Indian Patents. 277986:CANCER CELL BINDING RECOMBINANT LECTIN WITH ANTITUMOR ACTIVITY AND METHOD OF PREPARATION

Title of Invention	CANCER CELL BINDING RECOMBINANT LECTIN WITH ANTITUMOR ACTIVITY AND METHOD OF PREPARATION
Abstract	The present invention discloses genes for new variants in E.coli that are more close to the wild type lectin sequence but still differ in surface charges. This recombinant version (exemplified Rec-2 and Rec-3) are cancer cell binding with anti tumor activity show blood group specificity similar to the native lectin but retains the stability and the solubility properties.

Full Text	FORM 2 THE PATENTS ACT, 1970 COMPLETE SPECIFICATION Section 10 Cancer Cell binding Recombinant Lectins with Antitumor Activity and Method of Preparation Unichem Laboratories Ltd., an Indian company of Mahalaxmi Chambers, 22 Bhulabhai Desai Road, Mumbai - 400026, Maharashtra, India and Karnatak University, Dharwad 580 003, Karnatak, India The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed: 1 8 FEB 2009 FIELD OF INVENTION The invention relates to the field of carbohydrate binding proteins, and more specifically to a protein which binds specifically to particular oligosaccharides associated with particular medical significance. The invention specifically relates to lectins, more particularly to recombinant fungal lectins having ability to recognize glycans of clinical importance and their antiproliferative effect on cancer cells finding applications in cancer diagnosis, therapy and research. BACKGROUND OF INVENTION Lectins are proteins, or glycoproteins that agglutinate erythrocytes of some or all blood groups in vitro. They are important group of bioactive proteins found in most organisms. Lectins are used as tools for diagnostic and therapeutic purpose in health care areas. Lectins are also used in the purification of glycoproteins, oligosaccharide analysis and in cell selection processes. Lectins can bind reversibly with monosaccharides or the sugar moiety of, glycoproteins or glycolipids or polysaccharides. Changes in carbohydrate structures on the cell surfaces are a hallmark during cancer. Such changes results in exposure of tumor associated carbohydrate structures. Amongst them, is the increased occurrence of Thomsen-Friedenreich antigen, a disaccharide Galβl->3GalNac-0-Ser/Thr. In normal cells, this antigen is masked by certain sugars, sialic acid or sulphates. Some of the lectins have ability to specifically recognize and bind to these altered structures and exerts various physiological effects such as apoptosis, cytotoxicity, proliferative/antiproliferative activity, metastasis, inhibition of cell adhesion, etc., Hence such lectins are currently being considered for use as cancer diagnostic and therapeutic agents and also for anti tumor activity. Several lectins have been isolated and characterized from variety of sources. They however differ in their physico-chemical properties such as molecular size and sugar specificities. There is also characterization of a few recombinant proteins. The recombinant mushroom lectin from Marasmius oreades has a molecular size of 33 kDa and shows high affinity for Galαl, 3Gal and Galcαl, 3Galβl, 4GlcNAc. 2 (R.P.Kruger et al, J.Biol.Chem., 277, 15002-15005, 2002; H.C.Winter et al. J.Biol.Chem ,277, 14996-15001, 2002., I.J. Goldstein, etal. US Patent :6958321) Peanut lectin that shows affinity to Thomsen-Friedenreich antigen has been cloned (V.Sharma and A.Surolia, Gene (Amst), 148, 299-304, 1994) and subsequently some mutants of this protein with a subunit molecular mass of ~28kDa has also been expressed in E.coli (V.Sharma, M.Vijayan and A.Surolia, J. Biol. Chem., 271, 21200-21213,1996) with some difference in its preference for sugar specificity. A much smaller lectin of molecular mass of 11.73 kDa with high binding affinity to L-fucose from Pseudomonas aeruginosa has also been expressed in E.coli as a Yellow fluorescent Protein-lectin fusion (BioTechniques, 41, 327-332, 2006). A mushroom lectin (ABL) from Agaricus bisporus, which has antiproliferative activity on human colon cancer cells and its curative effect in psoriasis is reported by Jonathan M. Rhodes; US patent # 5607679. Sclerotium rolfsii lectin (SRL) purified from the sclerotial bodies of the phytopathogenic fungus Sclerotium rolfsii exhibit complex carbohydrate specificity and exhibit specificity towards TF antigen. TF antigen is an oncofetal antigen, a disaccharide Galβl, 3GaNAc α ser/thr and is known to expressed on cell surfaces in different cancers. SRL apart from binding to TF antigen, it also bind to sialylated and sulfated disaccharide. The crystal structure of this lectin and its complete amino acid sequence is determined (Demetres 2007 and Satisha 2008). Binding studies indicated that SRL binds to different human cancer tissues and also to cultured cells. Further binding of SRL to cultured human leukemic cells and colon cells results in their antiproliferation and in colon cells effect is shown to be mediated by inducing apoptosis. However the purified native lectin has solubility problems for its use in various applications, hence there is need of forms of this lectin with improved stability for practical applications. Lectins thus have enormous potential in the area of medical assays. The instant application proceeds to provide certain lectins with novel characteristics so as to be useful in the medical field and improved properties. 3 SUMMARY OF THE INVENTION The present invention discloses recombinant lectins having specificity to certain sugar chains uniquely found on certain cancer cells. The present invention further provides method for preparing two recombinant lectins (designated as UC-SS/CSR-1803 and UC-SS/CSR-1805 or simply Rec-2 and Rec-3 respectively) expressed in E.coli. The method comprises the synthesis of lectin genes whose sequences are derived by deliberate modifications made to the original amino acid sequence of a lectin from Sclerotium rolfsii (Sathisha et.al., Amino Acids; 2008, 35, 309-320 ), a soil borne phytopathogenic fungus, and its cloning in E.coli. The recombinant lectins expressed as soluble proteins and are purified by ion exchange and gel filtration chromatographic techniques. The present invention specifically provides two recombinant lectins (Rec-2 and Rec-3) having approximate subunit masses (Mr 16.1 kDa), which are specific to certain O-glycosidically linked glycans occurring in glycoproteins and on cell surfaces, but does not show specificity to N-Hnked glycans.. The property of recognizing O-glycans but not N-glycans and showing no specificity to human blood groups makes them different from the other recombinant lectin disclosed in copending patent application 30/MUM/2008. It is also found that the recombinant lectins Rec-2 & Rec-3 does not bind to fetuin but exhibit very strong affinity for asialofetuin, there by making it different from that of said recombinant lectin (patent application 30/MUM/2008) and also the native lectin. By virtue of this recognition property they bind to certain cell surface molecules expressed during malignancy (Fig. 1) and thus bind to human Breast cancer, colon cancer, ovarian cancer tissues (Fig .2) and also cultured cells and leukemic cell lines (table 1). Apart from binding to cultured human cancer cells, recombinant lectins exert in vitro antiproliferative activity on the cells (Table 2) contemplating their diagnostic and therapeutic properties. The invention discloses the DNA sequences for Rec-2 and Rec-3 coding for 141 amino acids long lectins in each case similar to the native lectin (S. rolfsii lectin), but with few deliberate modifications synthesized chemically and cloned into E. coli (SEQ ID 1.1 and SEQ ID 2.1). The cloned lectins were expressed after induction with the inducer IPTG- The expressed protein appeared as soluble and in active form in 4 each case. The recombinant lectins were purified to essential homogeneity by resorting to anion exchange and molecular sieve chromatography. The mass spectrometric and SDS PAGE estimate of molecular weights show a value of approximate 16.1 kDa. The isoelectric focusing experiment shows the PI value of 6.614 whereas the native lectin has 6.219, the property markedly helped in improving the solubility and stability of recombinant proteins. Improved solubility, , stability and the binding to desialylated TF antigen of the recombinant lectins have greater implications of their applications in various fields as reagents apart from ease of production. The present invention provides a method for preparing a recombinant lectin expressed in a host cell such as E.coli or yeast. This method comprises the synthesis of a lectin gene based on amino acid sequence derived from the MALDI MS/MS of the Sclerotiam rotlsit lectin (SEQ ID. I), a soil borne pfryfopatfrcgerrrc Fungus, and its cloning in the host cell. In order to obtain desired properties the amino acid sequence of the native lectin was modified deliberately and recombinant lectins were obtained expressing in E-coli. The present invention discloses two recombinant lectins with modified amino acid sequences but have close resemblances with native lectin with respect to their binding properties towards cancer tissues, in vitro cultured cells, and their apoptotic activity to cultured cells. However these two lectins have better solubility and stability and desired sugar specificity properties for applications as compared to native lectin. The recombinant lectin expressed as a soluble protein is purified by ion exchange and gel filtration chromatographic techniques. The amino acid sequence SEQ ID 2 and 3 represents such lectin having all the above characteristics. The nucleic acid molecule encoding the amino acid for the protein of the above characteristics is also within the scope of the application. The DNA sequence of SEQ ID 1.1 and 2.1 represents such nucleic acid encoding the lectin having the above characteristics. The sequences also envisage any change in the nucleotide or amino-acid sequences including but not limited to any substitution, deletion, addition or modification such as acetylation, nitration, glycation 5 ,sulphonation etc. in any of the positions of the entire sequence and also to include truncations at 5' or 3' end of the sequences which do not alter the properties disclosed in the instant application. In one aspect this recombinant lectin in a biotinylated form or modified with a fluorogenic chromophore can be used for the detection of cancer cells or cancer associated specific antigens. The growth modulatory effect of the recombinant lectin can have potential application in cancer therapy as anti tumor agents. In another aspect the recombinant lectin can be used in cancer diagnosis, therapy and research. The application particularly discloses a DNA sequence (SEQ ID 1.1 and 2.1 ) coding for a 141 amino acids long lectin similar to the lectin (S. rolfsii), but with a few modifications was chemically synthesized and cloned into E.coli. The method for producing said recombinant lectin has also been disclosed. The cloned lectin is expressed after induction with the inducer IPTG BRIEF DESCRIPTION OF FIGURES Figure 1: Comparative profiles of native SRL receptors isolated from the membranes of human normal resting PBMCs (1), PHA-activated PBMCs (2), Molt-4 (3) and Jurkat cells (4). Membrane proteins (40 \|ig) were resolved by SDS-PAGE (10% gel) and blotted on to nitrocellulose membrane. SRL interacting proteins were probed with biotinylated lectin and compared with standard molecular weight protein markers (M) after visualization with streptavidin-HRP reaction. Figure 2: Binding pattern of Rec 2 and Rec 3 and native lectin to human cancerous tissues of breast and ovary in comparison with H & E staining. Biotinylated lectins were used for the immunohistochemistry 6 Figure 3: The Amino acid sequence of lectin protein isolated from S. rolfsii (SEQ ID 1); Protein Sequence of the recombinant lectin Rec 2 of the instant application. The number of amino acid coding for this lectin is 141 and is represented by SEQ ID 2. The altered/modified positions have been highlighted in the sequence. Protein Sequence of the recombinant lectin Rec 3 of the instant application. The number of amino acid coding for this lectin is 141 and is represented by SEQ ID 3. The altered/modified positions have been highlighted in the sequence. Multiple sequence alignment of the SEQ ID I -3. Figure 4: A DNA sequence encoding the lectin of the instant application, SEQ ID 1.1 and 2.1 The length of the optimized sequence is —-- nucleotides which includes the 5* end Nde f (CATATG) and 3 end BamHT (GGATCC) restriction sites.SEQ fD: 1.1; GENE NAME; UC- SS/CSR—1803 (Rec-2); SEQ ID: 2.1; GENE NAME: UC-SS/CSR-1805(Rec-3) DETAILED DESCRIPTION OF THE INVENTION The present invention discloses cloned genes for two new variants, designated here as Rec-2 and Rec-3, in E-coli that are more close to the native S. rolfsii lectin in amino acid sequence, but still differ in surface charges. Surface charge was altered deliberately in order to enhance the better solubility and stability of the proteins as compared to native lectin. Two lectin genes coding S. rolfsii lectin with few alterations are chemically synthesized and expressed in E-coli. Recombinant lectins are purified by conventional chromatographic methods and are shown to have molecular mass approximate 16,000 Da. The two recombinant lectins described here are different in blood group specificity, recognize all the human blood groups and have exclusive ability to recognize TF antigen and its cryptic forms. The two blood group non specific recombinant lectins are more closely resemble with native S. rolfsii lectin in sugar binding, cell binding and in bringing apoptotic properties. However the recombinant lectins have better stability and solubility properties compared to native lectin making them ideal for various Applications. 7 The various applications to the use of the recombinant lectins may be in the following: Considering the property of cancer cell binding Rl and R2 can be used as drug delivery agents for cancer treatment and may also find application as diagnostic tools in the detection of microbial / viral pathogens. These recombinant lectins can be used for the detection of certain glycoproteins and glycoconjugates present in biological fluids. The lectins exhibits substrate specificity towards cancer associated cell surface carbohydrate such as desialylated TF antigen, they can have application as affinity matrices for the purification of glycoproteins and glycoconjugates of physiological and biochemical significance that bind to these recombinant lectins. Recombinant lectins can be used as fusion partners for the purification of recombinant lectins. The invention would now be disclosed by way of preferred methodology and embodiments: Lectin Gene construct and cloning: The amino acid sequence derived from the MALDI MS/MS and x-ray crystallography data of the lectin purified from the soil fungus Sclerotium rolfsii formed the basis for the gene construct. The gene corresponding to this sequence SEQ ID 1 was chemically synthesized by following the standard protocols. The assembled gene was first inserted into a pUC57 vector. The insert was released by digesting the plasmid with Ndel & BamHI and then recloned in to pET20b previously digested Ndel and BamHI. After ligation the plasmid was used to transform E.coli DE3 (GOLD) host strain for expression. The recombinant clones were analyzed for release of insert after digestion with Nde I and Bam HI. The SDS-PAGE analysis of recombinant E.coli upon induction with IPTG showed the expected ~16kDa protein. ■8 Cell growth A single colony of the recombinant E.coli was inoculated into 5ml LB-Amp allowed to grow at 370C overnight with shaking. The overnight grown culture was inoculated into the Fermentation Media and grown at 370 C till an OD of ~2.0 was reached, the cultures were then induced with 250uM IPTG (final concentration) and grown overnight at 200C. Cell extract preparation The overnight grown cultures were centrifiiged at 8000rpm for l0min and the pelleted cells (4gms) were suspended in 40ml of 50mM Tris-HCl pH8.0 containing ImM PMSF and ImM EDTA. The cell suspension was sonicated for 20min using the sonicator. The cells were centrifiiged at 12000rpm, l0min at 40C. The supernatant was used for further purification. Purification of the protein DEAE-cellulose chromatography: The supernatant (400mg of protein) in 50 mM Tris-HCl buffer pH 8.0 was loaded on 25ml DEAE (BIORAD) column equilibrated in 50mM Tris-HCl buffer pH 8.0. The column was washed with 2 column volumes of buffer followed by step-wise elution of the column with buffer b containing 75mM NaCI and 200mM NaCI. The lectin was eluted with buffer containing 300mM NaCI. This fraction contained 216 mg of protein. PEI chromatography: The DEAE elute (216mg) was loaded on to the NUCLEOSIL 4000-7-PEI (250 x 10mm) column, equilibrated with 50 mM Tris-acetate buffer pH 8.0. The bound protein was eluted by applying a linear gradient of NaCI from 0-100% in 15min at 2ml/min flow rate using the FPLC system and the protein eluted at 98%B (60mg). The eluted protein was dialyzed against water and repurified on the same PEI column using ammonium bicarbonate buffer. The PEI column was equilibrated with 20mM ammonium bicarbonate buffer pH 8.0 and the protein was loaded on the column. The bound protein was eluted using buffer 9 gradient from 20-500mM ammonium bicarbonate buffer. The eluted protein (30mg) was dialyzed against water, lyophilized and stored at -200C. Gel filtration chromatography: The final purification of the recombinant protein was achieved by gel filtration chromatography on Superdex G-75 equilibrated with 25 mM TBS, pH 7.2 on AKTA Prime plus purification system. The expression of the protein and the purification of the protein were checked on 15% SDS PAGE and the results are given here. There was a major Coomassie blue stainable protein band corresponding to a molecular size of 16kDa. EXAMPLES Solubility studies: Purified recombinant lectins were dialyzed extensivefy against water and lyophilized. Lyophyllized samples were suspended in buffers of different pH, the protein content and the hemagglutination activity were determined in the clear supernatant after centrifugation. Solubility and the hemagglutinating activities were compared with native lectin. Histochemical studies: Binding of the lectins to tissue sections of confirmed cases of human colon epithelial cancer were performed using biotinylated lectins and the binding was observed by light microscopy. Tissue sections were stained routinely using hematoxylin and eosin and lectin histochemical studies were carried out by standard protocols using five μm tissue sections of cancerous tissues. Sections of colon cancer tissues each of 5 μm thickness used for histochemical studies were obtained using rotary microtome semi-automatic (Leica RM 2145) and deparaffinised by heating on a slide warmer at 60 °C for 1 h. The tissue sections were rehydrated by keeping them in two changes of xylene for five minutes each which were then transferred to absolute and 95% alcohol for three minutes each. As a routine procedure to confirm the state of tissues being in normal or cancerous condition, tissue sections were stained with hematoxylin and eosin (H and E) stains. Tissue sections, after rehydration were treated with basophilic 10 hematoxylin stain for 20-25 min, followed by 1% HCI, acid-alcohol dip and the tissue sections were gently washed under running tap water. Then, sections were counterstained with eosin for 2 minutes, washed with water, dried and dipped in xylene and mounted with DPX (Distrene 80 Dibutyl Phthalate Xylene), a nonaqueous permanent mounting medium and observed using light microscope (Carl zeiss Jenalumar) and photographed. Histochemical staining was carried out, using biotinylated lectins as described by Danguy and Gabius, in Gabius and Gabius (1993). All the reagents were equilibrated to room temperature prior to the staining. At no time the tissue sections were allowed to dry during the staining procedure. Study also included control slides prepared by excluding the biotinylated Jectin. To block the endogenous peroxidase activity, tissue sections after rehydration were incubated with methanol containing 0.3% hydrogen peroxide for 30 minutes at room temperature. Sections were then rinsed with water for 5 minutes and then with TBST (pH 7.2) for 5 minutes. After the tissue sections were rinsed with PBS pH 7.2, sections were incubated with 1% periodate treated BSA in 0.1 M PBS solution for 15 min to block nonspecific binding sites by BSA. Sections were then rinsed with TBST for 5 min. Biotinylated lectins (20μg/ml in PBS) were carefully applied to cover the sections completely and incubated for 1 h, so as to facilitate the complete binding. Sections were then rinsed with TBST, for 5 minutes. These tissue sections were then incubated with streptavidin-HRP (1—>1000 dilution) in PBS, pH 7.2, for 30 minutes at room temperature. Sections were then rinsed with two changes of TBST, pH 7.2, for 5 minutes. After streptavidin-HRP conjugation, tissue sections were developed in DAB Chromogen/H202 substrate in buffered solution (pH 7.5), which was prepared according to the guidelines of manufactures. Tissue sections were incubated for 10 minutes and then gently rinsed with water for 5 minutes. The slides were allowed to dry and dipped in xylene and mounted in DPX. Sections were then examined under light microscope (Carl zeiss Jenalumar) to observe and study the staining intensity patterns by the normal and cancer tissues and photographed. Identification of cell surface antigens: 11 Identification of neoglycans expressed on activated and transformed lymphocytes in comparison with normal lymphocytes by the recombinant lectin; In order to identify the neoglycoproteins expressed on lymphocytes upon activation by PHA and transformed cells, the interaction studies were carried out with respective cell membrane proteins with biotinylated lectin. Isolation of Cell Membrane Proteins: Human peripheral lymphocytes were prepared from the blood collected from a healthy donor. The blood sample (20ml) collected in to glass centrifuge tube containing EDTA (12.5 mM final concentration) and the normal lymphocytes were separated by centrifiigation in Ficoll-Hypaque (Pharmacia) gradient. A portion of normal lymphocytes were activated by incubating with PHA (2.5 μg/ml PHA) in RPMI-1640 medium supplemented with 10% FCS for 72 hours in C02 incubator. Activated cells were harvested by centrifiigation and the activation was confirmed by checking for the expression ofCD25 receptors. Normal lymphocytes, activated lymphocytes and leukemic cell lines; Molt-4 and Jurkat were finally maintained in RPMI 1640 culture medium, supplemented with glutamine (2 mM), heat inactivated FCS (10%), penicillin (100U/ml) and streptomycin (100-ug/mI). The cultures were maintained at 37oC in humidified atmosphere (95% air and 5% C02). Membrane proteins from these cultured cells were isolated by using "Pierce Membrane Protein Extraction Reagent kit" method (Mem-PER, Prod #89826) and the protein contents were estimated using Bio-Rad's DC-Protein estimation Kit. Isolated membrane proteins were finally concentrated by chloroform-methanol precipitation, and the precipitated proteins were air dried and stored at 40C till further use. Electrophoresis & western Blotting: Polyacrylamide gel electrophoresis was performed in the presence of SDS using a minigel system (Hoefer Scientific Instruments, USA) in 10% gel for 60 mins at 120 volts. The proteins were transferred from polyacrylamide gels to Nitro Cellulose membranes in a semidry blotting equipment using transfer buffer (240mM Glycine, 25mM Tris, 0.1% SDS) containing 20% methanol at 75mA for 3hr at 4 OC. Membranes were saturated after transfer with blocking solution (3% p-BSA) and 12 washed with TBST. Lectin binding was carried out at room temperature for 4 hours with biotin-coupled lectin at the final concentration of 20μg/ml (prepared in TBST). After thorough washing, blot was incubated with Streptavidin-HRP (1:1000 in TBST) at room temperature for 1hr and excess Streptavidin-HRP was washed with TBST. Finally the lectin-glycoproteins bands were visualized by developing with DAB chromogenic system. The results of these findings show that the present recombinant lectin has the ability to bind to some of the uniquely expressed neoglycans on the leukemic cells and also activated lymphocytes. SDS PAGE and lectin blotting Proteins from the ovarian cyst fluids samples were separated by SDS PAGE in 10 % gels as described by Laemmli et al using Hoefer Scientific Instruments, San Francisco, USA. Electrophoresis was carried out for 70 min.l using 110 volts. Immediately after the electrophoresis fractionated protein bands were resolved were blotted onto nitro cellulose membrane using semidry blot assembly. Blotted membrane was washed with distilled water and stained with Ponceau reagent to confirm the efficiency of blotting and to mark the location of standard proteins. Finally the blot was washed with TBS containing 0.1 % Tween -20 (TBST) and treated with 3 % P-BSA in TBST for overnight to prevent non-specific binding. After washing with TBST, blot was incubated with biotinylated lectins (20 ug/ ml in TBST) at 370 C for 4-6 hours. Excess and unbound lectin was removed by washing the membrane with TBST and then the membranes were incubated with Streptavidin-HRP (lμg/ml in TBST) for one hour. Unbound Streptavidin-HRP was removed by washing with TBST. The lectin binding glycoproteins were visualized by staining for peroxidase activity using DAB system. Proliferative and Antiproliferative activity on PBMCs and cultured leukemic and colon cancer cells Proliferative/antiproliferative activity of SRL on human peripheral blood mononuclear cells (PBMCs) was determined by tritiated thymidine incorporation assay, using freshly isolated human PBMCs as described by Wang et al., [1992] with some modifications. PBMCs were isolated from the freshly collected blood samples 13 of healthy donors and the cells were maintained in RPMI 1640 complete medium as described previously. The cells were diluted with RPMI medium containing 10% FCS and then seeded (1x105 cells/50 μl/well) in 96-well microplates (NUNC Denmark). Various concentrations (10, 5, 2.5, 1.25 and 0.625 μg/ml) of SRL in RPMI 1640 medium was added to each well in a final volume of 100 μl and incubated at 37°C in a humidified, 5% C02 atmosphere for 72 h. Eighteen hours prior to the total incubation period, 1 uCi of tritiated thymidine was added to each well in a final volume of 10 μ1 and the incubation was continued for another 18 h. Finally, cells were harvested onto a glass filter paper (Amersham Biosciences) using cell harvester (NUNC, Denmark), washed thoroughly with distilled water and dried in micro-oven. Filter discs were transferred to scintillation vials (Tarsons, India) containing 600 μl of toluene-based scintillation cocktail. Finally the incorporated tritiated thymidine was measured by β-scintillation counter (Packard Bioscience Ltd.) as counts per minute (CPM). In order to confirm the lectin-receptor mediated proliferative activity, assays were performed using SRL preincubated with mucin. SRL (10 ug/ml) was preincubated with mucin (12.5 μg in 100 μl) for 1 h at 37°C and used for cell proliferation assay. Appropriate controls containing cells without lectin and cells with mucin alone were included. Results are representative of three independent experiments done in triplicate. Proliferative/antiproliferative effect of SRL on Molt-4 leukemic cell line was determined by tritiated thymidine incorporation assay as described previously. Proliferative/antiproliferative effect of SRL on Molt-4 leukemic cell line was determined by tritiated thymidine incorporation assay as described previously. Cells were seeded (1x105 / well) in a 96-well tissue culture plate and allowed to acclimatize for 3h before lectin treatment Serial concentrations of SRL from 6.25 μg/ml to 100 μg/ml in 50 μl of RPMI medium was added to each well and incubated for 72 h. All other steps of assay remained same as described earlier. For lectin blocking assay, SRL (100 ug/ml) was preincubated with 12.5 μg of mucin in a total volume of 100 μl for 1 h at 37°C and used for assay. Recombinant Sequences 14 The gene sequences and the proteins having the following biological activity: recognize all the human blood groups and have exclusive ability to recognize TF antigen and its cryptic forms, resemble with native S. rolfsii lectin in sugar binding, cell binding and in bringing apoptotic properties, have better stability and solubility properties compared to native lectin are covered within the scope of the application. The nucleotide molecules encoding the recombinant molecules may be DNA or RNA. The proteins of the above mentioned characteristics whether obtained by deliberate or random mutations are covered within the concept of the instant application. Binding of recombinant lectins to different cultured cells are presented in table 1. Table; 1, Binding of Recombinant lectins to Cultured leukemic (Molt 4& Jurkat), Ovarian (PA 1) and colon (COLO 205) cells and the inhibition of binding by mucin Cell lines % positive cells Rec2 Rec3 Native Molt 4 Lectin 84.03 99.87 88.45 Lectin+Mucin 0.24 0.30 0.24 Jurkat Lectin 81.35 97.85 33.84 Lectin+Mucin 0.89 1.02 0.50 PA-1 Lectin 97.97 97.58 96.72 Lectin+Mucin 2.5 4.28 1.63 COLO 205 Lectin 66.53 90.29 91.66 Lectin+Mucin 0.87 1.08 0.99 In each case binding of R2 and R3 with Molt 4, Jurkat (leukemic cells), PA-1 (ovarian cells) and COLO 205 (colon cells) is greater than 90 % and the binding can be abolished by mucin. As another embodiment the immunomodulatory effect on these cultured cells is disclosed. Both R2 and R3 exerted anti proliferative activity on the cultured cells, 15 highest effect (60%) on leukemic cells as compared to ovarian cells (50%) and colon cells (40%). their growth modulatory effect can have potential application in cancer therapy, (table 2) 16 Table; 2, Anti proJiferative activity of recombinant lectins on cultured cells by MTT assay Cell lines Decrease in cell viability (%) Rec 2 (25 μg/ml) Rec 3 (25 μg/ml) Native (25 μ.g/ml) Molt 4 60.68 61.10 57.93 PA-1 46.56 50.60 53.85 COLO-205 26.67 40.43 23.44 In one of the preferred embodiments, the expressed lectin protein (SEQ ID 2 and 3) differs from the native lectin protein in terms of at least one amino acid modification/substitution at positions. Of these, positions changes are indicated in Table 3 Table: 3: Amino acid positions changed for Rec 2 and Rec 3 compared to native lectin sequence Lectin Amino acid positions changed 1 14 34 113 123 Native Ac-T N T E E S1 T D T Q Q S2 V D S Q Q The recombinant lectin sequences can be biotinylated or attached with fluorogenic chromophore by using commonly known methods to be used for the detection of cancer cells or cancer associated specific antigens. The growth modulatory effect of the recombinant lectin finds its application in cancer therapy. Further the use of recombinant lectin as a drug delivery agent for cancer treatment is envisaged in the present application. 17 While the present invention has been described with respect to certain preferred embodiments, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the scope of the invention as defined in the following claims. Indian Patent application No. 30/MUM/2008 is herewith referred in its entirety, made by the same applicant, wherein another recombinant lectin with different functions has been claimed, by way of reference. 18 We Claim: 1. A lectin protein comprising of an amino acid sequence of SEQ ID 1 altered /modified to have at least one of the following biological activity; having specificity to cancer associated cell surface antigen such as TF antigen or its modified forms attached to proteins in general O~linked chains and lipids and having better solubility and stability properties as compared to lectin of SEQ ID 1, 2. The lectin protein as claimed in claim 1 wherein said lectin further has specificity to O-glycosidically linked glycans occurring in glycoproteins and on cell surfaces, but does not show specificity to N-linked glycans; does not bind to fetuin but exhibit very strong affinity for asialofetuin; 3. The lectin protein as claimed in claim 1 wherein said lectin binds to certain cell surface molecules expressed during malignancy and bind to human Breast cancer, colon cancer, ovarian cancer tissues and also cultured cells and leukemic cell lines; exert in vitro antiproliferative activity on the cells 4. The lectin protein as claimed in claim 1 wherein said lectin has a molecular weight of approximate 16.1 kDa and PI value of 6.614 5. The lectin protein as claimed in claims 1-4 wherein said alteration/modification of SEQ ID 1 is at any one of the following positions 14(N-D), 113(E-Q)and 123(E-Q). 6. The lectin protein as claimed in claims 1-5 wherein said alteration/modification of SEQ ID 1 is at any one of the following positions l(Ac-T-V)and34(T-S) 7. The lectin protein as claimed in any preceding claim wherein said altered/modified sequence is SEQ ID No.2 or 3. 19 8. The lectin protein as claimed in any preceding claim as and when used in detection of cancer cell, cancer diagnosis or cancer therapy. 9. A nucleic acid molecule encoding a lectin protein having the following biological activities: having specificity to cancer associated cell surface antigen such as TF antigen or its modified forms attached to proteins in general O-linked chains and lipids and have better solubility and stability properties as compared to lectin of SEQ ID 1. 10. The lectin protein as claimed in claim 9 wherein said lectin has specificity to O-glycosidically linked glycans occurring in glycoproteins and on cell surfaces, but does not show specificity to N-linked glycans; does not bind to fetuin but exhibit very strong affinity for asialofetuin; 11. The lectin protein as claimed in claim 9 wherein said lectin binds to certain cell surface molecules expressed during malignancy and bind to human Breast cancer, colon cancer, ovarian cancer tissues and also cultured cells and leukemic cell lines; colon epithelial cells etc.,., exert in vitro antiproliferative activity on the cells 12. The lectin protein as claimed in claim 9 wherein said lectin has a molecular weight of about 16.1 kDa and PI value of about 6.614 13. The nucleic acid molecule as claimed in claim 9 wherein said nucleic acid molecule is DNA or RNA. 14. The nucleic acid molecule as claimed in claim 9 wherein said lectin protein is altered/modified Sclerotium rolfsii lectin protein of the amino acid sequence of SEQ ID 1.1 or 2.1.. 20 15. The nucleic acid molecule as claimed in claim wherein said lectin protein comprises the amino acid sequence of SEQ ID 2 or SEQ ID 3. 16. A recombinant vector comprising operatively linked in the 5' to 3' direction: a promoter which functions in a host cell; a structural nucleic acid sequence as claimed in claims 9-15 wherein said nucleic acid sequence encodes a lectin protein; and a termination signal. 17. A recombinant vector as claimed in claim 16 wherein said recombinant vector is capable of being replicated, transcribed, translated and expressed in a unicellular organism. 18. A transformed host cell containing the vector as claimed in claim 16. 19. The transformed host cell as claimed in claim 18 wherein said host cell is Escherichia coli bacterium or yeast cell. 20. A process for producing a recombinant Sclerotium rolfsii lectin protein comprising: • culturing a host cell containing the recombinant vector of claim 16 coding for the lectin protein; • expressing the recombinant lectin protein • isolating said lectin protein from the culture. 21. A lectin protein, a nucleic acid molecule encoding said lectin protein, a vector comprising said nucleic acid molecule and a host cell containing said vector substantially as herein described with reference to accompanying drawings and examples. Dated this February 2009 Anand and Anand Agents of the Applicant 21 ABSTRACT Cancer Cell Binding Recombinant Lectins with Antitumor Activity and Method of Preparation The present invention discloses genes for new variants in E.coli that are more close to the wild type lectin sequence but still differ in surface charges. This recombinant version (exemplified Rec-2 and Rec-3) are cancer cell binding with anti tumor activity show blood group specificity similar to the native lectin but retains the stability and the solubility properties. 1 8 FEB 2009 22

Full Text

FORM 2
THE PATENTS ACT, 1970 COMPLETE SPECIFICATION
Section 10
Cancer Cell binding Recombinant Lectins with Antitumor Activity and Method
of Preparation

Unichem Laboratories Ltd., an Indian company of Mahalaxmi Chambers, 22 Bhulabhai Desai Road, Mumbai - 400026, Maharashtra, India
and Karnatak University, Dharwad 580 003, Karnatak, India

The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed:

1 8 FEB 2009

FIELD OF INVENTION
The invention relates to the field of carbohydrate binding proteins, and more specifically to a protein which binds specifically to particular oligosaccharides associated with particular medical significance. The invention specifically relates to lectins, more particularly to recombinant fungal lectins having ability to recognize glycans of clinical importance and their antiproliferative effect on cancer cells finding applications in cancer diagnosis, therapy and research.
BACKGROUND OF INVENTION
Lectins are proteins, or glycoproteins that agglutinate erythrocytes of some or all blood groups in vitro. They are important group of bioactive proteins found in most organisms. Lectins are used as tools for diagnostic and therapeutic purpose in health care areas. Lectins are also used in the purification of glycoproteins, oligosaccharide analysis and in cell selection processes. Lectins can bind reversibly with monosaccharides or the sugar moiety of, glycoproteins or glycolipids or polysaccharides.
Changes in carbohydrate structures on the cell surfaces are a hallmark during cancer. Such changes results in exposure of tumor associated carbohydrate structures. Amongst them, is the increased occurrence of Thomsen-Friedenreich antigen, a disaccharide Galβl->3GalNac-0-Ser/Thr. In normal cells, this antigen is masked by certain sugars, sialic acid or sulphates. Some of the lectins have ability to specifically recognize and bind to these altered structures and exerts various physiological effects such as apoptosis, cytotoxicity, proliferative/antiproliferative activity, metastasis, inhibition of cell adhesion, etc., Hence such lectins are currently being considered for use as cancer diagnostic and therapeutic agents and also for anti tumor activity.
Several lectins have been isolated and characterized from variety of sources. They however differ in their physico-chemical properties such as molecular size and sugar specificities. There is also characterization of a few recombinant proteins. The recombinant mushroom lectin from Marasmius oreades has a molecular size of 33 kDa and shows high affinity for Galαl, 3Gal and Galcαl, 3Galβl, 4GlcNAc.
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(R.P.Kruger et al, J.Biol.Chem., 277, 15002-15005, 2002; H.C.Winter et al. J.Biol.Chem ,277, 14996-15001, 2002., I.J. Goldstein, etal. US Patent :6958321)
Peanut lectin that shows affinity to Thomsen-Friedenreich antigen has been cloned (V.Sharma and A.Surolia, Gene (Amst), 148, 299-304, 1994) and subsequently some mutants of this protein with a subunit molecular mass of ~28kDa has also been expressed in E.coli (V.Sharma, M.Vijayan and A.Surolia, J. Biol. Chem., 271, 21200-21213,1996) with some difference in its preference for sugar specificity. A much smaller lectin of molecular mass of 11.73 kDa with high binding affinity to L-fucose from Pseudomonas aeruginosa has also been expressed in E.coli as a Yellow fluorescent Protein-lectin fusion (BioTechniques, 41, 327-332, 2006).
A mushroom lectin (ABL) from Agaricus bisporus, which has antiproliferative activity on human colon cancer cells and its curative effect in psoriasis is reported by Jonathan M. Rhodes; US patent # 5607679.
Sclerotium rolfsii lectin (SRL) purified from the sclerotial bodies of the phytopathogenic fungus Sclerotium rolfsii exhibit complex carbohydrate specificity and exhibit specificity towards TF antigen. TF antigen is an oncofetal antigen, a disaccharide Galβl, 3GaNAc α ser/thr and is known to expressed on cell surfaces in different cancers. SRL apart from binding to TF antigen, it also bind to sialylated and sulfated disaccharide. The crystal structure of this lectin and its complete amino acid sequence is determined (Demetres 2007 and Satisha 2008). Binding studies indicated that SRL binds to different human cancer tissues and also to cultured cells. Further binding of SRL to cultured human leukemic cells and colon cells results in their antiproliferation and in colon cells effect is shown to be mediated by inducing apoptosis. However the purified native lectin has solubility problems for its use in various applications, hence there is need of forms of this lectin with improved stability for practical applications.
Lectins thus have enormous potential in the area of medical assays. The instant application proceeds to provide certain lectins with novel characteristics so as to be useful in the medical field and improved properties.
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SUMMARY OF THE INVENTION
The present invention discloses recombinant lectins having specificity to certain sugar chains uniquely found on certain cancer cells. The present invention further provides method for preparing two recombinant lectins (designated as UC-SS/CSR-1803 and UC-SS/CSR-1805 or simply Rec-2 and Rec-3 respectively) expressed in E.coli. The method comprises the synthesis of lectin genes whose sequences are derived by deliberate modifications made to the original amino acid sequence of a lectin from Sclerotium rolfsii (Sathisha et.al., Amino Acids; 2008, 35, 309-320 ), a soil borne phytopathogenic fungus, and its cloning in E.coli. The recombinant lectins expressed as soluble proteins and are purified by ion exchange and gel filtration chromatographic techniques.
The present invention specifically provides two recombinant lectins (Rec-2 and Rec-3) having approximate subunit masses (Mr 16.1 kDa), which are specific to certain O-glycosidically linked glycans occurring in glycoproteins and on cell surfaces, but does not show specificity to N-Hnked glycans.. The property of recognizing O-glycans but not N-glycans and showing no specificity to human blood groups makes them different from the other recombinant lectin disclosed in copending patent application 30/MUM/2008. It is also found that the recombinant lectins Rec-2 & Rec-3 does not bind to fetuin but exhibit very strong affinity for asialofetuin, there by making it different from that of said recombinant lectin (patent application 30/MUM/2008) and also the native lectin. By virtue of this recognition property they bind to certain cell surface molecules expressed during malignancy (Fig. 1) and thus bind to human Breast cancer, colon cancer, ovarian cancer tissues (Fig .2) and also cultured cells and leukemic cell lines (table 1). Apart from binding to cultured human cancer cells, recombinant lectins exert in vitro antiproliferative activity on the cells (Table 2) contemplating their diagnostic and therapeutic properties.
The invention discloses the DNA sequences for Rec-2 and Rec-3 coding for 141 amino acids long lectins in each case similar to the native lectin (S. rolfsii lectin), but with few deliberate modifications synthesized chemically and cloned into E. coli (SEQ ID 1.1 and SEQ ID 2.1). The cloned lectins were expressed after induction with the inducer IPTG- The expressed protein appeared as soluble and in active form in
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each case. The recombinant lectins were purified to essential homogeneity by resorting to anion exchange and molecular sieve chromatography. The mass spectrometric and SDS PAGE estimate of molecular weights show a value of approximate 16.1 kDa. The isoelectric focusing experiment shows the PI value of 6.614 whereas the native lectin has 6.219, the property markedly helped in improving the solubility and stability of recombinant proteins. Improved solubility, , stability and the binding to desialylated TF antigen of the recombinant lectins have greater implications of their applications in various fields as reagents apart from ease of production.
The present invention provides a method for preparing a recombinant lectin expressed in a host cell such as E.coli or yeast. This method comprises the synthesis of a lectin gene based on amino acid sequence derived from the MALDI MS/MS of the Sclerotiam rotlsit lectin (SEQ ID. I), a soil borne pfryfopatfrcgerrrc Fungus, and its cloning in the host cell.
In order to obtain desired properties the amino acid sequence of the native lectin was modified deliberately and recombinant lectins were obtained expressing in E-coli. The present invention discloses two recombinant lectins with modified amino acid sequences but have close resemblances with native lectin with respect to their binding properties towards cancer tissues, in vitro cultured cells, and their apoptotic activity to cultured cells. However these two lectins have better solubility and stability and desired sugar specificity properties for applications as compared to native lectin.
The recombinant lectin expressed as a soluble protein is purified by ion exchange and gel filtration chromatographic techniques.
The amino acid sequence SEQ ID 2 and 3 represents such lectin having all the above characteristics. The nucleic acid molecule encoding the amino acid for the protein of the above characteristics is also within the scope of the application. The DNA sequence of SEQ ID 1.1 and 2.1 represents such nucleic acid encoding the lectin having the above characteristics. The sequences also envisage any change in the nucleotide or amino-acid sequences including but not limited to any substitution, deletion, addition or modification such as acetylation, nitration, glycation
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,sulphonation etc. in any of the positions of the entire sequence and also to include truncations at 5' or 3' end of the sequences which do not alter the properties disclosed in the instant application.
In one aspect this recombinant lectin in a biotinylated form or modified with a fluorogenic chromophore can be used for the detection of cancer cells or cancer associated specific antigens.
The growth modulatory effect of the recombinant lectin can have potential application in cancer therapy as anti tumor agents.
In another aspect the recombinant lectin can be used in cancer diagnosis, therapy and research.
The application particularly discloses a DNA sequence (SEQ ID 1.1 and 2.1 ) coding for a 141 amino acids long lectin similar to the lectin (S. rolfsii), but with a few modifications was chemically synthesized and cloned into E.coli.
The method for producing said recombinant lectin has also been disclosed. The cloned lectin is expressed after induction with the inducer IPTG
BRIEF DESCRIPTION OF FIGURES
Figure 1: Comparative profiles of native SRL receptors isolated from the membranes of human normal resting PBMCs (1), PHA-activated PBMCs (2), Molt-4 (3) and Jurkat cells (4). Membrane proteins (40 |ig) were resolved by SDS-PAGE (10% gel) and blotted on to nitrocellulose membrane. SRL interacting proteins were probed with biotinylated lectin and compared with standard molecular weight protein markers (M) after visualization with streptavidin-HRP reaction.
Figure 2:
Binding pattern of Rec 2 and Rec 3 and native lectin to human cancerous tissues of breast and ovary in comparison with H & E staining. Biotinylated lectins were used for the immunohistochemistry
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Figure 3:
The Amino acid sequence of lectin protein isolated from S. rolfsii (SEQ ID 1);
Protein Sequence of the recombinant lectin Rec 2 of the instant application. The
number of amino acid coding for this lectin is 141 and is represented by SEQ ID 2.
The altered/modified positions have been highlighted in the sequence.
Protein Sequence of the recombinant lectin Rec 3 of the instant application. The
number of amino acid coding for this lectin is 141 and is represented by SEQ ID 3.
The altered/modified positions have been highlighted in the sequence.
Multiple sequence alignment of the SEQ ID I -3.
Figure 4: A DNA sequence encoding the lectin of the instant application, SEQ ID 1.1 and 2.1 The length of the optimized sequence is —-- nucleotides which includes the 5* end Nde f (CATATG) and 3 end BamHT (GGATCC) restriction sites.SEQ fD: 1.1; GENE NAME; UC- SS/CSR—1803 (Rec-2); SEQ ID: 2.1; GENE NAME: UC-SS/CSR-1805(Rec-3)
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses cloned genes for two new variants, designated here as Rec-2 and Rec-3, in E-coli that are more close to the native S. rolfsii lectin in amino acid sequence, but still differ in surface charges. Surface charge was altered deliberately in order to enhance the better solubility and stability of the proteins as compared to native lectin. Two lectin genes coding S. rolfsii lectin with few alterations are chemically synthesized and expressed in E-coli. Recombinant lectins are purified by conventional chromatographic methods and are shown to have molecular mass approximate 16,000 Da. The two recombinant lectins described here are different in blood group specificity, recognize all the human blood groups and have exclusive ability to recognize TF antigen and its cryptic forms. The two blood group non specific recombinant lectins are more closely resemble with native S. rolfsii lectin in sugar binding, cell binding and in bringing apoptotic properties. However the recombinant lectins have better stability and solubility properties compared to native lectin making them ideal for various Applications.
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The various applications to the use of the recombinant lectins may be in the following:
Considering the property of cancer cell binding Rl and R2 can be used as drug delivery agents for cancer treatment and may also find application as diagnostic tools in the detection of microbial / viral pathogens.
These recombinant lectins can be used for the detection of certain glycoproteins and glycoconjugates present in biological fluids.
The lectins exhibits substrate specificity towards cancer associated cell surface carbohydrate such as desialylated TF antigen, they can have application as affinity matrices for the purification of glycoproteins and glycoconjugates of physiological and biochemical significance that bind to these recombinant lectins.
Recombinant lectins can be used as fusion partners for the purification of recombinant lectins.
The invention would now be disclosed by way of preferred methodology and embodiments:
Lectin Gene construct and cloning:
The amino acid sequence derived from the MALDI MS/MS and x-ray crystallography data of the lectin purified from the soil fungus Sclerotium rolfsii formed the basis for the gene construct. The gene corresponding to this sequence SEQ ID 1 was chemically synthesized by following the standard protocols. The assembled gene was first inserted into a pUC57 vector. The insert was released by digesting the plasmid with Ndel & BamHI and then recloned in to pET20b previously digested Ndel and BamHI. After ligation the plasmid was used to transform E.coli DE3 (GOLD) host strain for expression. The recombinant clones were analyzed for release of insert after digestion with Nde I and Bam HI. The SDS-PAGE analysis of recombinant E.coli upon induction with IPTG showed the expected ~16kDa protein.
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Cell growth
A single colony of the recombinant E.coli was inoculated into 5ml LB-Amp allowed to grow at 370C overnight with shaking. The overnight grown culture was inoculated into the Fermentation Media and grown at 370 C till an OD of ~2.0 was reached, the cultures were then induced with 250uM IPTG (final concentration) and grown overnight at 200C.
Cell extract preparation
The overnight grown cultures were centrifiiged at 8000rpm for l0min and the pelleted cells (4gms) were suspended in 40ml of 50mM Tris-HCl pH8.0 containing ImM PMSF and ImM EDTA. The cell suspension was sonicated for 20min using the sonicator. The cells were centrifiiged at 12000rpm, l0min at 40C. The supernatant was used for further purification.
Purification of the protein
DEAE-cellulose chromatography:
The supernatant (400mg of protein) in 50 mM Tris-HCl buffer pH 8.0 was loaded on
25ml DEAE (BIORAD) column equilibrated in 50mM Tris-HCl buffer pH 8.0. The
column was washed with 2 column volumes of buffer followed by step-wise elution
of the column with buffer b containing 75mM NaCI and 200mM NaCI. The lectin was
eluted with buffer containing 300mM NaCI. This fraction contained 216 mg of
protein.
PEI chromatography:
The DEAE elute (216mg) was loaded on to the NUCLEOSIL 4000-7-PEI (250 x 10mm) column, equilibrated with 50 mM Tris-acetate buffer pH 8.0. The bound protein was eluted by applying a linear gradient of NaCI from 0-100% in 15min at 2ml/min flow rate using the FPLC system and the protein eluted at 98%B (60mg). The eluted protein was dialyzed against water and repurified on the same PEI column using ammonium bicarbonate buffer.
The PEI column was equilibrated with 20mM ammonium bicarbonate buffer pH 8.0 and the protein was loaded on the column. The bound protein was eluted using buffer
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gradient from 20-500mM ammonium bicarbonate buffer. The eluted protein (30mg) was dialyzed against water, lyophilized and stored at -200C.
Gel filtration chromatography: The final purification of the recombinant protein was achieved by gel filtration chromatography on Superdex G-75 equilibrated with 25 mM TBS, pH 7.2 on AKTA Prime plus purification system.
The expression of the protein and the purification of the protein were checked on 15% SDS PAGE and the results are given here. There was a major Coomassie blue stainable protein band corresponding to a molecular size of 16kDa.
EXAMPLES
Solubility studies:
Purified recombinant lectins were dialyzed extensivefy against water and lyophilized. Lyophyllized samples were suspended in buffers of different pH, the protein content and the hemagglutination activity were determined in the clear supernatant after centrifugation. Solubility and the hemagglutinating activities were compared with native lectin.
Histochemical studies:
Binding of the lectins to tissue sections of confirmed cases of human colon epithelial cancer were performed using biotinylated lectins and the binding was observed by light microscopy. Tissue sections were stained routinely using hematoxylin and eosin and lectin histochemical studies were carried out by standard protocols using five μm tissue sections of cancerous tissues. Sections of colon cancer tissues each of 5 μm thickness used for histochemical studies were obtained using rotary microtome semi-automatic (Leica RM 2145) and deparaffinised by heating on a slide warmer at 60 °C for 1 h. The tissue sections were rehydrated by keeping them in two changes of xylene for five minutes each which were then transferred to absolute and 95% alcohol for three minutes each. As a routine procedure to confirm the state of tissues being in normal or cancerous condition, tissue sections were stained with hematoxylin and eosin (H and E) stains. Tissue sections, after rehydration were treated with basophilic
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hematoxylin stain for 20-25 min, followed by 1% HCI, acid-alcohol dip and the tissue sections were gently washed under running tap water. Then, sections were counterstained with eosin for 2 minutes, washed with water, dried and dipped in xylene and mounted with DPX (Distrene 80 Dibutyl Phthalate Xylene), a nonaqueous permanent mounting medium and observed using light microscope (Carl zeiss Jenalumar) and photographed.
Histochemical staining was carried out, using biotinylated lectins as described by Danguy and Gabius, in Gabius and Gabius (1993). All the reagents were equilibrated to room temperature prior to the staining. At no time the tissue sections were allowed to dry during the staining procedure. Study also included control slides prepared by excluding the biotinylated Jectin. To block the endogenous peroxidase activity, tissue sections after rehydration were incubated with methanol containing 0.3% hydrogen peroxide for 30 minutes at room temperature. Sections were then rinsed with water for 5 minutes and then with TBST (pH 7.2) for 5 minutes. After the tissue sections were rinsed with PBS pH 7.2, sections were incubated with 1% periodate treated BSA in 0.1 M PBS solution for 15 min to block nonspecific binding sites by BSA. Sections were then rinsed with TBST for 5 min. Biotinylated lectins (20μg/ml in PBS) were carefully applied to cover the sections completely and incubated for 1 h, so as to facilitate the complete binding. Sections were then rinsed with TBST, for 5 minutes. These tissue sections were then incubated with streptavidin-HRP (1—>1000 dilution) in PBS, pH 7.2, for 30 minutes at room temperature. Sections were then rinsed with two changes of TBST, pH 7.2, for 5 minutes. After streptavidin-HRP conjugation, tissue sections were developed in DAB Chromogen/H202 substrate in buffered solution (pH 7.5), which was prepared according to the guidelines of manufactures. Tissue sections were incubated for 10 minutes and then gently rinsed with water for 5 minutes. The slides were allowed to dry and dipped in xylene and mounted in DPX. Sections were then examined under light microscope (Carl zeiss Jenalumar) to observe and study the staining intensity patterns by the normal and cancer tissues and photographed.
Identification of cell surface antigens:
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Identification of neoglycans expressed on activated and transformed lymphocytes in comparison with normal lymphocytes by the recombinant lectin; In order to identify the neoglycoproteins expressed on lymphocytes upon activation by PHA and transformed cells, the interaction studies were carried out with respective cell membrane proteins with biotinylated lectin.
Isolation of Cell Membrane Proteins:
Human peripheral lymphocytes were prepared from the blood collected from a healthy donor. The blood sample (20ml) collected in to glass centrifuge tube containing EDTA (12.5 mM final concentration) and the normal lymphocytes were separated by centrifiigation in Ficoll-Hypaque (Pharmacia) gradient. A portion of normal lymphocytes were activated by incubating with PHA (2.5 μg/ml PHA) in RPMI-1640 medium supplemented with 10% FCS for 72 hours in C02 incubator. Activated cells were harvested by centrifiigation and the activation was confirmed by checking for the expression ofCD25 receptors.
Normal lymphocytes, activated lymphocytes and leukemic cell lines; Molt-4 and Jurkat were finally maintained in RPMI 1640 culture medium, supplemented with glutamine (2 mM), heat inactivated FCS (10%), penicillin (100U/ml) and streptomycin (100-ug/mI). The cultures were maintained at 37oC in humidified atmosphere (95% air and 5% C02). Membrane proteins from these cultured cells were isolated by using "Pierce Membrane Protein Extraction Reagent kit" method (Mem-PER, Prod #89826) and the protein contents were estimated using Bio-Rad's DC-Protein estimation Kit. Isolated membrane proteins were finally concentrated by chloroform-methanol precipitation, and the precipitated proteins were air dried and stored at 40C till further use.
Electrophoresis & western Blotting:
Polyacrylamide gel electrophoresis was performed in the presence of SDS using a minigel system (Hoefer Scientific Instruments, USA) in 10% gel for 60 mins at 120 volts. The proteins were transferred from polyacrylamide gels to Nitro Cellulose membranes in a semidry blotting equipment using transfer buffer (240mM Glycine, 25mM Tris, 0.1% SDS) containing 20% methanol at 75mA for 3hr at 4 OC. Membranes were saturated after transfer with blocking solution (3% p-BSA) and
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washed with TBST. Lectin binding was carried out at room temperature for 4 hours with biotin-coupled lectin at the final concentration of 20μg/ml (prepared in TBST). After thorough washing, blot was incubated with Streptavidin-HRP (1:1000 in TBST) at room temperature for 1hr and excess Streptavidin-HRP was washed with TBST. Finally the lectin-glycoproteins bands were visualized by developing with DAB chromogenic system.
The results of these findings show that the present recombinant lectin has the ability to bind to some of the uniquely expressed neoglycans on the leukemic cells and also activated lymphocytes.
SDS PAGE and lectin blotting
Proteins from the ovarian cyst fluids samples were separated by SDS PAGE in 10 % gels as described by Laemmli et al using Hoefer Scientific Instruments, San Francisco, USA. Electrophoresis was carried out for 70 min.l using 110 volts. Immediately after the electrophoresis fractionated protein bands were resolved were blotted onto nitro cellulose membrane using semidry blot assembly. Blotted membrane was washed with distilled water and stained with Ponceau reagent to confirm the efficiency of blotting and to mark the location of standard proteins. Finally the blot was washed with TBS containing 0.1 % Tween -20 (TBST) and treated with 3 % P-BSA in TBST for overnight to prevent non-specific binding. After washing with TBST, blot was incubated with biotinylated lectins (20 ug/ ml in TBST) at 370 C for 4-6 hours. Excess and unbound lectin was removed by washing the membrane with TBST and then the membranes were incubated with Streptavidin-HRP (lμg/ml in TBST) for one hour. Unbound Streptavidin-HRP was removed by washing with TBST. The lectin binding glycoproteins were visualized by staining for peroxidase activity using DAB system.
Proliferative and Antiproliferative activity on PBMCs and cultured leukemic and colon cancer cells
Proliferative/antiproliferative activity of SRL on human peripheral blood mononuclear cells (PBMCs) was determined by tritiated thymidine incorporation assay, using freshly isolated human PBMCs as described by Wang et al., [1992] with some modifications. PBMCs were isolated from the freshly collected blood samples
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of healthy donors and the cells were maintained in RPMI 1640 complete medium as described previously. The cells were diluted with RPMI medium containing 10% FCS and then seeded (1x105 cells/50 μl/well) in 96-well microplates (NUNC Denmark). Various concentrations (10, 5, 2.5, 1.25 and 0.625 μg/ml) of SRL in RPMI 1640 medium was added to each well in a final volume of 100 μl and incubated at 37°C in a humidified, 5% C02 atmosphere for 72 h. Eighteen hours prior to the total incubation period, 1 uCi of tritiated thymidine was added to each well in a final volume of 10 μ1 and the incubation was continued for another 18 h. Finally, cells were harvested onto a glass filter paper (Amersham Biosciences) using cell harvester (NUNC, Denmark), washed thoroughly with distilled water and dried in micro-oven. Filter discs were transferred to scintillation vials (Tarsons, India) containing 600 μl of toluene-based scintillation cocktail. Finally the incorporated tritiated thymidine was measured by β-scintillation counter (Packard Bioscience Ltd.) as counts per minute (CPM). In order to confirm the lectin-receptor mediated proliferative activity, assays were performed using SRL preincubated with mucin. SRL (10 ug/ml) was preincubated with mucin (12.5 μg in 100 μl) for 1 h at 37°C and used for cell proliferation assay. Appropriate controls containing cells without lectin and cells with mucin alone were included. Results are representative of three independent experiments done in triplicate.
Proliferative/antiproliferative effect of SRL on Molt-4 leukemic cell line was determined by tritiated thymidine incorporation assay as described previously. Proliferative/antiproliferative effect of SRL on Molt-4 leukemic cell line was determined by tritiated thymidine incorporation assay as described previously. Cells were seeded (1x105 / well) in a 96-well tissue culture plate and allowed to acclimatize for 3h before lectin treatment Serial concentrations of SRL from 6.25 μg/ml to 100 μg/ml in 50 μl of RPMI medium was added to each well and incubated for 72 h. All other steps of assay remained same as described earlier. For lectin blocking assay, SRL (100 ug/ml) was preincubated with 12.5 μg of mucin in a total volume of 100 μl for 1 h at 37°C and used for assay.
Recombinant Sequences
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The gene sequences and the proteins having the following biological activity: recognize all the human blood groups and have exclusive ability to recognize TF antigen and its cryptic forms, resemble with native S. rolfsii lectin in sugar binding, cell binding and in bringing apoptotic properties, have better stability and solubility properties compared to native lectin
are covered within the scope of the application. The nucleotide molecules encoding the recombinant molecules may be DNA or RNA. The proteins of the above mentioned characteristics whether obtained by deliberate or random mutations are covered within the concept of the instant application.
Binding of recombinant lectins to different cultured cells are presented in table 1.
Table; 1, Binding of Recombinant lectins to Cultured leukemic (Molt 4& Jurkat), Ovarian (PA 1) and colon (COLO 205) cells and the inhibition of binding by mucin

Cell lines % positive cells
Rec2 Rec3 Native
Molt 4 Lectin 84.03 99.87 88.45
Lectin+Mucin 0.24 0.30 0.24
Jurkat Lectin 81.35 97.85 33.84
Lectin+Mucin 0.89 1.02 0.50
PA-1 Lectin 97.97 97.58 96.72
Lectin+Mucin 2.5 4.28 1.63
COLO 205 Lectin 66.53 90.29 91.66
Lectin+Mucin 0.87 1.08 0.99
In each case binding of R2 and R3 with Molt 4, Jurkat (leukemic cells), PA-1 (ovarian cells) and COLO 205 (colon cells) is greater than 90 % and the binding can be abolished by mucin.
As another embodiment the immunomodulatory effect on these cultured cells is disclosed. Both R2 and R3 exerted anti proliferative activity on the cultured cells,
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highest effect (60%) on leukemic cells as compared to ovarian cells (50%) and colon cells (40%). their growth modulatory effect can have potential application in cancer therapy, (table 2)
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Table; 2, Anti proJiferative activity of recombinant lectins on cultured cells by MTT assay

Cell lines Decrease in cell viability (%)

Rec 2 (25 μg/ml) Rec 3 (25 μg/ml) Native (25 μ.g/ml)
Molt 4 60.68 61.10 57.93
PA-1 46.56 50.60 53.85
COLO-205 26.67 40.43 23.44
In one of the preferred embodiments, the expressed lectin protein (SEQ ID 2 and 3) differs from the native lectin protein in terms of at least one amino acid modification/substitution at positions. Of these, positions changes are indicated in Table 3
Table: 3: Amino acid positions changed for Rec 2 and Rec 3 compared to native lectin sequence

Lectin Amino acid positions changed

1 14 34 113 123
Native Ac-T N T E E
S1 T D T Q Q
S2 V D S Q Q
The recombinant lectin sequences can be biotinylated or attached with fluorogenic chromophore by using commonly known methods to be used for the detection of cancer cells or cancer associated specific antigens. The growth modulatory effect of the recombinant lectin finds its application in cancer therapy. Further the use of recombinant lectin as a drug delivery agent for cancer treatment is envisaged in the present application.
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While the present invention has been described with respect to certain preferred embodiments, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the scope of the invention as defined in the following claims.
Indian Patent application No. 30/MUM/2008 is herewith referred in its entirety, made by the same applicant, wherein another recombinant lectin with different functions has been claimed, by way of reference.
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We Claim:
1. A lectin protein comprising of an amino acid sequence of SEQ ID 1 altered /modified to have at least one of the following biological activity; having specificity to cancer associated cell surface antigen such as TF antigen or its modified forms attached to proteins in general O~linked chains and lipids and having better solubility and stability properties as compared to lectin of SEQ ID 1,
2. The lectin protein as claimed in claim 1 wherein said lectin further has specificity to O-glycosidically linked glycans occurring in glycoproteins and on cell surfaces, but does not show specificity to N-linked glycans; does not bind to fetuin but exhibit very strong affinity for asialofetuin;
3. The lectin protein as claimed in claim 1 wherein said lectin binds to certain cell surface molecules expressed during malignancy and bind to human Breast cancer, colon cancer, ovarian cancer tissues and also cultured cells and leukemic cell lines; exert in vitro antiproliferative activity on the cells
4. The lectin protein as claimed in claim 1 wherein said lectin has a molecular weight of approximate 16.1 kDa and PI value of 6.614
5. The lectin protein as claimed in claims 1-4 wherein said alteration/modification of SEQ ID 1 is at any one of the following positions 14(N-D), 113(E-Q)and 123(E-Q).
6. The lectin protein as claimed in claims 1-5 wherein said alteration/modification of SEQ ID 1 is at any one of the following positions l(Ac-T-V)and34(T-S)
7. The lectin protein as claimed in any preceding claim wherein said altered/modified sequence is SEQ ID No.2 or 3.
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8. The lectin protein as claimed in any preceding claim as and when used in detection of cancer cell, cancer diagnosis or cancer therapy.
9. A nucleic acid molecule encoding a lectin protein having the following biological activities: having specificity to cancer associated cell surface antigen such as TF antigen or its modified forms attached to proteins in general O-linked chains and lipids and have better solubility and stability properties as compared to lectin of SEQ ID 1.
10. The lectin protein as claimed in claim 9 wherein said lectin has specificity to O-glycosidically linked glycans occurring in glycoproteins and on cell surfaces, but does not show specificity to N-linked glycans; does not bind to fetuin but exhibit very strong affinity for asialofetuin;
11. The lectin protein as claimed in claim 9 wherein said lectin binds to certain cell surface molecules expressed during malignancy and bind to human Breast cancer, colon cancer, ovarian cancer tissues and also cultured cells and leukemic cell lines; colon epithelial cells etc.,., exert in vitro antiproliferative activity on the cells
12. The lectin protein as claimed in claim 9 wherein said lectin has a molecular weight of about 16.1 kDa and PI value of about 6.614
13. The nucleic acid molecule as claimed in claim 9 wherein said nucleic acid molecule is DNA or RNA.
14. The nucleic acid molecule as claimed in claim 9 wherein said lectin protein is altered/modified Sclerotium rolfsii lectin protein of the amino acid sequence of SEQ ID 1.1 or 2.1..
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15. The nucleic acid molecule as claimed in claim wherein said lectin protein comprises the amino acid sequence of SEQ ID 2 or SEQ ID 3.
16. A recombinant vector comprising operatively linked in the 5' to 3' direction: a promoter which functions in a host cell; a structural nucleic acid sequence as claimed in claims 9-15 wherein said nucleic acid sequence encodes a lectin protein; and a termination signal.
17. A recombinant vector as claimed in claim 16 wherein said recombinant vector is capable of being replicated, transcribed, translated and expressed in a unicellular organism.

18. A transformed host cell containing the vector as claimed in claim 16.
19. The transformed host cell as claimed in claim 18 wherein said host cell is Escherichia coli bacterium or yeast cell.
20. A process for producing a recombinant Sclerotium rolfsii lectin protein comprising:

• culturing a host cell containing the recombinant vector of claim 16 coding for the lectin protein;
• expressing the recombinant lectin protein
• isolating said lectin protein from the culture.
21. A lectin protein, a nucleic acid molecule encoding said lectin protein, a vector
comprising said nucleic acid molecule and a host cell containing said vector
substantially as herein described with reference to accompanying drawings
and examples.
Dated this February 2009
Anand and Anand Agents of the Applicant
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ABSTRACT
Cancer Cell Binding Recombinant Lectins with Antitumor Activity and Method
of Preparation
The present invention discloses genes for new variants in E.coli that are more close to the wild type lectin sequence but still differ in surface charges. This recombinant version (exemplified Rec-2 and Rec-3) are cancer cell binding with anti tumor activity show blood group specificity similar to the native lectin but retains the stability and the solubility properties.
1 8 FEB 2009
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Patent Number

277986

Indian Patent Application Number

350/MUM/2009

PG Journal Number

51/2016

Publication Date

09-Dec-2016

Grant Date

07-Dec-2016

Date of Filing

18-Feb-2009

Name of Patentee

UNICHEM LABORATORIES LTD.

Applicant Address

22 BHULABHAI DESAI ROAD,MUMBAI

Inventors:

#	Inventor's Name	Inventor's Address
1	BALE MURUGI SWAMY	DEPT OF BIOCHEMISTRY, KARNATAK UNIVERSITY, DHARWAD 580003,
2	SHASHIKALA RAMACHANDRA INAMDAR	DEPT OF BIOCHEMISTRY, KARNATAK UNIVERSITY, DHARWAD 580003,
3	HEMALATHA VENKAT	UNICHEM LABORATORIES LTD, MAHALAXMI CHAMBERS 22, BHULABHAI DESAI ROAD, MUMBAI 400026,
4	VISHWANATH BASAVARAJ CHACHADI	DEPT OF BIOCHEMISTRY, KARNATAK UNIVERSITY, DHARWAD 580003,
5	NAGARAJA NARAYAN NAGRE	DEPT OF BIOCHEMISTRY, KARNATAK UNIVERSITY, DHARWAD 580003,
6	CANDADAI SESHADRI RAMADOSS	UNICHEM LABORATORIES LTD, MAHALAXMI CHAMBERS 22, BHULABHAI DESAI ROAD, MUMBAI 400026,

PCT International Classification Number

A61K47/48; C07K14/34; C07K14/415

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA