CN1262643C - Monocolone antibody, encoding gene, its uses and hybrid nodule cell line for cecreting same - Google Patents

Monocolone antibody, encoding gene, its uses and hybrid nodule cell line for cecreting same Download PDF

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CN1262643C
CN1262643C CN 03120599 CN03120599A CN1262643C CN 1262643 C CN1262643 C CN 1262643C CN 03120599 CN03120599 CN 03120599 CN 03120599 A CN03120599 A CN 03120599A CN 1262643 C CN1262643 C CN 1262643C
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antibody
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monoclonal antibody
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CN1530440A (en
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阎锡蕴
刘琴
林芸
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Institute of Biophysics of CAS
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Institute of Biophysics of CAS
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Abstract

The present invention provides a hybridoma cell line capable of secreting a monoclonal antibody. The preservation number is CGMCC0491. The present invention also provides a monoclonal antibody secreted by the hybridoma cell line, or a single-chain antibody derived from the monoclonal antibody, a Fab segment, or a human-mouse chimeric antibody, a gene for encoding the antibodies and purposes thereof.

Description

A kind of monoclonal antibody, its encoding gene, purposes and the hybridoma cell line of secreting it
Invention field
The present invention relates to a kind of hybridoma cell line of secrete monoclonal antibody, a kind of monoclonal antibody perhaps is derived from the single-chain antibody of this monoclonal antibody, Fab fragment, perhaps people-mouse chimeric antibody, encode their gene and their purposes.This antibody-like stops early pregnancy by suppressing tumor growth and transfer with the CD146 interaction of molecules.Therefore, they can be used for diagnosis and treatment with the associated angiogenesis disease, are novel contraception of a class and miscarriage medicine.
Background of invention
Tumour is a kind of and disease associated angiogenesis, is the No.1 killer of current harm humans life and health.The number of the infected about 1,600,000 of the annual malignant tumour of China.Die from cancer person and account for more than 20% of total toll, occupy first (according to statistical information in 1996) of city resident's cause of death.Lack the method for tumour being carried out early stage specific diagnosis at present.In oncotherapy, three big routine＜radiotherapies, chemotherapy and operation〉be first-selected, pharmacological agent is an importance.Because most of tumor chemotherapeutic drugs are not specific effects at tumour cell, so side effect is big and be easy to generate resistance.The factor that influences the tumor pharmacother effect in addition also comprises the mode of transport of medicine and perviousness etc., and this is that present tumor therapeutics is badly in need of the key issue that solves.This research finds that first the CD146 molecular specific is distributed in the new vessel endotheliocyte, plays important effect in the new vessel generative process.Vasculogenesis all can take place under physiology and pathological conditions.Vasculogenesis under the pathological conditions comprises tumour, diabetic retinopathy, rheumatoid arthritis etc., and the vasculogenesis under the physiological condition comprises fetal development and wound healing etc.
One of characteristic of the present invention is expressed in tumor neogenetic blood vessels with being to find the CD146 molecular selectivity, so it can be used as the diagnosis that a tumor vessel characteristic mark is used for tumour.Aspect treatment, the strategy that the present invention taked is by disturbing CD146 in the effect aspect the vasculogenesis, thereby the nutrition and the oxygen supply of blocking-up tumor area make the tumour cell ischemic necrosis [1]The present invention compares with traditional anti-tumour antibody, and its characteristics are that anti-CD146 antibody is that a class is efficient, wide spectrum, nontoxic or low toxicity, the new type anticancer medicine that has no drug resistance.The mechanism of action is as follows, and (1) tumor vascular endothelial cell is relatively stable, and sudden change is few, so the antineoplastic vascular medicine can not resemble easy generation resistance the antitumor cell medicine; (2) the specific target molecule difference of dissimilar solid tumors, and the new vessel structure that they are depended on for existence is roughly the same, so anti-tumor neovascularization antibody has broad spectrum, promptly a kind of antineoplastic vascular medicine can be applied to the treatment of kinds of tumors; (3) thousands of tumour cells depend on a capillary vessel and obtain oxygen and nutritive substance, even small amount of drug also can cause thrombosis for the incomplete destruction of blood vessel, cause the death of massive tumor cell ischemic.Therefore dosage of the present invention is few, the efficient height; (4) the tumor vessel medicine directly acts on blood vessel endothelium, need not to be penetrated into the tumour deep, has avoided caused drug osmotic of tumor tissues partial high pressure and distribution problem; (5) antibody optionally destroys tumor vessel but not normal blood vessels, and the poison that this height drug targeting specificity will significantly reduce medicine is paid effect.
Two of characteristic of the present invention is to find that the CD146 molecular specific is distributed in the trophocyte, implants and placenta is set up and played important effect in the process the embryo.The mechanism of action of contraceptive mainly is by intervening the blastocyst implantation process at present.Blastocyst invades the intrauterine process of parent (be called for short and implant), is one and is subjected to parent progesterone and estrogenic common control process.What the embryo implanted carries out smoothly, requires blastocyst to hatch out from zona pellucida on the one hand, is in active state, also requires the uterus of parent to be in receive status on the other hand, can accept the implantation of blastocyst.This two aspect is harmonious time and space requirement height, otherwise will cause the blastocyst graft failure, causes miscarriage [2,3]The key of development novel contraceptive medicine is to seek the newtype drug target molecule that plays a crucial role in embryo's implantation process.Have the contraceptive target spot of application prospect will possess following 2 primary conditions (1) specificity as one: contraception drug candidate target spot must only play a crucial role in the blastocyst implantation process, and other organs are not had vital role, otherwise can increase Side effects of pharmaceutical drugs; (2) versatility:, therefore preferably select for use a contraception target spot can be applied to a plurality of kind implantation process because the blastocyst of different kinds is implanted and the mode of early stage placentation is widely different.
We find that first CD146 is a very ideal contraceptive target molecule, possess above-mentioned two conditions: (1) CD146 is a very conservative general target molecule, the CD146mRNA sequence homology of people and mouse reaches more than 82%, and it all plays an important role in the blastocyst of people and mouse is implanted; (2) the CD146 molecule is blood vessel endothelium and embryo trophocyte [4]Last high expression level, this has guaranteed the specificity of CD146 as the contraception target molecule.We find that anti-CD146 antibody can effectively block the mouse blastocyst and implant, and cause the mouse abortion ratio to reach more than 80%, and this exploitation that is found to be contraceptive provides new approach and candidate new medicine.Further analyze the mechanism of action of anti-CD146 antibody, we find: (1) anti-CD146 antibody inhibition mouse trophocyte is sticked and is moved; (2) suppress mouse trophocyte secrete metalloproteinases, thereby the trophocyte who suppresses the blastocyst periphery invades the parent intrauterine; (3) vasculogenesis of inhibition deciduomata causes fetal development to lag behind.In experimentation on animals, behind the anti-CD146 antibody of Gestation period injected in mice, not only cause the miscarriage phenomenon, and the vessel density in the parent uterine decidua is compared obvious minimizing with control group.These test-results provide new approaches and novel targets for the exploitation of contraceptive, and anti-CD146 antibody is a class novel contraceptive medicine.
The present invention adopts two kinds of technological lines to prepare the antibody of anti-CD146 molecule: (1) adopts the tumor cell culture supernatant to induce Human umbilical vein endothelial cells, makes it become the vascular endothelial cell of propagation.The latter is used for immune mouse and obtains monoclonal antibody.This novel method has not only improved the abundance of hyperplasia vascular endothelial cell sign, has kept the native conformation of membrane antigen, but also can find the new target molecule of proliferative phase blood vessel.(2) utilize modern molecular biology and antibody engineering technology, develop the genetic engineering antibody of various different structure forms.These antibody than former generation mouse monoclonal antibody have bigger using value, show that mainly immunogenicity is lower, toxic side effect is little; Be easy to combine and form immunocomplex efficiently with other functional molecular; Antibody gene is easy to preserve and help carrying out the transformation of antibody structure and function, to be adapted to clinical application better.
Summary of the invention
An object of the present invention is to provide a kind of hybridoma cell line of secreting the monoclonal antibody of specific combination CD146 molecule.
Another object of the present invention provides a kind of monoclonal antibody of specific combination CD146 molecule, perhaps is derived from the single-chain antibody of this monoclonal antibody, Fab fragment, perhaps people-mouse chimeric antibody.
Another purpose of the present invention provides this monoclonal antibody of a kind of coding, perhaps is derived from the single-chain antibody of this monoclonal antibody, Fab fragment, the perhaps nucleotide sequence of people-mouse chimeric antibody.
A further object of the present invention provides a kind of monoclonal antibody, perhaps is derived from the single-chain antibody of this monoclonal antibody, Fab fragment, the perhaps purposes of people-mouse chimeric antibody.
The invention provides a kind of hybridoma cell line of secrete monoclonal antibody, secreted monoclonal antibody specific combination CD146 molecule.This clone has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on September 19th, 2000, the preservation centre address is, China, and Beijing, the Zhong Guan-cun, its deposit number is CGMCC0491.
The present invention also provides by described hybridoma cell line excretory monoclonal antibody, perhaps is derived from the single-chain antibody of this monoclonal antibody, Fab fragment, perhaps people-mouse chimeric antibody.The variable region of heavy chain of described monoclonal antibody can have the aminoacid sequence shown in the SEQ ID NO:1, and the variable region of light chain of described antibody can have the aminoacid sequence shown in the SEQ ID NO:2.
The present invention also provides the Fab fragment of described monoclonal antibody, and wherein, the segmental Fab light chain of this Fab can have the aminoacid sequence shown in the SEQ ID NO:3, and the segmental Fab heavy chain of this Fab can have the aminoacid sequence shown in the SEQ ID NO:4.
The present invention also provides the single-chain antibody of described monoclonal antibody, and wherein, this single-chain antibody can have the aminoacid sequence shown in the SEQ ID NO:5.
The present invention also provides coding monoclonal antibody of the present invention, perhaps is derived from the single-chain antibody of this monoclonal antibody, Fab fragment, the perhaps nucleotide sequence of people-mouse chimeric antibody.
The present invention also provides monoclonal antibody of the present invention, perhaps is derived from the single-chain antibody of this monoclonal antibody, the Fab fragment, and perhaps people-mouse chimeric antibody is used for the application of the medicine of diagnosing tumor and targeted therapy in preparation.
The present invention also provides monoclonal antibody of the present invention, perhaps is derived from the single-chain antibody of this monoclonal antibody, Fab fragment, the perhaps application of people-mouse chimeric antibody in drug screening and tumor vaccine.
The present invention also provides monoclonal antibody of the present invention, perhaps is derived from the single-chain antibody of this monoclonal antibody, Fab fragment, the perhaps application of people-mouse chimeric antibody in the medicine of preparation prevention early pregnancy and contraception.
The present invention also provides monoclonal antibody of the present invention, perhaps is derived from the single-chain antibody of this monoclonal antibody, Fab fragment, the perhaps application of people-mouse chimeric antibody in the medicine of preparation treatment and new vessel diseases associated.
In sum, we find that first anti-CD146 antibody not only can suppress embryo's implantation and cause miscarriage, and can suppress tumor growth and transfer.Therefore, this antibody-like can be used for the diagnosis and the treatment of tumour and other and associated angiogenesis disease, is novel contraception of a class and miscarriage medicine.
Brief Description Of Drawings
Fig. 1: flow cytometry CD146 antibody combines with various cells, anti-CD146 antibodies Human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cell (mvec) (HMVEC), and debond epidermic cell (A431) and mammary epithelial cell (HBL100).
Fig. 2: immunohistochemical methods is represented the new vessel in the tissues such as anti-CD146 antibodies cancer of the stomach, liver cancer, lung cancer, ovarian cancer, leiomyosarcoma.
Fig. 3: SDS-PAGE analyzes the CD146 antibody affinity chromatography and separates and the purifying target molecule.1-protein standard molecular weight; 2-human umblilical vein endothelial protein crude extract, 3-chromatography column effluent liquid; 4-chromatography column elutriant, immunoglobulin (Ig) protein ingredient is afterwards removed in 5-elutriant and Protein A reaction.
Fig. 4: the aminoacid sequence of antibody target molecule and CD146 molecule relatively.
Fig. 5: gel analysis antibody chain variable region (VL) and variable region of heavy chain (VH) are through the gene product behind the pcr amplification.1-1Kb DNA standard molecular weight; The 2-carrier; The 4-VL gene; The 5-VH gene.
Fig. 6: monoclonal antibody gene and construction of recombinant plasmid.1-100bp DNA standard molecular weight; 2-light chain of antibody gene; 3-carrier pComb3-k SacI/XbaI; The 4-antibody chain variable region; 5-light chain of antibody gene; 6-heavy chain of antibody Fd; The 7-variable region of heavy chain.
Fig. 7: SDS-PAGE and immunoblotting detect monoclonal antibody and single-chain antibody scFv.
Fig. 8: ELISA detects monoclonal antibody (IgG), and monoclonal antibody and single-chain antibody scFv are active to the combination of CD146 molecule.
Fig. 9: gel electrophoresis analysis contains the recombinant plasmid of single-chain antibody gene.1,2,4-represents different clones respectively; The 3-DNA standard molecular weight.
Figure 10: the test of chick chorioallantoic membrane vasculogenesis: A homotype mouse IgG does not have influence to the chorioallantoic membrane angiogenic growth.The anti-CD146 antibody of B has the obvious suppression effect to chicken embryo vasculogenesis.
Figure 11: anti-CD146 antibody inhibiting tumor growth, inhibiting rate is respectively leiomyosarcoma (SK-LMS-1) 50%, liver cancer (7721) 71.9%, carcinoma of the pancreas (SW1990) 48.7%.With 131During the anti-CD146 antibody coupling matter treatment of I-carcinoma of the pancreas, tumor control rate reaches 82%.
Figure 12: animal experiment is represented the situation of lotus human pancreas cancer nude mice after anti-CD146 antibody of process and homotype murine antibody (control group) treatment.Compare with control group, anti-CD146 antibody has the obvious suppression effect for growth of tumor.
Figure 13: the pathologic finding of treatment back tumour.A-compare group homotype murine antibody (on) and anti-CD146 Antybody therapy after the tumour size; Tumour pathological analysis behind the anti-CD146 Antybody therapy of B-finds that tumor tissues medium vessels density reduces the visible cavity necrosis of tumour cell around the visible embolism of part blood vessel, blood vessel.
Figure 14: anti-CD146 antibody is to the influence of mouse ectoplacenta awl adherent.
Figure 15: anti-CD146 antibody is to the influence of mouse ectoplacenta awl expansion.
Figure 16: the growth in vitro situation of mouse ectoplacenta awl in the presence of anti-CD146 antibody or homotype mouse IgG.A-is the not outwards expansion of mouse ectoplacenta awl in the presence of anti-CD146 antibody, and the expansion of B-mouse ectoplacenta awl in the presence of homotype mouse IgG is unaffected.
Figure 17: the mouse ectoplacenta awl adherent influence of anti-CD146 antibody to stimulating through FGF.
Figure 18: the influence that anti-CD146 antibody is expanded the mouse ectoplacenta awl that stimulates through FGF.
Figure 19: the mouse ectoplacenta awl adherent influence of anti-CD146 antibody to stimulating through VEGF.
Figure 20: the influence that anti-CD146 antibody is expanded the mouse ectoplacenta awl that stimulates through VEGF.
Figure 21: anti-CD146 antibody is to the influence of mouse ectoplacenta awl metalloprotease excretory.(a) and (d) for cultivating the zymogram result of 24 hours nutrient solutions, (b) with (e) for cultivating the zymogram result of 48 hours nutrient solutions, (c) and (f) EDTA treatment group.
Figure 22: the influence that anti-CD146 antibody is implanted the mouse blastocyst.Anti-CD146 antibody causes the embryo's miscarriage more than 80%.Figure 22-A is to be contrast with PBS, and Figure 22-B is to be contrast with homotype mouse IgG.
Figure 23: animal experiment is observed CD146 antibody and is caused the pregnant mouse miscarriage.Mouse Uterus is divided the left and right sides, after the anti-CD146 antibody treatment of left side process, and indivedual remaining dysontogenesis or deformities.Handle through irrelevant mouse IgG for contrast on the right side, and fetal development is normal.
Embodiment
Embodiment one: anti-CD146 MONOCLONAL ANTIBODIES SPECIFIC FOR and evaluation
Use hybridoma technology and produce monoclonal anti CD146 antibody [5], concrete operations are as follows: adopt the culture supernatant of liver cancer cell to induce Human umbilical vein endothelial cells (HUVEC) hyperplasia, as immunogen BALB/C mice is carried out immunization five times with outgrowth HUVEC.Each inoculation position is peritoneal injection and subcutaneous injection, and the total cellular score of injection is approximately 10 7Immunization was got spleen after three days the last time, and splenocyte is suspended in the RPMI1640 substratum.In the presence of polyoxyethylene glycol (PEG), splenocyte and P3-X63-Ag8.635 rat bone marrow tumour cell are merged, and hybridoma is screened with the HAT selective medium.
The method that people such as utilization Douillard introduce [6], with hyperplasia HUVEC cell inoculation overnight incubation in 96 well culture plates.With 0.5% glutaraldehyde fixed cell of fresh configuration 15 minutes, give a baby a bath on the third day after its birth time with PBS.Add bovine serum albumin sealing 1 hour.The hybridoma culture supernatant, enzyme labelled antibody and the substrate that add secretory antibody then carry out the ELISA screening.Select only combine with immunogen and not with former generation HUVEC bonded monoclonal antibody.The positive colony that filters out is further screening by the immunohistochemical methods method.To normal hepatocytes and the dyeing of liver cancer tissue frozen section, only select with the liver cancer vascular reaction not the hybridoma cell strain that reacts with normal hepatocytes with each clone's culture supernatant.Through the screening of above two kinds of methods, the specific antibody of Huo Deing only reacts with hyperplasia HUVEC and with the liver cancer blood vessel endothelium at last, not with the normal hepatocytes vascular reaction.
Repeatedly clone the hybridoma of secreting specific antibody with restricted dilution method, obtain the hybridoma HE2A5 (preserving number CGMCC0491) of secretion new vessel specific antibody at last.The secreted antibody recognition target antigen of HE2A5 hybridoma is the CD146 molecule.
The preparation method of ascites is summarized as follows: six week BALB/C mice abdominal injection pristane in age (Pristane) 0.5ml/ only.After 10 days, with the hybridoma suspension inoculation in BALB/C mice abdominal cavity, 1 * 10 7/ ml/ only after about ten days, collects ascites, the centrifuging and taking supernatant.By the albumin A affinity chromatography, monoclonal antibody purification from culture supernatant or ascites.With monoclonal antibody purification sterile filtration, and refrigeration or freezing preservation.Use Zymed company antibody subtype identification kit, the ELISA method identifies anti-CD146 antibody and belongs to IgG type antibody.
It is active to the combination of various clones to adopt the FACS cell sorter to analyze anti-CD146 antibody.With 1 * 10 6Cell and anti-CD146 antibody culture supernatant were hatched 1 hour altogether.Give a baby a bath on the third day after its birth time with PBS, add the FITC-sheep anti-mouse igg antibody, floating educating 45 minutes.Give a baby a bath on the third day after its birth all over after, cell is suspended among the 500 μ l PBS again.On FACS, carry out immunofluorescence analysis and measure mean fluorecence density.Experimental results reduction is in table 1 and Fig. 1.
Combining of the normal and tumor cell line of table 1 facs analysis CD146 antibody and people
Clone Human umbilical vein endothelial cells HUVEC people navel venule endothelial cell HMVEC HCC ALEX osteosarcoma cell Saos-2 epidermal cell A431 MC A375 MC SBcl lung carcinoma cell 1 A549 ovarian cancer cell LS-174 oophoroma SW1116 embryo kidney cancer cell 293 leiomyosarcoma SK-LMS-1 HCCs 7721 CD146 antibody++---+------- Clone transitional cell bladder carcinoma cell line T24 leukaemia T cell Jurkat pulpefaction leukaemia granulocyte KG-1 pancreatic cancer cell SW1990 fibrosarcoma cell HT1080 galactophore epithelial cell HBL100 ovarian cancer cell SKOV3 PBLC fibroblast prostate gland cancer cell Pc-1 Choriocarcinoma cell line JAR pancreatic cancer cell SW1990 cervical cancer cell HeLa CD146 antibody-------------
Utilize frozen tissue section and immunohistochemical methods method [7]Identify the reaction of anti-CD146 antibody and human normal tissue and tumor tissues, the result shows the high specific reaction of the capillary blood vessel of anti-CD146 antibody and many tumor tissues, and with the reaction of healthy tissues very limited (table 2, Fig. 2).
Combining of the normal and tumor tissues blood vessel of table 2 immunohistochemical analysis CD146 antibody and people
Healthy tissues The positive/example Tumor tissues The positive/example
Liver
0/14 Liver cancer 20/20
Brain 0/6 Brain tumor 9/9
Suprarenal gland 0/4 Kidney 2/2
Pancreas 0/5 Carcinoma of the pancreas 6/6
Stomach 2/2 Cancer of the stomach 10/10
Colon 2/2 Colorectal carcinoma 15/15
Mammary gland 1/1 Mammary cancer 20/20
Lung 2/2 Lung cancer 18/18
Ovary 2/2 Ovarian cancer 9/9
Cardiac muscle 0/2 Leiomyosarcoma 13/13
Tiroidina 0/2 Thyroid carcinoma 16/20
Lymphoglandula 0/2 Laryngocarcinoma 4/4
Marrow 0/2 The squama cancer 3/3
Sinusitis 0/2 Fibrohemangioma 4/4
Carcinoma of endometrium 15/15
Other 24/25
In order further to study the physico-chemical property of antibody target molecule, we use biotin labeling HUVEC epicyte protein, immunoprecipitation, methods analyst such as SDS-PAGE and immunoblotting (Xiyun Yan, YunLin, Dongling Yang, et al., A novel anti-CD146 monoclonal antibody, AA98, inhibits angiogenesis and tumor growth, Blood, February 27,2003.), determine that the antibody target molecule is that a molecular weight is (Fig. 3) about 97kD, be expressed in the membranin of cell surface, antigenic determinant is a conformational structure.Utilize affinity chromatography separation and purification antibody target molecule, show that by the amino acid sequence analysis result 20 amino acid of N end and people's cell adhesion molecule CD146 of target molecule is identical, confirm CD146 molecule (Fig. 4).
The CD146 molecule has another name called MUC18, A32 antigen, MCAM, Mel-CAM and S-Endo-1 [8-10], be accredited as the member of Ig gene superfamily recently.It is a kind of glycoprotein of striding film, and its extracellular region has the class Ig structural domain of a typical V-V-C-C-C, only has one to stride the film district, and the kytoplasm part is shorter, and the recognition site of some potential protein kinases is arranged.CD146 is the protein of a high glycosylation, and about 35% molecular weight is made up of carbohydrate, comprises sialic acid and other carbohydrate.At present, understand very few to the CD146 function.There is test to show that it participates in cell adhesion, migration and cell signaling [11-13]The present invention has explained the part biological function of CD146 molecule, has found CD146 molecule and antibody thereof the using value aspect diagnosing tumor and treatment and contraceptive.
Embodiment two: the preparation of anti-CD146 people-mouse chimeric antibody
Because mouse monoclonal antibody is identified as foreign matter in the human immune system, bring out the immunological rejection of body, thereby be not suitable for human body therapy.In order to overcome this shortcoming.The present invention adopts modern molecular biology technique, has developed people/mouse chimeric antibody chiAA98.The variable region of this antibody (antigen binding domain) is from mouse source protein (account for antibody molecule 1/3), and other constant regions are from human IgG (account for antibody molecule 2/3).This antibody has not only kept the specificity of mouse monoclonal antibody conjugated antigen, can compete and suppress combining of monoclonal anti CD146 antibody and its target antigen; And reduced the mouse derived components in the antibody molecule, and lowered people's immunological rejection, increased the biological effector function of antibody, be convenient to clinical application.The operation steps of development chimeric antibody is as follows:
Separation and purification mRNA from hybridoma HE2A5, the synthetic first chain cDNA.The design primer: the variable region of light chain primer is 5 ' gg tct aga gag ctc gtg atg aca 3 ' (23mer) (SEQID NO:6) and 5 ' at gga tcc agc ccg ttt tat ttc c, 3 ' (24mer) (SEQID NO:7); The variable region of heavy chain primer is 5 ' ccg ctc gag gag gtg cag ctg ctg gaatct 3 ' (30mer) (SEQ ID NO:8) and 5 ' ccc aag ctt tga gga gac ggtga, 3 ' (23mer) (SEQ ID NO:9).With PCR method clone and the light and heavy chain variable region gene (Fig. 5) of amplification antibody, and be cloned into respectively on the T carrier (U.S. Promega company) and carry out gene sequencing.Derive the variable region of heavy chain of this coded by said gene according to the gene order that is obtained and be made up of 118 amino acid, sequence is shown in SEQ ID NO:1.Anti-CD146 antibody chain variable region is made up of 112 amino acid, and sequence is shown in SEQID NO:2.
Then the light chain of anti-CD146 antibody is connected with weight chain constant area gene with people's light chain respectively with heavy chain variable region gene, make up the fusion gene (Wu Xiaoping of the complete chimeric antibody of codified, the research of new antitumoral angiogenesis gene antibody, the candidate for doctorate of Chinese Academy of Sciences academic dissertation, 2000.7); PDHL (U.S. Promega company) is carried in the expression that this fusion gene is inserted into selective mark and gene-controlled area (as promotor, enhanser, terminator) [14]In; This expression vector of amplification in bacterium, separation and purification contains the expression vector of chimeric antibody gene.The expression vector that will contain chimeric antibody gene with lipofectin (American I nvitrogen company) imports among the mammalian cell CHO.With MTX (American I nvitrogen company) screening transformant, obtain transgenosis cell strain CHO-chiAA98.Stability (Wu Xiaoping, the research of new antitumoral angiogenesis gene antibody, the candidate for doctorate of Chinese Academy of Sciences academic dissertation, 2000.7) with ELISA screening secretory antibody and identification of cell strain CHO-chiAA98.Detect the antigen-binding specificity and the effector function of chimeric antibody, the result shows that anti-CD146 chimeric antibody keeps the specificity of the anti-CD146 antibody of parental generation.
The preparation of embodiment three anti-CD146 antibody-Fab
Utilize the tumor vascular specificity of antibody recognition, with the antibody recognition function fragment with have immunocompetence or anti-tumor active substance is connected, develop dual intensity and reach fusion rotein with identification and lethal effect.Wherein antibody component mainly is Fab.The operation steps of producing the monoclonal antibody function fragment is as follows:
At first design a cover primer: antibody Fd primer is (mH1) (SEQ ID NO:10) and 5 ' gat atc act agt ggg ccc gct ggg ctc3 ' (forIgG2a/2b) (SEQ ID NO:11) of 5 ' agg tcc agc tgc tcg agt ctg g3 '; The light chain of antibody primer is (t/a) ct cc (SEQ ID NO:12) and 3 ' primer, 5 ' gc tct aga aag ctta tta aca ctc att cct gtt gaa (SEQ ID NO:13) of 5 ' primer gat att gag ctc gtgatg ac (c/a) ca (g/a).With quick preparation, purified mRNA test kit (Promega) from about 2 * 10 7Extract and purified mRNA among the individual hybridoma HE2A5.MRNA with purifying is a template, and cDNA is synthesized in reverse transcription.Using PCR method, is template with cDNA, adds primer in the PCR reaction system respectively, carries out 30 round-robin pcr amplifications.Each round-robin condition is: 94 ℃ of sex change 30s, and 55 ℃ of annealing 90s, 72 ℃ are extended 90s, and reaction proceeds to last circulation back and be incubated 10min in 72 ℃.Reaction is identified amplified production (Fig. 6) with agarose gel electrophoresis after finishing.Fd (antibody variable region and the constant region CH1) gene and the light chain gene of the anti-CD146 antibody of amplification are cloned into pComb3H (U.S. Promega company) [15]On the expression vector (Wu Xiaoping, the research of new antitumoral angiogenesis gene antibody, the candidate for doctorate of Chinese Academy of Sciences academic dissertation, 2000,7).The Fab-pCom3H recombinant plasmid is changed over to intestinal bacteria XL-Blue (DE3) (U.S. Promega company), with containing penbritin selective medium screening reorganization bacterium.Induce the reorganization bacterium to express the anti-CD146 antibody-Fab of solubility with IPTG.The anti-CD146 antibody-Fab of separation and purification (Fig. 7) from born of the same parents' pericentral siphon.The ELISA and the immune marking are identified the specificity (Fig. 8) of the anti-CD146 antibody-Fab of solubility.
Analyze the gene order of anti-CD146 antibody-Fab and derive anti-CD146 light chain of antibody and be made up of 217 amino acid, sequence is shown in SEQ ID NO:3.Anti-CD146 heavy chain of antibody Fd is made up of 222 amino acid, and sequence is shown in SEQ ID NO:4.
Embodiment four: the preparation of anti-CD146 small molecules single-chain antibody
Anti-CD146 small molecules single-chain antibody is specificity and the bioactive smallest molecule that keeps antibody.Because the little and immunogenicity of its molecular weight is low, therefore help carrying medicine and toxin carries out immunotherapy as carrier.The preparation method is as follows:
The design primer: the variable region of light chain primer is 5 ' primer for VL (25mer) ccg gag ctcgtg atg aca caa tct c (SEQ ID NO:14) and 3 ' primer for VL (26mer) gc tct aga ccg ttt tat ttc cag ctt (SEQ ID NO:15); Heavy chain chain variable region primer is 5 ' primer for VH (27mer) ccg ctc gag tct gga gct gag ctg gtg (SEQ ID NO:16) and 3 ' primer for VH (26mer) gg act agt tga gga gacggt gac cgt (SEQ ID NO:17).With anti-CD146 antibody-Fab gene is template, with increase the respectively weight chain variable region gene of antibody of PCR method.30 circulations of PCR reaction carrying out.Each round-robin condition is: 94 ℃ of sex change 30s, and 55 ℃ of annealing 90s, 72 ℃ are extended 90s, and reaction proceeds to last circulation back and be incubated 10min in 72 ℃.Reaction is identified amplified production with agarose gel electrophoresis after finishing.The antibody variable gene of amplification is cloned on the pComb3H expression vector, make up VH-linker-VL fusion gene (Wu Xiaoping, the research of new antitumoral angiogenesis gene antibody, the candidate for doctorate of Chinese Academy of Sciences academic dissertation, 2000.7) (Fig. 9).Make up soluble single-chain antibody efficient expression vector.The reorganization bacterium of penbritin screening secretory antibody, the IPTG inducing culture, centrifugal collection contains the supernatant of soluble antibody function fragment.Chromatographic separation purification of single stranded antibody (Fig. 7).ELISA identifies the specificity and the biological activity (Fig. 8) of the anti-CD146 single-chain antibody of solubility.Strand anti-CD146 antibody nucleotide sequence analysis and the aminoacid sequence derived are shown in SEQ ID NO:5.
Embodiment five, anti-CD146 antibody suppress vasculogenesis
We adopt chick chorioallantoic membrane vasculogenesis (CAM) model to detect the influence of CD146 antibody to vasculogenesis.Method is as follows [16]: fertilization Lay Hangzhoupro egg is cultivated after three days for 37 ℃ and is divested eggshell, in semicircle container, continued sterile culture three days, to resist on CD146 antibody and control antibodies solution absorbs to 2 * 2mm filter paper, then filter paper is lain against and hatch 24 to 48 hours on the chorioallantoic membrane vasoganglion, dissecting microscope is observed vasculogenesis down and is taken a picture.
Found that the 6th day at chick embryo development, with PBS, anti-CD146 antibody, homotype murine antibody application of sample are cultivated after 24 hours and are observed continuously in the chorioallantoic membrane blood vessel.In every group of 20 application of sample eggs, it is 16/20 that CD146 antibody suppresses vasculogenesis.Homotype mouse IgG does not have any reaction to the chorioallantoic membrane angiogenic growth.At the avascular area of the visible about 6 * 6mm of CD146 antibody sample application zone, wherein do not observe hemorrhage and inflammatory reaction.Compare with control group PBS or homotype murine antibody, anti-CD146 antibody all has the obvious suppression effect to chicken embryo vasculogenesis.(Figure 10).
The growth of embodiment six, anti-CD146 antibody inhibiting tumor and transfer
Lotus people knurl nude mice animal experiment is divided into liver cancer with people's tumor model, 3 groups of carcinoma of the pancreas and leiomyosarcomas.The tumor cell inoculation amount is respectively liver cancer cell 7721 (American type culture collection (American Type Culture Collection, Rockville, MD)) 1 * 10 7Individual, pancreatic cancer cell SW1990 (American type culture collection (American Type Culture Collection, Rockville, MD)) 2 * 10 6Individual and leiomyosarcoma cell SK-LMS-1 (American type culture collection (American Type Culture Collection, Rockville, MD)) 0.5 * 10 6Individual, only begin intraperitoneal administration 10mg/kg/ during to the about 1cm of diameter at tumor growth, 2 times weekly.The liver cancer treatment group is tumour cell and antibody administration simultaneously.Control group replaces anti-CD146 antibody with PBS or homotype mouse IgG respectively.Measure the knurl major diameter weekly, minor axis, and calculate the knurl volume. when dead mouse appears in control group, stop observing, by formula calculate tumour inhibiting rate, tumour inhibiting rate (%)=1-(experimental group knurl volume/control group knurl volume).
When tumor growth arrived the about 1cm of diameter, beginning abdominal injection antibody 10mg/kg/ only 2 times weekly, observed 3-4 week.We find that anti-CD146 antibody uses separately three kinds of tumours is all had in various degree therapeutic action, and tumor control rate is respectively leiomyosarcoma 50%, liver cancer 71.9%, carcinoma of the pancreas 48.7%.When we anti-CD146 antibody with 131The I coupling is used 131During the anti-CD146 antibody coupling matter treatment of I-carcinoma of the pancreas, only observe 10-18 days tumor control rates behind the single administration and reach 82%-86%.This result show anti-CD146 antibody and antibody thereof- 131The I conjugate all can effectively suppress the kinds of tumors growth, and the latter's tumor-inhibiting action stronger (Figure 11).
In therapeutic process, we observe the whole body situation of mouse.Find that CD146 Antybody therapy group mouse spirit is good, appetite is normal, and body weight does not have and subtracts, and does not have metastases and death.And control group causes mouse listless because gross tumor volume is big and ulcer and necrosis occur, and obviously becoming thin and emaciation phenomenon (Figure 12) appears in poor appetite.Lung and lymphoglandula in the 60-80% control group mice are found the metastases kitchen range, and 20% mouse is in treatment death in 14-16 days.
To the further pathological analysis of tumor tissues, find to be reduced to 1/3 of control group through the tumor tissues medium vessels density of CD146 Antybody therapy, the visible cavity necrosis of tumour cell (Figure 13) around the visible embolism of part blood vessel, blood vessel.Because anti-CD146 antibody selective binding vascular endothelial cell but not the tumour cell that is used to inoculate, so we think that the tumor-inhibiting action of CD146 antibody may realize by suppressing tumor-blood-vessel growth.
The influence that embodiment seven anti-CD146 antibody stick and expand mouse ectoplacenta awl
For study CD146 the Gestation period Mouse Uterus and the embryo in expression, we with the methods analyst of in situ hybridization the expression of CD146 in the whole Gestation period Mouse Uterus.Experimental result shows that CD146 mainly is expressed in implantation site, embryo trophocyte, uterine decidua blood vessel etc. and implants closely-related position with blastocyst.What cause especially that we note is the high expression level of CD146 molecule on the trophocyte.For further confirmation, we adopt immunofluorescence methods analyst CD146 molecule trophocyte's in mouse ectoplacenta awl distribution.The result shows, the strong trophocyte of anti-CD146 antibody specific combination transport property, and prompting CD146 may participate in trophocyte's transition process.
Therefore, we have separated mouse ectoplacenta awl, study the influence that anti-CD146 antibody sticks and expands the mouse trophocyte in external level, concrete grammar is as follows, the mouse post-coitum detects first day of cloudy bolt as gestation first day, from the 8.5th day female mouse uterine decidua of gestation, take out the embryo, at the embryonic ectoderm place ectoplacenta awl is separated, after cleaning, move in the culture dish at random and cultivate, nutrient solution is a Ham ' s F-12 substratum, and replenishes 3% foetal calf serum, 1.6mg/ml sodium bicarbonate, 0.3mg/ml glutamine, 0.24mg/ml calcium lactate and 400U/ml gentamicin.24 well culture plates are pressed 10 μ l/ hole beddings with the 0.1mg/ml ln in advance, and room temperature is air-dry more than 3 hours in super clean bench.Add anti-CD146 antibody (20 μ g/ml) as experimental group in the ectoplacenta awl nutrient solution, the control group normal mouse IgG of same concentrations.Cultivated 48 hours, observed once under inverted microscope every 12 hours, rock culture plate gently, motionless person is adherent ectoplacenta awl, move out of trophocyte's the ectoplacenta that is considered to expand awl around the adherent ectoplacenta is bored, experimental result shows that anti-CD146 antibody capable suppresses ectoplacenta awl sticking and the rate of spread (Figure 14 at ln, 15), wherein more remarkable to the inhibition of ectoplacenta awl expansion.In control group, the trophocyte normally moves and expands, and in the experimental group, trophocyte's migration and extended capability are suppressed, and the circular stretch-like unlike control group of cell (Figure 16) experiment repeats 3 times.In addition, we have also detected the effect of sticking and expanding of anti-CD146 antibody to boring through the mouse ectoplacenta of bFGF and VEGF stimulation, find that anti-CD146 antibody also has inhibition effect (Figure 17～20) to it, concrete grammar is not add foetal calf serum in ectoplacenta awl nutrient solution, and bFGF or the VEGF of adding 10ng/ml, other steps are the same.
Embodiment eight CD146 antibody suppress mouse ectoplacenta awl secrete metalloproteinases
Separate the 8.5th day mouse ectoplacenta of gestation awl, after cultivating 24 and 48 hours, collect nutrient solution, measure the protein concentration of nutrient solution with the Bradford method.Carry out acrylamide gel electrophoresis then, with 10% acrylamide gel that contains the 1mg/ml gelatin as separation gel, 4% acrylamide gel is as concentrating glue, every swimming lane 10-20 μ l nutrient solution is equivalent to 6 μ g gross proteins, hatches half hour with non-reducing sample buffer 37 degree, go up the sample electrophoresis again, electrophoresis carries out in ice bath, and the voltage that concentrates in the glue is 80 volts, and the voltage in the separation gel is 120 volts.Gel washs in 2.5%TritonX-100 for several times behind the electrophoresis, at Incubating Solution (150mmol/L NaCl, 5mmol/L CaCl 2And 50mmol/L Tris-HCl, pH 7.6) in 37 degree hatched 16-18 hour, conventional Xylene Brilliant Cyanine G G-250 dyeing, quantitative analysis is carried out at Bio-rad Chemdoc 2000 gel imaging instrument in the decolouring back.Whether add EDTA (20mmol/L) chelating calcium ion in the Incubating Solution, be the metalloprotease role to identify.The result shows that anti-CD146 antibody has obvious suppression (Figure 21), quantitative analysis significant difference, p＜0.01 to trophocyte's secrete metalloproteinases 9 and metalloprotease 2 in the mouse ectoplacenta awl.
Embodiment nine anti-CD146 antibody cause the miscarriage of pregnant mouse
In experimentation on animals, whether we observe anti-CD146 antibody can termination of pregnancy.Experiment is divided into 3 groups, every group of 9 mouse.Inject anti-CD146 antibody for one group, one group is contrast with PBS, and another group is contrast with homotype mouse IgG.The purpose of injection homotype mouse IgG is in order to get rid of the influence that mouse IgG itself implants blastocyst.At pregnant the 3rd day, its left side horn of uterus (being the junction of uterus and ovary) is injected anti-CD146 antibody (2 μ g antibody are dissolved in 10 μ lpH7.4 phosphoric acid buffers) with microsyringe, its right side horn of uterus is injected the PBS of equal volume or the normal mouse IgG of same concentrations.Put to death mouse after five days, the successful implantation rate of statistics mice embryonic under anatomical lens.The result shows that the implantation of mouse blastocyst is blocked through injecting the uterus, right side of anti-CD146 antibody, and inhibiting rate has dysplasia and odd-shaped picture now up to (Figure 22) remaining embryo more than 80%, and control sides uterus blastocyst is implanted uninfluenced (Figure 23).This phenomenon explanation CD146 molecule is implanted and is kept the embryo and plays important effect aspect pregnant, and anti-CD146 antibody can influence that the embryo implants and embryo's growth.
Reference
1.Folkman J.Tumor angiogenesis：therapeutic implication.N Engl J Med，1971，285(21)：1182～1186
2.Paria，B.C.et al.Deciphering the cross-talk of implantation：Advance and challenges.Science，2002，296(21)：2185-2188
3.Cross，J.C.et al.Implantation and the placenta：Key pieces of the development puzzle.Science，1994，266(2)：1508-1518
4.Shih，I.M.，Wang，T.L，Wu，T.C.et al.Expression of Mel-CAM in implantation siteintermediate trophoblastic cell line，IST-1，limits its migration on uterine smooth musclecells.J.Cell sci.111，2655-2664(1998).
5.Kohler and Milstein.Nature，1975，256：495
6.Enzyme-linked Immunosorbent Assay for Screening monoclonal antibody productionusing enzyme-labeled second antibody.Meth.Enzymol，1983，92：168-74.
7.Ivan Lefkovits.Immunology Methods Manual.1997
8.Shih，I.M.，Elder，D.E.，Speicher，D.et al.Isolation and functional characterization ofthe A32 melanoma-associated antigens.Cancer Res54，2514-2520(1994)
9.Bardin，N.，Frances，V.，Lesaule，G.et al.Identification of the S-Endoendothelial-associated antigen.Biochem.Biophys.Res.Commun.218，210-216(1996).
10.Shih，I.E.，Nesbit，M.，Herlyn，M.et al.A new Mel-Cam(CD146)-specific monoclonalantibody，MN-4，on paraffin-embedded tissue.Mod.Pathol.11，1098-1106(1998).
11.Shih，I.M.，Speicher，D.，Hsu，M.Y.et al.Melanoma cell-cell interactions are mediatedthrough heterophilic Mel-CAM/ligand adhesion.Cancer Res.57，3835-3840(1997).
12.Anfosso，F.，Bardin，N.，Frances，V.et al.Activation of human endothelial cells viaS-Endo-1 antigen(CD 146)stimulates the tyrosine phosphorylation of focal adhesionkinase p125 FAK.J.Biol.Chem.273，26852-26856(1998).
13.Anfosso，F.，Bardin，N.，Vivier，E.et al.Outside-in signalling pathway linked to CD146engagement in human endothelial cells.J Biol Chem(2000).
14. Zhu winter jasmine etc., cell and molecular immunology magazine 16:355-358,2000
15.Kang AS，et al.Linkage of recognition and replication functions by assemblycombinatorial antibody Fab libraries along phage surfaces.Proc Natl Acad Sci USA，88：363-366，1991.
16.Auerbach R，Kubai L，Knighton D.A simple procedure for the long-term cultivation forchicken embryo.Dev Biol，41：391-394，1974.
Sequence table
＜110〉Institute of Biophysics, Academia Sinica
＜120〉a kind of monoclonal antibody, its encoding gene, purposes and the hybridoma cell line of secreting it
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ggactagttg aggagacggt gaccgt 26

Claims (16)

1. the hybridoma cell line of a secrete monoclonal antibody, its deposit number is CGMCC0491.
2. one kind by the described hybridoma cell line excretory of claim 1 monoclonal antibody, wherein, the variable region of heavy chain of described antibody has the aminoacid sequence shown in the SEQ ID NO:1, and the variable region of light chain of described antibody has the aminoacid sequence shown in the SEQ ID NO:2.
3. single-chain antibody that is derived from the described hybridoma cell line excretory of claim 1 monoclonal antibody, wherein, this single-chain antibody has the aminoacid sequence shown in the SEQ ID NO:5.
4. Fab fragment that is derived from the described hybridoma cell line excretory of claim 1 monoclonal antibody, wherein, the segmental Fab light chain of this Fab has the aminoacid sequence shown in the SEQ ID NO:3, and the segmental Fab heavy chain of this Fab has the aminoacid sequence shown in the SEQ ID NO:4.
5. nucleotide sequence of the described monoclonal antibody of claim 2 of encoding.
6. nucleotide sequence of the described single-chain antibody of claim 3 of encoding.
7. the coding described Fab of claim 4 segmental nucleotide sequence.
8. the application of the described monoclonal antibody of claim 2 in the preparation tumor vaccine.
9. the application of the described single-chain antibody of claim 3 in the preparation tumor vaccine.
10. the application of the described Fab fragment of claim 4 in the preparation tumor vaccine.
11. the application of the described monoclonal antibody of claim 2 in the medicine of preparation prevention early pregnancy and contraception.
12. the application of the described single-chain antibody of claim 3 in the medicine of preparation prevention early pregnancy and contraception.
13. the application of the described Fab fragment of claim 4 in the medicine of preparation prevention early pregnancy and contraception.
14. the application of the described monoclonal antibody of claim 2 in the medicine of preparation treatment and new vessel diseases associated.
15. the application of the described single-chain antibody of claim 3 in the medicine of preparation treatment and new vessel diseases associated.
16. the application of the described Fab fragment of claim 4 in the medicine of preparation treatment and new vessel diseases associated.
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