Title of Invention	"A PROCESS FOR THE PREPARATION OF A NOVEL BIOMARKER SPECIFIC FOR O-ACETYLATED SIALIC ACID USEFUL FOR THE DIAGNOSIS, MONITORING OUTCOME OF TREATMENT AND PREDICTION OF RELAPSE."
Abstract	A process for preparation of a novel protein biomarker specific for 0-acetylated sialic add useful for the diagnosis, monitoring outcome of treatment and prediction of relapse which comprises, (i) separating serum from blood collected from patients of acute lymphoblastic leukemia by known methods/emoving low molecular weight fractions • and galactose binding proteins (non-specific protein) from the serum by column chromatography on affinity matrix, (ii) collecting unbound fraction from affinity matrix (iii) passing the galactose free protein fraction obtained in step (ii) over another affinity matrix to capture 0-acetyl sialic add specific protein fraction (IV) eluting specific protein fraction with a buffer at alkaline pH in the range of 8.0 -11.0, immediately neutralizing the fraction (v) passing Oacetyl sialic acid specific protein obtained in step (iv) over Protein G- agarose or protein A agarose or protein A Sepharose or protein G Sepharose or anti - human immunoglobulin or only IgG / IgM coupled to Sepharose or agarose column to get Oacetyic sialic acid specific protein immunoglobulin and , eluted with a buffer at addic pH in the,range.of 2.0-6.5 immediately neutralizing the fraction and dialyzing to get novel biomarker.

Title of Invention

"A PROCESS FOR THE PREPARATION OF A NOVEL BIOMARKER SPECIFIC FOR O-ACETYLATED SIALIC ACID USEFUL FOR THE DIAGNOSIS, MONITORING OUTCOME OF TREATMENT AND PREDICTION OF RELAPSE."

Abstract

A process for preparation of a novel protein biomarker specific for 0-acetylated sialic add useful for the diagnosis, monitoring outcome of treatment and prediction of relapse which comprises, (i) separating serum from blood collected from patients of acute lymphoblastic leukemia by known methods/emoving low molecular weight fractions • and galactose binding proteins (non-specific protein) from the serum by column chromatography on affinity matrix, (ii) collecting unbound fraction from affinity matrix (iii) passing the galactose free protein fraction obtained in step (ii) over another affinity matrix to capture 0-acetyl sialic add specific protein fraction (IV) eluting specific protein fraction with a buffer at alkaline pH in the range of 8.0 -11.0, immediately neutralizing the fraction (v) passing Oacetyl sialic acid specific protein obtained in step (iv) over Protein G- agarose or protein A agarose or protein A Sepharose or protein G Sepharose or anti - human immunoglobulin or only IgG / IgM coupled to Sepharose or agarose column to get Oacetyic sialic acid specific protein immunoglobulin and , eluted with a buffer at addic pH in the,range.of 2.0-6.5 immediately neutralizing the fraction and dialyzing to get novel biomarker.

Full Text	The present invention relates to a process for the preparation of a novel biomarker specific for O-acetylated sialic acid useful for the diagnosis, monitoring outcome of treatment and prediction of relapse and biomarker prepared thereby. Broadly the main usage of this invention is to provide-A process..for the preparation of a novel biomarker specific for O-acetylated sialic acid useful for the diagnosis, monitoring outcome of treatment and prediction of relapse and biomarker prepared thereby. The leukemia are a heterogeneous group of neoplasm arising from the malignant transformation of hematopoietic i.e. biood forming cells. Leukemia can be broadly classified accordingly to the cell type involved primarily (myeloid or lymphoid) and as acute and chronic depending on the natural history of the disease. Acute lymphoblastic leukemia (ALL) is the commonest type of leukemia in childhood. It is primarily a disease of children and young adults, it occurs in all races with a peak incidence in children between 3 and 5 years of age. ALL is diagnosed in 2000 -3000 new cases of children leukemia in United States each year, whereas Acute myelogenous leukemia is diagnosed in only 500 children and Chronic myeloid leukemia fewer than 100. About 40 million children in USA are effected under the group of 15 years about 3/4 th of these have ALL. Pediatric hematopoietic malignancies rank first in cancer incidence arid mortality in children and are responsible for roughly 40% of childhood related death. The causes are not known but environmental agents including irradiation, chemical carcinogens, cytogenetic abnormalities and retrovirus Infections are known to play an important role in the etiology of leukemia. For instance, individual with occupational radiation exposure, patients receiving radiation therapy or Japanese survivors of the atomic bomb explosions have a predictable and dose related increased incidence of leukemia. For oncologist, Acute iymphoblastic leukemia (ALL) represent a major therapeutic success as this can be achieved in nearly 65% of patients (Pui CH, Crist W M Jr. Current Opinion in Oncology, Vol7, p 36, 1995 and Pui CH, Crist W M Jr. Lancet ,Vol 347, p1783,1996 ). However, relapse and eventually treatment failure occur in nearly many cases receiving identical treatment and this area is a major challenge for leukemia specialists (Pui CH, Crist W M Jr. New England Journal of Medicine, Vol 332, p1618, 1995). At the time of diagnosis, the leuKemic mass is usually between 1011-1012 cells and available chemotherapeutic agents produce a fractional cell kill capable of a 3 to 5 log kill resulting in the elimination of 99.99 to 99.999% of leukemia cells. The persistence of this remaining 0.01 to 0.001% leukemic cells tantamount to the persistence of 108 to 109 cells respectively. What is important is these persisting leukemic cells are not detectable by standard morphology in bone marrow or peripheral blood, it is these ceils that are responsible for relapse, if post induction chemotherapy fails to eradicate them. To eliminate, this non-detectable yet existing leukemic cell mass, maintenance therapy is given for an extended period (2-2.5 years) with the purpose of reducing the possible relapse. It is defined as the presence of leukemic cells not detectable by morphology. Assays to detect residual blast cells is the need of the hour as these will help the clinician to assess the effect of treatment on tumor burden and allow anticipation of relapse with greater precision (Brisco MJ, Condan J, Hughes E.etal. Lancet, Vo! 343, p196,1994). Currently no specific marker is available to pinpoint the dose of chemotherapy and duration of maintenance therapy in acute lymphoblastic leukemia. Existing methods for the detection of leukemia blast cells are i) cytomorphology and karyotyping ii) Immunological methods iii) molecular detection. Cytomorphology and karyotyping : Acquired non-random chromosomal translocation occur in 30-70% of ALL patients and can serve as marker of disease. But the approach has limited sensitivity (1-5%) primarily due to the paucity of leukemic cells during clinical remission (Campana D, Pui CH, Blood,Vol 85, p1416, 1995). Fluorescent in situ hybridization using chromosome specific or locus specific probes allows to identify abnormally ties in cells at metaphase (Le Beau MM, Blood, Vol 81, p1979, 1993). However, sensitivity remains at the 1% level. Immunological Methods: Immunological Methods based on the recognition of leukemia associated phenotypes not usually found in normal bone marrow have had promising result. Immunophenotyping has been complemented by flow cytometric analysis where a combination of markers have been able to quantify MRD with a sensitivity is usually in the order of 102 to 103 which Is inferior to available to DNA based method (Meydan N et al. Nature, Vol 379, p645, 1996). Molecular approaches: In the majority of cases, relapse of acute lymphoblastic leukemia is thought to involve the same leukemic clone as the original disease (Bunin NJ et.al., Leukemia,Vol 4, p727, 1990) around 80% of cases of childhood acute lymphoblastic leukemia are due to clonal expansion of precursor B cells and have rearrangement of JgH gene, from which specific DNA probes have been generated. Several PCR methods (Brisco M J, Condan J, Hughes E, Lancet, Vol 343, p196,1994; Veelken H, Tyeko 8, Sklar J., Blood, Vol78, p1318, 1991; Wasserman R, Galili N, Ito J, et al., Journal of Clinical Oncology, VoUO, p1879, 1992) have been reported. This technique which detects leukemia specific DNA sequences such as fusion regions of immunogiobulin (Ig) and TcR genes with a sensitivity of 10~5 for the detection of residual disease in childhood acute lymphoblastic leukemia. All these published methods are successful in only half of the patients since different individuals can show different rearrangement of immunogiobulin genes or T cell Receptor genes. The principle Drawbacks of the PCR methods for routine follow up of the patients are: (i) occurrence of false positives due to contamination- of reaction mix with previously employed samples (ii) occurrence of false negatives results owing to degraded RNA or DNA or clonal evolution in approximately 20% of cases (iii) not all leukemic specific gene rearrangements are amenable to initial amplification of PCR using universal primers (iv) a heterogeneous distribution of residual leukemic cells may result in sampling error since gene rearrangements may be different in different individuals (V). Therefore, the reliability of PCR assays depends on the use of stringent quantitation protocols and analysis of multiple genetic targets to prevent false negative results due to changes in the pattern of gene rearrangement during the disease course. These mandatory technical requirements further complicate an already laborious procedure and also make it expensive thereby limiting its suitability for routine clinical use . In comparison, use of this novel biomarker effectively will serve as an index to reflect the clinical status of individual patients following therapy. Since PCR undetectable residual disease is necessary for cures in most patients, it can be proposed that molecular remission defined as PCR undetectable disease,is a milestone and target for achieving cure. Evidently all these problems are unlikely to be exploited for commercial basis, In India, the research in the field of acute lymphoblastic leukemia is mainly carried out at the Tata Memorial Hospital, Bombay. Their interest in acute lymphoblastic leukemia is focused on cytogenetics (Gladstone D et. al., Indian Journal of Medical Research, Vol 99, p264, 1994), infection analysis (Raje B et.al. Pediatric Hematology and Oncology,Voi11, p271, 1994) and central nervous system relapse (Iyer A et.al., Leukemia and Lymphoma, Vol 13, p183, 1994): - Sialic acids are a family of derivatives of N-acetyl or N-glycolyl neuraminic acids and are very important constituents of cell surface architecture. Sialic acids piay an important role in receptors for viruses, peptide hormones and toxins. Sialic acids also function as masking agents on antigens, receptors and other recognition sites of the cell surface (Varki A, Glycobiology, Vol2, p25, 1992). The O-substituted sialic acids exhibit species and tissue specific distribution in animals (Schauer R, Advanced Carbohydrate Chemistry Biochemistry, Vol40, p131, 1982). Changes in siaiic acid and the degree of O-acetyiation of sialic acid residues have been reported in transformed-and malignant cells. Recently, in our laboratory, exploiting the restricted specificity of 9-O-acetylated sialic acid binding lectin, specific biomarkers namely 9-O-acetyl sialoglycocongugates on lymphoblast of 87 children suffering from ALL have been identified (Sinha D, Mandal C and Bhattacharyya D et a/., Leukemia,Vol13, p119:125,1999) and assessed their differential expression at different phases of therapy (Sinha D, Mandal C and Bhattacharya D. 1999 Leukemia 13 (2) 309-312; Mandal C, Sinha D, Sharma V and Bhattacharya D, Indian Journal of Biochemistry Biophysics, Vol34, p82, 1997). A blood based lymphoproliferation assay to monitor the treatment outcome of ALL patients (n = 203) have been successfully developed (Sinha D, Mandal C and Bhattacharyya D. Leukemia Research, 1999 (in press); and Mandai C et al UK patent 1999 PATENT APP in Europe filo No St8V 2509/D/96 allowed; Sinha D, MandalC and Bhattacharyya D. Leukemia Research, 1999 (in press). An extensive world wide patent search has not shown any patent which claims for serum based reagent for monitoring treatment outcome in ALL. Considering the drawbacks of the above mentioned methods, we felt to produce a reagent and we are successful not only in detection and purifying the reagent but also we have developed a diagnostic assay which is simple, sensitive and specific to detect and monitor treatment outcome of ALL. We have recently detected a novel biomarker specific for O-acetylated sialic acid in patients serum against 9-O-acetyl sialoglycocongugates which appeared on lymphoblasts. There is no serum based methods are available for diagnosis, monitoring the treatment outcome and prediction of relapse of ALL patients. There is presently no group, nationally or internationally who have detected specific reagent from the serum and used it for monitoring treatment outcome of ALL employing a sialoglycoprotein namely bovine submaxillary mucin. The selectivity of this biomarker specific for O-acetylated sialic acid has allowed not only the identification but also monitoring cancer cells using serum from the ALL patients. The main objective of the present invention is to provide a process for the preparation of a novel biomarker specific for O-acetylated sialic acid useful for the diagnosis, monitoring outcome of treatment and prediction of relapse and biomarker prepared thereby which obviates the drawbacks as detailed above. Another objective of the present invention is to provide a novel biomarker by the process of present invention and also provide a process for the quantification of a novel biamarfrer specific for O-acetylated sialic acid present in the serum of ALL patient with the help of a simple, specific, sensitive, non-invasive and economical bovine submaxillary mucin ELISA which allows the assessment of the treatment outcome of ALL patients. This will specially be helpful for the detection of a novel biomarker specific for O-acetylated sialic,acid which will reflect chemotheraputic outcome in these children at different clinical stages of the disease. So, this invention provides an indicator as to when and how long should chemotherapy be continued and to predict the probability of relapse. Therefore, this reagent will serve as an effective measure in the battle for monitoring treatment outcome in patients suffering from acute lymphoblastic leukemia. Accordinglyrthe present invention provides a process for preparation of a novel protein biomarker specific for O-acetylated sialic acid useful for the diagnosis, monitoring outcome of treatment and prediction of relapse which comprises, (i) separating serum from blood collected from patients of acute lymphoblastic leukemia by known methods removing low molecular weight fractions and galactose binding protetnsjfrom the serum by column chromatograpny on affinity matrix, (ii) collecting unbound fraction from affinity matrix (iii) passing the galactose free protein fraction obtained, in step,(ii) over another affinity matrix to capture O-acetyl sialic acid specific protein fractions (IV) eluting specific protein fraction with a buffer at alkaline pH in the range of 8.0 -11.0, immediately neutralizing the fraction (v) passing O-acetyl sialic acid specific protein obtained in step (iv) over Protein G agarose or protein A agarose or protein A Sepharose or protein G Sepharose or anti - human immunoglobulin or only IgG / IgM coupled to Sepharose or agarose column to get O-acetyl sialic acid specific protein immunoglobulin and eluted with a buffer at acidic pH in the range of 2.0-6.5, immediately neutralizing the fraction and dialyztng to get novel biomarker. In an embodiment of the present invention the serum may be separated from the blood of patients of acute lymphoblastic leukemia in which blood have been collected either in absence or in presence of any anti-coagulants such as alsevers solution, potassium oxaiate, ammonium oxalate, heparin, dextrose etc. In another embodiment of the present invention the solvent used for removal of albumin and other low molecular weight proteins may be saturated ammonium sulfate solution in the range of 15% to 40%. in another embodiment of the present invention the galactose binding proteins may be removed by using any galactose containing glycoproteins such as asialo bovine submaxillary mucin, asialo sheep submaxillary mucin, asialo human chronic gonadotropin, asialo equine gastric mucin or hog gastric mucin etc. coupled with Sepharose or agarose as an affinity matrix. In another embodiment of the present invention the affinity matrix, for capturing O acetyl sialic acids specific fractions, used may be any available sialogiycoconjugates having terminal O acetyl sialic acids such as bovine submaxiilary mucin or any gangliosides having terminal O acetyl sialic acids such as GD3. In another embodiment of the present invention the solvent used for eiuting O acetyl sialic acids specific proteins may be any buffer such as ammonium hydroxide, borste buffer, carbonate bicarbonate buffer and ammonium bicarbonate buffer with high pH*from 8 to 11 even expensive sugar such as O-acetylated sialic acid (as a monocaccharide or disaccharides or higher oligosaccharides with subterminal galactose or N acetyl galactosamine) may be used as eiuting sugar. Molarity of buffer may be in the range of 0.02-0.5M In another embodiment of the present invention neutralizing may be carried out with sodium acetate or monosodium phosphate for better stability of the eluted protein. If sugars are used as eiuting solution it may be removed simply by dialysis. Dialysis may be carried out at cold temperature between 4-25°C for 24 to 72 hours to get novel biomarker. Accordingly, the present invention provide a novel biomarker by the process of present invention and used the same for diagnosis, monitoring outcome of treatment and prediction of relapse using a bovine submaxillary mucin-Enzyme linked immunosorbent assay (ELISA). The novelty of this process is to capture the novel biomarker specific for O-acetylated sialic acid by using bovine submaxillary mucin as an affinity matrix after careful removal of albumin, other low molecular weight fractions and galactose binding proteins from ALL serum. Characterization of this novel biomarker by several biochemical, immunochemical techniques it reveals that (I) biomarker is specific to leukemic blast cells in acute lymphoblastic leukemia, (ii) Irrespective of the lymphocytic origin of cancer cells such as acuts lymphoblastic leukemia of B lymphocytes or acute lymphoblastic leukemia of T lymphocytes this novel biomarker shows equal specificity. Therefore, it may be consider as common biomarker and used for diagnosis of both types of acute lymphablastic leukemia, (iii) The amount of a novel biomarker specific for O-acetylated sialic acid is determined and has been exploited to correlate the status of patients with regards to their blast cells which stay in peripheral blood even after chemotherapy. The quantity of a novel biomarker specific for O- acetylated sialic acid has been established to be directly proportional to presence" of leukemic blast cells i.e. cancer cells, therefore, by measuring the amount of novel biomarker by newly developed ELiSA it is possible to know the status of different stages of the disease, (iv) Thus its correlation with the status of the disease is assessed and confirmed that the novel biomarker has both diagnostic and prognostic potential, (v) The immunoglobulin .G (IgG) subclass distribution of these antibodies reacting with O-AcSA in patients in comparison to normal healthy individuals has been characterized by the ELISA. Amongst the four human IgG subclasses, IgG, and lgG2 are significantly increased in patients as compared to normal individuals. However, their lgG3 and lgG4 levels are unchanged and comparable to normal healthy individuals. Therefore measuring both IgGi and lgG2 specific for O- Acetyl sialic acid in patients by an isotype ELISA is very much beneficial. The disease specific IgM antibody has also been found to be potentially important. Detection of this novel biomarker specific for O-acetylated sialic acid in patients serum has been carried out by bovine submaxillary mucin-ELISA for estimation of this novel biomarker specific for O-acetylated sialic acid: Preparation of bovine submaxillary mucin : Bovine submaxillary mucin was prepared according to the method of Murphy & Gottschalk, Biochimica et Biophysica Acta.,vo\ 52, p 349, 1961. Briefly, tissues were homogenized and extracted thrice with an equal amount of water by centrifugation at 10,000 g for 15 minutes at 4°C. The supernatant was collected, pH adjusted to 4.5 and the resulting precipitate removed by centrifugation at SOOOg for 20 minutes. The supernatant was then neutralized (pH 6.0) and dialyzed against water. Barium acetate was slowly added to the dialysate to make it 0.1 M followed by precooled methanol to give an alcohol concentration of 64% (v/v) and incubated overnight at 4°C. The precipitate formed was retrieved by centrifugation, dissolved in 0.1M EDTA, dialyzed extensively against water and stored at -20°C until use. Bovine submaxillary mucin and asialo-bovine submaxillary mucin were separately coupled to sepharose 4B using the method of Kohn and Wilchek . Asialo- bovine subrriaxillary mucin was prepared by acid hydrolysis of bovine submaxillary mucin with 0.05M H2SO4 at 80°C for one hour. De-O-acetylated Bovine submaxiliary mucin was prepared by incubation with 0.2N NaOH for 45 minutes at 4°C followed by immediate neutralization. Bovine submaxiilary mucin, having high percentage of 9-O-acetyiated sialic acid, has been used to successfully capture a novel biomarker specific for O-acetylated sialic acid from the patients serum which in turn can be detected by horse radish peroxidase conjugated goat anti human IgG and detected by using a substrate and optical density measured in an ELISA reader. Briefly, in bovine submaxiilary mucin coated plate, wells were coated with bovine submaxiilary mucin. Following three washes with buffer the-wells were blocked. Purified O-acetylated sialic acid specific biomarker or patient sera was incubated for some time at cold and its binding to the coating material' was measured colorimetrically using horse radish peroxidase conjugated goat anti human antibodies and azino-bis thio-sulfonic acid as the substrate. The optical density was measured in an ELISA reader. The amount of a novel biomarker specific for O-acetylated sialic acid was determined and has been exploited to correlate the status of patients with regards to their blast cells which stay in peripheral blood even after chemotherapy. Thus its correlation with the status of the disease was assessed. The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention. Example 1: Approximately 2-3 ml of blood was collected from T- acute lymphoblastic leukemia patients, blood was allowed to clot and the serum was separated by centrifugaiion. Purification of a novel biomarker specific for O-acetylated siaiic acid: Patient serum (5mi) pooled from two T- ALL patients containing 184mg of total protein was used to purify the polyclonal antibody fraction with preferential affinity for O-AcSA. Briefly, 100% saturated solution of ammonium sulphate solution was added slowly to the patient's serum at 4°C in such a way so that final concentration of ammonium sulphate in the solution become 33%. The solution was kept at cold for overnight for complete precipitation. Next day solution was centrifuged and diaiyzed extensively against phosphate buffer saline to remove trace amount of ammonium sulphate fractionation. Now the" total content of proteins is 138mg. This protein (138mg) was passed over an asialo- bovine submaxiilary mucin -Sepharose 4B (3.6 mg/ml) column to remove galactose binding proteins. The galactose specific • fractions remain bind to the column and unbound fractious do not have affinity for galactose. Therefore, they do not bind to the column. These unbound free fractions (70mg) was then loaded onto an another column, namely bovine submaxiilary mucin -Sepharose 4B column (5.7 mg/ml) which had been previously equilibrated with phosphate buffered saline (PBS, pH 7.2). Following removal of non-specifically bound proteins by extensive washings in PBS, specific protein was eluted with 0.1M NH4OH, pH 11.0 and immediately neutralized with 0.2 N acetic acid. The eluted protein fraction (4mg) was then passed over a Protein G-agarose column (2 ml, Pierce), previously equilibrated with PBS, eluted with 0.1M citric acid from pH 6.5 was carried out and the fractions immediately neutralized with 2M Tris followed by extensive dialysis against PBS. The yield of the product, novel biomarker, anti-Oacetyl sialic acid antibody, lgG1 was 0.140mg. The biological activity and affinity of this a" novel biomarker specific for O-acetylated sialic acid was confirmed by the bovine submaxillary mucin -ELISA and Its specificity for O-acetylated sialic acids validated using de-O-acetylated Bovine submaxiiiary mucin as the coating agent in bovine submaxillary mucin - ELISA . Example 2. The use of this biomarker for the diagnosis of acute lymphoblastic leukemia was carried out by developing a bovine submaxillary mucin -ELISA. Diagnosis of T- ALL: In Bovine submaxillary mucin- ELISA, ELISA plate was coated with bovine submaxillary mucin (5 ng/ml, 100 (4.1/weil) in 0.02M phosphate buffer, pH 7.4 and left it for overnight at 4°C. Following three washes with phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T), the wells were blocked with 2% bovine serum albumin for 2 hours at 25°C. Patient sera from T ALL patients having 90% leukemic blasts or purified O-acetylated sialic acids specific biomarker was incubated overnight at 4°C in 1:10 dilutions and its binding to bovine submaxillary mucin was measured coiorimetrically using horse radish peroxidase (HRP) conjugated protein A (1:5000, Sigma, St. Louis, MO, USA) and azino-bis thio-sulfonic acid (ABTS) as the substrate. Example 3. Diagnosis of B- ALL by developing a bovine submaxillary mucin -ELISA. In Bovine submaxiiiary mucin- ELISA, ELISA plate was coated with bovine submaxillary mucin (5 jig/ml, 100 nl/well) in 0.02M phosphate buffer, pH 7.4 and left it for overnight at 4°C. Following three washes with phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T), the wells were blocked with 2% dry milk powder for 2 hours at 25°C. Patient from B ALL having 80% leukemic blasts or purified O-acetylated siaiic acids specific biomarker was incubated overnight at 4°C in 1:10 dilutions and its binding to bovine submaxillary mucin was measured coiorimetrically using horse radish peroxidase (HRP) conjugated goat artti human lgG1 (1:5000, Sigma, St. Louis, MO, USA) and azino- bis thio-sulfonic acid (ABTS) as the substrate. Example 4.Diagnosis of ALL having both T and B leukemic blasts by developing a bovine submaxillary mucin -ELISA.: In bovine submaxillary mucin- ELISA, ELISA plate was coated with bovine submaxiliary mucin (5 ng/ml, 50 nl/weil) in 0.02M phosphate buffer, pH 7,4 and left it for 6 hours at 4°C. Following three washes with phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T), the wells were blocked with 2% bovine serum albumin for 1 hours at 37°C. Patient sera of mixed lineage having 80% leukemic blasts in both B and T lymphocytes or purified O-acetylated sialic acids specific biomarker was incubated overnight at 4°C in 1:10 dilutions and its binding to bovine submaxillary mucin was measured colorimetrically using horse radish peroxidase (HRP) conjugated goat anti human lgG2 (1:5000, Sigma, St. Louis, MO, USA) and azino-bis thio-sulfonic acid (ABTS) as the substrate. Example 5: Approximately 2-3 ml of blood was collected from B - acute lymphoblastic leukemia patients, blood was allowed to clot and the serum was separated by centrifugation. 4ml sera pooled from two B-ALL'patients containing 170mg of total protein was used to purify the novel biomarker. Briefly, 100% saturated solution of ammonium sulphate solution was added slowly to the patient's serum at 4°C in such a way so that final concentration of ammonium sulphate in the solution become 30%. The solution was kept at cold for,'overnight for complete precipitation. Next day solution was centrifuged and diaiysed extensively against phosphate buffer saline to remove trace amount of ammonium sulphate fractionation. Now the total content of proteins is 12Qmg. This protein (120mg) was passed over an asialo-, bovine sheep submaxillary mucin -Sepharose 4B (3.6 mg/ml) column to remove galactose binding proteins. The galactose specific fractions remain bind to the column and unbound fractions do not have affinity for galactose. Therefore, they do not bind to the column. These unbound free fractions (65mg) was then loaded onto an another column, namely bovine submaxillary mucin -Sepharose 4B column (5.7 mg/ml) which had been previously equilibrated with phosphate buffered saiine (PBS, pH 7.2). Following removal of non-specifically bound proteins by extensive washings in PBS, specific protein was eluted with 0.2M NH4OH, pH 10.0 and immediately neutralized with 0.4N acetic acid. The eluted protein fraction (3mg) was then passed ovor a Protein G-agarose column (1.50 ml), previously equilibrated with PBS, eluted with 0.1 M citric acid from pH 3.5 was carried out and the fractions immediately neutralized with 2M Tris followed by extensive dialysis against PBS. The yield of the product, novel biomarker, anti-O acetyl sialic acid antibody, lgG2, was0.125mg. Example 6: Approximately 2-3 ml of blood was collected from mixed lineage B&T - acute lymphoblastic leukemia patients, blood was allowed to clot and the serum was separated by centrifugation. 4ml sera pooled from two patients containing 165mg of total protein was used to purify the novel biomarker. Briefly, 100% saturated solution of ammonium sulphate solution was added siowly to the patient's serum at 4°C in such a way so'that final concentration of ammonium sulphate in the solution become 40%. The solution was'kept at cold for overnight for complete precipitation. Next day solution was centrifuged and dialyzed extensively against phosphate buffer saline to remove trace amount of ammonium sulphate fractionation. Now the total content of proteins is 130mg. This protein (130mg) was passed over an asialo- bovine submaxillary mucin -Sepharose 4B (3.6 mg/mi) column to remove galactose binding proteins. The galactose specific fractions remain bind to the column and unbound fractions do not have affinity for galactose. Therefore, they do not bind to the column. These unbound free fractions (60rng) was then loaded onto an another column, namely bovine submaxillary mucin -Sepharose 48 column (5.7 mg/ml) which had been previously equilibrated with phosphate buffered saline (PBS, pH 7.2). Following removal of non-specifically bound proteins by extensive washings in PBS, specific protein was eluted with 0.05M NH4OH, pH 11.0 and immediately neutralized with 0.15 N acetic acid. The eiuted protein fraction (3.5mg) was then passed over a Protein A-agarose column (2 ml, Pierce), previously equilibrated with PBS, eluted with giycin HCI buffer pH 2.5 and the fractions immediately neutralized with 1M Tris followed by extensive dialysis against PBS. The yield of the product, novel biomarker, anti-O acetyl sialic acid antibody, was 0.140mg. Example 7: Approximately 5 ml of blood was collected from normal human volunteers and the serum was separated by centrifugation. Crude serum (80mg) following a 33% ammonium sulphate fractionation (55mg) was passed over an asialo- bovine submaxillary mucin -Sepharose 4B (3.6 mg/ml) column to remove gaiactose binding proteins. The resulting eluate (10mg) was then loaded onto a bovine submaxillary mucin -Sepharose 48 column (5.7 mg/ml) which had been previously equilibrated with phospnate buffered saline. Following removal of non-specifically bound proteins by extensive washings in PBS, specific protein was eluted with 0.1 M NH4OH, pH 11.0 and immediately neutralized with 0.2 N acetic acid. The yield of this O-acetylated sialic acids specific biomapker was which was 10 fold less than ALL serum, The eluted protein (0.40mg) fraction was then passed over a Protein G-agarose column, previously equilibrated with PBS, eluted with 0.1M citric acid, pH 2.5 followed by immediate neutralization with 2M Tris and extensively diaiysed against PBS. No significant amount of novel biomarker could be detected. Example 8: Approximately 5 ml of blood was collected separately from five different blood related diseases (Non hodgkin lymphoma, Chronic Myloblastic Leukemic, Acute Myloblastic Leukemic, Thalassemmia and Aplastic Anemia) served as negative controls and the serum was separated by centrifugation and processed separately for purification of O-acetylated sialic acids specific biomarker (if any). Serum following a 33% ammonium sulphate fractionation was passed over an asialo-bovine submaxillary mucin -Sepharose 4B (3.6 mg/m!) column. The unbound fraction was then loaded onto a bovine submaxiJIary mucin -Sepharose 4B column (5.7 mg/ml) in phosphate buffered saline. Following removal of non-specifically bound proteins by extensive washings in PBS, No specific protein could be eiuted with 0.1M NH4OH, pH 11.0. Therefore, O-acetyiated sialic acids specific biomarker is absent in theses patients. The main advantages of the present invention are : 1 To provide a reagent, a novel biomarker specific for O-acety!ated sialic acid, by an simple process with a significant high yield. 2. The reagent has an amplified biological potency to bind with cancer cells, therefore it has tremendous potential in terms of its detection capability of ALL patients under different phases of treatment eg. Potential for diagnosis and prognosis of ALL patients. 3. Furthermore, the process for purification of a novel biomarker specific for O-acetylated sialic acid is not a complicated one and comprises only a few simple steps including removal of other non -O-acetylated proteins by using asialo bovine submaxillary mucin affinity column followed by capturing novel O-acetylated sialic acid specific biomarker using a bovine submaxillary mucin - Sepharose column. Thus a novel biomarker specific for O-acetylated sialic acid purified from these patient's serum is of significant and maximum potency in detecting ALL patients under different phases of treatment i.e. detection of minimal residual disease in ALL, which is a challenging problem for leukemia specialist. 4. To provide a reagent by a process" with a significant commercial application and can predict relapse. 5 For diagnosis of ALL patients of different lineages e.g. T cell ALL, B cell ALL and mixed lineage ALL having cancer in both B and T lymphocytes by this novel biomarker specific for O-acetylated sialiclcid reflecting its general expression. 6. It can distinguish between different stages of acute lymphoblastic leukemia which correlate well with the clinical status of the disease. 7. It detects the extent to which the patient has responded to chemotherapy. 8. Serum from other hematological disorder do not possess this biomarker 9. Normal human serum contain very insignificant amount of this biomarker. We Claim: 1. A process for preparation of a novel protein biomarker specific for O-acetylatecl sialic add useful for the diagnosis, monitoring outcome of treatmen and prediction of relapse which comprises, (i) separating serum from blood collected from patients of acute ly nphoblastic leukemia by known methods,removing low molecular weight fractions and galactose binding proteins (non-specific protein) from the serum by column chromatography on affinity matrix, (ii) collecting unbound fraction from affinity matrix (iii) passing the galactose free protein fraction obtained in step (ii) over another affinity matrix to capture 0-acetyl sialic acid specific protein fraction (IV) eluting specific protein fraction with a buffer at alkaline pH in the range of 8.0 -11.0, immediately neutralizing the fraction (v) passing Oacetyl sialic acid specific protein obtained in step (iv) over Protein G- agarose or protein A agarose or protein A Sepharose or protein G Sepharose or anti - human immunoglobulin or only IgG / IgM coupled to Sepharose or agarose column to get 0-acetyic sialic add specific protein immunoglobulin and eluted with a buffer at acidic pH in the,range.of 2.0-6.5 immediately neutralizing the fraction and dialyzing to get novel biomarker. 2. A process as daimed in daim 1 wherein a novel biomarker specific for 0-acetylated sialic acid is purified from the patients suffering from acute lymphoblastic leukemia patients at different stages of the disease. 3. A process as daimed in daims and 2 wherein whole blood is collected in a container in presence or absence of any available anticoagulants such as alsevers solution or hepan'n, dextrose etc. 4. A process as daimed in claims I to 3 wherein blood was incubated for dotting at room temperature for a short penod of time or plasma may be collected from blood. 5. A process as claimed in claims I to 4 wherein nonspecific proteins are removed from specific affinity matrix such as bovine submaxillary mudn -Sepharose 4B by extensive washing of unbound non 0-acetyl sialic add specific fractions with buffer such as phosphate buffer saline or Tris buffer salhe. , 6. A process as daimed in daims 1 to 5 wherein specific protein is eiuted with buffer of alkaline pH in the range of 8.0 -11.0 from bovine suBmaxillary mucin-Sepharose 48. 7. A process as daimed in daim 1 to 6 wherein eluted specific protein is immediately neutralized with sodium acetate or monosodium phosphate for better stability of the eluted protein. 8. A process for the -preparation of a novel protein biomarkerspecific for O-acetyiated sialic acid useful for the diagnosis monitoring outcome of treatment and prediction of relapse as subsiantially describeed reference to the examples.

Full Text

The present invention relates to a process for the preparation of a novel biomarker specific for O-acetylated sialic acid useful for the diagnosis, monitoring outcome of treatment and prediction of relapse and biomarker prepared thereby.
Broadly the main usage of this invention is to provide-A process..for the preparation of a
novel biomarker specific for O-acetylated sialic acid useful for the diagnosis, monitoring outcome of
treatment and prediction of relapse and biomarker prepared thereby. The leukemia are a
heterogeneous group of neoplasm arising from the malignant transformation of hematopoietic i.e.
biood forming cells. Leukemia can be broadly classified accordingly to the cell type involved
primarily (myeloid or lymphoid) and as acute and chronic depending on the natural history of the
disease. Acute lymphoblastic leukemia (ALL) is the commonest type of leukemia in childhood. It is
primarily a disease of children and young adults, it occurs in all races with a peak incidence in
children between 3 and 5 years of age. ALL is diagnosed in 2000 -3000 new cases of children
leukemia in United States each year, whereas Acute myelogenous leukemia is diagnosed in only
500 children and Chronic myeloid leukemia fewer than 100. About 40 million children in USA are
effected under the group of 15 years about 3/4 th of these have ALL. Pediatric hematopoietic
malignancies rank first in cancer incidence arid mortality in children and are responsible for roughly
40% of childhood related death.
The causes are not known but environmental agents including irradiation, chemical carcinogens, cytogenetic abnormalities and retrovirus Infections are known to play an important role in the etiology of leukemia. For instance, individual with occupational radiation exposure, patients receiving radiation therapy or Japanese survivors of the atomic bomb explosions have a predictable and dose related increased incidence of leukemia.
For oncologist, Acute iymphoblastic leukemia (ALL) represent a major therapeutic success as this can be achieved in nearly 65% of patients (Pui CH, Crist W M Jr. Current Opinion in Oncology, Vol7, p 36, 1995 and Pui CH, Crist W M Jr. Lancet ,Vol 347, p1783,1996 ). However, relapse and eventually treatment failure occur in nearly many cases receiving identical treatment and this area is a major challenge for leukemia specialists (Pui CH, Crist W M Jr. New England Journal of Medicine, Vol 332, p1618, 1995).
At the time of diagnosis, the leuKemic mass is usually between 1011-1012 cells and available chemotherapeutic agents produce a fractional cell kill capable of a 3 to 5 log kill resulting in the
elimination of 99.99 to 99.999% of leukemia cells. The persistence of this remaining 0.01 to 0.001% leukemic cells tantamount to the persistence of 108 to 109 cells respectively. What is important is these persisting leukemic cells are not detectable by standard morphology in bone marrow or peripheral blood, it is these ceils that are responsible for relapse, if post induction chemotherapy fails to eradicate them. To eliminate, this non-detectable yet existing leukemic cell mass, maintenance therapy is given for an extended period (2-2.5 years) with the purpose of reducing the possible relapse. It is defined as the presence of leukemic cells not detectable by morphology. Assays to detect residual blast cells is the need of the hour as these will help the clinician to assess the effect of treatment on tumor burden and allow anticipation of relapse with greater precision (Brisco MJ, Condan J, Hughes E.etal. Lancet, Vo! 343, p196,1994).
Currently no specific marker is available to pinpoint the dose of chemotherapy and duration of maintenance therapy in acute lymphoblastic leukemia. Existing methods for the detection of leukemia blast cells are i) cytomorphology and karyotyping ii) Immunological methods iii) molecular detection.
Cytomorphology and karyotyping : Acquired non-random chromosomal translocation occur in 30-70% of ALL patients and can serve as marker of disease. But the approach has limited sensitivity (1-5%) primarily due to the paucity of leukemic cells during clinical remission (Campana D, Pui CH, Blood,Vol 85, p1416, 1995). Fluorescent in situ hybridization using chromosome specific or locus specific probes allows to identify abnormally ties in cells at metaphase (Le Beau MM, Blood, Vol 81, p1979, 1993). However, sensitivity remains at the 1% level.
Immunological Methods: Immunological Methods based on the recognition of leukemia associated phenotypes not usually found in normal bone marrow have had promising result. Immunophenotyping has been complemented by flow cytometric analysis where a combination of markers have been able to quantify MRD with a sensitivity is usually in the order of 102 to 103 which Is inferior to available to DNA based method (Meydan N et al. Nature, Vol 379, p645, 1996). Molecular approaches: In the majority of cases, relapse of acute lymphoblastic leukemia is thought to involve the same leukemic clone as the original disease (Bunin NJ et.al., Leukemia,Vol 4, p727, 1990) around 80% of cases of childhood acute lymphoblastic leukemia are due to clonal expansion of precursor B cells and have rearrangement of JgH gene, from which specific DNA probes have been generated. Several PCR methods (Brisco M J, Condan J, Hughes E, Lancet, Vol 343,
p196,1994; Veelken H, Tyeko 8, Sklar J., Blood, Vol78, p1318, 1991; Wasserman R, Galili N, Ito J,
et al., Journal of Clinical Oncology, VoUO, p1879, 1992) have been reported. This technique which
detects leukemia specific DNA sequences such as fusion regions of immunogiobulin (Ig) and TcR
genes with a sensitivity of 10~5 for the detection of residual disease in childhood acute lymphoblastic
leukemia. All these published methods are successful in only half of the patients since different
individuals can show different rearrangement of immunogiobulin genes or T cell Receptor genes.
The principle Drawbacks of the PCR methods for routine follow up of the patients are: (i) occurrence
of false positives due to contamination- of reaction mix with previously employed samples (ii)
occurrence of false negatives results owing to degraded RNA or DNA or clonal evolution in
approximately 20% of cases (iii) not all leukemic specific gene rearrangements are amenable to
initial amplification of PCR using universal primers (iv) a heterogeneous distribution of residual
leukemic cells may result in sampling error since gene rearrangements may be different in different
individuals (V). Therefore, the reliability of PCR assays depends on the use of stringent quantitation
protocols and analysis of multiple genetic targets to prevent false negative results due to changes in
the pattern of gene rearrangement during the disease course. These mandatory technical
requirements further complicate an already laborious procedure and also make it expensive thereby
limiting its suitability for routine clinical use . In comparison, use of this novel biomarker effectively
will serve as an index to reflect the clinical status of individual patients following therapy.
Since PCR undetectable residual disease is necessary for cures in most patients, it can be proposed that molecular remission defined as PCR undetectable disease,is a milestone and target for achieving cure. Evidently all these problems are unlikely to be exploited for commercial basis,
In India, the research in the field of acute lymphoblastic leukemia is mainly carried out at the Tata Memorial Hospital, Bombay. Their interest in acute lymphoblastic leukemia is focused on cytogenetics (Gladstone D et. al., Indian Journal of Medical Research, Vol 99, p264, 1994), infection analysis (Raje B et.al. Pediatric Hematology and Oncology,Voi11, p271, 1994) and central nervous system relapse (Iyer A et.al., Leukemia and Lymphoma, Vol 13, p183, 1994): -
Sialic acids are a family of derivatives of N-acetyl or N-glycolyl neuraminic acids and are very important constituents of cell surface architecture. Sialic acids piay an important role in receptors for viruses, peptide hormones and toxins. Sialic acids also function as masking agents on antigens, receptors and other recognition sites of the cell surface (Varki A, Glycobiology, Vol2, p25, 1992). The
O-substituted sialic acids exhibit species and tissue specific distribution in animals (Schauer R, Advanced Carbohydrate Chemistry Biochemistry, Vol40, p131, 1982). Changes in siaiic acid and the degree of O-acetyiation of sialic acid residues have been reported in transformed-and malignant cells. Recently, in our laboratory, exploiting the restricted specificity of 9-O-acetylated sialic acid binding lectin, specific biomarkers namely 9-O-acetyl sialoglycocongugates on lymphoblast of 87 children suffering from ALL have been identified (Sinha D, Mandal C and Bhattacharyya D et a/.,
Leukemia,Vol13, p119:125,1999) and assessed their differential expression at different phases of therapy (Sinha D, Mandal C and Bhattacharya D. 1999 Leukemia 13 (2) 309-312; Mandal C, Sinha D, Sharma V and Bhattacharya D, Indian Journal of Biochemistry Biophysics, Vol34, p82, 1997). A blood based lymphoproliferation assay to monitor the treatment outcome of ALL patients (n = 203) have been successfully developed (Sinha D, Mandal C and Bhattacharyya D. Leukemia Research, 1999 (in press); and Mandai C et al UK patent 1999 PATENT APP in Europe filo No St8V 2509/D/96 allowed; Sinha D, MandalC and Bhattacharyya D. Leukemia Research, 1999 (in press). An extensive world wide patent search has not shown any patent which claims for serum based reagent for monitoring treatment outcome in ALL.
Considering the drawbacks of the above mentioned methods, we felt to produce a reagent and we are successful not only in detection and purifying the reagent but also we have developed a diagnostic assay which is simple, sensitive and specific to detect and monitor treatment outcome of ALL. We have recently detected a novel biomarker specific for O-acetylated sialic acid in patients serum against 9-O-acetyl sialoglycocongugates which appeared on lymphoblasts. There is no serum based methods are available for diagnosis, monitoring the treatment outcome and prediction of relapse of ALL patients.
There is presently no group, nationally or internationally who have detected specific reagent from the serum and used it for monitoring treatment outcome of ALL employing a sialoglycoprotein namely bovine submaxillary mucin. The selectivity of this biomarker specific for O-acetylated sialic acid has allowed not only the identification but also monitoring cancer cells using serum from the ALL patients.
The main objective of the present invention is to provide a process for the preparation of a novel biomarker specific for O-acetylated sialic acid useful for the diagnosis, monitoring outcome of treatment and prediction of relapse and biomarker prepared thereby which obviates the drawbacks
as detailed above.
Another objective of the present invention is to provide a novel biomarker by the process of present
invention and also provide a process for the quantification of a novel biamarfrer specific for O-acetylated sialic acid present in the serum of ALL patient with the help of a simple, specific, sensitive, non-invasive and economical bovine submaxillary mucin ELISA which allows the assessment of the treatment outcome of ALL patients. This will specially be helpful for the detection of a novel biomarker specific for O-acetylated sialic,acid which will reflect chemotheraputic outcome in these children at different clinical stages of the disease. So, this invention provides an indicator as to when and how long should chemotherapy be continued and to predict the probability of relapse. Therefore, this reagent will serve as an effective measure in the battle for monitoring treatment outcome in patients suffering from acute lymphoblastic leukemia.
Accordinglyrthe present invention provides a process for preparation of a novel protein biomarker specific for O-acetylated sialic acid useful for the diagnosis, monitoring outcome of treatment and prediction of relapse which comprises, (i) separating serum from
blood collected from patients of acute lymphoblastic leukemia by known methods removing low
molecular weight fractions and galactose binding protetnsjfrom the serum by column chromatograpny
on affinity matrix, (ii) collecting unbound fraction from affinity matrix (iii) passing the galactose free protein fraction obtained, in step,(ii) over another affinity matrix to capture O-acetyl sialic acid specific protein fractions (IV) eluting specific protein fraction with a buffer at alkaline pH in the range of 8.0 -11.0, immediately neutralizing the fraction (v) passing O-acetyl sialic acid specific protein obtained in step (iv) over Protein G agarose or protein A agarose or protein A Sepharose or protein G Sepharose or anti - human immunoglobulin or only IgG / IgM coupled to Sepharose or agarose column to get O-acetyl sialic acid specific protein immunoglobulin and eluted with a buffer at acidic pH in the range of 2.0-6.5, immediately neutralizing the fraction and dialyztng to get novel biomarker. In an embodiment of the present invention the serum may be separated from the blood of patients of acute lymphoblastic leukemia in which blood have been collected either in absence or in presence of any anti-coagulants such as alsevers solution, potassium oxaiate, ammonium oxalate, heparin, dextrose etc.
In another embodiment of the present invention the solvent used for removal of albumin and other low molecular weight proteins may be saturated ammonium sulfate solution in the range of 15% to
40%.
in another embodiment of the present invention the galactose binding proteins may be removed by
using any galactose containing glycoproteins such as asialo bovine submaxillary mucin, asialo sheep
submaxillary mucin, asialo human chronic gonadotropin, asialo equine gastric mucin or hog gastric
mucin etc. coupled with Sepharose or agarose as an affinity matrix.
In another embodiment of the present invention the affinity matrix, for capturing O acetyl sialic acids
specific fractions, used may be any available sialogiycoconjugates having terminal O acetyl sialic acids such as bovine submaxiilary mucin or any gangliosides having terminal O acetyl sialic acids such as GD3.
In another embodiment of the present invention the solvent used for eiuting O acetyl sialic acids specific proteins may be any buffer such as ammonium hydroxide, borste buffer, carbonate bicarbonate buffer and ammonium bicarbonate buffer with high pH*from 8 to 11 even expensive sugar such as O-acetylated sialic acid (as a monocaccharide or disaccharides or higher oligosaccharides with subterminal galactose or N acetyl galactosamine) may be used as eiuting sugar. Molarity of buffer may be in the range of 0.02-0.5M
In another embodiment of the present invention neutralizing may be carried out with sodium acetate or monosodium phosphate for better stability of the eluted protein. If sugars are used as eiuting solution it may be removed simply by dialysis. Dialysis may be carried out at cold temperature between 4-25°C for 24 to 72 hours to get novel biomarker.
Accordingly, the present invention provide a novel biomarker by the process of present invention and used the same for diagnosis, monitoring outcome of treatment and prediction of relapse using a bovine submaxillary mucin-Enzyme linked immunosorbent assay (ELISA).
The novelty of this process is to capture the novel biomarker specific for O-acetylated sialic acid by using bovine submaxillary mucin as an affinity matrix after careful removal of albumin, other low molecular weight fractions and galactose binding proteins from ALL serum. Characterization of this novel biomarker by several biochemical, immunochemical techniques it reveals that (I) biomarker is specific to leukemic blast cells in acute lymphoblastic leukemia, (ii) Irrespective of the lymphocytic origin of cancer cells such as acuts lymphoblastic leukemia of B lymphocytes or acute lymphoblastic leukemia of T lymphocytes this novel biomarker shows equal specificity. Therefore, it may be consider as common biomarker and used for diagnosis of both types of acute lymphablastic
leukemia, (iii) The amount of a novel biomarker specific for O-acetylated sialic acid is determined
and has been exploited to correlate the status of patients with regards to their blast cells which stay in
peripheral blood even after chemotherapy. The quantity of a novel biomarker specific for O-
acetylated sialic acid has been established to be directly proportional to presence" of leukemic blast
cells i.e. cancer cells, therefore, by measuring the amount of novel biomarker by newly developed
ELiSA it is possible to know the status of different stages of the disease, (iv) Thus its correlation with
the status of the disease is assessed and confirmed that the novel biomarker has both diagnostic and
prognostic potential, (v) The immunoglobulin .G (IgG) subclass distribution of these antibodies
reacting with O-AcSA in patients in comparison to normal healthy individuals has been characterized
by the ELISA. Amongst the four human IgG subclasses, IgG, and lgG2 are significantly increased in
patients as compared to normal individuals. However, their lgG3 and lgG4 levels are unchanged and
comparable to normal healthy individuals. Therefore measuring both IgGi and lgG2 specific for O-
Acetyl sialic acid in patients by an isotype ELISA is very much beneficial. The disease specific IgM
antibody has also been found to be potentially important.
Detection of this novel biomarker specific for O-acetylated sialic acid in patients serum has been carried out by bovine submaxillary mucin-ELISA for estimation of this novel biomarker specific for O-acetylated sialic acid: Preparation of bovine submaxillary mucin :
Bovine submaxillary mucin was prepared according to the method of Murphy & Gottschalk, Biochimica et Biophysica Acta.,vo\ 52, p 349, 1961. Briefly, tissues were homogenized and extracted thrice with an equal amount of water by centrifugation at 10,000 g for 15 minutes at 4°C. The supernatant was collected, pH adjusted to 4.5 and the resulting precipitate removed by centrifugation at SOOOg for 20 minutes. The supernatant was then neutralized (pH 6.0) and dialyzed against water. Barium acetate was slowly added to the dialysate to make it 0.1 M followed by precooled methanol to give an alcohol concentration of 64% (v/v) and incubated overnight at 4°C. The precipitate formed was retrieved by centrifugation, dissolved in 0.1M EDTA, dialyzed extensively against water and stored at -20°C until use. Bovine submaxillary mucin and asialo-bovine submaxillary mucin were separately coupled to sepharose 4B using the method of Kohn and Wilchek . Asialo- bovine subrriaxillary mucin was prepared by acid hydrolysis of bovine submaxillary mucin with 0.05M H2SO4 at 80°C for one hour. De-O-acetylated Bovine submaxiliary
mucin was prepared by incubation with 0.2N NaOH for 45 minutes at 4°C followed by immediate
neutralization.
Bovine submaxiilary mucin, having high percentage of 9-O-acetyiated sialic acid, has been used to successfully capture a novel biomarker specific for O-acetylated sialic acid from the patients serum which in turn can be detected by horse radish peroxidase conjugated goat anti human IgG and detected by using a substrate and optical density measured in an ELISA reader. Briefly, in
bovine submaxiilary mucin coated plate, wells were coated with bovine submaxiilary mucin. Following three washes with buffer the-wells were blocked. Purified O-acetylated sialic acid specific biomarker or patient sera was incubated for some time at cold and its binding to the coating material' was measured colorimetrically using horse radish peroxidase conjugated goat anti human antibodies and azino-bis thio-sulfonic acid as the substrate. The optical density was measured in an ELISA reader. The amount of a novel biomarker specific for O-acetylated sialic acid was determined and has been exploited to correlate the status of patients with regards to their blast cells which stay in peripheral blood even after chemotherapy. Thus its correlation with the status of the disease was assessed. The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.
Example 1: Approximately 2-3 ml of blood was collected from T- acute lymphoblastic leukemia patients, blood was allowed to clot and the serum was separated by centrifugaiion. Purification of a novel biomarker specific for O-acetylated siaiic acid: Patient serum (5mi) pooled from two T- ALL patients containing 184mg of total protein was used to purify the polyclonal antibody fraction with preferential affinity for O-AcSA. Briefly, 100% saturated solution of ammonium sulphate solution was added slowly to the patient's serum at 4°C in such a way so that final concentration of ammonium sulphate in the solution become 33%. The solution was kept at cold for overnight for complete precipitation. Next day solution was centrifuged and diaiyzed extensively against phosphate buffer saline to remove trace amount of ammonium sulphate fractionation. Now the" total content of proteins is 138mg. This protein (138mg) was passed over an asialo- bovine submaxiilary mucin -Sepharose 4B (3.6 mg/ml) column to remove galactose binding proteins. The galactose specific •
fractions remain bind to the column and unbound fractious do not have affinity for galactose.
Therefore, they do not bind to the column. These unbound free fractions (70mg) was then loaded onto an another column, namely bovine submaxiilary mucin -Sepharose 4B column (5.7 mg/ml) which had
been previously equilibrated with phosphate buffered saline (PBS, pH 7.2). Following removal of non-specifically bound proteins by extensive washings in PBS, specific protein was eluted with 0.1M NH4OH, pH 11.0 and immediately neutralized with 0.2 N acetic acid. The eluted protein fraction (4mg) was then passed over a Protein G-agarose column (2 ml, Pierce), previously equilibrated with PBS, eluted with 0.1M citric acid from pH 6.5 was carried out and the fractions immediately neutralized with 2M Tris followed by extensive dialysis against PBS. The yield of the product, novel biomarker, anti-Oacetyl sialic acid antibody, lgG1 was 0.140mg.
The biological activity and affinity of this a" novel biomarker specific for O-acetylated sialic acid was confirmed by the bovine submaxillary mucin -ELISA and Its specificity for O-acetylated sialic acids validated using de-O-acetylated Bovine submaxiiiary mucin as the coating agent in bovine submaxillary mucin - ELISA .
Example 2. The use of this biomarker for the diagnosis of acute lymphoblastic leukemia was carried out by developing a bovine submaxillary mucin -ELISA. Diagnosis of T- ALL:
In Bovine submaxillary mucin- ELISA, ELISA plate was coated with bovine submaxillary mucin (5 ng/ml, 100 (4.1/weil) in 0.02M phosphate buffer, pH 7.4 and left it for overnight at 4°C. Following three washes with phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T), the wells were blocked with 2% bovine serum albumin for 2 hours at 25°C. Patient sera from T ALL patients having 90% leukemic blasts or purified O-acetylated sialic acids specific biomarker was incubated overnight
at 4°C in 1:10 dilutions and its binding to bovine submaxillary mucin was measured coiorimetrically using horse radish peroxidase (HRP) conjugated protein A (1:5000, Sigma, St. Louis, MO, USA) and azino-bis thio-sulfonic acid (ABTS) as the substrate.
Example 3. Diagnosis of B- ALL by developing a bovine submaxillary mucin -ELISA.
In Bovine submaxiiiary mucin- ELISA, ELISA plate was coated with bovine submaxillary mucin (5 jig/ml, 100 nl/well) in 0.02M phosphate buffer, pH 7.4 and left it for overnight at 4°C. Following three washes with phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T), the wells were blocked with 2% dry milk powder for 2 hours at 25°C. Patient from B ALL having 80% leukemic blasts
or purified O-acetylated siaiic acids specific biomarker was incubated overnight at 4°C in 1:10 dilutions
and its binding to bovine submaxillary mucin was measured coiorimetrically using horse radish peroxidase (HRP) conjugated goat artti human lgG1 (1:5000, Sigma, St. Louis, MO, USA) and azino-
bis thio-sulfonic acid (ABTS) as the substrate. Example 4.Diagnosis of ALL having both T and B leukemic blasts by developing a bovine submaxillary
mucin -ELISA.: In bovine submaxillary mucin- ELISA, ELISA plate was coated with bovine submaxiliary mucin (5
ng/ml, 50 nl/weil) in 0.02M phosphate buffer, pH 7,4 and left it for 6 hours at 4°C. Following three
washes with phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T), the wells were
blocked with 2% bovine serum albumin for 1 hours at 37°C. Patient sera of mixed lineage having 80% leukemic blasts in both B and T lymphocytes or purified O-acetylated sialic acids specific biomarker was incubated overnight at 4°C in 1:10 dilutions and its binding to bovine submaxillary mucin was measured colorimetrically using horse radish peroxidase (HRP) conjugated goat anti human lgG2 (1:5000, Sigma, St. Louis, MO, USA) and azino-bis thio-sulfonic acid (ABTS) as the substrate.
Example 5: Approximately 2-3 ml of blood was collected from B - acute lymphoblastic leukemia patients, blood was allowed to clot and the serum was separated by centrifugation. 4ml sera pooled from two B-ALL'patients containing 170mg of total protein was used to purify the novel biomarker. Briefly, 100% saturated solution of ammonium sulphate solution was added slowly to the patient's serum at 4°C in such a way so that final concentration of ammonium sulphate in the solution become 30%. The solution was kept at cold for,'overnight for complete precipitation. Next day solution was centrifuged and diaiysed extensively against phosphate buffer saline to remove trace amount of
ammonium sulphate fractionation. Now the total content of proteins is 12Qmg. This protein (120mg) was passed over an asialo-, bovine sheep submaxillary mucin -Sepharose 4B (3.6 mg/ml) column to remove galactose binding proteins. The galactose specific fractions remain bind to the column and unbound fractions do not have affinity for galactose. Therefore, they do not bind to the column. These unbound free fractions (65mg) was then loaded onto an another column, namely bovine submaxillary mucin -Sepharose 4B column (5.7 mg/ml) which had been previously equilibrated with phosphate buffered saiine (PBS, pH 7.2). Following removal of non-specifically bound proteins by extensive washings in PBS, specific protein was eluted with 0.2M NH4OH, pH 10.0 and immediately neutralized with 0.4N acetic acid. The eluted protein fraction (3mg) was then passed ovor a Protein
G-agarose column (1.50 ml), previously equilibrated with PBS, eluted with 0.1 M citric acid from pH 3.5 was carried out and the fractions immediately neutralized with 2M Tris followed by extensive
dialysis against PBS. The yield of the product, novel biomarker, anti-O acetyl sialic acid antibody, lgG2, was0.125mg.
Example 6: Approximately 2-3 ml of blood was collected from mixed lineage B&T - acute
lymphoblastic leukemia patients, blood was allowed to clot and the serum was separated by
centrifugation. 4ml sera pooled from two patients containing 165mg of total protein was used to purify
the novel biomarker. Briefly, 100% saturated solution of ammonium sulphate solution was added
siowly to the patient's serum at 4°C in such a way so'that final concentration of ammonium sulphate
in the solution become 40%. The solution was'kept at cold for overnight for complete precipitation.
Next day solution was centrifuged and dialyzed extensively against phosphate buffer saline to
remove trace amount of ammonium sulphate fractionation. Now the total content of proteins is
130mg. This protein (130mg) was passed over an asialo- bovine submaxillary mucin -Sepharose 4B
(3.6 mg/mi) column to remove galactose binding proteins. The galactose specific fractions remain
bind to the column and unbound fractions do not have affinity for galactose. Therefore, they do not
bind to the column. These unbound free fractions (60rng) was then loaded onto an another column,
namely bovine submaxillary mucin -Sepharose 48 column (5.7 mg/ml) which had been previously
equilibrated with phosphate buffered saline (PBS, pH 7.2). Following removal of non-specifically
bound proteins by extensive washings in PBS, specific protein was eluted with 0.05M NH4OH, pH
11.0 and immediately neutralized with 0.15 N acetic acid. The eiuted protein fraction (3.5mg) was
then passed over a Protein A-agarose column (2 ml, Pierce), previously equilibrated with PBS, eluted
with giycin HCI buffer pH 2.5 and the fractions immediately neutralized with 1M Tris followed by
extensive dialysis against PBS. The yield of the product, novel biomarker, anti-O acetyl sialic acid
antibody, was 0.140mg.
Example 7: Approximately 5 ml of blood was collected from normal human volunteers and the serum was separated by centrifugation. Crude serum (80mg) following a 33% ammonium sulphate fractionation (55mg) was passed over an asialo- bovine submaxillary mucin -Sepharose 4B (3.6 mg/ml) column to remove gaiactose binding proteins. The resulting eluate (10mg) was then loaded onto a bovine submaxillary mucin -Sepharose 48 column (5.7 mg/ml) which had been previously equilibrated with phospnate buffered saline. Following removal of non-specifically bound proteins by extensive washings in PBS, specific protein was eluted with 0.1 M NH4OH, pH 11.0 and immediately neutralized with 0.2 N acetic acid. The yield of this O-acetylated sialic acids specific biomapker was
which was 10 fold less than ALL serum, The eluted protein (0.40mg) fraction was then passed over a Protein G-agarose column, previously equilibrated with PBS, eluted with 0.1M citric acid, pH 2.5 followed by immediate neutralization with 2M Tris and extensively diaiysed against PBS. No significant amount of novel biomarker could be detected.
Example 8: Approximately 5 ml of blood was collected separately from five different blood related diseases (Non hodgkin lymphoma, Chronic Myloblastic Leukemic, Acute Myloblastic Leukemic, Thalassemmia and Aplastic Anemia) served as negative controls and the serum was separated by centrifugation and processed separately for purification of O-acetylated sialic acids specific biomarker (if any). Serum following a 33% ammonium sulphate fractionation was passed over an asialo-bovine submaxillary mucin -Sepharose 4B (3.6 mg/m!) column. The unbound fraction was then loaded onto a bovine submaxiJIary mucin -Sepharose 4B column (5.7 mg/ml) in phosphate buffered saline. Following removal of non-specifically bound proteins by extensive washings in PBS, No specific protein could be eiuted with 0.1M NH4OH, pH 11.0. Therefore, O-acetyiated sialic acids specific biomarker is absent in theses patients. The main advantages of the present invention are :
1 To provide a reagent, a novel biomarker specific for O-acety!ated sialic acid, by an simple process with a significant high yield.
2. The reagent has an amplified biological potency to bind with cancer cells, therefore it has
tremendous potential in terms of its detection capability of ALL patients under different phases of
treatment eg. Potential for diagnosis and prognosis of ALL patients.
3. Furthermore, the process for purification of a novel biomarker specific for O-acetylated sialic
acid is not a complicated one and comprises only a few simple steps including removal of other
non -O-acetylated proteins by using asialo bovine submaxillary mucin affinity column followed by
capturing novel O-acetylated sialic acid specific biomarker using a bovine submaxillary mucin -
Sepharose column. Thus a novel biomarker specific for O-acetylated sialic acid purified from these
patient's serum is of significant and maximum potency in detecting ALL patients under different
phases of treatment i.e. detection of minimal residual disease in ALL, which is a challenging
problem for leukemia specialist.
4. To provide a reagent by a process" with a significant commercial application and can predict relapse.
5 For diagnosis of ALL patients of different lineages e.g. T cell ALL, B cell ALL and mixed lineage ALL having cancer in both B and T lymphocytes by this novel biomarker specific for O-acetylated sialiclcid reflecting its general expression.
6. It can distinguish between different stages of acute lymphoblastic leukemia which correlate well
with the clinical status of the disease.
7. It detects the extent to which the patient has responded to chemotherapy.
8. Serum from other hematological disorder do not possess this biomarker
9. Normal human serum contain very insignificant amount of this biomarker.

We Claim:
1. A process for preparation of a novel protein biomarker specific for O-acetylatecl sialic add useful for the diagnosis,
monitoring outcome of treatmen and prediction of relapse which comprises, (i) separating serum from blood
collected from patients of acute ly nphoblastic leukemia by known methods,removing low molecular weight fractions
and galactose binding proteins (non-specific protein) from the serum by column chromatography on affinity matrix, (ii)
collecting unbound fraction from affinity matrix (iii) passing the galactose free protein fraction obtained in step (ii) over another affinity
matrix to capture 0-acetyl sialic acid specific protein fraction (IV) eluting specific protein fraction with a buffer at alkaline pH in the
range of 8.0 -11.0, immediately neutralizing the fraction (v) passing Oacetyl sialic acid specific protein obtained in step (iv) over
Protein G- agarose or protein A agarose or protein A Sepharose or protein G Sepharose or anti - human immunoglobulin
or only IgG / IgM coupled to Sepharose or agarose column to get 0-acetyic sialic add specific protein immunoglobulin and
eluted with a buffer at acidic pH in the,range.of 2.0-6.5 immediately neutralizing the fraction and dialyzing to get novel
biomarker.
2. A process as daimed in daim 1 wherein a novel biomarker specific for 0-acetylated sialic acid is purified from the patients
suffering from acute lymphoblastic leukemia patients at different stages of the disease.
3. A process as daimed in daims and 2 wherein whole blood is collected in a container in presence or absence of any
available anticoagulants such as alsevers solution or hepan'n, dextrose etc.
4. A process as daimed in claims I to 3 wherein blood was incubated for dotting at room temperature for a short penod of
time or plasma may be collected from blood.
5. A process as claimed in claims I to 4 wherein nonspecific proteins are removed from specific affinity matrix such as
bovine submaxillary mudn -Sepharose 4B by extensive washing of unbound non 0-acetyl sialic add specific fractions with
buffer such as phosphate buffer saline or Tris buffer salhe. ,
6. A process as daimed in daims 1 to 5 wherein specific protein is eiuted with buffer of alkaline pH in the range of 8.0 -11.0 from
bovine suBmaxillary mucin-Sepharose 48.
7. A process as daimed in daim 1 to 6 wherein eluted specific protein is immediately neutralized with sodium acetate or
monosodium phosphate for better stability of the eluted protein.
8. A process for the -preparation of a novel protein biomarkerspecific for O-acetyiated sialic acid useful for the diagnosis
monitoring outcome of treatment and prediction of relapse as subsiantially describeed reference to the examples.

Documents:

1192-del-1999-abstract.pdf

1192-del-1999-claims.pdf

1192-del-1999-complete specification (granted).pdf

1192-del-1999-correspondence-others.pdf

1192-del-1999-correspondence-po.pdf

1192-del-1999-description (complete).pdf

1192-del-1999-form-1.pdf

1192-del-1999-form-2.pdf

1192-del-1999-form-3.pdf

1192-del-1999-form-4.pdf

1192-del-1999-form-9.pdf

1192-del-1999-petition-138.pdf

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Patent Number

191209

Indian Patent Application Number

1192/DEL/1999

PG Journal Number

40/2003

Publication Date

04-Oct-2003

Grant Date

06-May-2004

Date of Filing

08-Sep-1999

Name of Patentee

COUNCIL OF SCEINTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI 110001, INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	CHITRA MANDAL	INDIAN INSTITUTE OF CHEMICAL BIOLOGY CALCUTTA-700032, INDIA
2	MITALI CHATTERJEE	INDIAN INSTITUTE OF CHEMICAL BIOLOGY CALCUTTA-700032, INDIA
3	SANTANU PAL	INDIAN INSTITUTE OF CHEMICAL BIOLOGY CALCUTTA-700032, INDIA

PCT International Classification Number

A61K 31/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA