JP4467973B2 - Diagnosis and treatment of antibodies, cancer and other symptoms corresponding to non-functional P2X7 receptor - Google Patents

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

  The present invention relates to diagnosis and treatment of diseases including cancer. Disease types relevant to the present invention include cancers derived from epithelial cells and malignant lymphomas. The invention also relates to other symptoms such as abnormal tissue generation / growth, irritable bowel syndrome and viruses and other infections. The present invention also has a great potential to be applied to diseases and symptoms other than those described above.

Adenosine triphosphate (ATP) can activate a ligand-gated purine activation receptor known as the P2X receptor. P2X ₇ from P2X ₁ as a subtype of receptor has been identified. It is known that there are different types of P2X receptor subtypes in many cells, including epithelial cells, leukocytes, lymphocytes, thymocytes, macrophages and dendritic cells.

  The P2X receptor is permeable to some cations such as calcium and others such as phosphorus and sodium. Influx of calcium into cells via the P2X receptor may be associated with cell death.

P2X ₇ subtype are thought to be related to apoptosis, or programmed cell death in a variety of cell. When ATP is present, P2X ₇ receptor expressed on the cell surface, within one second, it is possible to open calcium channels through the cell membrane. Continuous exposure to ATP can lead to the formation of large pores, which can saturate cells with excess calcium and cause apoptosis within seconds to tens of seconds.

P2X ₇ amino acid sequence of the receptor of human and rat, for example, are known, as referenced in FIG. 1 (e.g., see Patent Document 1.).

For example, in epithelial cancer cells, exposure to ATP usually does not result in apoptosis. In such a cell it is found to express P2X ₇ receptor which can not form a pore. These are considered non-functional receptors.

Similar animal cell lines, including hybridoma of rodent, human cancer cell lines, such as prostate PC3 and the like milk MCF7, P2X ₇ receptor is found in the conformation of the non-functional at the cell surface.

Malignant lymphoma patients B- cells expressing a non-functional P2X ₇ receptors. Lymphoma develops from a malignant clone that escapes cytolytic destruction. This process proceeds further leading to the accumulation of malignant B-lymphocytes, ie lymph node injury and / or splenomegaly.
US Pat. No. 6,133,434

First, the present invention is a probe for detecting diseases and symptoms, the probe being adapted to distinguish between a functional P2X ₇ receptor and a non-functional P2X ₇ receptor. Preferably, the probe identifies a non-functional P2X ₇ receptor and functional P2X ₇ receptors by detecting change for binding to P2X ₇ receptors of adenosine triphosphate (ATP), or in the P2X ₇ receptor A functional P2X ₇ receptor is distinguished from a non-functional P2X ₇ receptor by detecting changes in the binding of one or more proteins necessary for pore formation. In yet another embodiment, the probe is functional P2X ₇ receptors and non-functional P2X ₇ identify the receptor by detecting a portion or portions of the reveal of P2X ₇ receptor when bound ATP does not exist To do. Such a portion (of the P2X ₇ receptor) may comprise a P2X ₇ monomer.

The present invention also provides identifying and using the probes described above and functional P2X ₇ receptor nonfunctional P2X ₇ receptors, providing a expression profile of receptor, and the expression profile of receptor obtained in the above step There is also provided a method of discovering a disease or condition comprising the step of comparing to a normal profile. Differences (in comparison results) may be detected in relation to the probe itself, for example, as suggested above.

The probe may be natural or man-made. The probe is preferably an antibody, and may be a polyclonal antibody, a monoclonal antibody, a recombinant antibody, an antibody adapted to the human body, a human antibody, and appropriate fragments thereof. Antibody is preferably one that corresponds to an epitope located in the extracellular region adjacent to (a P2X ₇ receptor) ATP binding site. If receptor is P2X ₇ receptor in humans, the probe includes a functional receptor which proline at amino acid 210 has a sequence that is trans (trans) conformation, local amino acid 210 is a cis (cis) conformation It is preferably adapted to distinguish from non-functional receptors having sequences that greatly mutate the protein structure.

  The probe may be prepared using any suitable technique apparent to those skilled in the art.

The scope of the invention also includes probes to identify by detecting conformational changes other than those described above in the ATP binding position, and a functional P2X ₇ receptors and non-functional P2X ₇ receptors. For example, the detected change may be in an amino acid other than proline as described above. An example of such an amino acid is Pro199, which greatly mutates the local protein structure in the cis conformation. As another example, the detected change may be from another perspective.

The probe may be adapted to detect an area that does not vary with the functional state of the P2X ₇ receptor. Conformation of monomer subunits lacking bound ATP can be detected using probes, such as by specifically detecting the shape of the surface region of the receptor that is accessible only when the selected epitope is not bound by ATP. Is possible. The probe may detect changes in binding of one or more proteins necessary for pore formation, such as ancillary or other proteins. Examples of such proteins include, but are not limited to laminin, integrin, β-actin, α-actin and supervillin.

In the present invention, pre-neoplastic cells, very early neoplastic tissue, the tumor tissue on or in which the progression, or, expressed in a non-functional P2X ₇ on any neoplastic cell which are optionally expressed receptor to specifically detect binding or it non-functional P2X ₇ receptors are, it may be used an antibody specific for P2X ₇ subtype. That, P2X ₇ receptor may have been expressed in normal cell membranes, even possible by the state to function as a partial channel, the gate is closed, or a non-functional configuration only when the conformations detected or Combined.

  Furthermore, the conformation of monomer subunits lacking bound ATP can also be detected by antibodies. This is because the selected epitope specifically detects the shape of the surface area that is accessible only when ATP is not bound.

In the present invention, non-functional P2X ₇ receptors can be detected or bound by using an antibody which corresponds to an epitope to cause a conformational change from the structure present in functional receptors. It is known that the amino acid sequence of a non-functional receptor can be identical to the amino acid sequence of a functional receptor, and thus conformational changes in the receptor are related to the correlation between the receptor and ATP. As described above, since ATP molecules are receptor agonists, when ATP is bound to a receptor, the receptor can open a channel through the cell membrane for influx of calcium ions. That is, non-functionality occurs due to the lack of proper binding of the ATP agonist to the receptor, which is due to insufficient production or lack of locally available ATP due to increased degradation rates. Possible phenomena are included. If ATP binding to the receptor is prevented, the conformation of the receptor is altered. This can be detected by using an antibody specifically designed to bind to a region of the protein affected by ATP binding.

For P2X ₇ receptor in humans, specific sequences involved in the conformational change may include Pro210, which conformational changes and no ATP bound from the transformer (trans) form to the cis (cis) form. Thus, in the case of the human receptor, the sequence of a suitable epitope that produces an antibody includes Pro210 and may extend to both sides of this residue to the extent necessary to elicit an antibody response. A non-limiting example may include a portion extending from Gly200 to Cys216. Furthermore, homologous portions of other mammals such as rats that cross-react with human tissues can also be used. For example, there are two amino acid substitutions compared to the human sequence, but the same Gly200 to Cys216 part of rat can also be used (see, for example, Patent Document 2).
US Pat. No. 6,133,434

  In the case of non-human receptors, the specific sequence can be confirmed by appropriate experimentation.

Detection of non-functional P2X ₇ receptors according to the present invention may show a distribution pattern in which functional P2X ₇ receptors (ie normal cells) remain unlabeled. However, the non-functional conformation of the P2X ₇ receptor can be detected at an early stage of pre-tumor formation, initially in the cell nucleus and cytoplasm. For example, in the case of epithelial cell carcinoma, the use of the method according to the present invention can be performed several years prior to the detection of normal cancer pathological symptoms by biopsy hematoxylin and iocin (“H & E”) stained slides. It is also possible to detect tumor formation. That is, it is possible to detect the prostate, skin, breast cancer, etc. much earlier than the present and bring about the benefit of starting treatment early.

The complete range of illnesses and symptoms detected by the probes and methods of the present invention has not yet been confirmed. However, prostate cancer, breast, skin, lung, cervix, uterus, stomach, esophagus, bladder, colon, vagina and other epithelial cell carcinomas, blood cancers including malignant lymphomas, irritable bowel syndrome, and HIV or others, humans Can be confident that it includes infection with pathogenic viruses such as Mycobacterium tuberculosis. Infections directly by inhibiting cofactor required for functionality, or by upregulating the cofactors such as to inhibit P2X ₇ function on epithelial or other cells, the non-functional receptors It may be expressed and make infected cells less susceptible to apoptosis destruction.

  Unless otherwise stated, the phrase “disease or symptom” is intended to include herein all the specific illnesses and symptoms mentioned above.

In the particular case of irritable bowel syndrome ( "IBS"), in patients with this condition, the stroma beneath the epithelium, of the gastrointestinal tract which normally expresses P2X ₇ receptors in the lymphocytes present widely distributed It has been found that the mucosa is up-regulated. In infected patients, this increased expression can be observed in the mucosa between the duodenum and the rectum. Increased expression may be seen in isolated regions or, in more extreme cases, may increase overall over the entire length of the intestinal tract.

In the most mild cases, but overall P2X ₇ receptor is up-regulated, they are all functional receptors, and does not penetrate into the epithelium. In more serious cases, and has a non-functional receptors in locations most affected in the expression of the overall P2X ₇ receptor is much higher, and the gastrointestinal tract. For example, they may be concentrated in the mucosa of the cecum or may invade the epithelium. The most severe cases may have been further increased expression of overall P2X ₇ receptor, and most of the receptor is the case invading more to epithelial cells in non-functional.

As described above, non-functional P2X ₇ receptors is caused by the appropriate binding to receptor of ATP agonist lacking. The cause may include a shortage of locally available ATP due to underproduction or increased rate of enzymatic degradation of ecto-ATPase ATP. If binding of ATP to the receptor is hindered, the receptor conformation changes as described above, and this can be detected using the probes of the present invention. However, to detect the distribution of all P2X ₇ receptor, a method of using the other epitopes of the extracellular region of P2X ₇ receptor that is not affected by ATP binding is most preferred. The probe may be able to detect a region of the P2X ₇ receptor that does not change with functional status by detecting epitopes common to functional and non-functional conformations such as Val65-Lys81.

The scope of the present invention, the distribution of all P2X _7, in order to distinguish the percentage of non-functional receptors expressed in the mucosa of the gastrointestinal tract, one specific for P2X ₇ subtype or two antibodies It is also included to use. Thus, the two antibodies can be used together to detect both the total number of receptors and the receptor channel that is present only in a closed or non-functional conformation. The first antibody is adapted to detect the total number of P2X ₇ receptor. A probe comprising or linked to an antibody of the present invention can provide a second antibody that detects IBS, not only distinguishing between functional and non-functional P2X ₇ receptors, Other regions of the receptor that do not change with sex can also be detected. These antibodies may be used separately or simultaneously. Preferably, they are used in combination.

The non-functional P2X ₇ receptors separately sensing a total P2X ₇ receptor determines the severity of the symptoms. Expression of non-functional P2X ₇ receptors in the mucosa of the gastrointestinal portion appears as a pattern in a state in which normal cells are not labeled. Subsequently, the non-functional P2X ₇ (receptor) conformation was first accompanied by a patch invading the epithelium from a partial patch in the subepithelial stroma, with a mild symptom case, and It is detected between the extent of non-functional P2X ₇ receptor expression in a wide range over the entire length of the gastrointestinal tract.

The present invention also provides a method of diagnosing irritable bowel syndrome, the method detects the P2X ₇ expression profile of cells and / or tissues, and compared to a predetermined expression profile of normal cell and / or tissue Including that. Detection of P2X ₇ expression profile preferably comprises the use of one or more antibodies. Moreover, this antibody is different from the probe of the present invention, it is preferable that such does not detect a change for binding to P2X ₇ receptors of ATP. The preparation of such antibodies is obvious to those skilled in the art.

  The present invention includes the use of one or more antibodies to diagnose irritable bowel syndrome.

  The treatment regimen for this condition is described below in connection with the third aspect of the present invention.

  The diagnostic method can be used in conventional immunohistochemical techniques using standard microscopes. Diagnostic methods can also be used in vivo.

  Diagnosis using the probes and methods of the present invention can also be performed using in situ imaging techniques that detect distribution within body tissue. In addition, standard microscopes, confocal microscopes and fluorescent active cell sorting methods may be used. Normal immunohistochemical techniques for examining lymph, prostate, breast, skin, lung, uterus, bladder, vagina, stomach, esophagus, and similar biopsy, and fine needle aspiration of chest and other tissues, Also, cell smears (specimens) such as those collected for the detection of vaginal cancer can be used.

For in-vivo diagnosis, the probe is a human antibody or domain, and preferably produced without containing animal components. The antibody is preferably labeled with a short-lived radioactive label and is preferably detectable by scanning techniques such as positron emission tomography (PET scanner). Such imaging methods can detect aggregates of labeled antibodies everywhere in the body, thus signaling the presence of a non-functional (P2X ₇ ) receptor associated with the presence of a tumor. Ideally, such a test should be done only to check for the presence of a second cancer after the discovery of the first cancer, or a general screen from a blood test (see below) Should be done after discovering the possibility of the presence of one or more tumors.

The probes and methods of the present invention may be applied to provide a non-functional P2X ₇ receptor, ie, a blood test to detect cancer or precursor symptoms of cancer. For example, fluorescently labeled antibody (monoclonal or polyclonal) probes to detect binding of non-functional receptors in a wide variety of gated leukocytes such as T lymphocytes, B lymphocytes, or macrophages In addition, some of the patient's blood cells can be used for flow cytometry.

  In another blood test format, the probe preferably takes the form of a labeled antibody linked to a matrix provided in the kit. By doing in this way, it becomes possible to detect by the presence or absence of the color reaction of the antibody couple | bonded with the positive leukocyte. Such a kit would be suitable for use by a medical practitioner.

In similar blood tests, antibody probes of the present invention, to screen patient as if not cancer missing functionality of P2X ₇ they are in one or more white blood cells normal cell destruction journey is inhibited It can also be used as a diagnostic tool. Such patients, macrophages are not express non-functional P2X ₇ receptors, such macrophages, for example, Mycobacterium tuberculosis, or destroy the infected cells by other infectious organisms, including malaria and HIV This suggests that the function is inhibited. These (infectious) organisms, the missing functions of P2X ₇ is in one or more white blood cells, normal cell destruction journey is suitably grown in such patients is inhibited.

  The probes and methods according to the present invention may be used with other techniques.

The present invention provides an antibody for treating a disease or condition, wherein the antibody distinguishes between a functional P2X ₇ receptor and a non-functional P2X ₇ receptor and is adapted to bind only to a non-functional P2X ₇ receptor. Has been. Preferably, the antibody by detecting the changes associated with binding to receptors of adenosine triphosphate (ATP), or binding of one or more proteins necessary to form pores in the P2X ₇ receptor Preferably, the functional P2X ₇ receptor is discriminated from the non-functional P2X ₇ receptor by detecting the change in and is adapted to bind only to the non-functional P2X ₇ receptor. In another embodiment, the antibody identifies a functional and non-functional P2X ₇ receptors by detecting a portion of the receptor which becomes the dew when bound ATP does not exist.

  The antibody for treating a disease or symptom may be the same as the antibody for diagnosing the disease or symptom. Such antibodies can be used, for example, in the treatment of local skin cancer. For planned treatment of cancer, the antibody or active portion thereof should be of a human or human domain to minimize undesirable side effects of the immune response.

  The antibody of the present invention can be used to treat diseases and symptoms in mammals including humans. Examples of diseases and symptoms that can be applied are as described above in connection with the probes of the present invention.

The present invention also provides an epitope capable of generating the antibody of the second aspect of the present invention. This epitope preferably includes Pro210 and preferably includes a portion of Gly200 to Cys216 (in the P2X ₇ sequence of the human receptor). The epitope is preferably linked to the C-terminal of a Cys residue (Cys216), which Cys residue is diphtheria toxin via a chemical cross-linker called maleimidocaproyl-N-hydroxysuccinimide (MCS). Is cross-linked. By doing so, the conformation of the linked epitope peptide occupies a stable cis proline configuration.

This particular peptide conformation is intended to be given to a human or animal with one or more illnesses or symptoms. Examples of the disease and symptom include epithelial cancer such as prostate, breast, skin, lung, neck, uterus, stomach, esophagus, bladder, colon and vaginal cancer, malignant lymphoma, irritable bowel syndrome and HIV, etc. It is an infection caused by other pathogenic organisms such as viruses and Mycobacterium tuberculosis. Patients causes an immune response by the administration of (above) the composite epitope and generate antibodies that recognize the non-functional P2X ₇ receptors present on infected on cells by doing so, that bind to these infected cells It is preferable that appropriate immune cells are stimulated to destroy complex cells. Other cells that have previously undergone cell death may also be affected.

The sequences referred to above are not intended to limit the scope of the invention, and the invention includes alternative sequences and those that create similar specific immune responses, such as carriers and cross-linkers. Preferably, the immune response reacts only with non-functional P2X ₇ receptors and desirably does not react with all functional P2X ₇ receptors, ie avoids side effects.

In the present invention a second aspect, the aberrant or non-functional P2X ₇ receptors are expressed on the cell surface to regulate programmed cell death by targeting, and normal (functional) expressing receptor There is also provided a method of using the antibody of the present invention as a therapeutic medium for treating a disease or a symptom of a patient without touching the living cells. The invention also includes the use of an epitope of the invention that generates antibodies, as described above.

The present invention also provides a pharmaceutical composition for treating or preventing a disease or condition of a patient. The compositions may contain pharmaceutically effective amount of wherein the antibody or epitope to produce such amounts (antibodies), abnormal or non-functional P2X ₇ receptors were expressed on the surface are cells of the program It is possible to regulate cell death.

  The pharmaceutically effective amount of the antibody or epitope will vary from patient to patient and depending on the nature of the disease or condition. These changing values can be ascertained by one skilled in the art.

  The pharmaceutical composition of the present invention may be applied together with a pharmaceutically acceptable carrier, and such carrier is well known in the art or can be produced in the future and is suitable for the intended use. Either is acceptable. Similar to the carrier, the pharmaceutical composition of the present invention may contain other components such as pigments, preservatives, buffers and antioxidants.

  The pharmaceutical composition of the present invention may be in any form, and may be administered in the form of, for example, a coating, cream, liquid, suspension, powder, tablet, capsule, suppository or vaginal suppository.

  The pharmaceutical compositions of the present invention may be administered in any suitable manner, and may include, for example, oral, parenteral, intravenous, intramuscular, subcutaneous or topical administration.

  The present invention also provides a method for treating or preventing a disease or condition in a patient. This method involves administering to a patient a pharmaceutical composition of the present invention.

  The present invention also provides the use of the pharmaceutical composition of the present invention in the treatment or prevention of a patient's disease or condition.

  It will be apparent to those skilled in the art that the method of use of the pharmaceutical composition of the present invention also needs to be adjusted to obtain the maximum effect. It may be necessary to consider the severity and nature of the disease or condition.

A third aspect of the present invention, for example in patients with malignant lymphoma B- cell in such focus on the expression of ATP platinum loop (enzymes) that controls supply of the P2X ₇ receptor in ATP. Opening of P2X ₇ receptors of leukocytes Kyan'neru is completed by hydrolysis of rapid ATP agonist by ecto--ATP platinum loop and ecto--ATP di phosphotransferase hydrolase (ecto -ATPD platinum loop). These enzymes regulate many biological processes that depend on ATP. The substrate-specific activity of ecto-ATPase and ecto-ATPDase suggests the presence of multiple types including CD39 on the surface of white blood cells. Proliferation of these ecto -ATP platinum loop and ecto -ATPD platinum loop is limited supply of ATP needed to control the pore formation of the P2X _7, followed B- in programmed cell death required for regulating the number of cells May be provided.

Similarly, in the case of IBS, proliferation of ATP platinum loop is believed may be involved in the lack of appropriate binding of the P2X ₇ receptor of ATP agonist.

Accordingly, in this third aspect, the present invention provides a formulation for the treatment or prevention of a disease or condition in a patient. This preparation includes one or more substances adapted to regulate the expression of ATP platinum loop for controlling the supply of ATP to the patient's cells or tissues P2X ₇ receptor in. The present invention also provides a method of treating or preventing patients with the disease or condition, the method is adapted to regulate the expression of ATP platinum loop for controlling the supply of ATP to the patient's cells or tissues P2X ₇ receptor of Administering a formulation comprising one or more substances.

  An example of such an ATPase may be CD39 or CD73.

Such materials may be in the form of analogue of ATP, preferably with hydrolysis disabled, and is specific for P2X _7, or ATP reducing the feed rate of ATP to a binding site of the P2X ₇ platinum loop Other substances that interfere with the local activity of may also be used. Preparations may be in the form of a specifically corresponding human antibody to the non-functional P2X ₇ receptors.

Substances such as ATP analogs bind to P2X _7, maintaining the P2X ₇ to conformation pores open, i.e. forcing a state having a pore of functionality. In this state, it is possible to ingest (P2X ₇ is) of both large and small cation-permeable enzyme. In this way, use of a synthetic agonist of the aforementioned is the restore function of receptors, simultaneously, it is also possible to control the growth brought about by the role as a calcium channel P2X ₇ on cells.

  The disease or condition (to which the present invention is applied) is preferably malignant lymphoma or IBS, but may be other disease or condition including other epithelial cells or blood cancer or viruses and other pathogenic infections.

In the case of malignant lymphoma, ATP platinum loop controls the local supply of ATP to P2X ₇ receptors, this reduces the amount of ATP can be coupled to the P2X ₇ receptor by, inactivating these receptors. This leads to a dramatic reduction in programmed B-cell death. These ATPases are specifically expressed on the B-cell surface and appear to be even more expressed in malignant lymphomas. Application of certain ATP platinum loop inhibitors preferably used to regulate the feed rate of ATP P2X ₇ on receptors to regulate programmed B- cells.

For the treatment of malignant lymphomas, aforementioned substances is a P2X ₇ agonist of hydrolysis not form may include synthetic agonist capable of interfering with ATP platinum loop or ATPD platinum loop.

In connection with irritable bowel syndrome, administration of the formulations of the present invention is intended to restore receptor function that may have been reduced by muscle overactivity under the area representing the mucosal symptoms. ing. The preparation of the present invention may act directly on the mucous membrane to remove non-functional receptors and thereby restore the normal normal digestive tract secretion mechanism. Therapeutic regimen aims to restore the local supply of ATP to the non-functional receptor, thereby restoring normal receptor function. The consequences of controlling receptor function include restoring normal control of gastrointestinal secretion and peristalsis. This specific synthesis agonists P2X _7, preferably, intestinal soluble or agonists which are not hydrolyzed by ATP platinum loop applies a systemic supply, or antibodies directed against non-functional P2X ₇ receptors, preferably, It can be accomplished by systemically administering small specific human antibodies that remove non-functional receptors and leave only functional receptors.

If if abnormal peristalsis under smooth muscle is reduced local feed rate of ATP binding to normal functional P2X ₇ receptors, treatment may, by limiting the movement of the digestive tract It may include restoring this natural agonist supply using methods that limit the uptake or use of ATP by smooth muscle.

The present invention also provides a pharmaceutical composition for the treatment of a disease or condition, which composition is adapted to regulate the expression of ATPase (enzyme) that controls the supply of ATP to the P2X ₇ receptor. A pharmaceutically effective amount of one or more substances.

In the present invention all aspects, the virus to non-functional P2X ₇ receptors in cells infected with the virus are expressed more is protected, or such even normal cell status ( non-functional), such as receptor is increased expression, aberrant P2X ₇ receptor posed by viral infection extends to be applied similarly to other pathological conditions involving.

  The present invention also provides a method of treating irritable bowel syndrome, the method comprising administering to a patient a pharmaceutical composition as defined above.

  The present invention also provides the use of such pharmaceutical compositions in the treatment of irritable bowel syndrome.

  The pattern of using one or more pharmaceutically effective compositions described above may have to be changed as appropriate to obtain the best effect.

If expressed in another form, the present invention provides a method of treating irritable bowel syndrome, the method includes administering a composition adapted to restore P2X ₇ receptor function. The function of the receptor may be depleted by overactivity of the muscles underneath the areas of the mucous membrane that are becoming sensitive. The composition may be the same as the substance contained in the preparation of the present invention described above.

  From a further aspect, the present invention provides a method for distinguishing different conformations of a protein. This method uses specific epitopes (active / passive immunization) by binding to all aggregates of proteins with a selected conformation using epitopes that can generate antibodies, or antibodies themselves. Is done by An example of this is the prion protein conformation that leads to vCJD symptoms. Abnormal forms of the protein can be targeted by specific antibodies or epitopes that produce antibodies. (Epitope) is preferably human and of small size for best pharmaceutical effect.

(Brief description of the figure)
FIG. 1 (SEQ ID NO: 1) shows the amino acid sequence of the human P2X7 receptor (prior art). Arrays 65 to 81 and 200 to 216 are highlighted and are referenced below.

(Detailed Description of the Invention)
To generate an antibody corresponding to the non-functional P2X7 receptor, the epitope of sequences 200 to 216, including Cys in sequence 216, shown in FIG. 1 (SEQ ID NO: 1) was used.

To generate an antibody corresponding to the promiscuous P2X7 receptor, the epitope of sequences 65 to 81 shown in FIG. 1 (SEQ ID NO: 1) was used, to which was added the N-terminal Cys. This antibody cannot be discriminated whether the receptor is non-functional, but is designed to detect all (P2X7) receptors and provides staining obtained using two different antibodies separately. By comparison, the proportion of receptors that are functional can be determined.

  In order to maintain conformational stability and provide a larger antigenic structure, the Cys residue of the epitope is a maleimide caproyl to a diphtheria toxin (DT) carrier with 10 peptide epitopes attached to each DT carrier. Conjugated via -N-hydroxysuccinimide (MCS) crosslinker. These complex epitopes were injected as antigens into several animals (sheep, rabbits and mice) in order to generate antibodies specific for the epitope.

  The process of generating antibodies by use of antigen / adjuvant mixtures injected into animals at specific times is described in the well-known art. More specific examples of generating antibodies are shown below.

Example 1
<Sheep anti-P2X ₇ antibody>
Complex of 500 [mu] g (P2X ₇ epitopes of about 100 [mu] g) are diluted to 0.8mL in phosphate buffered saline (PBS), and emulsified in Freund's complete adjuvant of 1.2 mL. The sheep were injected with the antigen / adjuvant emulsion at multiple sites, both subcutaneously and muscle. Eight weeks later, the same sheep were injected with the same amount of the emulsified complex in multiple places with Freund's incomplete adjuvant. This operation was repeated 4 weeks later and blood was collected from the jugular vein. Collected sera were tested for antibody specificity. To provide a pool of serum containing specific antibodies, the sheep were infused and bled regularly at 8 week intervals.

  Other sheep were injected with the same amount of conjugated antibody similar to the schedule above, but different adjuvants were used. For these animals, 0.7 mL of diluted antigen was applied to 0.1 mL of Quill A / DEAE Dextran solution (2.5 mg Quill A / mL PBS + 25 mg Of DEAE Dextran and 1.2 mL of ISA50V Montanide. This emulsion was injected at multiple sites both subcutaneously and muscle. Antibodies obtained using this adjuvant produced the same specificity as antibodies made using Freund's adjuvant.

(Example 2)
<Rabbit anti-P2X ₇ antibody>
In rabbits, antibodies were generated using two adjuvants similar to sheep and an injection schedule. The only difference was that 300 μg of the complex was injected. The antibody produced had the same specificity as that produced in sheep and could reliably identify the corresponding epitope.

(Example 3)
<Anti-P2X ₇ antibody of a mouse>
Identifying in mice and antibodies to complex epitopes, antibodies against complex and non epitopes nonfunctional P2X ₇ epitopes were generated (which can not be formed pores, i.e. the receptor which can not cause apoptosis Is possible).

In these experiments, ImmunEase ^™ , a product of QAIGEN Pty Ltd, containing the immunostimulatory product CpG DNA (registered trademark of Coley Pharmaceutical Group Inc.) was used.

5 μg of epitope or complex epitope was diluted with 70 μL PBS and 30 μL ImmunEasy ^™ adjuvant. Mice were injected subcutaneously and into multiple sites. This was done again after 2 weeks and again after another 2 weeks. Mice were bled 8 weeks after the third injection. Antibodies generated in mice by this method, it is possible to identify the different P2X ₇ epitopes and antibodies corresponding to P2X ₇ non-functional epitope also gave the same result as antibodies produced in sheep and rabbits.

As the above examples show, antibodies against various epitopes of the P2X ₇ receptor can be consistently generated using different species and different adjuvants. In particular, antibodies against P2X ₇ receptor epitopes that identify non-functional P2X ₇ receptors that cannot form pores under normal physiological conditions and cannot function in apoptosis are also routinely generated. be able to.

Example 4
Antibody to detect the non-functional P2X ₇ is tested by binding the antibody to cells P2X ₇ is expressing P2X 7 _(human) having a known function, represented by the ability to exploit the ethidium or rubidium It was done. These P2X ₇ protein channels Shirezu be mutated in base pairs 1513, channel if it does not form pores that cause apoptosis. These and similar non-functional P2X ₇ receptors expressed on malignant B leukocytes also bind antibodies in flow cytometry and normal immunohistochemistry, while normal functional P2X ₇ (squeezing large amounts of calcium, ethidium and rubidium Could not bind to this antibody. This is because the functional receptor did not have the epitope chosen to detect the non-functional receptor. Pro210 adopted a cis conformation at the non-functional receptor, and it was this conformation in particular that was fixed in the complex epitope used to generate the antibody. In receptors that appear functional, Pro210 has a trans conformation. This was a result of the binding to the P2X ₇ receptor of ATP (adenosine triphosphate). When ATP was bound, the portion of Pro210 directly adjacent to the ATP binding site took the trans configuration.

  This was confirmed using site-directed mutagenesis that caused Pro210 to be changed to Ala (alanine) fixed in the trans configuration. This mutated protein was found to be fully functional and could not bind to the antibody produced to detect the non-functional receptor.

(Example 5)
Further evidence for the specificity of the antibody to detect the non-functional receptors were obtained in experiments labeled macrophages expressing P2X _7. Macrophages bound to the antibody When using common antibody to all P2X _7, but the antibodies directed against non-functional P2X _7, did not bind to contact with cancer cells as mouse hybridoma cells. Contact between macrophages and hybridoma cells caused expression of non-functional P2X ₇ on macrophages, which was detected by antibodies to non-functional P2X ₇ as well as antibodies common to all P2X ₇ .

Macrophages and extracted from patients with malignant lymphoma B- cell leukocytes were tested, all of these cells common antibodies similar to all P2X _7, bound to the antibody to non-functional P2X _7. This is non-functional in all cancer cells in which P2X ₇ has been detected, and pores leading to the apoptosis formed by functional P2X ₇ cannot be formed, thus causing apoptosis in cancer cells. It proves that it can not be done.

All epithelial cell carcinomas in humans such as prostate, breast, bladder, skin, stomach, neck and other parts, and malignant lymphoma, chronic lymphocyte leukemia and brain tumor, and tested dog milk and prostate, cat skin, mouse all the same tumors in other mammals, including hybridoma cells and mouse fibrosarcoma cells express the same non-functional P2X _7. The sequence similarity of the epitopes selected for humans, rats, cats, dogs and mice is sufficient to make an undeniable identification in all of the above cases. This is the same cancer mechanism in these mammals, and all cancer cells express a non-functional P2X ₇ receptor that is unable to form pores that when activated activate normal cells that die. That is. In this way, apoptosis is switched off and the cancer cells become immortal.

(Example 6)
Cancer cells, such as infected B- Serurinpa balls as further evidence of the inability to cause apoptosis by P2X ₇ function, B cells of leukemia patients have the non-functional P2X ₇ receptors are in cultures 2 Incubated with 5 mM ATP for hours. The result was that all non-functional receptors were opened by excess ATP, causing apoptosis and killing infected cells.

(Example 7)
As further evidence that the antibody selectively binds to the cancer cells, the patient's skin with basal cell carcinoma (BCC) is, by antibodies against non-functional P2X ₇ receptors, suspended in an inert cream underground Processed and applied to the lesion and its surroundings (see Example 10 below). Within one week of applying topical antibody every day, all symptoms of BCC disappeared and normal skin did not have a (non-functional) receptor, so the surrounding skin was not affected at all.

(Application to diagnosis)
Animals with specific non-functional P2X ₇ antibody, and a description of the common application of the probes and methods of the present invention to diagnosis of most cancers in humans and other mammals, described by way of example.

  In mammalian and human prostate tissue, such as cats and dogs, when the antibodies of the invention are used for diagnosis, the label will not be obtained without a cancerous or precancerous disorder. However, the diagnostic method of the present invention can reveal the first signs of neoplastic changes without the appearance of morphological changes associated with the tumor that can be detected by H & E staining.

  At this point, it is necessary to stain the first receptor unit that appears in the epithelial cell nucleus. These move to the cytoplasm at a later stage of the disease and have a field effect throughout the prostate, so it is sufficient to biopsy a smaller amount of tissue to determine that the tumor is present. In the second half of the disease, staining is more restricted to the epithelial apex.

  Similarly, other epithelial cell carcinomas such as breast, lung, colon and skin in humans and other mammals such as cats and dogs can be detected with sufficient margin because there is no longer a clear field effect in these other tissues. can do.

  Progression of the same stage as above is seen in these other tissues such as milk and vagina, and there is staining of cell nuclei prior to cytoplasmic staining, and normal tissues are not stained. Tubes and leaflets of infected milk tissue are easily detected due to local field effects within each infected tube system, even when the normal morphology indicates that there is no cancer. Adjacent uninfected tubes are not stained. Similarly, infected lymph glands that directly drain the tissue containing the tumor also show a tumor signature due to the field effect of the infected lymphocytes. Therefore, the sentinel lymph node can be detected without any cancer metastasis to the cell to the (lymph) node.

  Skin cancers such as basal cell carcinoma, squamous cell carcinoma, dysplasia, and malignant melanoma are well beyond the non-functional receptors and normal skin that is not labeled in both the epidermis and deep dermis. Positive for staining of channel components (monomers) in keratinocytes and melanocytes.

All mammalian cancer cell lines tested, such as human prostate (PC3) and milk (MCF7) and rodent hybridomas, are positive for cell surface non-functional receptors, and thus apoptosis in these cancer cells It is suppressed. A common application of this diagnosis is that it shows the universal nature of this receptor in other animal species than humans through the same label in mouse hybridoma cells. Normal human B- Serurinpa sphere are shown to express functional P2X ₇ receptors on the cell surface, thus when needed but apoptosis occurs in patients with malignant lymphoma human B- Serurinpa sphere Non functional P2X ₇ receptors have shown that is expressed on the cell surface and thus apoptosis is hindered.

(Application to treatment)
Expressed on the surface of cancerous cells, it attempts to generate this obviously apoptosis universal P2X ₇ non-functional type as the target can be used to treat most cancers in humans and other mammals. Examples are given below:

(Example 8)
Based on macrophages mouse hybridoma cells, avidity for non-functional P2X ₇ was grown in the absence and condition antibodies reinforced exists. During 4 days, the number of cells increased from 1 × 10 ⁴ to 7 × 10 ⁴ for cells incubated with purified normal IgG, and the number of cells by incubating with non-functional P2X ₇ antibody The number remained at 1.5 × 10 ⁴ .

Example 9
This example shows that antibodies generated in response to non-functional epitopes of the P2X ₇ receptor can suppress tumor formation in vivo.

As indicated above, the antibody corresponding to the non-functional P2X ₇ epitope produced in sheep recognized the non-functional P2X ₇ apoptosis receptor on the surface of this mouse hybridoma cell. Addition of this antibody to hybridoma cell cultures slowed cell growth. Mouse hybridoma cells form tumors when injected into prepared inbred mouse strains.

In this experiment, 10 Balb-c female mice in each of the 3 groups each received the following treatment:
Group 1: On day one, 10 mice were injected (IP) into peritoneal tissue in 0.5 mL cell culture medium each containing 1 × 10 ⁶ hybridoma cells. On days 2 and 3, the mice were injected with 0.5 mL of cell culture medium into the peritoneal tissue.
Group 2: On day 1, 10 mice each were injected (IP) into 0.5 mL of cell culture medium containing 1 mg of sheep IgG and in the presence of 1 × 10 ⁶ hybridoma cells. On days 2 and 3, the mice were injected into peritoneal tissue with 0.5 mL of cell culture medium containing 1 mg of purified sheep IgG.
Group 3: day 1, contained 10 mice anti P2X ₇ non-functional epitope IgG sheep of purified 1mg respectively, through the 0.5mL of the cell culture medium 1 × 10 ⁶ hybridoma cells are present Peritoneal tissue was injected (IP). On days 2 and 3, the mice were injected into peritoneal tissue with 0.5 mL of cell culture medium containing 1 mg of purified sheep anti-P2X ₇ IgG.
All groups of mice were killed on day 11 and examined for the presence of tumors. Tumors were removed and surveyed.
The results are as follows:

A random analysis showed a significant difference in tumor weight between groups (probability P <0.01). Experimental group that received treatment with anti-P2X ₇ non-functional antibodies was significantly different as compared to two control groups (P <0.01). That is, treatment with antibodies against P2X ₇ non-functional epitope is that significantly reduced the amount of tumor in the experimental animals.

(Example 10)
Purified antibodies with specific affinity (to greatly improve specificity) administered to 3 human basal cell carcinomas ("BCC") either in liquid state for 7 days or floated in dimethicone cream base It was done. After treatment, there was no evidence of BCC pathology and the control skin was not affected at all due to the absence of the target protein.

(Example 11)
Skin damage in the form of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) (both primary and secondary tumors), including recurrent tumors and dysplasias, has been purified in further studies. And either purified or regular IgG were treated using a mixture in a dimethicone cream base or an osmotic cream base. Since non-functional receptors are not present in normal skin, no side effects were detected in any normal skin. All types of cancer responded to the presence of antibody and disappeared in a period of 36 hours to 1 week with twice daily treatment. There was no recurrence for 12 months. The size of the treated tumors ranged from 3 mm in diameter and not raised to 5 cm in diameter and 4 mm in thickness. A total of 34 pathologically qualified tumors were successfully removed within a treatment period of less than a week.

Treatment to patients generally involves the formation of human monoclonal antibodies (such as herceptin), so internal cancers can be treated as well as the efficacy obtained with local treatments. it is conceivable that. To avoid side effects, normal functional P2X are expressed on the cell surface, such as a lymphocyte ₇ should not all subject to any action. Thus, the antibody should bind only to proteins expressed on the cell surface that have been attempted but cannot initiate apoptosis. In other words, all of the target cells, including cancer cells, are only cells that are trying to self-destruct by programmed cell death. P2X ₇ receptors of these cells, particularly cancer cells, would be in the state or ATP is nonfunctional is lacking.

(Active immunization)
Active immunization can also be used for therapeutic purposes. In this case, a human or other mammal is immunized against the epitope (s) specific to a conformation similar to that taken only by non-functional (ATP-deficient) forms of receptors on the cell surface. Need to be done. Conformational flexibility including partial exposure of the epitope form present in the functional receptor should be avoided. For example, the cis configuration of the epitope Gly200-Cys216 should be fixed before being used by an appropriate method. Additional evidence that this idea is valid is the observation that many animals, including mice, rabbits and sheep used to generate antibodies, have not compromised the immune system. Many of these animals did not develop any tumor.

The following specific examples illustrate this:
(Example 12)
Protocol: The experiment was based on a mouse tumor model. Forty female 10-week-old female inbred Balb C mice were used and divided into two groups of 20 animals each. Group 1 is for experiments and group 2 is a control group.

Day 1: Twenty experimental animals from group 1 were injected with 0.1 mg peptide epitope (hP2X ₇ sequence 200-216) conjugated to diphtheria toxin via MCS crosslinker. This contained about 0.02 mg of peptide epitope. The peptide conjugate was emulsified with QUILL A / DEAE Dextran / Montanide ISA 50V adjuvant mix and injected into 0.1 mL subcutaneous and multiple sites of muscle.

  Twenty mice from the control group, Group 2, were injected with 0.1 mL of the adjuvant mix without peptide conjugate subcutaneously and at multiple sites in the muscle.

Day 8: 20 mice from group 1 were injected with 0.01 mg peptide epitope (hP2X ₇ sequence 200-216) conjugated to diphtheria toxin via MCS crosslinker (approximately 0.002 mg peptide Including epitopes). The peptide was contained in a phosphate buffered saline solution and was mixed with the commercially available CpG DNA adjuvant ImmunoEasy (sold by Qiagen) according to the protocol. Each mouse was injected with 0.1 mL of peptide conjugate / adjuvant solution subcutaneously and at multiple sites in the muscle.

  Twenty group 2 mice were injected with the equivalent of phosphate buffered saline / CpG DNA adjuvant mix. This was injected into each mouse at 0.1 mL subcutaneous and multiple locations in the muscle.

Day 26: 20 mice from group 1 were injected with 0.025 mg peptide epitope (hP2X ₇ sequence 200-216) conjugated to diphtheria toxin via MCS crosslinker (approximately 0.005 mg peptide Including epitopes). This was contained in a phosphate buffered saline solution and mixed with Qiagen CpG DNA adjuvant ImmunEasy. Again, 0.1 mL of this mixture was injected into each mouse subcutaneously and at multiple locations in the muscle. The control group was injected as on day 8.

  Day 29: All mice were injected with tumor cells contained in 0.1 mL tissue culture medium into one subcutaneous site located behind the neck. The tumor cells used were the mouse hybridoma cell line designated cell line WEHI164 and made at the Walter and Eliza Hall Institute in Melbourne, Australia.

  (Tumor) cells were injected in both experimental and control group mice at two concentrations. Each group was further divided into two. Ten mice in each experimental and control group were injected with 160,000 cells per mouse, and 10 mice in each group were injected with 320,000 cells.

The cells of the cell line, previously had been tested for the presence of non-functional P2X ₇ epitope on the cell surface. This was done using an antibody specifically recognized by the sheep that produced a non-functional form of the receptor.

Day 38: All mice were killed, blood for analysis of antibodies to non-functional P2X ₇ epitope was collected. All mice were surveyed and tumors were excised and surveyed.

  A random analysis of the results showed statistically significant differences between the control and treated (experimental) groups, and between the low dose and high dose groups (P = 0.0003). ). The low dose group showed a greater difference because the small amount of tumor had less impact on the immune system's ability to counteract the mouse.

(ATP analog)
Synthetic agonists are used to efficiently bind to the ATP binding site of P2X ₇ pores, making the pores functional, thereby restoring the function of the receptor, and at the same time the advantages of P2X ₇ on the cell The effect of regulating is shown in the following culture experiment. Neoplastic B-cells were collected from patients with CLL, mixed with an equal number of similar cells from normal patients, and treated with 2.5 mM ATP for 4 hours. No tumor cells remained and only normal cells remained. In vivo (in vivo), benzene is ATP or more selective P2X ₇ agonist, the use of benzene ATP is not appropriate. Thus, the selective ATP analogs that can selectively bind to P2X ₇ with much stronger binding force than ATP or BzATP, may be designed to reverse the process of apoptosis within the infected tumor cell types.

(Industrial applicability)
The invention is applicable in all aspects to the fields of human and veterinary medicine and health, has the potential to enable early and accurate diagnosis of disease, and in many cases is much less invasive than the prior art or trauma It will not be.

It shows the sequence of the human P2X ₇ receptor.

Claims (26)

An isolated P2X ₇ receptor having the amino acid sequence shown in SEQ ID NO: 1, wherein the proline at position 210 is in cis conformation.   Use of a peptide having residues 200 to 216, including a proline at position 210 in the cis conformation of the amino acid sequence of the receptor according to claim 1 in the manufacture of a vaccine for the prevention or treatment of a disease or condition.   A vaccine for the prevention or treatment of a disease or symptom comprising a peptide having residues 200 to 216 including a proline at position 210 in the cis conformation of the amino acid sequence of the receptor according to claim 1.   4. The vaccine of claim 3, wherein the peptide is conjugated to a carrier.   The vaccine according to claim 4, wherein the carrier is diphtheria toxin.   The vaccine according to any one of claims 3 to 5, further comprising an adjuvant.   7. The vaccine of claim 6, wherein the adjuvant comprises quill A / DEAE dextran / montanide.   Use of a vaccine according to any one of claims 3 to 7 in the manufacture of a medicament for treating or preventing a disease or condition in a patient.   Use according to claim 8, wherein the disease is cancer.   10. Use according to claim 9, wherein the cancer is selected from the group consisting of prostate, breast, skin, lung, cervix, uterus, stomach, esophagus, bladder, colon, vagina, and blood cancer, and malignant lymphoma. . Sheep for binding to P2X ₇ receptor having the amino acid sequence shown in SEQ ID NO: 1, rabbit, an antibody of a mouse or rat, in the case 2 10-position of proline Ru system controller conformation near, including 210-position proline the binding to an extracellular region of Li Scepter, when 210-position of the proline Ru trans conformation near, not bound to the fine extracellular region, an antibody. If 210-position of the proline Ru system controller conformation near, SEQ ID NO P2X ₇ binding to an extracellular region of the receptor having the amino acid sequence shown in, position 210 proline trans conformation near containing position 210 proline that if, in the manufacture of the antibodies that do not bind to the fine extracellular region, compositions prepared for application to the skin for the prevention or treatment of a disease or condition. If 210-position of the proline Ru system controller conformation near, SEQ ID NO P2X ₇ binding to an extracellular region of the receptor having the amino acid sequence shown in, position 210 proline trans conformation near containing position 210 proline case, containing antibodies which do not bind to the fine extracellular region, compositions prepared for application to the skin that.   14. The composition of claim 13, wherein the antibody is a sheep antibody.   15. The composition of claim 13 or 14, further comprising a compound that promotes penetration of the antibody into the skin.   The composition according to any one of claims 13 to 15, further comprising a compound for moisturizing the skin.   17. A composition according to claim 16, wherein the skin moisturizing compound is dimethicone.   18. A composition according to any one of claims 13 to 17, wherein the composition is prepared as a cream, lotion, ointment, gel, aerosol or spray.   Use of a composition according to any one of claims 13 to 18 in the manufacture of a medicament for treating or preventing a disease or condition in a patient.   20. Use according to claim 19, wherein the disease or condition is skin cancer.   21. Use according to claim 20, wherein the skin cancer is selected from the group consisting of basal cell carcinoma, squamous cell carcinoma, malignant melanoma, and dysplasia. If 210-position of the proline Ru system controller conformation near, SEQ ID NO P2X ₇ binding to an extracellular region of the receptor having the amino acid sequence shown in, position 210 proline trans conformation near containing position 210 proline case, use of the antibodies that do not bind to the fine extracellular region, the preparation of suitable reagents for detecting the disease or condition by in vivo imaging techniques that.   23. Use according to claim 22, wherein the in vivo imaging technique is positron emission tomography. When the 210-position of the proline is in the system controller conformation, Ru SEQ ID NO P2X ₇ binding to an extracellular region of the receptor having the amino acid sequence shown in, position 210 proline trans conformation near containing position 210 proline case, an antibody that does not bind to the fine extracellular region, contacting the collected cells or tissue from the patient; and the cell or tissue, comprising the step of determining whether bound by the antibody, An in vitro method for detecting whether a patient has a disease or symptom.   25. The in vitro method of claim 24, wherein the disease or condition is cancer.   26. The in vitro method of claim 25, wherein the cancer is selected from prostate, breast, skin, lung, cervix, uterus, stomach, esophagus, bladder, colon, vagina, and blood cancer, and malignant lymphoma. .

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