CN1351888A

CN1351888A - Method for producing anti-osteopontic antibody medicine and use

Info

Publication number: CN1351888A
Application number: CN01140535A
Authority: CN
Inventors: 韩梅; 温进坤
Original assignee: Hebei Medical University
Current assignee: Hebei Medical University
Priority date: 2001-11-10
Filing date: 2001-11-10
Publication date: 2002-06-05

Abstract

The present invention relates to the field of biological preparation technology. By means of bioengineering process, the humanized anti-osteopontin single chain antibody, anti-osteopontin monoclonal antibody and anti-osteopontin polyclonal antibody are produced and made into various medicament forms and are used in preventing and treating vascular re-stenosis, atheoscleorsis and osteoporosis. The present invention has the features of novel materials, unique and reasonable production process, high curative effect and no toxic side-effect, and wide application prospect.

Description

Method for producing anti-osteopontic antibody medicine and purposes

Technical field

The present invention relates to the technical field of pharmaceuticals of bio-pharmaceuticals and treatment of vascular restenosis, atherosclerosis and osteoporosis.

Background technology

The cardiovascular diseases is one of the most serious disease of harm humans life and health, and its sickness rate accounts for first of all kinds of diseases, and coronary heart disease then is the main cause that causes such death.Percutaneous intracavity arteria coronaria plasty (PTCA) is one of important method for the treatment of at present coronary heart disease, and the annual whole world has the millions of people to accept this treatment approximately.But owing to there is the patient of 30%-50% restenosis to occur in 6 months after surgery, and so far still the definite medicine of inefficacy come out.Placing endovascular stent is the major measure of prevention of restenosis, and Stent can stop the arterial elasticity retraction, prevents reinventing of blood vessel wall, thereby reduces the restenosis incidence rate; But it has the vascular damaged of increasing and thrombotic danger, especially the damage of blood vessel endothelium and fibrolaminar fracture make the various kinds of cell factor directly act on blood vessel wall middle level smooth muscle cell, make it hyper-proliferative and migration under inner membrance, new intima forms, and causes restenosis.Theory and practice proves, hypertension, and cardiovascular diseasess' such as atherosclerosis, vascular restenosis morbidity is relevant with migration with the abnormality proliferation of vascular smooth muscle cell.Therefore, suppress vascular smooth muscle cell proliferation and the suitable main policies that becomes treatment and prevent above-mentioned cardiovascular diseases of moving, verified, the abnormality proliferation of vascular smooth muscle cell and migration are because due to some gene expression dysregulations.Therefore, promote or suppress the function of these expression of gene and/or increase or blocking gene product albumen matter, can reach control is the cardiovascular diseases's of main pathological characters purpose with the vascular smooth muscle cell hyper-proliferative.The method that is subjected to the principle of gene regulation to carry out cardiovascular diseases's treatment based on vascular smooth muscle cell proliferation is worked as base because of treatment at present.The exogenous gene that is imported mostly is coding and suppresses the gene of vaso-active substance (NOS, atrial natriuretic peptide, kallikrein etc.) of smooth muscle cell proliferation or the transcription factor gene (P53, RB, P21, Gax, Caspase) relevant with vascular smooth muscle cell proliferation and apoptosis etc.Yet as genomic medicine, they give expression to product after must entering cell, can bring into play therapeutical effect.Route of administration, transfer method and the efficient of source gene and some technical barriers in the safety are failed fine solution owing to be out at present, and therefore, the gene therapy of such disease still is in the laboratory research stage, does not extend to clinical as yet.Up to now, still there is not a kind of medicine that effectively presses down the vascular smooth muscle cell abnormality proliferation, prevention for vascular restenosis can only rely on hypertrophy and the tube chamber restenosis that the endovascular stent of placing costliness stops inner membrance, and atherosclerosis and prevention and treatment of osteoporosis are not had gratifying medicine yet.Along with organizing, the human protein learns going deep into of research, in cardiovascular system, the new protein relevant with vascular smooth muscle cell proliferation, migration and apoptosis constantly is found, but, in these protein, which kind of composition is to the propagation of vascular smooth muscle cell and moved main regulating action and it be unclear that, and therefore, to reach the purpose of treatment of vascular smooth muscle cell proliferation and migration at some proteinic specific antibody or antagonist still unrealistic by using for desire.The iuntercellular adhesion molecule is interactional main molecules between mediated cell and cell and cell and the extracellular matrix, and is closely related with vascular smooth muscle cells migration.Utilize between solubility adhesion molecule or anti-adhesion molecule antibody blocking vascular smooth muscle cell and the extracellular matrix and interact, can play the effect that suppresses vascular smooth muscle cells migration, but, China still is being in a backward condition aspect the research and development of solubility adhesion molecule and antibody thereof at present, there is no open report aspect laboratory research and the drug development.Look into new both at home and abroad; do not retrieve with OPN as tissue growth promoter and will resist the OPN single-chain antibody or the small-molecular peptides antagonist as the research report of blocking-up vascular smooth muscle cells migration and propagation, still unmanned to from phage antibody library screening human antibody OPN single-chain antibody with screen the OPN antagonist from phage random small peptide storehouse and carry out patent protection.

Summary of the invention

The object of the invention provides a kind of novel anti-osteopontin antibody class medicine, production method and purposes, this medicine adopts thorugh biologic engineering method production, technology is reasonable, be used for treatment of vascular restenosis, atherosclerosis and osteoporosis and have curative effect preferably, and have recurrence, safe and reliable, the characteristics that have no side effect of being difficult for.

The present invention is achieved in that

A kind of anti-osteopontin antibody class medicine is characterized in that this medicine behaviour source anti-osteopontin single-chain antibody, anti-osteopontin monoclonal antibody or anti-osteopontin polyclonal antibody, is made into various pharmaceutical dosage forms.

The present invention's two is a kind of anti-osteopontin antibody class medicine production methods, it is characterized in that:

A, at first make up the phage human antibody library, separate patient's peripheral blood lymphocytes behind the PTCA, with rat OPN be antigen after external sensitization, transform through EBV, the B cell of enrichment secreting specificity antibody provides total RNA; Heavy chain of antibody, chain variable region gene segment are fitted together through recombinant PCR with the DNA joint of coding pentadecapeptide (GIY4Ser) 3, make up phage antibody library;

B, from phage antibody library, screen anti-OPN single-chain antibody with KOPNH, getting nitrocellulose filter is soaked in the OPN solution, dry up, put into the polypropylene plastics ware, getting the phage suspension adds in the ware, jog under the room temperature, with the PBS flush away not in conjunction with phage, use elution buffer instead,, collect eluent with eluting and the bonded phage of OPN, regulate PH to 7.0-8.0 with neutralization buffer, the phage of institute's eluting increases to phage in order to infect the E.coli of exponential phase, promptly makes people source anti-osteopontin single-chain antibody.

The present invention's three is purposes of a kind of above-mentioned anti-osteopontin antibody class medicine, it is characterized in that being used for the treatment of vascular restenosis, arterial pulse is atherosis and osteoporosis.

The present invention is human antibody OPN antibody or the OPN antagonist of developing according to the pathomechanism that restenosis behind the PTCA takes place, with the extracellular matrix is that target not only can suppress vascular smooth muscle cell migration under inner membrance, propagation and matrix reconstruction, and utilize its anti-adhesion effect, also can effectively stop the mononuclear cell in the circulation blood to adhere to, build up, with anti thrombotic action at damage location.If in Stent, local coating drug stent associating intravenous injection medication can reach the therapeutic effect that suppresses restenosis.Osteoporosis is old people's a common disease, and its sickness rate occupies first of each disease, does not still have the active drug of this disease of treatment at present.The effect that suppresses calcium deposition, promotes molten bone in view of OPN has is used in the OPN micromolecule antagonist and endogenous OPN, can be used as prevention of osteoporosis and medicine for treatment.The OPN inhibitor medicine series of this project development a kind of stromatin that to be seminar filter out on the basis of research work for many years.As base stock, adopt biological high-tech means to develop, pharmaceutically-active target is at extracellular matrix, and the terminal of effect then is a cell, by blocking-up endogenous OPN activity, reaches the purpose that suppresses cell adhesion, migration and propagation.Its pharmacological mechanism is different with clinical existing similar medicine, the latter many with vascular smooth muscle cell as target.Succeeding in developing and clinical practice of such medicine started the new way of cardiovascular disease therapies theoretically.On the mentality of designing of drug development, utilize the high homology of people, Mus OPN structure, with Mus OPN is antigen, activate the sensitization person monocytic cell, and then structure phage human antibody library, and therefrom filter out high activity and specific people source OPN single-chain antibody, simplified the preparation procedure of human antibody.

Medicine novelty of the present invention, available biological engineering method production, its technology is reasonable, is used for the treatment of vascular restenosis, tremulous pulse medicated porridge is waved sclerosis and sclerotin disease has curative effect preferably, and have be difficult for to recur, safe and reliable, the characteristics that have no side effect.

The specific embodiment

Below the present invention is illustrated, but not as a limitation of the invention.

Embodiment 1: the production of the anti-OPN single-chain antibody in people source.

1, the structure of phage human antibody library

Separate patient's peripheral blood lymphocytes behind the PTCA, with rat OPN be antigen after external sensitization, transform through EBV, the B cell of enrichment secreting specificity antibody extracts total RNP.Heavy chain of antibody, chain variable region gene segment are fitted together through recombinant PCR with the DNA joint of coding pentadecapeptide (Gly4Ser) 3, make up phage antibody library.

2, personnel selection OPN screens anti-OPN single-chain antibody and gets in the people OPN solution that nitrocellulose filter is soaked in (70ug/ml) from phage antibody library, room temperature is placed and is dried up, put into the polypropylene plastics plate, getting 2ml phage suspension adds in the plate, jog is 1～2 hour under the room temperature, not in conjunction with phage, uses the 2ml elution buffer with the PBS flush away instead, at room temperature incubation is 10 minutes, with eluting and the bonded phage of OPN.Collect eluent, regulate pH to 7.0～8.0, repeat eluting with neutralization buffer, the phage of institute's eluting is in order to infect the E.coli of exponential phase, phage is increased, get supernatant and carry out ELISA, the activity of the phage display single-chain antibody that the detection elutriation goes out and the specificity of antibody.

3, the expression of chain antibody and evaluation

The phage suspension infects E.coliHB2151, with the LB culture fluid amplification culture that contains penicillin, glucose, is that derivant induces single-chain antibody to express with IPTG, extracts periplasm protein, carries out purification by SephadexG-75.Identify the molecular weight of antibody with SDS-PAGE.

4, the screening of OPN antagonist and the evaluation of biologic activity in the small peptide storehouse

Wash in a pan sieve phage dodecapeptide storehouse with immobilization OPN, adopt the ELISA screening positive clone, suppress its activity of experimental identification, suppress its specificity of experimental identification and affinity with competition with cell adhesion and migration.

Be conventional method about production anti-OPN monoclonal antibody, anti-OPN polyclonal antibody method.

By animal experiment and pharmacological testing, the present invention has definite curative effect.1. the people OPNcDNA highly effective eukaryon expression carrier that has successfully constructed, and confirm can stably express and secretion in Chinese hamster ovary celI, recyclable OPN product amount reaches 2mg/ml from three days culture medium of serum-free culture, through the DEAE-cellulose column chromatography purification, capillary electrophoresis identifies that purity reaches 98%, SDS-PAGE shows that the molecular weight of purification OPN is 66kDa, its aminoacid of efficient liquid phase chromatographic analysis is formed consistent with standard OPN, simultaneously, can obtain high yield OPN (1.6g/ml) too from people and the rat smooth muscle cells culture fluid cultivated, and set up and to have improved the OPN secretory volume, the cell culture condition that foreign protein is few.2. the adherent influence of OPN pair cell: utilize 96 orifice plates to carry out the cell adhesion test, observe the influence of OPN pair cell adhesive capacity.The result shows, within the specific limits, the adhesion rate of vascular smooth muscle cell raises along with the increase of OPN concentration and the prolongation of action time, have that tangible amount one is imitated and the time one effect relationship; After the anti-OPN antibody of adding sealed its avtive spot, the cell adhesion rate descended, and prompting OPN is the active factors of short cell adhesion.3. the influence of OPN on cell migration: stimulated smooth muscle cell 24 hours with the OPN of 32ug/ml, the plane migration can take place in cell, and the distance of being moved is 2.1 times of stimulating group not.4. the chemical chemotaxis of OPN: detect with the Boyden cell and to confirm that the chemical chemotaxis of the OPN amount of presenting is under certain condition imitated dependence.5. successfully constructed phage primary antibody storehouse, total man source.Through 3 affine naughty sieves, the special phage of recovery is by the clone of ELISA evaluation and screening with the people source OPN of purification,, with 3 times of the A405 numerical value that read to the positive standard of negative control.Select 5 strain positive phage clones; It is special to OPN that 2 clones are wherein arranged, and all the other 3 clones all respond to other matrix components, PCR identify mix phage the antibody gene fragment length be total length ScFV gene (～800bp).6. utilize by the human B lymphocyte of Mus source OPN sensitization and made up people's antibody library.7. 5 strain positive colonies have been set up with hybridoma technology.8. the foundation of animal model and efficacy of medicine observing, strip off art with Balloon endothelial and set up the vascular restenosis model, the anti-OPN monoclonal antibody of postoperative intravenous injection immediately, the next day once, treated 30 days, put to death animal, pathological section confirms that compared with the control, medication group vascellum endometrial hyperplasia is not obvious, the treatment effective percentage reaches 80%, cure rate 20%.9. normal person and tumor patient blood plasma OPN horizontal checkout, get 20 health adults and digestive system carcinoma patient 40 people (gastric cancer 10 people, rectal cancer 8 people, colon cancer 7 people, the esophageal carcinoma 10 people, hepatocarcinoma 5 people), venous blood samples detects blood plasma OPN level with anti-OPN monoclonal antibody, and the result detects in the normal human blood less than OPN, oncosis human blood OPN level is 12～48nmol/L, and then the rising of OPN level is more obvious with neoplasm metastasis person.10. internal and external test confirms, anti-OPN antibody can suppress the function of osteoclast, protect against osteoporosis.

Annotate: OPN osteopontin CHO Chinese hamster ovary cell SCFV strand Hangzhoupro body

PBS phosphate buffer PCR polymerase chain reaction

Claims

1, a kind of anti-osteopontin antibody class medicine is characterized in that this medicine is human antibody osteopontin single-chain antibody, anti-osteopontin monoclonal antibody or anti-osteopontin polyclonal antibody, is made into various pharmaceutical dosage forms.

2, a kind of anti-osteopontin antibody class medicine production method is characterized in that:

A, at first make up the phage human antibody library, separate patient's peripheral blood lymphocytes behind the PCTA, with rat OPN be antigen after external sensitization, transform through EBV, the B cell of enrichment secreting specificity antibody provides total RNA; Heavy chain of antibody, chain variable region gene segment are fitted together through recombinant PCR with the DNA joint of coding pentadecapeptide (GIY4Ser), make up phage antibody library;

B, from the somatic antibody storehouse, screen anti-OPN single-chain antibody with KOPN, getting nitrocellulose filter is soaked in the OPN solution, dry up, put into the polypropylene plastics ware, getting the phage suspension adds in the ware, jog under the room temperature, with the PBS flush away not in conjunction with phage, use elution buffer instead,, collect eluent with eluting and the bonded phage of OPN, regulate PH to 7.8-8.0 with neutralization buffer, the phage of institute's eluting increases to phage in order to infect the E.coli of exponential phase, promptly makes people source anti-osteopontin list and connects antibody.

3, a kind of purposes of anti-osteopontin antibody class medicine according to claim 1 is characterized in that being used for treatment of vascular restenosis, atherosclerosis and osteoporosis.