Title of Invention

A CELL FREE IMMUNOGEN PREPARATION / PROPHYLACTIC VACCINE

Abstract

ABSTRACT A cell free preparations of tumor antigen adapted for immunotherapy of cancer and/or prophylactic vaccine obtained thereof for destroying cancer cells as soon as these appear in the body. Also discloses is a diagnostic kit and a method of early detection and treatment of cancer involving the aforesaid ceff free preparation of tumor antigen. The developed cell free immunogen preparation/vaccine comprise cell free form of tumor antigen covalently bound with antigen presenting fraction of major histocompatiability complex (MHC) which is immunologically antigenic and capable of sensitizing lymphocytes and at the same time free from any of the cell surface markers which may cause auto-immune diseases in the immunized subjects. The diagnostic kit involving the above cell free preparations of tumor antigen provide for a simple, effective and early detection of cancer and a method for carrying out such detection of cancer The cell free preparation is also directed to serve in the method of treatment for making all the cancer cells in the body vulnerable to destructible by an activated immune surveillance system in the host and as a prophylactic vaccination adapted to destroy cancer cells as soon as these appear in the body To The Controller of Patents The Patent Office At Mumbai.

Full Text

FORM - 2 THE PATENTS ACT, 1970 (39 OF 1970) COMPLETE SPECIFICATION (See Section 10) 1. TITLE OF INVENTION A CELL FREE IMMUNOGEN PREPARATION / PROPHYLACTIC VACCINE ORIGINAL 502/MUMNP/03 2 a) SAHASRABUDHE Dr. Madhao Bhalchandra, b) 41, Saras Baug, Sion-Trombay Road, Deonar, Mumbai - 400 088, State of Maharashtra, India, c) an Indian national The following specification particularly describes the nature of the invention and the manner in which it is to be performed. GRANTED 18/5/2005 This application has been divided out of parent application No. 358/MUM/2003, dated 10th April 2003. FIELD OF THE INVENTION The present invention relates to cell free preparations of tumor antigen adapted for immunotherapy of cancer and/or prophylactic vaccine obtained thereof for destroying cancer cells as soon as these appear in the body. The invention also discloses a diagnostic kit and a method of early detection and treatment of cancer involving the aforesaid cell free preparation of tumor antigen. BACKGROUND ART Initiation of cancer is an extremely stealthy process and its progression in the body, at least in early stages, goes on so silently that even the host carrying the cancer cells is completely oblivious of its presence. In early stages cancer is symptomless; there are no aches or pains, no rise in temperature or discomfort of any kind even to motivate the person to seek medical advice. When the person himself is blissfully unaware of the presence of cancer cells in his/her body, it is extremely difficult even to identify the person at risk of cancer from amongst the normal healthy population, let alone detect his/her cancers early. Cancer cells thus appear in the body mostly without symptoms and hence escape early detection. Non invasive techniques such as MRI, CT, Radioimmuno scintigraphy, however advanced, can not detect lesions of the order of less than 2 mm in diameter which account for 8 million cancer cells. Thus the current diagnostic methods detect cancers only when the tumour load exceeds a million cells. It is too late by then. Cancer is curable only if it is detected early. At present there is no method to detect cancer early to guaranty cures. Currently the emphasis is more on clinical examination when the person approaches the physician for possible early detection. As mentioned above, most of the modern diagnostic methods require a minimum size of 2 mm diameter of tumour mass (tumour load more than a million cells) to be detected. Tumours with less than this size are not detectable by the methods in vogue. Some attempts have been made in the past to use the presence of degradation products of tumours in blood or urine, as the test for early detection. But these attempts have two major drawbacks. (i) Newly formed early cancer cells are unlikely to disintegrate by themselves. Only those tumours which have aged to some extent or which have become necrotic because of being situated 200 microns or more away from blood supply (that is the diameter of tumour mass has to be more than 400 microns) will become necrotic and release degradation products in the circulation. Tumour masses with diameters more than 400 microns would contain more than million cells, a situation already too late. Besides any exfoliated product released in blood stream or in urine, would be uniformly distributed in the total volume of blood in the human body. Assuming 7 litres as the total blood volume, whatever is released by tumours would be diluted 7000 fold; thus determining minute quantities in few ml of blood is extremely difficult unless the tumour is sufficiently large. Lymphocytes, which are constantly circulating in the body, however have the uncanny capacity of "noticing" anything "alien" and storing this information. If the said "alien" entity happens to be on the cell surface, such as the tumour cell surface antigens, the lymphocytes would have detected the presence of cancer cells right from the time the first cancer cell makes its appearance in the body. However, tumor cell surface antigens are found to be immunologically non-antigenic or at best antigenically very weak and hence the sensitization of lymphocyte is also weak. . Powerful probe is therefore required to specifically detect weak sensitization and pick out the feebly sensitized lymphocytes. Need therefore exists in the art to develop tumour antigens which would be immunologically antigenic and antigenically sufficiently strong to pick out sensitized lymphocytes to possibly retrieve the information from sensitized lymphocytes in the blood stream of a person carrying cancer cells to thereby favour early and effective detection of cancer. In my Indian Patent No. 174538 dated January 19, 1993 there is disclosed the process for exposing tumor-mimetic cell surface (TuMiCeS) antigens and proto-oncogene-products on normal non-malignant cell surfaces and its use in early detection of cancer. The process disclosed comprises exteriorizing tumor mimetic cell surface (TuMiCeS) antigens on normal cell surfaces by controlled in situ hydrophilic hydrophobic inter-conversion on normal cell surfaces using reagents like fluoro dinitro benzene and related compounds and isolating the tumor mimetic antigens in purified form by immuno-affinity chromatography process. The said tumor mimetic cell surface (TuMiCeS) antigen is found to be between 200 to 500 times stronger than the antigens normally seen on tumor cell surfaces. The tumor mimetic cell surfaces (TuMiCeS) of my above co-pending application are adapted to pick out only those lymphocytes which have been sensitized to tumor cell surface markers in cancer patients. However, since cancer cells appear and grow in the body extremely stealthily, simple detection does not serve to arrest the rapid growth of such cancer cells and possibly to make the body respond to the need for early detection of new generations of such harmful cancer cells and also control the residual cancer cells. Most of the currently used anti-cancer drugs are targeted to act against symptoms of cancer rather than the disease proper. Even the newly introduced anti-angiogenesis drugs are no exception. Since no property or characteristic which is exclusively present only in cancer cells and not on any of the normal cells, has so far been found, development of drug acting selectively only on cancer cells has not been possible. Under the circumstance immunotherapy appears to be the best possible alternatives. In the selection of immunogen for immunotherapy, one has to be extremely careful and guard against the danger of inadvertent induction of autoimmune diseases in the recipient host. Cancer cells automatically get disqualified because they carry tumour markers, which are commonly seen on some normal cells of the body. Besides, cancer cells have already lost the property to be immunogenic, because the tumour antigens are not appropriately presented in association with MHC. Also, it is likely that the use of cancer cells, even in attenuated forms, as immunogens in human cancer patients could lead to serious problems on ethical grounds. Moreover, because the tumour cell surface antigens are not appropriately presented by MHC such tumour antigens are found to be immunologically non-antigenic or very weakly antigenic to sensitize the lymphocytes. Importantly, it is because of such problems and limitations of tumour antigens in cancer cells that the same cannot serve as an effective probe even for specifically sensitised lymphocytes. OBJECTS OF THE INVENTION It is thus the basic objective of the present invention to develop a cell free immunogen preparation/vaccine with the tumour antigen appropriately presented by antigen presenting fraction of MHC to thereby make it immunologically antigenic and capable of sensitizing lymphocytes and at the same time free from any of the cell surface markers which may cause auto immune diseases in the immunized subjects. Another object of the invention is directed to provide cell free immunogen preparation, appropriately presented tumor antigens free of all HLA antigens which could be simply and effectively used in immunization of cancer patients and also make the cancer cells vulnerable to destruction without any danger of inducing auto-immune diseases in cancer patients. Yet another object of the present invention is to provide tumor antigen based cell free preparation which would selectively conjugate with sensitized B lymphocytes elaborating the specific antibodies and in the process carry the same and deposit/tag on to cancer cells to make them recognizable /vulnerable to destruction by the immune surveillance system of the host. Another aspect of the present invention is to provide for a diagnostic kit involving a cell free preparations of tumor antigen which would involve simple, effective and early detection of cancer and a method for carrying out such detection of cancer. Yet another object of the present invention is directed to a method of treatment for making all the cancer cells in the body vulnerable to destructible by an activated immune surveillance system in the host. Yet further object of the present invention is directed to providing a prophylactic vaccination adapted to destroy cancer cells as soon as these appear in the body. Summary of the Invention In accordance with the basic aspect of the present invention there is provided a cell free immunogen preparation/prophylactic vaccine comprising cell free form of tumor antigen covalently bound with antigen presenting fraction of major histocompatiability complex (MHC). It is found by way of the present invention that it is possible to selectively provide the tumor antigens covalently bound to freely floating transiently formed antigen presenting fraction of MHC which are free from MHC firmly anchored on the hydrophobic layer of the cell membrane. The isolated cell free form of such tumor antigens is found to be fully immunogenic and has the capabilities of inducing specific immune response in recipient cancer patients. In accordance with another aspect of the present invention there is provided a method of producing the cell free preparation of tumor antigens covalently bound with antigen-presenting fraction comprising: i. providing FDNB-tagged normal human WBCs; ii. immunizing animals with said FDNB-tagged cells; iii. freeing the antiserum from all species specific, HLA or other undesirable antibodies; iv. isolation of immunoglobuling free of contaminants; v. insolubalization of specific immonoglobuling on selective support system to thereby set up immuno-affinity chromatography column; and vi. separating the tumor antigen covalently bond with antigen presenting fraction of MHC as the cell free preparation. In accordance with a preferred aspect the separation of tumor antigen covalently bound with antigen presenting fraction of MHC comprise: a) isolating WBCs from normal "0" group healthy persons; b) tagging the isolated WBCs with FDNB preferably 104 molecules of FDNB per cell; c) disintigrating the FDNB-tagged cells and passing the homogenate on the immuno-affinity column; d) discard the washing; and e) eluting the absorbed tumour antigen covalently bound with antigen-presenting fraction of MHC, by selective eluent at appropriate pH. It is thus possible by way of the present invention to avoid the the difficulty in achieving immunogen in cell free form by developing an immuno-affinity chromatography column, which will selectively pick out in the cell-free preparation, only the appropriately presented tumour antigens, free of all HLA antigens, from the homogenates of FDNB-tagged cells. This antigen preparation thus obtained is suitable to use in immunization of cancer patients and also making all the cancer cells vulnerable to destruction by the above induced immune surveillance system in the host, without any danger of inducing autoimmune disease in the cancer patients. Thus, in accordance with yet another aspect of the present invention there is provided a method of immunization of cancer patients or vaccination comprising. inducing the immune response in cancer patients by treating with the cell free preparation of tumor antigen covalently bond with antigen presenting fraction of MHC. Following the above method it is possible to make all the cancer cells in body vulnerable to destruction by the immune surveillance system of the host. It is possible to immunize the cancer patients with the cell-free immunogen. The sensitized B lymphocytes elaborate the specific antibodies within themselves. These conjugate with the appropriately presented tumour antigens and carry the same in circulation and deposit/tag on to cancer cells, thus make them recognizable/vulnerable to destruction by the immune surveillance system of the host. The above, therefore, provides for a possible immunotherapy of cancer patients without risking the danger of inducing autoimmune disease against cells with d, ferent HLA profiles. The cell free preparation uses only those cells which derive newly acquired antigenic properties, which appear only on transformed cells as a direct consequence of carcinogenesis process. Thus it is possible to pick out true tumour antigens, distinct from surface markers seen on normal cells. It is for this reason it is possible to pick out cancer patients harboring any type of cancers involving the cell free preparation of the invention. The method thus would be applicable for detection as well as for immunotherapy in all types of cancer located anywhere in the body. Importantly the cell free preparation of the invention takes care of problems of tumour antigens in cancer cells being not appropriately presented by antigen presenting function of MHC, and consequential failure to be recognizable to immune surveillance system of the host and evading destruction by it. The cell free preparation thus serves as an effective immune system to make all the cancer cells in the body recognizable and therefore destructible by the immune surveillance system of the host. This is specifically achieved by the invention by labeling in situ, all cancer cells in the body with an auxiliary tumour antigen appropriately presented by the antigen-presenting fraction of MHC ( the cell-free preparation ). The sensitized B lymphocytes of the body, act as lyposomes, to carry the tumour antigens covalently bound with antigen-presenting fraction of MHC, on to cancer cells. This is possible because of the unexpected finding of the present invention that tumour antigen in the present preparation is immunologically identical to the common tumour antigens expressed on all types of cancer cells In accordance with yet another aspect of the present invention there is provided a diagnostic kit for early detecton of cancer comprising: a) tumor antigen covalently bound with antigen-presenting fraction of MHC in cell free form; b) means for labeling of the antigen;and c) instruction to carry out the test. Preferably, the kit of the invention comprises: a) tumor antigen covalently bound with antigen-presenting fraction of MHC in cell free form; b) means for labeling of the antigen; c) means for withdrawal of blood sample to be tested; and d) instruction to carry out the test. In accordance with yet another aspect of the present invention there is provided a method of producing the diagnostic kit for early detection of cancer comprising. a) providing tumor antigen covalently bound with antigen-presenting fraction of MHC in cell free form; b) providing means for labeling of the antigen; c) optionally providing means for withdrawal of blood sample to be tested; d) providing instruction to carry out the test. In accordance with yet another preferred aspect the invention provides for a method for early detection of cancer using the above kit comprising: a) labelling the tumour antigen covalently bound with antigen-presenting fraction of MHC with suitable enzyme to provide a selective ELLISA reagent; b) withdrawing peripheral blood from the person to detect the presence of sensitized B lymphocytes in the collected blood specimen by the ELISA method ; c) reacting the sample with the ELISA reagent; d) washing the cell preparation free of excess of un-reacted ELISA reagent; e) determine the number of cells carrying the ELISA reagent. DETAILED DESCRIPTION It is thus possible by way of the above invention to provide a new and an alternative source of tumour antigens specifically presented in association with MHC, in FDNB-tagged normal cells, and also develop a method of isolating it by immuno-affimty chromatography. Importantly, while Immuno-affinity-chromatography is commonly used technology for one-step separation of desired antigens the specificity of isolation, the invention achieves the desired specificity and novelty of immunoglobulin used in setting up the immuno-affinity-column. Importantly, all other non-essential antibodies from the immunoglobulin used are effectively removed in the making of the immuno-affinity-column. Thus in the invention the affinity column is developed to isolate specific tumour antigens only. If the tumour antigen is covalently bound to antigen-presenting fraction of MHC macromolecule, which is it firmly anchored on hydrophobic layer of the cell membrane, freeing the tumour antigen from the MHC macromolecule is extremely difficult. It is by way of the finding of the present invention that the tumour antigens in FDNB-tagged cells are covalently bound to freely floating transiently formed antigen-presenting fraction of MHC, which are free from the clutches of the hydrophobic layer of the cell membrane that it is possible to isolate tumour antigens covalently bound to antigen-presenting fraction of MHC. The present antigen preparation therefore is fully immunogenic and has the capabilities of inducing specific immune responses in the recipient cancer patients. It is based on the aforesaid that it is possible by way of the invention to successfully isolate a fully functional immunogen in cell-free form. The isolated tumour antigens covalently bound with antigen-presenting segment of MHC, from FDNB-tagged normal cells, by immuno-affinity chromatography of the invention could be used as prophylatic vaccine to build up immuno response in the host before the first cancer clones make their appearance in the body. Importantly, cancer cells encountered in cancer patients in the clinics, acquire the capabilities of evading the immune surveillance system of the host. Such cancer cells are thus incapable of inducing immune response in the hosts. It is, therefore important to re-activate the immune surveillance system of the host. This is possible with the tumour antigens covalently bound with antigen preseting segment of MHC of the present invention. The preparation also assists in making all the cancer cells in the body, vulnerable to in vivo destruction by immune surveillance system of the host. The active immunogen in the "vaccine" of the invention is a cell free macromolecule, with comparatively small molecular weight. Most importantly it is found by way of the invention that this cell free preparation by coincidence selectively readily gets attached, by antibody-antigen reaction, to sensitized B lymphocytes circulating in the body of immunized patients. Since sensitized B lymphocytes are known to continuously elaborate specific antibodies within the cell, the sensitized B lymphocytes still maintain an affinity to combine with cancer cells, through another set of antibody-antigen reactions. This property also provides to sensitize B lymphocytes, as natural "lypsomes" to carry cell free small molecular weight, appropriately presented tumour antigens, on to cancer cells and thus make them vulnerable to destruction by the immune surveillance system of the host. The cell free "vaccine" of the invention thus plays a dual role. First it acts as an immunogen to reactivate immune responses in the host and secondly it assists in making all cancer cells vulnerable to destruction by immune surveillance system of the host. Since the sensitized B lymphocytes, are patient's own cells, it is easier to make all cancer cells vulnerable to destruction, wherever these may be located, including those located in the brain beyond the impervious blood brain barrier. It is thus also possible by way of the invention to develop an in vitro screening method for picking out person at risks of cancers, long time before the tumour load attains 1000 cell mass (0.1 mm in diameters). Detecting the presence of lymphocytes specifically sensitized to tumour antigens, in the circulating blood, is the most reliable method, with immunological specificities, for detection of cancers in the body. Since only 5 ml of peripheral blood is all that is required form the person to. be screened, the method can be adapted for use during periodic health check-ups of general population. This would help in picking out persons harbouring even symptom-less cancers, six months to one year before the cancer is barely detectable by any of the currently used diagnostic methods. EXAMPLES The details of the invention, its objects, and advantages are explained hereunder in greater details in relation to the non-limiting exemplary illustrations. Example I: Preparation of the Cell-Free Immunogen A) Setting up immuno-affinitv column :- Step 1. Immunize rabbits/horses/or any other suitable animal with FDNB-tagged normal human WBCs (104 molecules of FDNB per cell) Step 2 Withdrawal of immunized blood and separating the antiserum free of blood cell:, complements etc. Step 3 Absorption of antiserum with pooled RBCs and WBCs from large number of normal humans, to free the antiserum of all species specific or HLA antibodies and other undesirable antibodies Step 4 Isolation of immunoglobulins free from all other constituents of blood by Ammonium sulphate precipitation followed by iso-electric precipitation. Step 5 Insolubalization of specific immunoglobulins on suitable support system to set up immuno-affinity chromatography column. B) Separation of tumour tumour antigen covalently bound with antigen-presenting fraction of MHC. Step 1 Isolate WBCs from normal "O" group healthy persons. Step 2 Tag the isolated WBCs with FDNB (104 molecules of FDNB per cell) Step 3 Disintigrate the FDNB-tagged cells and pass the homogenate on the immuno-affinity column. Step 4 Discard the washings; Step 5 Elute the absorbed tumour antigen covalently bound with antigen-presenting fraction of MHC, by suitable eluent at appropriate pH to thereby obtain the cell free immunogen. Example II :Uso of the cell free preparation for early detection of cancer Step 1 Label the preparation of the invention obtained under Example I above the tumour antigen covalently bound with antigen-presenting fraction of MHC, with suitable enzyme. Step 2 Withdraw 5 ml peripheral blood from the person to detect the presence of sensitized B lymphocytes in the collected blood specimen by our ELISA method. Step 3 Sensitized B lymphocyte would form a complex with our ELISA reagent Step 4 Wash the cell preparation free of excess of un-reacted ELISA reagent. Step 5 Determine the number of cells carrying the ELISA reagent. Example III Use of the preparation in Re-induction specific immune response against cancer cells in cancer patients. The cell-free preparation of tumour antigen covalently bound with antigen-presenting fraction of MHC as per Example I, can be used for re-activating specific immune surveillance system of host. Using the cell free preparation it is possible to make all the cancer cells in the body vulnerable to destruction by the" re-induced immuhe surveillance system in cancer patients. Immunized cancer patients would have a fairly good number of sensitized B lymphocytes circulating in the body. Covalently bound tumour antigens simultaneously circulating in the blood stream, get tagged on to sensitized B-lymphocytes through the inherent antigen-antibody reaction. Since the sensitized B-lymphocytes are continuously elaborating within itself, tumour specific antibodies, these (the sensitized B lymphocytes) have the capabilities of getting tagged on to tumour antigens present on all cancer cells, wherever these may be present in the body. This provides for additional tumour antigen appropriately presented by antigen-presenting function of MHC on all cancer cells wherever these may be present. This makes all the cancer cells recognizable and therefore vulnerable to destruction by immune surveillance system of the host. This is possible because the tumour antigen in the present cell-free preparation, is immunologically identical to tumour antigens expressed on all cancer cells. It will thus be possible to destroy the residual cancer cells, which invariably remain in the body, even after exhaustive chemotherapy is completed and which are chiefly responsible for recurrences of the disease. Since body's own immune surveillance system and its own circulating B lymphocytes are used in making all the cancer cells in the body, vulnerable to destruction by immune surveillance system of the host, it is possible that cancer cells located in brain beyond the blood-brain barrier, would also be destroyed, Example IV Prophylactic vaccination for destroying cancer cells the moment these appear in the body. Cancer cell clones are immunogenic as well as recognizable and therefore vulnerable to destruction by immune surveillance system of the host, within the first few days of their appearance in the body. But the immune response is not sufficiently strong to destroy the newly formed cancer cell clones. By the time the immune response acquires sufficient teeth to destroy the cancer cells, the cancer cells have already acquired the capabilities of evading the immune response of the body. This can be avoided by pre-inducing immune response by prophylactic vaccination with the cell-free preparation of Example I above, in persons in cancer risk groups, so that the immune surveillance system of the body is ready to destroy the tumour cells the moment these make their appearance in the body. The above go to demonstrate cell-free preparation of tumor antigen of the invention, common for all cancers, covalently bound to antigen-presenting fraction of MHC, and its use in immunotherapy of cancer to control cancer cells located anywhere in the body and also for prophylactic vaccination for destroying cancer cells the moment these appear in the body. I CLAIM: 1. A cell free immunogen preparation/prophylactic vaccine specific for all cancers comprising cell free tumor antigen derived from newly acquired non-self tumor antigen from normal cells and antigen presenting fraction of MHC (major histocompatiability complex) wherein the said tumor antigen and antigen presenting fraction of MHC are covalently bound. 2. A cell free immunogen preparation/prophylactic vaccine as claimed in claim 1 wherein the tumor antigen is immunologically identical to the common tumor antigens expressed on all types of cancer cells. 3. A cell free immunogen preparation/prophylactic vaccine as claimed in claim 1 or 2 wherein the said active immunogenic tumor antigen is a cell free macromolecule, with comparatively small molecular weight adapted to selectively get attached, to sensitized B lymphocytes circulating in the body of immunized patients. 4. A diagnostic kit for early detection of cancer comprising : a) Cell free tumor antigen which is covalently bound with antigen-presenting fraction of MHC in cell free form as claimed in claim 1; b) means for labeling of the antigen preferably suitable enzymes; and c) instruction to carry out the ELISA test 5. A cell free immunogen preparation/prophylactic vaccine comprising cell free form of tumor antigen covalently bond with antigen presenting fraction of major histocompatiability complex (MHC), and a diagnostic kit using the same substantially as herein described and illustrated with reference to the accompanying examples. Dated this 14th day of May 2003 Dr. Sanchita Ganguli Of S.MAJUMDAR & CO. Applicant's Agents 8. A diagnostic kit as claimed in claim 7 comprising: a) tumor antigen covalently bound with antigen-presenting fraction of MHC in cell free form; b) suitable enzyme for labeling of the antigen; c) means for withdrawal of blood sample to be tested; and d) instruction to carry out the ELISA test. 9. Use of the isolated tumour antigens covalently bound with antigen-presenting segment of MHC as claimed in anyone of claims 1 to 6 from FDNB-tagged normal cells, by immuno-affinity chromatography as prophylactic vaccine to build up immuno response in the host. 10. Use of the tumour antigens covalently bound with antigen-presenting segment of MHC as claimed in anyone of claims 1to 6 in making all the cancer cells in the body, vulnerable to in vivo destruction by immune surveillance system of the host. 11. Use of the cell free preparation as claimed in anyone of claims 1 to 6 as an immunogen to reactivate immune responses in the host and in making all cancer cells vulnerable to destruction by immune surveillance system of the host. 12. A cell free immunogen preparation/prophylactic vaccine comprising cell free form of tumor antigen covalently bond with antigen presenting fraction of major histocompatiability complex (MHC), and a diagnostic kit using the same substantially as herein described and illustrated with reference to the accompanying examples. Dated this 14th day of May 2003 ANJAN SEN Of S. MAJUMDAR & CO Applicants' Agent

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502-mum-2003-abstract(18-05-2005).doc

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502-mum-2003-claims(granted)-(18-05-2005).doc

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502-mum-2003-correspondence(21-03-2007).pdf

502-mum-2003-correspondence(ipo)-(26-12-2005).pdf

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