CN103130896A - Monoclonal antibody for resisting cell surface ectopic expression, and preparation method and application thereof - Google Patents
Monoclonal antibody for resisting cell surface ectopic expression, and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biological pharmacy and provides a monoclonal antibody for resisting cell surface ectopic expression. The amino acid sequence of a light chain variable region at the Fab fragment antigen-binding site of an antibody molecule is shown as SEQ ID NO:2, and the amino acid sequence of a heavy chain variable region is shown as SEQ ID NO:4. The invention also provides application of the monoclonal antibody in preparation of medicaments for treating and preventing digestive tract tumors or digestive tract tumor metastasis. The invention also provides a detection kit and medicinal composition containing the monoclonal antibody. The invention also provides a preparation method of the monoclonal antibody. A series of in vivo and in vitro tests prove that the purified and degermed monoclonal antibody has high affinity for PALP and/or IALP antigen for resisting extracellular ectopic expression of tumor cells and has a biological activating function of inhibiting growth and migration of tumor cells.
Description
Technical field:
The invention belongs to field of biological pharmacy, relate in particular to a kind of monoclonal antibody, is the monoclonal antibody and its production and use of the alkaline phosphatase of the digestive tract tumor cell surface ectopic expressions such as a kind of anti-cancer of the stomach specifically.
Background technology:
Cancer of the stomach (Gastric Cancer, GC) is modal alimentary system malignant tumour, is worldwide the second largest reason of Tumor-assaciated death.China is one of country that the incidence gastric cancer rate is the highest in the world, and annual new cases are nearly 400,000, accounts for globally 42%, and a lot of patient has belonged to late period when medical.At present, the early screening of cancer of the stomach and make a definite diagnosis the biopsy of main dependence gastroscope conjunctive tissue pathology, although the specificity of this early screening diagnostic means, susceptibility, accuracy and security in recent years all had raising to a certain degree, but due to the existence of universal difficulty and many interfering factorss, clinical application effect is very not satisfactory.Therefore, often be developed to late period during most patients with gastric cancer first visit, main operative treatment, radiotherapy, the systemic chemotherapy of relying on, but these traditional treatment measures are somewhat expensive not only, increased individual, social economical burden, and the specific aim for the treatment of is not strong, curative effect is usually barely satisfactory, patient's five year survival rate is no more than 10% usually, so the clinical treatment of cancer of the stomach still allows of no optimist.In recent years, although the enforcement of some new medicines and combined treatment increases patient's therapeutic response rate, in the random clinical study of having carried out, all do not reach the therapeutic goal of median survival interval more than 1 year.Therefore because stomach cancer cell is generally insensitive to chemotherapy and radiotherapy, and still lack at present effective cancer of the stomach second line treatment scheme, the research and development of new curing gastric cancer strategy and medicine in recent years become the focus of basis and clinical study.NCI-PRG suggestion in 2002 is carried out the definition of molecule aspect to tumour, thereby realizes that effectively the individualized treatment of tumour patient, recurrence detect and prognosis evaluation.Under the guidance of these new concepts, the molecular targeted therapy of cancer of the stomach arises at the historic moment, and becomes new biology methods for the treatment of.
Molecular targeted therapy is as target spot with tumour cell overexpression or specific expressed some significant molecule.The nineties in last century, the different kinds of molecules target therapeutic agent entered the treatment that clinical trial is used for kinds of tumors in succession so far, and some drugs demonstrates curative effect preferably.These drug mains will comprise two large class, i.e. macromole monoclonal antibody and small molecule tyrosine kinase inhibitors.Due to the identification of antibody high degree of specificity and conjugated antigen, so the antibody target of tumour treatment (Antibody-Targeted Therapy, ATT) shows out unique charm at therapeutic field of tumor.With with the closely-related molecule of tumor development as target spot, agonist (Agonist) or competitive antagonism (Competitive Antagonism) by monoclonal antibody, and with the carrier of antibody as the transmission medicine, or by some amynologic mechanism (as ADCC), realization suppresses, kills and wounds the specificity of tumour cell or the specific aim of tumor growth microenvironment is disturbed, therefore compare with the traditional treatment measure, effect is more obvious and toxic side effect reduces greatly.The important supplement that the antibody drug targeted therapy of tumour will become the traditional treatment mode even might replace existing methods for the treatment of.
The antibody drug targeted therapy development in recent years of cancer of the stomach is rapid.Preclinical study and clinical studies show, monoclonal antibody medicine Cetuximab take EGF-R ELISA (EGFR) as target spot, with epidermal growth factor acceptor 2 (EGFR2, HER2) being the monoclonal antibody medicine Trastuzumab of target spot and the monoclonal antibody medicine Bevacizumab(Avastin take vascular endothelial growth factor (VEGF) as target spot) the anti-cancer of the stomach that all has to a certain degree is active, but it is not high all to exist specificity, the problem that targeting is not strong, the actual clinical result for the treatment of is not very good.The core of stomach cancer target treatment monoclonal antibody medicine research is to seek the antibody of identifying the cancer of the stomach specific antigens and preparing the natural epi-position of anti-specific antigens.At first Brichory etc. are incorporated into proteomics method evaluation and screening tumor associated antigen and antitumor autoantibody field.In the present genome times afterwards comprehensively, this strategy is widely used in kinds of tumors research, comprise cancer of the stomach, and disclose numerous possible new cancer of the stomach molecular markers, as glucose regulated protein (Glucose Regulated Protein, GRP78), heat shock protein(HSP) (Heat Shock Protein, HSP27,70,60) and fibrinopeptide A (Fibrin Peptide A).But because the ideal candidates target spot of tumour monoclonal antibody targeted therapy is the cell surface specific antigens, and some technology that adopts in proteomics research be difficult to guarantee the native conformation of antigen, limited cancer of the stomach molecular marker that evaluation and screening obtains and the further research of corresponding antibodies and used.Undoubtedly, identify cancer of the stomach or other tumor cell surface specific antigens under native state, and realize that the specific antibody that high-throughput prepares anti-cancer of the stomach or other tumor surface molecular marker will identify and the research and development of stomach cancer target treatment monoclonal antibody medicine provide powerful for the cancer of the stomach molecular marker.
Alkaline phosphatase (ALP) belongs to heterodimeric protein, and molecular weight is 56kDa.Each monomer is comprised of 449 amino acid, and complete ALP molecule presents the topological framework of typical α/β.ALP is a kind of glucoproteinase that contains zinc.People's ALP presents the highest vigor and can be hydrolyzed phosphate monoester compound substrate at the alkaline environment of pH8.0.Normal ALP is by the pho-A genes encoding.It is a kind of secretory protein, the monomer precursor of synthesizing amino end secreting signal peptide in endochylema, and secreting signal peptide guiding precursor is rear cut across the inner membrance transportation, and the homodimer of formation is secreted into outside born of the same parents.
ALP has a plurality of hypotypes and is that a class can be with the dephosphorylized enzyme of corresponding substrate or isozyme.ALP in normal human serum generally is less than half of gross activity mainly from liver from the ALP of osseous tissue.The hypotype of present known ALP has Placenta Type (PALP), visible peristalsis visible intestinal peristalsis (IALP), sexual cell type (GALP) and non-specific tissue-type (TNSALP).And TNSALP adds the secondary isozymes such as the formed liver of a plurality of modifications, kidney, bone after genetic expression;
In Human Physiology sexual abnormality or pathologic situation, ALP mainly is discharged to peripheral blood through liver, thereby serum levels of ALP increases.Mainly there are the tissues such as the bone, liver, kidney of body in ALP.Hepatobiliary system disease comprises that the ALP such as liver and gall malignant tumour, skeleton development are abnormal, pregnant woman can increase, but also be not reported at present the report that raises in the digestive tract tumor such as cancer of the stomach, in also exchanging without any document, patent or meeting, the hypotype of report ALP can be expressed at tumor cell surface by atopy.
Summary of the invention:
In order to solve the problems of the technologies described above, the invention provides monoclonal antibody of the surperficial ectopic expression of a kind of anti-cell and its production and use, the monoclonal anti physical efficiency of the surperficial ectopic expression of described this anti-cell effectively suppresses growth and the transfer of digestive tract tumor cell.
The invention provides the monoclonal antibody of the surperficial ectopic expression of a kind of anti-cell, the light chain variable region amino acid sequence of its antibody molecule Fab section antigen-binding site is as shown in SEQ ID NO:2, and the weight chain variable region amino acid sequence of its antibody molecule Fab section antigen-binding site is as shown in SEQ ID NO:4.
Further, the cDNA sequence of its variable region of light chain is as shown in SEQ ID NO:1, and the cDNA sequence of its variable region of heavy chain is as shown in SEQ ID NO:3.
Further, the monoclonal antibody of the surperficial ectopic expression of above-mentioned anti-cell is a kind of immunoglobulin (Ig) of mouse IgG hypotype.
The present invention also provides the above-mentioned purposes of monoclonal antibody in the medicine of preparation treatment, prevention digestive tract tumor or transfer.
Further, described digestive tract tumor includes but are not limited to: squamous cell carcinoma of stomach, adenocarcinoma of stomach, small cell carcinoma, adenosquamous carcinoma, carcinoid, the esophageal carcinoma, cancer of the stomach, duodenal cancer, carcinoma of the pancreas, cholangiocarcinoma, carcinoma of gallbladder, liver cancer, colorectal carcinoma, colorectal cancer.
The present invention also provides a kind of detection kit, contains the monoclonal antibody of the above-mentioned surperficial ectopic expression of a kind of anti-cell.
Further, above-mentioned detection kit also contains the marker of being combined with the monoclonal antibody of the surperficial ectopic expression of above-mentioned anti-cell, and described marker is fluorescent marker or radioactively labelled substance or enzyme mark marker.
The present invention also provides a kind of pharmaceutical composition, contains the monoclonal antibody of the above-mentioned surperficial ectopic expression of a kind of anti-cell.
Further, above-mentioned pharmaceutical composition also comprises pharmaceutically acceptable carrier, vehicle or mixture.
The present invention also provides the application in serology, pathological diagnosis, x-ray tomography video picture or the positron emission computerized tomography of the monoclonal antibody of above-mentioned a kind of anti-cell surface ectopic expression at digestive tract tumor-x-ray tomography video picture.
the present invention also provides the preparation method of the monoclonal antibody of above-mentioned a kind of anti-cell surface ectopic expression, it is characterized in that: adopt four kinds of grades to make up in proportion the stomach cancer cell line SGC7901 of (mixing), BGC823, MKN28, MKN45 viable cell multiple spot immune mouse, mouse spleen cell after three subnormal immunity and booster immunization and murine myeloma cell SP2/0 merge by the PEG chemistry, filter out can secrete can be in conjunction with above-mentioned four kinds of cancer of the stomach viable cell surface antigens not with the hybridoma cell strain of people's normal circumference blood mononuclear cell phase reaction, after this hybridoma cell strain subclone, the hybridoma supernatant that acquisition is cultivated, namely obtain monoclonal antibody of the present invention through affinity purification.
Further, by immunoaffinity chromatography method purifying Hybridoma Cell Culture supernatant liquor.
The present invention sets up the method for an effective tumor living cell high flux screening, merge by the SGC-7901 viable cell immune mouse that mixes and the SP2/0 clone of selecting high immunoreactive mouse spleen and mouse the antibody hybridoma that produces, by to the high flux screening of the anti-gastric carcinoma cell lines of hybridoma antibody and to normal people's peripheral blood mononuclear white corpuscle (PBMC) thus contrasting to screen has obtained the monoclonal antibody that this strain MS17-57 reacts GI tumour cell high specific.
The present invention has adopted the method with the reverse Screening and Identification antigen of anti-Anti-gastric Cancer Monoclonal Antibody of preparation, is intended to realize that " single stage method " prepares simultaneously the MS17-57 monoclonal antibody of the surperficial native conformation antigen of the anti-stomach cancer cell of specificity and identify its specificity cancer of the stomach molecular marker ALP endoglin expression albumen.
be difficult to obtain the antibody of the surperficial native conformation antigen of identification viable cell due to traditional antibody screening preparation method, so the present invention has adopted the immunity of " shotgun " viable cell, hybridoma technology is in conjunction with the method for flow cytometry high throughput testing, directly screen the MS17-57 specific antibody of the surperficial native conformation antigen of anti-cell in the viable cell level, after the correlation analysis of the evaluation of carrying out MS17-57 antibody and MS17-57 antibody and clinicopathologic features mathematic(al) parameter, adopt Western blotting, immuno-precipitation and protein spectrometry are completed the quick evaluation of prepared MS17-57 antibodies specific in conjunction with target antigen albumen, thereby screen and found the stomach cancer cell surface molecular mark that this ALP is new.
MS17-57 monoclonal antibody of the present invention can high degree of specificity ground to approximately IALP and/or the PALP molecular reaction naturally expressed of the tumor cell surface of 56kDa of molecular weight.In the own special monoclonal antibody hybridoma screening design of the present invention, expressed monoclonal antibody is to be secreted by the monoclonal antibody hybridoma cell strain of called after MS17-57 to produce, and the hypotype of mouse monoclonal antibody is to belong to IgG1 heavy chain and κ light chain.
The present invention is intended to realize the new cancer of the stomach molecular marker of Screening and Identification in the anti-stomach cancer cell surface molecular mark specific antibody of preparation by oppositely identify the strategy of antigen with antibody.Monoclonal antibody is a kind of good proteomics research instrument, it is hybridoma method that traditional monoclonal antibody prepares approach, usually adopt non-molecular marker immunity, the high-throughput screening method of a large amount of bed boards after also less employing is merged, the probability that therefore obtains the natural epitope antibodies of anti-cell antigen is lower.The present invention adopts similar " shotgun " (" shot gun " manner) by the viable cell immune mouse, adopt the method that unique hybridoma high fusion rate method merges, bed board (at every turn more than 50-60 piece plate) in enormous quantities screens in conjunction with the FACS-HTS high throughput testing, directly the monoclonal antibody of finally selecting the specificity high-affinity with the antibody of conformational epitope (Conformational Epitopes) molecular marker, and is identified in screening anti-cell surface by check on the viable cell level.
The present invention compares with prior art, and its technical progress is significant.the present invention is after the Clinicopathological Parameters correlation analysis that carries out digestive tract tumor such as prepared anti-cancer of the stomach cell mixing strain monoclonal antibody specific and cancer of the stomach, IALP and/or PALP molecularity functional analysis that the MS17-57 monoclonal antibody is acted on mutually at tumor cell surface, find that the MS17-57 monoclonal antibody produces for the surperficial PALP/IALP antigen of lineup's stomach cancer cell, but and inducing specific, the effect biological respinse, can specific recognition cancer of the stomach etc. digestive tract tumor and carry out targeted therapy, can be applied in the diagnosis and image of digestive tract tumor simultaneously.The present invention has set up one and has overlapped the completely new approach that high-throughput prepares anti-cancer of the stomach and the surperficial native conformation antigen-specific antibodies of digestive tube solid tumor cell, and with the MS17-57 monoclonal antibody that obtains as the proteomics research instrument, the reverse digestive tube solid tumor cell surface molecular target mark such as Screening and Identification cancer of the stomach.If with the biological agent that can develop after described chimericization of monoclonal antibody human/mouse or humanization for human gastrointestinal tract's oncotherapy.
Description of drawings:
Fig. 1 has shown the detection that FACS reacts four kinds of mice serum and SGC7901 and BGC823 cell strain titres of normal pbmc, cancer of the stomach of mixing after the stomach cancer cell line immunity.
Fig. 2 shown FACS to the MS17-57 monoclonal antibody respectively with the stomach cancer cell line association reaction in various degree of 4 kinds of immune use.
Fig. 3 is that the MS17-57 monoclonal antibody detects with the FACS of normal pbmc, Fetal Stomach mucous epithelium transformed cells GES-1 and stomach cancer cell line AGS association reaction respectively.
Fig. 4 is that the MS17-57 monoclonal antibody detects with the FACS of normal pbmc, stomach cancer cell MKN74, TMK-1, KKLS, ST-8 and ST-9 clone association reaction respectively.
Fig. 5 is MS17-57 monoclonal antibody and the protein bound ELISA detection reaction of cancer of the stomach BGC823 cytolemma extracting.
Fig. 6 is MS17-57 monoclonal antibody and the protein bound ELISA detection reaction of cancer of the stomach MKN45 cytolemma extracting.
Fig. 7 is that the ELISA of MS17-57 monoclonal antibody and the reaction of GES-1 cytolemma extracting protein binding detects.
Fig. 8 is that the MS17-57 monoclonal antibody detects in the immunohistochemical reaction of stomach mucous membrane transformant GES-1 and stomach cancer cell BGC823, MKN45.
Fig. 9 is that the MS17-57 monoclonal antibody carries out the immunoprecipitation of direct method and indirect method to cancer of the stomach BGC823 and MKN45 cytolemma extracting target protein.
Figure 10 is PALP and the qRT-PCR reaction of IALP on the mRNA level of various stomach cancer cell lines.
Figure 11 is that the MS17-57 monoclonal antibody is respectively to cancer of the stomach BGC823 and MKN45 Growth of Cells increment inhibited reaction.
Figure 12 is that the MS17-57 monoclonal antibody is to the restraining effect of Gastric cancer cell MKN45 locomotory movement; A. cell culture fluid contrast; B.5 μ g/mL MS17-57 monoclonal antibody; C.10 μ g/mL MS17-57 monoclonal antibody; D.20 μ g/mL MS17-57 monoclonal antibody; E.5 μ g/mL homotype contrasts monoclonal antibody; F.20 μ g/mL homotype contrasts monoclonal antibody.
Figure 13 is that the MS17-57 monoclonal antibody is to the restraining effect of stomach cancer cell BGC823 locomotory movement; A. cell culture fluid contrast; B.5 μ g/mL MS17-57 monoclonal antibody; C.10 μ g/mL MS17-57 monoclonal antibody; D.20 μ g/mL MS17-57 monoclonal antibody; E.5 μ g/mL homotype contrasts monoclonal antibody; F.20 μ g/mL homotype contrasts monoclonal antibody.
Figure 14 is that the MS17-57 monoclonal antibody is to inhibiting quantitative Analysis and the comparison of stomach cancer cell BGC823 locomotory movement.
Figure 15 is that the MS17-57 monoclonal antibody can suppress Gastric cancer cell MKN45 in the growth of mouse interior tumor; The A.MS17-57 monoclonal antibody suppresses the experimental group of MKN45 cell strain tumor growth, and 4 average each mouse of mouse have 1.5 tumours and diameter in 0.30cm size left and right; B. homotype contrasts monoclonal antibody to the control group of MKN45 cell strain tumor growth, and 4 average each mouse of mouse have 8.5 tumours and diameter in 0.31cm size left and right; C. the control group of blank PBS damping fluid to MKN45 cell strain tumor growth, 4 mouse of blank group, on average each mouse has 9.0 tumours and diameter in 0.28cm size left and right.
Figure 16 is that the MS17-57 monoclonal antibody can suppress stomach cancer cell BGC823 in the growth of mouse interior tumor; The A.MS17-57 monoclonal antibody suppresses the experimental group of BGC823 cell strain tumor growth, and 4 average each mouse of mouse have 1 tumour and diameter in 0.27cm size left and right; B. homotype contrasts monoclonal antibody to the control group of BGC823 cell strain tumor growth, and 4 average each mouse of mouse have 7 tumours and diameter in 0.31cm size left and right; C. the control group of blank PBS damping fluid to BGC823 cell strain tumor growth, 4 mouse of blank group, on average each mouse has 6.5 tumours and diameter in 0.3cm size left and right.
Embodiment:
The ordinary articles that the reagent that following embodiment adopts and experimental apparatus are this area can be bought on market or can obtain by regular approach.
Adopt the 4 strain stomach cancer cells (BGC823, MKN28, MKN45 and SGC7901 all derive from Shanghai Inst. of Cytobiology, Chinese Academy of Sciences) of four kinds of equal proportions mixing through in containing the RPMI1640 substratum of 10%FBS, 5%CO
2, cultivate under 37 ℃ of environment after, the mixing viable cell of collecting in the PBS damping fluid as immunogen, (purchase in Nanjing University's experimental animal models center the A/J-JAX mouse, mouse derives from The Jackson Laboratory, the U.S.) immunity is carried out in the subcutaneous and intraperitoneal injection in back, each every mouse 1 * 10
7Individual cell, immunity every other week is once; After the 3rd 1 week of immunity, get mice serum, flow cytometry high throughput system (FACS-HTS) detects the response situation of serum and 4 strain stomach cancer cells, healthy volunteer PBMC is cell [healthy volunteer's peripheral blood separates peripheral blood mononuclear cells (Peripheral Blood Mononuclear Cells, PBMC) and screens as the normal cell antigen control at fluorescence flow cytometer high flux screening (FACS-HTS) through Ficoll liquid] in contrast.Select higher and lower with the control cells PBMC cross reaction degree mouse (under same Dilution ratio, the difference of the average fluorescent strength value of serum and stomach cancer cell and control cells＞500) of serum titer to merge front booster immunization.
Get the mouse spleen that booster immunization is crossed, be prepared into single cell suspension with the spleen cell of the DMEM nutrient solution of serum-free No. 1 mouse that immunoreactivity is higher; At 50%PEG(pH7.4) under condition, splenocyte and SP2/0 murine myeloma cell are merged bed board (50 blocks of plates) in enormous quantities and cultivate 10 days to antibody hybridoma cell clone's formation with the HAT selective medium after merging.Collect and mix above-mentioned four kinds of stomach cancer cell lines of cultivating in enormous quantities as the screening object, separate normal people's peripheral blood PBMC cell as the screening control cells, two kinds of cells are resuspended in the 1.5%BSA/PBS of precooling confining liquid and evenly distribute (approximately every hole 1～2x10 respectively all
5Cell) respectively add in 51 blocks of 96 U-shaped plates in hole [102 blocks of plates, two the 51st block of plates are respectively positive (mice serum after immunity and gradient dilution) and feminine gender (normal mouse serum and gradient dilution and HAT selectivity nutrient solution) control board] altogether.
supernatant liquor (being equivalent to first antibody is original monoclonal antibody) each 70 microlitres/hole that 50 antibody hybridoma cells are merged plates (96 orifice plate) moves into respectively in corresponding screen plate and control board and concussion mixes, reaction and with after confining liquid washing in ice bath, the cell fluorescence Coloration experiment reaction that adds again the fluorescently-labeled sheep anti mouse of FITC in 100 microlitres/hole and carry out, fluorescence fluidic cell mark high flux screening (FACS-HTS) thus, first with the Cell regulate FACS parameter of blank hole and homotype control wells and as background, cell sample to each hole of U-shaped plate, two group of 96 hole carries out the FACS detection one by one.That satisfies simultaneously following two conditions is decided to be the positive cell hole: (1) and 4 strain stomach cancer cell surface antigens have association reaction (namely compare with homotype control signal peak, sample signal peak offset amplitude greater than a logarithmic value); (2) with the PBMC cell without association reaction (namely compare with homotype control signal peak, sample signal peak offset amplitude is less than 5-10%'s).Selected and picking is in conjunction with the hybridoma high flux screening cell of uniqueness, through subclone and the repeatedly detection of antagonist Hybridoma Cell Culture supernatant of the transition of selective medium and ordinary culture medium, hybridoma, the antibody affinity purification of the hybridoma supernatant that the MS17-57 monoclonal antibody is selected and 4 ℃ of Preservation in sterile conditions and add 50% glycerine to preserve for a long time in-20 ℃ after 0.2 micron membranes filters.
Use Qiagen(Valencia, California, USA) RNeasy test kit is extracted total RNA from MS17-57 monoclonal antibody hybridoma cell strain, use Invitrogen(Grand Island, New York, United States) SuperScript III First-Strand test kit the mRNA reverse transcription is become the cDNA library of MS17-57 monoclonal antibody.Utilize the ProgenBiotechnik company of Germany " Mouse IgG Library Primer Set " (F2010) 23 primers of test kit carry out 21 PCR reactions (not comprising the reaction of lambda light chain), special light, the translation that the heavy chain product carries out DNA sequencing, amino acid polypeptide sequence that produces and CDRs(epiope zone) and the FW(skeleton regional) identification.
Table-1(a), the variable region cDNA sequence of MS17-57 monoclonal antibody light chain (SEQ ID NO:1):
5’-atgtctgcatctccaggggaaaaggtcaccatgacctgcagggccagctcaagtat
aatttccagttacttgcactggttccagcagaagtcaggtgccccccccaaactct
ggatttatagcacatccaacttggcttctggagtccctgctcgcttcagtggcagt
gggtctgggacctcttactctctcacaatcagcagtgtggaggctgaagatgctgc
cacttattactgccagcagttcagtggttcccctatcacgttcggtgctggaccaa
gctggaactga-3’
Table-1(b), the variable region amino acid sequence of MS17-57 monoclonal antibody light chain (SEQ ID NO:2):
MSASPGEKVTMTC
RASSSIISSYLHWFQQKSGAPPKLWIY
STSNLASG
VPARFSGSGSGTSYSLTISSVEAEDAATYYC
QQFSGSPITFGAGPSWNX
Table-2(a), the variable region cDNA sequence of MS17-57 monoclonal antibody heavy chain (SEQ ID NO:3):
5’-gaggtccaagctgcagcagtctggaactgaactggtaaagcctggggcttcagtga
agttgtcctgcaaggcttttgactacaccttcacaaactacgatattaactgggtg
aagcagaggcctggacagggacttgagtggattggatggatttatcctggaagtgg
tagtactgaatacggtgagaagttcaaggggaaggccacactgactgcagacaaat
cctccagcacagtctacatgctcctcagcagcctgacagctgaggattctgcggtc
tatttctgtgcaagatcgagtaactggtacttcgatgtctggggtatagggaccac
ggtcaccgtctcctcagccaaaacgacacccccatctgactat-3’
Table-2(b), the variable region amino acid sequence of MS17-57 monoclonal antibody heavy chain (SEQ ID NO:4):
RSKLQQSGTELVKPGASVKLSCKAF
DYTFTNYDINWVKQRPGQGLEWIG
WIYPGSGSTE
YGEKFKGKATLTADKSSSTVYMLLSSLTAEDSAVYFCAR
SSNWYFDVWGIGTTVTVSSA
KTTPPSDY
(a) and (b) DNA and the aminoacid sequence of table-1 and table-2 are reciprocity.Black italic and underscore in aminoacid sequence are the positions that shows the CDR zone, are by CDR1, CDR2 and CDR3 is arranged sequentially and regional sequence between them is skelemin sequence (FW).
On the template of 96 holes " U ", with about 200,000 different stomach cancer cells/100 microlitres and adding in U-shaped plate in 1%BSA/PBS allotment average every hole, respectively the serum after the immunity of cancer of the stomach viable cell is become 5 times of titre serial dilutions, then every hole adds respectively 100 microlitres, after mixing, on ice or 4 ° of C reacted 20 minutes, the sheep anti-mouse igg Fc-FITC100 μ L/ hole that adds again the 1:333 dilution after 2 washings, after 4 ° of C reaction and washing, read the MFI value in each hole on the LSR-II fluorescence flow cytometer of BD company-HTS machine.
Result shows: the titre significant reaction that No. 1 mice serum that immunoreactivity is higher is combined with stomach cancer cell is higher than the titre of being combined with normal pbmc, and this mouse boosting cell is used for the fusion experiment that MS17-57 monoclonal antibody hybridoma produces.(as shown in Figure 1).
MS17-57 monoclonal antibody after affinity purification through and the embodiment 3 described U orifice plate cell dyeing methods of explanation, the stomach cancer cell line of four kinds of immune use is carried out in conjunction with staining reaction, and read the MRI value on LSR-II FACS instrument.Other experimental procedure is identical with embodiment 3.
Result shown FACS to the MS17-57 monoclonal antibody respectively with the stomach cancer cell line association reaction in various degree of 4 kinds of immune use, wherein the MS17-57 monoclonal antibody is the highest to the reaction of MKN-45 stomach cancer cell line, and relatively low to the SGC7901 cell response, with other two cell strain reactivities between middle (as shown in Figure 2).
MS17-57 monoclonal antibody after affinity purification carries out in conjunction with staining reaction normal pbmc, stomach cancer cell line GES-1, AGS, and read the MRI value on LSR-II FACS instrument through with embodiment 3, described U orifice plate cell dyeing methods being described.
Presentation of results MS17-57 monoclonal antibody all has higher binding reactive to GES-1 and ags cell strain, and with normal pbmc without association reaction, the irrelevant monoclonal antibody of simultaneously homotype contrast is all the negative control (as shown in Figure 3) without association reaction.
MS17-57 monoclonal antibody after affinity purification carries out in conjunction with staining reaction normal pbmc and each stomach cancer cell line, and read the MFI value on LSR-II FACS instrument through with figure-1, described U orifice plate cell dyeing method being described.
Result shown the MS17-57 monoclonal antibody respectively with the association reaction of normal pbmc, stomach cancer cell MKN74, TMK-1, KKLS, ST-8 and ST-9 clone, and result all presents feminine gender namely without association reaction (as shown in Figure 4).
MS17-57 monoclonal antibody after affinity purification and stomach cancer cell line BGC823 cytolemma extracting albumen (being coated in the Immunlon-II ELISA Sptting plate of U.S. Fisher Scientific company in advance) carry out the serial enzymes connection and close reaction, and read to read on the plate instrument OD value and mapping at ELISA.
The ELISA result has shown that MS17-57 monoclonal antibody and cancer of the stomach BGC823 cytolemma extracting albumen have association reaction to a certain degree, illustrate the MS17-57 monoclonal antibody can with degraded after in conjunction with target protein reaction, this has done preparing experiment (as shown in Figure 5) for the antibody mediated immunity co-precipitation work of back.
MS17-57 monoclonal antibody after affinity purification and stomach cancer cell line MKN45 cytolemma extracting albumen (being coated in the Immunlon-II ELISA Sptting plate of U.S. Fisher Scientific company in advance) carry out the serial enzymes connection and close reaction, and read to read on the plate instrument OD value and mapping at ELISA.
The ELISA result has shown that MS17-57 monoclonal antibody and cancer of the stomach MKN45 cytolemma extracting albumen have association reaction to a certain degree.The same with the experiment of Fig. 5, the MS17-57 monoclonal antibody can with degraded after in conjunction with target protein reaction, this has done preparing experiment (as shown in Figure 6) for the antibody mediated immunity co-precipitation work of back.
MS17-57 monoclonal antibody after affinity purification and Fetal Stomach mucous epithelium transformed cells GES-1 membranin film extracting albumen (being coated in the Immunlon-II ELISA Sptting plate of U.S. Fisher Scientific company in advance) carry out the serial enzymes connection and close reaction, and read to read on the plate instrument OD value and mapping at ELISA.
The ELISA result has shown has lower association reaction (as shown in Figure 7) to MS17-57 monoclonal antibody and GES-1 cytolemma extracting albumen.
Stomach mucous membrane transformant GES-1 and stomach cancer cell BGC823, MKN45 after centrifugal on Cell sheet glass (Cytospin) in the catalase staining reaction of being combined with the MS17-57 monoclonal antibody and show that target point protein all distributes and the reaction of the immunohistochemical method of surface of cell membrane result (magnification is respectively 40x and 100x).
The immunohistochemical method detected result has shown that the MS17-57 monoclonal antibody can be incorporated on the surface of cell membrane of stomach mucous membrane transformant GES-1 and stomach cancer cell BGC823, MKN45, and this is also the evidence (as shown in Figure 8) to MS17-57 monoclonal antibody target protein location.
Embodiment 11
Immuno-precipitation is MS17-57 monoclonal antibody and stomach cancer cell film extracting albumen test indirectly, and the Fc end of antibody is with Protein-A magnetic bead specific binding and wash away non-specific adsorption albumen, then by add thermal dissociation together with the SDS-PAGE sample-loading buffer; The direct immunization precipitator method are directly to be coupled on the Dynabeads magnetic bead of American I nvitrogen company (Grand Island, New York, United States) activation with the MS17-57 monoclonal antibody, and a few step reactions then are with immuno-precipitation is identical indirectly.Facts have proved and just can obtain special and target spot band clearly with direct method for the immuno-precipitation of MS17-57 monoclonal antibody.Follow-up repeatedly high quick mass spectroscopy has determined that the corresponding target spot of MS17-57 monoclonal antibody is PALP and/or IALP albumen.
The SDS-PAGE sex change AgNOR stain band of the direct immunization precipitator method shows, the MS17-57 monoclonal antibody only has a band on BGC823 cell strain extracting membranin be IALP albumen, and two bands (namely having the IALP band that the PALP protein band is arranged) are arranged on MKN45 cell strain extracting membranin, other residual heavy, light chain antibody decomposes band all on normal position.The co-immunoprecipitation method is because amplification procedure and the too high susceptibility of method have been brought very strong background and non-specific indirectly.(as shown in Figure 9, A is non-direct immunization precipitation, and B is the direct immunization precipitation.）
The qRT-PCR method: total RNA uses Qiagen(Valencia, California, USA) the RNeasy test kit extract from cell.Use Invitrogen(Grand Island, New York, United States) SuperScript-III First-Strand test kit the mRNA reverse transcription is become cDNA.Use Applied BioSystems(Foster City, California, USA) primer, probe and TaqMan(Invitrogen) genetic expression Master Mix(Invitrogen) amplification, detect relevant gene with StepOnePlus instrument (Invitrogen).GAPDH is detected simultaneously as reference gene.
Relative?expression=2
ΔCt?sample?(gene?of?interest-GAPDH)/2
ΔCt?reference (gene?of?interest-GAPDH)
By the analysis that IALP and PALP divide other qRT-PCR that the rna level of various stomach cancer cell lines is expressed, IALP has in various degree expression at MKN45, SGC7901, AGS and GES-1 cell, but PALP only expresses higher at the BGC823 cell strain.These data and IP and MASS some coincide as a result, but also there are differences, be the observations (as shown in figure 10) on two different RNA and protein level after all.
Embodiment 13
Adopt Dojindo (the Santa Clara of company, California, USA) CCK-8 cell dyeing counting reagent kit (CellCounting Kit-8) is by the cell proliferation experiment of the multiple hole CCK-8 dyeing of each condition 5, different MS17-57 monoclonal antibody dosage control and add in each cell hole (every porocyte be controlled at 500 cells left and right), every block of plate contains various various dose, condition and each 5 multiple hole, spreads 8 blocks of plates.Take out a plate every day and carry out the CCK-8 dyestuff viable cell that exists in the hole is carried out staining reaction, measures OD value and calculating with microplate reader under OD450nm hatch 4 hours in 37 ° of C cell culture incubators after, thus the propagation situation of indirect proof cell.
CCK8 dyestuff detected result shown the MS17-57 monoclonal antibody under two various dose respectively to cancer of the stomach BGC823(such as Figure 11 A) and MKN45(such as Figure 11 B) the increment inhibited reaction of Growth of Cells.The MS17-57 monoclonal antibody is to Approach of gastric carcinoma cells proliferation inhibited or highstrung, and the MS17-57 monoclonal antibody concentration of 2～3 μ g/mL can have remarkable inhibited reaction.
Stomach cancer cell line MKN45 is spread in the cell that does not contain matrigel, each cell spreads into 50,000 cell, then add respectively MS17-57 monoclonal antibody and homotype control antibodies with the concentration gradient of 20 μ g/mL, 10 μ g/mL, 5 μ g/mL and substratum contrast liquid respectively, 37 ° of C observe the MS17-57 monoclonal antibody and suppress the effect that stomach cancer cell line moves after standing 48 hours.The experimental result of experimental group and control group shows that the MS17-57 monoclonal antibody has the shift function that suppresses stomach cancer cell.
Result shown the MS17-57 monoclonal antibody the Transwell cell in the Gastric cancer cell MKN45 locomotory movement is had a different concns gradient and in various degree restraining effect, homotype antibody has shown good negative control (as shown in figure 12).
Embodiment 15
Experimental procedure as embodiment 14, stomach cancer cell line BGC823 is spread in the cell that does not contain matrigel, each cell spreads into 50,000 cell, then add respectively MS17-57 monoclonal antibody and homotype control antibodies with the concentration gradient of 20 μ g/mL, 10 μ g/mL, 5 μ g/mL and substratum contrast liquid respectively, 37 ° of C observe the MS17-57 monoclonal antibody and suppress the effect that stomach cancer cell line moves after standing 48 hours.The experimental result of experimental group and control group shows that the MS17-57 monoclonal antibody has the shift function that suppresses stomach cancer cell.
Result shown the MS17-57 monoclonal antibody the Transwell cell in stomach cancer cell BGC823 locomotory movement is had a different concns gradient and in various degree restraining effect, homotype antibody has shown good negative control (as shown in figure 13).
Embodiment 16
Experimental procedure as embodiment 14, Transwell cell after the effect of different concns MS17-57 monoclonal antibody is worn stomach cancer cell line BGC823 cell and film collection and the dyeing that film is crossed, then read optical density value under OD450nm, make this histogram after calculation result and batch interior error amount.
Result shown the MS17-57 monoclonal antibody in the Transwell cell to the restraining effect functional response of stomach cancer cell BGC823 locomotory movement and the relative inhibition quantitative Analysis of having carried out many experiments of holes again.Result shows that the locomotory movement that the MS17-57 monoclonal antibody can suppress stomach cancer cell BGC823 can reach 20-30% (as shown in figure 14).
Embodiment 17
The experimental group of Figure 15 A, to the PBS liquid of 2.0 milliliters of MS17-57 monoclonal antibodies of mouse (nude mice) injection and MKN45 cytomixis, it has comprised MS17-57 monoclonal antibody (100 microgram antibody protein) and the 1x10 of 50 μ g/mL
6The MKN45 cell, every two week injections are (abdominal cavity and tail vein) totally 3 times once, the 6th week after initial injection is opened mouse peritoneal and observe the tumour that Gastric cancer cell MKN45 grows.The homotype monoclonal antibody control group of figure-15B, to the PBS liquid of 2.0 milliliters of homotype contrast monoclonal antibodies of mouse (nude mice) injection and MKN45 cytomixis, it has comprised homotype contrast monoclonal antibody (100 microgram antibody protein) and the 1x10 of 50 μ g/mL
6The MKN45 cell.Every two weeks are injected once (abdominal cavity and tail vein) totally 3 times, and the 6th week after initial injection is opened mouse peritoneal and observe the tumour that Gastric cancer cell MKN45 grows; The control group of the blank PBS damping fluid of figure-15C, to 2.0 milliliters of blank PBS damping fluids of mouse (nude mice) injection and MKN45 cell mixture, it has also comprised the MKN45 cell of 1x106, every two weeks are injected once (abdominal cavity and tail vein) totally 3 times, and the 6th week after initial injection is opened mouse peritoneal and observe the tumour that Gastric cancer cell MKN45 grows.
Result has shown that the MS17-57 monoclonal antibody can suppress Gastric cancer cell MKN45 in the function of mouse interior tumor growth.
Embodiment 18
The experimental group of Figure 16 A, PBS liquid to 2.0 milliliters of MS17-57 monoclonal antibodies of mouse (nude mice) injection and BGC823 cytomixis, it has comprised the MS17-57 monoclonal antibody (100 microgram antibody protein) of 50 μ g/mL and the BGC823 cell of 1x106, every two weeks are injected once (abdominal cavity and tail vein) totally 3 times, and the 6th week after initial injection is opened mouse peritoneal and observe the tumour that stomach cancer cell BGC823 grows.The homotype monoclonal antibody control group of Figure 16 B, to the PBS liquid of 2.0 milliliters of homotype contrast monoclonal antibodies of mouse (nude mice) injection and BGC823 cytomixis, it has comprised the homotype contrast monoclonal antibody (100 microgram antibody protein) of 50 μ g/mL and the BGC823 cell of 1x106.Every two weeks are injected once (abdominal cavity and tail vein) totally 3 times, and the 6th week after initial injection is opened mouse peritoneal and observe the tumour that stomach cancer cell BGC823 grows; The control group of the blank PBS damping fluid of Figure 16 C, to 2.0 milliliters of blank PBS damping fluids of mouse (nude mice) injection and BGC823 cell mixture, it has also comprised 1x10
6The BGC823 cell, every two week injections are (abdominal cavity and tail vein) totally 3 times once, the 6th week after initial injection is opened mouse peritoneal and observe the tumour that stomach cancer cell BGC823 grows.
Result has shown that the MS17-57 monoclonal antibody can suppress stomach cancer cell BGC823 in the function of mouse interior tumor growth.
Claims (13)
1. the monoclonal antibody of anti-cell surface ectopic expression, it is characterized in that: the light chain variable region amino acid sequence of its antibody molecule Fab section antigen-binding site is as shown in SEQ ID NO:2, and the weight chain variable region amino acid sequence of its antibody molecule Fab section antigen-binding site is as shown in SEQ ID NO:4.
2. the monoclonal antibody of anti-cell as claimed in claim 1 surface ectopic expression, it is characterized in that: the cDNA sequence of its variable region of light chain is as shown in SEQ ID NO:1, and the cDNA sequence of its variable region of heavy chain is as shown in SEQ ID NO:3.
3. the monoclonal antibody of the surperficial ectopic expression of a kind of anti-cell as claimed in claim 1, is characterized in that: the immunoglobulin (Ig) that is a kind of mouse IgG hypotype.
4. the purposes of monoclonal antibody claimed in claim 1 in the medicine of preparation treatment, prevention digestive tract tumor.
5. the purposes of monoclonal antibody claimed in claim 1 in preparation treatment, prevention digestive tract tumor diversion medicaments.
6. purposes as described in claim 4 or 5, described digestive tract tumor includes but are not limited to: squamous cell carcinoma of stomach, adenocarcinoma of stomach, small cell carcinoma, adenosquamous carcinoma, carcinoid, the esophageal carcinoma, cancer of the stomach, duodenal cancer, carcinoma of the pancreas, cholangiocarcinoma, carcinoma of gallbladder, liver cancer, colorectal carcinoma or colorectal cancer.
7. a detection kit, is characterized in that: the monoclonal antibody that contains the surperficial ectopic expression of a kind of anti-cell claimed in claim 1.
8. a kind of detection kit as claimed in claim 7, it is characterized in that: also contain the marker of being combined with the monoclonal antibody of the surperficial ectopic expression of described anti-cell, described marker is fluorescent marker or radioactively labelled substance or enzyme mark marker.
9. a pharmaceutical composition, is characterized in that: the monoclonal antibody that contains the surperficial ectopic expression of a kind of anti-cell claimed in claim 1.
10. a kind of pharmaceutical composition as claimed in claim 9, is characterized in that: also comprise pharmaceutically acceptable carrier, vehicle or mixture.
11. the monoclonal antibody of the surperficial ectopic expression of described a kind of anti-cell of claim 1 is in serology, pathological diagnosis or the x-ray tomography video picture of digestive tract tumor or the application in positron emission computerized tomography-x-ray tomography video picture.
12. the preparation method of the monoclonal antibody of the surperficial ectopic expression of described a kind of anti-cell of claim 1, it is characterized in that: the stomach cancer cell line SGC7901 that adopts four kinds of equal proportions to mix, BGC823, MKN28, MKN45, viable cell multiple spot immune mouse, mouse spleen cell after three subnormal immunity and booster immunization and murine myeloma cell SP2/0 merge by the PEG chemistry, filter out can secrete can be in conjunction with above-mentioned four kinds of cancer of the stomach viable cell surface antigens not with the hybridoma cell strain of people's normal circumference blood mononuclear cell phase reaction, after this hybridoma cell strain subclone, the hybridoma supernatant that acquisition is cultivated, namely obtain monoclonal antibody of the present invention through affinity purification.
13. the preparation method of the monoclonal antibody of a kind of anti-cell as claimed in claim 12 surface ectopic expression is characterized in that: by immunoaffinity chromatography method purifying Hybridoma Cell Culture supernatant liquor.
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