE 6 Assay results of EC50 of bispecific antibodies blocking binding of IL-4 to IL-4Rα 4E9-V0 4E9V10-2B3V2 dupilumab hIgG EC50(nM) 4.447 3.653 4.321 2.038

TABLE-US-00007 TABLE 7 Assay results of EC50 of bispecific antibodies blocking binding of IL-5 to IL-5R 2B3 4E9V10-2B3V2 reslizumab hIgG EC50(nM) 6.911 3.612 6.208 27387599489

[0097] The results show that the humanized bispecific antibody was non-attenuated and slightly advantageous in blocking IL-4/IL-4Rα or IL-5/IL-5R receptor-ligand binding than the non-humanized antibody. Compared with the corresponding commercial drugs, the humanized bispecific antibody has almost the same and slightly advantageous blocking ability.

Example 10

[0098] Neutralization Assay for IL-4-Induced or IL-13-Induced TF1 Cell Proliferation by Bispecific Single Domain Antibodies

[0099] Referring to Example 2 for the assay method.

[0100] The assay results are shown in Tables 8 and 9 and FIGS. 7 and 8.

TABLE-US-00008 TABLE 8 Assay results of bispecific single domain antibodies neutralizing IL-4-induced TF-1 cell proliferation 4E9-V0 4E9V10-2B3V2 dupilumab hIgG EC50(nM) 0.2812 0.2618 0.1297 0.3750

TABLE-US-00009 TABLE 9 Assay results of bispecific single domain antibodies neutralizing IL-13-induced TF-1 cell proliferation 4E9-V0 4E9V10-2B3V2 dupilumab hIgG EC50(nM) 0.2291 0.2154 0.1783 0.1312

[0101] The results show that the humanized bispecific antibody was non-attenuated and slightly advantageous in neutralizing IL-4-induced TF-1 cell proliferation than the non-humanized antibody. Compared with the corresponding commercial drugs, the humanized bispecific antibody has almost the same blocking ability.

Example 11

[0102] Assay for Human Recombinant IL-5 Protein-Induced TF1 Cell Proliferation and Tool Antibody (Tab) Neutralizing Proliferation

[0103] A. Assay for Human Recombinant IL-5 Protein-Induced TF1 Cell Proliferation:

[0104] (1) TF-1 cells passaged 3-4 times after recovery were plated in a 96-well plate at 10000 cells/well.

[0105] (2) The human IL-5 protein was formulated into a solution with a maximum concentration of 500 ng/mL, and subjected to 5-fold gradient dilution.

[0106] (3) The IL-5 protein solution subjected to gradient dilution was added into cell culture wells in an equal volume to the cell culture solution.

[0107] (4) After incubation for 72 h, the cell viability was detected with a luminescence cell viability assay kit.

[0108] (5) The EC80 concentration for IL-S-induced TF-1 cell proliferation was calculated according to the assay results, and the calculated result was 2.96 ng/mL.

[0109] B. Assay for Tab Neutralizing Human IL-S-Induced TF1 Cell Proliferation

[0110] (1) TF-1 cells passaged 3-4 times after recovery were plated into a 96-well plate at 10000 cells/well.

[0111] (2) The Tab was formulated into a solution of 10 μg/mL, and subjected to 5-fold gradient dilution.

[0112] (3) The Tab subjected to gradient dilution was mixed with the IL-5 at the EC80 concentration obtained in the proliferation assay at 1:1 to prepare a mixed solution.

[0113] (4) The mixed solution was added into cell culture wells in an equal volume to the cell culture solution.

[0114] (5) After incubation for 72 h, the cell viability was detected with a luminescence cell viability assay kit.

[0115] (6) The EC50 concentration of the Tab neutralizing the IL-S-induced TF-1 cell proliferation was calculated according to the assay results.

Example 12

[0116] Assay for Humanized Bispecific Single Domain Antibodies Neutralizing IL-S-Induced TF1 Cell Proliferation.

[0117] (1) TF-1 cells passaged 3-4 times after recovery were plated into a 96-well plate at 10000 cells/well.

[0118] (2) The Tab and the aforementioned humanized bispecific single domain antibody were formulated into a solution of 10 μg/mL, and subjected to 5-fold gradient dilution.

[0119] (3) The Tab and the humanized antibody subjected to gradient dilution were mixed respectively with the IL-5 protein of the EC80 concentration obtained in example 13-A at 1:1 to prepare a mixed solution.

[0120] (4) The mixed solution was added into cell culture wells in an equal volume to the cell culture solution.

[0121] (5) After incubation for 72 h, the cell viability was detected with a luminescence cell viability assay kit.

[0122] (6) The EC50 concentrations of different single domain antibodies neutralizing the IL-5-induced TF-1 cell proliferation were calculated according to the assay results, and the assay results are shown in Table 10 and FIG. 9.

TABLE-US-00010 TABLE 10 Assay results of bispecific antibodies neutralizing IL-5-induced TF-1 cell proliferation reslizumab hIgG 4E9V10-2B3V2 2B3 EC50(nM) 0.1221 0.1080 0.1753 0.3362

[0123] The results show that the humanized bispecific antibody was non-attenuated and slightly advantageous in neutralizing IL-5-induced TF-1 cell proliferation than the non-humanized antibody. Compared with the corresponding commercial drugs, the humanized bispecific antibody has almost the same blocking ability.

Example 12

[0124] Affinity kinetic assay for the humanized bispecific antibody was carried out by the assay method shown in example 4, and the assay results are shown in Table 11.

TABLE-US-00011 TABLE 11 Binding antigen KD(M) Ka(1/Ms) Ka Error Kd(1/s) Kd Error R.sup.2 4E9V10-2B3V2 IL-4Rα 2.33E−10 1.98E+05 1.24E+03 4.60E−05 6.73E−06 0.994 4E9V10-2B3V2 IL-5 6.63E−10 2.13E+05 1.29E+03 1.41E−04 5.84E−06 0.9905

[0125] The results show that the affinity of the bispecific antibody targeting IL4Rα and IL-5 was determined with two antigens respectively, and the corresponding affinities were both below 1 nM.

[0126] The foregoing displays and describes basic principles, main features, and advantages of the present disclosure. A person skilled in the art may understand that the present disclosure is not limited to the foregoing embodiments. Descriptions in the embodiments and this specification only illustrate the principles of the present disclosure. Various modifications and improvements are made in the present disclosure without departing from the spirit and the scope of the present disclosure, and these modifications and improvements shall fall within the protection scope of the present disclosure. The protection scope of the present disclosure is subject to the appended claims and equivalents thereof.