CN105273084A - Targeting HER2 humanized non-internalized single chain antibody P2h2 and application thereof - Google Patents
Targeting HER2 humanized non-internalized single chain antibody P2h2 and application thereof Download PDFInfo
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Abstract
The invention discloses a targeting HER2 humanized non-internalized single chain antibody P2h2 and an application thereof, and belongs to the technical field of antitumor. The amino acid sequence of the humanized non-internalized single chain antibody P2h2 is represented by SEQ. ID. NO.1. The antibody is obtained through the following steps: transplanting CDR of a mouse single chain antibody e23sFv to FR of a human body consensus sequence with highest homology, selectively mutating 13 important amino acid residues maintaining the spatial conformation of the antibody in the FR to mouse amino acid residues in the e23sFv, constructing a single chain antibody phage display database with the database capacity of 213, and screening the humanized non-internalized single chain antibody P2h2 with high affinity activity. A good foundation is laid for application in clinic diagnosis and treatment fields.
Description
Technical field
The invention belongs to antitumor technical field, relate to a kind of anti-human HER2 single-chain antibody, in particular to a kind of through the anti-human HER2 single-chain antibody of humanization modified non-internalization, comprise its aminoacid sequence and nucleotide sequence and preparation method for antibody with it to the diagnosis and treatment purposes of HER2 positive tumor cell.
Background technology
HER2 (humanepidermalgrowthfactorreceptor-2, ErbB2/P185) be second member of Human epidermal growth factor receptor (EGFR) family, encoded by proto-oncogene erbB2/Her2, encoding gene is positioned human chromosome 17q21, molecular weight is 185kDa, is a kind of tyrosine kinase receptor membrane glycoprotein.HER2, by forming heterodimer with HER1, HER3 or HER4, causes the signal path activation such as downstream MAPK and PI3K, excessively conducts growth signals thus causes undesired cell proliferation.In mankind's kinds of tumors, comprise 25-30% mammary cancer, 35-45% carcinoma of the pancreas and 90% colorectal carcinoma, the nonsmall-cell lung cancer of 16-57%, the cancer of the stomach etc. of 9-38% all find that there is the amplification of HER2 proto-oncogene or protein overexpression phenomenon, be called HER2 positive tumor clinically, main manifestations is that malignancy is high, easy progress and transfer, insensitive to chemicotherapy, easily recur, the survival of patients phase is short.
1975, Kohler and Milstein invented the hybridoma technology being used for preparing monoclonal antibody (Monoclonalantibody, mAb), and monoclonal antibody medicine develops rapidly and is applied to clinical afterwards.In recent years, the antibody drug of target HER2 has also become HER2 positive tumor and has treated new focus.Herceptin (He Saiting, English name Trastuzumab/Herceptin) be the anti-HER 2 humanized monoclonal antibody drug that Genetech company develops, U.S. FDA was in approval listing in 1998, current Herceptin combined chemotherapy medicine (taxol etc.) has become the first-line treatment scheme of HER2 process LAN advanced breast cancer and late gastric cancer, in the treatment of HER2 positive metastatic mammary cancer, clinical effective rate can reach 38%, Europe EMEA has ratified the first-line treatment scheme of Trastuzumab combined chemotherapy medicine as the positive advanced gastric carcinoma for the treatment of HER2.
Although the clinical application of Trastuzumab achieves challenging result, still have a large amount of patient reactionless to treatment, or recurrence after treatment.Studies have reported that mouse monoclonal antibody is applied to the mankind and has stronger immunogenicity, body immune system can be caused the immunological rejection of this foreign protein, human anti-murine antibodies (Humananti-mouseantibody, HAMA) react, when reusing, even can cause the anaphylactic shock of serious patient.And due to the existence of HAMA, mouse monoclonal antibody can be removed very soon in human body, and the transformation period is very short, because which limit its clinical application.Trastuzumab is human mouse chimeric antibody, but its variable region portion is mouse, and can cause human body and produce HAMA, the research report having metastatic breast cancer prolonged application Trastuzumab to treat points out that cardiac toxic appearred in the patient of nearly 25%; Third stage test-results shows, HER2 positive breast cancer patient only has about 1/3 pair of Trastuzumab single-dose treatment to have definite curative effect; Clinical, even if combined chemotherapy, most patient is treated in 1 year will produce resistance at use Trastuzumab.
Along with understanding in depth relation between all kinds of antibody structure and function, antibody engineering technical antagonism body structure is utilized to transform, by the monoclonal antibody human source (Monoclonalhumanization) of animal-origin, to reduce the treatment that the immunogenicity produced after these monoclonal antibodies are applied to human body repeatedly makes it for human diseases, it is a focus of research at present.The main policies of antibody humanization's transformation has following several:
1. chimeric antibody
The constant region of antibody is the position that in antibody molecule structure, immunogenicity is the strongest, use genetic engineering technique to be spliced mutually with the constant region of people's antibody mouse monoclonal antibody variable region, namely become the humanized most simple form people/mouse chimeric antibody (chimericantiboay) of mouse monoclonal antibody.Chimeric antibody greatly reduces the immunogenicity of mouse source monoclonal antibody, remains again affinity and the specificity of mouse monoclonal antibody simultaneously.Because chimeric antibody still remains the mouse source sequence of original mouse source antibody about about 30%, though its immunogenicity decreases, but still HAMA reaction in various degree can be caused.
2. complementary determining region (complementarydeterminingregion, CDR) humanized antibody of transplanting
Light, the variable region of heavy chain of each antibody molecule Fab section respectively have 3 antigen complementary determining regions (CDR1, CDR2 and CDR3), framework region (the frameworkregion of Stability Analysis of Structures effect has been had between them, FR), CDR directly can contact antigen, determine the specificity of antibody, FR is as supporting that its three-dimensional conformation of support of CDR is very conservative.CDR grafted antibody is the antibody that the skeleton district FR that the complementary determining region of mouse monoclonal antibody (CDR) is transplanted to people's monoclonal antibody is built, and prove through clinical trial, its immunogenicity is significantly reduced.
3. the reconstruct of people source FR template
1. template is replaced, and searching in existing antibody sequence storehouse has the people source FR of maximum homology in order to replace with mouse mAbFR.Gently, heavy chain usually from different people's antibody, can make there is better homology between mouse source CDR and people's template like this.Should closely-related amino-acid residue be retained in the people source FR of replacement with CDR by mouse FR.In order to keep the space conformation of CDR, the accumulation residue below original antibodies CDR and the residue around CDR be paid special attention to.2. Compensation Transformation, Compensation Transformation can think the further extension that template is replaced, in people FR selection, there is interaction by with CDR, have substantial connection or the residue played a crucial role is folded to FR space structure change with affinity of antibody, transplant to compensate complete CDR.3. reservation is located, the simplest situation template (minimalpositionaltemplate) can confirm which position keeps the antigen-binding domains integrity of antibody necessary in antibody variable region, when selecting to be used for humanized people FR, first from existing human antibody conserved sequence, search the sequence the most similar to mouse FR; Secondly the key position residue of mouse variable region is determined according to the simplest situation template; Finally retain all these positions and by rest part humanization.
4. surface is reinvented
The amino-acid residue in the non-surperficial inhuman source of Human monoclonal antibody variable region (Fv) is replaced with the amino-acid residue of humanized, make the surperficial humanization in non-human source antibodies Fv district, reduce its immunogenicity, do not affect the overall space conformation in Fv district simultaneously, thus retain the structure of its antigen-binding site.
5. epitope orthoselection (epitope-guidedselection)
The heavy chain of the light chain of murine antibody or heavy chain library and people's antibody or light chain libraries are matched, be built in " people one mouse hybrid antibody storehouse ", screen the clone be combined with antigen, be separated the heavy chain or light chain gene that obtain people's antibody, again their light chains with people or heavy chain library are mixed, with antigen selection, the human antibodies specific be combined with antigen just can be obtained.
Summary of the invention
The object of the present invention is to provide humanization modified non-internalization single-chain antibody P2h2 and the application of a kind of target HER2.
The humanization modified non-internalization single-chain antibody P2h2 of a kind of target HER2 disclosed by the invention, its aminoacid sequence is as shown in SEQ.ID.NO.1.
Encode the nucleotide sequence of humanization modified non-internalization single-chain antibody P2h2 of above-mentioned target HER2 as shown in SEQ.ID.NO.2.
The humanization modified non-internalization single-chain antibody P2h2 that the invention also discloses above-mentioned target HER2 is preparing the application in antitumor drug and/or healthcare products, and the application of the humanization modified non-internalization single-chain antibody P2h2 of target HER2 in preparation HER2 positive tumor diagnostic reagent.
Described medicine and/or healthcare products are medicine and/or the healthcare products of anti-HER2 positive tumor cell.
Described HER2 positive tumor cell is breast cancer cell, stomach cancer cell, non-small cell lung cancer cell or ovarian cancer cell etc.
Compared with prior art, the present invention has following useful technique effect:
1, the present invention carries out humanization modified first to mouse anti-human HER2 single-chain antibody e23sFv, e23 is avidity and the best clone of Tumor suppression efficiency in the hybridoma prepared after HER2 molecule extracellular fragment immune mouse, still belongs to the first time to the humanization modified of single chain antibody format e23sFv of e23.
2, the FR of human antibody consensus sequence FR and the e23sFv the highest with VL, VH homology of e23sFv is replaced, select 13 FR sites that antagonist conformation plays an important role simultaneously, sport the amino-acid residue of original e23sFv, correct folding with bonding chain antibody conformation to greatest extent.
3, building storage capacity is 2
13mutant single-chain antibody library, utilize display technique of bacteriophage to filter out the Humanized single chain antibody strain P2h2 high to HER2 avidity, ensure can have higher avidity through humanization modified single-chain antibody.
4, kinds of experiments means are utilized to verify from molecule to cell levels through humanization modified P2h2 to the affinity of HER2 molecule and whether have internalization activity, compare itself and the immunogenic size of mouse source single-chain antibody e23sFv, guarantee that the molecule of last screening has higher affinity and lower immunogenicity, for the antitumor diagnosis and treatment New Policy researching and developing target HER2 further lays the foundation.
Accompanying drawing explanation
Fig. 1 is that full-length human single-chain antibody hue23sFv aminoacid sequence builds FB(flow block);
Fig. 2 is the mutant primer sequence results of Humanized single chain antibody mutant;
Fig. 3 is multisite mutation PCR Gel electrophoresis results figure;
Fig. 4 is that elutriation result figure taken turns by the structure and 4 of humanization single chain antibody phage antibody library;
Fig. 5 is that PhageELISA screens the highest single-chain antibody strain result of avidity;
Fig. 6 is prokaryotic expression and the purification result of 4 strain Humanized single chain antibodies;
Fig. 7 is that SPR measures Humanized single chain antibody to recombinant human HER2 molecule affinity costant;
Wherein (a) is for e23sFv is to the affinity costant KD4.512*10 of recombinant human HER2 molecule
-8m; B () is for P2h2 is to the affinity costant KD1.559*10 of recombinant human HER2 molecule
-9m; C () is e23sFv, P1h2, P1h3, P2h2, P2h5 distributing in conjunction with dissociation capability to recombinant human HER2 molecule.
Fig. 8 is that ELISA measures Humanized single chain antibody to the affinity of recombinant human HER2 molecule;
Fig. 9 is the specificity that Flow cytometry Humanized single chain antibody combines HER2 positive tumor cell;
Figure 10 is that laser confocal microscope detection Humanized single chain antibody internalization enters HER2 positive tumor cell activity;
Wherein, (a) enters HER2 positive breast cancer cells BT-474 for e23sFv, P2h2 internalization; B () enters HER2 positive ovarian cancer cells SKOV-3 for e23sFv, P2h2 internalization; C () internalization can not enter HER2 negative breast cancer cells MCF-7 for e23sFv, P2h2.
Figure 11 is the level that ELISPOT detects that the stimulation human PBMC of Humanized single chain antibody produces IFN-γ;
Figure 12 is human peripheral multiple cytokine checking and appraising Humanized single chain antibody immunogenicity result;
Wherein, (a) is the level of e23sFv, P1h2, P1h3, P2h2, P2h5 stimulation Healthy People PBMC TNF secretion-α; B () is the level of e23sFv, P1h2, P1h3, P2h2, P2h5 stimulation Healthy People PBMC secretion of gamma-IFN; C () is the level of e23sFv, P1h2, P1h3, P2h2, P2h5 stimulation Healthy People PBMC secretion IL-10; D () is the level of e23sFv, P1h2, P1h3, P2h2, P2h5 stimulation Healthy People PBMC secretion IL-2; E () is the level of e23sFv, P1h2, P1h3, P2h2, P2h5 stimulation Healthy People PBMC secretion IL-4; F () is the level of e23sFv, P1h2, P1h3, P2h2, P2h5 stimulation Healthy People PBMC secretion IL-5; G () is the level of e23sFv, P1h2, P1h3, P2h2, P2h5 stimulation Healthy People PBMC secretion IL-17a; H () is the level of e23sFv, P1h2, P1h3, P2h2, P2h5 stimulation Healthy People PBMC secretion IL-21.
Specific embodiments:
One, the structure of Humanized single chain antibody and mutant bacteriophage antibody library thereof
1, the determination of Humanized single chain antibody sequence
BLAST is carried out in the variable region of light chain of e23sFv, find the human antibody light variable region sequences the highest with its homology, replace the FR of e23sFv with the framework region (FR) of subclass consensus sequence (concensussequence) belonging to this human antibody light variable region, build the light chain huVL of full-length human.The heavy chain of e23sFv is carried out to the replacement of same procedure, build the heavy chain huVH of full-length human, and be connected with Linker with huVL, build the hue23sFv of full-length human, as shown in Figure 1.
2, the determination of Humanized single chain antibody mutant sequence
Carry out e. coli codon optimization to the gene order of hue23sFv, full genome synthesizes, and is built in pUC19 carrier.13 sites antagonist structure in the FR of hue23sFv played an important role, adopt the method for multipoint mutation, synthesize mutant primer respectively, mutant primer sequence as shown in Figure 2.Each sports the amino-acid residue in original mouse source to utilize Multi-sitedirectedmutationkit (Takara), builds 2
13individual mutant, as shown in Figure 3.
3, the structure of Humanized single chain antibody mutant bacteriophage antibody library
With SfiI and NotI for restriction enzyme site, hue23sFv and mutant enzyme are cut and connects into pCANTAB5E phage vector, Electroporation-competent cells TG1, be transferred to 2*YTAG substratum amplification cultivation to logarithmic growth after date, add helper phage M13K07 and infect 1h, centrifugally abandon supernatant, resuspended with 2*YTAK, 37 DEG C of overnight incubation, collect supernatant and be the phage library that surface display has single-chain antibody, measuring its phage titre is 5*10
11cFU.
Two, the screening of high-affinity Humanized single chain antibody and qualification
The phage antibody library of Humanized single chain antibody and the elisa plate 37 DEG C being coated with HER2 molecule are hatched 2h altogether, 0.05%Tween20PBS washs 5 times, add logarithmic phase TG1 bacterium liquid and hatch 1h in 37 DEG C of slow shaking, make the scFv-phage in conjunction with HER2 infect TGI again and be able to enrichment.Get the bacterial cultures infecting ScFv-phage and measure titre, the secondary ScFv-phage library of preparation after simultaneously infecting with helper phage M13K07, again carry out elutriation, and progressively increase washing times, carry out four-wheel elutriation altogether, measure the phage titre of often wheel elutriation input and output and calculate yield, as shown in Figure 4.
ELISAScreening is carried out to the phage be combined with HER2 after four-wheel screening, filter out the phage antibody that 20 strain avidity are higher, 4 strains that wherein avidity is the highest be P1h2, P1h3, P2h2, P2h5 as shown in Figure 5, P2h2 aminoacid sequence is as shown in SEQ.ID.NO.1.
Three, the prokaryotic expression of Humanized single chain antibody, purifying and renaturation
The 4 strain phage single-chain antibody sequence clones that elutriation obtains are entered former expression vector pET28a, 1mMIPTG induction is expressed with inclusion bodies, inclusion body with 6MGuCl sex change through Ni-NTA affinitive layer purification, the method removing denaturing agent slowly to dialyse makes protein renaturation thus obtains the activated single-chain antibody of tool, SDS-PAGE detects single-chain antibody purity and reaches more than 90%, as shown in Figure 6.
Four, surface plasma resonance technology (SPR) measures the avidity of Humanized single chain antibody to HER2 molecule
BiacoreT100 (GE) is utilized to measure the affinity costant KD of Humanized single chain antibody to HER2 molecule.CM5 chip coupling capture antibody antihumanIgG (Fc), further bag reorganized people HER2 albumen (Fctag), preparing concentration is respectively that the single-chain antibody of 32nM, 16nM, 8nM, 4nM, 2nM is as moving phase, single loop kinetics model measures it and is combined with HER2 molecule the size causing response value (RU), thus calculating e23sFv and Humanized single chain antibody are to HER2 affinity constant.As shown in Figure 7, the affinity constant KD4.512*10 of e23sFv
-8the affinity constant of M, P2h2 is 1.559*10
-9the affinity constant of M, P2h2 is apparently higher than e23sFv.
Five, ELISA detects Humanized single chain antibody to the affinity of cell surface HER2 molecule
96 orifice plates are spread, every porocyte number 1*10 with HER2 positive tumor cell BT-474
4, cultivate 24h for 37 DEG C, abandon culture supernatant, 4% paraformaldehyde room temperature fixes 20min, 5% skim-milk room temperature closes 1h, add 3 times of doubling dilution Humanized single chain antibodies, maximum concentration 3uM, 4 DEG C of overnight incubation, PBST washes 6 times, add the primary antibodie incubated at room 2h of the anti-His of 1:1000 dilution, PBST washes 6 times, add two anti-incubated at room 1h of the HRP mark of 1:2500 dilution, PBST washes six times, TMB color development at room temperature 10min, OD450 is measured after color development stopping, as shown in Figure 8, single-chain antibody P2h2 comparatively mouse source e23sFv more early arrives plateau and platform numerical value is larger, point out P2h2 to HER2 molecule affinity higher than e23sFv.
Six, Flow cytometry Humanized single chain antibody specificity that HER2 positive tumor cell is combined
Fluorescein Dylight488 marks single-chain antibody respectively, measures labeling effciency and is respectively e23sFv73.3%, P1h274.6%, P1h372.1%, P2h273.8%, P2h575.2%.Hatched altogether with HER2 positive tumor cell BT-474 and SKOV-3 respectively by fluorescein-labeled Humanized single chain antibody P2h2, HER2 negative tumor cells MCF-7 is as negative control.Simultaneously by unlabelled e23sFv with two kinds of different final concentration (125nM, 250nM) mix with fluorescein-labeled Humanized single chain antibody P2h2 after and BT-474 and SKOV-3 hatch, 1h on ice, streaming wash liquid three times, 1000rpm/min is centrifugal, 500ulPBS is resuspended, upper machine testing.As shown in Figure 9, P2h2 can be blocked by the e23sFv of different concns the keying action of HER2 in conjunction with it with BT-474 and SKOV-3 surface HER2 molecule, and blocking effect presents dose-dependent feature, prompting P2h2 in conjunction with HER2 molecule epi-position comparatively parent e23sFv not there is considerable change.
Seven, laser confocal microscope detects Humanized single chain antibody to the internalization activity of HER2 positive tumor cell
The cell climbing sheet of preparation HER2 positive tumor cell BT-474 and SKOV-3,4h is hatched altogether at 37 DEG C with fluorescein-labeled single-chain antibody, after mounting, confocal laser scanning microscope each strain single-chain antibody internalization enters the activity of HER2 positive tumor cell, and HER2 negative tumor cells MCF-7 is as negative control.As shown in Figure 10, have no obvious fluorescence distribution in BT-474 and the SKOV-3 born of the same parents that P2h2 is hatched, prompting P2h2 internalization can not enter HER2 positive tumor cell.
Eight, ELISPOT evaluates Humanized single chain antibody immunogenicity
Take 12 routine healthy human peripheral bloods, lymphocyte separation medium is separated mononuclearcell (PBMC), respectively with 3uMe23sFv, P1h2, P1h3, P2h2, P2h5 (PBS dilution) stimulates 24h, ELISPOT to detect PBMC secretion of gamma-IFN level in 37 DEG C.The positive cell frequency that result prompting Humanized single chain antibody P2h2 stimulates PBMC to produce IFN-γ comparatively parent e23sFv all has obvious minimizing, and difference has statistical significance, as shown in figure 11.
Nine, human peripheral cytokine profiles checking and appraising Humanized single chain antibody immunogenicity
Take 12 routine healthy human peripheral bloods, lymphocyte separation medium is separated mononuclearcell (PBMC), 24h is stimulated in 37 DEG C respectively with 3uMe23sFv and Humanized single chain antibody P1h2, P1h3, P2h2, P2h5, collecting cell culture supernatant, LEGENDplex kit measurement cytokine IL-2, IL-4, IL-5, IL-10, IL-17a, IL-21, IFN-γ, TNF-α concentration, compare the change of e23sFv and P1h2, P1h3, P2h2, P2h5 stimulation PBMC cytokine secretion amount.The level that result prompting Humanized single chain antibody P2h2 stimulates PBMC to produce IL-2, IL-10, TNF-α, IFN-γ comparatively parent e23sFv all has obvious minimizing, prompting is after humanization modified, single-chain antibody P2h2 initiation human body produces immunoreactive ability and obviously declines, difference has statistical significance, as shown in figure 12.
In sum, the invention provides the humanization non-internalization single-chain antibody P2h2 of a kind of target HER2, the acquisition of this antibody is the FR CDR of mouse single-chain antibody e23sFv being transplanted to the highest people's antibody consensus sequence of homology, to be optionally the mouse amino-acid residue in e23sFv to 13 the important amino acid residue mutations maintaining antibody space conformation in FR again, building storage capacity be 2
13single chain antibody phage shows storehouse, filters out the humanization non-internalization single-chain antibody P2h2 that 1 strain has high-affinity, for laying a solid foundation in the application in clinical diagnosis and treatment field.
Claims (5)
1. a humanization modified non-internalization single-chain antibody P2h2 of target HER2, is characterized in that, the aminoacid sequence of described humanization modified non-internalization single-chain antibody P2h2 is as shown in SEQ.ID.NO.1.
2. the humanization modified non-internalization single-chain antibody P2h2 of target HER2 as claimed in claim 1, it is characterized in that, the nucleotide sequence of the non-internalization single-chain antibody P2h2 of encoding humanized transformation is as shown in SEQ.ID.NO.2.
3. humanization modified non-internalization single-chain antibody P2h2 according to claim 1 is preparing the application in antitumor drug and/or healthcare products.
4. apply as claimed in claim 3, it is characterized in that, described medicine and/or healthcare products are medicine and/or the healthcare products of anti-HER2 positive tumor cell.
5. the application of humanization modified non-internalization single-chain antibody P2h2 in preparation HER2 positive tumor diagnostic reagent of target HER2 according to claim 1.
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