CN1234405A - High-effective broad-spectrum antibody for inhibiting growth and metastasis of tumour - Google Patents

High-effective broad-spectrum antibody for inhibiting growth and metastasis of tumour Download PDF

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CN1234405A
CN1234405A CN 99107586 CN99107586A CN1234405A CN 1234405 A CN1234405 A CN 1234405A CN 99107586 CN99107586 CN 99107586 CN 99107586 A CN99107586 A CN 99107586A CN 1234405 A CN1234405 A CN 1234405A
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CN1124284C (en
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阎锡蕴
袁玖
李力
吴小平
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Institute of Biophysics of CAS
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Institute of Microbiology of CAS
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Abstract

The new type antibody for inhibiting tumor growth and metastasis, including murine monoclonal antibody AA98, human-murine chimeric antibody Chi AA98 and Fab and Fv functional fragments of antibody AA98, is characterized by that it can make specific recognition of neogenetic blood vessel of tumor, possess the capability for inducing ADCC and CDC, and can specifically and broad-spectrally inhibite the tumor growth and metastasis. These antibodies or antibody combination can be made into the high-effective low-toxic new type medicine with broad-spectrum anti-cancer function, at the same time can be used for early diagnosis of tumor and basic research of mutual interaction of tumor and its blood vessel.

Description

High-efficiency broad spectrum suppresses the novel antibody of tumor growth and transfer
The invention belongs to molecular biology and antibody engineering technical field.Specifically, the present invention relates to a class can the most of common human tumor new vessel of specific recognition and suppress tumor growth and the novel antibody of transfer.
Cancer 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.
Tumour three big conventional (radiotherapy, chemotherapy and operation〉in the treatment, pharmacological agent is an importance.The factor that influences at present the tumor pharmacother effect is many-sided, comprises specificity, mode of transport, perviousness and the induced tumor resistance of medicine.The specificity of its Chinese traditional medicine and resistance are the key issues that tumor therapeutics is badly in need of solution.
Characteristic of the present invention is that it is that a class is TS, wide spectrum, nothing or low chemical sproof new type anticancer medicine.The strategy that the present invention taked is lifeline-nutrition and the blood supply by the blocking-up tumour cell, thereby makes the tumour cell ischemic necrosis.This anticancer New Policy be based on 2 theories 1. tumour depend on blood vessel and the theory of growing.2. tumor neogenetic blood vessels neither resembles and lacks genetic stability the tumour cell, is different from normal blood vessels again.The renewal of normal blood vessels endothelium needs more than 100 day, and tumor vessel only needs a couple of days, and has the unexistent new mark of some normal blood vessels endotheliums.The distinctive mark of these tumor vessels is the new target of tumor drug screening.
The present invention compares with traditional anti-tumour antibody, and its characteristics are wide spectrum, special, efficient, the difficult resistance that produces.The mechanism of action is as follows, and 1. tumor vascular endothelial cell is relatively stable, and sudden change is few, thereby can not resemble and be easy to produce resistance the tumour cell; 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; 3. a thousands of tumour cell 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.
By document and IBM patent data library inquiry neovascularization inhibitor (angiogenesisinhibitor) and new vessel antibody (angiogenesis antibody), do not see that document relevant with the present invention and patent deliver.
The present invention relates to the novel antibody that a class acts on the human tumor blood vessel, comprise mouse monoclonal antibody AA98 and people-mouse chimeric antibody chiAA98 and small molecules function antibody AA98-Fab and AA98-scFv, they act on various tumor vessels, comprise liver cancer, cancer of the stomach, large bowel cancer, mammary cancer, lung cancer, ovarian cancer and leiomyosarcoma, and any reaction is taken place in healthy tissues hardly.Monoclonal antibody itself or antibody conjugates all have the effect that suppresses multiple human body tumor growth.
The present invention and other antibody relatively have unique advantage: the first, and special: antibody recognition tumor tissues and non-normal tissue, this high degree of specificity will significantly reduce the poison of medicine and pay effect in clinical application.The second, wide spectrum: dissimilar solid tumor cells contain different specific antigens, as breast cancer antigen difference and colon cancer antigen; New vessel forms so that the nutrition of the mad growth of tumour to be provided but tumor growth all needs to stimulate on every side.Though the tumor tissues difference, and tumour stimulates outgrowth new vessel all to have some common features, the present invention develops at these common traits, so its antitumous effect is a wide spectrum.The 3rd, efficient: antibody can be divided into five classes according to its constructional feature, and great majority only have recognition function, and minority not only has recognition function and also has lethal effect.Monoclonal antibody of the present invention belongs to the IgG2a hypotype, is a kind of rare lethal antibody.Itself gets final product independent kill cancer cell, and its mechanism of action is because some antibody in the presence of body effcct cell and serum, has induce antibody dependent cell toxic action (ADCC) and complement-dependent cytotoxicity (CDC), thus the kill tumor cell.The 4th, antibody is because the special new vessel endothelium that is distributed in of target antigen that they are discerned participates in growth of tumor and transfer to tumor neovasculature high selectivity.The 5th, antibody and binding substances thereof are that a class has wide spectrum, efficient, low toxicity, low chemical sproof new type antineoplastic medicine.The 6th, antibody can be used for the diagnosis and the location of tumour, detects cancer by the inside and outside method.The 7th, antibody is as a kind of good research tool, be used to study new vessel forms and with the interaction of tumor growth and transfer.
The innovation part of the present invention aspect technological line is: 1. adopt the tumor cell culture supernatant to induce Human umbilical vein endothelial cells, make it become the vascular endothelial cell of propagation.The latter is used for immune mouse and obtains monoclonal antibody AA98.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 new target molecule.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.
The present invention includes a cover tumor vascular monoclonal antibody of identification and a genetic engineering antibody, the latter comprises chimeric antibody, monoclonal antibody and single-chain antibody.Wherein monoclonal antibody and chimeric antibody all have antibody dependent cellular cytotoxicity (ADCC) and CDC (CDC) activity, and this is the basis of their vivo antitumor effects.Itself just has therapeutic action these antibody.And monoclonal antibody and single-chain antibody do not show ADCC and CDC activity in vivo, can be used for carrier, connect chemotherapeutics, toxin, cytokine, enzyme and radio isotope.Therefore, these small molecular antibodies can be used as one of composition of various immune conjugates, are used for the diagnosis and the treatment of tumour.
In order more fully to understand and apply the invention, provide the following example.
Embodiment one: the preparation of monoclonal antibody AA98 and evaluation
Use hybridoma technology manufacture order clonal antibody AA98 (Kohler and Milstein, Nature2 56:495,1975; Yeh et al, Proc.Natl.Acad.Sci.USA, 1979; Yeh etal., Int.J.Cancer, 1982).Be summarized 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.The total cellular score of per injection is approximately 10 7Immunization was got spleen after three days the last time, and splenocyte is suspended in the RPMI 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.
Utilization Douillard, et al., Enzyme-linked Immunosorbent Assay forScreening monoclonal antibody production using enzyme-labeledsecond antibody, Meth.Enzymol, .92:168-74,1983 methods of introducing seed cells into 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 successively add secretory antibody then.By the ELISA repeated screening, obtain to take place the antibody of the high-affinity of specific reaction at last with hyperplasia HUVEC.
Repeatedly clone the hybridoma HE2A5 of secretion specific antibody AA98 with dilution method.A large amount of amplified hybridization oncocyte HE2A5 that cultivate collect the culture supernatant that contains antibody A A98 and are used for the antibody function evaluation.In addition hybridoma HE2A5 is made cell suspension, be used to prepare antibody ascites.
The preparation method of ascites is summarized as follows: six week BALB/C mice abdominal injection pristane in age (Pri stane) 0.5ml/ only.After 10 days, with the hybridoma suspension inoculation in BALB/C mice abdominal cavity, 1 * 10 7/ ml/ only after 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 antibody A A98 and belongs to IgG homotype antibody.
Utilize frozen tissue section and immunohistochemical methods method (Immunology Methods Manual, Edited by Ivan Lefkovits, 1997) reaction of evaluation antibody A A98 and human normal tissue and tumor tissues, the result shows the high specific reaction of the capillary blood vessel of antibody A A98 and many tumor tissues, and very limited with the reaction of healthy tissues.Table 1 has reacted the immunohistochemical staining situation of antibody A A98 to various tumor tissues and healthy tissues.In immunohistochemical methods section, clearly visible antibody is only to the reaction of the tiny blood vessels in the tumor tissues, and not with big medium vessels and healthy tissues in vascular reaction.
Table 1: immunohistochemical methods is identified antibody A A98 and human normal tissue
Reaction with new vessel in the tumor tissues
Organize routine number number positive Organize routine number number positive
Liver 10 0 Liver cancer 10 10
Stomach 60 Cancer of the stomach 66
Large intestine 50 Large bowel cancer 77
Mammary gland 50 Mammary cancer 88
Lung 40 Lung cancer 44
Ovary 11 Ovarian cancer 55
Unstriated muscle 60 Leiomyosarcoma 10 10
Cardiac muscle 20 Kidney 10
Spleen 22 Brain 10
Adopt FACS cell sorter analysis list clonal antibody and Fab thereof active to the combination of various clones.With 1 * 10 6Culturing cell is suspended in the substratum that 100 μ l contain 10 μ g/ml antibody A A98, floating educating 1 hour.Give a baby a bath on the third day after its birth time with substratum, add the FITC-sheep anti-mouse igg antibody of nutrient solution dilution, floating educating 45 minutes.Give a baby a bath on the third day after its birth all over after, cell is suspended among the 200 μ l-500 μ lPBS again.On FACS, carry out immunofluorescence analysis and measure mean fluorecence density.Experimental result show antibody A A98 can specific combination the vascular endothelial cell of propagation.
Adopt MTT method research antibody A A98 to the THE NATURAL CYTOTOXIC EFFECT of target cell and the cytotoxicity of complement-mediated.Observed beyond thought result in experiment, both antibody A A98 not only had the cytotoxicity of complement-mediated, and was not adding under the situation of any modification, and antibody itself can kill and wound target cell separately, and its kill rate strengthens with the increase of antibody dosage.Killing-efficiency further improves after adding complement.Experimental results reduction is in table 2.
Table 2. antibody A A98 is to the lethal effect of hyperplasia vascular endothelial cell
Kill rate (%) antibody dosage (μ g/ml) antibody antibody+complement complement 1.0 4.83 51.42 0.0010.0 15.72 50.76 0.00100.0 35.32 58.33 0.00
Experimentation on animals shows that antibody A A98 uses separately or label isotope 131I all can suppress the growth and the transfer of human body kinds of tumors.Illustrate antibody A A98 below and do not adding the experimental result that suppresses the human pancreas cancer growth under the situation of any modification.
Average knurl volume is 130mm before 40 of the BALB/C-NU mouse of lotus human pancreas cancer (middle differentiation duct adenocarcinoma Fwk-3), administration 3, random packet.(1) antibody A A98, (4) human IgG, (5) not treatment group.Intraperitoneal injection is administered twice weekly.Measured long, the shortest diameter of tumour in per three days, according to formula (knurl volume=(minor axis 2* major diameter) * and π/6) the calculating gross tumor volume.Calculate inhibition rate of tumor growth according to following formula.Inhibition rate of tumor growth (%)=(1-treatment group knurl volume/non-treatment group knurl volume) * 100%.Observations as shown in Figure 1.
Embodiment two: the preparation of people-mouse chimeric antibody chiAA98
Because mouse monoclonal antibody lacks the biological effect function in the human immune system, also be identified as foreign matter simultaneously, brings out the immunological rejection of body, thereby is 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 kept the specificity of mouse monoclonal antibody conjugated antigen, can compete and suppress combining of monoclonal antibody AA98 and its target antigen; And reduced the mouse derived components in the antibody molecule, increased the biological effector function of antibody.The operation steps following 1 of development chimeric antibody) separation and purification mRNA from hybridoma HE2A5, the synthetic first chain cDNA; 2) (23mer) and 5 ' at gga tcc agc ccg ttt tat ttc c of design primer:variable region of light chain 5 ' gg tct aga gag ctc gtg atg aca 3 ', 3 ' (24mer); Variable region of heavy chain 5 ' ccg ctc gag gag gtg cag ctg ctg gaa tct 3 ' is and 5 ' ccc aag ctt tga gga gac ggt ga (30mer), 3 ' (23mer) 3) clone and light, the heavy chain variable region gene of amplification antibody with PCR method; 4) sequential analysis people antibody variable gene is derived the aminoacid sequence of this coded by said gene; Antibody A A98 variable region of heavy chain is made up of 118 amino acid; Sequence is as follows: 1 Glu Val Gln Leu Leu Asp Ser Gly Ala Glu Leu Val Arg Pro Gly Ala, 16 17 Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe Thr Asn Tyr, 32 33 Trp Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu Glu Trp Ile, 48 49 Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe, 64 65 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala Tyr, 80 81 Met Leu Leu Ser Ser Leu Lys Ser Glu Asp Ser Ser Val Tyr Phe Cys, 96 97 Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr, 112113 Thr Val Thr Val Ser Ser, 118 antibody A A98 variable region of light chain are comprised of 112 amino acid; Sequence is as follows: 1 Glu Leu Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly, 1617 Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser, 3233 Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro, 4849 Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala, 6465 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His, 8081 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg, 9697 Glu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala 1125) the weight chain variable region gene of antibody A A98 is connected with people's light weight chain constant area gene respectively, makes up the fusion of the complete chimeric antibody of codified; 6) this fusion gene is inserted among the expression vector pdHL2.4 of selective mark and gene-controlled area (as promotor, enhanser, terminator); 7) this expression vector of amplification in bacterium, the expression that separation and purification contains chimeric antibody gene is carried
Body;
8) expression vector that will contain chimeric antibody gene with lipofectin imports mammalian cell
Among the CHO;
9) screen transformant with MTX, obtain transgenosis cell strain CHO-chiAA98;
10) with the stability of ELISA screening secretory antibody and identification of cell strain CHO-chiAA98;
11) antigen-binding specificity and the effector function of detection chimeric antibody.
Embodiment three: the preparation of antibody recognition function fragment AA98-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:
1) design one cover primer: 5 ' agg tcc agc tgc tcg agt ctg g3 ' (mH1) and 5 ' gat
Atc act agt ggg ccc gct ggg ctc3 ' (for IgG2a/2b), light chain of antibody
Primer 5 ' primer gat att gag ctc gtg atg ac (c/a) ca (g/a) is ct (t/a)
Cc and 3 ' primer, 5 ' gc tct aga aag ctt a tta aca ctc att cct gtt
gaa
2) with preparation fast, purified mRNA test kit (Promega) is from about 2 * 10 7Individual hybridization
Extract and purified mRNA among the oncocyte HE2A5.
3) mRNA with purifying is a template, and cDNA is synthesized in reverse transcription.
4) using PCR method, is template with cDNA, adds primer in the PCR reaction system respectively,
Carry out 30 round-robin pcr amplifications.Each round-robin condition is: 94 ℃ of sex change 30s, 55
℃ annealing 90s, 72 ℃ are extended 90s, and reaction proceeds to last circulation back in 72 ℃
Insulation 10min.Reaction is identified amplified production with agarose gel electrophoresis after finishing.
5) Fd (antibody variable region and constant region CH1) gene and the light chain gene of amplification antibody A A98
Be cloned on the pComb3H expression vector.
6) the Fab-pCom3H recombinant plasmid is changed over to intestinal bacteria XL-Blue (DE3), with containing ammonia benzyl green grass or young crops
Mycin selective medium screening reorganization bacterium.
7) induce the reorganization bacterium to express soluble antibody AA98-Fab with IPTG.
8) separation and purification antibody A A98-Fab from born of the same parents' pericentral siphon.
9) ELISA and the immune marking are identified solubility AA98-Fab antibody fragment.
10) gene of nucleotide sequence analysis antibody A A98-Fab and to derive its amino acid sequence as follows:antibody A A98 light chain is made up of 217 amino acid; ： 1 Glu Leu Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 16 17 Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser 32 33 Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro 48 49 Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala 64 65 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 80 81 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg 96 97 Glu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala 112113 Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu 128129 Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro 144145 Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn 160161 Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr 176177 Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His 192193 Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile 208209 Val Lys Ser Phe Asn Arg Asn Glu Trp 217AA98Fd222,： 1 Glu Val Gln Leu Leu Asp Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 16 17 Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe Thr Asn Tyr 32 33 Trp Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu Glu Trp Ile 48 49 Ala Arg Ile Tyr Pro Gly Thr Asp lle Thr Tyr Tyr Asn Glu Lys Phe 64 65 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala Tyr 80 81 Met Leu Leu Ser Ser Leu Lys Ser Glu Asp Ser Ser Val Tyr Phe Cys 96 97 Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 112113 Thr Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 128129 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 144145 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 160161 Ser Gly Ser Leu Ser Ser Gly Cys Ala His Leu Pro Ala Val Leu Gln 176177 Ser Asp Leu Tyr Thr Leu Ser Ser Leu Met Thr Val Thr Ser Ser Thr 192193 Trp Ala Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 208209 Thr Lys Val Asp Lys Lys Ile Glu Pro Asn Gly Ala Thr Ser 222
Embodiment four: the preparation of single-chain antibody AA98-scFv
The characteristics of small molecules single-chain antibody AA98-scFv are that to keep specificity and the molecular weight of antibody A A98 little, and immunogenicity is little, and penetration power is strong, helps carrying medicine and toxin as carrier.The preparation method is as follows:
1) design primer: variable region of light chain primer 5 ' primer for VL (25mer) ccg gag ctc
Gtg atg aca caa tct c and 3 ' primer for VL (26mer) gc tct aga ccg
Ttt tat ttc cag ctt heavy chain chain variable region primer 5 ' primer for VH (27mer)
Ccg ctc gag tct gga gct gag ctg gtg and 3 ' primer for VH (26mer)
gg?act?agt?tga?gga?gac?ggt?gac?cgt。
2) be template with antibody A A98-Fab gene, increasing the weight chain of antibody respectively with PCR method can
Become district's gene.30 circulations of PCR reaction carrying out.Each round-robin condition is: 94 ℃ of sex change
30s, 55 ℃ of annealing 90s, 72 ℃ are extended 90s, and reaction proceeds to last circulation back and exists
Be incubated 10min in 72 ℃.Reaction is identified amplified production with agarose gel electrophoresis after finishing.
3) antibody variable gene of amplification is cloned on the pComb3H expression vector, makes up VH-
The linker-VL fusion gene.
4 ) AA98： 1 Glu Leu Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 16 17 Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser 32 33 Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro 48 49 Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala 64 65 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 80 81 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg 96 97 Glu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ser 112113 Arg Arg Gly Gly Gly Ser Arg Gly Gly GLy Pro Gly Gly GLy GLy Ser 128129 Glu Val Gln Leu Leu Asp Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 144145 Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe Thr Asn Tyr 160161 Trp Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu Glu Trp Ile 176177 Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe 192193 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala Tyr 208209 Met Leu Leu Ser Ser Leu Lys Ser Glu Asp Ser Ser Val Tyr Phe Cys 224225 Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 240241 Thr Val Thr Val Ser Ser5 ) 。 6) the reorganization bacterium of penbritin screening secretory antibody, the IPTG inducing culture, centrifugal collection contains the supernatant of soluble antibody function fragment.7) ELISA, the immune marking and immunohistochemical methods identify soluble antibody AA98-scFv fragment.8) chromatographic separation purification of single stranded antibody.

Claims (15)

1. a class has the antibody and the function fragment thereof of specific reaction to new vessel, it is characterized in that: this antibody-like comprises authentic monoclonal antibody and genetic engineering antibody, they can specific recognition new vessel endotheliocyte membrane antigen, has the ability of bringing out ADCC and CDC, this antibody-like itself and binding substances thereof all can suppress the formation of new vessel, have special and antitumous effect wide spectrum.
2. antibody according to claim 1 is characterized in that this antibody-like can be a kind of mouse monoclonal antibody, called after AA98.
3. antibody according to claim 2 is characterized in that antibody can be a kind of AA98 antibody that is produced by hybridoma HE2A5.
4. hybridoma HE2A5 according to claim 3 is characterized in that inducing the Human umbilical vein endothelial cells hyperplasia with the tumor cell culture supernatant, and with proliferation of vessels endothelial cell immunotoxin mouse, obtains the method for secretory antibody AA98 hybridoma.
5. antibody according to claim 1, it is characterized in that antibody can be a kind of people-mouse embedding antibody ChiAA98, it contains the variable region of the described antibody A A98 of claim 2 and the constant region of people's antibody, and its antigen binding domain competition suppresses antibody A A98 and combines with the specificity of its target antigen.
6. according to the described antibody ChiAA98 of claim 5, it is characterized in that this antibody is to be produced by CHO-AA98 engineering cell system.
7. according to claim 2 or 5 described antibody, it is characterized in that the aminoacid sequence that antibody heavy chain variable region and chain variable region gene are led.
Antibody heavy chain variable region is made up of 118 amino acid; ：1 Glu Val Gln Leu Leu Asp Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1617 Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe Thr Asn Tyr 3233 Trp Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu Glu Trp Ile 4849 Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe 6465 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala Tyr 80 81 Met Leu Leu Ser Ser Leu Lys Ser Glu Asp Ser Ser Val Tyr Phe Cys 96 97 Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 112113 Thr Val Thr Val Ser Ser 118112,： 1 Glu Leu Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1617 Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser 3233 Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro 4849 Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala 6465 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 8081 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg 9697 Glu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala 112
8. antibody as claimed in claim 1 is characterized in that antibody can be a kind of Fab function fragment (AA98-Fab), and the aminoacid sequence that this antibody gene is derived is as follows,
Antibody A A98-Fab light chain is made up of 217 amino acid; ： 1 Glu Leu Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 16 17 Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser 32 33 Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro 48 49 Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala 64 65 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 80 81 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg 96 97 Glu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala 112113 Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu 128129 Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro 144145 Lys Asp Ile Asn Val Lys Trp Lys Ile Asp GIy Ser Glu Arg Gln Asn 160161 Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr 176177 Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His 192193 Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile 208209 Val Lys Ser Phe Asn Arg Asn Glu Trp 217AA98-FabFd222,： 1 Glu Val Gln Leu Leu Asp Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 16 17 Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe Thr Asn Tyr 32 33 Trp Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu Glu Trp Ile 48 49 Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe 64 65 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala Tyr 80 81 Met Leu Leu Ser Ser Leu Lys Ser Glu Asp Ser Ser Val Tyr Phe Cys 96 97 Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 112113 Thr Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 128129 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 144145 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 160161 Ser Gly Ser Leu Ser Ser Gly Cys Ala His Leu Pro Ala Val Leu Gln 176177 Ser Asp Leu Tyr Thr Leu Ser Ser Leu Met Thr Val Thr Ser Ser Thr 192193 Trp Ala Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 208209 Thr Lys Val Asp Lys Lys Ile Glu Pro Asn Gly Ala Thr Ser 222
9. antibody as claimed in claim 1; AA98-scFv,： 1 Glu Leu Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 16 17 Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser 32 33 Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro 48 49 Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala 64 65 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 80 81 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg 96 97 Glu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ser 112113 Arg Arg Gly Gly Gly Ser Arg Gly Gly GLy Pro Gly Gly GLy GLy Ser 128129 Glu Val Gln Leu Leu Asp Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 144145 Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe Thr Asn Tyr 160161 Trp Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu Glu Trp lle 176177 Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe 192193 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala Tyr 208209 Met Leu Leu Ser Ser Leu Lys Ser Glu Asp Ser Ser Val Tyr Phe Cys 224225 Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 240241 Thr Val Thr Val Ser Ser 246
10. as any one described antibody in the claim 2,5,8,9, it is characterized in that wherein any antibody can be a kind of recombinant protein, it contains any one described antibody aminoacid sequence in the claim 2,5,8,9, and the antibody derivatives with similar functions.
11. as any one described antibody and derivative thereof in the claim 2,5,8,9,10, it is characterized in that antibody conjugates, wherein any one described antibody and its bonded material can be a kind of antitumor drugs, a kind of toxin, a kind of radioactivity agent, a kind of enzyme or another kind of antibody.
12. specific recognition new vessel antibody according to claim 1 is characterized in that this antibody-like such as claim 2,5,8,9,10 described any antibody and derivative thereof can be applicable to human tumor imaging level diagnosis.
13., it is characterized in that the respective target molecular antigen that any antibody of the present invention is discerned as claim 2,5,8,9,10 described any antibody and derivatives thereof.
14. target antigen as claimed in claim 13 is characterized in that the target antigen that any antibody of the present invention is discerned can be applied to drug screening and tumor vaccine.
15., it is characterized in that wherein any antibody can be applicable to relevant immunological effect of new vessel and fundamental research as claim 2,5,8,9,10 described any antibody and derivatives thereof.
CN 99107586 1999-05-25 1999-05-25 High-effective broad-spectrum antibody for inhibiting growth and metastasis of tumour Expired - Fee Related CN1124284C (en)

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CN103159824A (en) * 2013-02-06 2013-06-19 中国科学院生物物理研究所 Full sealed pipelined protein purification system and application on preparation of sterile apyrogeneity protein pharmaceutical medicine
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CN109111528A (en) * 2018-09-20 2019-01-01 杭州普略生物科技有限公司 Using MSLN as the Chimeric antigen receptor of target spot
CN109111528B (en) * 2018-09-20 2021-06-22 杭州普略生物科技有限公司 Chimeric antigen receptor with MSLN as target point

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