WO2017096124A1

WO2017096124A1 - Heterodimeric vascular endothelial growth factor and use thereof

Info

Publication number: WO2017096124A1
Application number: PCT/US2016/064554
Authority: WO
Inventors: Alan Yueh-Luen LEE; Jui-Ling TSAI
Original assignee: National Health Research Institutes; Kung, Hsing-Jien
Priority date: 2015-12-03
Filing date: 2016-12-02
Publication date: 2017-06-08
Also published as: CN108699562A; JP6936808B2; EP3384029A4; JP2019500900A; US20180355004A1; EP3384029B1; TW201726715A; US10851142B2; TWI746491B; CN108699562B; EP3384029A1

Abstract

A fusion protein, comprising: (i) a first vascular endothelial growth factor (VEGF) isoform, and (ii) a second VEGF isoform, and (iii) a dimerization domain between the first isoform and the second isoform, wherein the first isoform and the second isoform are selected from VEGF_12i and VEGF₁₆₅.

Description

HETERODIMERIC VASCULAR ENDOTHELIAL GROWTH FACTOR

AND USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to U.S. Provisional Application No. 62/262,630, filed on December 3, 2015, the entire content of which is hereby incorporated by reference herein.

BACKGROUND

Angiogenesis is a critical rate-limiting process during tumor progression, which is induced by tilting the balance toward proangiogenic factors to drive vascular growth. Cancer cells in a microenvironment count on angiogenesis to supply oxygen and nutrients. Thus, agents targeting angiogenic pathways have been investigated as potential cancer drugs.

Initial efforts have primarily focused on targeting endothelial and tumor-derived vascular endothelial growth factor A (VEGF- A)/VEGFR signaling. Several different strategies have been designed to inhibit this signaling as monotherapy or adjuvant therapies, e.g., small molecules inhibitors of VEGFR signaling, VEGF-Trap, anti-VEGFR antibodies, and the anti- VEGF- A monoclonal antibody Bevacizumab (Avastin), the first VEGF- A targeted antibody approved by the US FDA in 2004.

However, a significant number of patients either do not respond to antiangiogenic agents or rapidly develop resistance to them. Tumors may develop resistance to antiangiogenic agents via adaptive responses, e.g., upregulating alternative proangiogenic signaling, co-opting normal peritumoral blood vessels, suppressing immune surveillance by recasting immune cells and bone-marrow-derived proangiogenic cells, and activating invasiveness phenotype. These adaptive responses are induced by intratumoral hypoxia that results from tumor vessel pruning and extensive suppression of angiogenesis.

It remains a major challenge to efficiently inhibit VEGF-A/VEGFR signaling and, at the same time, alleviate resistance to antiangiogenic therapy.

SUMMARY

In one aspect, provided herein is a fusion protein that contains (i) a first vascular endothelial growth factor (VEGF) isoform, and (ii) a second VEGF isoform, and (iii) a dimerization domain between the first isoform and the second isoform. The first isoform and the second isoform are selected from VEGF121 and VEGF1₆5. VEGF121 can have an amino acid sequence that is at least 80% (e.g., at least 99%, 98%, 97%, 96%, 95%, 90%, or 85%) identical to the sequence of SEQ ID NO: 2. VEGFi₆5 can have an amino acid sequence that is at least 80% (e.g., at least 99%, 98%, 97%, 96%, 95%, 90%, or 85%) identical to the sequence of SEQ ID NO: 4.

In one embodiment, the dimerization domain contains two Fc regions and a linker between the two Fc regions. The linker can be a flexible linker consisting of 15 to 30 amino acids. For example, the linker can be (Gly-Gly-Gly-Gly-Ser)_n, n being 3, 4, 5, or 6.

In one embodiment, the fusion protein includes, in the direction from the N-terminus to the C-terminus, VEGF121, one of the two Fc regions, the linker, the other of the two Fc regions, and VEGF1₆5.

In another embodiment, the fusion protein contains, in the direction from the N- terminus to the C-terminus, VEGF1₆5, one of the two Fc regions, the linker, the other of the two Fc regions, and VEGF121.

The fusion protein can have an amino acid sequence that is at least 80% (e.g., identical to the sequence of SEQ ID NO: 11.

In another aspect, provided herein is a nucleic acid molecule that includes a nucleic acid sequence encoding the fusion protein described in this disclosure. In one embodiment, the nucleic acid sequence encodes an amino acid sequence that is at least 80% (e.g., at least 99%, 98%, 97%, 96%, 95%, 90%, or 85%) identical to the sequence of SEQ ID NO: 11.

In yet another aspect, provided herein is a pharmaceutical composition that contains the fusion protein and a pharmaceutically acceptable carrier. The pharmaceutical composition can be used to treat cancer or inhibit angiogenesis in a subject in need thereof.

The details of one or more embodiments are set forth in the accompanying drawing and the description below. Other features, objects, and advantages of the embodiments will be apparent from the description and drawing, and from the claims. BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic representation of the VEGF isoforms.

FIG. 2 is a schematic representation of a VEGF121-VEGF1₆5 fusion protein

FIG. 3 is a set of graphs showing the effect of the VEGF121-VEGF1₆5 protein on cell proliferation. A total of 1 x 10⁴ 3B-11 cells (A) and HCT-15 cells (B) were treated with the fusion protein (42, 83, 125 pM) in the presence of VEGF1₆5 (222 pM). Cell proliferation was measured by a CCK-8 kit (t-test, *P < 0.05, **P < 0.01, n = 3). 1: Control; 2: VEGF₁₆₅; 3: VEGF₁₆₅ + VEGF₁₂i-VEGF₁₆₅ (42 pM); 4: VEGF₁₆₅ + VEGF₁₂i-VEGF₁₆₅ (83 pM);

5: VEGF₁₆₅ + VEGF₁₂i-VEGF₁₆₅ (125 pM).

FIG. 4 is a set of graphs showing the effect of the VEGF121-VEGF1₆5 protein on tube formation. (A) 3B-11 (8 x 10⁵) cells were inoculated on Matrigel and treated with VEGF121- VEGFies (42, 83, 125 pM) in the presence of VEGFi₆₅ (222 pM). Tube formation was quantified by counting the connected cells in randomly selected fields at lOOx magnification. 1: Control; 2: VEGF₁₆₅; 3: VEGF₁₆₅ + VEGF₁₂i-VEGF₁₆₅ (42 pM); 4: VEGF₁₆₅ + VEGF₁₂₁- VEGF₁₆₅ (83 pM); 5: VEGF₁₆₅ + VEGF₁₂i-VEGF₁₆₅ (125 pM). (B) VEGF₁₂₁-VEGF₁₆₅ plasmid was transfected into 3B-11 cells. Tube formation assay was carried out using the 3B- 11 cells. Data are presented as the means + SEM based on three independent experiments. 1: Control; 2: VEGF₁₆₅ (222 pM); 3: VEGF₁₂₁-VEGF₁₆₅ plasmid. ^*P < 0.05, ^**P < 0.01, ***p < 0.001.

FIG. 5 is a graph showing the effect of the VEGFi₂i-VEGFi₆5 protein on 3B-11 cell migration. VEGFi₂i-VEGFi₆5 plasmid was transfected into 3B-11 cells. Cell migration was determined using the transfected 3B-11 cells. Cell migration ability of 3B-11 cells was enhanced in the presence of VEGF1₆5 (222 pM) but inhibited by the presence of the VEGFm- VEGF1₆5 plasmid.

FIG. 6 is a set of graphs showing the effect of the VEGFi₂i-VEGFi₆5 protein on HCT- 15 cell migration. (A) HCT-15 cells were inoculated in Trans well™ permeable inserts and treated with the VEGFi₂i-VEGFi₆₅ protein (42, 83, 125 pM) or in the presence of VEGFi₆₅

(222 pM). The distance between the gap was analyzed in Transwell™ permeable inserts. Untreated HCT-15 cells were used as controls. 1: Control; 2: VEGF₁₆₅; 3: VEGF₁₆₅ + VEGF₁₂₁-VEGF₁₆₅ (42 pM); 4: VEGF₁₆₅ + VEGF₁₂₁-VEGF₁₆₅ (83 pM); 5: VEGF₁₆₅ + VEGF₁₂₁-VEGF₁₆₅ (125 pM). (B) VEGF₁₂₁-VEGF₁₆₅ plasmid was transfected into HCT-15 cells. Cell migration was determined using the transfected HCT-15 cells. Cell migration ability of HCT-15 cells was enhanced in the presence of VEGF1₆5 (222 pM) but inhibited by the presence of VEGF₁₂₁-VEGF₁₆₅ plasmid. i-test, ^*P < 0.05, ^**P < 0.01, ***p < 0.001, n = 3.

FIG. 7 is a graph showing the effect of the VEGFi₂i-VEGFi₆5 protein on cell invasion. HCT-15 cells were treated with different concentrations of VEGF i₂i-VEGFi₆₅ (42, 83, 125 pM) in the presence of VEGF1₆5 (222 pM). Cell invasion was determined by the transwell chamber assay. After migrating for 48 h, the number of cells passing through the filter was counted after staining with crystal violet (original magnification: 400x). 1: Control; 2: VEGF₁₆₅; 3: VEGF₁₆₅ + VEGF₁₂i-VEGF₁₆₅ (42 pM); 4: VEGF₁₆₅ + VEGF₁₂i-VEGF₁₆₅ (83 pM); 5: VEGF₁₆₅ + VEGF₁₂i-VEGF₁₆₅ (125 pM). i-test, ^*P < 0.05, ^**P < 0.01, *** p < 0.001.

FIG. 8 is a set of graphs showing the effect of the VEGF121-VEGF1₆5 protein in a xenograft tumorigenesis assay. HCT-15 cells were injected subcutaneously into the dorsal flank of nude mice (1 site per mouse). Injected mice were examined every two days for tumor formation. Different concentrations of VEGF121-VEGF1₆5 protein (10, 50, or

250 ng/ml) or a PBS control were directly injected into the tumors in the mice. (A) The body weight of injected mice was monitored. (B) Tumor volume was estimated from its length and width, as measured by a 6-inch-dial caliper, using the formula: tumor volume = 1 x w² x 0.52.

DETAILED DESCRIPTION

It was surprisingly discovered that a heterodimeric vascular endothelial growth factor (VEGF) composed of two different VEGF isoforms reduced proliferation, migration, invasion, and tube formation in endothelial and cancer cells through competing with VEGF1₆5 homodimer in a paracrine and an autocrine manner.

Therefore, described herein is a fusion VEGF protein containing isoform VEGF121 and isoform VEGF1₆5 linked by a dimerization domain.

The VEGF-A gene includes eight exons, which can give rise to alternatively spliced variants, i.e., VEGF121, VEGF145, VEGFi₆₅, VEGFi₈₃, VEGFi₈₉, and VEGF₂₀₆. See Fig. 1 VEGF121 is a freely soluble and weakly acidic polypeptide that lacks a heparin- binding domain. A VEGF₁₂i nucleic acid sequence (SEQ ID NO: 1) and the amino acid sequence it encodes (SEQ ID NO: 2) are provided herewith. The sequence of SEQ ID NO: 2 includes an N-terminal signal peptide, which is not present in the mature form of VEGF121.

VEGF1₆5 contains basic amino acids and a heparin-binding domain that binds the

VEGF receptor to induce signal transduction and stimulate endothelial cell proliferation. A VEGF1₆5 nucleic acid sequence (SEQ ID NO: 3) and the amino acid sequence it encodes (SEQ ID NO: 4) are provided herewith. The sequence of SEQ ID NO: 4 includes an N- terminal signal peptide, which is not present in the mature form of VEGF1₆5.

The fusion VEGF further includes a dimerization domain positioned in between the two VEGF isoforms such that the fusion VEFG forms a heterodimer. In one embodiment, the dimerization domain consists of two Fc regions linked by a linker. The Fc region can be a human IgG Fc region. For example, the Fc region can have the amino acid sequence of SEQ ID NO: 6, which is encoded by the nucleic acid sequence of SEQ ID NO: 5.

The linker between the two Fc regions can be any flexible linker known in the art. The linker can have between 15 and 30 amino acids. A flexible linker can be a Gly- and Ser- rich linker. For example, the linker can be (Gly-Gly-Gly-Gly-Ser)_n (SEQ ID NO:7), n being an integer (e.g., 1, 2, 3, 4, 5, 6, 7, or 8).

The fusion protein can further include a signal peptide at the N-terminus. The signal peptide can be the signal peptide endogenous to the VEGF isoforms. For example, the signal peptide can have the sequence of SEQ ID NO: 9 (encoded by the nucleic acid sequence of SEQ ID NO: 8). The C-terminal VEGF isoform in the fusion protein may or may not include a signal peptide.

In addition, the fusion protein can include a C-terminal tag to facilitate isolation or identification of the fusion protein. Such tag can be a poly(His) tag, HA tag, Myc tag, V5, or FLAG tag.

The fusion protein can contain, in the direction from the N-terminus to the C- terminus, VEGFi₆5, an Fc region, a linker, an Fc region, and VEGF121. Alternatively, the fusion protein can contain, in the direction from the N-terminus to the C-terminus, VEGF121, an Fc region, a linker, an Fc region, and VEGF1₆5. Each isoform can be linked to an Fc region directly or indirectly via a linker, which can be different or identical to the linker between the two Fc regions. In one embodiment, the fusion protein has an amino acid sequence that is at least 80% (e.g., at least 99%, 98%, 97%, 96%, 95%, 90%, or 85%) identical to the sequence of SEQ ID NO: 11, which is encoded by the sequence of SEQ ID NO: 10.

Conventional methods, e.g., recombinant technology, can be used to make the fusion protein. For example, an expression construct encoding the protein can be generated and introduced into suitable host cells (e.g., mammalian cells). The fusion protein expressed in the host cells can then be isolated.

The fusion protein can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition. The composition can be administered to a subject in need thereof to treat cancer or inhibit angiogenesis. The fusion protein can also be used in a combination therapy with other cancer treatments. The fusion protein can also be conjugated to or encapsulated in moieties (e.g., lipids, carbohydrates, polymers or nanoparticles) designed to target the fusion protein to tumors and/or their associated vasculature.

The composition can be formulated with a pharmaceutically acceptable carrier such as 5 a phosphate buffered saline, a bicarbonate solution, and/or an adjuvant. Suitable

pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are known in the art. This composition may be prepared as an injectable, liquid solution, emulsion, or another suitable formulation.

Examples of adjuvants include, but are not limited to, alum-precipitate, Freund's0 complete adjuvant, Freund's incomplete adjuvant, monophosphoryl-lipid A/trehalose

dicorynomycolate adjuvant, water in oil emulsion containing Corynebacterium parvum and tRNA, and other substances that accomplish the task of increasing immune response by mimicking specific sets of evolutionarily conserved molecules including liposomes, lipopoly saccharide (LPS), molecular cages for antigen, components of bacterial cell walls, 5 and endocytosed nucleic acids such as double-stranded RNA, single-stranded DNA, and unmethylated CpG dinucleotide-containing DNA. Other examples include cholera toxin, E. coli heat-labile enterotoxin, liposome, immune- stimulating complex (ISCOM),

immunostimulatory sequences oligodeoxynucleotide, and aluminum hydroxide. The composition can also include a polymer that facilitates in vivo delivery.

o An effective amount of the composition described above may be administered

parenterally, e.g., subcutaneous injection, intravenous injection, or intramuscular injection. Other routes of administration may also be used. A skilled practitioner would be able to determine the appropriate dosage and route of administration.

Cancers that can be treated with the fusion protein include solid tumors such as 5 glioblastoma, colorectal cancer, lung cancer, renal cancer, liver cancer, kidney cancer,

neuroendocrine tumors, breast cancer, esophageal cancer, gastrointestinal stromal tumors, melanoma, ovarian cancer, cervical cancer, pancreatic cancer, prostate cancer, stomach cancer, and head and neck cancer.

The specific example below is to be construed as merely illustrative, and not o limitative of the remainder of the disclosure in any way whatsoever. Without further

elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.

EXAMPLE

We generated a novel chimeric dimer of VEGF121-VEGF1₆5 fused with two Fc regions of human IgGl as a powerful antiangiogenic modulator. It was found that the chimeric VEGF121-VEGF1₆5 recombinant protein reduced tube formation of 3B-11 endothelial cells and inhibited invasiveness of HCT-15 cancer cells. Furthermore, we found that the VEGF121- VEGFies protein attenuated VEGFR2-HIF-la signaling through the PI3K-AKT-mTOR pathway in cancer cells. The data demonstrated that the chimeric VEGF121-VEGF1₆5 protein antagonizes angiogenesis and HIF-Ια signaling, and suggested that it could combat drug resistance to antiangiogenic therapy.

Construction and characterization of a VEGF₁₂i-VEGF₁₆₅ fusion protein

A VEGF121-VEGF1₆5 fusion was generated by fusing a human IgGl Fc nucleic acid sequence to the 3' terminus of a VEGF121 sequence and the 5' terminus of a VEGF1₆5 sequence, respectively, and the two Fc sequences were connected by a linker sequence. The VEGF121-VEGF1₆5 fusion nucleic acid sequence was cloned into the pcDNA3.1 vector, yielding the expression vector for VEGF121-VEGF1₆5. The integrity of the final construct was confirmed by DNA sequencing. The deduced protein includes a putative 26-aa signal peptide. See Fig. 2. The plasmid containing the VEGF121-VEGF1₆5 fusion gene was transfected into 293T cell line. The expression and secretion of VEGF121-VEGF1₆5 fusion protein were confirmed by Western blot. We found a single band of -120 kDa for VEGF121-VEGF1₆5 fusion protein in samples from cultured medium and cell lysates of transfected 293T cells (data not shown). The recombinant protein VEGF121-VEGF1₆5 was expressed as a His-tag fusion protein in 293T cells and purified using nickel affinity chromatography. The purity and the molecular weight of the purified fusion protein were determined by Western blot (data not shown). These results indicated that VEGF121-VEGF1₆5 formed a dimer covalently linked by IgGl Fc fragments and a polypeptide linker.

VEGF121-VEGF1₆5 fusion protein inhibited cell proliferation induced by VEGF1₆5

Since endothelial cell proliferation is required for early angiogenic response, we examined whether the VEGF121-VEGF1₆5 protein affected proliferation of VEGF1₆5- stimulated 3B-11 cells, a convenient endothelial cell model for tube formation assay. See Zhou et al., Methods 2008, 44(2): 190-195. VEGF₁₆₅-induced cell growth of 3B-11 was blocked in a concentration-dependent manner by VEGF121-VEGF1₆5. See Fig. 3, panel A. The effect of VEGF1₆5 at a concentration of 222 pM on 3B-11 cell proliferation was easily inhibited by the recombinant VEGF121-VEGF1₆5 at 42 pM, suggesting that the VEGF121- VEGF1₆5 protein was able to efficiently block the activity of VEGFi₆5-induced proliferation. Moreover, the VEGF121-VEGF1₆5 protein exhibited similar potency at inhibiting VEGF1₆5- induced growth of HCT-15 cancer cells. See FIG. 3, panel B. We also found that VEGF121- VEGF1₆5 exhibited similar activity in inhibiting cell proliferation of 3B-11 and HCT-15 cells in an autocrine manner when the plasmid of the fusion gene was transfected into these cells, although the inhibition was not statistically significant (data not shown). The results suggest that VEGF121-VEGF1₆5 inhibit the increase in cell number due to suppression of proliferation, not cytotoxicity. Furthermore, VEGF121-VEGF1₆5 inhibited cell transformation of HCT-15 in an autocrine manner (data not shown). Our results indicate that the VEGF121-VEGF1₆5 recombinant protein can block cell proliferation of 3B-11 as well as cell proliferation and transformation of colon cancer cell HCT-15 in autocrine and paracrine manners.

VEGF121-VEGF1₆5 fusion protein inhibited tube formation induced by VEGF1₆5

Later stages of angiogenesis require morphological alterations of endothelial cells, which result in lumen formation. We examined tube formation in vitro in the presence of the VEGF121-VEGF1₆5 protein. An in vitro tube formation assay was employed by using 3B-11 endothelial cells that were induced to invade a three-dimensional collagen gel where they formed a network of capillary-like tubes. See Zhou et al., Methods 2008, 44(2): 190-195.

The results showed that 3B-11 cells could form a tube network under normal condition, and VEGF1₆5 increases numbers of tube formation. See Fig. 4, panel A. However, the numbers of tube-like structure formation in 3B-11 cells were inhibited by the addition of the VEGF121-VEGF1₆5 protein in a concentration-dependent manner. See Fig. 4, panel A. Furthermore, the VEGF121-VEGF1₆5 protein significantly inhibited VEGFi₆5-induced tube formation in a paracrine manner (Fig. 4, A) and in an autocrine manner (Fig. 4, B). These results demonstrated that the VEGF121-VEGF1₆5 chimeric protein can inhibit VEGF1₆5- induced angiogenesis in vitro.

VEGF121-VEGF1₆5 fusion protein inhibited cell migration Cell migration is a critical process in angiogenesis and tumor metastasis. We examined whether cell migration was affected by the VEGF121-VEGF1₆5 chimeric protein. Cell migration was examined by a gap-closure migration assay. Consistently, the VEGF121- VEGF1₆5 protein significantly inhibited migration of 3B-11 cells in an autocrine manner. See Fig. 5. In addition, the results showed that cell migration of HCT-15 induced by VEGF1₆5 was inhibited by the addition of the VEGF121-VEGF1₆5 protein in a concentration-dependent manner. See Fig. 6, panel A. The VEGF121-VEGF1₆5 protein significantly inhibited cell migration in a paracrine (Fig. 6, A) and an autocrine manner (Fig. 6, B). These data suggested that VEGF121-VEGF1₆5 chimeric protein can inhibit migration of endothelial cells and tumor cells.

VEGF121-VEGF1₆5 fusion protein impaired tumor invasion

To validate VEGF121-VEGF1₆5 chimeric protein's effect on metastasis of tumor cells, we examined the effect of the protein on cell invasion by using the Transwell assay. In the absence of VEGF121-VEGF1₆5, VEGF1₆5 induced invasive capability as indicated by intensive penetration. See Fig. 7. However, VEGFi₆5-induced cell invasion was inhibited by the addition of VEGF₁₂i-VEGF₁₆₅ in a concentration-dependent manner in HCT-15 cancer cells.

The number of penetrated cells was significantly decreased when treated with increased concentrations of the VEGF121-VEGF1₆5 protein as compared with VEGF1₆5 only. See Fig. 7.

These results indicated that the invasion of cancer cells was markedly suppressed by the addition of the VEGF121-VEGF1₆5 protein, suggesting a role of the protein in suppressing cancer metastasis.

VEGF₁₂i-VEGF₁₆₅ chimeric protein attenuated autocrine VEGFR2-HIF-la- VEGF165/Lon signaling through PI3K-AKT-mTOR pathway

To check whether VEGF121-VEGF1₆5 attenuates HIF-Ια signaling to decrease the resistance to anti- angiogenic therapy, we first examined the effect of VEGF121-VEGF1₆5 on the VEGFR2-HIF-la-VEGFi₆5 axis in tumor cells. Lon is upregulated by the hypoxia inducible factor- la (HIF-la) and involved in response to low oxygen availability, which adapt cancer cells to a hypoxic environment. We examined whether the fusion protein influenced the expression of Lon protease. The expression of HIF-la, VEGF1₆5, and Lon was determined by Western blot analysis. The results showed that VEGFR2-HIF-la-

VEGFi₆5/Lon signaling in HCT-15 cancer cells was activated by VEGF1₆5 treatment and hypoxia stimulated by cobalt chloride (C0CI₂) (data not shown). However, the signaling activation was inhibited by the addition of VEGF₁₂₁-VEGF₁₆₅ in a concentration-dependent manner under normoxia and hypoxia conditions. The recombinant VEGF₁₂₁-VEGF₁₆₅ protein reduced the level of HIF-Ια, VEGF₁₆₅, Lon, and phospho-VEGFR2 induced by VEGF₁₆₅ and/or hypoxia (data not shown). Mechanically, the VEGF₁₂₁-VEGF₁₆₅ protein inhibited the activation of VEGFR2-HIF-la-VEGFi₆5/Lon signaling through repressing PBK-AKT-mTOR pathway (data not shown), suggesting that the VEGF₁₂₁-VEGF₁₆₅ protein overcame survival mechanism triggered by the PI3K-AKT-mTOR to VEGFR2-HIF-la- VEGFi₆5/Lon axis under hypoxia. These data suggest that VEGF₁₂₁-VEGF₁₆₅ can inhibit autocrine VEGFR2-HIF-la-VEGFi₆₅/Lon signaling through PBK-AKT-mTOR pathway in cancer cells.

VEGF₁₂₁-VEGF₁₆₅ fusion protein impaired tumor growth in vivo

Our data demonstrated that the chimeric VEGF₁₂i-VEGF₁₆₅ protein can arrest tube formation of endothelial cells and interfere with tumor cell growth, migration and invasion in vitro. A xenograft tumorigenesis assay was performed to demonstrate the inhibitory effect of the VEGF₁₂i-VEGF₁₆₅ protein on tumors in vivo. BALB/c nude mice (6-8 weeks old) were used in the assay. lxlO⁶ HCT-15 cells suspended in 0.2 ml of Matrigel were injected subcutaneously into the dorsal flank of nude mice (1 site per mouse). The mice were examined every two days for tumor formation. Different concentrations of the VEGF121- VEGF₁₆₅ protein (10, 50, or 250 ng/ml) or a PBS control were directly injected into the tumors in the mice. See Fig. 8, panel B. The body weight of the mice was monitored. See Fig. 8, panel A. The mice were then sacrificed by CO₂ euthanasia.

The body weight of the mice treated with the VEGF₁₂₁-VEGF₁₆₅ chimeric protein did not change significantly, suggesting that the protein was not toxic to the animals. See Fig. 8, panel A. The chimeric protein reduced tumor growth in the mice in a dose-dependent manner. See Fig. 8, panel B. Thus, the study demonstrated that the VEGF₁₂₁-VEGF₁₆₅ chimeric protein can suppress tumor growth in vivo.

Materials and Methods

Cell lines and cell cultures: 3B-11 cells were purchased from ATCC (#CRL-2160, Manassas, VA, USA). 3B-11 cells were maintained in DMEM and HCT-15 cells in RPMI-

1640 supplemented medium with 10% (v/v) heat-inactivated FBS (fetal bovine serum qualified; Invitrogen), 1% PSA (penicillin-streptomycin amphotericin B ; Biological industries, NY, USA) in a 37°C humidified incubator with 5% C0₂.

Purification of VEGF₁₂₁-VEGF₁₆₅ recombinant proteins: The plasmids were transfected into 293T cells using the Biomics transfection reagent as described in the instruction manual of the pcDNA3.1 vector helper- free system (Biomics). After incubation for 36 hours, the supernatant of the 293T cell culture was collected and purified on Ni-NTA resin, eluted with 250 mmol/L imidazole according to the instruction manual. The recombinant protein was concentrated by the Microcon Centrifugal Filter Unit (Millipore, Bedford, MA, USA). Finally, the purified protein was confirmed by 10% SDS-PAGE and Western Blotting.

Cell proliferation assay: The proliferation of 3B-11 cells and HCT-15 cells was assessed using CCK-8 dye reduction assay (Enzo, USA). 3B-11 or HCT-15 cells were pre- treated with different concentrations of VEGF₁₂i-VEGF₁₆₅ (42, 83, 125 pM) for 30 min, a commercial VEGF₁₆₅ was added (222 pM, Abeam, Cambridge, MA, USA), and then the cells were incubated for 24 hours. At the end of the treatment, 10 μΐ of the CCK-8 solution was added to each well of the plate and the plate was incubated for 2~4 hours in the incubator. After shaking the plate for 10 seconds, cell viability was assessed by measuring the absorbance at 450 nm using a microplate reader. All measurements were performed three times. The Γ-test was used to compare groups. Data are presented as mean + SD.

Colony formation assay: Clonogenic assay is an in vitro transformation assay based on the ability of a single cell to grow into a colony. To examine this, the plasmid of

VEGF₁₂₁-VEGF₁₆₅ was transfected into HCT-15 in a 10 cm dish overnight and treated with VEGF₁₆₅ recombinant protein 222 pM as a positive control. Next day, the treated HCT-15 cells (~ lxlO³ per well) were plated in 6-well plates and incubated in a 37 °C incubator. Fresh RPMI medium containing 10% FBS was added every 48 hours. At the end of the 14th day, cells were washed twice with ice cold PBS, fixed with methanol for 10 minutes and then stained with 1% crystal violet in methanol for 15 minutes followed by washing with deionized water. Colonies with more than 50 cells were scored and counted under the microscope at 200x.

Cell migration assay: Cell migration assay was determined by gap closure assay. 3B-

11 cells or HCT-15 cells were treated with different concentrations (42, 83, 125 pM) of the recombinant proteins for 16 h (37 °C, 5% CO₂). These cells were trypsinized and resuspended in serum- free DMEM or RPMI-1640 medium. A total of 8xl0⁵ cells in 70 μΐ serum-free DMEM or RPMI-1640 were seeded in medium in each well (8xl0⁵ cells/ well) and incubated at 37 °C, 5% CO_2. Next day, the ibidi culture-insert (Applied Biophysics, USA) was gently removed by using sterile tweezers, and each well was then filled with 2 ml 0.1% FBS medium. Cell migration was monitored for 48 h by microscope.

Tube formation assay: Corning Matrigel® Matrix (BD Biosciences, San Jose, CA, USA) solution was thawed on ice overnight and 50 μΐ aliquots were coated onto a 96-well plate and incubated at 37°C for lh to solidify. 50 μΐ of DMEM supplemented with 10% FBS medium containing about 8xl0⁵ 3B-11 cells was seeded onto the plated Matrigel Matrix and incubated at 37°C. These cells were treated as previously described. The assay was done in triplicate and was incubated at 37 °C with 5% CO₂. Images of the formation of capillary-like structures were obtained after 2 h with a computer-assisted microscope (Olympus, Tokyo, Japan) at 200x magnification. Tubular structures were quantified by manually counting the numbers of connected cells in randomly selected fields at 200x magnification. Total tube numbers of network formation were counted.

Cell invasion assay: Cell invasion was evaluated using a transwell chamber (Corning Costar; Cambridge, MA, USA) equipped with a Matrigel-coated filter membrane (8 μιη pores). Briefly, the filters were pre-coated with 200 μg/ml basement membrane proteins (Matrigel; BD Biosciences, San Jose, CA, USA) and allowed to dry overnight at 37°C with 5% C0₂. HCT-15 cells (8xl0⁵) in FBS-free medium were seeded in the upper chambers, and lower wells were filled with 10% FBS medium. After incubation at 37°C for 48h, non- migratory cells on the upper side of the insert were removed with a cotton swab. The cells that had passed through the filter were fixed in methanol and stained with crystal violet.

Randomly selected fields on the lower side of the photograph under microscopy were counted.

Immunoblotting: HCT-15 cells were seeded onto a 10 mm dish at a density of 1.5xl0⁶ cells in 10 ml medium for 24 h under normoxia and hypoxia (C0CI₂, Cobalt dichloride; 150 μΜ) and treated as previously described. Total protein concentrations were determined using a BCA protein assay. Equal quantities of total protein were resolved using 10% SDS-PAGE and electroblotted onto polyvinylidene fluoride membranes. Membranes were blocked with

5% skimmed milk and probed overnight at 4°C with primary antibodies. Membranes were then probed with the appropriate HRP-conjugated secondary antibodies (GeneTex, Hsinchu, Taiwan) and the immunoreactive bands were visualized using an enhanced chemiluminescence method (Bio-Rad, Hercules, CA, USA). Antibodies used in this study were purchased or produced as indicated. Antibody to human Lon was produced as described previously. See Wang et al., Cancer Sci 2010, 101(12):2612-2620; and Cheng et al., Cell death & disease 2013, 4, e681. Phospho-PI3K (Tyr458/ Tyrl99, #4228), phospho- AKT (Ser473, #4060), and phoshpo-mTOR (Ser2448, #2971) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA); HIF-la (#610958) antibodies was obtained from BD Biosciences (Franklin Lakes, NJ); phospho-VEGFR2 (Tyrl054/Tyrl059, ab5473), VEGF-165A (ab69479) antibodies were from Abeam (Cambridge, MA, USA); beta-actin antibody was fromfrom GeneTex (GTX109639, Hsinchu, Taiwan).

Statistical methods: Parametric Student's t test was used in this study to judge the significance of difference between conditions of interest. In general, a P value of <0.05 was considered as statistically significant (Student's t test, *p < 0.05, **p < 0.01, and ***p < 0.001). OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any

combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the described embodiments, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

Claims

WHAT IS CLAIMED IS:

1. A fusion protein, comprising:

(i) a first vascular endothelial growth factor (VEGF) isoform, and

(ii) a second VEGF isoform, and

(iii) a dimerization domain between the first isoform and the second isoform.

wherein the first isoform and the second isoform are selected from VEGFm and VEGF1₆5.

2. The fusion protein of claim 1, wherein the dimerization domain contains two Fc regions.

3. The fusion protein of claim 2, further comprising a linker between the two Fc regions.

4. The fusion protein of claim 3, wherein the fusion protein contains, in the direction from the N-terminus to the C-terminus, VEGF121, one of the two Fc regions, the linker, the other of the two Fc regions, and VEGF1₆5.

5. The fusion protein of claim 3, wherein the fusion protein contains, in the direction from the N-terminus to the C-terminus, VEGF₁₆₅, one of the two Fc regions, the linker, the other of the two Fc regions, and VEGF121.

6. The fusion protein of any of claims 3-5, wherein the linker is a flexible linker consisting of 15 to 30 amino acids.

7. The fusion protein of claim 6, wherein the linker is (Gly-Gly-Gly-Gly-Ser)_ni wherein n is 3.

8. The fusion protein of claim 7, wherein each of the Fc regions is a human IgGl Fc region.

9. The fusion protein of claim 8, further comprising a peptide tag.

10. The fusion protein of claim 9, wherein the tag is a C-terminal 6X-His tag.

11. The fusion protein of claim 10, further comprising an N-terminal signal peptide.

12. The fusion protein of claim 11, wherein the fusion protein has an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 11.

13. The fusion protein of claim 12, wherein the fusion protein has the sequence of SEQ ID NO: 11.

14. A nucleic acid molecule, comprising a nucleic acid sequence that encodes the fusion protein of any of claims 1-13.

15. A host cell, comprising the nucleic acid molecule of claim 14.

16. A pharmaceutical composition, comprising the fusion protein of any of claims 1-13 and a pharmaceutically acceptable carrier.

17. A method of treating a cancer in a subject, the method comprising administering to a subject in need thereof the composition of claim 16.