VASCULAR ENDOTHELIAL GROWTH FACTOR FUSION PROTEIN

Abstract

The present invention relates to a fusion protein binding to a vascular endothelial growth factor (VEGFR) and/or a placental growth factor (PIGF). The fusion protein of the present invention comprises (a) a Fc domain of IgG1, wherein two heavy chains are linked by disulfide bond, and (b) four immunoglobulin domain2s of the VEGFR1, wherein two immunoglobulin domain2s are sequentially fused to each heavy chain of the Fc domain of (a). The present fusion protein has excellent activities of inhibiting cell migration and cell invasion, and has highly enhanced growth inhibition effects to various carcinomas and fibroblasts. Therefore, The fusion protein of the present invention can be used in the preparation of an agent for treating cancers or ocular diseases.

Claims

1. A fusion protein of immunoglobulin-like domain binding to vascular endothelial growth factor (VEGF), comprising (a) Fc domain of IgG1, wherein two heavy chains are linked by disulfide bond; and (b) four immunoglobulin domain2s of VEGFR1, and wherein one immunoglobulin domain2 of the VEGFR1 and another immunoglobulin domain2 of the VEGFR1 are sequentially fused to each heavy chain of the Fc domain of (a).

2. An isolated nucleic acid molecule encoding a fusion protein, wherein the fusion protein comprises, (a) Fc domain of IgG1, wherein two heavy chains are linked by disulfide bond; and (b) four immunoglobulin domain2s of VEGFR1, wherein one immunoglobulin domain2 (SEQ ID No.: 1) of the VEGFR1 and another immunoglobulin domain2 (SEQ ID No.: 1) of the VEGFR1 are sequentially fused to each heavy chain (SEQ ID No.: 2) of the Fc domain of (a), and wherein the isolated nucleic acid molecule comprises the nucleic acid sequence encoding the Fc domain and the nucleic acid sequence encoding four immunoglobulin domain2s of the VEGFR1.

3. A recombinant expression vector comprising the isolated nucleic acid molecule of claim 2.

4. The recombinant expression vector of claim 3, which is any one selected from the group consisting of YAC (yeast artificial chromosome), YEp (yeast episomal plasmid), YIp (yeast integrative plasmid) and recombinant virus.

5. A host cell comprising the recombinant expression vector of claim 4.

6. The host cell of claim 5, which is any one selected from the group consisting of bacteria, yeast, insect cell and mammalian cell.

7. The host cell of claim 6, wherein the mammalian cell is a CHO (chinese hamster ovary) cell.

8. A method for producing a fusion protein of immunoglobulin-like domain binding to VEGF, which comprises, (a) incubating the host cell of claim 5 under conditions for the production of a polypeptide; and (b) recovering the polypeptide produced in the above (a).

9. A fusion protein of immunoglobulin-like domain produced according to the method of claim 8.

10. A pharmaceutical composition for treating angiogenic tumor, which comprises the fusion protein of claim 1 as a pharmacologically active ingredient and pharmaceutically acceptable excipients.

11. The pharmaceutical composition for treating angiogenic tumor of claim 10, wherein the tumor is colon cancer, pancreatic cancer, rectal cancer, stomach cancer, kidney cancer, prostate cancer, uterine sarcoma, leukemia, or skin cancer.

12. A pharmaceutical composition for treating angiogenic ocular disease, which comprises the fusion protein of claim 1 as a pharmacologically active ingredient and pharmaceutically acceptable excipients.

13. The pharmaceutical composition for treating angiogenic ocular disease of claim 12, wherein the ocular disease is senile macular degeneration, exudative senile macular degeneration, choroidal neovascularization, pathologic myopia, diabetic retinopathy, diabetic macular edema, retinal vascular occlusions, retinopathy of prematurity or angiogenic glioma.

14. A method for producing a fusion protein of immunoglobulin-like domain binding to VEGF, which comprises, (a) incubating the host cell of claim 6 under conditions for the production of a polypeptide; and (b) recovering the polypeptide produced in the above (a).

15. A fusion protein of immunoglobulin-like domain produced according to the method of claim 14.

16. A method for producing a fusion protein of immunoglobulin-like domain binding to VEGF, which comprises, (a) incubating the host cell of claim 7 under conditions for the production of a polypeptide; and (b) recovering the polypeptide produced in the above (a).

17. A fusion protein of immunoglobulin-like domain produced according to the method of claim 16.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 illustrates fusion protein constructs including the fusion protein of the present invention and other fusion proteins of the prior arts.

[0035] FIGS. 2A, 2B and 3 illustrate the structures of aflibercept and KP-VR2 (VEGFR fusion protein) of the present invention.

[0036] FIG. 4 shows the molecular weight of KP-VR2 fusion protein confirmed by SDS-PAGE and western blot.

[0037] FIG. 5 shows binding affinities of the fusion protein of the present invention, aflibercept and bevacizumab for VEGF-A165 or VEGF-A121.

[0038] FIG. 6 shows binding affinities of the fusion protein of the present invention, aflibercept and bevacizumab for PIGF.

[0039] FIG. 7 shows the maximum binding interactions between the present fusion protein or aflibercept and VEGF-A165.

[0040] FIGS. 8A and 8B show pharmacokinetic profiles upon administration routes of the present fusion protein, comparing to aflibercept.

[0041] FIGS. 9A and 9B show the effects of the fusion protein of the present invention, aflibercept and bevacizumab on HUVEC invasion.

[0042] FIGS. 10A and 10B show the effects of the fusion protein of the present invention, aflibercept and bevacizumab on Ea-hy926 migration.

[0043] FIGS. 11A and 11B show the ability of the fusion protein of the present invention to inhibit proliferations of 9 (nine) carcinomas, comparing to aflibercept.

[0044] FIGS. 12 and 13 show the inhibitory ability of the fusion protein of the present invention to cell viabilities of anti-VEGF resistant carcinomas and fibroblast, comparing to aflibercept.

[0045] FIGS. 14, 15A, 15B, 16A, 16B, 17A and 17B represent comparison of tumor growth and tumor weights in human cancer models ((HT-29, LOVO and SKUT1B).

[0046] FIG. 18 shows the recombinant expression vector for KP-VR2.

[0047] FIG. 19 shows the recombinant expression vector for KP-VR3.

DETAILED DESCRIPTION

[0048] The present invention is generally directed to 4 valent Fc-fusion proteins, and more particularly, to Fc-fusion proteins. Fc-fusion proteins are typically formed by fusion of the Fc fragment of immunoglogulin (Ig) to a ligand-binding region of receptor and have a structure similar to that of an antibody.

[0049] A wide variety of immunoadhesion molecules are suggested in the literature. However, immunoadhesion molecules according to the present invention have different structure with the conventional immunoadhesion molecules, and there is also no prior art predicting or describing preparations of the immunoadhesion molecules according to the present invention.

[0050] Hereinafter, the present invention will be described in more detail with reference to the examples. It should be understood that these examples are not to be in any way construed as limiting the present invention.

Examples

Example 1: Production of the VEGFR Fusion Protein KP-VR2

[0051] Human VEGFR2 domain3 fused to human IgG1 Fc domain (KP-VR1); human VEGFR1 domain2 (SEQ No.: 1) fused to human IgG1 Fc domain (SEQ No.: 2) (KP-VR2); human VEGFR2 domain3 (SEQ No.: 3) and VEGFR1 domain2 fused to human IgG1 Fc domain (KP-VR3); human VEGFR1 domain 2 and VEGFR2 domain 3 fused to human IgG1 Fc domain (KP-VR4, aflibercept) were cloned into pCHO 1.0 vector (Invitrogen), respectively.

[0052] The CHO-S cells (Invitrogen) were transfected with the KP-VR1, KP-VR2, KP-VR3 and KP-VR4 constructs, respectively, and the transfected cells were selected by using methotrexate and puromycin. The KP-VR2 and KP-VR4 (VEGF-Trap; aflibercept; Eylea) were purified with protein A-sepharose affinity chromatography. The purified proteins were quantified by HPLC analysis, and then SDS-PAGE and western blot were performed sequentially. The FIG. 1 shows each construct. The FIG. 3 represents the results of western blot. As shown in the FIG. 3, the human VEGFR1 domain2 fused to human IgG1 Fc domain (KP-VR2) had about 120 kDa band size.

Example 2: Determination of Binding Affinities of the KP-VR2 of the Present Invention, Aflibercept and Bevacizumab to VEGF-A

[0053] The binding affinities of the KP-VR2 of the present invention, aflibercept and bevacizumab to VEGF-A were compared in this experiment. ELISA assay was carried out in accordance with the BEVACIZUMAB Summary Validation Report (Feb. 28, 2014). The 96 well plate (Nunc) was coated with 50 ng/ml VEGFA165(I) or VEGFA121(III) (R&D systems), and then KP-VR2, KP-VR4 (aflibercept) or Avastin (bevacizumab) was added to the well in gradually increasing amount from 0.0122 to 1,000 ng/ml. Next, the plate was washed and reacted with peroxidase-conjugated anti-human Fc antibody. Then, 3,3,5,5-tetramethylbenzidine (TMB) solution (Roche) was added, and thereafter absorbance was detected at 450 nm by using ELISA reader (spectrophotometer, Biorad). The FIGS. 5 (I and III) shows the results.

[0054] Furthermore, the 96 well plate was coated with 2 nM of KP-VR2 or KP-VR4 (aflibercept), and then VEGF165(II) or VEGF121(IV) (R&D systems) was added thereto in gradually increasing amount from 0.03125 to 64 nM, to compare the binding amount of KP-VR2 or KP-VR4 (aflibercept) to VEGF-A. Next, the plate was washed and reacted with the anti-human VEGF antibody for 1 hour. Then, the plate was washed again and reacted with peroxidase-conjugated anti-goat Ig antibody. Next, 3,3,5,5-tetramethylbenzidine solution was added, and thereafter absorbance was detected at 450 nm by using ELISA reader (spectrophotometer, Biorad). The FIGS. 5 (II and IV) shows the results.

Example 3: Comparison of Neutralization Activity for PLGF (Placental Growth Factor) of the KP-VR2 of the Present Invention, Aflibercept or Bevacizumab

[0055] ELISA was performed for the detection of binding affinities of the KP-VR2 of the present invention, aflibercept or bevacizumab to PIGF (placental growth factor). The 96 well plate was coated with 50 ng/ml of PIGF(I), and then KP-VR2 (0.0122˜1,000 ng/ml KP-VR4 (aflibercept) (0.390625˜16,000 ng/ml) or Avastin (bevacizumab) (0.0122˜1,000 ng/ml) was added to the well in gradually increasing amount. Then, the plate was washed and reacted with peroxidase-conjugated anti-human Fc antibody. Next, 3,3,5,5-tetramethylbenzidine solution was added, and absorbance was detected at 450 nm by using ELISA reader (spectrophotometer, Biorad).

[0056] Further, the 96 well plate was coated with 400 ng/ml of KP-VR2 or KP-VR4 (aflibercept), and then PIGF was added thereto in increasing amount from 0.8 to 12,500 ng/ml, to compare the binding amount of KP-VR2 or KP-VR4 (aflibercept) to PIGF. Next, the plate was reacted with biotinylated-PIGF antibody for 1 hour, and then was washed. Thereafter, the plate was further reacted with peroxidase-conjugated anti-goat Ig antibody (R&D systems). Next, 3,3,5,5-tetramethylbenzidine (TMB) solution was added, and then absorbance was detected at 450 nm by using ELISA reader.

[0057] Further, the 96 well plate was coated with 50 ng/ml of PIGF, and then KP-VR2 or aflibercept was added thereto in gradually increasing amount from 2 to 31,250 ng/ml, to compare the binding amount of KP-VR2 and KP-VR4 (aflibercept) to PIGF. Next, the plate was washed and reacted with anti-human Fc antibody. Thereafter, 3,3,5,5-tetramethylbenzidine (TMB) solution (Roche) was added, and absorbance was detected at 450 nm by using ELISA reader. The FIGS. 6 (I, II and III) shows the results.

[0058] As shown in the FIG. 6 (I), the titer of the KP-VR2 to PIGF increased about 42 times comparing to the KP-VR4 (aflibercept), and the maximum binding amount of the KP-VR2 to PIGF increased about 54.3% in comparison to the KP-VR4 (aflibercept) (FIG. 6 (II)). These results support that the KP-VR2 has significantly higher binding affinity to the PIGF. Meanwhile, the KP-VR2 of the present invention showed similar binding affinity to the PIGF as the KP-VR4 (aflibercept).

Example 4: Comparison of Total Avidity of the Present Fusion Protein of KP-VR2 to its Ligand (VEGFA165, VEGFA121 or PLGF)

[0059] The avidity of the KP-VR2 and KP-VR4 (aflibercept) to VEGFA165, VEGFA121 or PIGF were determined by ELISA, and the results were shown in Table 1 (the comparison of avidity of KP-VR2 and aflibercept to VEGF and PIGF) below.

TABLE-US-00001 TABLE 1 ligand binding affinity ligand Bmax (OD) note KP-VR2 VEGF-A165 1.93 ± 0.01  54.4% increased aflibercept VEGF-A165 1.25 ± 0.05 KP-VR2 VEGF-A121 2.10 ± 0.03 44.83% increased aflibercept VEGF-A121 1.45 ± 0.03 KP-VR2 PLGF 3.42 ± 0.02  54.3% increased aflibercept PLGF 2.21 ± 0.03

[0060] As shown in the Table 1, the KP-VR2 had about 45˜55% higher binding affinity to VEGFA than the aflibercept, and had about 55% higher binding affinity to PIGF than the aflibercept.

Example 5: Comparison of Binding Affinities of the Present Fusion Protein of KP-VR2 and Aflibercept to VEGF-A165

[0061] The binding affinity comparison experiment using SPR (surface plasmon resonance) was carried out following the reference (Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab. Angiogenesis. 2012, 15(2):171-185). Firstly, SPR chip was stabilized with HBST buffer (50 nM HEPES, 150 nM NaCl, 0.1% Tween 20) and then protein A was bound thereto at a density of 1,000 RU (resonance unit). The chip was washed by using 10 mM glycine buffer, and the remaining unbound protein A was removed therefrom. Next, KP-VR2, KP-VR4 (aflibercept) and bevacizumab (2 nM) were added and bound to the chip, respectively. The VEGFA165 (0.5˜8 nM) was flown over the chip. The results were shown in FIG. 7 and Table 2 (binding affinity of the KP-VR2, KP-VR4, and KP-VR3 to VEGF).

TABLE-US-00002 TABLE 2 Kinetic binding parameters ligand Rmax (RU) note KP-VR2 VEGF 119.55 ± 6.12 47.5% increased aflibercept VEGF ~87.44 avastin VEGF ~74.66

[0062] As shown in the FIG. 7, the amount of KP-VR2 increased in proportion to the concentration of VEGFA165, while KP-VR4 (aflibercept) and bevacizumab reached the maximum binding value at 4 nM. Further, as shown in the Table 2, the maximum binding value of KP-VR2 to VEGF-A was about 120 RU, while the maximum binding Values of KP-VR4 (aflibercept) and bevacizumab (Avastin) were 87 RU and 75 RU, respectively. Therefore, it was confirmed that the KP-VR2 of the present invention had significantly increased total avidity to VEGF-A, comparing to KP-VR4 (aflibercept) and bevacizumab.

Example 6. The Pharmacokinetic Profile of the Present KP-VR2 in a Rat

[0063] Rats were classified into two groups to assess the pharmacokinetic profiles of the VR-2 and KP-VR4 (aflibercept). 1 mg/kg of the KP-VR2 and 1 mg/kg of the KP-VR4 (aflibercept) were i.v. (intravenous) injected into one group of rats, and they were s.c. (subcutaneous) injected into another group of rats.

[0064] As for the i.v. injection group, the blood was gathered from the rats jugular veins at 0 min, 5 min, 15 min, 45 min, 3 hours, 5 hours, 24 hours, 48 hours, 72 hours, 96 hours and 168 hours after the respective caudal vein injection of the KP-VR2 and KP-VR4 (aflibercept) to 6 week-old female SD (Sprague-Dawley) rats weighing 180˜200 g (VEGF-TRAP: A VEGF BLOCKER WITH POTENT ANTI TUMOR EFFECTS. PNAS. 2002, 99(17):139311398), and thereafter the changed amount of the injected materials was assessed. Next, as for the s.c. injection group, the blood was gathered from the rat jugular vein at 0 hour, 1 hour, 2 hours, 4 hours, 6 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 48 hours, 96 hours, 168 hours, 240 hours and 336 hours after the respective injection of the KP-VR2 and KP-VR4 (aflibercept) on the back area of the rat neck of 6 week-old female SD (Sprague-Dawley) rats weighing 180˜200 g, and thereafter the changed amount of the injected materials was assessed. The results were shown in FIGS. 8A and 8B. As shown in the FIGS. 8A and 8B, the KP-VR2 showed similar in vivo half-life and pharmacokinetic profiles to the KP-VR4 (aflibercept).

Example 7: Analysis of Cell Migration and Invasion Inhibition by KP-VR2 in Endothelial Cells

Example 7-1. Detection of Cell Invasion Inhibition Induced by the VEGF-A after Treating HUVEC (Human Umbilical Vein Endothelial Cells) with the KP-VR2 and KP-VR4 (Aflibercept)

[0065] The cell migration was assessed by using chemotactic motility assay in Transwell system, to detect cell migration and invasion of endothelial cells by the KP-VR2. The growth factor reduced Matrigel (Millipore) was coated on the Transwell system, and then cell invasion was determined by using the invasion assay. The quantitative analysis of cell migration and cell invasion assay were performed as follows. The bottom of the Transwell was coated with 10 μl of 0.1% gelatin and dried for 24 hours. The QCM 24-well cell invasion assay kit (Corning) was used for this experiment. The detached cells were gathered with cell invasion assay medium (endothelial cell basal medium, EBM, 0.1% FBS). The cell number was adjusted to 5×104 cells/300 μl cell invasion medium, and the cells were seeded on each insert. 6 wells were treated with 0˜200 nM KP-VR2, KP-VR4 (aflibercept) (Eylea, trademark), or bevacizumab (Avastin, trademark). The VEGFs (350 ng/ml) in the 6 wells were treated and then incubated for 48 hours at 37 . After the incubation, the remaining cells and medium were removed and the insert was moved to a new well. Next, the insert was placed in 225 μl of the cell isolation solution and further incubated for 30 minutes, at 37 in an incubator.

[0066] The insert was stirred to detach the remaining cells. Thereafter, 75 μl of the lysis buffer/dye solution was further added to the mixed solution of the cell isolation solution and the cells, and the resulting solution was placed for 15 minutes at room temperature. Next, 200 μl of the solution was moved into 96 well and 595 nm fluorescence image was obtained. The results were shown in the FIGS. 9A and 9B and Table 3 (the analysis of the inhibition of endothelial cell migration and invasion by KP-VR2).

TABLE-US-00003 TABLE 3 endothelial cell migration inhibition rate VEGF inhibitor ligand IC.sub.50 (nM) note KP-VR2 VEGF-A.sub.165 11.00 ± 1.40 aflibercept VEGF-A.sub.165 20.08 ± 3.95 bevacizumab VEGF-A.sub.165 21.28 ± 2.72

[0067] As shown in the FIGS. 9A and 9B, while VEGF induced cell migration and invasion in HUVEC (Lonza), KP-VR2, KP-VR4 (aflibercept) and bevacizumab inhibited the induced cell migration and invasion. The KP-VR2 of the present invention showed higher inhibitory activity than KP-VR4 (aflibercept) and bevacizumab at the same concentration (13 nM). Further, as shown in the Table 3, the 1050 of KP-VR2, KP-VR4 (aflibercept) and bevacizumab were about 11 nM, 20 nM and 21 nM, respectively, which explains that the KP-VR2 has outstanding inhibitory activity to cell invasion.

Example 7-2. Detection of Cell Invasion Inhibition Induced by the VEGF-A after Treating Ea-Hy926 Cell Line with KP-VR2, KP-VR4 (Aflibercept) or Bevacizumab

[0068] EA-hy926 cell lines (ATCC CRL-2922™) (3.5×105 cells/ml) were seeded on 96 well plate in DMEM (Dulbecco's Modified Eagle's Medium Sigma) having 0.1% FBS. Next, the plate was treated with the KP-VR2 (1,500 μg/ml), KP-VR4 (aflibercept) (3,000 μg/ml), and bevacizumab (3,000 μg/ml), respectively, at 37° C., 5% CO2 condition for 24 hours. As a negative control, a medium not treated with the fusion proteins was used. The results were shown in FIGS. 10A and 10B. As shown in the FIGS. 10A and 10B, the KP-VR2 of the present invention much effectively inhibited the growth and proliferation of EA-hy926 cells induced by VEGF at lower concentration than KP-VR4 (aflibercept) and bevacizumab.

Example 8: Comparison of Carcinoma Cell Proliferation Inhibitory Abilities Between KP-VR2 and KP-VR4

[0069] MTS cell proliferation colorimetric assay was performed for 9 (nine) kinds of carcinoma cell lines to determine the inhibitory ability of KP-VR2 against carcinoma cell division. The Table 4 shows 9 carcinoma cell lines used in this experiment.

TABLE-US-00004 TABLE 4 name Bio-marker origins note HT29 VEGF positive Human colorectal adenocarcinoma ATCC HTB-38 ™ LoVo VEGF positive Human colorectal adenocarcinoma ATCC CCL-229 ™ PLGF positive AGS PLGF positive Human gastric adenocarcinoma ATCC CRL-1739 ™ VEGF positive SKUT1b VEGF positive Human uterine sarcoma ATCC HTB-115 ™ VEGFR1 positive Caki-1 VEGF positive Renal Clear Cell carcinoma ATCC HTB-46 ™ VEGFR1 positive Hy-926 VEGF positive Human Endothelial ATCC CRL-2922 ™ VEGFR1 positive HCT 116 VEGF positive Human colorectal carcinoma ATCC CCL-247 ™ PANC VEGF positive Human Pancreas Adenocarcinoma ATCC CRL-2553 ™ 02.03 SW480 VEGF positive Human colorectal adenocarcinoma ATCC CCL-228 ™ PLGF positive PC-3 VEGF positive Human prostate adenocarcinoma ATCC CRL-1435 ™ PLGF positive

[0070] The 9 kinds of carcinoma cell lines, HT-29 (ATCC HTB-38™), LOVO (ATCC CCL-229™), HCT116 (ATCC CCL-247™), SKUT1b (ATCC HTB-115™), Caki-1 (ATCC HTB-46™), AGS (ATCC CRL-1739™), Panc0203 (ATCC CRL-2553™) SW480 (ATCC CCL-228™), and PC-3 (ATCC ORL-1435™) were incubated to reach to full confluency at 37° C., 5% CO2 condition. Next, the carcinoma cell lines were removed and seeded (3×103 cells/well) on RPM11640 medium (Sigma) comprising 10% FBS. Thereafter, the cell lines were incubated at 37° C. for 24 hours. Next, the medium was substituted with RPM11640 medium (Sigma) having 0.5% FBS.

[0071] Subsequently, the cell lines were treated with 2, 4 or 6 nM of KP-VR2 or KP-VR4 (aflibercept), and further incubated for 72 hours. The MTS reagent (Qiagen) was added to the plate and absorbance was detected at 490 nm. The results were shown in FIG. 11A, 11B and Table 5. As shown in the FIGS. 11A and 11B, the KP-VR2 of the present invention showed significantly excellent cell division inhibition activity to all carcinoma lines employed in this experiment.

TABLE-US-00005 TABLE 5 in vitro efficacy in vitro efficacy Types test method KP-VR4 KP-VR2 EA-hy926 in vitro efficacy ~17% growth ~33% growth inhibition inhibition HT29 in vitro efficacy — ~44% growth inhibition LoVo in vitro efficacy ~35% growth ~65% growth inhibition inhibition HCT116 in vitro efficacy — ~44% growth inhibition SKUT1b in vitro efficacy — ~26% growth inhibition CaKi-1 in vitro efficacy — ~67% growth inhibition PANC0203 in vitro efficacy — ~37% growth inhibition SW480 in vitro efficacy — ~38% growth inhibition AGS in vitro efficacy — ~34% growth inhibition PC-3 in vitro efficacy — ~20% growth inhibition

[0072] Further, as disclosed in the Table 5 (cell growth inhibition activity), the KP-VR2 of the present invention showed highly enhanced effect of cell growth inhibition to the 9 carcinomas targeting VEGF and PIGF.

Example 9: Comparison of Activities of Cell Division Inhibition of KP-VR2 and Aflibercept in Anti-VEGF Resistant Carcinomas and Fibroblast Cell Lines

[0073] MTS cell growth assay was performed for carcinoma cells and fibroblast cell lines resistant to anti-VEGFA or anti-VEGFR, to assess inhibitory activity of KP-VR2 to carcinoma cell division. The Table 6 shows 4 kinds of carcinoma cell lines and 2 kinds of fibroblast cell lines used in this experiments.

TABLE-US-00006 TABLE 6 types Biomarker Origin note CT26 A model resistant to Mouse Colon ATCC CRL-2638 ™ anti-VEGFR carcinoma B16-F10 A model resistant to Mouse Skin ATCC CRL-6475 ™ FOLFIRY(p38) melanoma EL4 Anti-VEGF-A Mouse lymphoma ATCC TIB-39 ™ refractory LCC1 Anti-VEGF-A Mouse lung ATCC CRL-1642 ™ refractory carcinoma NIH3T3 fibroblast Mouse embryo KCLB NO21658 fibroblast Hs27 fibroblast Human skin ATCC CRL-1634 ™ fibroblast

[0074] The 4 kinds of carcinoma cell lines, (EL-4(ATCC TIB-39™), LLC1(ATCC TIB-39™), B16F10 (ATCC CRL-6475™), CT-26 (ATCC CRL-2638™), and PC-3 (ATCC CRL-1435™); and fibroblast cell line NIH3T3 (KCLB No. 21658) and Hs27 (ATCC CRL-1634™) were incubated to reach to full confluency at 37° C., 5% CO2 condition. Next, the carcinoma and fibroblast cell lines were removed and seeded (3×103 cells/well) on RPM11640 medium (Sigma) comprising 10% FBS. Thereafter, the cell lines were incubated at 37° C. for 24 hours. Then, the medium was substituted with RPM11640 medium (Sigma) having 0.5% FBS and the cell lines were treated respectively with 2, 4 or 6 nM of the KP-VR2 or KP-VR4 (aflibercept). Subsequently, the treated cell lines were further incubated for 72 hours. Next, the MTS reagent (Qiagen) was added to the plate and absorbance was detected at 490 nm. The results were illustrated in FIG. 12, FIG. 13 and Table 7 (carcinoma and fibroblast cell lines).

TABLE-US-00007 TABLE 7 kinds test method KP-VR4 KP-VR2 CT26 in vitro efficacy — ~56% growth inhibition B16-F10 in vitro efficacy — ~30% growth inhibition EL4 in vitro efficacy ~49% growth inhibition ~67% growth inhibition LLC1 in vitro efficacy ~12% growth inhibition ~70% growth inhibition NIH3T3 in vitro efficacy ~41% growth inhibition ~52% growth inhibition Hs27 in vitro efficacy ~15% growth inhibition ~25% growth inhibition

[0075] As shown in the FIG. 12, 13 and Table 7, the KP-VR2 of the present invention showed significantly enhanced activity of cell growth inhibition not only for the cancer cells (see Experiment 8), but also for the anti-VEGF/VEGFR resistant carcinoma cell lines (EL-4, LLC1, B16F10 and CT-26) and the fibroblast cell lines (Hs27 and NIH3T3).

Example 10: Comparison of Tumor Growth Inhibition of KP-VR2 and Aflibercept in a Nude Mouse

[0076] The various kinds of tumor xenograft models (HT-29, LOVO & SKUT1b) were employed in BALB/c nude mice to perform in vivo experiments for the assessment of tumor growth inhibition activity of KP-VR2.

Experiment 10-1. HT-29 In Vivo Xenograft

[0077] HT-29 cells (ATCC HTB-38™) (5×106 cells/0.2 ml) were subcutaneously injected into the back area of a nude mouse (Orient bio, female, 4 week-old). When the volume of tumor became larger than 200 mm.sup.3, the KP-VR2 (1 mg/kg or 2 mg/kg) and KP-VR4 (aflibercept) (2 mg/kg or 3 mg/kg) were injected, respectively, into the abdominal cavity of the mouse twice a week, and the same amount of PBS (phosphate buffered saline) was injected into the abdominal cavity of the negative control mouse at the same time and period. The volume of the tumor was measured every 3 to 4 days, and the results were disclosed in FIG. 14. As shown in the FIG. 14, the KP-VR2 of the present invention showed about 40% increased growth inhibition activity for the HT-29 colon cancer cell line at dose of 2 mg/kg, in comparison to the same amount of the aflibercept (KP-VR4). Further, similar effects were observed between the KP-VR2 (1 mg/kg) and the aflibercept (KP-VR4) (3 mg/kg).

Example 10-2. LOVO In Vivo Xenograft

[0078] The same experiment as the Example 10-1 was performed using LOVO (ATCC CCL-229™) cell lines instead of HT-29 cell lines. The LOVO cell lines (5×106 cells/0.2 ml) were subcutaneously injected into the back area of a nude mouse (Orient bio, female, 4 week-old). When the volume of the tumor became larger than 200 mm.sup.3, KP-VR2 (1 mg/kg) and KP-VR4 (aflibercept) (1 mg/kg) were injected, respectively, into the abdominal cavity of the nude mouse twice a week, and the same amount of PBS (phosphate buffered saline) was injected into the abdominal cavity of the negative control mouse at the same time and period. The volume of the tumor was measured every 3 to 4 days, and the tumor was extracted at the last 42th day to compare the weights. The results were disclosed in FIGS. 15A and 15B. As shown in the FIGS. 15A and 15B, the KP-VR2 of the present invention had 50%˜60% increased growth inhibition activity for the rectal cancer cell line (LOVO) at dose of 1 mg/kg, comparing to the same amount of the aflibercept (KP-VR4) (P<0.05). The tumor growth inhibition activity of the KP-VR2 of the present invention was observed from the initial time of injection, and the difference in inhibition activity between the KP-VR2 and the aflibercept (KP-VR4) has increased since the injection. Further, the injection of the present KP-VR2 resulted in about 50% reduced tumor weight, comparing to the aflibercept at the same injection dose.

Example 10-3. SKUT1B In Vivo Xenograft

[0079] The uterus cancer SKUT1B cell line (ATCC HTB-115™), which is a VEGFR1-positive cell line, was injected into the nude mouse. The SKUT1B cell lines (1×107 cells/0.2 ml) were subcutaneously injected into the back area of the nude mouse, and the KP-VR2 (2 mg/kg) and KP-VR4 (aflibercept) (2 mg/kg) were injected, respectively, into the abdominal cavity of the nude mouse twice a week from 1 day to 32 day after the injection. The same amount of PBS (phosphate buffered saline) was injected into the abdominal cavity of the negative control mouse at the same time and period. The volume of the tumor was observed every 3 to 4 days, and the tumor was extracted at the last 37th day to compare the weights of tumors. The results were disclosed in FIGS. 16A and 16B. As shown in the FIGS. 16A and 16B, the KP-VR2 of the present invention had 60%˜70% increased growth inhibition activity for the cell lines used in this experiment at dose of 2 mg/kg, comparing to the same amount of the aflibercept (KP-VR4) (P<0.05). Further, the injection of the present KP-VR2 resulted in about 70% reduced tumor weight, comparing to the aflibercept at the same injection dose.

INDUSTRIAL APPLICABILITY

[0080] As described above, the (VEGFR) fusion protein of the present invention comprises (a) a Fc domain of IgG1, wherein two heavy chains are linked by disulfide bond, and (b) four immunoglobulin domain2s of the VEGFR1, wherein two immunoglobulin domain2s are sequentially fused to each heavy chain of the Fc domain of (a). Accordingly, the present invention enables to provide a pharmaceutical composition for treating cancers and/or ocular diseases caused by angiogenesis.