Title of Invention

AN IMPROVED METHOD OF ISOLATION AND PURIFICATION OF GLYCOPROTEIN TIITS HAVING IMMUNOSTIMULATORY AND ANTITUMOR PROPERTY

Abstract

In order to establish the mechanism(s) of immunomodulatory and anti-tumor properties of sheep erythrocytes (SRBC), the T11 target structure (T11TS) or CD58 molecule as a pertinent component of SRBC was isolated, purified and charactered and finally administered in rats with experimentally induced brain tumor. Results showed inhibition and/or abrogation of tumor growth as evidenced through histological evidences and improved survival Subsequent studies on cellular immunity also revealed enhanced lymphocyte mediated cytotoxity, FMN and MF mediated phagocytosis at peripheral immune system in T11TS-treated tumor bearing animals, suggesting potentiation of the cell-mediated immune response. IL-2R expression on splenic lymphocytes has been shown to occur in T11TS-treated tumor bearing animals, suggesting lymphocyte activation. Flowcytometric analysis of CD25 expression has also provided evidences for infiltration of activated lymphocytes in the brain tumor tissues. The analysis of data suggests that T11TS or sheep form of LFA3 is capable of inhibiting/preventing tumor growth in rat brain by way of immunopotentiation (CMI) at the peripheral immune system and thereby facilitating infiltration of the activated lymphocytes into the brain cavity through the blood brain barrier. The results suggest that the purified TUTS fraction has favorable indications as an effective immunotherapeutic module to treat specific diseased conditions such as brain tumor/cancer.

Full Text

2) Field of invention The present invention relates to an immunotherapeutic pharmaceutical preparation in particular a selective glycopeptide active as an immunotherapeutic module especially as an antitumor /cancer immunotherapeutic agent The selective glycopeptide molecule in the immunotherapeutic pharmaceutical preparation can be isolated and characterized from sheep red blood cells (SRBC). The immunotherapeutic pharmaceutical preparation of the selective glycopeptide apart from serving as a broad spectrum immunostimulant favoring cancer load reduction is also directed to serve as a therapeutic probe in other immunodeficiency diseases such as AIDS, retrovirally induced anergic states like hepatits, encephalomyelitis and the like(10,30). 3) Background of invention with regard to drawbacks associated with known art Tumors of the central nervous system (CNS) occupy a unique position within the spectrum of human cancers, and represent often "difficult to treat" neoplasm. Nevertheless, the conventional therapeutic approaches such as surgery, radio and chemotherapy cause a generalized immunesuppression in addition to the lesion to the brain. Several anticancer drugs/compounds have been identified with or from raw chemical compounds or of herbal origin or derived from the organisms like bacteria. The chemical compounds known to favor cancer therapy are broadly termed as chemotherapeutics which serve as the major cancer killer. This identify the functionally discrete domain of the cancer cells as targets like antimetabolite, cytotoxic, cytostatic activity to reduce the tumor load. Compounds derived from plants mostly find the similar mode of activities involving the cancer cell cycle. A typical example vincristine derived from Nayantara plants (Catharanthus roseus). Physical agents like ionizing radiation, laser beam and under specialized circumstances UV radiation (for melanoma and other external malignancy) are also used to minimize the tumor burden. Use of immunological components for the prupose although started much earlier (viz. Bacillus Calmette Guerin by Mathe, 1889., C.parvum by Halpern,1960), was abundant most possibly for the lack of knowledge towards appropriate dosimetry, target specific presentation and immunological activation processes in many case such components were used or advised to use together with chemotherapeutic or radiotherapeutic probes. In recent years bacterial membrane products, cytokines and tumor products have also been tried for immunotherapeutic purpose DRAWBACK OF KNOWN ART The component used so far have been found to be effective in reducing the tumor load in many cases but with a concomitant decrease in the immunological reactivity in the host concerned. The chemotherapeutic & radiotherapeutic manoeuvre are .mostly damaging to the bone marrow aplasia with a simultaneous reflection at the peripheral end and thereby a severe immunosuppression. This resulted in random infective procedures shortening the life expectancy in the individual concerned. Thus use of such materials always contraindicated warning the life span. A generalised functional derangement involving several organ systems also intensify the complications and the patients die mostly due to the secondary manifestation rather than the disease itself. Furthermore, the high price of the chemotherapeutics. cytokine and growth factor drugs etc do not allow a permissible management programme in amongst the cancer patients of low income group. 4) Object of invention The basic object of the present invention is the pharmaceutical preparation of an immunotherapeutic module in particular against cancer and other immunodeficiency diseases. which would have the following advantages/features; i) simple and easy to obtain with appreciable therapeutic effects ; ii) simple and easy to administer ; iii) safe free of any "no bone marrow toxicity and immunosuppression ; iv) helps in preventing also the secondary infections . v) is cost effective and thus affordable to the common people ;. vi) no pathogenic manifestation and adverse effect in the subject (4) Another object of the present invention is directed to provide for a selective glycopeptide which would serve as an immunomodifier in the host concerned especially for treating tumor/cancer and /or other immune disorders such as AIDS and other retrovirally induced anergic states like hepatitis, encephalomyelitis. Yet another object of the present invention is to provide for a selective glycopeptide which would serve as an immunotherapeutic module in particular for immunotherapy against cancer and the like and determine the selective timing, dosimetry target specific presentation to boost immuno-logical activation and make possible the administration of the selective glycopeptide for treating such immunodeficient diseases / conditions in human and non-human mammals. Yet further object of the present invention is directed to provide an immunotherapeutic module for treating cancer and the like comprising of administration of a selective bio-molecule in selective non toxic effect amounts which would not involve any pathogenic manifestations in the host. Thus according to one aspect of the present invention there is provided an pharmaceutical preparation as immunotherapeutic module in particular against cancer and the like comprising : 5) A summary of invention a selective glycopeptide (T11TS) having functional character of stimulating immune-effector cells for tumor killing. The above selective peptide as the active component can be found and isolated from sheep red blood cells. Importantly, it has surprisingly been identified by way of extensive studies under the present invention that the said selective glycopeptide molecule have specific pharmaceutical application in treatment of immunodeficient conditions such as cancer. HIV and the like Surprisingly and importantly it is found by way of the present invention that the selective glycopeptide component (TUTS) such as isolated from SRBC shows a higher immunostimulatory effect when compared to SRBC as a whole used for the same purpose The properties of SRBC when applied to tumor bearing experimental animals were characterised by stimulation of particular cellular components in immunity viz Lymphocytes, neutrophils macrophages and dendritic cells It is identified by way of the present invention that such properties could be boosted when the identified active said biomolecule. per-se is administered in tumor bearing animals. The resultant effect was nearly complete removal of the tumor load. The selective glycopeptide thus showed an uninterrupted, significant immunoboosting and antitumor property compared to the whole SRBC In accordance with another aspect of the present invention there is provided an immuno-therapeutic pharmaceutical preparation as immunotherapeutic module in particular immunotherapeutic module against cancer and the like comprising : said selective glycopeptide having functional characteristics of immune stimulation of cancer inhibition alongwith a pharmaceutically acceptable carrier Suitable carriers that can be used along with the active glycopeptide include any aqueous base or saline only In accordance with yet further aspect of the present invention provided for identification of the timing, dosimetry, target timing, dosimetry target specific to achieve the discussed immuno-therapeutic activity of said selective glycopeptide. comprising the active a selective glycopeptide having above functional character in the dose range of 0.41 mg/kg body weight In accordance with yet further aspect of the present invention there is provided a method of treatment of immunodeficient conditions in human or non-human mammals especially in immunodeficient conditions such as cancer. AIDS Hepatitis comprising administration of an effective non-toxic amount of the selective glycopeptide with or without any water soluble pharmaceutically acceptable carrier thereof 6) Brief description of the accompanying drawing Fig. 1. Fluorescent spectral analysis of brain cell kinetics in normal (N), following ENU mediated tumor induction (8 months) (E) and after T11TS administration in ENU (E) animals. The hyperkinetic "E" cells were brought back to near normal level with T11TS (ET). Fig. 2 (a) to 2(f) Effects of T11TS administration (ET1-ET3) on IL-2R (CD25) expression on splenic lymphocytes (2a:N, 2b:NT, 2c:E, 2d:ETl, 2e:ET2, 2f:ET3): cells with ET1 showed maximum CD25 expression as revealed from flowcytometric analysis (FACS-Calibur, Beckton -Dickinson, USA). Flg.3 (a) to 3 (e) T11TS administration in vivo showing activation of brain infiltrated lymphocytes (3a - N, 3b - E, 3c - ETC, 3d - ET2, 3e - ET3). Fig.3( f ) to 3( J ) Flowcytometric analysis of brain infiltrated lymphocytes. Fig. 4 In-vivo effects of TUTS administration on rosette forming lymphocytes of different groups of rats. Second booster dose (ET2) produced maximum rosette forming efficacy in splenic lymphocytes of ENU treated animals. Fig. 5 In-vivo effects of T11TS on cytotoxic efficacy of splenic lymphocytes. Third booster dose (ET3) was found to induce CTL at its maximum ENU induced animals. Fig.6 Effects of TUTS administered in vivo on PMN mediated phagocytosis. The most significant effect was observed with ET1 with highest phagocytic burst by PMN in tumor bearing animals. Fig. 7 In vivo effects of TUTS on macrophage mediated phagocytosis in different groups of rats. ET1 showed the most significant stimulation of MF for tumor phagocytosis under the event. Fig.8 Molecular weight of the purified T11TS fraction as determined by SDS- PAGE gel electrophoresis. Fig.9 Survival data of normal (N). ethylnitrosourea induced (ENU), and ENU induced tumor bearing animal group treated with different doses of T11TS fraction (ET1, ET2, ET3) as well as ENU-SRBC group. Wherein T11TS treated ENU-induced tumor bearing animal groups the values are higher than the ENU- group. ET1 and SRBC exhibited nearly equal level of efficacy. Fig.I Histological studies show normal glial cell populations in white matter of cerebral cortex with few astrocytes oligodendrocytes and a few neurons. Fig II. The effect of ENU showed grade 4 oligodendroglioma with mitotic figure, giant ceils and absence of intercellular spacing. Fig. III. Effect of first dose of T11TS fraction showed reduced glial cell population with enlarged nucleic due to increased permeability of nuclear membrane to water. Most importantly reduction in the number of oligodendroglioma cells with spongiosis and appototic figures, lymphocyte infiltration and marginatum have been observed with this dose. Fig. IV. The hypocellularity with oedema and degenerative changes and presence of lymphocytes have been observed following administration of second dose of T11TS fraction Fig. V. The third dose of T11TS fraction shows return to the normal glial features with gliosis. Evidence of necrosis and calcification was noted. Fig. VI. Finally the effect of SRBC has been observed through reduced oligodendroglial cell population with gliosis nearly same that of first dose of T11TS fraction (Fig.2f). 7) Detailed description of the invention with reference to drawing The details of the invention, its objects and advantages are explained hereunder in greater details in relation to non-limiting exemplary embodiments as discussed hereunder Isolation of active glycopeptide (T11TS) from sheep red blood cell The method followed steps slightly modified from that of Kitao et al (21) 1 ml volume of packed sheep red blood cells have been incubated for one hour at 37°C in the presence of trypsinphosphate buffer (100µg/ml). The red tinted supernatant was removed and then treated with onequarter volume of 25% trichloroacetic acid to precipitate the non-specific proteins. The clear supernatant was obtained by centrifugation, neutralized with NaOH. and dialyzed against distilled water Since the glycoprotein is acidic in nature, it was separated from neutral peptides by ion exchange chromatography on a DEAE-Cellulose column (15x8 cm) previously equilibrated with 0 05M formate buffer. pH 6 8 The acidic glycopeptide was then eluted with a five chamber gradient system containing 1 ml each of (1) water. (2) 0.05M formic acid (3) 0 2M formic acid (4) 0 4M formic acid and (5) 0.4M formic acid in 0 3M sodium chloride Protein Content of elutes The elutes from the DEAE-cellulose column, were collected as a series of fraction EF I. EF II. EF III. EF IV and EF V and analysed for absorbance at 280 mm which showed a higher O D value for fraction EF III (0 346) compared to those for another fractions. The results are illustrated in TABLE - 1 hereunder TABLE - I Elute Fractions O D 280 EF1 0 001 EFII 0 153 EFIII 0 346 EFIV 0 319 EFV C 13 Table - 1 Spectrophotometric reading (O.D at 280 nm) of different elute fractions (EF) isolated from SRBC membrane EFIII showed the highest OD The results indicated the total extent of protein content from the fraction EM III To estimate the protein content qualitatively from this fraction, Lowry's method (27) was performed and the amount determined was found to be 50 µg/ml Consequently, the amount of TUTS administrable per Kg body weight was calculated to be 0 41 mg/Kg body weight which represent the content in 1 ml Rosette formation Lymphocytes were separated from spleen cell suspension on a percoll density gradient elution method (1). 0.25 ml of 3 - 4 x 106 lymphocytes were mixed with 0.25 ml of 1% (PCV/saline volume) sheep erythrocytes (SRBC) and incubated at 37 °C for 15 minutes. Following brief centrifugation, the preparation was kept at 4 °C overnight. Number of rosettes formed were counted per 200 lymphocytes and expressed as rosette %(34) Rosette inhibition assay Selection of the elute by maximum CD2 ligand binding assay was performed with each elute fraction (I - V). 0 25 ml of 3 - 4 x 106/ ml lymphocytes (splenic/blood) were mixed with 0 5 ml, 1 ml and 1.5 ml portions of glycopeptide fractions in different tubes They were then incubated at 37 °C for 15. 30 and 60 minutes, washed off twice with PBS. and finally, pellets were suspended in 0.25 mi of media (RPMI-1640) without FBS fetal bovine serum). The treated lymphocytes were then subjected to resetting technique described as above. The elute fraction forming minimum number of rosettes was considered as the richest source of T11TS. Rosette Inhibition Assay Results Rosette inhibition assay carried out for the identification of TUTS glycoprotein part from the elute fractions (EF I to EF V) and the minimum number of rosette forming elute was determined as the chosen elute fraction peak Although rosette inhibition was found in all the fractions. EF III exhibited complete inhibition a: represented in TABLE - II hereunder' TABLE - II Fraction Normal Control i Rosette/200 lymphocytes ' 38 % Rosettes 19% 1% 3.5% 0% 4.5% 5.5% .._ EFI EFII EFIIl EFIV EFV Table - II Represents "rosette inhibition assay* by incubating EFI to EFV in vitro with lymphocytes for 60 minutes EFIIl showed complete inhibition of rosette formation and was considered the fraction of choice The immuno-therapeutic effect of selected glycopeptide T11TS for the treatment of tumour/cancer indications was tested as detailed hereunder. E-Rosette Formation The normal animal group (Gr I) showed its rosette forming capacity to be 19%±4.3t and in T11TS injected normal animals it was 24%±3.92. The ENU animal groups (Grill) showed significant decrease in rosette forming capacity, which is 2%±1 03 (p Animals Healthy rats of Drukrey strain of both sexes (strain originally supplied by Central Drug Laboratory, Calcutta. India and maintained subsequently in the laboratory) formed the materials of the whole investigations. They were grouped as follows (I) Normal untreated controls (N), (II) Normal-T11TS: Normal animals receiving (1 ml. i.p.) of T11TS fraction. (Ill) ENU: Animals received 80 mg/kg body wt. of ethyl nitrosourea (ENU) 7-10 days after birth (i.p). Group (IV). (V) and (VI) received 1ml. 2ml and 3ml of T11TS fraction respectively at the end of 7th month of ENU administration and assigned as ET1, ET2 and ET3 respectively. Group (VII) consisted of rats injected with 0.5ml of 7% SRBC (PCV/VoI. The animals, comprising of-20 in each group were weaned at 30 days of age and housed separately in groups of five animals in isolated cages. All animals were fed autoclaved Hind-Lever pellet and water ad libitum Rats were examined daily and weighed weekly throughout the experimental period. ENU-administration for brain tumor induction ENU was freshly prepared by dissolving 10 mg/ml. in sterile saline and adjusting the pH to 4.5 with crystalline ascorbic acid. ENU was injected intraperitoneally (i.p.) to 100 animals with an acute dose of 80 mg/kg body weight in the first week after birth (12,22.23,26). Tumor was detected after 5-7 months after ENU administration. Cytokinetic study of brain tumors The whole brain homogenate was prepared as described before (7) from each group of animals, namely the normal (N). the ENU treated (E) and those having both ENU and T11TS (11 dose) (ETI) The cells as prepared in a homogeneous suspension were counted in the order of 107 /ml and then stained with 10 µ of HO-33342 fluorochrome dye for 15 minutes in PBS Following several washes the cells were resuspended in PBS and fluorescence activity measured in a spectrofluorimeter (Hitachi. Japan) Hyperkinetic malignant cells of brain are regulated by T11TS Compared to the healthy normal control, the high fluorescent spectra (six times) of ENU treated rat brain cells presented a hyperkinetic malignant feature of brain cells concerned The results are illustrated in accompanying Figure 1 The results clearly indicate that administration of T11TS in vitro was able to limit the cytokinetic process of the ENU treated brain cells at normal physiology. Cell surface immunofluorescence and fluorescence activated cell sorting Splenic lymphocytes separated on a percoll gradient (1.5) were washed twice with PBS and final volume adjusted to 1 x 107 cells, incubated in dark with FITC conjugated CD25 monoclonal antibody (5 µ) (Beckton Dickinson, USA). After 30 minutes of incubation the cells were washed thrice with PBS, and analysed on FACS calibur (Becton Dickinson. USA) (Argon Laser, excitation at 488 nm. 515 kband pan filter). Whole brain cells were also tagged with anti-CD25-FITC conjugate and in both the preparations isotype control (IgGka) was maintained. In total 10,000 events were acquired and data were analysed using cell Quest software (Beckton Dickinson, USA). IL-2R (CD25) as an indicator for T-Cell Activation The effects of T11TS fraction on CD25 expression on splenic T lymphocytes in animals with experimentally induced tumor (E) (with ENU) revealed interesting results. The CD25(IL-2R) expression on splenic T cells of normal group of animals (Gr I) was 2.66%. The lymphocytes of the group III animals (ENU-group) showed a marked decrease in IL-2R expression in comparison to the normal ones The group IV (ETI), i e., the tumor bearing animals that received a single booster dose of 1 ml of T11TS-fraction. showed a highly significant increase in IL-2R expression on splenic lymphocytes (97 06%) whereas group V and VI (ET1, ET3) were found poorly responding (3 46% and 1 34 respectively) The results are illustrated in accompanying Figs 2(a) - 2(g) Flowcytometric analysis (FACS) carried out also suggested the diminished expression of IL-2R on tumour infiltrating lymphocytes could be due to the presence of some soluble blocking factor, such as IL2 blocking factor and/or some other inhibitory effects encountered in that microenvironment By administration of T11TS of subsequent booster dose it was possible to however remove the microenvironmental interference to re-express CD25 (IL-2R) This is clearly illustrated in the results under Figures 3(a) to 3(e) Lymphocytes Mediated Cytotoxicity: CT' Assay This was found only using a fluorochrome dye Hoechest 33342 (H033342. Sigma, USA) HO-33342 binds to DNA of cells irreversibly without leakage until lysed. Tumor cells (target) were labelled with H033342 fluorochrome dye (6 µ/106/ml) for fifteen minutes at 37°C and excess dye was washed off. Cytotoxicity assay was performed by maintaining an effector - target ratio at 10.1 through an incubation (37°C, 4% CO2 - air environment) period of 18 hours. Fluorochrome released as per target lysis measured in a spectrofluorimeter (Hitachi, Tokyo) provided an index of cytotoxic efficacy of effectors (8) Enhancement of cell mediated cytotoxic activity as observed with elute fraction III In vitro functional assay for CTL activity with the various features EF1 to EFV and control were studied and the results represented in Fig. 5. As illustrated in Table - III above with EF III (incubated and washed lymphocytes) the CTL activity has been found to be 6%±0.62. Cytotoxic activity of lymphocytes of normal group of animals was 30%±3.62 which decreased significantly to 7%± 1 69 (p PMN - mediated phagocytosis Splenic tissues was obtained from group I to VII animal groups Single cell suspensions were prepared by teasing splenic tissues with forceps, followed by percoll density gradient centrifugation 3 ml of cell suspensions was layered on 5 ml of percoll gradient (density 1 089) and centrifuged for 20 minutes at 800 g The PMN layer was then removed from the interface washed thrice with PBS and finally suspended in 1 ml of media. Then neutrophils (PNMs) were allowed to react with target tumor cells in presence of NBT for 18 hours. Reduction of yellow NBT to blue formazan indicated the extent of phagocytic burst by effectors concerned (6) Phagocytic activity of the polymorphonuclear neutrophil has been observed in T11TS administered animal The phagocytic activity of PMN was significantly reduced in ENU induced tumor group (Gr.lII animals), as revealed from reduced colour density of the phagosomes (0 016+0.001) where as it was 0.03±0.002 in normal control group (Fig.6). The normal group receiving T11TS showed an elevation of phagocytic activity (0.053±0.003). Further, it was significantly increased with the single booster dose of T11TS fraction in ETI animals. (0.033 ±0 002) which restored the value back to its normal level The tumor bearing animals that received the second booster dose of 1 ml of T11TS fraction (F.T2) showed still significantly higher phagocytic activity (0.065 + 0.006), (p Macrophage (M0) mediated phagocytosis Single cell suspension was prepared from the splenic tissue as described above The cell suspensions were incubated in plastic petridish (Corning, USA) in a CO2incubator for 30 minutes for adherence The non-adherent cells were then washed off and M0 were separated by washing thrice with PBS-EDTA. The cells were then washed thrice with PBS and suspended in 1 ml of media The macrophages were then allowed to react with the target tumor cells in presence of NBT as described above. induction of macrophage-mediated phagocytosis observed in T11TS treated animal The phagocytic activity of normal macrophages (Mc i as determined through NBT reduction assay (O.D. of reduced phagosome was found to be 0 037 -0 002 (Fig.6). ENU treated animals showed significant decline (p In-Vitro Studies with elute fraction III Lymphocytes, PMN and macrophages (purified, as described before) from the normal splenic tissue (group I) were incubated each separately with 0.5 ml of elute fraction EF-III for 60 minutes. Cells were washed thrice with PBS and suspended in 1 ml of media (RPMI-1640). Each type of cells were then used as effector cells in functional assays for immune parameter such as CTL, PMN and MF mediated phagocytosis. In vitro effects of T11TS on the functional efficacy of immunocyte of normal rats: Rosetting and cytotoxic capacity of lymphocytes and phagocytic efficacy of PMN and M0 were all inhibited drastically as further represented in Table - III hereunder; TABLE - III Parameters Normal In vitro T11TS Rosette (%) 19% 0% CTL(%) 30% 6±0.62% PMN (O.D.) 0.030±0.002 - MF(O.D.) 0.037±0.002 - Table-III In vitro effects of T11Ts on the functional efficacy of immunocyte of normal rats : Rosetting and cytotoxic capacity of lymphocytes and phagocytic efficacy of PMN and M0 were all inhibited drastically Determination of Molecular Weight by SDS-PAGE gel electrophoresis Molecular weight of the T11TS-fraciion (glycoprotein) was determined by analytical sodium dodecyl sulphatic-polyacrylamide gel electrophoresis SDS-PAGE, 40µl. of T11TS-fraction and 40ui. of electrophoresis sample buffer (2.3 ml 10% SDS. 1ml. 0.625 M-tris-HCl. Ph 6.8. 1 ml.glycerol, and 5.2 ml. distilled water) was mixed. To this 10 mg. Of bromophenol blue and a 2.5 % solution of ß-mercaptoethanol was added. The sample was placed in a boiling water bath for 5 minutes . cooled to room temperature and electrophoresed in 4% staking gel over a 10% resolving gel at 200V for 40 minutes. Protein bands were visualized by staining with 0.2% coomassre brilliant blue (Sigma. U.S.A.) in 50% ethanol 7% acetic acid for 4-5 hr. and destained in 50% ethanol and 7% acetic acid Standard molecular weight markers (Bio-Rad, CA. US A.) of molecular weight 90. 80, 32. 20. 18 and 15 kDa (Bio-Rad, CA, U.S.A.) were used. On the basis of SDS-PAGE analysis the molecular weight of the T11TS- glycoprotein was found to be 17 Kd approximately Survival studies Of all the animals prepared as above . observations were made to account for the total number of da\s survived by individual animals and the mean survival time in each group were determined Further, progressive neurologic signs and weight loss were taken into account in selecting the animals for the tumor development study at the initial phase. Moreover , histological studies of brain of such animals were made to ascertain the study. The average survival for the normal control animal was found to be 705+35 days(n= 10 ). In contrast . the average survival value for the ENU treated animals was significantly ( p 18 Histological Studies Portions of brain tissues from respective group of animals were prepared for routine histological studies tissues were Fixed in 10% formal-buffer overnight and finally dehydrated and embedded in paraffin through histokinet processing Sections were cut at 5µ, thickness and finally stained with routine haematoxilinc / cosine. Histological studies show normal glial cell populations in white matter of cerebral cortex (Fig. ) with few astrocytes oligodendrocytes and a few neurons. The effect of ENU showed grade 4 oligodendroglioma with mitotic figure, giant cells and absence of intercellular spacing (Fig. ). Effect of first dose of T11TS fraction showed reduced glial cell population with enlarged nucleic due to increased permeability of nuclear membrane to water. Most importantly reduced in the number of oligodendroglioma cells with spongiosis and appototic figures, lymphocyte infiltration and margination have been observed with this dose (Fig ). The Hypocellularity with oedema and degenerative changes and presence of lymphocytes have been observed following administration of second dose of T11TS fraction (Fig. ). The third dose of T11Ts fraction shows return to the normal glial features with gliosis. Evidence of necrosis and calcification is noted (Fig. ). Finally the effect of SRBC has been observed through reduced oligodendroglial cell population with gliosis nearly same that of first dose of T11TS fraction (Fig.). The first effective outcome of T11TS administration (0.41 mg/ Kg body weight) in ENU treated animals was the abrogation or inhibition of brain tumor induction in rats This has long been proved that N-ethyl-Nitrosourea (ENU) is the most potent of neuro carcinogens and produce tumors with morphological and biological similarities to naturally occurring neural neoplasms in man and animals (59). Histological studies have provided evidence for neoplastic growth following five months of ENU administration. When compared to normal rat brain it clearly demonstrates grade IV oligodendroglioma with multiple mitotic figures and oligodendroglial cells in close apposition with no intercellular spaces (60) This is corroborated with the findings that the foci of oligodendrocytes have been reported in the cortex and basal ganglia in rats treated transplacentally with ENU (61) a dual pathogenesis for ENU-induced gliomas seems to be the most plausible explanation (62) Oligodendroglioma thus develop from these early oligodendrocyte proliferation while all other gliomas owe their origins to the subependymal plate cells, in the present study administration of T11TS fraction in ENU treated animals demonstrated anti-tumor activity in a dose dependent manner as evidenced through the reversion of neoplastic glial features to normal glial features. Infiltration of lymphocytes and margination of the endothelial lining in the brain the hallmark of the first dose of T11TS administration in ENU animals. The hypocellularity observed in the first dose gives way to apoptotic and enlarged nuclei due to enhanced permeability of the ruptured nuclear membrane Finally, the third dose leads to the total clearance of the neoplastic cells with necrosis and calcification. It seems likely that T11TS fraction has the capability to inhibit or block tumor proliferation not only throgh the potentiation of the immune network, but also by interfering with the mitotic rate of the malignant cells It is assumed that it possibly do that by 1) inducing a cytostatic effect 2) differentiation induction 3) blocking DNA synthesis and growth factor interactions and 4) interactin on cellular oncogene expression Recently molecular biology of ENU induced rat brain tumor identified a new oncogene, neu which is detectable by transfection into mouse NIH 3TC cells (62 ). The data obtained from fluoroscopic analysis further directed the investigation towards the causative factors responsible for tumor inhibition (Fig 1). The role of cellular immune components as direct killer of tumor cells was considered at the first instance However, the role of T11TS in inhibiting brain tumor in a direct or cytokine induced manner cannot be ruled out under the event 20 We Claim 1. An improved method of isolation and purification of T11TS glycoprotein from Sheep Red Blood Cell (SRBC) having the immunostimulatory and antitumor property where at first the SRBC was centrifuged in 2000-2500 r.p.m. for 10 - 15 minutes to obtain the packed cell volume. The packed cell volume was then subjected to enzymatic digestion at 37°C. Then the digested SRBC was centrifuged in 2000-2500 r.p.m. for 15 - 20 minutes. After the centrifugation the red tinted supernatant was taken and treated with an organic acid of specific strength to precipitate the non-specific proteins. After the nonspecific proteins being precipitated a clear supernatant was obtained by centrifugation at 1500-2000 r.p.m. for 5 -7 minutes. The acidic supernatant was neutralized with an alkali of specific strength. Next, the neutralized supernatant was subjected to chromatographic process, where the column was previously set at a certain pH with organic buffer. The said supernatant was charged for 20-30 minutes on the column for proper binding with the column material. After that the column was eluted with distilled water and organic acid of increasing ionic strengths. The elute fraction were neutralized with N/10 NaOH. The glycoprotein was identified from elute fraction III obtained through DEAE-cellulose column by a functional immunological assay called rosette inhibition assay. In addition the amount of T11TS glycoprotein was 50ug/ml of elute fraction as determined by Lowry's method. 2. An improved method of isolation and purification of T11TS glycoprotein from Sheep Red Blood Cell (SRBC) as claimed in Claim 1, wherein the said glycoprotein was isolated by column chromatography using Di-Ethyl Amino Ethyl (DEAE) Cellulose as adsorbant and the column length of 8 cm and radius of 1.5cm. 21 3. An improved method of isolation and purification of T11TS glycoprotein from Sheep Red Blood Cell (SRBC) as claimed in Claim 1, wherein the said glycoprotein was isolated in a column previously set at pH 6.8 by organic acid buffer which in this case was formate buffer of 0.05 M strength. 4. An improved method of isolation and purification of T11TS glycoprotein from Sheep Red Blood Cell (SRBC) as claimed in Claim 1, wherein the said glycoprotein was isolated from SRBC by digesting the packed cell volume of SRBC using trypsin enzyme. 5. An improved method of isolation and purification of T11TS glycoprotein from Sheep Red Blood Cell (SRBC) as claimed in Claim 1, wherein the said glycoprotein was isolated from SRBC by digesting the packed cell volume of SRBC with the enzyme trypsin dissolved in phosphate buffer (trypsin-phosphate buffer) having a strength of 1000 mg of trypsin (approximately 1000BAEE units/mg) /ml of phosphate buffer. 6. An improved method of isolation and purification of T11TS glycoprotein from Sheep Red Blood Cell (SRBC) as claimed in Claim 1, wherein the said glycoprotein was isolated from SRBC by digesting the packed cell volume of SRBC with equal volume of trypsin phosphate buffer with an incubation An improved method of isolation and purification of T11TS glycoprotein from Sheep Red Blood Cell (SRBC) as claimed in Claim 1, wherein the red tinted supernatant obtained by centrifugation of trypsin digested SRBC in the process of isolation of the said glycoprotein was treated with the organic acid, which in this case was one-quarter volume of 25% tri-chloro acetic acid (TCA). 22 8. An improved method of isolation and purification of T11TS glycoprotein from Sheep Red Blood Cell (SRBC) as claimed in Claim 1, wherein the said glycoprotein was isolated by eluting the DEAE cellulose column with distilled water and organic acid of increasing ionic strength which in this case were 0.05 M formic acid, 0.2M formic acid, 0.4M formic acid and 0.4M formic acid in 0.3 M NaCI respectively. 9. An improved method of isolation and purification of T11TS glycoprotein from Sheep Red Blood Cell (SRBC) as claimed in Claim 1, wherein the said glycoprotein was isolated by eluting the DEAE cellulose column with distilled water and organic acid of increasing ionic strength of same volume of the neutralized clear supernatant subjected to column chromatography. In order to establish the mechanism(s) of immunomodulatory and anti-tumor properties of sheep erythrocytes (SRBC), the T11 target structure (T11TS) or CD58 molecule as a pertinent component of SRBC was isolated, purified and charactered and finally administered in rats with experimentally induced brain tumor. Results showed inhibition and/or abrogation of tumor growth as evidenced through histological evidences and improved survival Subsequent studies on cellular immunity also revealed enhanced lymphocyte mediated cytotoxity, FMN and MF mediated phagocytosis at peripheral immune system in T11TS-treated tumor bearing animals, suggesting potentiation of the cell-mediated immune response. IL-2R expression on splenic lymphocytes has been shown to occur in T11TS-treated tumor bearing animals, suggesting lymphocyte activation. Flowcytometric analysis of CD25 expression has also provided evidences for infiltration of activated lymphocytes in the brain tumor tissues. The analysis of data suggests that T11TS or sheep form of LFA3 is capable of inhibiting/preventing tumor growth in rat brain by way of immunopotentiation (CMI) at the peripheral immune system and thereby facilitating infiltration of the activated lymphocytes into the brain cavity through the blood brain barrier. The results suggest that the purified TUTS fraction has favorable indications as an effective immunotherapeutic module to treat specific diseased conditions such as brain tumor/cancer.

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00202-cal-2001 form-3.pdf

00202-cal-2001 pa.pdf

202-cal-2001-granted-abstract.pdf

202-cal-2001-granted-acceptance publication.pdf

202-cal-2001-granted-claims.pdf

202-cal-2001-granted-correspondence.pdf

202-cal-2001-granted-description (complete).pdf

202-cal-2001-granted-description (provisional).pdf

202-cal-2001-granted-drawings.pdf

202-cal-2001-granted-form 1.pdf

202-cal-2001-granted-form 13.pdf

202-cal-2001-granted-form 2.pdf

202-cal-2001-granted-form 3.pdf

202-cal-2001-granted-letter patent.pdf

202-cal-2001-granted-pa.pdf

202-cal-2001-granted-specification.pdf