WO2003020762A1 - Anticorps au recepteur p2x7 non fonctionnel, diagnostic et traitement de cancers et d'autres conditions - Google Patents

Anticorps au recepteur p2x7 non fonctionnel, diagnostic et traitement de cancers et d'autres conditions Download PDF

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Publication number
WO2003020762A1
WO2003020762A1 PCT/AU2002/001204 AU0201204W WO03020762A1 WO 2003020762 A1 WO2003020762 A1 WO 2003020762A1 AU 0201204 W AU0201204 W AU 0201204W WO 03020762 A1 WO03020762 A1 WO 03020762A1
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WIPO (PCT)
Prior art keywords
receptors
functional
antibody
probe
condition
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PCT/AU2002/001204
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English (en)
Inventor
Angus Gidley-Baird
Julian Alexander Barden
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Intreat Pty Limited
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/fr
Priority to JP2003525032A priority Critical patent/JP4467973B2/ja
Priority to CA2459348A priority patent/CA2459348C/fr
Priority to AU2002322192A priority patent/AU2002322192B2/en
Application filed by Intreat Pty Limited filed Critical Intreat Pty Limited
Publication of WO2003020762A1 publication Critical patent/WO2003020762A1/fr
Priority to US10/622,313 priority patent/US7326415B2/en
Priority to ZA2004/02630A priority patent/ZA200402630B/en
Priority to US11/968,607 priority patent/US7531171B2/en
Priority to US12/417,989 priority patent/US7888473B2/en
Priority to US12/975,341 priority patent/US8080635B2/en
Priority to US13/298,222 priority patent/US8399617B2/en
Priority to US13/766,630 priority patent/US8709425B2/en
Priority to US14/218,935 priority patent/US20140323693A1/en
Priority to US14/726,391 priority patent/US9663584B2/en
Priority to US15/498,301 priority patent/US10450380B2/en
Priority to US16/568,072 priority patent/US20200071419A1/en

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    • 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

Definitions

  • Antibodies to non-functional P2X 7 receptor diagnosis and treatment of cancers and other conditions
  • This invention concerns diagnosis and treatment of diseases, including cancers.
  • diseases including cancers.
  • the types of diseases with which this invention is concerned include cancers derived from epithelial cells and malignant lymphoma.
  • the invention also concerns other conditions, such as preneoplastic states, irritable bowel syndrome and viral and other infections. It is quite possible that the invention is also applicable to other diseases and conditions.
  • Adenosine triphosphate can activate ligand-gated purinergic receptors known as P2X receptors.
  • Receptor subtypes P2X j to P2X 7 have been identified. It is known that different P2X receptor subtypes are present in many cells, including epithelial cells and leukocytes, including lymphocytes, thymocytes, macrophages and dendritic cells.
  • P2X receptors are permeable to calcium ions as well as some other cations, such as potassium and sodium. An influx of calcium ions into a cell via a P2X receptor can be associated with cell death.
  • the P2X 7 subtype is involved n apoptosis, or programmed cell death, in many cell types.
  • the P2X 7 receptor expressed on the surface of a cell is capable, within a second, of opening calcium channels through the cell membrane.
  • ATP apoptosis
  • Continued exposure to ATP can lead to the formation of large pores, within a few seconds to tens of seconds, that enable the cell to be flooded with excess calcium, inducing apoptosis.
  • the amino acid sequences of the human and rat P2X 7 receptors are known, for example, from US patent No. 6,133,434 (Buell et al). Refer also to Figure 1 herein. Exposure to ATP does not generally result in apoptosis in the case of epithelial cancer cells, for example. It has been found that such cells express P2X 7 receptors that are unable to form pores. These are regarded as non-functional receptors.
  • the P2X 7 receptor is found on the cell surface in a non-functional conformation.
  • the B-cells of patients with malignant lymphoma express non-functional P2X 7 receptors. Lymphoma develops from malignant clones that escape cytolytic destruction. This process leads to the progressive accumulation of malignant B- lymphocytes and thus lymphadenopathy and/or splenomegaly.
  • this invention provides a probe for detection of a disease or condition, the probe being adapted to distinguish between functional P2X 7 receptors and non-functional P2X 7 receptors.
  • the probe distinguishes between functional and non-functional P2X 7 receptors by detecting change in relation to binding of adenosine triphosphate (ATP) to the receptors or by detecting change in binding of one or more proteins necessary for pore formation in P2X 7 receptors.
  • the probe detects one or more parts of the P2X 7 receptor exposed in the absence of bound ATP.
  • Such receptor part may include a P2X 7 monomer.
  • the invention also provides a method for detecting a disease or condition, the method including the steps of using the probe of the invention to distinguish between functional P2X 7 receptors and non- functional P2X 7 receptors, providing a receptor expression profile, and comparing the receptor expression profile with that of a normal profile.
  • the change may be detected, for example, as indicated above in connection with the probe itself.
  • the probe may be natural or artificial.
  • the probe is an antibody, which may be poIyclonaL monoclonal, recombinant, a humanised antibody, a human antibody or an appropriate fragment thereof.
  • the antibody is preferably directed against an epitope located in an extracellular domain adjacent to a site for binding ATP.
  • the probe is preferably adapted to distinguish between functional receptors having a sequence in which proline at amino acid 210 is in the trans conformation and non-functional receptors having a sequence in which the proline at amino acid 210 is in the cis conformation that acts to impart a significant alteration in the local protein structure.
  • the probe may be prepared using any suitable technique, as will be readily apparent to one skilled in the art.
  • the probe may distinguish between functional and non-functional receptors through detection of other conformational changes occurring at a site for binding ATP.
  • the change detected may be in an amino acid other than the proline referred to above.
  • An example of such an amino acid is Prol99 which, when in the cis conformation, significantly alters the local protein structure.
  • the change detected may be in some other respect.
  • the probe may also be adapted to detect other regions of the P2X 7 receptor unchanged by functional state.
  • the conformation of the monomeric subunits lacking bound ATP may be detectable using the probe, as the epitope chosen may specifically detect the shape of a region of the surface of the receptor accessible only when ATP is not bound.
  • the probe may detect change in binding of one or more proteins, such as accessory or other proteins, necessary for pore formation.
  • proteins such as accessory or other proteins, necessary for pore formation.
  • Non-limiting examples of such proteins are laminin, integrin, beta-actin, alpha- actinin and supervillin.
  • a P2X 7 subtype-specific antibody can be used to specifically detect or bind to non-functional P2X 7 receptors expressed in or on cells forming part of preneoplastic tissue, very early neoplastic tissue, advanced neoplastic tissue and on any neoplastic cell expressing non-functional P2X 7 receptors.
  • the P2X 7 receptor is detected or bound only when in the close- gated or non-functional conformation, even though it may be normally expressed in the cell membranes and may otherwise be partially able to function as a channel.
  • the conformation of the monomeric subunits lacking bound ATP is also detectable with the antibody, because the epitope chosen specifically detects the shape of a region of the surface accessible only when ATP is not bound.
  • the non-functional P2X 7 receptors can be detected or bound by using an antibody directed against an epitope that undergoes a conformational change from the structure present in functional receptors. It has been found that the amino acid sequence of the non-functional receptors can be identical to the amino acid sequence of functional receptors, so that the cause of the conformational change in the receptors relates to the interaction of the receptors with ATP. As set out above, the ATP molecules act as receptor agonists, so that when ATP is bound to the receptors, they are able to open a channel through the cell membrane for the inflow of calcium ions.
  • Non-functionality is therefore caused by a lack of appropriate binding of the ATP agonists to the receptors, for reasons that may include a deficit in the local availability of ATP through production deficit or increase in the rate of degradation. If ATP binding to the receptors is disrupted, the receptor conformation is altered. This can be detected by using an antibody specially designed to bind to the region of the protein affected by the binding of the ATP.
  • the specific sequence involved in the conformational change may include Pro210, which undergoes a change in conformation from the trans form to the cis form in the absence of bound ATP.
  • an appropriate epitope sequence against which an antibody must be raised may include Pro210, and may extend either side of this residue, to an appropriate extent necessary to induce an antibody response.
  • this may include a segment extending from Gly200 to Cys216.
  • a homologous segment from other mammals, such as rat may be used where this cross-reacts with human tissue.
  • the same segment Gly200 to Cys216 in rat may be used, although there are two amino acid substitutions in the rat sequence compared with the human sequence (refer US patent No. 6,133,434, for example).
  • the specific sequence may be ascertained by suitable experiment.
  • non-functional P2X 7 receptors may show a distribution pattern in which functional receptors (and hence normal cells) may remain essentially unlabelled.
  • non-functional conformations of P2X 7 receptors may be detected, initially in the nuclei and cytoplasm of cells, at a very early stage in preneoplasia.
  • preneoplasia For example, in the case of epithelial cell cancer, using the method of the invention it may be possible to detect preneoplasia several years prior to the normal pathological appearance of cancer as detected by haematoxylin and eosin ("H & E") stained slides of biopsied tissues.
  • H & E haematoxylin and eosin
  • epithelial cell cancers such as prostate, breast, skin, lung, cervix, uterus, stomach, oesophagus, bladder, colon and vaginal cancers
  • blood cancers including malignant lymphoma, irritable bowel syndrome and infection by viruses such as HIV or other pathological organisms, such as Mycobacterium tuberculosis.
  • Infection may cause non-functional receptors to be expressed either directly through inhibition of co-factors required for functionality, or through the up-regulation of co-factors acting to inhibit P2X 7 function on epithelial or other cells, so rendering the infected cell less amenable to destruction by apoptosis.
  • IBS irritable bowel syndromes
  • total P2X 7 receptors are up-regulated, but these are all functional and they do not penetrate into the epithelium.
  • total P2X 7 receptor expression is even higher, and the most affected areas of the gut exhibit receptors that are non-functional. These may be localised to caecal mucosa, for example, and may penetrate into the epithelium.
  • the most severe cases are those in which total P2X 7 receptor expression is further increased and most of the receptors are non-functional with increased epithelial cell penetration.
  • non- functionality of P2X 7 receptors is caused by lack of appropriate binding of the ATP agonist to the receptors.
  • the reasons for this may include a deficit in the local availability of ATP through production deficit or increase in rate of degradation through ecto-ATPase enzymatic degradation of ATP.
  • ATP binding to the receptors is disrupted, the receptor conformation is altered as already discussed, and this can be detected using the probe of the invention.
  • the detection of total P2X 7 receptor distribution is best achieved using an epitope to other regions of the extracellular domain of the P2X 7 receptor that is not affected by ATP binding.
  • the probe may be capable of detecting regions of the P2X 7 receptor unchanged by functional state, by detecting an epitope common to both functional and non-functional conformations, such as Val65-Lys81.
  • P2X 7 subtype-specific antibodies to specifically distinguish between total P2X 7 distribution and the proportion of receptors that are non-functional and expressed in gut mucosa.
  • the two antibodies used together can detect both total receptor count and those receptor channels present only in a close-gated or non-functional conformation.
  • the first antibody is adapted to detect total P2X 7 receptor expression.
  • the probe comprising or attached to the antibody of the invention can provide the second antibody for detection of IBS, not only distinguishing between functional and nonfunctional P2X 7 receptors, but also allowing for detection of other regions in which the receptor is unchanged by functional state.
  • the antibodies may be used separately or together. Preferably, they are used in combination.
  • Expression of non- functional P2X 7 receptors in the gastrointestinal mucosa occurs in a pattern in which normal cells remain essentially unlabelled. Thereafter, the non- functional conformation of P2X 7 is first detected in the stroma underneath the epithelium ranging from isolated patches in mild cases of the syndrome to extensive expression throughout the length of the gastrointestinal tract with isolated patches of infiltration of nonfunctional receptors into the epithelium.
  • the invention also provides a method of diagnosing irritable bowel syndrome, comprising detecting the P2X 7 expression profile of cells and/or tissue and comparing the profile with a predetermined expression profile of normal cells and or tissue.
  • the detection of the P2X expression profile includes use of one or more antibodies.
  • antibody or antibodies are different from the probe of the invention in that they do not detect change in relation to binding of ATP to the P2X 7 receptors.
  • the preparation of such antibodies will be readily apparent to one skilled in the art.
  • the invention also includes use of one or more antibodies to diagnose irritable bowel syndrome.
  • the diagnostic can be used in standard microscopy employing standard immunohistochemical techniques.
  • the diagnostic may also be used in vivo.
  • Diagnosis using the probe and method of the invention may be carried out using in situ imaging techniques to detect distribution in body tissues.
  • standard microscopy, confocal microscopy and fluorescence activated cell sorting may be used.
  • Normal immunohistochemical techniques for testing lymph, prostate, breast, skin, i ⁇ ng, uterus, bladder, cervix, stomach, oesophagus and similar biopsies, also fine needle aspirates of breast and other tissue and cell smears such as those taken for the detection of cervical cancer, may be used.
  • the probe is a human antibody or domain, manufactured with no animal components.
  • the antibody is preferably labelled with a short-lifetime radiolabel, detectable by means of scanning technology such as positron emission tomography (PET scanner).
  • PET scanner positron emission tomography
  • Such imaging can detect the aggregation of labelled antibody anywhere in the body, thus signalling the presence of non- functional receptors, associated with the presence of any tumour.
  • PET scanner positron emission tomography
  • Such a test should be conducted only after detection of primary cancer and for the purpose of checking for secondary cancer, or after a general screen by means of a blood test (refer below) has detected the likelihood of the presence of one of more tumours.
  • the probe and method of the invention may be employed to provide a blood test for detecting non-functional P2X 7 receptors and hence cancer or pre-cancerous conditions.
  • the probe in the form of a fluorescent labelled antibody can be used in flow cytometry against blood cell fractions of the patient in order to detect binding to non-functional receptors on various gated leukocytes, including T lymphocytes, B lymphocytes or macrophages.
  • the probe preferably takes the form of a labelled antibody attached to a matrix provided in a kit, enabling detection by the presence of a colour reaction to the binding of the fixed antibody to positive white blood cells.
  • a kit may be suitable for use by medical practitioners.
  • the antibody probe of the invention may be used as a diagnostic tool for screening patients who may not have cancer but in whom the normal cell killing pathways are inhibited through lack of function in P2X 7 on one or more leukocytes.
  • patients may express non-functional receptors on macrophages, indicating inhibition of the ability of those macrophages to kill infected cells, such as those infected by organisms like Mycobacterium tuberculosis, or other infectious agents including malaria and HIV.
  • Such organisms preferentially proliferate in patient? for whom the normal cell killing pathways are inhibited through lack of function in P2X 7 on one or more leukocytes.
  • This invention provides an antibody for treating a disease or condition, the antibody being adapted to distinguish between functional P2X 7 receptors and nonfunctional P2X 7 receptors and being adapted to bind only to non- functional receptors.
  • the antibody distinguishes between the functional and nonfunctional receptors by detecting change in relation to binding of adenosine triphosphate (ATP) to the receptors, or by detecting change in binding of one or more proteins necessary for pore formation in P2X 7 receptors and being adapted to bind only to non-functional receptors.
  • the antibody distinguishes between the functional and non-functional receptors by detecting parts of the receptor exposed in the absence of bound ATP.
  • the antibody for treating diseases and conditions may be the same as the antibody which may be used as the probe for diagnosing diseases and conditions.
  • Such an antibody could be used to treat skin cancers topically, for example.
  • the antibody or its active fragments should be human or a human domain, in order to minimise undesirable immune response side effects.
  • the antibody of the invention may be used to treat diseases or conditions in mammals, including humans. Examples of the diseases or conditions have been set out above in connection with the probe of the invention.
  • the invention also provides an epitope capable of causing the generation of the antibody of the second aspect of the invention.
  • the epitope preferably includes Pro210 and encompasses the segment Gly200 to Cys216 (in the P2X 7 sequence of the human receptor).
  • the epitope should preferably have attached to the C-terminal end a Cys residue (Cys216) that is cross-linked to diphtheria toxin via the chemical cross-linker maleimidocaproyl-N-hydroxysuccinirnide (MCS), so that the conformation adopted by the attached epitope peptide occupies a stable cis proline configuration.
  • This specific peptide conformation is intended to be presented to humans or animals with one or more diseases or conditions, especially epithelial cell cancers, such as prostate, breast, skin, lung, cervix, uterus, stomach, oesophagus, bladder, colon and vaginal cancers, as well as malignant lymphoma, irritable bowel syndrome and infection by viruses such as HIV or other pathological organisms, such as Mycobacterium tuberculosis.
  • the patient will preferably mount an immune response to the applied conjugated epitope and so generate antibodies recognising the non-functional P2X 7 receptors present on the surface of the affected cells, thus binding to them and alerting the appropriate immune cell to destroy the complexed cells. Other cells primed for cell death may also be affected.
  • sequence referred to above is not limiting on the scope of the invention, which includes alternate sequences and carriers and cross- • linkers that similarly produce a specific immune response, preferably against only non-functional P2X 7 receptors, preferably ignoring all functional receptors expressed on cell surfaces, and so avoiding side effects.
  • the invention in this second aspect, also provides for the use of the antibody of the invention as a therapeutic vehicle for treatment of a disease or condition in a patient to regulate programmed cell death by targeting aberrant or non-functional P2X 7 receptors expressed on the surface of cells, while leavmg all cells expressing normal (functional) receptors untouched.
  • the invention also covers the use of the epitope of the invention to cause the generation of the antibody, as above.
  • the invention also provides a pharmaceutical composition for treatment or prevention of a disease or condition in a patient, the composition including a pharmaceutically effective amount of an antibody, or an epitope to cause the generation of such an amount, capable of regulating programmed cell death of cells having expressed on their surface aberrant or non-functional P2X 7 receptors.
  • the pharmaceutically effective amount of the antibody or epitope will vary according to the patient and the nature of the disease or condition. These variables can be ascertained by one skilled in the art.
  • composition of the invention may be administered in conjunction with a pharmaceutically acceptable carrier, which may be any of those known in the art or devised hereafter and suitable for the intended use.
  • a pharmaceutically acceptable carrier which may be any of those known in the art or devised hereafter and suitable for the intended use.
  • the pharmaceutical compositions of the invention may include other ingredients, including dyes, preservatives, buffers and antioxidants, for example.
  • the pharmaceutical composition of the invention may take any desired form and may be administered, for example, in the form of an ointment, cream, solution, suspension, powder, tablet, capsule, suppository or pessary.
  • the pharmaceutical composition of the invention may be administered in any suitable way, which may include oral, parenteral, intravenous, intramuscular, subcutaneous or topical administration.
  • the invention also provides a method of treating or preventing a disease or condition in a patient, the method including administering to the patient a pharmaceutical composition according to the invention.
  • the invention also provides the use of the pharmaceutical composition of the invention, in the treatment or prevention of a disease or condition, in a patient.
  • the third aspect of the invention focuses on the expression of ⁇ TPases (enzymes) that control the supply of ATP to P2X 7 receptors, for example in the B-cells of a patient having malignant lymphoma.
  • ⁇ TPases enzymes that control the supply of ATP to P2X 7 receptors
  • Channel opening of P2X 7 receptors on leukocytes is terminated through the rapid hydrolysis of ATP agonist by ecto- ATPases and ecto-ATPdiphosphohydrolases (ecto-ATPDases).
  • ecto-ATPDases ecto-ATPDases
  • These enzymes regulate numerous physiological processes that are dependent on ATP.
  • Substrate specificity of ATPase and ATPDase activity on lymphocytes indicates the presence on the lymphocytes of more than one type on the cell surface, including CD39. Proliferation of one or more of these ATPases or ATPDases could limit the supply of ATP needed to control
  • proliferation of ATPases may contribute to lack of appropriate binding of the agonist ATP to the P2X 7 receptors.
  • the invention provides a preparation for treatment or prevention of a disease or condition in a patient, the preparation including one or more substances adapted to regulate the expression of ATPases that control the supply of ATP to P2X 7 receptors in the patient's cells or tissues.
  • the invention also provides a method of treating or preventing a disease or condition in a patient, the method including the step of administering to the patient a preparation including one or more substances adapted to regulate the expression of ATPases that control the supply of ATP to P2X 7 receptors in the cells or tissue of the patient.
  • Examples of such ATPases may be CD39 or CD73.
  • Such a substance may take the form of an ATP analogue, preferably non- hydrolysable, and specific for P2X 7 , or another substance that inhibits the action of local ATPases depleting the availability of ATP for the P2X 7 binding site.
  • the preparation may be in the form of a human antibody directed specifically against non-functional P2X 7 receptors.
  • a substance such as an ATP analogue may bind to the P2X 7 and hold it in open pore configuration, thus forcing the pore to assume a functional state, in which it is able to take up both large and small cation permeants.
  • the use of such a synthetic agonist may act to restore receptor function, at the same time as controlling the growth advantage that P2X 7 provides cells in its role as a calcium channel.
  • the disease or condition is preferably malignant lymphoma or IBS but the invention may also extend to other diseases or conditions, including other epithelial cell or blood cancers or viral and other pathological infections.
  • the ATPases control the local supply of ATP to the P2X 7 receptors so as to reduce the concentration of ATP available for binding to the P2X 7 receptors and so deactivate them leading to a significant reduction in programmed B-cell death.
  • These ATPases may be specifically expressed on the surface of the B-cells and appear to be up-regulated in malignant lymphoma.
  • application of a specific ATPase inhibitor may be used to regulate the availability of ATP on the P2X 7 receptors, so regulating programmed B-cell death.
  • ⁇ _t substance may include a synthetic agonist capable of blocking ATPases or ATPDases, of the form of non- hydrolysable P2X 7 agonist.
  • administration of the preparation of the invention is intended to restore receptor function that may be depleted through overactivity of the muscle underlying the affected region of mucosa.
  • the preparation of the invention may act on the mucosa directly to remove these nonfunctional receptors and thereby restore local normal gastrointestinal secretory mechanisms.
  • Therapeutic treatment is aimed at restoring the local supply of ATP to the non-functional receptors, so that normal receptor function is restored.
  • the consequences of control of receptor function include restoration of normal control of gastrointestinal secretions and peristalsis.
  • treatment may involve restoration of this natural supply of agonist by means of a limit on the uptake or use of ATP by the smooth muscle through application of a treatment to temporarily limit gut motility.
  • the invention also provides a pharmaceutical composition for treatment of a disease or condition, the composition including a pharmaceutically effective amount of one or more substances adapted to regulate the expression of ATPases (enzymes) that control the supply of ATP to P2X 7 receptors.
  • ATPases enzymes
  • the invention in all its aspects extends to such similar applications that could be made in other medical conditions in which aberrant P2X 7 receptors are involved as a result of viral infection where the virus is protected in the infected cell by up- regulating non-functional P2X 7 receptor or where such receptors are up-regulated from the normal cell condition.
  • the invention also provides a method of treating irritable bowel syndrome, comprising administering to a patient a pharmaceutical composition as defined above.
  • the invention also provides the use of such a pharmaceutical composition in the treatment of irritable bowel syndrome.
  • the pattern of use of one or more of me above pharmaceutically effective agents may need to be altered for optimum effect.
  • the invention provides a method of treating irritable bowei syndrome, the method including administering a composition adapted to restore P2X 7 receptor function.
  • the receptor function may have been depleted through overactivity of the muscle underlying the affected region of mucosa.
  • the composition may be the same as that set out above for the substance included in the preparation of the invention.
  • the invention provides a method for distinguishing between different conformations of proteins by using an epitope capable of causing the generation of an antibody, or the antibody itself, to effect specific pharmaceutical outcomes (active as well as passive immunisation) from binding to all members of the proteins with a selected conformation.
  • an epitope capable of causing the generation of an antibody, or the antibody itself
  • specific pharmaceutical outcomes active as well as passive immunisation
  • An example of this would be prion proteins in the conformation that leads to the condition vCJD.
  • the abnormal form of the protein could be targeted by a specific antibody or epitope causing the generation of the antibody, preferably human and reduced in size for optimum pharmacological effect.
  • Figure 1 shows the amino acid sequence of the human P2X 7 receptor (prior art). Sequences 65 to 81 and 200 to 216 are highlighted and are referred to below.
  • the epitope used was the sequence 200 to 216 in Figure 1, containing a Cys at 216.
  • the epitope used was the sequence 65 to 81 in Figure 1, to which was added an N-te ⁇ ninal Cys. This antibody could not detect whether the receptor was non-functional but was designed to detect all receptor so that the proportion of receptor that was functional could be determined by comparing the staining obtained by using the two antibodies separately.
  • the Cys residues on the epitopes were coupled via a maleimidocaproyl-N- hydroxysuccinimide (MCS) cross linker to diphtheria toxin (DT) carrier with ten peptide epitopes attached to each DT carrier, to maintain conformational stability and idc a larger antigcnic structure
  • MCS maleimidocaproyl-N- hydroxysuccinimide
  • DT diphtheria toxin
  • conjugate 500 ⁇ g was diluted in phosphate-buffered saline (PBS) to 0.8 mL and was emulsified with 1.2 mL of Freund's Complete adjuvant. Sheep were injected at multiple sites both subcutaneously and intramuscularly with the antigen/adjuvant emulsion. Eight weeks later the sheep were again injected with the same amount of conjugate emulsified with Freund's Incomplete adjuvant at multiple sites. This was repeated 4 weeks later and the animals were bled from the jugular vein. The serum collected was tested for antibody specificity. The sheep were then routinely injected and bled at eight week intervals to provide a pool of serum containing the specific antibodies.
  • PBS phosphate-buffered saline
  • Antibodies were raised in rabbits using the same two adjuvants as with the sheep and the same injection schedules, the only difference being that 300 ⁇ g amounts of the conjugate were used for the injection.
  • the antibodies raised had the same specificities as those produced in the sheep and could readily discriminate between the epitopes against which they were raised.
  • Antibodies were raised in mice against the conjugated epitopes and also against the unconjugated epitope of the non-functional P2X 7 epitope (which is able to discriminate receptors that cannot from pores and thus fail to be apoptotic).
  • the adjuvant used was the QAIGEN Pty Ltd product, ImmunEasyTM which contains the immuno-stimulatory product CpG DNA (trademark of Coley Pharmaceutical Group Inc.)
  • mice 5 ⁇ g of epitope or conjugated epitope was diluted in 70 ⁇ L of PBS and 30 ⁇ L of ImmunEasyTM adjuvant. Mice were injected at multiple sites subcutaneously and intramuscularly. This regime was repeated two weeks later and again at a further two weeks. Mice were bled eight days after the third injection. Antibodies raised in mice by this method were again able to discriminate between the different P2X 7 epitopes and the antibodies against the P2X 7 non-functional epitope gave the same results as those raised in sheep and rabbits.
  • antibodies to various epitopes of the P2X 7 receptor in different species and using different adjuvants may be raised consistently.
  • antibodies to an epitope of the P2X 7 receptor which identifies the receptor in the non-functional state, in which it cannot form a pore and carry out its apoptotic function under normal physiological conditions may be raised rountinely.
  • the antibody detecting non- functional P2X 7 was tested by binding the antibody to cells expressing P2X 7 (human) with known function as revealed through the ability of the P2X 7 to take up ethidium or rubidium. These P2X 7 protein channels may have been mutated at base pair 1513, such that the channels would not form apoptotic pores. These and similar non-functional P2X 7 receptors expressed on malignant B lymphocytes also bound the antibody in flow cytometry and in standard immunohistochemistry while cells expressing normal functional P2X 7 (capable of taking up calcium, ethidium and rubidium with large fluxes) were unable to bind the antibody, because the epitope chosen to detect the nonfunctional receptors was unavailable in functional receptors.
  • the Pro210 adopted a cis conformation in the non- functional receptors and it was specifically this conformation that was stabilised in the conjugated epitope used to raise the antibody.
  • the Pro210 was in the trans conformation in the receptors that were shown to be functional. This was a result of the binding of ATP (adenosine triphosphate) to the P2X 7 receptor. When ATP was bound, the Pro210 on a segment immediately adjacent to the ATP binding site adopted a trans configuration.
  • ATP adenosine triphosphate
  • the macrophages and B-cell lymphocytes extracted from patients with malignant iymphoma were tested ana ail these cells bound the antibody to universal P2X 7 as well as the antibody to the non- functional P2X 7 receptors, verifying that P2X 7 was non-functional in all the cancer cells detected, with the apoptotic pore formed by functional P2X 7 unable to form and thus induce apoptosis in cancer cells.
  • All such cancer cells from all epithelial cell cancers in humans such as prostate, breast, bowel, skin, stomach, cervix and others as well as malignant lymphoma, chronic lymphocytic leukaemia and brain mmours, as well as the same mmours in other mammals that were tested, including breast and prostate in dog and skin in cat as well as all mouse hybridoma cells and mouse fibrosarcoma cells, all express the same non-functional P2X 7 . Sequence similarity between human, rat, cat, dog and mouse at the chosen epitopes is sufficient for positive identification to be made in all the above cases.
  • B cells from leukaemia patients containing non-functional P2X 7 receptors were incubated with 5 mM ATP for 2 hours in culture. The results were that all the non-functional receptors were forced by the excess ATP to open and induce apoptosis that killed the affected cells.
  • BCC basal cell carcinomas
  • epithelial cell cancers like breast, lung, colon and skin in humans and in other mammals, such as cats and dogs, can be detected with margins as there is no longer a clear field effect in these other tissues.
  • Skin cancers such as basal cell carcinoma, squamous cell carcinoma and dysplastic naevi as well as malignant melanomas show positive staining for non-functional receptors and channel components (monomers) in keratinocyte and melanocyte layers with clear margins beyond which normal skin is unlabelled on both epidermis and deep within the dermis.
  • AU tested mammalian cancer cell lines such as human prostate (PC3) and breast (MCF7) and rodent hybridomas are positive for the non-functional receptors on the cell surface so that apoptosis is inhibited in these cancer cells.
  • PC3 human prostate
  • MCF7 breast
  • rodent hybridomas are positive for the non-functional receptors on the cell surface so that apoptosis is inhibited in these cancer cells.
  • the general application of this diagnostic is seen by way of the same label on mouse hybridoma cells showing the ubiquitous nature of the receptor in other ariimal types besides human.
  • No ⁇ nal human B-cell lymphocytes show that functional P2X 7 receptors are expressed on the cell surface, so enabling apoptosis when necessary, while human B-cell lymphocytes from patients with malignant lymphoma show that nonfunctional P2X 7 receptors are expressed on the cell surface, so curtailing apoptosis.
  • Targeting this apparently ubiqitous P2X 7 non-functional conformer expressed on the cell surface of cancer cells attempting to undergo apoptosis may be used to treat most cancers in humans and other mammals. Examples are set out below:
  • Mouse hybridoma cells were grown on a macrophage base both in the presence and absence of affinity purified antibody to non- unctional P2X 7 .
  • Group 1 10 mice each injected intraperitoneally (IP) with 1 x 10* hybridoma cells in 0.5 mL of cell culture medium on Day 1. On Days 2 and 3, they received an mtraperitoneal injection of 0.5 mL of cell culture medium.
  • IP intraperitoneally
  • Group 2 10 mice each injected intraperitoneally (IP) with 1 x 10 6 hybridoma cells in 0.5 mL of cell culture medium ⁇ >nt ⁇ ining 1 mg of purified sheep IgG on Day 1. On Days 2 and 3, they were injected with 0.5 mL of cell culture medium containing 1 mg of purified sheep IgG.
  • IP intraperitoneally
  • mice each injected intraperitoneally (IP) with 1 x 10° hybridoma cells in 0.5 mL of cell culture medium containing 1 mg of purified sheep anti-P2X 7 non- functional epitope IgG on Day 1. On Days 2 and 3, they received a further injection of 0.5 ml of cell culture medium containing 1 mg of purified sheep anti-P2X 7 IgG.
  • mice from all the groups were killed on Day 11 and examined for the presence of tumour.
  • the tumours were excised and weighed.
  • BCC human basal cell carcinomas
  • BCC basal cell carcinoma
  • SCC squamous cell carcinoma
  • the size of the tumours treated ranged from 3mm diameter with no raised border to 5cm diameter and up to 4mm thick. A total of thirty four histologically confirmed tumours have been successfully eliminated within one week treatment periods. It is believed that application to patients in general would involve production of a human monoclonal antibody (such as herceptin) so that internal cancers could be treated with the same efficacy as is revealed with topical application. All normal functional P2X 7 expressed on the cell surfaces of cells such as lymphocytes would need to remain unaffected by the presence of the antibody to avoid side effects. The antibody should therefore only bind to proteins expressed on the cell surface of cells attempting to but unable to initiate apoptosis. Thus all cells targeted would be only those attempting to kill themselves through programmed cell death, including cancer cells. The P2X 7 receptors on these cells, particularly cancer cells, would be in a non-functional or ATP-depleted state.
  • Active immunisation may also be used for therapeutic purposes.
  • the humans or other mammals need to be immunised against a specific epitope or epitopes that are in a conformation that mimics the conformation adopted only by the receptors in their non-functional (ATP-depleted) shape on the cell surface.
  • Conformational flexibility that includes partial exposure of an epitope shape that is present in functional receptors should be avoided.
  • the cis configuration of the epitope Gly200-Cys2 l6 as an example should be fixed before use by appropriate means. As added proof that this concept is sound is the observation that numerous animals including mice, rabbits and sheep used to raise the antibodies have not been immuno-compromised. None of these many animals have ever developed any mmours.
  • the experiment was conducted on the basis of a mouse mmour model. Forty ten-week old female inbred Balb C mice were used, and divided into two groups of twenty, Group 1 being experimental and Group 2 being the control group. Day 1: The twenty experimental animals in Group 1 were injected with 0.1 mg of the peptide epitope (hP2X 7 sequence 200-216) conjugated to diphtheria toxin via the MCS crosslinker. This contained approximately 0.02 mg of the peptide epitope.
  • the peptide conjugate was emulsified with a QUILL A/DEAE Dextran/Montanide ISA 50V adjuvant mix and injected in a volume of 0.1 mL at multiple subcutaneous and intramuscular sites.
  • mice in the control group were injected with 0.1 mL of the adjuvant mix without peptide conjugate at multiple subcutaneous and intramuscular sites.
  • Day 8 The twenty Group 1 mice were injected with 0.01 mg of the peptide epitope (hP2X 7 sequence 200-216) conjugated to diphtheria toxin via the MCS crosslinker (containing approximately 0.002 mg of the peptide epitope).
  • the peptide was contained in a phosphate buffered saline solution and mixed according to the protocol with the commercially available CpG DNA adjuvant ImmunEasy (from Qiagen). A volume of 0.1 mL of peptide conjugate/adjuvant solution was injected at multiple subcutaneous and intramuscular sites in each mouse.
  • mice w eie injected with the comparable phosphate buffered saline/ CpG DNA adjuvant mix. This was injected in a volume of 0.1 mL in each mouse at multiple subcutaneous and intramuscular sites.
  • mice were injected at two concentrations into both the experimental and control groups of mice. Each group was subdivided into two. Ten mice from each of the experimental and control groups received 1 0,000 cells per mouse and ten mice from each group received 320,000 cells per mouse.
  • the cells from this cell line had previously been tested for the presence of the nonfunctional P2X 7 epitope on their cell surface. This was done using an antibody raised in sheep which specifically recognises the non-functional form of the receptor.
  • the invention in all its aspects has application to the fields of human and veterinary medicine and health, with the potential to enable early and accurate diagnosis of diseases and effective treatIER, which in many cases is far less invasive or traumatic than those available in the prior art.

Abstract

La présente invention concerne une grande variété de maladies et de conditions, y compris les cancers. L'invention propose une sonde destinée à la détection d'une telle maladie ou condition. La sonde est capable de distinguer entre des récepteurs fonctionnels de P2X7 et de récepteurs non fonctionnels de P2X7. La sonde peut établir cette distinction de diverse manières, dont une consiste en la détection d'une modification par rapport à la liaison de l'adénosine triphosphate (ATP) aux récepteurs. L'invention propose également un procédé permettant la détection de la maladie ou condition, utilisant la sonde. L'invention concerne en outre un traitement de la maladie ou condition, au moyen d'un anticorps, ou d'un épitope capable de générer l'anticorps, pouvant établir la distinction entre les récepteurs fonctionnels et non fonctionnels de P2X7. Enfin, l'invention concerne des procédés de traitement, des compositions pharmaceutiques et l'utilisation de la sonde et de l'anticorps
PCT/AU2002/001204 2001-01-17 2002-09-03 Anticorps au recepteur p2x7 non fonctionnel, diagnostic et traitement de cancers et d'autres conditions WO2003020762A1 (fr)

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JP2003525032A JP4467973B2 (ja) 2001-09-03 2002-09-03 非機能性ｐ２ｘ７リセプターに対応する抗体、癌及びその他の症状の診断及び治療
CA2459348A CA2459348C (fr) 2001-09-03 2002-09-03 Anticorps au recepteur p2x7 non fonctionnel, diagnostic et traitement de cancers et d'autres conditions
AU2002322192A AU2002322192B2 (en) 2001-09-03 2002-09-03 Antibodies to non-functional P2X7receptor, diagnosis and treatment of cancers and other conditions
US10/622,313 US7326415B2 (en) 2001-01-17 2003-07-17 Antibodies to non-functional P2X7 receptor
ZA2004/02630A ZA200402630B (en) 2001-09-03 2004-04-02 Antibodies to non-functional p2x7 receptor diagnosis and treatment of cancers and other conditions
US11/968,607 US7531171B2 (en) 2001-01-17 2008-01-02 Antibodies to non-functional P2X7 receptor
US12/417,989 US7888473B2 (en) 2001-01-17 2009-04-03 Non-functional P2X7 receptor
US12/975,341 US8080635B2 (en) 2001-01-17 2010-12-21 Non-functional P2X7 receptor
US13/298,222 US8399617B2 (en) 2001-01-17 2011-11-16 Non-functional P2X7 receptor
US13/766,630 US8709425B2 (en) 2001-01-17 2013-02-13 Antibodies to non-functional P2X7 receptor
US14/218,935 US20140323693A1 (en) 2001-01-17 2014-03-18 Antibodies To Non-Functional P2X7 Receptor
US14/726,391 US9663584B2 (en) 2001-01-17 2015-05-29 Antibodies to non-functional P2X7 receptor
US15/498,301 US10450380B2 (en) 2001-01-17 2017-04-26 Polypeptide immunogen for generating an antibody to non-functional P2X7 receptor
US16/568,072 US20200071419A1 (en) 2001-01-17 2019-09-11 Antibodies to non-functional p2x7 receptor

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CN106310245A (zh) * 2010-09-10 2017-01-11 生物权威(澳大利亚)有限责任公司 陪伴动物治疗
WO2012031333A1 (fr) 2010-09-10 2012-03-15 Biosceptre International Limited Traitements d'animaux de compagnie
AU2011301153B2 (en) * 2010-09-10 2014-11-27 Biosceptre International Limited Companion animal treatments
EP2613808A4 (fr) * 2010-09-10 2014-01-22 Biosceptre Int Ltd Traitements d'animaux de compagnie
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US10245308B2 (en) 2011-07-01 2019-04-02 Biosceptre (Aust) Pty Ltd Combination therapy utilizing P2X7 peptides
US10543262B2 (en) 2011-07-01 2020-01-28 Biosceptre (Aust) Pty Ltd Combination therapy utilizing P2X7 peptides
WO2017041143A1 (fr) 2015-09-11 2017-03-16 Ctm@Crc Ltd. Récepteurs d'antigènes chimériques et leurs utilisations
EP3816294A1 (fr) 2015-09-11 2021-05-05 Biosceptre (UK) Limited Récepteurs d'antigènes chimériques et leurs utilisations
US11260131B2 (en) 2016-10-21 2022-03-01 Biosceptre (Aust) Pty Ltd Cytotoxic particles for targeting P2X7 receptor

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MY142283A (en) 2010-11-15
JP4467973B2 (ja) 2010-05-26
CA2459348C (fr) 2013-06-18
NZ565994A (en) 2010-02-26
AU2002322192B2 (en) 2008-05-01
CN100497386C (zh) 2009-06-10
NZ549019A (en) 2008-05-30
CN101445555B (zh) 2013-07-03
JP2005513416A (ja) 2005-05-12
ZA200402630B (en) 2005-06-29
CN101445555A (zh) 2009-06-03
CN1625565A (zh) 2005-06-08
CA2459348A1 (fr) 2003-03-13

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