NZ565994A - Antibodies to non-functional P2X7 receptor, diagnosis and treatment of cancers and other conditions - Google Patents

Abstract

Disclosed is an isolated P2X7 receptor having an amino acid sequence that has at least about 70% identity to the sequence shown in Figure 1, wherein the amino acid sequence of the isolated P2X7 receptor contains a proline corresponding to proline 210 shown in Figure 1, said proline being in a cis conformation. Also disclosed is the use of said receptor for the prevention or treatment of cancer. (62) Divided Out of 549019

Description

RECEIVED at IPONZ on 21 December 2009 - .* " 4* 565994 Patent No. 5 - Complete Specification No. 565994 Date: 3 September 2002 Antibodies to non-functional P2X7 receptor, diagnosis and treatment of cancers and other conditions We, Intreat Pty Limited, of Level 10, 26 O'Connell Street, Sydney, New South Wales 2000, Australia, hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement INTELLECTUAL PROPERTY OFFICE OF N.Z. 2 t DEC M RECEIVED Freehills l\3660331 Page 1 16/02/2008 16:5® feehl I Is 12346S78 7/38 *10055383203* 56 5 9 U I Antibodies to non-functional P2X? receptor, diagnosis and treatihent of cancers and other conditions This application is a di visional of New Zealand Patent Application No. 532054.

TECHNICAL FIELD This invention concerns diagnosis and treatment of diseases, inchidmg cancers.

The types of diseases with which this invention is concerned include cancers derived from epithelial cells sad malignant lymphoma. The invention also concerns other conditions, such as preneoplastic states, irritable bowel syndrome and viral tad ether infections. It is quite possible that the invention is also applicable toother diseases and conditions, BACKGROUND Adenosine triphosphate (ATP) can activate iigand-gated purinergic receptors known as P2X receptors. Receptor subtypes P2Xj to P2X? have been identified. It is toovm 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 ceil via a P2X receptor can be associated wittLcoH death.

It is believed that the P2X? subtype is involved in apoptosis, or programmed cell death, in many cell types. In the presence of ATP, the P2X7 receptor expressed on the surface of a cell is capable, within a second, of opening calcium channels through the cell membrane. 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; receptors are known, for example, from US patent No. 6,133,434 (Buell et al). Refer also to Figure 1 herein.

IPONZ 15 FEB 2008 /02/2008 15:68 565994 fresh Ills 1 £34-5678 B/3S 2 Exposure to ATP does not generally result in apoptosis in the ease of epithelial cancer cells, for example. It has been-found that such cells express P2X7 receptors that are unable to form pores. These arc rogaided as non-ftnctioaal receptors.

In human cancer cell lines, such as prostate PC3 sod breast MCF7, &s well as in animal cell lines including rodent hybridomas, the P2X7 receptor is found on the cell surface in a non-functional conformation.

The B-cells of patients with malignant lymphoma express non-functional F2X7 receptors. Lymphoma develops from malignant clones that escape cytolytic destruction. This process leads to the progressive aooimulation of malignant B-lymphocytes and thus lymphadenopathy and/or splenomegaly.

SUMMARY OF THE INVENTION In a first aspect, this invention provides a probe for detection of a disease or condition, the probe being adapted to distinguish between functional P2X, receptors and noa-functional P2X, receptors. Preferably, the probe distinguishes between functional and non-functional P2X, 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 P2X7 receptors. In an alternate embodiment, fee probe detects me or more parts of the P2X7 receptor exposed in the absence of bound ATP. Such receptor part may include a P2X7 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 P2X7 receptors and non-functional ?2Xy 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 io connection with the probe itself. 1 5/02/2008 1 ft:5 6 f r e eh I i Is 12345678 9/39 565994 3 The probe may be natural or artificial. Preferably, the probe is an antibody, which may be polyclonal, monoclonal, recombinant, a hintMmi«d 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. In the case of human P2X7 receptors, 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 reccptors having a sequence in which the proline at amino acid 210 is in the as conformation that acts to impart a significant alteration in the local protein structm*.

The probe may be prepared using any suitable technique, as will be readily apparent to one skilled in the art It is within the scope of the invention that the probe may distinguish between functional and non-functional receptors through detection of other conformational changes occurring at a site for binding ATP. For example, the change detected may be in an amino acid other than the proline referred to above. An example of such an amino acid is Pro 199 which, when in the cis conformation, significantly alters the local protein structure. As another example, the change detected may be in some other respect.

The probe may also be adapted to detect other regions of the F2XT receptor unchanged by functional state. The conformation of the monomelic 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. Non-limiting examples of such proteins are laminin, integrin, beta-actin, alpha-actinin and supervilliii. is the present invention, a P2X, subtypc-specific antibody can be used to specifically detect or bind to non-fiinctiooal P2X, reccptors expressed in or on cells /02/200 B 18:56 •f reehl lis 123+5878 10/39 565994 4 fanning pan of preneoplastic tissue, very early neoplastic tissue, advanced neoplastic tissue and on any neoplastic cell expressing non-functional P2X? receptors. Thus, the P2X? 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.

Further, the conformation of the monomelic submits lacking bound ATP is also detectable with the antibody, because the epitope chosen specifically detects the shape of a region of the surface accessiblc only when ATP is not bound.

In the present invention, die nonfunctional F2X? 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 . hem found that the amino acid sequence of the noo-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 tons. 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.

In the case of human P2X, receptors, the specific sequence involved in the conformational change may include Pro210, which undergoes a fth&ngg in conformation ftom the trans form to the cis form in the absence of bound ATP-Thus, in the case of human receptors, an appropriate epitope sequence against which an antibody must be raised may include Pro2l0, and may extend either side /02/2008 16:57 freehills 12345678 11/S9 565994 of this residue, to an appropriate extent necessary to induce an antibody response. By way of non-limiting example, this may include a segment extending from -Gly200 to Cys216. Further, a homologous segment from other mammals, such as rat, may be used where this cross-reacts with human tissue. As an example, the same segment Gly2Q0 to Cys2l6 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).

In the case of non-human reccptors, the specific sequence may he ascertained by suitable experiment The detection of non-functional P2X7 receptors according to the invention may show a distribution pattern in which functional receptors (and hence normal cells) may remain essentially unlabelled However, non-functional conformations of P2X7 receptors may he detected, initially in the nuclei and cytoplasm of cdls, at a very early stage in 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 haematoxyiin and eosin {"H & E") stained slides of biopsied tissues. Thus, cancers such as prostate, skiq and breast may be detected far earlier than is currently die -case, with the advantages of introduction of early therapy.

The full scope of the diseases and conditions which may be detected by the probe and method of the invention has not yet been ascertained. However, it is believed that these include epithelial cell cancers, such as prostate, breast, skin, lung, cervix, uterus, stomach, oesophagus, bladder, colon and vaginal cancers, as well as 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 /02/2008 18:57 565994 freehl I Is 12345678 12/39 6 up-iegulatkm of co-factors acting to inhibit P2X7 function on epithelial or other cells, so rendering the infected cell less amenable to destruction by apoptosis.

Unless otherwise indicated, the term "disease or condition" as used herein is intended to include all those specific diseases and conditions set out in the In the specific case of babble bowel syndromes HBS")» it has now been found that, is patients with Ms condition, the gut mucosa, tot normally expresses P2X7 receptors to the widely distributed lymphocytes present in the stroma beneath the can be observed from duodenum to rectal mucosa. The increased expression may be found in isolated regions, or to be generally increased over the entire length of the intestinal tract in more extreme cases.

In the least affected cases, total P2X, receptors are up-regulated, but these are all functional and they do not penetrate into the epithelium. In more severe cases, total P2X-> 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 penelxate into the epithelium. The most severe cases are those in which total P2X? receptor expression is further increased and most of the receptors are non-functional with increased epithelial cell penetration.

As already discussed, non-functionaiity of P2X7 receptors is caused by lack of appropriate binding of the ATP agonist to the receptors. The reasons for this may include a deficit in die local availability of ATP through production deficit or increase in rats of degradation through ecto-ATPase enzymatic degradation of ATP. If ATP binding to the receptors is disrupted, the recq>tor conformation is altered as already discussed, and this can be detected using the probe of the invention. However, the detection of total P2X-, receptor distribution is best achieved using an epitope to other regions of the extracellular domain of the P2X7 rcceptor that is not affected by ATP binding. The probe may be capable of /0Z/20Q8 15:57 freehllls 12345878 19/39 565994 7 detecting regions of &e P2X? receptor unchanged by functional state, by detecting an epitope common to both functional and non-functional conformations, such as Val65-Lys81.

It is within the scope of this invention to use one or two P2X7 subtype-specific antibodies to specifically distinguish between total P2X3 distribution and the proportion of receptors that arc non-functional and expressed in gut mucosa. Thus the two antibodies used together can detect both total receptor count and those receptor channelspresent only in a close-gated or noor&nctjonalconformation. The first antibody is adapted to detect total P2X, 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 fractional and nonfunctional P2X7 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.

The detection of allP2X, receptors, separately from non-functional P2X? receptors, determines the severity of the condition. Expression of non-functional P2X? receptors in the gastrointestinal mucosa occurs in a pattern in which normal cells remain essentially unlabelled. Thereafter, the non-functional conformation of P2X, is first detected in the stroma underneath the epithelium ranging from isolated patches in mild cases of the syndrome to extensive expression throughout die length of the gastrointestinal tract with isolated patches of infiltration of nonfunctional receptors into the epitheiium.

The invention also provides a method of diagnosing irritable bowel syndrome, comprising detecting the P2X7 expression profile of cells and/or tissue and comparing the profile with a predetermined expression profile of normal cells and/or tissue. Preferably, the detection of the P2X7 expression profile includes use of one or more antibodies. Further, it is preferred that such antibody or antibodies are different from the probe of the invention in that they do not detect change in 565994 8 relation to binding of ATP to the P2Xi receptors. Hw preparation of such antibodies wiB be readily apparent to one skilled in the art The invention also includes use of one or more antibodies to diagnose irritable bowel syndrome.

Therapeutic treatment for this condition is discussed below, in connection with the third aspect of this invention.

The diagnostic can be used in standard microscopy employing standard immuoohistochemical 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. In addition, standard microscopy, confocal microscopy and fluorescence activated cell sorting may be used. Normal immunohistocheznical techniques for testing lymph, prostate, breast, skin, iung, uterus, bladder, cervix, stomach, oesophagus and similar biopsies, also fine needle aspirates of breast and other tissue and ceil smears such as those taken for the detection of cervical cancer, may be used.

For in vivo diagnosis, it is preferred that 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). 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. Ideally, 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, receptors and hence cancer or precancerous /0Z/2C08 ,S:57 t re«h I I Is 1 2345678 15/39 565994 9 conditions. By my of wimple, the probe m the fonn of a fluorescent labelled antibody (monockmal or polyclonal) 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. in another form of blood test, the probe preferably takes fee form of & 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. Such a kit may be suitable for use by medical practitioners.

In a similar blood test, 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 tailing pathways are inhibited through lack of function in P2X7 on one or more leukocytes. Such patients may express non-fimctional 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 patients fox whom tbe normal cell killing pathways are inhibited through lack of function in P2X7 on one or more leukocytes.

Other techniques may be used with the probe and method of the invention.

This invention provides an antibody for treating a disease or condition, the antibody being adapted to distinguish between functional P2X7 receptors and nonfunctional P2X7 receptors and being adapted to bind only to non-functional receptors. Preferably, the antibody distinguishes between fee 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 P2X7 receptors and being adapted to bind only to noa-functional receptors. In another embodiment, the antibody /02/2008 16:57 freehltls 12J+5S78 18/39 565994 w distinguishes between the functional and non-functional receptors by detecting The antibody for treating diseases and conditions may be fee 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. For systemic treatment of cancer, the antibody or its activc fragments should be human or a toimsm domain, in order to minimise undesirable immune response side effects.

The antibody of the invention may be used to treat diseases or conditions in mqpfmoU, 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? sequence of the human receptor). The epitope should preferably have attached to the C-terminal end a Cys residue (Cys2!6) that is cross-linked to diphtheria toxin via the chemical cross-linker maleimidocaproyl-N-hydroxysuccinimide {MCS), so that the conformauon aciopicd 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 welt 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 P2Xr receptors present on the surface of the affected cells, thus binding to them and alerting the appropriate immune cell to destroy the cotnpiexed cells. Other cells primed for cell death may also be affected. /02/2008 18:57 565994 •f'reehl I Is 12345678 1 7/39 11 It is to be understood that the sequence referred to above is not limiting on the scope of the invention, which includes alternate sequences and carriers and cross- • linkeES that similarly produce a specific immune response, preferably against only nonfunctional P2X7 receptors, preferably ignoring all ftmctbnal receptors expressed on cell surfaces, and so avoiding side effects.

The invention, in this second aspect, also provides for fee 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, receptors expressed on the surface of cells, while leaving 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, die composition including a pharmaceutically effective amount of an antibody, or an epitope to cause die generation of such an amount, capable or regulating programmed cell death of cells having expressed on their surface aberrant or non-functional P2X7 receptors.

The pharmaceutical^ effective amount oi the antibody or epitope wiii vary according to the patient and the nature of the disease or condition. These variables can be ascertained by one skilled in the ait.

The pharmaceutical 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 die intended use. As well as earners, the pharmaceutical compositions of the invention may include other ingredients, including dyes, preservatives, buffos 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, capsulc, suppository or pessary. 565994 12 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 It will be apparent to one skilled in the ait that the pattern of use of the pharmaceutical composition of the invention may need to be altered for optimum effect U may be necessary to take into account the nature of the disease or condition as well as its severity.

The third aspect of the invention focuses on the expression of ATPases (enzymes) that control the supply of ATP to P2X, receptors, for example in the B-cells of a patient having malignant lymphoma. Channel opening of P2X7 receptors on leukocytes is terminated through the rapid hydrolysis of ATP agonist by ecto-ATPases and ecto-ATPdiphosphohydrolases (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 moie of these ATPases or ATPDases could limit the supply of ATP needed to control P2X, pore formation and the subsequent programmed cell death needed to regulate B-ccll numbers.

Similarly, it is believed that, in the case of IBS, proliferation of ATPases may contribute to lack of appropriate binding of the agonist ATP to the P2XT reccptors.

Accordingly, in this third aspect, the invention provides a preparation for treatment or prevention of a disease or condition in a patient, the preparation including one or /02/2008 16:57 565994 1reehllis 12345678 19/39 13 more substances adapted to regulate the expression of ATPases that control the supply of ATP to P2X7 receptors in die patient's cells or tissues. The invention also provides & method of treating or preventing a disease or condition in a patient, the method including die step of administering to the patient a preparation including one or more substances adapted to regulate the expression of ATPases that control ihe supply of ATP to P2X7 receptors in the cells or tissue of the patient Examples of such ATPases may be CD39 or CD73.

Such a substancc may take the form of an ATP analogue, preferably non-hydrolysable, and specific for P2X7> oi another substance that inhibits the action Of local ATPases depicting the availability of ATP for the P2X7 binding site. The preparation may be in the form of a human antibody directed specifically against non-functional P2X? receptors.

A substance such as an ATP analogue may bind to the P2X? 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. In this way the use of such a synthetic agonist may act to restore receptor function, at the same time as controlling the growth advantage that F2X, provides cells ia 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.

In the case of malignant lymphoma, the ATPases control ihe local supply of ATP to the P2X? receptors so as to reduce the concentration of ATP available for binding to the P2X7 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-cclls and appear to be up-regulated in malignant lymphoma. Preferably, application of a specific ATPase inhibitor may be used to 565994 14 regular the availability of ATP on the P2X7 receptors, so regulating programmed B-cell death.

For treatment of malignant lymphoma, the substance may include a synthetic agonist capable of blocking ATPases ot ATPDasea, of the fonn of non-bydiolysablc P2X7 agonist.

In relation to irritable bowel syndrome, administration of the preparation of the invention is intended to restore receptor function that may be depleted through overactivity of the musclc underlying the affected region of mucosa. The preparation of the invention may act on the mucosa directly to remove these non-functional 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. This maybe achieved by application of enteral or systemic supply of synthetic P2Xr specific agonist, preferably not!-hydrolysable by ATPases, by systemic application of an antibody directed against non-functional P2X> receptors, preferably a small human specific antibody to remove the non-functional receptors, leaving only functional receptors.

If abnormalities of peristalsis in the underlying smooth muscle are responsible for depleting the local availability of ATP for binding to the normal P2X, receptors, 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, die 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 P2X7 receptors. fteahllls 1234 567 S 21/39 /02/2008 16:58 565994 The invention in all its aspects extends to such similar applications that could be made in other medical conditions in which abenft&t P2X7 reccptors are involved as a result of viral infection where the virus is protected in the infected cell by up-regulating non-functional P2XT 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 *ie above pharmaceutically effective agents may need to be altered for optimum effect.

Expressed another way, the invention provides a method of treating irritable bowei syndrome, the method including administering a composition adapted to restore P2X7 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.

In a further aspect, 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 example of this would be prion proteins is 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. 565994 16 BRIEF DESCRIPTION OFTHBDRAWING Figure 1 shows the amino acid sequence of the human P2Xt receptor (prior art). Sequences 65 to 8i and 200 to 216 are highlighted and ate referred to below.

DETAILED DESCRIPTION OF THE INVENTION To raise the antibody specifically to non-functional P2X7,fhe epitope used was the sequence 200 to 216 in Figure ^containing a Cys at 216.

To raise the antibody to non-discriminatory P2X?,the epitope used was the sequence 65 to 81 in Figure 1, to which was added aft N-terminal Cys. This antibody could not detect whether the receptor was ticm-fimctional 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-hydroxysuccinimidc (MCS) cross linker to diphtheria toxin (DT) carrier with ten peptide epitopes attached to each DT carrier, to maintain conformational stability end provide a larger antigenic structure. These conjugated epitopes vcrc used ns the antigens for injection into several animal species (sheep, rabbit and mouse) to raise antibodies specific to the epitopes, m the usual maimer.

The procedure for raising antibodies is well documented in the prior art by use of antigen/adjuvant mixtures injected into animals at particular times. Specific examples for raising the antibodies are set out below: Example 3 Sheep anti-P2X7 antibodies 500 ng of conjugate (approximately 100 fig ofP2X7 epitope) was diluted in phosphate-buffeted saline (PBS) to 0.£ mL and was emulsified with 1.2 mL of Freund's Complete adjuvant Sheep were injected at multiple sites both f r e eh 11 Is 1234567B 23/39 /02/2008 l»:58 565994 17 subcutaneously aod intramuscularly with the antigen/adjuvant emulsion. Eight weeks later the sheep were again injected with the same amount of conjugate emulsified withFreund's Incomplete adjuvant at multiple rites. This was repeated 4 weeks laterand the animals were bled from the jugular vein. The serwn 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.

Other sheep were injected with the same dose of corrugated antigen similar to the schedule above but a different adjuvant wasused. In these animals, 0.7 mL of the diluted antigen was mixed with 0.1 mL of a Quail A / DEAE Dextran solution (2-5 mg Quill A+25 trig DEAE Dextian per mL of PBS) and 12 mL of ISA 50V Montanide. The emulsion was injected at multiple sites both subcutaneously and intramuscularly. The antibodies produced wing this adjuvant produced the same specificities as those produced using Freund's adjuvant gyatflple 2 Rabbit anti-P2Xv antibodies Antibodies were raised in rabbits using the same two adjuvants as with die sheep and the same injection schedules, die only difference being that 300 fig 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 Exaiflpfc 3 Mice anti-P2X7 antibodies Antibodies were raised in mice against the conjugated epitopes and also against the unconjugated epitope of the non-functional P2XT epitope (which is able to discriminate receptors that cannot from pores and thus fail to be apoptotic). ,02/2006 16:58 565994 freahllls 12345678 24/3J 18 la these experiments, tbe adjuvant used was the QAIGEN Pty Ltd product, IrmuunEasy™ which contains the unmuno-stiniulaloiy product CpG DNA (trademark of Coley Pharmaceutical Group lac.) fig of epitope or conjugated epitope was diluted in 70 jiL of PBS and 30 pL of ImmunEasy™ 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 ia mice by this method were again able to discriminate between die different P2X? epitopes and the antibodies against the P2X7 non-functional epitope gave the same results as those raised in sheep and rabbits.

As the above Examples illustrate, antibodies to various epitopes of the P2X7 receptor in different species and using different adjuvants may be raised consistently. In particular, antibodies to an epitope of the P2X7 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.

Example 4 The antibody detecting non-functional P2X, was tested by binding the antibody to cells expressing P2X7 (human) with known function as revealed through the ability of the P2X7 to take up ethidium or rubidium. These P2X7 protein channels may have been mutated at base pair 1513, such that the channels would not form apoptotic pores. These and similar non-functional P2Xj receptors expressed on malignant B lymphocytes also bound the antibody in flow cytometry and in standard immunohistochemistry while cells expressing normal fiiwctitwai P2X7 (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 aon-fuactional receptors and it was specifically this f r eehI 1 Is 1 23 + 5078 25/99 565994 19 conformation that was stabilised in the conjugated epitope used to raise the antibody. The Pto210 was in the trans conformation ia fee receptors that were shown to be functional. This was a result of the binding of ATP (adenosine triphosphate) to the P2X7 receptor. When ATP was bound, the Pro210 on a segment immediately adjacent to the ATP binding site adopted a trans configuration.

This was verified using site directed mutagenesis to change die Pro210 to an Ala that was fixed in die trans configuration and this mutant protein was found to be folly functional and unable to bind the antibody raised to detect the non-functional receptor.

Example 5 Further verification of the specificity of the antibody to detect the non-functional receptor came in experiments that labelled macrophages expressing P2X7. The macrophages bound antibody to the P2X7 receptors using the P2X, universal antibody but did not bind the antibody to non-functional P2X7 until they had been exposed to cancer ceils such as mouse hybridoma cells. Contact between the macrophages, and the hybridoma cells induced the expression on the nwcroph'igs? of non-functional P2X? that was detected by the antibody to non-functional P2X7 , as well as the universal P2Xj antibody.

The macrophages and B-cell lymphocytes extracted from patients with malignant lymphoma were tested ana all these cells bound the antibody to universal P2X? as well as die antibody to die non-functional P2X-, receptors, verifying that P2X7 was non-functional in all the cancer cells detected, with die apoptotic pore formed by functional P2X7 unable to form and thus induce apoptosis in cancer cells.

Ali such cancer cells from all epithelial eel! cancers in humans such as prostate, breast, bowel, skin, stomach, cervix and others as well as malignant lymphoma, chronic lymphocytic leukaemia and brain tumours, as well as fee same tumours in /02/2008 19:56 f r aehI I Is 12J4567B 26/39 565994 other mammals that were tested, including breast and prostate in dog and skin in cat as well as all moose hybridoma colls and mouse fibrosarcoma cells, all express the same non-functional P2X,. 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. This shows that the mechanism of cancer in these mammals is identical in that all cancer cells express non-functional P2X7 receptors unable to form apoptotic pores that would noimaUy kill the cell when activated. In this way the cancer cells become immortal, with apoptosis being switched off.

BhimM As further verification that the cancer cells such as affected B-cell lymphocytes are unable to induce apoptosis through P2X7 function, B cells from leukaemia patients containing non-functional P2X, 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 inducc apoptosis that killed the affected cells.

Example 7 As further verification thai the antibody selectively binds cancer cells, skin from patients with basal celt carcinomas (BCC) were treated with the antibody to the non-functional P2X7 receptors, suspended m an inert cream base and applied to the lesion and surrounding skin (refer Example 10, below). Within 1 week of daily application of the topical antibody, all trace of the BCCs had disappeared with no effect on surrounding skin since normal skin was devoid of the receptors.

DIAGNOSTIC APPLICATIONS Descriptions are provided here by way of example, using the specific nonfunctional P2X, antibody in animals and demonstrating the universal application of the probe and method of the invention to the diagnosis of most cancers in humans and other mammals. freehllls 12345878 27/39 /02/2008 18:58 565994 21 In prostata tissue from humans and mammals, such as cats and dogs, when the antibody of the invention is used for diagnosis, do labelling is obtained in the absence of cancer or pre-cancerous lesions. However, the diagnostic method of the invention reveals first signs of neoplastic change while there is still no accompanying morphological changes detectable by H&E stain.

At this stage, it is necessary to stain for the receptor units first appearing in the nuclei of epithelial celts. These migrate to the cytoplasm in later stages of the disease, acting as a field effect throughout the prostate, so that less tissue need be biopsied to be certain of the existence of a tumour. In later stages of fee disease, the staining becomes more confined to die apical epithelium.

Similarly, other 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.

The same stage development is seen in these other tissues, like breast and cervix, with nuclear stain preceding cytoplasmic stain, while normal tissue is unstained. Affected ducts and lobules in breast tissue are readily detected due to the local field effu-t ••viihi;! '.lis individual Effected dyes system in the breast even where normal morphology suggests there is no cancer. A^acent unaffected ducts appear unstained. Similarly, affected lymph nodes, directly draining tissue containing a tumour, show signs of the tumour through the field clfect of affected lymphocytes. Thus, sentinel nodes can be detected without there being any metastatic cellular spread to the node.

Skin cancers, such as basal ceil carcinoma, squamous cell carcinoma and dysplastic naevi as well as malignant melanomas show positive staining for non-functional reccptors 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. /02/2008 18:53 freehl I Is 1 2349678 28/39 565994 22 All 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. The general application of this diagnostic is seen by way of the same label on mouse hybridoma cells showing the ubiquitous nature of the reccptor in otber animal types besides Human Normal human B-cell lymphocytes show that functional P2X, reeeptors are expressed on the cell surface, so enabling apoptosis when necessary, while human B-cel! lymphocytes fiom patients with malignant lymphoma show that non-fonctiDiial P2X, receptors are expressed on the cell surface, so curtailing apoptosis.

THERAPEUTIC APPLICATIONS Targeting this apparently ubiqitous P2X7 non-functional confonner expressed on the cell surfacc of cancer cclls attempting to undergo apoptosis may be used to treat most cancezs ia. humans and other mammals. Examples are set out below: F.x ample 8 Mouse hybridoma cells were grown on a macrophage base both in the presence and absence of affinity purified antibody to n on-functional P2X7. Cell counts revealed that over 4 days while cells coincubated with purified normal IgG grew from I x 104 to 7 x 104, coiucubation with non-functional P2Xj antibody kept the cell count to only 1.5 x 104.

Example 9 This example shows that antibodies Taised against the non-functional epitope of the P2Xj receptor can inhibit tumour formation in vivo.

As shows above, antibodies raised in sheep against the non-functional P2X7 epitope identified this non-functional P2X, apoptotic receptor on the surface of mouse hybridoma cells. Addition of this antibody to hybridoma cell cultures /02/2008 18:58 f r'eehl I Is 12345SJB 29/39 565994 23 retarded the growth of the cells. Mouse hybridoma cclis when injected into prepared iabrced mouse strains will cause tumour formation.

Id this experiment, three groups of 10 BaTb-c female mice each received the following treatments; Group 1: 10 mice each injected intraperitoneally (IP) with 1 x 10s hybridoma cells in 0.5 mL of cell culture medium on Day 1. On Days 2 and 3, they received an intraperitoneal injection of 0.5 mL of cell culture medium.

Group 2: 10 mice each injected intraperitoneally {IP) with 1 x 10® hybridoma cells in 0.5 mL of cell culture medium containing 1 nig 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.

Group 3: 10 mice each injected intraperitoneally (IP) with 1 x 10s hybridoma celts is 0.5 mL of cell culture medium containing 1 mg of purified sheep anti-P2X7 non-functional epitope IgG on Day 1. On Days 2 and 3, they.received a further injection of 0.5 m! of cell culture medium containing 1 mg of purified sheep anti-P2Xj IgG.

Mice from all the groups were killed on Day 11 and examined for the presence of tumour. The tumours were excised and weighed.

The results were as follows: Groups Observations Mean Tumour Weight per mice (±SD)(g) 1: Control 1 9 out of 10 mice bod tumours. 3.98 ±1.1 /02/2008 IS:59 565994 f r«ahI I 11 12345678 30/39 24 2: Control 2 out of 10 mice had tumours 2.93 ±0.9 3: Experimental 9 out of 10 mice had tumours 1.13 ±0.4 An analysis of variance showed a significant difference ia tumour weight between flic groups (probability ? < 0.01). The experimental group treated with the anli-P2X7 nonfunctional antibodies was significantly different (P < 0.01) from the two control groups. That is, treatment with antibodies against the P2X7 non-functional epitope significantly reduced the amount of tumour in the experimental animals.

SxaipptelQ Specific affinity purified antibody (to greatly improve specificity) was applied to 3 human basal cell carcinomas ("BCC") eitheT as a liquid held in place for 7 days or suspended in a dimethicone cream base. No trace of the BCC lesions was detectable after treatment, while control skm was entirely unaffected due to the absence of the protein target.

Example 11 Skin lesions of the form of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) (both primary tumours and sccondaiy tumours), including relapsed tumours and dysplastjc naevi, were treated in a farther trial using purified antibody, IgG either affinity purified or not, mixed in dimethicone cream base or a penetrating cream base. Since there were no non-functional receptors present in the normal skin there were no side effects detected in normal skin of any kind. The cancers of all types all responded to the presence of the antibody by disappearing within a period from thirty si* hours to one week with twice daily applications. No relapse has occurred in periods of up to twelve months. 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 ihirty four histologically confirmed tumours have been successfully eliminated within one week treatment periods. /02/2008 16:59 -f r eeh I I Is 1 23 + 5678 31 /39 565994 It is believed that application to patients in general would involve production of a human monoclonal antibody fsuch as hetccptin) so that internal cancers could be treated with the same efficacy as is revealed with topical application. All normal functional P2X? expressed on the cell surfaces of cells such as lymphocyte* would nee4 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. Tims all cells targeted would be only those attempting to kill themselves through programmed cell death, inchidmg cancer cells. The P2X? Teceptors on these cells, particularly cancer cells, would be in a noa-ftmctioaal or ATP-depleted state.

ACTIVE IMMUNISATION Active immunisation may also be used for therapeutic purposes, ha this case 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 Gly2O0-Cys2l6 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-cotnpromised, None of these many animals have ever developed any tumours.

A specific example illustrates this: Example 12 Protocol: The experiment was conducted on fee basis of a mouse tumour model. Forty ten-week old female inbred Balb C mice were used, and divided into wo groups of twenty, Group 1 being experimental and Group 2 being the control group. 565994 26 Day 1: The twenly experimental animals in Group I were injected with 0.1 mg of the peptide epitope (hP2X7 sequence 200-216) conjugated to diphtheria toxin via the MCS crosslinker. This contained approximately 0.02 mg of Hie 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 find intramuscular sites.

The twenty mice in the control group, Group 2, 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, 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 conjugatetedjuvant solution was injected at multiple subcutaneous and intramuscular sites in each mouse.

The twenty Group 2 mice wsre injected with die 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.

Day 26: The twenty Group ] mice were injected with 0.025 mg of the peptide epitope (hP2X7 sequence 200-216; conjugated to diphtheria toxin via the MCS crosslinker (containing approximately 0.005 mg of the peptide epitope). This was contained in a phosphate buffered saline solution and mixed with the Qiagen CpG DNA adjuvant ImmunEasy. Again 0.1 mL of the mix was injected in each mouse at multiple subcutaneous and intramuscular sites. The control group was injected as before on Day 8, /Q2/200& 16:99 freehl I Is 1 23 + 5578 33/39 565994 27 Day 29: All mice received an injection of tumour cells at a single subcutaneous site located at the back of the neck in 0.1 mL of tissue culture media. The tumour cclls used were a mouse fibrosarcoma cell line developed by the Waher and Eliza Hall Institute io Melbourne Australia designated cell line WEHI164.

The cells were injected at two concentration* into both die experimental and control groups of mice. Each group was subdivided into two. Ten mice from each of the experimental and control groups received 160,000 cells per mouse and ten mice from each group received 320,000 cells per mouse.

The cells firom this cell line had previously been tested fertile presence of titte nonfunctional P2Xf epitope on their cell surface. This was done using an antibody raised in sheep which specifically recognises the non-functional form of the receptor.

Day 38: All mice were killed and blood collected for analysis of antibodies to the non-functional P2X7 epitope. All mice were weighed and the tumours were excised and weighed.

Results Group Control 160,000 cells Experimental 160,000 cells Control 320,000 cells Experimental 320,000 cells n Mean tumour wt (mg) 599 270 1147 750 SD 307 108 633 363 SEM 97 34 200 115 Analysis of variance of the results showed a statistically significant difference between control and treatment groups and between low and high dose groups 005111434 565994 28 (P=0.0003). The lower dose group showed a larger difference due to the lower tumour load having less effect on the ability of the mice immune system to cope.

ATP ANALOGUE The efficiency of use of a synthetic agonist to effectively bind to ATP binding sites on the P2X7 5 pore, to force the pore to enter the functional state, thereby acting to restore receptor function as well as controlling the growth advantage that P2X7 provides cells, is shown in the following experiment in culture. Tumour B-cells collected from a patient with CLL, when mixed with a similar number of like cells from a normal patient were treated with ATP at 2.5mM for four hours. No tumour cells remained, only normal cells. The use of ATP or the more selective P2X7 agonist 10 benzoyl, benzoyl ATP is not appropriate in vivo. Thus, a selective ATP analogue able to selectively bind P2X7 at much higher affinity than either ATP or BzATP may be designed to reinstate the process of apoptosis in a range of affected tumour cell types.

INDUSTRIAL APPLICABILITY The invention in all its aspects has application to the fields of human and veterinary medicine 15 and health, with the potential to enable early and accurate diagnosis of diseases and effective treatment, which in many cases is far less invasive or traumatic than those available in the prior art.

As used herein, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude other additives, components, integers 20 or steps.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in New Zealand or any other jurisdiction.

Claims (37)

565994 29 THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An isolated P2X7 receptor: having an amino acid sequence that has at least about 70% identity to the sequence shown in Figure 1 wherein the amino acid sequence of the isolated P2X7 receptor contains a proline corresponding to proline 210 shown in Figure 1, said proline being in a cis conformation.

2. The receptor of claim 1 wherein the receptor is a rodent receptor.

3. The receptor of claim 2 wherein the receptor is a mouse, rat or rabbit receptor.

4. An isolated receptor having an amino acid sequence shown in figure 1 in which proline at position 210 is in a cis conformation.

5. Use of: a receptor according to any one of claims 1 to 4; or a peptide having an amino acid sequence of a fragment of the receptor, said sequence including proline at position 210 in a cis conformation; in the manufacture of a vaccine for the prevention or treatment of a cancer.

6. A vaccine for prevention or treatment of a cancer including: the P2X7 receptor of any one of claims 1 to 4; or a peptide having an amino acid sequence of a fragment of the receptor, said sequence including proline at position 210 in a cis conformation.

7. The vaccine according to claim 6 wherein the receptor or peptide is conjugated to a carrier.

8. The vaccine according to claim 7 wherein the carrier is diphtheria toxin.

9. The vaccine according to any one of claims 6 to 8 further including an adjuvant. 3372343 565994 30

10. The vaccine according to claim 9 wherein the adjuvant includes QUILL A /DEAE Dextran /Montanide.

11. Use of a vaccine according to any one of the preceding claims in the manufacture of a medicament for the treatment of a cancer in an individual or for the prevention of a 5 cancer in an individual.

12. Use according to claim 11 wherein the cancer is selected from the group consisting of prostate, breast, skin, lung, cervix, uterus, stomach, oesophagus, bladder, colon, vaginal, ovary and blood cancer and lymphoma.

13. Use of an antibody that binds to an extracellular domain of a P2X7 receptor having an 10 amino acid sequence as shown in Figure 1 in which proline at position 210 is in a cis conformation, and wherein the antibody does not bind to an extracellular domain of a P2X7 receptor having an amino acid sequence as shown in Figure 1 in which proline at position 210 is in a trans conformation, in the manufacture of a composition formulated for application to skin for the prevention or treatment of a cancer. 15

14. A composition including an antibody that binds to an extracellular domain of a P2X7 receptor having an amino acid sequence as shown in Figure 1 in which proline at position 210 is in a cis conformation, and wherein the antibody does not bind to an extracellular domain of a P2X7 receptor having an amino acid sequence as shown in Figure 1 in which proline at position 210 is in a trans conformation, the composition being formulated for 20 application to skin.

15. The composition according to claim 14 wherein the antibody is a sheep antibody.

16. The composition according to any one of claims 14 to 15 further including a compound for assisting the antibody to penetrate skin.

17. The composition according to any one of claims 14 to 16 further including a compound 25 for moisturising skin.

18. The composition according to claim 17 wherein the compound for moisturising skin is dimethicone. 3372343 565994 31

19. The composition according to any one of claims 14 to 18 wherein the composition is formulated as a cream, lotion, ointment, gel, aerosol or spray.

20. Use of a composition according to any one of the preceding claims in the manufacture of a medicament for the treatment of a cancer in an individual or for the prevention of a 5 cancer in an individual.

21. Use according to claim 20 wherein the cancer is skin cancer.

22. Use according to claim 21 wherein the skin cancer is selected from the group consisting of basal cell carcinoma, squamous cell carcinoma, melanoma and dysplastic naevi.

23. Use of an antibody that binds to an extracellular domain of a P2X7 receptor having an 10 amino acid sequence as shown in Figure 1 in which proline at position 210 is in a cis conformation, and wherein the antibody does not bind to an extracellular domain of a P2X7 receptor having an amino acid sequence as shown in Figure 1 in which proline at position 210 is in a trans conformation in the manufacture of a reagent suitable for detecting a cancer by an in vivo imaging technique. 15

24. The use according to claim 23 wherein the in vivo imaging technique is positron emission tomography.

25. An in vitro method for determining whether an individual has a cancer including: contacting a cell or tissue of an individual with an antibody that binds to an extracellular domain of a P2X7 receptor having an amino acid sequence as shown in Figure 20 1 in which proline at position 210 is in a cis conformation, and wherein the antibody does not bind to an extracellular domain of a P2X7 receptor having an amino acid sequence as shown in Figure 1 in which proline at position 210 is in a trans conformation; and determining whether the cell or tissue is bound by the antibody. 25

26. The in vitro method according to claim 25 wherein the cancer is selected from prostate, breast, skin, lung, cervix, uterus, stomach, oesophagus, bladder, colon, vaginal, ovary and blood cancer and lymphoma. 3372343 5 10 15 565994 32

27. A vaccine according to any one of claims 5 to 9, substantially as hereinbefore described with reference to the examples.

28. A composition according to any one of claims 14 to 19, substantially as hereinbefore described with reference to the examples.

29. An isolated receptor according to any one of claims 1 to 4, for use in treating or preventing a disease or condition.

30. Use of an isolated receptor according to any one of claims 1 to 4, for the manufacture of a medicament for treating or preventing preneoplasia.

31. Use of an isolated receptor according to any one of claims 1 to 4, for the manufacture of a medicament for treating or preventing an epithelial cell cancer.

32. Use according to claim 31, wherein the epithelial cell cancer is selected from the group consisting of prostate, breast, skin, lung, cervix, uterus, stomach, oesophagus, bladder, colon and vaginal cancers.

33. Use of an isolated receptor according to any one of claims 1 to 4, for the manufacture of a medicament for treating or preventing a blood cancer.

34. Use according to claim 33, wherein the blood cancer is malignant lymphoma.

35. Use of an isolated receptor according to any one of claims 1 to 4, for the manufacture of a medicament for treating or preventing irritable bowel syndrome.

36. Use of an isolated receptor according to any one of claims 1 to 4, for the manufacture of a medicament for treating or preventing an infection by HIV or Mycobacterium tuberculosis.

37. Use according to any one of claims 30 to 36, substantially as hereinbefore described.