CN105131116B - The humanization modified of targeting HER2 is internalized by single-chain antibody P1h3 and preparation method and application - Google Patents
The humanization modified of targeting HER2 is internalized by single-chain antibody P1h3 and preparation method and application Download PDFInfo
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Abstract
Humanization modified the invention discloses targeting HER2 is internalized by single-chain antibody P1h3 and its preparation method and application, belongs to antitumor technical field.It is disclosed by the invention it is humanization modified be internalized by single-chain antibody P1h3, amino acid sequence is as shown in SEQ.ID.NO.1；The acquisition of the antibody is that the CDR of mouse single-chain antibody e23sFv is transplanted to the FR of the highest human antibody consensus sequence of homology, to be reselection the mouse amino acid residue in e23sFv to maintaining 13 important amino acid residue mutations of antibody space conformation in FR, structure storage capacity is 213Single chain antibody phage shows library, and filtering out 1 plant has high affine and internalization activity Humanized single chain antibody P1h3, lays a solid foundation for the application in clinical diagnosis and therapy field.
Description
Technical field
The invention belongs to antitumor technical fields, are related to a kind of anti-human HER2 single-chain antibodies, more particularly to a kind of through people source
Change the anti-human HER2 single-chain antibodies of transformation being internalized by, including its amino acid sequence and nucleotide sequence and preparation method for antibody
With its diagnosis and treatment purposes to HER2 positive tumor cells.
Background technology
HER2 (human epidermal growth factor receptor-2, ErbB2/P185) is human epidermal growth
Second member of factor acceptor (EGFR) family, is encoded by proto-oncogene erbB2/Her2, and encoding gene is positioned at people's dyeing
Body 17q21, molecular weight 185kDa are a kind of tyrosine kinase receptor membrane glycoproteins.HER2 by with HER1, HER3 or HER4
Heterodimer is formed, the signal paths such as downstream MAPK and PI3K is caused to activate, growth signals is excessively conducted and is disliked so as to cause cell
Property proliferation.In mankind's kinds of tumors, including 25-30% breast cancer, 35-45% cancers of pancreas and 90% colorectal cancer, 16-
57% non-small cell lung cancer, gastric cancer of 9-38% etc. have had been found that the amplification of HER2 proto-oncogenes or protein overexpression phenomenon,
Clinically it is known as HER2 positive tumors, is mainly shown as malignancy height, easily progress and transfer, it is insensitive to chemicotherapy,
Easily recurrence, patient survival are short.
1975, Kohler and Milstein invented for prepare monoclonal antibody (Monoclonal antibody,
MAb hybridoma technology), later monoclonal antibody medicine rapidly develop and be applied to clinic.In recent years, the antibody drug of HER2 is targeted
Have become HER2 positive tumors and treats new hot spot.Herceptin (herceptin, English name Trastuzumab/Herceptin)
It is the anti-HER 2 humanized monoclonal antibody drug of Genetech companies exploitation, U.S. FDA is bent at present in approval listing in 1998
Trastuzumab combined chemotherapy drug (taxol etc.) has become HER2 and is overexpressed advanced breast cancer and late gastric cancer
First-line treatment scheme, in the treatment of HER2 positive metastatic breast cancer, up to 38%, European EMEA has been criticized clinical effective rate
First-line treatment scheme of the quasi- Trastuzumab combined chemotherapies drug as treatment HER2 positive advanced gastric carcinomas.
Although Trastuzumab clinical application achieve it is encouraging as a result, have a large amount of patients to treat nothing
It is recurred after reaction, or treatment.Studies have reported that mouse monoclonal antibody, which is applied to the mankind, stronger immunogenicity, body can be caused to exempt from
Epidemic disease system to the immunological rejection of the foreign protei, generate human anti-mouse antibody (Human anti-mouse antibody,
HAMA it) reacts, the anaphylactic shock of serious patient can be even caused when reuse.And due to the presence of HAMA, mouse monoclonal antibody
It can be removed quickly in human body, half-life period is very short, therefore limits its clinical application.Trastuzumab is chimeric for people mouse
Antibody, but its variable region portion is mouse, can cause human body and generate HAMA, there is metastatic breast cancer prolonged application
The research report of Trastuzumab treatments points out that cardiac toxic occurred in about 25% patient；Third stage test result
Show that HER2 positive breast cancer patients only have about 1/3 pair of Trastuzumab single-dose treatment to have definite curative effect；In clinic, i.e.,
Make combined chemotherapy, most patient just will produce drug resistance being treated in 1 year using Trastuzumab.
As relationship is understood in depth between all kinds of antibody structures and function, antibody engineering technical antagonism body structure is utilized
It is transformed, by the monoclonal antibody human source (Monoclonal humanization) of animal origin, to reduce these monoclonal antibodies
The immunogenicity for being applied to generate after human body repeatedly is allowed to the treatment for human diseases, is the hot spot studied at present.It is anti-
The humanization modified main policies of body have following several：
1. chimeric antibody
The constant region of antibody is the strongest position of immunogenicity in antibody molecule structure, with technique for gene engineering that mouse is single
Anti- variable region and the constant region of human antibody are mutually spliced, and simplest form people/mouse chimeric antibody of mouse monoclonal antibody humanization is become
(chimeric antiboay).Chimeric antibody greatly reduces the immunogenicity of mouse source monoclonal antibody, while remaining mouse monoclonal antibody again
Compatibility and specificity.Since chimeric antibody still remains the mouse source sequence of original mouse source antibody about 30% or so, immunogene
Though property decreases, but still different degrees of HAMA can be caused to react.
2. the humanized antibody of complementary determining region (complementary determining region, CDR) transplanting
The light and heavy chain variable region of each antibody molecule Fab section respectively have 3 antigen complementary determining regions (CDR1, CDR2 and
CDR3), there is the framework region (framework region, FR) that stable structure acts between them, CDR can be in direct contact anti-
Original determines the specificity of antibody, its three-dimensional conformation is extremely conservative as the holder of support CDR by FR.CDR grafted antibody is mouse list
Anti- complementary determining region (CDR) is transplanted to the built-up antibody of skeleton area FR of people's monoclonal antibody, clinical tests prove that, it is immunized
Originality is significantly reduced.
3. the reconstruct of people source FR templates
1. template replace, in existing antibody sequence library search with mouse mAb FR have the people source FR of maximum homology to
It replaces.Light and heavy chain can make have better homology between mouse source CDR and people's template in this way usually from different human antibodies.Ying Jiang
The amino acid residue closely related with CDR is retained in the people source FR of replacement in mouse FR.In order to keep the space conformation of CDR,
Pay special attention to the residue around accumulation residue and the CDR below original antibodies CDR.2. Compensation Transformation, Compensation Transformation can be recognized
There to be interaction with CDR in people's FR selections to be the further extension that template is replaced, have close pass with affinity of antibody
System or the residue to play a crucial role to the folding of FR space structures are changed, and are transplanted with compensating complete CDR.3. positioning retains, most
Simple situation template (minimal positional template) can confirm which position is to maintain antibody in antibody variable region
Antigen-binding domains integrality necessary to, selection for humanization people FR when, first from existing human antibody
It is searched in conserved sequence and sequence most like mouse FR；Secondly determine that the key position of mouse variable region is residual according to most simple situation template
Base；Finally retain all these positions and by rest part humanization.
4. surface is remolded
The amino acid residue in the inhuman source in non-Human monoclonal antibody variable region (Fv) surface is replaced with to the amino acid residue of humanized,
The surface humanization for making the areas non-human source antibodies Fv, reduces its immunogenicity, while not influencing the overall space conformation in the areas Fv, to
Retain the structure of its antigen-binding site.
5. epitope M8003 line (epitope-guided selection)
The light chain of mouse antibody or heavy chain library and the heavy chain or light chain libraries of human antibody are matched, are built into that " people -- mouse is miscellaneous
Conjunction antibody library ", the clone of screening and antigen binding, separation obtain the heavy chain or light chain gene of human antibody, then by them with people's
Light chain or heavy chain library mixing, with antigen selection, can obtain the human antibodies specific with antigen binding.
Invention content
The purpose of the present invention is to provide a kind of targeting HER2 it is humanization modified be internalized by single-chain antibody P1h3 and its
Preparation method and application.
A kind of the humanization modified of targeting HER2 disclosed by the invention is internalized by single-chain antibody P1h3, amino acid sequence
As shown in SEQ.ID.NO.1.
The invention also discloses the nucleotide sequences for being internalized by single-chain antibody P1h3 of encoding humanized transformation, such as
Shown in SEQ.ID.NO.2.
The invention also discloses it is a kind of prepare targeting HER2 the humanization modified method for being internalized by single-chain antibody P1h3,
Include the following steps：
1) CDR of mouse single-chain antibody e23sFv is transplanted to the FR of the highest human antibody consensus sequence of homology；
2) the mouse amino in e23sFv will be sported to 13 amino acid residues of maintenance antibody space conformation in FR
Sour residue, structure storage capacity are 213Single chain antibody phage shows library；
3) the people source with high affine activity and high internalization activity is filtered out from obtained single chain antibody phage displaying library
Change single-chain antibody P1h3.
The invention also discloses the humanization modified single-chain antibody P1h3 of above-mentioned targeting HER2 to prepare antineoplastic
Application in object and/or health products, and the humanization modified single-chain antibody P1h3 of targeting HER2 are swollen in the preparation HER2 positives
Application in tumor diagnostic reagent.
The drug and/or health products is the drug and/or health products of anti-HER2 positive tumor cells.
The HER2 positive tumor cells are that breast cancer cell, stomach cancer cell, non-small cell lung cancer cell or oophoroma are thin
Born of the same parents etc..
Compared with prior art, the present invention has technique effect beneficial below：
1, the present invention has carried out people to the immune apoptosis molecule Immuno-Fdt-tBid single chain antibody portions e23sFv that promotees for the first time
Sourceization is transformed, and e23 is that HER2 molecule extracellular fragments are immunized affinity in the hybridoma prepared after mouse and inhibit tumour efficiency best
Clone, still belong to the first time to the humanization modified of single chain antibody format e23sFv of e23.
2, it will be replaced with the FR of the highest human antibody consensus sequence FR and e23sFv of VL, VH homology of e23sFv
It changes, while selecting 13 sites FR to play an important role to antibody conformation, the amino acid residue of original e23sFv is sported, with most
Limits ensure the correct folding of single-chain antibody conformation.
3, structure storage capacity is 213Mutant single-chain antibody library, filtered out using display technique of bacteriophage affine to HER2
The high Humanized single chain antibody strain P1h3 of power ensures can there is higher affinity by humanization modified single-chain antibody.
4, it is verified through humanization modified P1h3 to HER2 molecules from molecule to cellular level using kinds of experiments means
Affine activity and internalization compare itself and mouse source single-chain antibody e23sFv immunogenicities into the activity of HER2 positive tumor cells
Size, it is ensured that the molecule finally screened has higher affine, internalization activity and lower immunogenicity, for further structure target
To the immune rush apoptosis molecule of HER2 molecules, and realizes and lay the foundation in clinical application.
Description of the drawings
Fig. 1 is that full-length human single-chain antibody hue23sFv amino acid sequences build flow diagram；
Fig. 2 is the mutant primer sequence results of Humanized single chain antibody mutant；
Fig. 3 is multisite mutation PCR Gel electrophoresis results figures；
Fig. 4 is the structure and 4 wheel elutriation result figures of humanization single chain antibody phage antibody library；
Fig. 5 is that Phage ELISA screen the highest single-chain antibody strain result of affinity；
Fig. 6 is the prokaryotic expression and purification result of 4 plants of Humanized single chain antibodies；
Fig. 7 is that SPR measures Humanized single chain antibody to recombined human HER2 molecule affinity costants；
Wherein (a) is affinity costant KD 4.512*10s of the e23sFv to recombined human HER2 molecules-8M；(b) it is P1h3 counterweights
The affinity costant KD4.787*10 of group people's HER2 molecules-10M；(c) be e23sFv, P1h2, P1h3, P2h2, P2h5 to recombined human
The combination dissociation capability of HER2 molecules is distributed.
Fig. 8 is the affine activity that ELISA measures Humanized single chain antibody to recombined human HER2 molecules；
Fig. 9 is the specificity that Flow cytometry Humanized single chain antibody combines HER2 positive tumor cells；
Figure 10 is that laser confocal microscope detects Humanized single chain antibody internalization into HER2 positive tumor cell activity；
Wherein, (a) is that e23sFv, P1h3 internalization enter HER2 positive breast cancer cells BT-474；Be (b) e23sFv,
P1h3 internalizations enter HER2 positive ovarian cancer cells SKOV-3；(c) it cannot be internalized by into HER2 negative breasts for e23sFv, P1h3
Cancer cell MCF-7.
Figure 11 is the level for stimulation human PBMC's generation IFN-γ that ELISPOT detects Humanized single chain antibody；
Figure 12 is human peripheral multiple cytokine detection evaluation Humanized single chain antibody immunogenicity result；
Wherein, (a) is the level that e23sFv, P1h2, P1h3, P2h2, P2h5 stimulate Healthy People PBMC TNF secretions-α；(b)
The level of Healthy People PBMC secretion of gamma-IFN is stimulated for e23sFv, P1h2, P1h3, P2h2, P2h5；Be (c) e23sFv, P1h2,
P1h3, P2h2, P2h5 stimulate the level of Healthy People PBMC secretions IL-10；(d) it is that e23sFv, P1h2, P1h3, P2h2, P2h5 are pierced
Swash the level of Healthy People PBMC secretions IL-2；(e) it is e23sFv, P1h2, P1h3, P2h2, P2h5 stimulation Healthy People PBMC secretions
The level of IL-4；(f) level for being e23sFv, P1h2, P1h3, P2h2, P2h5 stimulation Healthy People PBMC secretions IL-5；(g) it is
E23sFv, P1h2, P1h3, P2h2, P2h5 stimulate the level of Healthy People PBMC secretions IL-17a；Be (h) e23sFv, P1h2,
P1h3, P2h2, P2h5 stimulate the level of Healthy People PBMC secretions IL-21.
Specific embodiment：
One, the structure of Humanized single chain antibody and its mutant bacteriophage antibody library
1, the determination of Humanized single chain antibody sequence
The light chain variable region of e23sFv is subjected to BLAST, is found and the highest human antibody light variable region of its homology
Sequence, with the framework region (FR) of the affiliated subclass consensus sequence in this human antibody light variable region (concensus sequence)
The FR for replacing e23sFv, builds the light chain huVL of full-length human.The replacement of same procedure, structure are carried out to the heavy chain of e23sFv
The heavy chain huVH of full-length human, and connect with Linker with huVL, the hue23sFv of full-length human is built, as shown in Figure 1.
2, the determination of Humanized single chain antibody mutant sequence
E. coli codon optimization is carried out to the gene order of hue23sFv, full genome synthesis is built into pUC19 carriers
In.13 sites that antibody structure plays an important role will be respectively synthesized using the method for multipoint mutation in the FR of hue23sFv
Mutant primer, mutant primer sequence are as shown in Figure 2.It is each using Multi-site directed mutation kit (Takara)
A amino acid residue for sporting original mouse source, structure 213A mutant, as shown in Figure 3.
3, the structure of Humanized single chain antibody mutant bacteriophage antibody library
Using Sfi I and Not I as restriction enzyme site, hue23sFv and mutant digestion are connected into pCANTAB5E bacteriophages
Carrier, Electroporation-competent cells TG1 after being transferred to 2*YTAG culture medium amplification cultivations to exponential phase, are added auxiliary and bite
Thalline M13K07 infects 1h, and centrifugation is abandoned supernatant, is resuspended with 2*YTAK, 37 DEG C of overnight incubations, and collection supernatant, which is surface display, to be had
The phage library of single-chain antibody, it is 5*10 to measure its phage titre11CFU。
Two, the screening and identification of high-affinity Humanized single chain antibody
The phage antibody library of Humanized single chain antibody and 37 DEG C of elisa plate for being coated with HER2 molecules are incubated 2h altogether,
0.05%Tween20PBS is washed 5 times, and exponential phase TG1 bacterium solutions are added and are incubated 1h in 37 DEG C of slow shake, make in conjunction with HER2's
ScFv- bacteriophages infect TG1 and are enriched with again.The bacterial cultures for having infected scFv- bacteriophages is taken to measure titre, while with auxiliary
Secondary ScFv- phage libraries are prepared after helper phage M13K07 infection, carry out elutriation again, and be stepped up washing times, altogether
Four-wheel elutriation is carried out, the phage titre of often wheel elutriation input and output is measured and calculates yield, as shown in Figure 4.
ELISA Screening are carried out to the bacteriophage that is combined with HER2 after four-wheel screens, filter out 20 plants it is affine
Highest 4 plants of the higher phage antibody of power, wherein affinity is P1h2, P1h3, P2h2, and P2h5 is as shown in figure 5, P1h3 amino
Acid sequence is as shown in SEQ.ID.NO.1.
Three, the prokaryotic expression of Humanized single chain antibody, purifying and renaturation
4 plants of phage single-chain antibody sequences that elutriation obtains are cloned into former expression vector pET28a, 1mM IPTG inductions
It is expressed with inclusion bodies, inclusion body, through Ni-NTA affinitive layer purifications, is removed with slow dialysis and become with 6M GuCl denaturation
Property agent method make protein renaturation to obtain active single-chain antibody, SDS-PAGE detects single-chain antibody purity up to 90%
More than, as shown in Figure 6.
Four, surface plasma resonance technology (SPR) measures affinity of the Humanized single chain antibody to HER2 molecules
Affinity costant KD of the Humanized single chain antibody to HER2 molecules is measured using Biacore T100 (GE).CM5 chips
Coupling capture antibody anti-human IgG (Fc) are further coated with recombined human HER2 albumen (Fc tag), prepare concentration respectively
For 32nM, 16nM, 8nM, 4nM, 2nM single-chain antibody as mobile phase, single cycle kinetics model measures itself and HER2 molecules
In conjunction with the size for causing response (RU), to calculate e23sFv and Humanized single chain antibody to HER2 affinity constants.Such as Fig. 7
Shown, the affinity constant of e23sFv is KD 4.512*10-8The affinity constant of M, P1h3 are KDP1h34.787*10-10M,
The affinity constant of P1h3 is apparently higher than e23sFv.
Five, ELISA detects affine activity of the Humanized single chain antibody to cell surface HER2 molecules
96 orifice plates are spread with HER2 positive tumor cells BT-474, per hole cell number 1*104, 37 DEG C are cultivated for 24 hours, are abandoned in culture
Clearly, 4% paraformaldehyde room temperature fixes 20min, and 5% skimmed milk power room temperature closes 1h, and it is anti-that 3 times of doubling dilution Humanized single chains are added
Body, maximum concentration 3uM, 4 DEG C of overnight incubations, PBST are washed 6 times, are added 1:The primary antibody of 1000 diluted anti-His is incubated at room temperature 2h,
PBST is washed 6 times, is added 1:The secondary antibody of 2500 diluted HRP labels is incubated at room temperature 1h, and PBST is washed six times, TMB color development at room temperature
10min measures OD450 after color development stopping, as shown in figure 8, single-chain antibody P1h3 reaches plateau at first, is higher than e23sFv, carries
Show that P1h3 has very strong affine activity to HER2 molecules and is higher than parent's mouse single-chain antibody e23sFv.
Six, the specificity that Flow cytometry Humanized single chain antibody combines HER2 positive tumor cells
Fluorescein Dylight 488 marks single-chain antibody, and it is respectively e23sFv 73.3% to measure labeling effciency,
P1h274.6%, P1h372.1%, P2h273.8%, P2h575.2%.By fluorescein-labeled Humanized single chain antibody P1h3
And mouse source single-chain antibody e23sFv is incubated with HER2 positive tumor cells BT-474 and SKOV-3 altogether respectively, HER2 negative tumours are thin
Born of the same parents MCF-7 is as negative control.Simultaneously by unlabelled e23sFv with two kinds of different final concentrations (125nM, 250nM) and fluorescein
Be incubated with BT-474 and SKOV-3 after the Humanized single chain antibody P1h3 mixing of label, on ice 1h, streaming wash liquid three times,
1000rpm/min is centrifuged, and 500ulPBS is resuspended, upper machine testing.As shown in figure 9, P1h3 can be with the surfaces BT-474 and SKOV-3
HER2 molecules combine, and it can be blocked the combination of HER2 by the e23sFv of various concentration, and dosage is presented in blocking effect
The characteristics of dependence, prompts the epitope of humanization modified single-chain antibody P1h3 combinations HER2 molecules not occur compared with e23sFv bright
Aobvious variation.
Seven, internalization activity of the laser confocal microscope detection Humanized single chain antibody to HER2 positive tumor cells
The cell climbing sheet for preparing HER2 positive tumor cells BT-474 and SKOV-3, exists with fluorescein-labeled single-chain antibody
37 DEG C are incubated 4h altogether, and each strain single-chain antibody internalization of confocal laser scanning microscope enters HER2 positive tumor cells after mounting
Activity, HER2 negative tumor cells MCF-7 is as negative control.As shown in Figure 10, BT-474 the and SKOV-3 born of the same parents that P1h3 is incubated
Interior visible apparent fluorescence distribution prompts P1h3 to have preferable internalization activity.
Eight, ELISPOT evaluates Humanized single chain antibody immunogenicity
12 healthy human peripheral bloods are taken, lymphocyte separation medium detaches mononuclearcell (PBMC), respectively with 3uM
For 24 hours in 37 DEG C of stimulations, ELISPOT detects PBMC secretion of gamma-IFN water by e23sFv, P1h2, P1h3, P2h2, P2h5 (PBS dilutions)
It is flat.As a result the positive cell frequency that prompt Humanized single chain antibody P1h3 stimulations PBMC generates IFN-γ has compared with parent e23sFv
It significantly reduces, difference has statistical significance, as shown in figure 11.
Nine, human peripheral cytokine profiles detection evaluation Humanized single chain antibody immunogenicity
12 healthy human peripheral bloods are taken, lymphocyte separation medium detaches mononuclearcell (PBMC), respectively with 3uM
E23sFv and Humanized single chain antibody P1h2, P1h3, P2h2, P2h5 are stimulated for 24 hours in 37 DEG C, collect cells and supernatant,
LEGEND plex kit measurement cell factors IL-2, IL-4, IL-5, IL-10, IL-17a, IL-21, IFN-γ, TNF-α are dense
Degree compares the variation of e23sFv and P1h2, P1h3, P2h2, P2h5 stimulation PBMC cytokine secretion amounts.As a result humanization is prompted
Single-chain antibody P1h2 stimulation PBMC generations IL-2, IL-10, TNF-α, the level of IFN-γ have compared with parent e23sFv obviously to be subtracted
It is few, it prompts after humanization modified, the ability that single-chain antibody P1h3 causes human body generation immune response is decreased obviously, difference tool
It is statistically significant, as shown in figure 12.
In conclusion the present invention provides a kind of single-chain antibody P1h3 that the humanization of targeting HER2 can be internalized by, the antibody
Acquisition is that the CDR of mouse single-chain antibody e23sFv is transplanted to the FR of the highest human antibody consensus sequence of homology, reselection
Property will in FR to maintain antibody space conformation 13 important amino acid residue mutations be e23sFv in mouse amino acid
Residue, structure storage capacity are 213Single chain antibody phage shows library, filters out 1 plant of people source with high-affinity and internalization activity
Change single-chain antibody P1h3, lays a solid foundation for the application in clinical diagnosis and therapy field.
Claims (4)
1. a kind of the humanization modified of targeting HER2 is internalized by single-chain antibody P1h3, which is characterized in that described humanization modified
The amino acid sequence for being internalized by single-chain antibody P1h3 as shown in SEQ.ID.NO.1.
2. a kind of nucleic acid molecules, which is characterized in that the nucleic acid molecule encoding is described in claim 1 humanization modified to be internalized by
Single-chain antibody P1h3, and the nucleotide sequence of the nucleic acid molecules is as shown in SEQ.ID.NO.2.
3. the humanization modified single-chain antibody P1h3 that is internalized by of targeting HER2 described in claim 1 is preparing antitumor drug
In application, which is characterized in that the antitumor drug be anti-HER2 positive tumor cells drug.
4. the humanization modified single-chain antibody P1h3 that is internalized by of targeting HER2 described in claim 1 swells in the preparation HER2 positives
Application in tumor diagnostic reagent.
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| CN101928345A (en) * | 2010-06-21 | 2010-12-29 | 中国科学技术大学 | Human source antibody and humanization remolding method thereof |
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| CN101928345A (en) * | 2010-06-21 | 2010-12-29 | 中国科学技术大学 | Human source antibody and humanization remolding method thereof |
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