PHARMACEUTICAL COMPOSITION CONTAINING, AS ACTIVE INGREDIENT, FUSION PROTEIN IN WHICH TUMOR-PENETRATING PEPTIDE AND ANTIANGIOGENESIS AGENT ARE FUSED, FOR PREVENTING AND TREATING CANCER OR ANGIOGENESIS-RELATED DISEASES

Abstract

A composition comprises, as an active ingredient, a fusion protein obtained by fusion of a tumor-penetrating peptide and an anti-angiogenic agent. The tumor-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 7.

Claims

1. A composition comprising, as an active ingredient, a fusion protein obtained by fusion of a tumor-penetrating peptide and an anti-angiogenic agent, wherein the tumor-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 7.

2. The composition of claim 1, wherein the tumor-penetrating peptide specifically binds to neuropilin 1.

3. The composition of claim 1, wherein the tumor-penetrating peptide and an anti-angiogenic agent are fused via a linker.

4. The composition of claim 3, wherein the linker comprises an amino acid sequence represented by SEQ ID NO: 8.

5. The composition of claim 1, wherein the fusion protein comprises a heavy chain defined by amino acid sequence of SEQ ID NO: 10, and a light chain defined by an amino acid sequence of SEQ ID NO: 11.

6. The composition of claim 1, wherein the anti-angiogenic agent is selected from the group consisting of: an anti-vascular endothelial growth factor (anti-VEGF) agent, an anti-platelet-derived growth factor (anti-PDGF) agent, an anti-placenta growth factor (anti-PlGF) agent, an anti-fibroblast growth factor (anti-FGF) agent, an anti-transforming growth factor (anti-TGF) agent, an anti-angiopoietin (anti-ANG) agent, an anti-hepatocyte growth factor (anti-HGF) agent, an anti-insulin-like growth factor (anti-IGF) agent, an anti-activin receptor-like kinase 1 (anti-ALK1) agent, an anti-ephrin A agent, an anti-interleukin agent, an anti-bone morphogenetic protein (anti-BMP) agent; antagonistic antibodies to receptors for VEGF, PDGF, PlGF, FGF, TGF, ANG, HGF, IGF, ALK1, ephrin A, or interleukin; and chimeric molecules based on the receptors.

7. The composition of claim 6, wherein the anti-vascular endothelial growth factor (anti-VEGF) agent is any one selected from the group consisting of ranibizumab, bevacizumab, aflibercept, conbercept, biosimilars thereof, variants thereof, and antibody-drug conjugates (ADCs) thereof.

8. A method for treating cancer or an angiogenesis-related disease in a subject in need thereof, the method comprising administering an effective amount of a composition to the subject in need thereof, the composition comprising, as an active ingredient, a fusion protein formed by fusion of a tumor-penetrating peptide and an anti-angiogenic agent, wherein the tumor-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 7.

9. The method of claim 8, wherein the cancer or angiogenesis-related disease is resistant or non-responsive to the anti-angiogenic agent.

10. The method of claim 8, wherein the angiogenesis-related disease is selected from the group consisting of rheumatoid arthritis, psoriasis, inflammation, endometriosis, and angioma.

11. The method of claim 8, wherein the cancer is selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, skin cancer, skin or intraocular melanoma, rectal cancer, perianal cancer, esophageal cancer, small intestine cancer, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland tumor, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, and head or neck cancer.

12. A method for inhibiting metastasis of cancer in a subject in need thereof, the method comprising administering an effective amount of a composition to the subject in need thereof, the composition comprising, as an active ingredient, a fusion protein formed by fusion of a tumor-penetrating peptide and an anti-angiogenic agent, wherein the tumor-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 7.

13. The method of claim 12, wherein the cancer is resistant or non-responsive to the anti-angiogenic agent.

14. The method of claim 12, wherein the cancer is selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, skin cancer, skin or intraocular melanoma, rectal cancer, perianal cancer, esophageal cancer, small intestine cancer, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland tumor, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, and head or neck cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0119] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0120] FIG. 1 is a schematic diagram of a fusion protein formed by genetic fusion of a tumor-penetrating peptide to the C-terminus of heavy chain of the anti-vascular endothelial growth factor antibody.

[0121] FIG. 2 shows SDS-PAGE results of confirming a fusion protein formed by fusion of a tumor-penetrating peptide and an anti-vascular endothelial growth factor (Lane 1: Avastin, Lane 2: Avastin-A22p, Lane 3: Avastin-TPP11).

[0122] FIGS. 3A and 3B show SE-HPLC results of confirming a fusion protein (Avastin-A22p) formed by fusion a tumor-penetrating peptide and an anti-vascular endothelial growth factor. (FIG. 3A: Avastin, FIG. 3B: Avastin-A22p).

[0123] FIGS. 4A and 4B show the results of evaluating the binding strength of Avastin-A22p with NRP1 or VEGF (FIG. 4A: binding strength with NRP1, FIG. 4B: binding strength with VEGF).

[0124] FIG. 5 shows the results of observing the distribution of proteins in cancer tissues through fluorescent microscopy at the elapsed time of 3, 8, and 16 h after the administration of Avastin and Avastin-A22p into SW620 xenograft animal models (Veh: saline-administered group).

[0125] FIG. 6 shows the results of evaluating the change of pericyte coverage in cancer cells in the treatment with Avastin-A22p (PECAM1: blood vessel staining, NG2: pericyte staining).

[0126] FIG. 7 shows the results of evaluating anticancer efficacy of Avastin-A22p in SW620 xenograft animal models.

[0127] FIGS. 8A and 8B show the results of confirming the migration inhibitory effect when HCT116/bev cells showing Avastin resistance were treated with VEGF to induce migration and then treated with 1 μM Avastin or Avastin-A22p (FIG. 8A: microscopic images, FIG. 8B: Microscopic observations results quantified and plotted as a graph).

[0128] FIGS. 9A and 9B show the results of confirming the protein phosphorylation of p130cas when U87MG cells were treated with 25 μg/ml Avastin or Avastin-A22p and, after 30 min, treated with 50 ng/ml PDGF, in order to investigate whether Avastin-A22p inhibits PDGE-mediated signaling (FIG. 9A: western blotting results, FIG. 9B: graph to quantify western blotting results, A22p+P: Avastin-A22p 25 ug/ml+PDGF 50 ng/ml, P: PDGF 50 ng/ml, C: control, A+P: Avastin 25 ug/ml+PDGF 50 ng/ml).

[0129] FIG. 10 shows SDS-PAGE results of confirming a fusion protein formed by fusion of a tumor-penetrating peptide and an anti-vascular endothelial growth factor (Lane 1: Avastin(V)-A22p, Lane 2: Avastin-A22p).

[0130] FIGS. 11A and 11B show SE-HPLC results of confirming a fusion protein formed by fusion of a tumor-penetrating peptide and an anti-vascular endothelial growth factor (FIG. 11A: Avastin(V)-A22p, FIG. 11B: Avastin-A22p).

[0131] FIGS. 12A and 12B show the results of evaluating the binding strength of Avastin(V)-A22p with NRP1 or VEGF, in comparison with Avastin-A22p (FIG. 12A: binding strength with NRP1, FIG. 12B: binding strength with VEGF).

[0132] FIGS. 13A and 13B show the result of inhibiting angiogenic effects of multiple proangiogenic growth factors in human umbilical vein endothelial cells (HUVECs) by Avastin-TPP11 (FIG. 13A: microscopic images, FIG. 13B: quantifying results by measurement of tube length using an angiogenesis analyzer. Data are presented as mean±SEM (n=3); *p<0.05 as compared with the vehicle group treated with the CM derived from each cancer cell line). FIGS. 13C and 13D show the results of evaluating the inhibition HUVEC migration induced by PlGF2- or VEGFB-compared to aflibercept by Avastin-TPP11 (FIG. 13C: PlGF2, FIG. 13D: VEGFB, data are presented as mean±SEM (n=3); ##p<0.01 as compared with the vehicle group; *p<0.05, .sup.**p<0.01 as compared with the growth factor alone group).

[0133] FIGS. 14A and 14B show the pharmacokinetics of Avastin-TPP11 as determined by enzyme-linked immunosorbent assay (FIG. 14A: the serum concentrations of Avastin-TPP11 and Avastin, FIG. 14B: the serum concentration of Avastin-TPP11 was determined based on the binding activity to immobilized VEGFA and determined using the biotinylated NRP1 b1b2 domain. Each symbol and error bar represent the arithmetic mean and the standard deviation of each drug at a given time point (n=5)).

[0134] FIGS. 15A and 15B show the results of observing the distribution of proteins in cancer tissues through fluorescent microscopy at the elapsed time of 3 h, 8 h, and 16 h after the administration of Avastin and Avastin-TPP11 into SW620 xenograft animal models (FIG. 15A: microscopic images, FIG. 15B: Microscopic observations results quantified and plotted as a graph. Data are presented as mean±standard error (SE).; *p<0.05, **p<0.01 as compared with the vehicle group).

[0135] FIG. 16 shows the results of evaluating anticancer efficacy of Avastin-TPP11 in BxPC-3 xenograft animal models. Data are presented as mean±SEM (n=7 per group); *p<0.05, **p<0.01 as compared with the vehicle group.

MODE FOR CARRYING OUT THE INVENTION

[0136] Hereinafter, the present invention will be described in detail.

[0137] The following Examples are merely for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1: Fabrication of Avastin-Tissue-Penetrating Peptide (TPP) Fusion Protein

[0138] For increasing efficacy and overcoming resistance with respect to an anti-VEGF agent, Avastin, the present inventors have attempted to fuse a tissue-penetrating peptide (TPP), which is capable of binding to both neuropilin 1 (NRP1) and neuropilin 2 or specifically binding only to neuropilin 1, to the C-terminus of Avastin. The amino acid sequences of TPP are shown in Table 1 below. Among the sequence listing on Table 1, TPPs having the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 4 can bind to both neuropilins 1 and 2, while TPPs having the amino acid sequence of SEQ ID NO: 5 to SEQ ID NO: 7 can bind specifically only to neuropilin 1. A schematic diagram of a fusion protein in which TPP is fused to Avastin is shown in FIG. 1.

[0139] Avastin as used herein is a peptide having the amino acid sequence of SEQ ID NO: 13 for a heavy chain and the amino acid sequence of SEQ ID NO: 11 for a light chain, and used for the present experiment after purchase from www.Drugbank.com.

[0140] Meanwhile, among TPPs shown in Table 1, A22p as TPP having the amino acid sequence of SEQ ID NO: 2 was obtained by modifying the C-terminus regions of VEGF165 as an intrinsic ligand of the neuropilin and class 3 semaphorin ligands; and TPP11 was obtained by isolating and identifying a peptide derived from clones selectively binding to the b1 domain of neuropilin 1 using both the b1b2 domain protein of neuropilin 1 and the b1b2 domain protein of neuropilin 2 as competitors. Here, Avastin as a linker was fused thereto to act as a bivalent on the neuropilin receptor, so that these peptides were designed to possess a capability of tissue penetration while having a similar affinity to VEGF and Sema3A ligands.

TABLE-US-00002 TABLE 1 TPP sequence information TPP#1 (SEQ ID NO: 1) HTPGNSNKWKHLQENKKGRNRR TPP#2 (SEQ ID NO: 2) HTPGNSNKWKHLQENKKGRPRR TPP#3 (SEQ ID NO: 3) REAPGAPRSPEPQDQKKPRNRR TPP#4 (SEQ ID NO: 4) REAPGAPRSPEPQDQKKPRPRR TPP#5 (SEQ ID NO: 5) HTPGNSNQFVLTSTRPPR TPP#6 (SEQ ID NO: 6) HTPGIATRTPR TPP#7 (SEQ ID NO: 7) HTPGNSKPTRTPRR

[0141] Specifically, fusion proteins in which TPP (A22p) having the amino acid sequence of SEQ ID NO: 2 and TPP (TPP11) having the amino acid sequence of SEQ ID NO: 7 were fused to the C-terminus of Avastin, respectively, and cell lines producing the fusion proteins were fabricated as described below.

[0142] <1-1> Construction of Expression Vector and Cell Transfection

[0143] Selection marker DHFR genes were inserted into pcDNA3.1(−) vectors, and heavy and light chains of Avastin-A22p and Avastin-TPP11 were cloned by restriction enzymes NotI and BamHI, respectively. A plasmid encoding the protein formed by fusion of each of the constructed antibody heavy constant regions and a peptide binding to NRP1 and a plasmid encoding a light chain protein were expressed for protein in CHO DG44 cells (obtained from Prof. Chasin) using Neon™ electroporesis. In T25 flask, 3×10.sup.6 cells transfected with each plasmid were inoculated and incubated at 37° C. Stable cells were secured using the selection marker, and then cultured in a floating state in serum-free SFM4CHO (Hyclone) for 7 days under conditions of 100 rpm, 37° C., pH 7.2, 50% DO2 in a bioreactor. The supernatant was separated from the cells through centrifugation, and sterilized using a 0.22-μm filter.

[0144] <1-2> Purification of Fusion Protein

[0145] The cultures of Avastin, Avastin-A22p, and Avastin-TPP11 were collected, and respective proteins thereof were purified while referring to a standard protocol. For the purification column, the protein A resin (MabselectSure resin, GE healthcare) was packed to 20 mL in the XK16/20 column (MabselectSure resin, GE healthcare), to which a linear speed of 200 cm/h was applied. 1 L of antibody was applied to the protein A column, and the column was washed with a 15-fold column volume of PBS (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na.sub.2HPO.sub.4, 2 mM KH.sub.2PO.sub.4). The antibody was eluted with 100 mL of 0.1 M glycine buffer at pH 3.0. The proteins were collected between 10 mAU and 10 mAU in the UV absorbance at 280 nm, and 60 mL of the proteins were neutralized to pH 7.0 using 0.7 mL of 1 M Tris buffer. The antibody fractions were filtered by a 0.2 μm-filter (Milipore), and then concentrated using an amicon (30 MWCO) concentrator (Milipore) at 3,500 rpm for 10 min, followed by exchange with PBS (pH 7.4) buffer. The purified fusion protein formed by fusion of the purified antibody heavy chain constant region and the selected peptide specifically binding to NRP1 was quantified using absorbance and absorption coefficient at a corrected wavelength of 280 nm. The purified fusion protein formed by fusion of the purified antibody heavy chain constant region and the selected peptide specifically binding to NRP1 was analyzed on 10% SDS-PAGE in reduction and non-reduction conditions.

[0146] The proteins were respectively purified from the cultures collected from the cell lines expressing Avastin, Avastin-A22p, and Avastin-TPP11, and separated by SDS-PAGE. FIG. 2 confirmed that Avastin-A22p and Avastin-TPP11 successfully formed antibodies, peptides were fused to Avastin in reduction conditions, and Avastin-A22p and Avastin-TPP11 were larger than Avastin in reduction conditions. In addition, it was confirmed that TPP fusion did not significantly affect antibody expression since Avastin-A22p and Avastin-TPP11 were expressed at similar levels.

[0147] In order to investigate the purity of Avastin-A22p purified protein, high performance lipid chromatography (HPLC) assay was performed. Agilent 1200 (Agilent) was used, and a size exclusion column (Biosuite 250, Waters) was used.

[0148] Specifically, a buffer solution (pH 6.2) containing 0.2 M potassium phosphate and 0.25 M potassium chloride was used as a mobile phase, and allowed to flow down at a flow rate of 0.35 ml/min for 20 min. The size of the protein was checked according to the retention time, and the purity thereof was checked according to the area and height.

[0149] As shown in FIG. 3, the purity of purified Avastin-A22p was analyzed using SE-HPLC. It was confirmed that the purity of Avastin-A22p was 97% or higher, and was larger than Avastin.

[0150] The present inventors, hereinafter, fabricated Avastin-A22p fusion protein in which A22p as a TPP having the amino acid sequence represented by SEQ ID NO: 2 was fused to Avastin, and evaluated activity thereof.

Example 2: Binding Ability of Avastin-Tissue-Penetrating Peptide (TPP) Fusion Protein to NRP1 and VEGF

[0151] In order to evaluate whether the N-terminus of the fusion protein Avastin-A22p as prepared in Example 1 may bind to VEGF in the same manner as Avastin while its C-terminus may bind to NRP1 to thus dually block the associated signaling, the binding of Avastin-A22p with NRP1 and VEGF was respectively evaluated.

[0152] The binding ability of the purified Avastin-A22p to Neuropilin 1-b1b2 domain was investigated in enzyme-linked immunosorbent assay (ELIA). Herceptin-A22p (HCT-A22p) as a positive control and Avastin (Roche co., Swiss) as a negative control were used. The target molecule Neuropilin 1-b1b2 domain (273-586) was reacted at 1 μg per well in the 96-well Maxibinding Immuno plate (SPL Life sciences., Korea) for 2 h at 37° C., and then washed with 0.1% PBST (0.1% Tween20, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na.sub.2HPO.sub.4, 2 mM KH.sub.2PO.sub.4) for 1 min three times. After reaction with 0.1% PBST containing 5% skim milk for 1 h, the reaction product was washed with 0.1% PBST for 1 min three times. Herceptin-A22p as a positive control and Avastin (Roche co., Swiss) as a negative control were used. Avastin-A22p, as experimental groups, was diluted with 0.1% PBST to 50 nM, 25 nM, 12.5 nM, and 6.25 nM, and the solutions were then treated at 37° C. for 1 h for each different concentrations, and washed with 0.1% PBST for 1 min three times. HRP-conjugated anti-kappa light chain antibody (Horseradish peroxidase-conjugated anti-human kappa light chain mAb, SIGMA-ALDRICH Co., USA) was 100,000-fold diluted with 0.1% PBST containing 5% skim milk, followed by treatment at 37° C. for 30 min. Thereafter, the reaction product was washed with 0.1% PBST for 1 min five times. For color development, the resultant product was treated with the TMB HRP colorimetric substrate (Surmodics co., USA), followed by reaction at room temperature for 10 min, treatment with the same volume of 1 N HCl (stop buffer), and then absorbance measurement at 450 nm. The binding ability of the expressed and purified Avastin-A22p to Neuropilin 1-b1b2 domain was confirmed through the obtained ELISA results.

[0153] The binding ability of the purified Avastin-A22p to Recombinant human VEGF 165 (R&D systems co. China) was investigated in ELISA. Avastin as a positive control and HCT-A22p as a negative control were used. The target molecule Recombinant human VEGF 165 was reacted at 0.2 μg per well in the 96-well Maxibinding Immuno plate for 2 h at 37° C., and then washed with 0.1% PBST for 1 min three times. After reaction with 1% PBST containing 5% skim milk for 1 h, the reaction product was washed with 0.1% PBST for 1 min three times. Avastin as a positive control and Herceptin-A22p as a negative control were used. Avastin-A22p, as experimental groups, was diluted with 0.1% PBST to 1.5 nM, 0.75 nM, 0.35 nM, and 0.17 nM, and the solutions were then treated at 37° C. for 1 h for each different concentrations, and washed with 0.1% PBST for 1 min three times. HRP-conjugated anti-kappa light chain antibody was 100,000-fold diluted with 0.1% PBST containing 5% skim milk, followed by treatment at 37° C. for 30 min. Thereafter, the reaction product was washed with 0.1% PBST for 1 min five times. For color development, the resultant product was treated with the TMB HRP colorimetric substrate, followed by reaction at room temperature for 10 min, treatment with the same volume of 1 N HCl, and then absorbance measurement at 450 nm. The binding ability of the expressed and purified Avastin-A22p to Recombinant human VEGF 165 was confirmed through the obtained ELISA results.

[0154] Related results are shown in FIG. 4.

[0155] As shown in FIG. 4, it was confirmed that Avastin-A22p bound to NRP1 at the same level as in HCT-A22p used as a control, and also bound to VEGF at the same level as in Avastin.

Example 3: Cancer Targeting Experiment of Avastin-A22p

[0156] It is known that Avastin is not a cancer-specific targeted drug, unlike other antibody medicines, and thus Avastin needs to be administered at a high dose for exerting efficacy and may be distributed in not only cancer tissues but also other tissues with a high level of VEGF, resulting in adverse side effects. Therefore, the use of the cancer-specific distribution of NRP1 is expected to increase the cancer targeting effect to reduce the dose, thereby increasing patient convenience and reducing dose-dependent side effects. The present inventors have attempted to evaluate whether Avastin-A22p can effectively target cancer.

[0157] In order to investigate whether Avastin-A22p is distributed better than Avastin in cancer cells by targeting NRP1 present in new blood vessels inside cancer tissues and cancer mass, when a tumor volume reached around 250 mm.sup.3 after SW620 (obtained from ATCC) was transplanted in nude mice, PBS, Avastin-A22p, or Avastin was intravenously administered at 5 mg/kg, respectively, and tumors were harvested at the elapsed time of 3, 8, and 16 h, and the distribution of proteins in the cancer tissue was investigated through immunohistochemistry using fluorescence microscopes.

[0158] More specifically, the harvested tumor was sectioned to a thickness of 8 μm by a paraffin section method, and perivascular cells were stained with, as primary antibody, NG2 antibody (Cell Signaling technology, USA) and secondary antibody conjugated to Alexa (red fluorescence, Life Technologies) recognizing the NG2 antibody. In order to observe Avastin and Avastin-A22p distributed in the tissue, an antibody conjugated to Alexa®488 (green fluorescence, Life Technologies) recognizing IgG was used.

[0159] Related results are shown in FIG. 5.

[0160] As shown in FIG. 5, it was confirmed that Avastin-A22p was higher in concentration in the cancer tissue than Avastin from 3 h after administration; as time passed, a greater amount of Avastin-A22p was also distributed in cells distant from the blood vessels; and Avastin-A22p also effectively reduced angiogenesis inside caner mass.

[0161] From the results of the present fluorescent microscopic observation, it is suggested that Avastin-A22p is expected to have excellent cancer targeting ability and tissue penetration ability in comparison with Avastin, while Avastin-A22p, unlike Avastin, may inhibit angiogenesis more effectively by inhibiting various angiogenic factors, such as VEGF-B, VEGF-C, PlGF as well as VEGF-A. Through these results, even a low dose of Avastin-A22p can target tumor, leading to reduced side effects. Furthermore, it could be expected that Avastin-A22p arrives at not only cancer cells close to blood vessels but also cancer tissues inside the cancer mass due to high penetration ability thereof, and thus inhibits the activity of various angiogenic factors, including VEGF-A, secreted in these cells, thereby inhibiting cell division and thus enhancing anticancer activity.

[0162] In conclusion, such results have verified that Avastin-A22p may be applied to the treatment of Avastin-non-responsive cases occurring due to relatively lower dependency on VEGF-A than the other angiogenic factors.

Example 4: Effect of Avastin-A22p7 on Pericyte Coverage

[0163] In angiogenesis, vascular growth occurs through cell division and growth at a high VEGF concentration, and then as the VEGF concentration decreases, the PDGF concentration is increased, and thus vascular normalization or reconstruction occurs. The normalization encompasses, together with a decrease in number and size of immature blood vessels, a process of reducing the vascular pressure by the interstitial fluid due to pericyte coverage of blood vessels. When the concentration of VEGF is lowered by Avastin administration, blood vessels increase pericyte coverage through normalization to weaken the penetration of a drug into the cancer tissue. It has been reported that the co-administration of Avastin and docetaxel into non-small cell lung cancer patients reduced the penetration of drugs into cancer (Arjaans et al., 2013). This fact indicates that the co-administration of Avastin and another drug has limitations, and may cause resistance to Avastin.

[0164] According to the report by Fan et al., the continuous treatment of primary or metastatic colorectal cancer cell lines with Avastin for three months resulted in cell lines adapted to Avastin from both the cell lines, and these cell lines showed the same cell growth as existing cell lines, but have increased cell migration, and thus showed faster metastasis than existing cancer cells in the xenograft. VEGFR1 and NRP1 were specifically highly expressed in such Avastin adapted cell lines, which were then transformed into cells that secrete various angiogenic factors, such as VEGF-A, VEGF-B, VEGF-C, and PlGF, in larger quantities, showing resistance to Avastin (Fan et al., 2011). Therefore, it was considered that the treatment with Avastin-A22p or Avastin-TPP11 capable of binding to NRP1 to inhibit signaling of various angiogenic factors in addition to VEGF-A will effectively inhibit the growth and metastasis of cell lines showing Avastin resistance.

[0165] In addition, on the basis of the report in which when anti-NRP1 antibody and a VEGF inhibitor are co-administered, the reduction of pericyte coverage of blood vessels by the anti-NRP1 antibody may be directly related to the enhancement of anti-cancer efficacy through the control of vascular normalization and may be involved in overcoming resistance, the present inventors conducted a fluorescent microscopic comparison test of a change of pericyte coverage in cancer tissues in a xenograft model administered with Avastin and Avastin-A22p, respectively.

[0166] In order to investigate angiogenesis and vascular stabilization inhibitory ability of Avastin-A22p in mouse models, SW620 was transplanted in nude mice, which were then administered with Avastin-A22p and Avastin, respectively, and the pericyte coverage in cancer tissue was monitored by fluorescence microscopy. When the tumor volume reached around 250 mm.sup.3 after SW620 cells were injected into Balb/c nude mice, PBS, Avastin, and Avastin-A22p were intravenously administered at 5 mg/kg for each mouse, respectively. After 16 h, tumors were harvested from the mice, followed by immunohistochemistry. The harvested tumor was sectioned to a thickness of 8 μm by a paraffin section method. Then vascular cells were stained with, as primary antibody, PECAM1 (Sigma) and secondary antibody conjugated to Alexa®488 recognizing PECAM1, while perivascular cells were stained with, as primary antibody, NG2 antibody and secondary antibody conjugated to Alexa®594 recognizing NG2 antibody.

[0167] Related results are shown in FIG. 6.

[0168] As shown in FIG. 6, it was confirmed that the pericyte coverage relative to blood vessels were distinctively lowered in Avastin-A22p administered group rather than Avastin-administered group, and thus the increase of the anticancer effect by Avastin-A22p can be expected to be involved in the pericyte coverage. These results are identical to the test results of animals co-administered with anti-NRP1 antibody developed by Genetech Inc., and Avastin. It was confirmed that the VEGF- and NRP1-inhibitory effects by Avastin-A22p were similar to those by respective independent antibodies. That is, it can be verified that Avastin-A22p is a protein in a concept of a dual specific antibody that inhibit VEGF and NRP1 at the same time.

[0169] These results may suggest that through the reduction of pericyte coverage by A22p, Avastin-A22p can increase the angiogenesis inhibitory effect and overcome the resistance occurring due to the continuous administration of Avastin.

Example 5: Evaluation of In Vivo Anticancer Efficacy of Avastin-A22p

[0170] In order to investigate anticancer efficacy of Avastin-A22p, a comparison test with Avastin was conducted on colon cancer cell line SW620 xenograft models. The SW620 cell line is a tumor cell line with 45% RGI to VEGF, and this animal experiment was conducted for the purpose of evaluating the anticancer efficacy against general tumors. It was confirmed through a pre-experiment that Avastin-A22p showed an efficacy equal to or higher than that of Avastin (results not shown), and in this experiment, SW620 cell-xenograft Balb/c-nude mice were intravenously administered with Avastin 5 mg/kg and Avastin-A22p 0.5, 1.25, 2.5 mg/kg twice a week for five weeks, respectively.

[0171] Related results are shown in FIG. 7.

[0172] As shown in FIG. 7, it was confirmed that the Avastin 5 mg/kg-administered group and the Avastin-A22p 1.25 mg/kg-administered group showed a similar inhibitory effect against cancer growth. That is, it can be seen that the Avastin-A22p fusion protein can exhibit an equivalent anticancer efficacy even with one quarter of the dose of Avastin.

[0173] The fact that Avastin-A22p fusion protein may exhibit an equivalent anticancer efficacy even with one quarter of the dose of Avastin is thought to result from the blocking of NRP1 signaling by A22p, suggesting that adverse side effects may be also reduced through the reduction of dose in the clinical applications.

Example 6: Test of Confirming Resistant Cell Line Migration Inhibition by Avastin-A22p

[0174] One of the causes of resistance and responsiveness to Avastin is that VEGF-A, as a target of Avastin, and other growth factors, such as FGF, notch ligand/receptor system, PlGF, and PDGF-BB are involved in signaling mechanisms. NRP1 is known to affect signaling by serving as a co-receptor together with VEGFR1, VEGFR3, C-met, and PDGFR as well as VEGFR2 binding with VEGF-A. The present inventors investigated whether A22p fused to Avastin inhibits the binding to NRP1 competitively with other angiogenic factors which bind to NRP1.

[0175] HCT116/bev cell lines (provided from L M Ellis, MD anderson), which had been obtained resistance to Avastin by continuous treatment of HCT116 cells (obtained from ATCC) with Avastin, were treated with Avastin and Avastin-A22p to investigate the migration inhibitory ability thereof, respectively. In comparison with its parent cells HCT116, HCT116/bev has higher expression levels of VEGF-A, VEGF-B, VEGF-C, and PlGF, with a better ability in cell migration and metastasis (Fan et al., 2011). Thus, if Avastin-A22p inhibits the migration of HCT116/bev cells, the possibility of Avastin-A22p to overcome resistance to Avastin and increase efficacy thereof can be confirmed. A specific test method was as follows.

[0176] HCT116 and HCT116/bev cells were each seeded at 4×10.sup.5 cells/well in the 6-well plate, and incubated for 2 days. Thereafter, a cross-shaped blank was made in each well. The medium was removed, and 50 ng/ml VEGF was added and dispensed to MEM-alpha medium supplemented with 1% FBS. Avastin and Avastin-A22p each were treated to become 1 μM. The blank was photographed immediately after the drug treatment (0 h), and the same position was photographed at 2 h intervals from 48 h. The cell migration inhibitory abilities of Avastin-A22p and Avastin were compared by calculating the width of the empty blank from the photographing results through the Image J program and inversely quantifying the degree of cell migration therefrom.

[0177] Results are shown in FIG. 8.

[0178] As shown in FIG. 8, the migration of HCT116 cells was increased from 51.7% to 67.4% by VEGF, whereas it was reduced to the same level of 61.2% and 61.7% for Avastin and Avastin-A22p treatment, respectively. On the contrary, as for HCT116/bev cells, the migration of HCT116/bev cells was 65.3% even in the group treated with no VEGF, indicating that HCT116/bev cells were more aggressive than HCT116 cells, while the migration of HCT116/bev cells was increased to 72.9% by VEGF treatment. It was observed that such migration was 74.1% by Avastin treatment which indicated no difference in migration, while such migration was 66.2% by Avastin-A22p treatment which indicated that the migration was suppressed to the same level as in the group treated with no VEGF.

[0179] The results that Avastin-adapted cell lines, HCT116/bev, showed increased migration in comparison with their parent cells, HCT116, regardless of the addition of VEGF-A indicated that the migration of HCT116/bev was increased dependent on not only VEGF-A but also other growth factors. The Avastin treatment did not suppress the migration, whereas the Avastin-A22p treatment reduced migration to the same level as in the treatment without VEGF-A. This means that Avastin-A22p inhibits signalings by other growth factors as well as VEGF-A, indicating that Avastin-A22p may contribute to overcoming of Avastin resistance. In addition, this result suggests that Avastin-A22p can promote anticancer effects and lower the possibility of resistance occurrence by inhibiting not only VEGF-A signaling but also other signaling procedures.

Example 7: Evaluation on PDGF Signaling Inhibition by Avastin-A22p

[0180] NRP1 acts as a co-receptor for PDGF receptor to enhance signaling by PDGF. It is known that the binding of PDGF to a receptor thereof in cancer cells increases the phosphorylation of p130cas protein and promotes the migration and invasion of cancer cells. The present inventors investigated whether A22p fused to Avastin inhibits PDGF signaling by binding to NRP1.

[0181] U87MG cells (obtained from ATCC) were treated with 25 μg/ml Avastin or Avastin-A22p and, after 30 min, treated with 50 ng/ml PDGF. After 5 min, proteins were extracted, and then the comparison of p130cas phosphorylation was carried out using anti-phospho-p130 antibody and anti-p130cas antibody.

[0182] Results are shown in FIG. 9.

[0183] As shown in FIG. 9, it was verified that p130cas phosphorylation was increased by PDGF, and Avastin treatment resulted in no difference, but the phosphorylation in the Avastin-A22p treatment group was suppressed to the level in the vehicle.

[0184] This result means that Avastin-A22p removes VEGF-A, and also suppresses PDGF signaling by binding to NRP1, and that Avastin-A22p can increase anticancer effects by suppressing migration and invasion by PDGF.

Example 8: Preparation of Avastin Variant-Tissue-Penetrating Peptide (TPP) Fusion Protein

[0185] In order to investigate whether a fusion protein, in which a tissue-penetrating peptide (TPP) was fused to a variant containing a frequently modified sequence in other antibody medicines while retaining its characteristics, it was attempted that a tissue-penetrating peptide (TPP) capable of binding to both neuropilin 1 (NRP1) and neuropilin 2 was fused to the C-terminus of the Avastin variant peptide (hereinafter, referred to as “Avastin(V)) with modifications of 362nd and 364th amino acids in the Avastin's amino acid sequence.

[0186] Specifically, a fusion protein in which TPP (A22p) having the amino acid sequence of SEQ ID: 2 was fused to C-terminus of Avastin (V), and a cell line producing the fusion protein were prepared, respectively. Selection marker DHFR genes were inserted into pcDNA3.1(−) vectors and heavy and light chains of Avastin(V)-A22p were cloned by restriction enzymes NotI and BamHI. A plasmid encoding the protein formed by fusion of each of the constructed antibody heavy constant regions and a peptide binding to NRP1 and a plasmid encoding a light chain protein were expressed for protein in CHO DG44 cells using Neon™ electroporesis. In T25 flask, 3×10.sup.6cells transfected with plasmid were inoculated, and incubated at 37° C. Stable cells were secured using the selection marker, and then cultured in a floating state in serum-free SFM4CHO (Hyclone) for 7 days under conditions of 100 rpm, 37° C., pH 7.2, 50% DO.sub.2 in a bioreactor. The supernatant was separated from the cells by centrifugation, and sterilized by a 0.22-um filter.

[0187] The culture of Avastin(V)-A22p was collected, and proteins were respectively purified while referring to a standard protocol. For the purification column, the protein A resin (MabselectSure resin, GE healthcare) was packed to 20 mL in the XK16/20 column (MabselectSure resin, GE healthcare), to which a linear speed of 200 cm/h was applied. 1 L of antibody was applied to the protein A column, and the column was washed with a 15-fold column volume of PBS (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na.sub.2HPO.sub.4, 2 mM KH.sub.2PO.sub.4). The antibody was eluted with 100 mL of 0.1 M glycine buffer at pH 3.0. The proteins were collected between 10 mAU and 10 mAU in the UV absorbance at 280 nm, and 60 mL of the proteins were neutralized to pH 7.0 using 0.7 mL of 1 M Tris buffer. The antibody fractions were filtered by a 0.2 μm-filter (Milipore), and then concentrated using an amicon (30 MWCO) concentrator (Milipore) at 3,500 rpm for 10 min, followed by exchange with PBS (pH 7.4) buffer containing 10% glycerol. The purified fusion protein formed by fusion of the purified the antibody heavy chain constant region and the selected peptide specifically binding to NRP1 was analyzed on SDS-PAGE in reduction and non-reduction conditions.

[0188] Respective proteins were purified from the cultures collected from the cell lines expressing Avastin(V)-A22p and Avastin-A22p, and isolated by SDS-PAGE. FIG. 10 confirmed that Avastin(V)-A22p successfully formed antibody, and has a similar molecular weight to Avastin-A22p.

[0189] In order to investigate the purity of Avastin(V)-A22p purified protein, high performance liquid chromatography (HPLC) assay was performed. Agilent 1200 (Agilent) was used, and a size exclusion column (Biosuite 250, Waters) was used.

[0190] Specifically, a buffer solution (pH 6.2) containing 0.2 M potassium phosphate and 0.25 M potassium chloride was used as a mobile phase, and allowed to flow down at a flow rate of 0.35 ml/min for 20 min. The size of the protein was checked according to the retention time, and the purity thereof was checked according to the area and height.

[0191] In FIG. 11, the purity of Avastin(V)-A22p was analyzed using SE-HPLC. It was verified that the purity of Avastin(V)-A22p was 97% or higher (FIG. 11A), which was the same as the purity of Avastin-A22p (FIG. 11B).

[0192] The present inventors, hereinafter, prepared an Avastin(V)-A22p fusion protein in which A22p as a TPP having the amino acid sequence represented by SEQ ID NO: 2 was fused to Avastin(V), and evaluated activity thereof.

Example 9: Binding Ability of Avastin(V)-Tissue-Penetrating Peptide (TPP) Fusion Protein to NRP1 and VEGF

[0193] In order to evaluate whether the N-terminus of the fusion protein Avastin(V)-A22p as prepared in Example 8 may bind to VEGF in the same manner as Avastin while its C-terminus may bind to NRP1 to thus dually block the associated signaling, the binding of Avastin(V)-A22p with NRP1 and VEGF was respectively evaluated.

[0194] The binding ability of the purified Avastin(V)-A22p to Neuropilin 1-b1b2 domain was investigated in enzyme-linked immunosorbent assay (ELISA). Avastin-A22p was used as a positive control. The target molecule Neuropilin 1-b1b2 domain (273-586) was reacted at 1 μg per well in the 96-well Maxibinding Immuno plate (SPL Life sciences., Korea) for 2 h at 37° C., and then washed with 0.1% PBST (0.1% Tween20, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na.sub.2HPO.sub.4, 2 mM KH.sub.2PO.sub.4) for 1 min three times. After reaction with 0.1% PBST containing 5% skim milk for 1 h, the reaction product was washed with 0.1% PBST for 1 min three times. Avastin(V)-A22p, as experimental groups, was diluted with 0.1% PBST to 50 nM, 25 nM, and 12.5 nM, and the solutions were then treated at 37° C. for 1 h for each different concentrations, and washed with 0.1% PBST for 1 min three times. HRP-conjugated anti-kappa light chain antibody (Horseradish peroxidase-conjugated anti-human kappa light chain mAb, SIGMA-ALDRICH Co., USA) was 100,000-fold diluted with 0.1% PBST containing 5% skim milk, followed by treatment at 37° C. for 30 min. Thereafter, the reaction product was washed with 0.1% PBST for 1 min five times. For color development, the resultant product was treated with the TMB HRP colorimetric substrate (Surmodics co., USA), followed by reaction at room temperature for 10 min, treatment with the same volume of 1 N HCl (stop buffer), and then absorbance measurement at 450 nm. The binding ability of the expressed and purified Avastin(V)-A22p to Neuropilin 1-b1b2 domain was confirmed through the obtained ELISA results.

[0195] The binding ability of the purified Avastin(V)-A22p to Recombinant human VEGF 165 (R&D systems) was investigated in ELISA. Avastin-A22p was used as a positive control. The target molecule Recombinant human VEGF 165 was reacted at 0.2 μg per well in the 96-well Maxibinding Immuno plate for 2 h at 37° C., and then washed with 0.1 PBST for 1 min three times. After reaction with 0.1% PBST containing 5% skim milk for 1 h, the reaction product was washed with 0.1% PBST for 1 min three times. Avastin(V)-A22p, as experimental groups, was diluted with 0.1% PBST to 6.25 nM, 3.12 nM, and 1.56 nM, and the solutions were then treated at 37° C. for 1 h for each different concentrations, and washed with 0.1% PBST for 1 min three times. HRP-conjugated anti-kappa light chain antibody was 100,000-fold diluted with 0.1% PBST containing 5% skim milk, followed by treatment at 37° C. for 30 min. Thereafter, the reaction product was washed with 0.1% PBST for 1 min five times. For color development, the resultant product was treated with the TMB HRP colorimetric substrate, followed by reaction at room temperature for 10 min, treatment with the same volume of 1 N HCl, and then absorbance measurement at 450 nm. The binding ability of the expressed and purified Avastin(V)-A22p to Recombinant human VEGF 165 was confirmed through the obtained ELISA results.

[0196] Results are shown in FIG. 12.

[0197] As shown in FIG. 12, it was confirmed that Avastin(V)-A22p bound to NRP1 at the same level as in Avastin-A22p used as a control (FIG. 12A), and also bound to VEGF at the same level as Avastin-A22p (FIG. 12B).

Example 10: Binding Ability of Avastin-TPP11 for VEGFA and NRP1

[0198] In order to evaluate whether the N-terminus of the fusion protein Avastin-TPP11 as prepared in Example 1 may bind to VEGF in the same manner as Avastin while its C-terminus may bind to NRP1 to thus dually block the associated signaling, the binding of Avastin-TPP11 with NRP1 and VEGF was respectively evaluated.

[0199] The binding ability of the purified Avastin-TPP11 for VEGFA and for the NRP1 extracellular domain (ECD) was investigated in Surface Plasmon Resonance (SPR) Spectroscopy using a Biacore T200 instrument (GE Healthcare).

[0200] The ligand VEGFA was immobilized on a CM5 chip at the level of 15 response unit (RU), and Avastin-TPP11 or Avastin was diluted and injected into the VEGFA-coated chip at a concentration of 3.125-200 nM for the analysis. In the NRP1-binding assays, the human NRP1 ECD was immobilized on CM5 chips at the level of 1600 or 1800 RU, respectively. Avastin-TPP11 was diluted and injected into the NRP1 ECD-coated chip at a concentration of 0.156-20.000 nM. Kinetic parameters were determined in the Biacore T200 evaluation software, ver. 3.0 (GE Healthcare). The Langmuir 1:1 model was employed for the binding analysis.

[0201] Related results are shown in Table 2 below:

TABLE-US-00003 TABLE 2 Immobilized Ag Protein Species Analyte K.sub.a (M.sup.−1S.sup.−1) K.sub.d (S.sup.−1) K.sub.D (M) NRP1 Human Avastin- 1.221 × 1.sup.05 1.921 × 10.sup.−3 1.574 × 10.sup.−8 ECD TPP11 VEGF165 Human Avastin- 2.917 × 10.sup.4 3.599 × 10.sup.−5 1.234 × 10.sup.−9 TPP11 VEGF165 Human Avastin 2.802 × 10.sup.4 2.930 × 10.sup.−5 1.046 × 10.sup.−9

[0202] As shown in Table 2, it was confirmed that equilibrium dissociation rate constant, KD, values of Avastin-TPP11 and Avastin toward VEGFA turned out to be 1.234×10.sup.−9 M and 1.046×10.sup.−9 M, respectively.

[0203] The binding affinity for VEGFA was almost the same between Avastin-TPP11 and Avastin, which means that the TPP11 fusion does not alter the affinity of the anti-human VEGF antibody for VEGFA.

[0204] The binding affinity of Avastin-TPP11 for the human NRP1 ECDs was found to be 1.574×10.sup.−8 M. This value is 10-fold weaker than the above-mentioned affinity for VEGFA (Table 2), suggesting that VEGFA-and-NRP1 dual-targeting Avastin-TPP11 binds 10-fold more strongly to VEGFA than to NRP1.

Example 11: Inhibition of Multiple Signaling Pathway of Proangiogenic Growth Factor by Avastin-TPP11

[0205] Neuropilin-1 acts as a co-receptor for a number of extracellular ligands such as semaphorins, VEGFs, and other proangiogenic growth factors. It has been reported that chronic exposure to Avastin facilitates tumor cell migration by means of compensatory pathways in colorectal cancer cells. Avastin-adapted cells secrete more VEGFA, VEGFB, VEGFC, and PlGF, which induce migration of tumor cells and angiogenesis of endothelial cells in this medium in comparison with the parental cells.

[0206] The inventors of the present application experimented whether Avastin-TPP11 could suppress the function of proangiogenic growth factors in HUVECs by competitive binding to NRP1.

[0207] Human endothelial cell tube formation was induced by a CM from Avastin-adapted cells (HCT116/Bev-A and SW480/Bev-A cells, which were provided from L M Ellis, MD anderson), which secrete a variety of proangiogenic growth factors, or by a CM from parental cells (HCT116 and SW480 cells obtained from ATCC). To prepare conditioned media (CMs) from cancer cells (HCT116, HCT116/Bev-A, SW480, and SW480/Bev-A cells), these cells were cultured with each basal medium plus 1% of FBS for 24 h, followed by treatment with vehicle, Avastin, or Avastin-TPP11. PBS was used as vehicle. After 24 h, the CM was collected and centrifuged to remove cell debris. The human endothelial cells were seeded on p-slide angiogenesis tool (ibidi)-coated Matrigel (Corning Life Sciences) with 50 μL of one of these CMs. At 24 h after the seeding, images were captured under a light microscope. Total tube length was measured and analyzed with an angiogenesis analyzer (Image J software).

[0208] Related results are shown in FIGS. 13A and 13B.

[0209] As shown in FIGS. 13A and 13B, Avastin-TPP11 and Avastin inhibited HUVEC tube formation induced by the parental-cell CM relative to vehicle at a concentration of 300 nM. Avastin-TPP11 also caused concentration-dependent inhibition of tube formation by HUVECs stimulated by the Avastin-adapted cell CM. In contrast, Avastin treatment failed to inhibit HUVEC mobility induced by the Avastin-adapted cell CM.

[0210] These results meant that Avastin-TPP11 can suppress signaling of other proangiogenic growth factors besides VEGFA and can break the resistance to Avastin by blocking the activity of these growth factors.

[0211] Furthermore, the inventors examined the effects of Avastin-TPP11 on VEGFB- or PlGF2-induced migration of HUVECs as compared to aflibercept. The latter is a recombinant Fc fusion protein composed of the binding domains of VEGFR1 and VEGFR2 and traps VEGF family members such as VEGFA, VEGFB, and PlGF.

[0212] Related results are shown in FIGS. 13C and 13D.

[0213] The addition of either PlGF2 or VEGFB significantly increased HUVEC migration as compared to a no-growth factor control group. Avastin-TPP11 significantly attenuated this migration under the influence of PlGF2 or VEGFB to the level similar to that of the group treated with aflibercept (FIGS. 13C and 13D).

[0214] These data suggested that Avastin-TPP11 might be a potent blocker of VEGF family ligands via binding to NRP1, although Avastin-TPP11 cannot trap the proangiogenic growth factors directly.

Example 12: Pharmacokinetics of Avastin-TPP11

[0215] The PK assay of Avastin-TPP11 was performed on Sprague-Dawley (SD) rats in comparison with Avastin. SD rats (males, eight weeks old) were acquired from Orient Bio Inc. (Seongnam, South Korea). Five SD rats were given a 10 mg/kg intravenous (i.v.) dose of either Avastin-TPP11 or Avastin, and blood samples (approximately 0.25 mL each) were collected at 5 min and 1 h, 2 h, 4 h, 8 h, 24 h, 32 h, 72 h, 120 h, 192 h, 240 h, and 360 h post-injection. The samples were centrifuged at 4° C. and 13,475 g for 5 min, and serum was collected and stored at −80° C. until analysis. The concentration of Avastin-TPP11 or Avastin in each serum sample was determined by two types of enzyme-linked immunosorbent assay (ELISA). One is a VEGFA-binding ELISA, where a target drug binds to VEGFA and is detected by means of an anti-human IgG-horseradish peroxidase (HRP) conjugate (Sigma Aldrich) to evaluate the binding affinity for VEGFA. The other is a dual-targeting ELISA, where a target drug binds to VEGFA and is detected by means of a biotinylated NRP1 b1b2 domain to test whether Avastin-TPP11 is capable of binding to both targets simultaneously. The NRP1 b1b2 domain was produced and purified, and the biotinylation was conducted according to the manufacturer's instructions (EZ-Link micro sulfo-NHS-biotinylation Kit, Thermo Fisher Scientific Inc.). The PK parameters (AUC.sub.all, AUC.sub.inf, t.sub.1/2, C.sub.0, CL, and V.sub.ss) were estimated by noncompartmental analysis in the Pheonix™ WinNonlin® software, ver. 7.0 (Pharsight) and were presented as mean±standard deviation.

[0216] Semi-log plots of serum concentration of Avastin-TPP11 or Avastin versus time after a single i.v. dose (10 mg/kg) are presented in FIGS. 14A and 14B.

[0217] As shown in FIGS. 14A and 14B, serum concentration-time profiles of Avastin-TPP11 and Avastin were similar.

[0218] Furthermore, none of the PK parameters had significant differences between the Avastin-TPP11 group and Avastin group (Table 3).

TABLE-US-00004 TABLE 3 AUC.sub.all AUC.sub.inf t.sub.1/2 C.sub.0 CL V.sub.ss Method Drug (μg/mL*day) (μg/mL*day) (days) (μg/mL) (mL/day/kg) (mL/kg) VEGFA Avastin 886.5 ± 71.8  1769.5 ± 110.4 14.7 ± 1.8  235.8 ± 4.4 5.7 ± 0.3 122.1 ± 13.0 binding Avastin- 868.1 ± 61.6  1722.5 ± 173.1 14.7 ± 2.0  264.1 ± 4.6 6.1 ± 0.7 122.2 ± 11.8 TPP11 Dual- Avastin- 947.9 ± 104.4 1384.2 ± 100.7 9.5 ± 0.6 270.8 ± 5.7 7.4 ± 0.5  99.4 ± 13.9 targeting TPP11 Values are expressed as mean ± standard deviation (n = 5) . AUC.sub.all: area under the time-serum concentration curve from time zero to the last quantifiable observation, AUC.sub.inf: area under the time-serum concentration curve from time zero to infinity; t.sub.1/2: elimination half-life, C.sub.0: initial serum concentration at time zero, C.sub.L: clearance, and V.sub.ss: the volume of distribution in the steady state.

[0219] Half-life (t.sub.1/2) of Avastin-TPP11 and Avastin was 14.7+2.0 and 14.7±1.8 days, respectively, i.e., within the range of values reported in another study on Avastin (12.3+3.2 days).

[0220] These results showed that the PK parameters and VEGFA-binding affinity of Avastin-TPP11 are comparable to those of the parental antibody.

[0221] Furthermore, the inventors examined whether Avastin-TPP11 can bind to VEGFA and NRP1 simultaneously in the sandwich ELISA (dual-targeting ELISA). Similar time profiles of Avastin-TPP11 in the VEGFA-binding ELISA and dual-targeting ELISA indicated that during circulation in the blood stream, TPP11 did not split from the heavy chain of the antibody or lose the binding affinity for NRP1 and supported the half-life of the antibody (FIG. 14B).

Example 13: Cancer Targeting Experiment of Avastin-TPP11

[0222] The present inventors have attempted to evaluate whether Avastin-TPP11 can effectively target cancer.

[0223] In order to investigate whether Avastin-TPP11 is distributed better than Avastin in cancer cells by targeting NRP1 present in new blood vessels inside cancer tissues and cancer mass, when a tumor volume reached around 250 mm.sup.3 after SW620 (obtained from ATCC) was transplanted in nude mice, PBS, Avastin-TPP11, or Avastin was intravenously administered at 5 mg/kg, respectively, and tumors were harvested at the elapsed time of 3 h, 8 h, and 16 h, and the distribution of proteins in the cancer tissue was investigated through immunohistochemistry using fluorescence microscopes.

[0224] More specifically, the harvested tumor was sectioned to a thickness of 8 μm by a paraffin section method, and perivascular cells were stained with, as primary antibody, CD34 antibody (Abcam) and secondary antibody conjugated to Alexa 594 (red fluorescence, Life Technologies) recognizing the CD34 antibody. In order to observe Avastin and Avastin-TPP11 distributed in the tissue, an antibody conjugated to Alexa® 488 (green fluorescence, Life Technologies) recognizing IgG was used.

[0225] Related results are shown in FIGS. 15A and 15B.

[0226] As shown in FIGS. 15A and 15B, it was confirmed that Avastin-TPP11 was higher in concentration in the cancer tissue than Avastin from 8 h after administration; as time passed, a greater amount of Avastin-TPP11 was also distributed in cells distant from the blood vessels.

[0227] From the results of the present fluorescent microscopic observation, it is suggested that Avastin-TPP11 is expected to have excellent cancer targeting ability and tissue penetration ability in comparison with Avastin, while Avastin-TPP11, unlike Avastin, is capable of inhibiting angiogenesis more effectively by inhibiting various angiogenic factors, such as VEGF-B, VEGF-C, PlGF as well as VEGF-A. Through these results, even a low dose of Avastin-TPP11 can target tumor, leading to reduced side effects. Furthermore, it could be expected that Avastin-TPP11 arrives at not only cancer cells close to blood vessels but also cancer tissues inside the cancer mass due to high penetration ability thereof, and thus inhibits the activity of various angiogenic factors, including VEGF-A, secreted in these cells, thereby inhibiting cell division and thus enhancing anticancer activity.

[0228] In conclusion, such results have verified that Avastin-TPP11 may be applied to the treatment of Avastin-non-responsive cases occurring due to relatively lower dependency on VEGF-A than the other angiogenic factors.

Example 14: Evaluation of In Vivo Anticancer Efficacy of Avastin-TPP11

[0229] In order to investigate anticancer efficacy of Avastin-TPP11, a comparison test with Avastin was conducted on pancreatic cancer cell line BxPC-3 xenograft models. The BxPC-3 cells (5×10.sup.6 cells/mouse) resuspended in 50% Matrigel (BD Biosciences) were inoculated subcutaneously into the right flank of the mice.

[0230] This animal experiment was conducted for the purpose of evaluating the anticancer efficacy of the inventive fusion protein Avastin-TPP11 against general tumors. It was confirmed that Avastin-TPP11 showed an efficacy equal to or higher than that of Avastin. When the mean tumor volume reached 150 mm.sup.3, the mice were randomized into five groups (vehicle, Avastin 10 mg/kg and Avastin-TPP11 1, 3, 10 mg/kg) and were i.v. injected with these agents via the tail vein twice a week for four weeks respectively.

[0231] Related results are shown in FIG. 16.

[0232] As shown in FIG. 16, Avastin treatment only negligibly inhibited tumor growth in BxPC-3-bearing mice, whereas Avastin-TPP11 treatment significantly retarded this tumor growth in a dose-dependent manner.

[0233] In addition, the analysis on day 30 revealed that the effects of Avastin treatment at a dose of 10 mg/kg biweekly were not statistically significantly different from those in the vehicle group. By contrast, Avastin-TPP11 treatment yielded around 52% tumor growth inhibition at the same dose.

[0234] The fact that the Avastin-TPP11 fusion protein can exhibit higher anticancer efficacy with the same dose of Avastin is thought to be due to the blockade of NRP1 signaling by TPP11, which suggests that side effects may also be reduced through dose reduction in clinical applications.

INDUSTRIAL APPLICABILITY

[0235] The pharmaceutical composition containing, as an active ingredient, a fusion protein formed by fusion of a tissue-penetrating peptide and an anti-angiogenic agent, for treating cancer or an angiogenesis-related disease may improve the tumor penetration of the anti-angiogenic agent and exert a cancer targeting effect, thereby exhibiting an excellent therapeutic effect with even a small dose and thus reducing side effects of the anti-angiogenic agent, while exhibiting an excellent therapeutic effect even on cancer or an angiogenesis-related disease having resistance to the anti-angiogenic agent by binding to an angiogenesis growth factor and NRP1 at the same time. Therefore, the present invention is highly industrially applicable.