FRAGMENTED GRS POLYPEPTIDE AND VARIANT THEREOF, AND USE THEREOF

Abstract

The present invention relates to a fragmented GRS polypeptide and a variant thereof, and a use thereof and, more specifically, to: an isolated polypeptide consisting of 8 to 170 consecutive amino acids including amino acids 531 to 538 in the amino acid sequence represented by SEQ ID NO: 1 or a polypeptide consisting of a variant having a sequence homology of 80% or more with the polypeptide; a fusion protein and a complex comprising the polypeptide; a polynucleotide coding for the polypeptide; and a use thereof for the prevention and treatment of neoplastic diseases, the detection of cancer cells, imaging, and drug delivery.

Claims

1. A polypeptide consisting of: an isolated polypeptide consisting of consecutive 8 to 170 amino acid residues, which contains amino acids at positions 531 to 538 of an amino acid sequence defined by SEQ ID NO: 1; or a variant having a sequence homology of 80% or more to the isolated polypeptide.

2. The polypeptide of claim 1, wherein the isolated polypeptide or the variant specifically targets a cancer cell.

3. The polypeptide of claim 1, wherein the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 2 to 6.

4. The polypeptide of claim 1, wherein the variant comprises a sequence having a sequence homology of 80% or more to any one amino acid sequence selected from the group consisting of SEQ ID NOS: 2 to 6.

5. The polypeptide of claim 1, wherein the variant comprises the amino acid sequence defined by SEQ ID NO: 7.

6. The polypeptide of claim 1, wherein the isolated polypeptide consists of 8 to 160 consecutive amino acids containing amino acids at positions 531 to 538 of the amino acid sequence defined by SEQ ID NO: 1.

7. The polypeptide of claim 1, wherein the isolated polypeptide consists of 8 to 100 consecutive amino acids containing amino acids at positions 531 to 538 of the amino acid sequence defined by SEQ ID NO: 1.

8. The polypeptide of claim 1, wherein the isolated polypeptide consists of 8 to 50 consecutive amino acids containing amino acids at positions 531 to 538 of the amino acid sequence defined by SEQ ID NO: 1.

9. The polypeptide of claim 1, wherein the isolated polypeptide consists of 8 to 20 consecutive amino acids containing amino acids at positions 531 to 538 of the amino acid sequence defined by SEQ ID NO: 1.

10. The polypeptide of claim 1, wherein the isolated polypeptide consists of 8 to 10 consecutive amino acids containing amino acids at positions 531 to 538 of the amino acid sequence defined by SEQ ID NO: 1.

11. The polypeptide of claim 1, wherein the isolated polypeptide or the variant is in a linear or cyclic form.

12. The polypeptide of claim 1, wherein the polypeptide is in a PEGylated form.

13. A fusion protein, comprising the polypeptide of claim 1 and a heterologous fusion partner.

14. The fusion protein of claim 13, wherein the heterologous fusion partner is an antibody or a fragment thereof.

15. The fusion protein of claim 14, wherein the fragment is selected from the group consisting of Fc, a diabody, Fab, Fab′, F(ab)2, F(ab′)2, Fv, and scFv.

16. A dimeric or multimeric complex, comprising at least one polypeptide of claim 1.

17. A polynucleotide encoding the polynucleotide of claim 1.

18. An expression vector comprising the polynucleotide of claim 17.

19. A host cell comprising the expression vector of claim 18.

20. A composition, comprising a physiologically acceptable carrier and at least one selected from the group consisting of: (i) the polypeptide of claim 1, (ii) a fusion protein comprising the polypeptide of (i) and heterologous fusion partner, (iii) a dimeric or multimeric complex comprising at least one polypeptide of (i), (iv) a polynucleotide encoding (i) to (iii), (v) an expression vector comprising (iv), and (vi) a host cell comprising (v).

21. A composition, comprising the polypeptide of claim 1 as an active ingredient.

22-23. (canceled)

24. A screening method for identifying an anticancer agent, the method comprising the steps of: (a) forming a reaction mixture containing (i) and (ii): (i) an ingredient selected from the group consisting of the polypeptide of claim 1, (ii) a test compound; and (b) determining an increase in an anti-cancer activity by the ingredient in the presence of the test compound, wherein a change in the anti-cancer activity in the presence of the test compound is determined compared to the anti-cancer activity in the absence of the test compound, thereby identifying an active test compound.

25. (canceled)

26. The composition of claim 21, wherein the polypeptide is labeled with at least one selected from the group consisting of a chromogenic enzyme, a radionuclide, a chromophore, a luminescent substance, a fluorescer, a super paramagnetic particle, or an ultrasuper paramagnetic particle.

27. A method for detecting cancer cells, the method comprising the steps of: (a) mixing the polypeptides of claim 1 with a biological sample; (b) removing the polypeptides that remain unbound or are non-specifically bound; and (c) determining whether and where the polypeptides are bound.

28-31. (canceled)

32. A composition comprising the polypeptide of claim 1 and an anticancer agent bound thereto as active ingredients.

33. The polynucleotide of claim 17, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 8 to 11.

34. (canceled)

35. A method for prevention and treatment of a neoplastic disease, the method comprising a step of administering to a subject in need thereof an effective amount of a composition comprising the polypeptide of claim 1 as an active ingredient.

36. The method of claim 35, wherein the neoplastic disease is selected from the group consisting of colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma, ovarian cancer, leukemia, myelodysplastic syndrome, polycythemia vera, lymphoma), cervical cancer, multiple myeloma, renal cell carcinoma, solid tumor, and angiogenesis-related diseases.

37. The method of claim 36, wherein the angiogenesis-related disease is selected from the group consisting of age-related macular degeneration, diabetic blindness, endometriosis, ocular neovascularization, hemangioma, nevus flammeus, nevus simplex, diabetic retinopathy, retinopathy of prematurity, neovascular glaucoma, erythrosis, proliferative retinopathy, psoriasis, hemophilic arthropathy, capillary proliferation in atheromatous atherosclerotic plaques, keloid, wound granulation, angiostenosis, rheumatoid arthritis, osteoarthritis, autoimmune diseases, Crohn's disease, restenosis, atheromatous arteriosclerosis, cat scratch disease, ulcers, glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy, glomerulopathy, and inflammation.

38. A method for detecting or imaging cancer cells, the method comprising a step of administering to a subject in need thereof an effective amount of a composition comprising the polypeptide of claim 1 as an active ingredient.

39. The method of claim 38, wherein the polypeptide is labeled with at least one selected from the group consisting of a chromogenic enzyme, a radionuclide, a chromophore, a luminescent substance, a fluorescer, a super paramagnetic particle, or an ultrasuper paramagnetic particle.

40. A method for cancer cell-specific drug delivery, the method comprising a step of administering to a subject in need thereof an effective amount of a composition comprising the polypeptide of claim 1 as an active ingredient.

41. The method of claim 40, wherein the drug is selected from the group consisting of paclitaxel, doxorubicin, vincristine, daunorubicin, vinblastine, actinomycin-D, docetaxel, etoposide, teniposide, bisantrene, homoharringtonine, Gleevec (STI-571), cisplain, 5-fluouracil, adriamycin, methotrexate, busulfan, chlorambucil, cyclophosphamide, melphalan, nitrogen mustard, nitrosourea, streptokinase, urokinase, alteplase, an angiotensin II inhibitor, an aldosterone receptor inhibitor, erythropoietin, an NMDA (N-methyl-d-aspartate) receptor inhibitor, lovastatin, rapamycin, celebrex, ticlopin, marimastat, and trocade.

42. A method for prevention or treatment of cancer, the method comprising a step of administering to a subject in need thereof an effective amount of a composition comprising the polypeptide of claim 1 and an anticancer agent bound thereto.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0281] FIG. 1a shows 3D structures of full-length GRS protein and representative fragments of significant importance (GRS-F4, GRS-F4-NT-1, GRS-F4-NT2, and GRS-F4-NT3), which are members of peptide fragments constructed from C-terminal regions of GRS protein.

[0282] FIG. 1b shows 3D structures and features of representative fragments (GRS-DP, GRS-DP-A linear, GRS-DP-B, GRS-DP-C, and GRS-DP-A cyclic), which are members of peptide fragments constructed from C-terminal regions of GRS protein and variants thereof and are of significant importance in connection with an anticancer motif.

[0283] FIG. 2 shows relative cancer cell apoptotic activities of peptides after SN12C (CDH6 positive renal cancer cell) is treated with predetermined concentrations thereof.

[0284] FIG. 3a shows Tm (melting temperature) of GRS-DP polypeptide of the present invention, as measured by thermal shift assay (full-length GRS protein serving as a control).

[0285] FIG. 3b shows Tm (melting temperatures) of GRS-DP-A cyclic, GRS-DP-A linear, GRS-DP-B, and GRS-DP-C, as measured by thermal shift assay (full-length GRS protein serving as a control).

[0286] FIG. 4 shows CD (Circular dichroism) spectra of the fragments.

[0287] FIG. 5a shows cell viability of CDH6- and pERK-positive cells (CDH6.sup.+/pERK.sup.+) or negative cells after treatment with GRS-DP (200 nM) to determine CDH-dependent action (doxorubicin 100 nM serving as a positive control).

[0288] FIG. 5b shows extents of dephosphorylation of pERK after the treatment of SN12C (CDH6 positive) cells with 100 nM or 200 nM of GRS-DP (GRS 100 nM serving as a positive control).

[0289] FIG. 5c shows extents of dephosphorylation of pERK after the treatment of RENCA (CDH6 negative) cells with 100 nM or 200 nM of GRS-DP (GRS 100 nM serving as a positive control).

[0290] FIG. 5d shows affinity of GRS-DP polypeptide of the present invention for CDH6 as measured by SPR (Surface Plasmon Resonance).

[0291] FIG. 5e shows affinity of GRS-DP-A linear polypeptide of the present invention for CDH6 as measured by SPR (Surface Plasmon Resonance).

[0292] FIG. 5f shows affinity of GRS-DP-A cyclic polypeptide of the present invention for CDH6 as measured by SPR (Surface Plasmon Resonance).

[0293] FIG. 6a is an experimental scheme for evaluating the ability of GRS, GRS-DP, and representative fragments derived therefrom (GRS-DP-B, or GRS-DP-A cyclic) to induce tumor regression in vivo by intra-tumor injection. Briefly, SN12C (CDH6 positive) cells were subcutaneously injected into BALB/c nude mice and then grown for 5 days to an average tumor size of 100 mm.sup.3. On day 5 and 7, PBS or test materials were each injected at a dose of 20 μg into tumors (n=5 animals/group). Tumor volumes were calculated according to “maximum diameter×minimum diameter 0.52”.

[0294] FIG. 6b is an image of tumors excised from experimental animals in each test group sacrificed on day 21 after xenotransplantation of tumor cells.

[0295] FIG. 6c shows monitoring results of tumor volumes in each test group with time.

[0296] FIG. 6d shows weights of tumors finally obtained from experimental animals in each test group sacrificed on day 21 after xenotransplantation of tumor cells.

[0297] FIG. 6e shows monitoring results of weights of experimental animals in each test group with time.

[0298] FIG. 7a is an image of tumors excised from experimental animals in each test group sacrificed on day 21 after xenotransplantation of tumor cells, showing results of evaluating the ability of GRS, GRS-DP, and representative fragments derived therefrom (GRS-DP-B, or GRS-DP-A cyclic) to induce tumor regression in vivo by intravenous injection. Briefly, SN12C (CDH6 positive) cells were subcutaneously injected into BALB/c nude mice and then grown for 5 days to an average tumor size of 100 mm.sup.3. On day 5 and 7, PBS or test materials were each intravenously injected at a dose of 5 MPK (n=5 animals/group). Tumor volumes were calculated according to “maximum diameter×minimum diameter 0.52”.

[0299] FIG. 7b shows monitoring results of tumor volumes in each test group with time.

[0300] FIG. 7c shows weights of tumors finally obtained from experimental animals in each test group sacrificed on day 21 after xenotransplantation of tumor cells.

[0301] FIG. 7d shows monitoring results of weights of experimental animals in each test group with time.

[0302] FIG. 8a is an image of tumors excised from test groups, showing comparison of abilities of GRS, GRS-DP, and representative fragments derived therefrom (GRS-DP-B or GRS-DP-A cyclic) to specifically target cancer cells in terms of fluorescence labeling intensity. In the experiment, B16F10 cells were subcutaneously injected into C57BL/6 mice and then grown for 14 days. On day 14, PBS or fluorescence-labeled test materials were each intravenously injected at a dose of 1 MPK, and tumors were excised 24 hours after injection.

[0303] FIG. 8b shows relative fluorescence ROI values of tumors excised from each of the test groups of FIG. 8a.

MODE FOR CARRYING OUT THE INVENTION

[0304] Below, a detailed description will be given of the present invention.

[0305] While the approaches to be utilized in the invention have been described above, the techniques that are utilized are described in greater detail below. These examples are provided to illustrate the invention, and should not be construed as limiting.

Example 1: Construction of GRS C Terminal-Derived Polypeptide and Assay for Anticancer Activity

[0306] 1-1. Construction of GRS C-Terminal Fragment and Assay for Cancer Cell Apoptotic Activity

[0307] From C-terminal regions of the full-length GRS protein (1-685, SEQ ID NO: 1), various polypeptide fragments were constructed. In Table 1, below, various polypeptide fragments (basically linear polypeptide) are listed. Among the various polypeptides in Table 1, some representative fragments of significant importance are depicted in FIGS. 1a and 1b in view of 3D structure. The polypeptide fragments were constructed by GL Biochem Shanghai Ltd, China (no 519 Ziyue road Minhang 200241 SHANGHAI SHANGHAI China) using solid phase synthesis.

[0308] The polypeptide fragments were assayed for cancer cell apoptotic activity. Concrete experiment procedures were as follows: H460, HCT116, MCF7, HeLa, SN12C or RENCA cells were each seeded at a density 5,000 cells/mL into 96-well plates (Corning, N.Y., USA) and incubated for 24 hours with 100 nM of each of the polypeptide fragments and the full-length GRS protein. Then, each sample was treated for 2 hours with 10 μl of CCK8 reagent (Dojindo Molecular Technologies Inc. Kumamoto, Japan) before absorbance was read at 570 nm on a microplate reader (TECAN, Mannedorf, Swiss).

TABLE-US-00001 TABLE 1 SEQ Poly- ID peptide Position # of NO: Form name (on SEQ ID NO: 1) aa  1 full GRS SEQ ID NO: 1(1-685) 685 length 14 truncated GRS-F4 511------------------------------------------- 175 685  6 truncated GRS-F4- 526-------------------------------------685 160 NT-1 37 truncated GRS-F4- 538----------------------------685 148 NT-2 38 truncated GRS-F4- 558--------------------685 128 NT-3  4 truncated GRS-DP 531---------------------600  70  3 truncated GRS-DP- 531--------555  25 A  2 truncated GRS-DP- 531-538   8 B 12 truncated GRS-DP- 538--552  15 C 39 truncated GRS-EX- 526--531   6 0 15 truncated GRS-EX- 531--540  10 1 16 truncated GRS-EX- 530--539  10 2 17 truncated GRS-EX- 529--538  10 3 18 truncated GRS-EX- 529----541  13 4 19 truncated GRS-EX- 531----545  15 5 20 truncated GRS-EX- 526---------545  20 6 21 truncated GRS-EX- 524-------538  15 7 22 truncated GRS-EX- 528-----540  13 8 23 truncated GRS-EX- 531----550  20 9 24 truncated GRS-EX- 519---------538  20 10 25 truncated GRS-EX- 525-------541  19 11 26 truncated GRS-EX- 511--------------540  30 12 40 truncated GRS-EX- 511------------535  25 13 27 truncated GRS-EX- 527---------------561  35 14 41 truncated GRS-EX- 540--------569  30 15 42 truncated GRS-EX- 556------------------685 130 16 43 truncated GRS-EX- 534----543  10 17 44 truncated GRS-EX- 525-----534  10 18 28 truncated GRS-EX- 516-------------------------565  50 19 29 truncated GRS-EX- 531-------------------580  50 20 30 truncated GRS-EX- 525---------------------574  50 21 31 truncated GRS-EX- 520----------------------569  50 22 32 truncated GRS-EX- 528--------------------------603  76 23 45 truncated GRS-EX- 577---------680 104 24 33 truncated GRS-EX- 529--------------------------628 100 25 46 truncated GRS-EX- 533------------569  37 26 34 truncated GRS-EX- 511--------------------------------------630 120 27 35 truncated GRS-EX- 530-----------------------------679 150 28 36 truncated GRS-EX- 515------------------------------------------ 170 29 684  5 truncated GRS-EX- 531--------------------------------685 155 30

[0309] The results of the cancer cell apoptotic assay exhibited that the polypeptide fragments GRS-F4, GRS-F4-NT-1, GRS-DP, GRS-DP-A (linear), GRS-DP-B, and those containing the regions thereof have cancer cell apoptotic activities similar to that of the full-length GRS protein, identifying the presence of an motif responsible for the anticancer activity in the 531-538 aa region of GRS-DP-B. In addition, this region showed that the anticancer activity motif is difficult to predict simply with structural analysis only.

[0310] 1-2. Construction of GRS C-Terminal Fragment Variant and Assay for Cancer Cell Apoptotic Activity

[0311] Variants were constructed by causing modifications (addition, deletion, or substitution) on the polypeptide fragments of Example 1-1. As such, peptides in cyclic forms as well as in linear forms were prepared according to modification methods.

[0312] First, linear polypeptide variants were synthesized by GL Biochem Shanghai Ltd, China (no 519 Ziyue road Minhang 200241 SHANGHAI SHANGHAI China) using a solid phase synthesis method. For cyclic variants, the linear peptides were allowed to undergo spontaneous cyclization by dissolving linear peptides at a concentration of 10.sup.−3-10.sup.−4 M in water and adjusting the acidity to a pH of 8 with diluted ammonia. The cyclized structures were detected by comparing mass spectra of peptides before and after the reaction.

[0313] As a representative example, the variant sequence CYTVFEHTFHVREGDEQRTFFSFPC (25 a.a, SEQ ID NO: 7) was prepared from the GRS-DP-A (linear) peptide and then cyclized. The resulting cyclic form was named “GRS-DP-A cyclic” herein. “GRS-DP-A cyclic” is a cyclic form in which a monosulfide linkage is formed between cysteine residues at the opposite termini.

[0314] 1-3. Comparison of In Vitro Anticancer Activity Among the Polypeptides

[0315] Comparison was made of cancer cell apoptotic activity among the polypeptide constructs of Examples 1-1 and 1-2. Each polypeptide was used in an amount of 1 μg or 2 μg, and the assay was performed in the same manner as in Example 1-1.

[0316] FIG. 2 shows experimental results of some representative polypeptides (GRS-DP, GRS-DP-A linear, GRS-DP-A cyclic, GRS-DP-B, and GRS-DP-C) among the various polypeptides. As can be seen in FIG. 2, the cancer cell apoptotic effect was not detected in GRS-DP-C, but remarkably in GRS-DP, GRS-DP-A linear, GRS-DP-A cyclic, and GRS-DP-B. Characteristically, the variant GRS-DP-A cyclic was superior to its origin peptide GRS-DP-A in terms of anticancer cell apoptotic effect.

[0317] The representative polypeptides GRS-DP-A linear, GRS-DP-A cyclic, GRS-DP-B, and GRS-DP-C, which show characteristically significant anticancer activity as shown in FIG. 2, are depicted for features in FIG. 1b. Briefly, “GRS-DP-A linear” is a 25-mer polypeptide fragment covering the 1.sup.st to 25.sup.th amino acids from the N-terminus of GRS-DP (corresponding to 531-555 aa on GRS), defined by SEQ ID NO: 3, and having a molecular weight of 3.079 kDa. “GRS-DP-A cyclic”, defined by SEQ ID NO: 7, was derived from GRS-DP-A linear by substituting each of the N-terminal methionine and the C-terminal alanine with cysteine. The cyclic form occurred as the cysteine residues at the opposite termini formed a monosulfide bond. It has a molecular weight of 3.083 kDa. “GRS-DP-B”, defined by SEQ ID NO: 2, is an 8-mer fragment covering the 1.sup.st to 8.sup.th amino acids from the N-terminus of GRS-DP-A linear (corresponding to 531-538 aa on GRS) and isolated from a region of alpha helix structure. “GRS-DP-C”, defined by SEQ ID NO: 12, is a 15-mer fragment covering the 8.sup.th to 22.sup.nd amino acids from the N-terminus of GRS-DP-A linear (corresponding to 538-552 aa on GRS) and isolated from a region of loop structure. “GRS-DP-B” and “GRS-DP-C” have molecular weights of 1.027 kDa and 1.855 kDa, respectively.

[0318] In full consideration of the result of Example 1-1 in which the anticancer activity motif is included in the region of 531-538 aa corresponding to GRS-DP-B (SEQ ID NO: 2) and the data of FIG. 2 in which GRS-DP-A cyclic showing anticancer activity is derived by substituting cysteine for the N-terminal methionine of GRS-DP-A linear (531-555 aa), the 532-538 aa region on full-length GRS protein (SEQ ID NO: 1) was finally identified as a motif critical for the anticancer activity.

[0319] 1-4. Melting Temperature (Tm) of the Polypeptides

[0320] Some representative polypeptides that were identified to have excellent anticancer activity in Example 1-3 were measured for melting temperature (Tm), using a thermal shift assay, with the full-length GRS polypeptide serving as a control. Concrete experiment methods are as follows.

[0321] The experiment was performed using ProteoStat Thermal Shift Stability Assay (Enzo Life Sciences, Farmingdale, N.Y.) according to the manufacturer's manual. In brief, the full-length GRS protein or the polypeptides of the present invention were each mixed at a concentration of 2 mg/mL with 10× PROTEOSTAT TS detection reagent. Each sample was heated in a linear gradient condition at 0.2° C./min from 25° C. to 99° C., and fluorescence was measured in triplicate using Thermal Cycler Dice™ Real Time system (Takara, Shiga, Japan) in the condition of 480 nm excitation and 615 nm emission.

[0322] As can be seen FIGS. 3a and 3b, GRS-DP, GRS-DP-A linear, GRS-DP-A cyclic, and GRS-DP-B showed Tm similar to or higher than that of the full-length GRS protein. In addition, GRS-DP-C serving as a control also showed Tm similar to that of GRS. Thus, GRS-DP-A linear, GRS-DP-A cyclic, GRS-DP-B, or respective polypeptides containing the same were identified to be as stable as or more stable than the full-length GRS protein.

[0323] 1-5. CD (Circular Dichroism) Spectroscopy for the Polypeptides

[0324] Some representative polypeptides that were identified to have excellent anticancer activity in Example 1-3 were analyzed for structural characteristic by CD (circular dichroism) spectroscopy. CD spectroscopy was conducted as follows. Far UV CD spectra for the full-length GRS protein and the polypeptide samples were recorded as averages using 0.1 cm path length quartz SUPRASIL cell (Hemlla, Germany). J-815 Circular Dichroism machine (JASCO, Oklahoma City, USA) with a spectral resolution of 1.0 nm at a bandwidth of 1 nm and 2.0 s. Spectra for 600 μl of each of 1.0 mg/mL samples were averaged of 3 scans after blank subtraction.

[0325] FIG. 4 shows CD analysis results for GRS-DP-A linear, GRS-DP-A cyclic, and GRS-DP-B, which are characteristically representative of the peptides and have excellent anticancer activity. Observation was made of an alpha-helix form structure in the full-length GRS protein, an N-extension helix form structure in both GRS-DP-A linear and GRS-DP-B, and the cyclic form structure intended in Example 1-1 in GRS-DP-A cyclic (see Ion mobility-mass spectrometry applied to cyclic peptide analysis: conformational preferences of gramicidin S and linear analogs in the gas phase Journal of the American Society for Mass Spectrometry Volume 15, Issue 6, June 2004, Pages 870-878/Chemical Synthesis and Folding Pathways of Large Cyclic Polypeptides: Studies of the Cystine Knot Polypeptide Kalata B1Biochemistry, Vol. 38, No. 32, 1999 10606)

Example 2: In Vitro Anticancer Activity Mechanism

[0326] 2-1. CDH6-Dependency

[0327] To examine whether the polypeptides of the present invention exhibits activity in a CDH6-dependent manner, CDH6 (Cadherin-6)- and pERK (Protein kinase R-like endoplasmic reticulum kinase)-positive cells (CDH6.sup.+/pERK.sup.+) or negative cells were measured for cell viability in the presence of the polypeptides of the present invention. As a representative of the peptides, GRS-DP (100 nM) was used in the experiment. Briefly, H460 (CDH6.sup.+/pERK.sup.+), HeLa (CDH6.sup.+/pERK.sup.+), SN12C (CDH6.sup.+/pERK.sup.+), RENCA (CDH6.sup.−/pERK.sup.+), MCF7 (CDH6.sup.−/pERK.sup.−) cells were incubated for 1 hour with full-length GRS (100 nM) or GRS-DP (100 nM). After incubation, the cells were washed twice with PBS and lysed in a lysis buffer (150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 10 mM NaF, 1 mM orthovandadate, 10% glycerol, protease cocktail). Thirty micrograms of each protein were run on SDS/PAGE, followed by western blotting with an ERK antibody purchased from Cell Signaling Technology (Danvers, Mass., USA) and an anti-cadherin-6 (K-cadherin) antibody purchased from Abcam (Cambridge, UK).

[0328] As shown in FIG. 5a, GRS-DP polypeptide reduced the cell viability of CDH6-positive cells only. Therefore, the polypeptides of the present invention were found to act in a CDH6-dependent manner.

[0329] 2-2. Anticancer Activity Mechanism of GRS-DP

[0330] To examine whether the polypeptides of the present invention dephosphorylates pERK by binding to CDH6 or not, SN12C (CDH6 positive) cells or RENCA (CDH6 negative) cells were treated with the polypeptides of the present invention (typically, 100 nM or 200 nM of GRS-DP) and dephosphorylation of pERK was analyzed. In this regard, full-length GRS 100 nM was used as a positive control. In brief, the cells were washed for 1 hour with full-length GRS or GRS-DP, washed twice with chilled PBS, and lysed in a lysis buffer (150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 10 mM NaF, 1 mM orthovandadate, 10% glycerol, protease cocktail). Thirty micrograms of each protein were run on SDS/PAGE, followed by western blotting with an ERK antibody purchased from Cell Signaling Technology (Danvers, Mass., USA) and an anti-cadherin-6 (K-cadherin) antibody purchased from Abcam (Cambridge, UK).

[0331] As shown in FIGS. 5b and 5c, GRS-DP polypeptide was found to mediate cancer cell apoptosis by inducing the CDH6-dependent dephosphorylation of pERK.

[0332] 2-3. Comparison of Interaction with CDH6

[0333] Comparison was made of the affinity of the peptides of the present invention for CDH6. The binding of GRS and GRS-derived peptides to cadherin6 (CDH6)-fc fusion protein was analyzed by surface plasmon resonance (SPR) using SR7500DC, Reichert Analytical Instrument (Depew NY). CDH6 was immobilized on a [CMDH chip] carboxymethyl dextran sensor chip via a free carboxyl group on the surface thereof, followed by injecting 0.1M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 0.05M N-hydroxysuccinimide at a flow speed of 5 μL/min to give an activated succinimide ester-modified surface. Various concentrations of GRS and GRS-derived peptides were injected at a flow speed of 30 μL/min into phosphate-based saline, after which a mobile-phase buffer was injected at the same flow speed to determine a dissociation rate. Data was analyzed using Software Scrubber 2.0 (Biological Software, Australia).

[0334] FIGS. 5d to 5f show representative examples of results of affinity assays for the peptides of the present invention. GRS-DP peptide was observed to have K.sub.D=83.9 nM while its shorter fragment GRS-DP-A linear showed better affinity with K.sub.D=61.2 nM. Specifically, GRS-DP-A cyclic, which is derived from GRS-DP-A linear, was measured to have K.sub.D=51.3 nM, exhibiting far better affinity for CDH6. Through the experiments, it was found that the polypeptide fragments containing the anticancer motif have higher affinity for CDH6 as their length is shorter. Particularly, when structured to have a cyclic form, they exhibited unpredictable effects.

Example 3: Comparison of In Vivo Anticancer Activity and Cancer Cell Targeting Ability Between the Peptides

3-1. Comparison of In Vivo Anticancer Activity Between the Polypeptides IT Injection

[0335] Some representative peptides (GRS-DP, GRS-DP-B, and GRS-DP-A cyclic) that were identified to have excellent anticancer activity in Example 1-3 were used as test substances while full-length GRS protein served as a control.

[0336] Experimental procedures of an assay for in vivo anticancer activity are schematically represented in FIG. 6a. Briefly, 1×10.sup.7 SN12C (CDH6 positive) cells were subcutaneously injected to the right flank of female BALB/c 8 weeks old (Dooyeol Biotech Co. Ltd, Seocho-gu, Seoul) and then grown for 5 days to an average tumor size of 100 mm.sup.3. On day 5 and 7, PBS (control) or test materials (including a control) were each injected at a dose of 20 μg into tumors (n=5 animals/group). After monitoring the tumors for 21 days, the mice were sacrificed on day 21 and the tumors were excised. Tumor volumes were calculated according to “maximum diameter×minimum diameter×0.52”. Test results are shown in FIGS. 6b to 6e.

[0337] As shown in FIGS. 6b to 6d, sizes (volumes) and weights of tumors were most remarkably reduced in the shortest fragment GRS-DP-B (8-mer). Particularly, such tumor regression effects were far higher, compared to those from full-length GRS protein and GRS-DP. Short fragments containing the anticancer motif (532-538 aa.) first revealed in the present invention, isolated from the full-length GRS protein, were found to have butter anticancer activity, compared to the intact protein. GRS-DP-A cyclic was also observed to exhibit a significant anticancer effect in vivo (see FIGS. 6b to 6d).

[0338] As shown in FIG. 6e, mice in each test group did not undergo a significant change in weight, compared to the control. Taken together, the data obtained in this test indicate that the polypeptide fragments constructed in the present invention are free of toxicity in vivo.

[0339] 3-2. In Vivo Anticancer Activity of Polypeptide IV Injection

[0340] As opposed to direct intratumoral injection in Example 3-1, efficacy was evaluated by intravenous injection in this Example. Some representative polypeptides (GRS-DP, GRS-DP-B, and GRS-DP-A cyclic) that had been identified to have excellent anticancer activity in Example 1-3 were used as test substances while full-length GRS protein served as a control.

[0341] Experimental procedures of an assay for in vivo anticancer activity are schematically represented in FIG. 6a. Briefly, 1×10.sup.7 SN12C (CDH6 positive) cells were subcutaneously injected to the right flank of female BALB/c 8 weeks old (Dooyeol Biotech Co. Ltd, Seocho-gu, Seoul) and then grown for 5 days to an average tumor size of 100 mm.sup.3. On day 5 and 7, PBS (control) or test materials (including a control) were each intravenously injected at a dose of 5 MPK (n=5 animals/group). After monitoring the tumors for 21 days, the mice were sacrificed on day 21 and the tumors were excised. Tumor volumes were calculated according to “maximum diameter×minimum diameter×0.52”. Test results are shown in FIGS. 7a to 7d.

[0342] As shown, sizes (volumes) and weights of tumors were most remarkably reduced in the GRS-DP-B group. Particularly, such tumor regression effects were far higher, compared to those from full-length GRS protein and GRS-DP (FIGS. 7a to 7c). In light of systemic administration such as intravenous injection, it is important to target a certain drug specifically toward a tumor site in order to achieve an advantageous anticancer effect without side effects when administered in a systemic manner. GRS-DP-A cyclic was also observed to have a significant level of anticancer effect in vivo (see FIGS. 7a to 7c).

[0343] As shown in FIG. 7d, mice in each test group did not undergo a significant change in weight, compared to the control. Taken together, the data obtained in this test indicate that the polypeptide fragments constructed in the present invention are free of toxicity in vivo.

[0344] 3-3. Ability of GRS-DP-Derived Small Fragments to Target Cancer Cells IV Injection

[0345] Even when administered in a systemic manner, GRS-DP-B was observed to exhibit the most remarkable anticancer effect in Example 3-2. Thus, the ability of GRS-DP-B to target tumors in practice was evaluated when it was administered in a systemic manner. For this, GRS-DP-B, GRS-DP-A cyclic, GRS-DP, and full-length GRS protein were assayed for tumor targeting ability in vivo. Briefly, B16F10 cells (5×10.sup.6 cells) were subcutaneously injected into the right flank of each of C57BL/6 mice 8 weeks old. On day 14, full-length GRS and the polypeptides of the present invention, each labeled with Alexa fluor 488, were intravenously injected at a dose of 1 mg/kg mouse weight, and the tumors were excised after 24 hours. Fluorescent signals of tumors were measured using IVIS Lumina Series III (PerkinElmer, Massachusetts, USA) to analyze regions of interest (ROI)

[0346] As can be seen in FIGS. 8a and 8b, remarkably high tumor-specific target ability was detected from GRS-DP-B, compared to full-length GRS protein and GRS-DP polypeptide. Therefore, GRS-DP-B per se can be used as an anticancer drug. In addition, the ability of the fragment to specifically targeting tumor sites allows the detection and imaging of tumors concurrently with the treatment of the tumors. Furthermore, when conjugated with an anticancer drug, the fragment exhibited a synergistic effect on anticancer treatment. Consequently, the polypeptides of the present invention have very advantageous values.

INDUSTRIAL APPLICABILITY

[0347] As described hitherto, the present invention relates to a fragmented GRS polypeptide, a variant thereof, and a use thereof and, more particularly, to an isolated polypeptide consisting of 8 to 170 consecutive amino acids, which contain the amino acids from positions 531 to 538 on the amino acid sequence defined by SEQ ID NO: 1; or a variant having a sequence homology of 80% or more to the isolated polypeptide, a fusion protein comprising the polypeptide, and a complex comprising the polypeptide, a polynucleotide coding for the polypeptide, and uses thereof in preventing and treating a neoplastic disease, in detecting cancer cells, in imaging cancer cells, and in delivering a drug to cancer cells.

[0348] The polypeptides (polypeptides isolated from GRS, and variants thereof) disclosed herein contain the GRS cell apoptotic motif first found in the present invention and are provided as truncated forms having certain lengths. The polypeptides exhibit far higher activity and targeting ability than the full-length protein and the domain, finding high industrial availability in the medicinal industry.