CN101445555B - Non-functional P2X7receptor antibodies, diagnosis and treatment of cancer and other diseases - Google Patents

Non-functional P2X7receptor antibodies, diagnosis and treatment of cancer and other diseases Download PDF

Info

Publication number
CN101445555B
CN101445555B CN2008102142942A CN200810214294A CN101445555B CN 101445555 B CN101445555 B CN 101445555B CN 2008102142942 A CN2008102142942 A CN 2008102142942A CN 200810214294 A CN200810214294 A CN 200810214294A CN 101445555 B CN101445555 B CN 101445555B
Authority
CN
China
Prior art keywords
cancer
carcinoma
acceptor
disease
functional
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
CN2008102142942A
Other languages
Chinese (zh)
Other versions
CN101445555A (en
Inventor
安格斯·吉德利-贝尔德
朱莉安·亚历山大·巴登
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biosceptre International Ltd
Original Assignee
Biosceptre International Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AUPR7430A external-priority patent/AUPR743001A0/en
Priority claimed from AUPR7431A external-priority patent/AUPR743101A0/en
Priority claimed from PCT/AU2002/000061 external-priority patent/WO2002057306A1/en
Application filed by Biosceptre International Ltd filed Critical Biosceptre International Ltd
Publication of CN101445555A publication Critical patent/CN101445555A/en
Application granted granted Critical
Publication of CN101445555B publication Critical patent/CN101445555B/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Oncology (AREA)
  • Engineering & Computer Science (AREA)
  • Communicable Diseases (AREA)
  • Hematology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention concerns a wide range of diseases and conditions, including cancers. The invention provides a probe for detection of such a disease or condition. The probe is able to distinguish between functional P2X7 receptors and non-functional P2X7 receptors. The probe can do this in various ways, one of which is detecting change in relation to binding of adenosine triphosphate (ATP) to the receptors. The invention also provides a method for detecting the disease or condition, using the probe. The invention extends to treatment of the disease or condition, using an antibody, or an epitope capable of generating the antibody, which can distinguish between functional and non-functional P2X7 receptors and bind to the non-functional receptors. Methods of treatment, pharmaceutical compositions and use of the probe and antibody are also included.

Description

Non-functional P2X 7The antibody of acceptor, the diagnosis of cancer and other disease and treatment
The application is that the denomination of invention of submitting on September 3rd, 2002 is " non-functional P2X 7The antibody of acceptor, the diagnosis of cancer and other disease and treatment " the dividing an application of No. 02821433.1 Chinese invention patent application.
Technical field
The present invention relates to diagnosis and treatment that disease comprises cancer.The disease type that the present invention relates to comprises and is derived from epithelial cancer and malignant lymphoma.The invention still further relates to other illness, as state before the tumour (preneoplastic states), irritable bowel syndrome (irritable bowelsyndrome) and virus infection and other infection.Very possible is that the present invention also can be applicable to other disease and illness.
Background
Triphosaden (ATP) can activate part gate purinoceptor (ligand-gatedpurinergic receptors), the known P2X acceptor that is called.Differentiated receptor subtype P2X 1To P2X 7Known different P2X receptor subtype is present in many cells, comprises epithelial cell and white corpuscle, comprises lymphocyte, thymocyte, scavenger cell and dendritic cell.
P2X is subjected to physical efficiency to see through calcium ion and some other positively charged ions, as potassium and sodium ion.Calcium ion flows into cell by the P2X acceptor can be relevant with necrocytosis.
Now confirm P2X 7Hypotype participates in the apoptosis of many cell types.In having the situation of ATP, at the P2X of cell surface expression 7Be subjected to physical efficiency in a second, to open calcium channel by cytolemma.Continue to be exposed to ATP and can cause forming macropore in a few seconds in second to tens, it makes pours into excessive calcium ion, inducing apoptosis in the cell.
Known person and P of Rats 2X 7The aminoacid sequence of acceptor for example from U.S. Patent No. 6,133, is known among 434 (the Buell et al).See that also the present invention is shown in Figure 1.
In the situation of for example epithelial cancer cells, be exposed to ATP and generally do not cause apoptosis.Have been found that this cell expressing can not form the P2X in hole 7Acceptor.Think that these acceptors are non-functional receptors.
Comprise in the rodent hybridoma at human cancer cell line such as prostate gland PC3 and mammary gland MCF7 and at animal cell line, be the P2X of non-functional conformation 7Acceptor is found at cell surface.
Malignant lymphoma patient's B cell expressing non-functional P2X 7Acceptor.Lymphoma produces from the malignant clone of escaping molten born of the same parents' destruction.Therefore this process causes the carrying out property of malignant B to be gathered and causes lymphadenopathy and/or splenomegaly.
Summary of the invention
Aspect first, the invention provides a kind of probe that detects disease or illness, described probe is fit to distinguish functional P2X 7Acceptor and non-functional P2X 7Acceptor.Preferably, described probe is by detecting the variation of Triphosaden (ATP) and receptors bind, perhaps by detecting at P2X 7The acceptor mesopore forms the variation of necessary one or more combination of proteins and distinguishes functional and non-functional P2X 7Acceptor.In another embodiment, the P2X that in the ATP of no combination situation, exposes of described probe in detecting 7One or more part of acceptor.This acceptor portion can comprise a P2X 7Monomer.
The present invention also provides a kind of method that detects disease or illness, said method comprising the steps of: use probe of the present invention to distinguish functional P2X 7Acceptor and non-functional P2X 7Acceptor provides the expression of receptor spectrum, and this receptor express spectra and acceptor normal expression spectrum are compared.Can detect variation wherein, for example above-mentioned with regard to the described variation of described probe itself.
Described probe can be natural or artificial.Preferably, described probe is a kind of antibody, and it can be polyclonal antibody, monoclonal antibody, recombinant antibodies, humanized antibody, people's antibody or its suitable fragment.Described antibody preferred pin is to being arranged in an epi-position of the contiguous ectodomain of ATP-binding site.At people P2X 7In the acceptor situation, described probe preferably is fit to distinguish functional receptor and non-functional acceptor, described functional receptor has a kind of sequence, wherein the proline(Pro) in the 210th amino acid position is transoid conformation, described non-functional acceptor has a kind of sequence, wherein the proline(Pro) in the 210th amino acid position is cisoid conformation, and it obviously changes local protein structure.
Any proper method preparation that described probe can use those skilled in the art to be easy to obtain.
Within the scope of the present invention, described probe can be distinguished functional and the non-functional acceptor by detecting other conformational change that occurs in ATP-binding site.For example, the variation that detects can be in an amino acid except above-mentioned proline(Pro).This seed amino acid for example is Pro199, obviously changes local protein structure when it is cisoid conformation.For example detected variation also can be variation in some other sides again.
Described probe can also be fit to the unaltered P2X of measuring ability state 7Other zone of acceptor.Can not use described probe in detecting in conjunction with the conformation of the monomer subunit of ATP, but because selected epi-position specific detection have only when ATP not in conjunction with the time just can and the shape in a zone of receptor surface.Described probe can detect the variation in one or more combination of proteins, and described protein such as hole form necessary attached protein or other protein.This protein non-limitative example is ln, integrin, beta-actin, α-actinine and super villin.
In the present invention, can use P2X 7The receptor subtype specific antibodies specific detects or in conjunction with non-functional P2X 7Acceptor, described P2X 7Acceptor is organized before forming tumour, unusual infantile tumour tissue, expression or at expression non-functional P2X in the cell of the part of late tumor tissue or on the cell 7Express on any tumour cell of acceptor.Therefore, P2X 7Even acceptor can be in cytolemma normal expression or can part as passage, close the door (close-gated) when it is in or just can be detected or combination during the non-functional conformation but have only.
In addition, do not have the conformation of monomer subunit of the ATP of combination can use antibody test yet because selected epitope specificity detect have only when ATP not in conjunction with the time just can and the shape in a zone on surface.
In the present invention, non-functional P2X 7The antibody that acceptor can use the structure that exists from functional receptor at a kind of experience to carry out the epi-position of conformational change detects or combination.The aminoacid sequence that has been found that the non-functional acceptor can be identical with the aminoacid sequence of functional receptor, so that the reason of conformational change is relevant with the interaction of acceptor and ATP in the acceptor.As mentioned above, the ATP molecule is as receptor stimulant, and when ATP and receptors bind, it can open the passage for the calcium ion inflow of cytolemma thus.Therefore non-functional causes by the suitable combination that lacks ATP agonist and acceptor, and reason can comprise that ATP produces that deficiency or degradation rate increase and the ATP part utilizability deficiency that causes.If the combination of ATP and acceptor is blocked, then receptor conformation is changed.This can be by using the specificity design be combined by ATP to influence the antibody of the regional combination of protein detect.
At people P2X 7In the situation of acceptor, the specific sequence that participates in conformational change can comprise Pro210, and it experiences a conformational change, does not exist under the ATP situation of combination from the trans cis that changes into.Therefore, under the situation of people's acceptor, Pro210 can be comprised for generation of one of a kind of antibody suitable epitope sequences, and the necessary suitable degree of antibody response of inducing can be extended to from the either side of this residue.Non-limitative example comprises a sections that extends to Cys216 from Gly200.In addition, can use the homology sections from other Mammals such as rat, itself and tissue cross reaction.For example, can use Gly200 to Cys216 sections identical in the rat, two aminoacid replacement be arranged (for example referring to U.S. Patent No. 6,133,434) although compare with the human sequence in the rat sequence.
In the situation of non-human acceptor, can determine specific sequence by suitable experiment.
To non-functional P2X of the present invention 7Acceptor detects a kind of distribution pattern can be shown, and wherein functional receptor (and normal cell therefore) can keep unmarked substantially.Yet, P2X 7The non-functional conformation of acceptor can be at first before tumour very early stages of cell nuclear and tenuigenin in detect.For example in the situation of cell carcinoma, use the method for the present invention can be at the h and E (H﹠amp by biopsy; E) pre-neoplastic formation appears detecting in several years before in the normal pathological manifestations of cancer of dyeing slide detection.Therefore, can earlier detect cancer than present detection method, as prostate cancer, skin carcinoma and mammary cancer, have thus can early treatment advantage.
Can also determine by the disease of probe of the present invention and method detection or the four corner of illness.Yet, be sure of that these diseases or illness comprise cell carcinoma, as prostate cancer, mammary cancer, skin carcinoma, lung cancer, cervical cancer, uterus carcinoma, cancer of the stomach, the esophageal carcinoma, bladder cancer, colorectal carcinoma and carcinoma of vagina, and the leukemia that comprises malignant lymphoma, irritable bowel syndrome and virus are as the infection due to HIV or other pathogenic micro-organism such as the mycobacterium tuberculosis.Infection can be directly by the required cofactor of inhibit feature or by just regulating the P2X that suppresses on epithelium or other cell 7The cofactor of function and cause the non-functional expression of receptor makes the cell of infection not be subject to the destruction of apoptosis thus.
Unless otherwise indicated, refer to be included in all these specific diseases and the illness described in the aforementioned paragraphs at this used term " disease or illness (disease andcondition) ".
In the situation of irritable bowel syndrome (IBS), find in having the patient of this illness normal expression P2X in the widely distributed lymphocyte that exists in the matrix under epithelium 7The intestinal mucosa of acceptor becomes up-regulated.In the patient that gets involved, the expression of this raising can observe to mucous membrane of rectum at duodenum.The expression of this raising can be found in separate areas, is perhaps more all generally improving on the small intestine total length in the extreme case.
In being involved minimum situation, total P2X 7Acceptor is up-regulated, but these all are functional and do not invade epithelial lining.In serious situation more, total P2X 7Expression of receptor is higher, and most of affected enteron aisles zone presents the acceptor of non-functional.These for example can be positioned at cecum mucosa, and can invade epithelial lining.Serious situation is total P2X wherein 7Expression of receptor further improves, and the most receptors right and wrong are functional, and its epithelial cell is invaded to be increased.
As disclosing P2X 7The non-functional of acceptor is because due to the suitable combination of shortage ATP agonist and acceptor.Reason can comprise ATP underproduce or since outer-ATPase enzymatic degradation ATP increase due to the local utilizability deficiency of ATP.If ATP and acceptor in conjunction with destroyed, then receptor conformation as revealed sample change, this can use probe in detecting of the present invention.Yet, detect total P2X 7Preferably by using an epi-position to realize, described epi-position is not to be subjected to ATP in conjunction with the P2X that influences in the acceptor distribution 7Other regional epi-position of the ectodomain of acceptor.Probe can be by the total epi-position of detection functionality and non-functional conformation the unaltered P2X of measuring ability state 7The zone of acceptor, described epi-position such as Val65-Lys81.
The present invention includes use one or two kind of P2X 7Subtype specificity antibody is distinguished total P2X with specificity 7The ratio of the non-functional acceptor that distributes and in intestinal mucosa, express.Therefore these two kinds of antibody that use together both can detect total acceptor number also can detect only be present in close the door or the non-functional conformation in those receptor channels.First kind of antibody is fit to detect total P2X 7Expression of receptor.The probe that comprises or invest antibody of the present invention can provide second kind of antibody to detect IBS, not only distinguishes functional and non-functional P2X 7Acceptor, but also can detect unaltered other zone of function of receptors state.Described antibody can use separately or together.Preferably, described antibody is used in combination.
Detect nand function P2X 7All P2X that acceptor separates 7Be subjected to physical efficiency to determine the severity of illness.Non-functional P2X 7The expression of acceptor in gastrointestinal tract mucous keeps unlabelled pattern to occur with normal cell substantially.Afterwards, P2X 7The non-functional conformation at first in subepithelial matrix, detect, the sheet of separating from slight syndrome situation distributes until distributing with the sheet that the non-functional acceptor is invaded the separation of epithelial lining simultaneously in gi tract total length wide expression.
The present invention also provides a kind of method of diagnosing irritable bowel syndrome, and described method comprises the P2X that detects cell and/or tissue 7Expression of receptor is composed, and the predetermined express spectra of this express spectra and normal cell and/or tissue is compared.Preferably, P2X 7The detection of expression of receptor spectrum comprises uses one or more antibody.In addition, preferred this antibody is different with probe of the present invention, and it does not detect ATP and P2X 7The variation of receptors bind.The preparation of this antibody will be apparent to those skilled in the art.
The present invention also comprises the application of one or more antibody in the diagnosis irritable bowel syndrome.
This treatment of conditions processing is discussed in conjunction with a third aspect of the present invention hereinafter.
Diagnostic reagent can use in the standard microscopy by the application standard immunohistochemical method.Diagnostic reagent also can use in vivo.
Use the diagnosis of probe of the present invention and method can use the original position imaging technique to carry out, to detect the distribution in body tissue.In addition, can use the standard microscopy, focus on the cell elutriation of microscopy and fluorescence-activation.Can the use test lymph, prostate gland, mammary gland, skin, lung, uterus, bladder, uterine cervix, stomach, the normal immunohistochemical method of oesophagus and similar biopsy reaches the fine needle aspiration thing of mammary gland and other tissue and those smears that cell smear obtains as the detection cervical cancer.
At in-vivo diagnostic, preferred probe is people's antibody or the structural domain of no animal component.Described antibody is preferably used short-decayed radio-labeling substance markers, and described radioactively labelled substance can detect by screening method such as positron emission computerized tomography (PET scanner).This imaging can detect the traget antibody of assembling at any position of body, therefore sends the signal that has the non-functional acceptor relevant with arbitrary tumour.Ideally, this test should only be carried out after detecting original cancer, and carries out for the purpose that detects the secondary cancer, perhaps carries out after having detected the general screening that may have one or more tumour by blood test (seeing the following stated).
Probe of the present invention and method can be used for providing a kind of blood test, to detect non-functional P2X 7Acceptor and cancer or precancerosis disease therefore.For example, the probe of fluorescent-labeled antibody (mono-clonal or polyclonal antibody) form can be used for patient's hemocyte component is carried out flow cytometry, comprise the T lymphocyte, the combination of the non-functional acceptor on bone-marrow-derived lymphocyte or the scavenger cell to detect with various gate white corpuscles.
In the blood test of another kind of form, described probe preferably adopts the traget antibody form that the matrix that provides in the test kit is provided, and makes to detect by there being the color producing reaction that immobilized antibody is combined with positive white corpuscle.This test kit can be suitable for the medical science practitioner to be used.
In a similar blood test, antibody probe of the present invention can be used as a kind of diagnostic tool, does not perhaps have cancer but its normal cell kills and wounds approach and lack P2X on one or more white corpuscle with screening 7Function and repressed patient.This patient can express the non-functional acceptor at scavenger cell, shows that the ability that those scavenger cells kill and wound the cell of infection (as mycobacterium tuberculosis, perhaps other infectant comprises the cell of infected by microbes such as malaria and HIV) is suppressed.This organism is is preferentially killed and wounded approach because the P2X on one or more white corpuscle of shortage at its normal cell 7Function and repressed patient's proliferation in vivo.
Other method also can be used with probe of the present invention and method.
The invention provides a kind of antibody for the treatment of disease or illness, described antibody is suitable for distinguishing functional P2X 7Acceptor and non-functional P2X 7Acceptor, and be suitable for only in conjunction with the non-functional acceptor.Preferably, described antibody is by the variation in the combination that detects Triphosaden (ATP) and acceptor or by detecting P2X 7The acceptor mesopore forms the variation in necessary one or more combination of proteins and distinguishes functional and the non-functional acceptor, and described antibody is suitable for only in conjunction with the non-functional acceptor.In another embodiment, described antibody is distinguished functional and the non-functional acceptor by a part that detects the acceptor that exposes in the situation of the ATP that does not have combination.
The antibody for the treatment of disease or illness can with as diagnose the illness or the antibody of the probe of illness identical.This antibody can for example be used for the topical treatment of skin cancer.At the systemic treatment cancer, described antibody or its active fragments should be people's antibody or people's antibody structure territory, so that unwished-for immunne response side effect is down to minimum.
Antibody of the present invention can be used for treating disease or the illness that Mammals comprises the people.The example of described disease or illness is above illustrating in conjunction with probe of the present invention.
The present invention also provides a kind of epi-position, and it can cause that the described antibody of second aspect present invention produces.Described epi-position preferably comprises Pro210 and contains Gly200-Cys216 (at people's acceptor P2X 7In the sequence) this sections.Described epi-position should preferably be adhered to a Cys residue (Cys216) at C-terminal; it is crosslinked by the amino caproyl-N-hydroxy-succinamide of chemical cross-linking agent maleimide (MCS) and diphtheria toxin, so that the conformation that accompanying epitope peptide adopts has a kind of stable cis-configuration.
This specific peptide conformation is had a mind to be and is passed the human or animal with one or more disease or illness, especially suffers from cell carcinoma such as prostate cancer, mammary cancer, skin carcinoma, lung cancer, cervical cancer, uterus carcinoma, cancer of the stomach, the esophageal carcinoma, bladder cancer, colorectal carcinoma and carcinoma of vagina, and malignant lymphoma, irritable bowel syndrome and virus are as the patient of HIV or other pathogenic micro-organism m tuberculosis infection.Described patient preferably produces immunne response to the applied epi-position of puting together, and therefore produces the non-functional P2X that exists on the affected cell surface of identification 7The antibody of acceptor, described antibody and described receptors bind also signal for suitable immunocyte, to destroy this compound cell.Other cell of trigger cell death also can be influenced.
Should understand and the invention is not restricted to above-mentioned sequence, but also comprise sequence and carrier and the linking agent of change, they similarly produce a specific specificity and reply, and are preferred only at non-functional P2X 7Acceptor, therefore the preferred all functions acceptor of expressing at cell surface of ignoring also avoids side effect.
The present invention also provides antibody of the present invention application as the treatment carrier in treatment disease that the patient takes a disease or illness in second aspect, and it is by being oriented in the unusual or non-functional P2X that expresses on the cell surface 7Acceptor and regulate apoptosis, all cells of expressing normal (functional) acceptor simultaneously is uninfluenced.The application that epi-position of the present invention causes that antibody produces has also been contained in the present invention, as mentioned above.
The present invention also provides a kind of pharmaceutical composition; with treatment or prevention disease that the patient is taken a disease or illness; described composition comprises the antibody of medicine effective quantity, perhaps causes the epi-position of the antibody that produces this amount, and it can be adjusted in unusual or non-functional P2X on the cell surface 7The cell programmatic death of expression of receptor.
The medicine effective quantity of described antibody or epi-position changes according to the character of patient and disease or illness.These variablees can be determined by those skilled in the art.
Pharmaceutical composition of the present invention can be co-administered with a kind of medicine acceptable carrier, and described carrier can be known in the art or described and be suitable for specifying any carrier of use later.Except carrier, pharmaceutical composition of the present invention can comprise other composition, for example comprises dyestuff, sanitas, buffer reagent and antioxidant.
Pharmaceutical composition of the present invention can be taked the form of any hope, and can be for example with ointment, missible oil (cream), and solution, suspension, powder, tablet, capsule, suppository or vaginal suppository form are used.
Pharmaceutical composition of the present invention can be used in any appropriate manner, comprise oral, non-enteron aisle, intravenously, intramuscular, subcutaneous or topical application.
The present invention also provides the method for a kind for the treatment of or prevention disease that the patient takes a disease or illness, and described method is included as the patient and uses pharmaceutical composition of the present invention.
The present invention also provides the application of pharmaceutical composition of the present invention in treatment or prevention disease that the patient takes a disease or illness.
Those skilled in the art recognize that the use pattern of pharmaceutical composition of the present invention can be according to the best use of effect and change.May essential character and the severity of considering disease or illness.
A third aspect of the present invention is at the expression of ATPases (enzyme), and P2X is supplied with in its control 7The ATP supply of acceptor is for example in malignant lymphoma patient's B cell.P2X on the white corpuscle 7The channel opener of acceptor is by being stopped by the quick hydrolysis to the ATP agonist of outer ATPase (ecto-ATPases) and outer ATP bisphosphate lytic enzyme (outer ATPDases).These enzymes are regulated numerous physiological processes that depends on ATP.The substrate specificity of ATPase and ATPDase activity shows in described more than one types of lymphocytic cell surface existence on the lymphocyte, comprises CD39.The propagation of one or more these ATPases or ATPDases can limit control P2X 7The hole forms required ATP supply, and the required cell programmatic death of adjusting B cell number subsequently.
Similarly, it is believed that in the situation of IBS, the propagation of ATPases can make agonist ATP and P2X 7The suitable combination of acceptor reduces.
Therefore, in this third aspect, the invention provides the preparation of a kind for the treatment of or prevention disease that the patient takes a disease or illness, described preparation comprises to be fit to be adjusted in to be controlled ATP and is supplied in P2X in patient's cell or tissue 7One or more material that the ATPase of acceptor expresses.The present invention also provides the method for a kind for the treatment of or prevention disease that the patient takes a disease or illness, and described method is included as the patient and uses a kind of preparation, and described preparation comprises to be fit to be adjusted in to be controlled ATP and be supplied in P2X in patient's cell or tissue 7One or more material that the ATPase of acceptor expresses.
This class ATPase for example is CD39 or CD73.
This material can be taked ATP analogue form, and preferred non-hydrolysable also is specific to P2X 7Material, perhaps suppress the another kind of material of local ATPases effect, described ATPases makes P2X 7The available ATP of binding site exhausts.Described preparation can be that specificity is at non-functional P2X 7People's antibody formation of acceptor.
A kind of material such as ATP analogue can with P2X 7In conjunction with and make it keep the open bore configuration, therefore make the hole present a kind of functional status, make big and small cation all energy is penetrating.By this way, the application of this synthetic agonist can recover function of receptors, controls P2X simultaneously 7Growth vigor as the cell that effect offers of calcium channel.
The preferred malignant lymphoma of described disease or illness or IBS, but the present invention also expands to other disease or illness, comprises other epithelial cell or hematologic cancers or virus and other pathogenic infection.
In the situation of malignant lymphoma, ATPases controls P2X 7The local supply of the ATP of acceptor is to reduce and P2X 7The concentration of the available ATP of receptors bind, and make described acceptor inactivation cause programmatic B necrocytosis obviously to reduce thus.In malignant lymphoma, these ATPases are can be on the B cell surface specific expressed and just show as and regulate.Preferably, the application of specific ATP ase inhibitor can be used for regulating P2X 7The utilizability of ATP on the acceptor is regulated programmatic B necrocytosis thus.
Be the treatment malignant lymphoma, described material can comprise a kind of P2X of non-hydrolysable 7The synthetic agonist of agonist form, it can block ATPases or ATPDases.
About irritable bowel syndrome, use preparation of the present invention and be intended to recover the function of receptors that to exhaust by the overactivity of the flesh layer under the mucous membrane involved area.Preparation of the present invention can directly work to remove these non-functional acceptors to mucous membrane, thereby recovers local normal gastro-intestinal secretion mechanism.The therapeutic treatment purpose is to recover the ATP part and is supplied in the non-functional acceptor, in order to recover normal function of receptors.The result of control function of receptors comprises that recovery is to the normal control of gastro-intestinal secretion and wriggling.This can be by the P2X7 specific agonist that enteron aisle is used or system's supply is synthetic, preferably can not be by the P2X of ATPases hydrolysis 7Specific agonist is by system applies non-functional P2X 7The antibody of acceptor, preferred small-sized human specific antibody to remove the non-functional acceptor, only stays functional receptor and realizes.
If the wriggling in unstriated muscle is unusual and the normal P2X of combination 7The local utilizability of the ATP that acceptor is required is exhausted relevant, and then treatment can comprise this natural supply that recovers agonist, and this limits the unstriated muscle picked-up or utilize ATP to carry out by the treatment of using the activity of temporary transient restriction enteron aisle.
The present invention also provides a kind of pharmaceutical composition for the treatment of part or illness, and the suitable adjusting control ATP that described composition comprises medicine effective quantity is supplied in P2X 7One or more material that the ATPases of acceptor (enzyme) expresses.
The present invention all extends to this similar application at it in aspect all, and it can be applied under other medical condition, for example unusual P2X 7Acceptor is the result of virus infection, wherein said virus in the cell that infects by just regulating non-functional P2X 7Acceptor is and protected, and perhaps wherein this acceptor is up-regulated under the normal cell condition.
The present invention also provides a kind of method for the treatment of irritable bowel syndrome, and described method is included as the patient and uses a kind of aforementioned pharmaceutical compositions.
The present invention also provides the application of this pharmaceutical composition in the treatment irritable bowel syndrome.
The use pattern of the effective preparation of above-mentioned one or more medicine can change according to the best use of effect.
In addition, the invention provides a kind of method for the treatment of irritable bowel syndrome, described method comprises uses the suitable P2X of recovery 7A kind of composition of function of receptors.Described function of receptors is exhausted by the overactivity of the flesh layer under the mucous membrane involved area.Described composition can be identical with the material that comprises in the invention described above preparation.
On the other hand, the invention provides a kind of method of distinguishing different protein conformations, by use epi-position can causing that antibody produces or antibody self with from all members' of protein of the conformation with selection combination realize that specific drugs learns result's (initiatively and passive immunization).Such example is to be the prion protein that causes vCJD illness conformation.The protein of this anomaly pattern can or cause that described antibody produces the epi-position orientation of (preferably people's antibody, and reduction size is to reach optimal drug effect) by specific antibody.
The accompanying drawing summary
Fig. 1 illustrates people P2X 7The aminoacid sequence of acceptor (prior art).The 65-81 and the 200-216 residue of sequence indicated especially and mentioned following.
Detailed Description Of The Invention
For generation is specific to non-functional P2X 7Antibody, used epi-position is the 200-216 residue of sequence shown in Figure 1, contains the 216th Cys.
For producing the P2X of no differentiation power 7Antibody, used epi-position is the 65-81 residue of sequence shown in Figure 1, wherein adds a N-terminal Cys.This antibody can not detect described acceptor, and whether right and wrong are functional, detect all acceptors so that the ratio of functional receptor can be determined by the dyeing that contrast uses two kinds of antibody to obtain respectively but be designed for.
Cys residue on these epi-positions is by amino caproyl-N-hydroxy-succinamide (MCS) linking agent of maleimide and the coupling of diphtheria toxin (DT) carrier; each DT carrier adheres to 10 epitope peptides, to keep conformational stability and a kind of bigger antigenicity structure is provided.These epi-positions of puting together are as antigen, are injected into that (sheep, rabbit and mouse) produces the antibody that is specific to described epi-position in some animal species in common mode.
Prior art has fully disclosed the method that produces antibody, is undertaken by in specific period antigen/adjuvant mixture being injected in the animal body.The specificity example that produces antibody is as described below:
Embodiment 1: sheep anti P2X 7Antibody
With 500 μ g conjugates (about 100 μ g P2X 7Epi-position) in phosphate buffered saline (PBS) (PBS) dilution for 0.8ml, and with the Freund ' s Freund's complete adjuvant emulsification of 1.2mL.With sheep at a plurality of positions through subcutaneous and intramuscularly antigen/adjuvant milk sap.After 8 weeks, described sheep is injected again the conjugate with the emulsification of Freund ' s Freund of same amount at a plurality of positions.Repeat this operation after 4 weeks and with animal via jugular vein blood sampling.The antibodies specific of the serum that test is collected.Then this sheep is regularly injected and blood sampling with 8 weekly intervals, so that the serum that contains specific antibody set to be provided.
With other sheep with isodose conjugated antigen through with above-mentioned similar scheme but use different adjuvants to inject.In these animals, the antigen that 0.7ml is diluted mixes with QuillA/DEAE Dextran solution (2.5mg Quill A+25mg DEAE Dextran/mLPBS) and the ISA50V Montanide of 1.2mL of 0.1ml.With this milk sap at a plurality of positions through subcutaneous and intramuscularly.The antibody that uses this adjuvant to produce produces the specificity identical with those antibody that use Freund ' s adjuvant to produce.
Embodiment 2: the anti-P2X of rabbit 7Antibody
Use two kind adjuvants and the identical infusion protocol identical with the experiment of carrying out in sheep to produce antibody in rabbit, unique difference is to use the conjugate of 300 μ g to inject.The antibody that produces has identical specificity with those antibody that produce in sheep, can be easy to distinguish the epi-position for generation of these antibody.
Embodiment 3: mouse anti P2X 7Antibody
In mouse at the epi-position of puting together and non-functional P2X 7The unconjugated epi-position of epi-position (it can distinguish the acceptor that can not form the hole and therefore can not cause programmatic death) produces antibody.
In these experiments, used adjuvant is the product I mmunEasy of QAJGEN Pty company limited TM, it contains immunostimulating product C pG DNA (trade mark of ColeyPharmaceutical Group company).
With 5 μ g epi-positions or the epi-position of puting together at 70 μ L PBS and 30 μ LImmunEasy TMDilute in the adjuvant.Mouse is carried out subcutaneous and intramuscularly at a plurality of positions.Repeat this scheme after two weeks and repetition again after another two weeks.Injecting back 8 days with mouse bloodletting blood sampling for the third time.The antibody capable that produces in mouse is by this method distinguished different P2X 7Epi-position, and P2X 7The antibody of non-functional epi-position illustrates the result identical with the antibody that produces in sheep and rabbit.
Shown in above embodiment, P2X 7The antibody of the various epi-positions of acceptor can and use different consistent generations of adjuvant in different plant species.Especially, can conventionally produce P2X 7An antibody of differentiating the epi-position of non-functional state acceptor of acceptor, this non-functional state acceptor can not form the hole under normal operation and can not carry out the dead function of its programmatic.
Embodiment 4:
Detect non-functional P2X 7Antibody by described antibody with express the P2X with known function 7The combination of the cell of (people) and testing, this ability by picked-up ethidium bromide or rubidium shows.These P2X 7The protein passage can be in base pair 1513 sudden changes, and this passage can not form the dead hole of programmatic thus.These of expressing in malignant B and similar non-functional P2X 7Acceptor also in conjunction with described antibody, and is expressed normal function P2X in flow cytometry and standard immunoassay histological chemistry 7Cell (can with big flow picked-up calcium, ethidium bromide and rubidium) can not be in conjunction with described antibody, because the epi-position of selected detection non-functional acceptor can not be utilized in functional receptor.Pro210 adopts a kind of cisoid conformation in the non-functional acceptor, and clear and definite this conformation is being stable for generation of puting together in the epi-position of antibody.Pro210 is to be transoid conformation in functional acceptor illustrating.This is ATP (Triphosaden) and P2X 7The result of receptors bind.When ATP was combined, the Pro210 on a sections of next-door neighbour's ATP-binding site adopted a kind of transoid conformation.
This is by using site-directed mutagenesis Pro210 is changed into the Ala that fixedly is transoid conformation and being confirmed, find this mutain have complete function and also can not with the antibodies that produces with detection non-functional acceptor.
Embodiment 5
Expressing P2X 7The scavenger cell of mark in experimentize with the specificity of further affirmation antibody test non-functional acceptor.Scavenger cell uses P2X 7Universal antibody is in conjunction with P2X 7The antibody of acceptor, but debond non-functional P2X 7Antibody, be exposed to cancer cells such as mouse hybridoma cell until them.Non-functional P2X is induced in contacting between scavenger cell and the hybridoma 7Expression on scavenger cell, this is by non-functional P2X 7Antibody and general P2X 7Antibody test.
The scavenger cell that test is extracted from the malignant lymphoma patient and bone-marrow-derived lymphocyte, all these cells are all in conjunction with general P2X 7Antibody and non-functional P2X 7The antibody of acceptor confirms P2X 7All right and wrong are functional in the cancer cells of all detections, by functional P2X 7The apoptosis hole that forms can not form, and therefore can not the inducing cancer cell apoptosis.
From all cell carcinomas of human body such as prostate cancer, mammary cancer, intestinal cancer, skin carcinoma, cancer of the stomach, cervical cancer and other cancer and malignant lymphoma, all these cancer cells of lymphocytic leukemia and cerebral tumor, and the identical tumour in other Mammals of testing is included in mammary cancer in the dog and prostate cancer and skin carcinoma and all mouse hybridoma cells and mouse fibrosarcoma cell in cat, all expresses identical non-functional P2X 7People in the epi-position of selecting, rat, cat, the sequence similarity between dog and the mouse is enough to carry out the positive and differentiates in above-mentioned situation.This mechanism that is illustrated in cancer in these Mammalss is identical, because all cancer cells are all expressed non-functional P2X 7Acceptor, described acceptor can not form the apoptosis hole, and described hole is common cell killing when activating.By this way, cancer cells becomes infinite multiplication, and the apoptosis program is closed.
Embodiment 6
For confirming that further cancer cells such as affected bone-marrow-derived lymphocyte can not pass through P2X 7The function inducing apoptosis will contain non-functional P2X 7The leukaemic's of acceptor B cell and 5mM ATP incubation were cultivated 2 hours.The result is that the apoptosis that kills affected cell is opened and induced to all non-functional acceptors owing to excessive ATP forces.
Embodiment 7
For further confirming antibody selective binding cancer cells, with rodent cancer (BCC) patient's skin non-functional P2X 7The antibody treatment of acceptor, described antibody are suspended in the inertia missible oil matrix and are applied to the skin lesion position and (see following examples 10) on every side.The described antibody of topical application every day in 1 week, all BCC vestiges all disappear, and illustrating does not simultaneously have effect to surrounding skin, because normal skin does not have described acceptor.
Diagnostic use
Be described in the special non-functional P2X of use in the animal for example at this 7Antibody and confirm probe of the present invention and the general application of method in diagnosis most of people and other animal cancer.
In the prostata tissue of humans and animals such as cat and dog, when antibody of the present invention is used for diagnosis, unmarked acquisition in the situation about before not having cancer or cancer, damaging.Yet diagnostic method of the present invention can disclose and pass through H﹠amp; The initial sign of the neoplastic when E dyeing still can not detect the morphological change of following.
In this stage, the receptor unit that must dye and at first in epithelial nucleus, occur.They migrated in the tenuigenin in the terminal stage of a disease, brought into play field-effect in prostate gland, thereby reduced the tissue that has required biopsy of determining tumour.In the terminal stage of a disease, dyeing becomes and more is limited to the top epithelium.
Similarly, other cell carcinoma such as the mammary cancer of people and other Mammals such as cat and dog, lung cancer, colorectal carcinoma and skin carcinoma can be used rim detection, because no longer include tangible field-effect in these other tissues.
In these other tissues such as mammary cancer and cervical cancer tissue, see identical phasic development, before kytoplasm dyeing, nuclear staining is arranged, and healthy tissues does not dye.Affected lactiferous ducts and lobule of mammary gland are easy to detect in mammary tissue because even do not have under the situation of cancer in the normal morphology prompting, the interior local field effect of indivedual affected mammary gland pipings also can be detected in the mammary gland.Adjacent unaffected lactiferous ducts shows as and is unstained.Similarly, directly drainage contains the affected lymphoglandula of the tissue of tumour, by affected lymphocytic field-effect the tumour sign is shown.Therefore, when sending out to lymphoglandula without any the cell that shifts, can detect the sentry post lymphoglandula.
Skin carcinoma such as rodent cancer, squamous cell cancer and heteroplasia mole and malignant melanoma are illustrated in the positive staining of non-functional acceptor and passage composition (monomer) in keratinocyte and the melanophore layer, and have sharp edge, the normal skin outside described sharp edge is all unmarked at the intradermal of epidermis and depths.
The mammalian cancer cells of all tests system all is positive as human prostate (PC3) and mammary gland (MCF7) and rodent hybridoma non-functional acceptor on cell surface, and apoptosis is suppressed in these cancer cells thus.The general application of this diagnosis illustrates acceptor omnipresence in other type of animal outside the people by the same tag on the mouse hybridoma cell.Normal people's bone-marrow-derived lymphocyte illustrates functional P2X 7Acceptor is expressed at cell surface, therefore can carry out apoptosis when needed, and malignant lymphoma patient's bone-marrow-derived lymphocyte illustrates non-functional P2X 7Therefore acceptor reduces apoptosis at cell surface expression.
Treatment is used
Express the P2X of this obvious omnipresence on the cancer cells surface of attempting to carry out apoptosis 7Non-functional conformation can be used for treating the most of cancers in people and other Mammals.Embodiment is as follows:
Embodiment 8
Mouse hybridoma cell is existed and the non-functional P2X that does not have affinity purification 7The scavenger cell matrix of antibody in grow.After cell counting shows 4 days, with the cell of the normal IgG incubation of purifying from 1 * 10 4Grow to 7 * 10 4Individual, and nand function P2X 7The antibody cell counting of incubation together only is 1.5 * 10 4Individual.
Embodiment 9
This embodiment illustrates at non-functional P2X 7The antibody that the epi-position of acceptor produces suppresses tumour in vivo and forms.
As implied above, in sheep at non-functional P2X 7The antibody capable that epi-position produces is identified in lip-deep this non-functional of mouse hybridoma cell P2X 7The apoptosis acceptor.This antibody is added the growth that postpones cell in the Hybridoma Cell Culture thing.Mouse hybridoma cell causes tumour to form when injecting the mouse species of the closed crossing for preparing.
In this experiment, each accepts following the processing with 3 groups of (10/group) Balb-c female mices:
The 1st group: with 10 mouse all the 1st day intraperitoneal (IP) be injected in 1 * 10 in the 0.5ml cell culture medium 6Individual hybridoma.At the 2nd day and the 3rd day, they accepted peritoneal injection 0.5ml cell culture medium.
The 2nd group: with 10 animals all the 1st day intraperitoneal (IP) be injected in 1 * 10 in the 0.5ml cell culture medium of the sheep IgG that contains the 1mg purifying 6Individual hybridoma.At the 2nd day and the 3rd day, contain the 0.5ml cell culture medium of the sheep IgG of 1mg purifying for their injections.
The 3rd group: with 10 mouse all the 1st day intraperitoneal (IP) be injected in the sheep anti P2X that contains the 1mg purifying 7In the 0.5ml cell culture medium of non-functional epi-position IgG 1 * 10 6Individual hybridoma.At the 2nd day and the 3rd day, they accepted the sheep anti P2X that another time injection contains the 1mg purifying 7The 0.5ml cell culture medium of IgG.
The mouse of all groups was all put to death at the 11st day, and detect the situation that exists of tumour.Tumor resection is also weighed.
The result is as follows:
Group Observe Every mouse average tumor weight (± SD) (g)
1: contrast 1 There are 9 to find tumour in 10 mouse 3.98±1.1
2: contrast 2 10 mouse are all found tumour 2.93±0.9
3: experimental group There are 9 to find tumour in 10 mouse 1.13±0.4
There were significant differences (probability P＜0.01) for tumor weight between variance analysis illustrated and respectively organizes.Use anti-P2X 7There were significant differences (P＜0.01) for the experimental group of non-functional antibody treatment and two control groups.Namely use P2X 7The antibody of non-functional epi-position is handled and significantly reduce dose,tumor in laboratory animal.
Embodiment 10
Placing 7 days liquid or to be suspended in form in the Simethicone missible oil matrix, the antibody (obviously improveing specificity) of specificity affinity purification is applied in 3 people's rodent cancer (BCC).Not having BCC infringement sign after handling can detect, and contrast skin is unaffected fully, because there is not the protein target position.
Embodiment 11
Skin lesion with rodent cancer (BCC) and squamous cell cancer (SCC) (primary tumo(u)r and secondary tumor) form, the tumour and the heteroplasia mole that comprise recurrence, further using the antibody purified of in Simethicone missible oil matrix or perviousness missible oil matrix, mixing in the experiment, affinity purification or the not IgG treatment of affinity purification.Therefore because in normal skin, there is not the non-functional acceptor, in the normal skin of any kind, detects and have no side effect.All types of cancers are all replied the existence of antibody, and this cancer of replying by using twice in every day in during 36 hours to 1 week disappears and shows.In 12 months time, there be not recurrence.The size of the tumour of being treated is thick to 5cm diameter and maximum 4mm from the 3mm diameter that does not have protruding border.Totally 34 tumours that confirm through histology were successfully eliminated in the treatment phase in 1 week.
It is believed that the application to the patient generally comprises generation human monoclonal antibodies (as herceptin), inner cancer can be treated equally effectively with topical application thus.All normal functional P2X in cell such as lymphocytic cell surface expression 7Need to keep not being subjected to the influence of antibody existence to avoid side effect.Therefore antibody should only be combined in to be attempted but can not cause expressed protein on the cell surface of apoptosis.Therefore all directed cells are those cells of attempting to kill by apoptosis himself, comprise cancer cells.These cells are the P2X on the cancer cells especially 7Acceptor is the state that non-functional or ATP exhaust.
Active immunity
Active immunity also can be used for the treatment of purpose.In this case, people or other Mammals need be at one or more specificity epitopes by immunizations, and the conformation of described epi-position is to simulate the conformation of only being taked by the acceptor of its non-functional on cell surface (ATP exhausts) form.The part that comprises that should avoid being present in the functional receptor exposes the receptor conformation flexibility of epi-position form.For example the cis-configuration of epi-position Gly200-Cys216 should be fixed by suitable mode before using.About this concept one further evidence is arranged, the many animals that namely observe for generation of antibody comprise mouse, and rabbit and sheep have not been non-responsivenesses.These animals do not have, and any tumour takes place again and again.
At this illustration a specificity embodiment:
Embodiment 12
Scheme: experimentize based on mouse tumor model.Use the female inbrde Balb C mouse in 40 10 ages in week, be divided into 2 groups, 20 every group, the 1st group is experimental group, and the 2nd group is control group.
The 1st day: the peptide epitopes (hP2X that 20 laboratory animal injection 0.1mg in the 1st group are puted together through MCS linking agent and diphtheria toxin 7Sequence 200-216).It comprises about 0.02mg peptide epitopes.With peptide conjugate usefulness QUILL A/DEAE Dextran/Montanide ISA50V adjuvant mixture emulsification and at a plurality of subcutaneous and intramuscular injection site injection 0.1ml.
With control group namely 20 mouse of the 2nd group at the 0.1ml adjuvant mixture of a plurality of subcutaneous and no peptide conjugates of intramuscular injection site injection.
The 8th day: the peptide epitopes (hP2X that 20 the 1st group of injected in mice 0.01mg are puted together through MCS linking agent and diphtheria toxin 7Sequence 200-216) (peptide epitopes that contains about 0.002mg).Described peptide is contained in the phosphate buffered salt solution and according to scheme and mixes with the CpGDNA adjuvant ImmunEasy (deriving from Qiagen) that is purchased.At a plurality of subcutaneous and intramuscular injection site injection 0.1mL of every mouse peptide conjugate/assist agent solution.
With 20 phosphate buffered saline (PBS)/CpG DNA adjuvant mixtures that the 2nd group of injected in mice is similar.This is at the described mixture of a plurality of subcutaneous and intramuscular injection site injection 0.1ml of every mouse.
The 26th day: the peptide epitopes (hP2X that 20 the 1st group of injected in mice 0.025mg are puted together through MCS linking agent and diphtheria toxin 7Sequence 200-216) (contain about 0.005mg peptide epitopes), it is contained in the phosphate buffered salt solution and with Qiagen CpG DNA adjuvant ImmunEasy and mixes.Again the mixture of 0.1mL is injected in every mouse in a plurality of subcutaneous and intramuscular injection site.Control group is as injecting as described in the 8th day.
The 29th day: all mouse all were accepted in tumor cell injection in the 0.1ml tissue culture medium (TCM) a subcutaneous location, and described position is positioned at nape portion.Used tumour cell is the mouse fibrosarcoma cell system that the Walter and Eliza Hall Institute by Melbourne, AUS provides, and the clone name is called WEHI164.
Described cell is injected in experimental mice and the control group mice with two kinds of concentration.Be further divided into two groups with every group.10 mouse of each experimental group and control group are accepted 160000 cell/mouse, and 10 mouse in addition of every group are accepted 320000 cell/mouse.
The cell of this clone is tested non-functional P2X on its cell surface in advance 7Epi-position have a situation.The antibody of this non-functional form by using the specific recognition acceptor produce in sheep carries out.
The 38th day: all mouse are all put to death and collect blood to analyze non-functional P2X 7The antibody of epi-position.All mouse are all weighed and tumor resection and weighing.
The result
Group Contrast 160000 cells Test 160000 cells Contrast 320000 cells Test 320000 cells
n 10 10 10 10
Average tumor weight (mg) 599 270 1147 750
SD 307 108 633 363
SEM 97 34 200 115
The result is carried out the difference (P=0.0003) that variance analysis is illustrated between control group and the treatment group and has statistical significance between low and high dose group.Than low dose group larger difference is shown, because lower tumour load is less to the effect of the ability of mouse immune system.
The ATP analogue
Using a kind of synthetic agonist effectively in conjunction with P2X shown in the following culture experiment 7ATP-binding site on the hole, thus and make described hole enter functional status recovery function of receptors and control P2X 7Offer the effectiveness of the growth vigor of cell.The tumour B cell of collecting from CLL patient and the similar cytomixis of similarity number purpose of collecting from normal patient use the ATP of 2.5mM to handle 4 hours, do not have tumour cell to keep getting off, only surplus normal cell.Be not suitable in vivo using ATP or having more optionally P2X 7The agonist benzoyl, benzoyl ATP.Therefore, can design a kind of selectivity ATP analogue with recovering programming cell death process in affected cell type, described ATP analogue can be with than ATP or the higher binding affinity selective binding P2X of BzATP 7
Industrial applicibility
All aspects of the invention all can be applicable to humans and animals medicine and health field, have the potentiality that can accurately diagnose the illness in early days and effectively treat, and its infringement and injury power more than those medicines that utilize in the prior art is low in many cases.

Claims (18)

1. the P2X of Fen Liing 7Acceptor, its aminoacid sequence as shown in Figure 1, the proline(Pro) that wherein is positioned at position 210 is cisoid conformation.
2.(a) P2X of claim 1 7Acceptor or (b) fragment of (a) acceptor for the preparation of the purposes in the vaccine of prevention or treatment disease or illness, described fragment comprises the aminoacid sequence of Gly200 to Cys216, the proline(Pro) that wherein is positioned at position 210 is cisoid conformation, and wherein said disease or illness are the cancers that is selected from prostate cancer, mammary cancer, skin carcinoma, lung cancer, cervical cancer, uterus carcinoma, cancer of the stomach, the esophageal carcinoma, bladder cancer, colorectal carcinoma, carcinoma of vagina, leukemia, lymphoma, the cancer of the brain and intestinal cancer.
3. be used for the vaccine of prevention or treatment disease or illness, it comprises
(a) P2X of claim 1 7Acceptor; Or
(b) (a) fragment of acceptor, described fragment comprises the aminoacid sequence of Gly200 to Cys216, and the proline(Pro) that wherein is positioned at position 210 is cisoid conformation,
Wherein said disease or illness are the cancers that is selected from prostate cancer, mammary cancer, skin carcinoma, lung cancer, cervical cancer, uterus carcinoma, cancer of the stomach, the esophageal carcinoma, bladder cancer, colorectal carcinoma, carcinoma of vagina, leukemia, lymphoma, the cancer of the brain and intestinal cancer.
4. the vaccine of claim 3, wherein said acceptor or fragment are coupled to carrier.
5. the vaccine of claim 4, wherein said carrier is diphtheria toxin.
6. the vaccine of one of claim 3-5 also comprises adjuvant.
7. the vaccine of claim 6, wherein said adjuvant comprises QUILL A/DEAEDextran/Montanide.
8. the vaccine of one of claim 3-7 is for the preparation of the disease in treatment or the object of prevention or the purposes in the composition of illness, and wherein said disease or illness are to be selected from prostate cancer, mammary cancer, skin carcinoma, lung cancer, cervical cancer, uterus carcinoma, cancer of the stomach, the esophageal carcinoma, bladder cancer, colorectal carcinoma, carcinoma of vagina, leukemia, lymphoma, the cancer of the cancer of the brain and intestinal cancer.
9.(a) P2X of claim 1 7Acceptor or (b) (a) thus the fragment of acceptor for the preparation of the purposes in the composition that gives skin prevention or treatment disease or illness, wherein said fragment comprises the aminoacid sequence of Gly200 to Cys216, the proline(Pro) that wherein is positioned at position 210 is cisoid conformation, wherein said disease or illness are to be selected from prostate cancer, mammary cancer, skin carcinoma, lung cancer, cervical cancer, uterus carcinoma, cancer of the stomach, the esophageal carcinoma, bladder cancer, colorectal carcinoma, carcinoma of vagina, leukemia, lymphoma, the cancer of the cancer of the brain and intestinal cancer.
10. composition, it comprises:
(a) acceptor of claim 1;
(b) (a) fragment of acceptor, described fragment comprises the aminoacid sequence of Gly200 to Cys216, the proline(Pro) that wherein is positioned at position 210 is cisoid conformation;
Described composition is the form that gives skin.
11. the composition of claim 10, it also comprises Simethicone.
12. the composition of claim 10 or 11, wherein said composition is mixed with ointment, missible oil, solution, suspension, powder, tablet, capsule, the form of suppository.
13. the composition of claim 10 or 11, wherein said composition is mixed with the form of vaginal suppository.
14. the composition of one of claim 10-13 is for the preparation of the disease in treatment or the object of prevention or the purposes in the medicine of illness, wherein said disease or illness are the cancers that is selected from prostate cancer, mammary cancer, skin carcinoma, lung cancer, cervical cancer, uterus carcinoma, cancer of the stomach, the esophageal carcinoma, bladder cancer, colorectal carcinoma, carcinoma of vagina, leukemia, lymphoma, the cancer of the brain and intestinal cancer.
15. the purposes of claim 14, wherein said disease or illness are skin carcinomas.
16. the purposes of claim 15, wherein said skin carcinoma is selected from rodent cancer, squamous cell cancer, melanoma and heteroplasia mole.
17.(a) acceptor of claim 1 or (b) fragment of (a) acceptor be fit to detect by scanning technique purposes in the reagent of disease or illness in preparation, wherein said fragment comprises the aminoacid sequence of Gly200 to Cys216, the proline(Pro) that wherein is positioned at position 210 is cisoid conformation, and wherein said disease or illness are the cancers that is selected from prostate cancer, mammary cancer, skin carcinoma, lung cancer, cervical cancer, uterus carcinoma, cancer of the stomach, the esophageal carcinoma, bladder cancer, colorectal carcinoma, carcinoma of vagina, leukemia, lymphoma, the cancer of the brain and intestinal cancer.
18. the purposes of claim 17, wherein said scanning technique is positron emission computerized tomography.
CN2008102142942A 2001-09-03 2002-09-03 Non-functional P2X7receptor antibodies, diagnosis and treatment of cancer and other diseases Expired - Lifetime CN101445555B (en)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
AUPR7430A AUPR743001A0 (en) 2001-09-03 2001-09-03 Diagnosis and treatment of irritable bowel syndrome
AUPR7431 2001-09-03
AUPR7431A AUPR743101A0 (en) 2001-09-03 2001-09-03 A cancer therapeutic
AUPR7430 2001-09-03
PCT/AU2002/000061 WO2002057306A1 (en) 2001-01-17 2002-01-17 Antibodies to non-functional p2x7 receptor diagnosis and treatment of cancers and other conditions
AUPCT/AU02/00061 2002-01-17

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CNB028214331A Division CN100497386C (en) 2001-09-03 2002-09-03 Antibodies of non-functional P2X7 receptor and its uses

Publications (2)

Publication Number Publication Date
CN101445555A CN101445555A (en) 2009-06-03
CN101445555B true CN101445555B (en) 2013-07-03

Family

ID=41706691

Family Applications (2)

Application Number Title Priority Date Filing Date
CNB028214331A Expired - Lifetime CN100497386C (en) 2001-09-03 2002-09-03 Antibodies of non-functional P2X7 receptor and its uses
CN2008102142942A Expired - Lifetime CN101445555B (en) 2001-09-03 2002-09-03 Non-functional P2X7receptor antibodies, diagnosis and treatment of cancer and other diseases

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CNB028214331A Expired - Lifetime CN100497386C (en) 2001-09-03 2002-09-03 Antibodies of non-functional P2X7 receptor and its uses

Country Status (9)

Country Link
JP (1) JP4467973B2 (en)
CN (2) CN100497386C (en)
AU (1) AU2002322192B2 (en)
CA (1) CA2459348C (en)
MY (1) MY142283A (en)
NZ (2) NZ549019A (en)
TW (1) TWI329648B (en)
WO (1) WO2003020762A1 (en)
ZA (1) ZA200402630B (en)

Families Citing this family (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK1961767T3 (en) 2001-01-17 2012-05-29 Biosceptre Int Ltd Diagnosis and treatment of cancers and other disorders
EP1570232B1 (en) * 2002-12-05 2016-11-02 KLA-Tencor Technologies Corporation Apparatus and methods for detecting overlay errors using scatterometry
US7344860B2 (en) 2003-04-03 2008-03-18 Bristol-Myers Squibb Company Polynucleotide encoding a novel human P2X7 splice variant, HBMYP2X7v
US8124730B1 (en) 2004-04-02 2012-02-28 Bristol-Myers Squibb Company Polynucleotide encoding a novel human P2X7 splice variant, HBMYP2X7v
US7767789B2 (en) * 2005-06-02 2010-08-03 University Hopitals of Cleveland Truncated proteins as cancer markers
WO2008043146A1 (en) * 2006-10-10 2008-04-17 Biosceptre International Limited Antibodies against non functional p2x7 receptor
JP2010505426A (en) * 2006-10-10 2010-02-25 バイオスセプター インターナショナル リミテッド Hybridoma producing antibody against non-functional P2X7 receptor
AU2013238152B2 (en) * 2007-09-14 2015-09-24 Biosceptre International Limited Purinergic (P2X) receptors in extra-cellular body fluid
ES2619681T3 (en) 2007-09-14 2017-06-26 Biosceptre (Aust) Pty Ltd Novel epitopes P2X7
NZ583896A (en) 2007-09-14 2011-05-27 Biosceptre Int Ltd Purinergic (p2x) receptors in extra-cellular body fluid
CN102143978B (en) * 2008-07-04 2015-02-18 生物权威国际有限公司 Anti- P2X7 peptides and epitopes
CN101469352B (en) * 2008-08-29 2011-12-21 苏州福英基因科技有限公司 In situ hybridization detection kit for early uterocarcinoma
EP2467404B1 (en) 2009-08-20 2015-09-30 Biosceptre International Limited Anti p2x7 receptor antibodies and fragments thereof
WO2011075789A1 (en) 2009-12-24 2011-06-30 Biosceptre International Limited Antibodies to non-functional oligomeric p2x7 receptors
AU2011301153B2 (en) * 2010-09-10 2014-11-27 Biosceptre International Limited Companion animal treatments
BR112013033974A2 (en) 2011-07-01 2017-02-14 Biosceptre Int Ltd combination therapy
EP2990800A1 (en) * 2014-08-29 2016-03-02 Fundació Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol Neprilysin as heartfailure (HF) prognostic marker
WO2017041143A1 (en) 2015-09-11 2017-03-16 Ctm@Crc Ltd. Chimeric antigen receptors and uses thereof
JP7127021B2 (en) * 2016-10-21 2022-08-29 バイオセプター・（オーストラリア）・ピーティーワイ・リミテッド cytotoxic particles
CN110054691B (en) * 2019-05-09 2021-09-07 潍坊医学院 Hybridoma cell line of anti-human P2RX7 monoclonal antibody

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6133434A (en) * 1997-04-28 2000-10-17 Glaxo Group Limited Purinergic receptor
DK1961767T3 (en) * 2001-01-17 2012-05-29 Biosceptre Int Ltd Diagnosis and treatment of cancers and other disorders

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BUELL G. ET AL..Blockade of human P2X7 receptor function with a monoclonal antibody.《BLOOD》.1998,3521-3528. *
GROSCHEL-STEWART U. ET AL.Localisation of P2X5 and P2X7 receptors by immunohistochemistry in rat stratified squamous epithelia.《CELL TISSUE RESEARCH》.1999,599-605. *
WILEY ET AL.A single nucleotide polymorphism is associated with loss of function of the monocyte P2X7 receptor.《BLOOD》.2000,17A. *

Also Published As

Publication number Publication date
NZ549019A (en) 2008-05-30
CN100497386C (en) 2009-06-10
TWI329648B (en) 2010-09-01
WO2003020762A1 (en) 2003-03-13
AU2002322192B2 (en) 2008-05-01
JP4467973B2 (en) 2010-05-26
JP2005513416A (en) 2005-05-12
MY142283A (en) 2010-11-15
CN101445555A (en) 2009-06-03
NZ565994A (en) 2010-02-26
CA2459348C (en) 2013-06-18
CN1625565A (en) 2005-06-08
ZA200402630B (en) 2005-06-29
CA2459348A1 (en) 2003-03-13

Similar Documents

Publication Publication Date Title
CN101445555B (en) Non-functional P2X7receptor antibodies, diagnosis and treatment of cancer and other diseases
US10450380B2 (en) Polypeptide immunogen for generating an antibody to non-functional P2X7 receptor
CN103987729B (en) The antibody of carcinomebryonic antigen relevant cell adhesion molecule (CEACAM)
CN105209489B (en) For the specific detection tool for the circulating tumor cell that interstitial and epithelial-mesenchymal convert
CN105050621B (en) The method for preventing and treating multiple sclerosis
AU2002322192A1 (en) Antibodies to non-functional P2X7receptor, diagnosis and treatment of cancers and other conditions
US20050136434A1 (en) Isolated heat-inducible cell surface protein and hyperthermia-based tumor immunotargeting therapy

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
ASS Succession or assignment of patent right

Owner name: BIOSCEPTRE INTERNATIONAL LIMITED

Free format text: FORMER OWNER: INTREAT PTY LTD.

Effective date: 20110916

C41 Transfer of patent application or patent right or utility model
TA01 Transfer of patent application right

Effective date of registration: 20110916

Address after: New South Wales, Australia

Applicant after: BIOSCEPTRE INTERNATIONAL Ltd.

Address before: New South Wales, Australia

Applicant before: Intreat Pty Ltd.

C14 Grant of patent or utility model
GR01 Patent grant
CX01 Expiry of patent term

Granted publication date: 20130703

CX01 Expiry of patent term