CLIAMS:We Claim,
1. A novelpeptide which is soluble, cell penetrable, anti-tumorigenic and capable of functionally targeting EWS-FLI1 protein exclusively expressed in Ewing’s sarcoma, the claimed peptide comprises essentially of an amino acid sequence of (SEQ ID NO: 1)CIEWS-PEP wherein the length of the peptide is 49 amino acid residues.
2. The peptide as claimed in claim 1 wherein the solubility of the said peptide is 99-100% soluble in water.
3. The peptide as claimed in claim 1 wherein the said peptide can be localized in the nucleus of Ewing’s Sarcoma Cells and cell penetration is 99.7%
4. The peptide as claimed in claim 1 wherein the said peptide inhibits cell proliferation of Ewing’s sarcoma cells
5. A pharmaceutical composition for use in treatment of Ewing’s Sarcoma comprises of therapeutically effective amount of peptide of claim 1 and atleast one pharmaceutical carrier, excipient or diluent.
6. The composition as claimed in claim 6, where in the composition shall be formulated into dosage forms selected from a group comprising of powder, a tablet, a capsule, a tablet matrix, a suppository, a controlled release formulation, a delayed release formulation, a slow release formulation, a sustained release formulation, a colonic release formulation, an oral formulation, parenteral formulation including but not limited to intravenous formulation, intramuscular formulation, a bead formulation, a microencapsulated delivery system, a fluid carrier, a solution, a gelatin capsule, a liposomal suspension or a nano-formulation with or without specific targeting moiety including antibody or ligands for oral or parenteral use.
,TagSPECI:Form 2

THE PATENT ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

“NOVEL CELL PENETRATING ANTI TUMORIGENIC POLYPEPTIDE (CIEWS-PEP) FOR POTENTIAL THERAPEUTIC APPLICATION IN TREATMENT OF EWING’S SARCOMA”

in the name of CANCER INSTITUTE an Indian National having address at CANCER INSTITUTE (WIA), ADYAR, CHENNAI – 600020, Tamil Nadu, India.

The following specification particularly describes the invention and the manner in which it is to be performed
FIELD OF INVENTION
The present invention relates to the field of therapeutic peptides. More particularly the present invention relates to novel cell penetrating anti tumorigenic polypeptide for potential therapeutic application in treatment of Ewing’s sarcoma, a bone cancer.
BACK GROUND OF THE INVENTION AND PRIOR ART:
The Ewing's sarcoma family of tumors (ESFT) is composed of highly malignant tumors of bone and soft tissue occurring in children, adolescents, and young adults, requiring novel, targeted therapies. ESFT are defined by the characteristic chromosomal translocation t(11:22) and its fusion protein product EWS-FLI1. The translocation brings central exons of the EWSR1 gene ("Ewing Sarcoma Breakpoint region 1") to the central exons of an ets family gene; FLI1 (“Friend Leukemia integration 1”; chromosome 11). This fusion protein is uniquely expressed in Ewing's sarcoma cells. Biochemical purification and characterization of EWS-FLI1 revealed it to be a disordered protein, and the intrinsic disorder of EWS-FLI1 is critical for its transcriptional activity. EWS-FLI1 functions as an oncoprotein and its activity is essential for the tumorigenicity of Ewing's sarcoma cells. Hence, EWS-FLI1 is an ideal therapeutic target protein in Ewing’s sarcoma due to its causative role in the process of tumorigenesis.EWS-Fli1 is known to interact with protein partners involved in transcription apparatus (FOS-JUN dimers, RNA polymerase holozyme II (Pol II), Creb-Binding protein (CBP), RNA Helicase A (RHA), NR01B), Spliceosome (snRNP U1C, TASR proteins). Transcriptional targets of EWS-FLI1 include NR01B, Enhancer of Zeste 2 (EZH2), caveolin, Aurora kinase A, hTERT, uridine phosphorylase, GSTM4, PLD2, GLI1, Cyclin D1, IGF1, MYC, NKX2-2, TGFßRII and IGFBP3.
Peptide therapeutics are a novel class of agents for cancer therapy. Therapeutic peptides (TPs) are capable of modulating important protein/protein interactions and eliciting a therapeutic response. In recent years peptide based targeting of drugable targets has greatly increased aided by discovery of new peptides, and the solid-phase peptide synthesis being optimized, allowing routine synthesis of large polypeptides and small proteins, this combined with rising number of side effects seen for small molecule therapeutics (eg., cancer chemotherapeutics or COX-2 inhibitors) has made TPs increasingly attractive. The number of peptide and protein new chemical entities has been continuously increasing during recent years as also our knowledge of biochemical pathways, and the resultant increase in the number of potential target proteins, nucleic acids, or lipidic structures whose surface offer themselves for agonist or antagonist development. In many cases a protein fragment, either part of the unprocessed protein or a cleaved piece, can be identified that serves as a ligand or the target itself.
Two studies of interest have investigated the role of peptides in inhibiting EWS-FLI1 activity, in one of the studies it was shown that the three-dimensional shape of EWS-FLI1 allows it to work together with another transcription factor called RNA Helicase A (RHA). A group of proteins that includes EWS-FLI1 and RHA were found responsible for forming Ewing’s Sarcoma. They then constructed a mimic of the portion of RHA that adheres to EWS-FLI1, called peptide E9R. E9R peptide could prevent EWS-FLI1 from working with RHA which in turn inhibited tumorigenic properties of EWS-FLI1 (Erkizan, H.V., et al.Nat Med15, 750-756 (2009)). In another study, using peptide phage display, peptides which can interact with the native EWS-FLI1 protein were identified. One of the peptides designated ESAP1 was used to test for anti-EWS-FLI1 activity. The peptide was found to interact with EWS-FLI1 protein and inhibited tumorigenic properties of Ewing's sarcoma cells (Erkizan, H.V., et al., Cell Cycle10, 3397-3408 (2011)).There are reports available in the literature for the existence of drugs in the treatment of Ewing’s sarcoma. However they suffers various drawbacks which includes lack of improved potency, stability, solubility, functional targeting, cell penetration, nuclear localization etc . Thus there exist a need in the state of art to develop a novel peptide which is devoid of above said drawbacks for the treatment of Ewing’s sarcoma.
OBJECT OF THE INVENTION
The main object of the invention is to design anovel, soluble therapeutic polypeptide comprises essentially of an amino acid sequence of (SEQ ID NO: 1)CIEWS-PEP capable of functionally targeting EWS-FLI1 protein exclusively expressed in Ewing’s sarcoma.
Another object of the present invention is to develop a novel therapeutic polypeptide possessing the property of cell penetration and nuclear localization of Ewing’s Sarcoma Cells
Yet another object of the present invention is to develop a novel therapeutic polypeptide that can inhibits cell proliferation of Ewing’s sarcoma cells
Yet another object of the present invention is to formulate a pharmaceutical composition comprising of the developed therapeutic polypeptide and atleast one pharmaceutical carrier, excipient or diluent
Further object of the present invention is to utilize the developed therapeutic polypeptide in treating Ewing’s sarcoma
BRIEF DESCRIPTION OF THE DRAWINGS:
Figure 1 illustrates the uptake of the peptide in Ewing’s sarcoma cells
Figure 2 illustrates the nuclear localization of the peptide in Ewing’s sarcoma cells.
Figure 3 illustrates the cytotoxicity in response to peptide treatment in Ewing’s sarcoma cells.
Figure 4 illustrates the inhibition of growth response to peptide treatment in Ewing’s sarcoma cells.
Figure 5 illustrates the inhibition of clonogenicity in response to peptide treatment in Ewing’s sarcoma cells.
Figure 6 illustrates the inhibition of cell cycle in response to peptide treatment in Ewing’s sarcoma cells
Figure 7 illustrates the inhibition of EWSFLI1 target genes in Ewing’s sarcoma cells.

SUMMARY OF THE INVENTION.
The present invention discloses a novel polypeptide sequence which is highly soluble, cell penetrable, anti-tumorigenic and capable of functionally targeting EWS-FLI1 protein exclusively expressed in Ewing’s sarcoma. The invention shall disclose the accurate amino acid composition of the peptide. The polypeptide CIEWS-PEP in the course of analysis has proved to be water soluble, capable of cell penetration, nuclear localization. It displays wide ranging anti tumorigenic effects in-vitro, like inhibition of cell proliferation, clonogenicity, cell cycle progression, and down-regulation of EWSFLI1 target genes known to be involved in tumorigenesis.
DETAILED DESCRIPTION OF THE INVENTION.
The present invention pertains to a novel polypeptide sequence designed to be soluble, cell penetrable, anti-tumorigenic and capable of functionally targeting EWS-FLI1 protein exclusively expressed in Ewing’s sarcoma.
Aminoacid composition of the peptide of the present invention starting from N-terminal to C-terminal designated CIEWS-PEP.
Sequence ID No 1
GlyArgLysLysArgArgGlnArgArgArgProGlnProLysLysLysArgLysValProSerGlnTyrSerGlnGlnSerSerSerTyrGlyGlnGlnAsnProSerTyrAspSerValArgArgGlyAlaTrpGlyAsnAsnMet
Abbreviations and Amino Acid Names
Ala = Alanine; Arg = Arginine; Asn = Asparagine; Asp = Aspartic Acid (Aspartate); Asx = Aspartic Acid or Asparagine; Cys = Cysteine; Glu = Glutamic Acid (Glutamate); Gln = Glutamine; Gix = Glutamine or Glutamic Acid; Gly = Glycine; His = Histidine; Ile = Isoleucine; Leu = Leucine; Lys = Lysine; Met = Methionine; Phe = Phenylalanine; Pro = Proline; Ser = Serine; Thr = Threonine; Trp = Tryptophan; Tyr = Tyrosine; Val = Valine.
Characterization of chemical and biophysical parameters of the developed polypeptide was carried out and the following results were observed.
The polypeptide of the present invention comprises of 49 amino acids, it has a molecular weight of 5849.5 Da, and theoretical pI of 11.92.
Atomic composition:
Carbon C 246
Hydrogen H 405
Nitrogen N 93
Oxygen O 72
Sulfur S 1

Formula:C246H405N93O72S1
Total number of atoms: 817
Extinction coefficients:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 9970
Abs 0.1% (=1 g/l) 1.704
The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro).
>20 hours (yeast, in vivo).
>10 hours (Escherichia coli, in vivo).
Aliphatic index: 13.88
Grand average of hydropathicity (GRAVY): -2.218

The solubility of the peptide was assessed using online solubility prediction tolls. The PROSO II tool (http://mips.helmholtz-muenchen.de/prosoII) predicted the peptide to be soluble with a solubility score of 0.953. Another prediction tool from university of Oklahoma (http://www.biotech.ou.edu/) also predicted that peptide is 99-100% soluble in water. The peptide was tested up to concentrations of 50 micro molar and no evidence of insolubility in the form of precipitate formation was observed indicating the soluble nature of the peptide.
The cell penetration property of the polypeptide into Ewing’s sarcoma cells was assessed.Ewing's sarcoma cell lines EWS502, and A673 were maintained in 25cm2 flasks. RPMI (Roswell Park Memorial Institute medium) and DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% FBS (Fetal Bovine serum) at 37oC in humidified incubator saturated in 5% Carbon dioxide. For experiments, exponentially growing cells in flasks which attained 70-80% confluence were used. The peptide transduction was performed following serum starvation. Exponentially growing cells was trypsinized following which the trypsin was inactivated using serum containing medium. The single suspension of cells is washed consecutively two times in serum free medium. Following which the cells were incubated in serum free medium at 37oC for 1 hr. After the incubation the cells were counted and requisite number of viable cells were mixed with the peptide and incubated at 37oC for 30 min. The cells were assessed for their peptide uptake using FACS (Fluorescence-activated cell sorting).The uptake of the peptide defined in terms of the number of cells positive for peptide was 99.7% indicating an uptake of the peptide in almost all the cells analyzed (Figure 1). The photographic insert shows the green fluorescence in the image of the cells indicates the presence of peptide in cells which is one of the design features of the invention.

Nuclear localization of CIEWS-PEP in Ewing’s sarcoma cells was determined as follows.Ewing's sarcoma cell lines EWS502 and A673 was grown on coverslips in a six well plate in RPMI (Roswell Park Memorial Institute medium) and DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% FBS (Fetal Bovine serum) at 37oC in humidified incubator saturated in 5% Carbon dioxide. The peptide transduction was performed following serum starvation. The cells were washed in phosphate buffered saline (PBS) and fixed in paraformaldehyde. The fixed cells were washed in PBS and stained with 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI). The coverslip was mounted onto a slide and analyzed by fluorescence microscopy. The results from fluorescence microscopy (Figure 2) indicate that co-localization of staining pattern between the nucleus staining DAPI reagent and the peptide. This indicates that the peptide localizes to the nucleus which is one of the design features of the invention.
Inhibition of growth and clonogenicity of Ewing’s sarcoma cells was next assessed.Cell proliferation was assessed using CellTiter 96 AQueous One Solution Cell Proliferation Assay (PromegaPvte Ltd. Singapore; Cat no: G3580), according to the manufacturer’s instructions. In order to assess effect of concentration of the peptide on cell survival, single cell suspension of exponentially growing Ewing's sarcoma cells (A673 cells) in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% FBS (Fetal Bovine serum) were prepared. After a cell count approximately 6X104cells were, incubated in serum free medium at 37oC for 30 minutes and then treated with peptide at 0, 10, 25, and 50micromolar concentration for one hour. All incubations were performed at 37oC in humidified incubator saturated in 5% Carbon dioxide.Cell proliferation was then assessed at 24 hour time point using the above mentioned assay in a 96 well plate at a seeding density of 2x103 cells per well.In order to assess time dependent survival of cells to peptide6x104 cells were, incubated in serum free medium at 37oC for 30 minutes and then treated with peptide at 50micromolar concentration for one hour. All incubations were performed at 37oC in humidified incubator saturated in 5% Carbon dioxide. Cell proliferation was then assessed at various time points using the above mentioned assay. Figures 3 and 4 describe results from the analysis.
The results from cell proliferation analysis (Figure 3) indicate that the peptide inhibits proliferation of Ewing’s sarcoma cells in concentration dependant manner. In the case of A673 the cell growth inhibition was observed at 24 hours was 88.52% (p = 0.0002) for 10 µM, 65.94% (p=0.0003) for 25 µM, and 55.79% (p<0.0001) for 50 µM, relative to the control cells whose survival was set at 100%. The error bars in the graph represent standard deviations of mean which was determined from the mean of four independent replicates. Statistical signifcance of the measurements was assessed from raw data using two tailed “t” test. The differences in survival was found to be highly significant.

The results from cell proliferation analysis (Figure 4) indicate that the peptide inhibits proliferation of Ewing’s sarcoma cells in time dependant manner. In the case of A673 the cell growth inhibition was observed at 24 hours (69.53%, p<0.0001), 48 hours (60.18%, p<0.0001), 72 hours (44.41%, p<0.0001), 120 hours (20.52%, p<0.0001) time points relative to the control cells whose survival was set at 100%. The error bars in the graph represent standard deviations of mean which was determined from the mean of five independent replicates.Statistical signifcance assessed was assessed from raw data using two tailed “t” test. The differences in survival was found to be highly significant.

Long term survival was assesed using clonogenic assay.Ewing's sarcoma cell lines EWS502 and A673 were grown in 6 well plate at a concentration of 20000 cells per well in RPMI (Roswell Park Memorial Institute medium) and DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% FBS (Fetal Bovine serum) at 37oC in humidified incubator saturated in 5% Carbon dioxide. The cells were incubated in serum free medium at 37oC for 1 hour and treated with 50 micromolar concentration of the peptide. After peptide treatment the untreated and peptide treated cells were incubated in RPMI (Roswell Park Memorial Institute medium) and DMEM (Dulbecco's Modified Eagle's Medium)supplemented with 10% FBS (Fetal Bovine serum) at 37oC in humidified incubator saturated in 5% Carbon dioxide for a period of 3 weeks. The colonies were stained in methylene blue and counted using colony counter. Figure 5 describes results from the clonogenic assay.The results (figure 5) indicate that in both Ewing’s sarcoma cell lines the colony formation was significantly inhibited. In the case of A673 cells 377 cells were present in untreated control cells whereas only 1 colony was evident in treated cells. In the case of EWS502 cells 68 colonies were estimated to be present in untreated control cells whereas only 5 colonies were estimated in peptide treated. This indicated that peptide can compromise long-term survival of Ewing’s sarcoma cells.

Inhibition of cell proliferation of Ewing’s sarcoma cells was established for the developed polypeptide. Cell growing in an exponential phase were used for the cell cycle analysis assay. The cells were trypsinized and ~5X105 cells of were used for the assay. The cells were subjected to serum starvation and treated with 50 micro molar of the peptide. Following treatment the cells are washed in 3%-BSA PBS two times. The cells were suspended in 500ul ice cold ethanol. The fixed cells were treated with RNA'aseenzyme and Propidium Iodide (PI) and immediately subjected to cell cycle analysis by FACS. Figure 6 describes the results from the FACS analysis. The results (Figure 6)indicate that the presence of the peptide inhibited cell proliferation of Ewing’s sarcoma cells. This was indicated by an increase in the cells in sub G1 phase (apoptotic/necrotic cells) by 6.97%, G1 phases by 6.77% of the cell cycle and decrease in the population in S phase by 8.3% and G2/M phase by 5.93%.

The inhibition of EWS-FLI1 target gene expression in Ewing’s sarcoma cells by the developed therapeutic polypeptide was next assessed.EWS-FLI1 target genes NR0B1, IGF1, EZH, CD99, Gli1, NKX2.2, CyclinD, and c-Myc were analyzed by Real-time Reverse transcriptase (RT) PCR analysis. For the analysis RNA isolation was performed from peptide treated and untreated cells. The 2.0ug of total RNA was reverse transcribed to c-DNA. The quantitative changes in gene expression was estimated using specific primers for the respective genes and SYBR green based chemistry in a real-time PCR machine. GAPDH gene expression was used as a control to normalize for equal loading of cDNA's. The changes in the gene expression pattern was used to infer on the functional effects of the peptide treatment. Figure 7 describes the data from the gene expression analysis.The results from EWSFLI1 target gene expression analysis indicates that the peptide is down-regulating the gene expression of 7 of the 8 target genes analyzed. NR0B1, IGF1, EZH, CD99, NKX2.2, CyclinD, and c-Mycare down regulated to various degrees as indicated by the histogram in the negative axis in the graph.This would indicate that the peptide functionally inhibits the transcriptional activity of the EWSFLI1 protein. This display of down-regulation of gene expression meets a functional criteria set for the invention.
In one of the preferred embodiment the present invention discloses a novelpeptide which issoluble, cell penetrable, anti-tumorigenic and capable of functionally targeting EWS-FLI1 protein exclusively expressed in Ewing’s sarcoma. The peptide comprises essentially of an amino acid sequence of (SEQ ID NO: 1)CIEWS-PEP.The length of the peptide is 49 amino acid residues.
As per the invention the peptide is 99-100% soluble in water.
According to the invention the peptide can be localized in the nucleus of Ewing’s sarcoma cells and cell penetration is 99.7%
In accordance with the invention the peptide inhibits cell proliferation of Ewing’s sarcoma cells
In another preferred embodiment the present invention shall disclose a pharmaceutical composition for use intreatment of Ewing’s Sarcoma comprises of therapeutically effective amount of polypeptide of the present invention and atleast one pharmaceutical carrier, excipient or diluent.
As per the invention the composition shall be formulated into dosage forms selected from a group comprising of powder, a tablet, a capsule, a tablet matrix, a suppository, a controlled release formulation, a delayed release formulation, a slow release formulation, a sustained release formulation, a colonic release formulation, an oral formulation, parenteral formulation including but not limited to intravenous formulation, intramuscular formulation, a bead formulation, a microencapsulated delivery system, a fluid carrier, a solution, a gelatin capsule, a liposomal suspension or a nano-formulation with or without specific targeting moiety including antibody or ligands for oral or parenteral use.
From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.
We Claim,
1. A novelpeptide which is soluble, cell penetrable, anti-tumorigenic and capable of functionally targeting EWS-FLI1 protein exclusively expressed in Ewing’s sarcoma, the claimed peptide comprises essentially of an amino acid sequence of (SEQ ID NO: 1)CIEWS-PEP wherein the length of the peptide is 49 amino acid residues.
2. The peptide as claimed in claim 1 wherein the solubility of the said peptide is 99-100% soluble in water.
3. The peptide as claimed in claim 1 wherein the said peptide can be localized in the nucleus of Ewing’s Sarcoma Cells and cell penetration is 99.7%
4. The peptide as claimed in claim 1 wherein the said peptide inhibits cell proliferation of Ewing’s sarcoma cells
5. A pharmaceutical composition for use in treatment of Ewing’s Sarcoma comprises of therapeutically effective amount of peptide of claim 1 and atleast one pharmaceutical carrier, excipient or diluent.
6. The composition as claimed in claim 6, where in the composition shall be formulated into dosage forms selected from a group comprising of powder, a tablet, a capsule, a tablet matrix, a suppository, a controlled release formulation, a delayed release formulation, a slow release formulation, a sustained release formulation, a colonic release formulation, an oral formulation, parenteral formulation including but not limited to intravenous formulation, intramuscular formulation, a bead formulation, a microencapsulated delivery system, a fluid carrier, a solution, a gelatin capsule, a liposomal suspension or a nano-formulation with or without specific targeting moiety including antibody or ligands for oral or parenteral use.
Dated this 27th day of January 2014 For CANCER INSTITUTE
By its Patent Agent

Dr.B.Deepa