# Patent

## Title
Fusion protein inhibiting angiogenesis or growth and use thereof

## Abstract

Provided are a fusion protein inhibiting angiogenesis or vascular growth, coding sequence thereof, vector comprising the coding sequence, host cell, pharmaceutical composition and use of the fusion protein. The fusion protein of the present invention has high thermostability, and has a dramatic decline in the protein aggregation formation rate in a fermentation process, and a significant increase in the purity and yield of the protein, and has better biological activity.

## Background
REFERENCE TO EARLIER FILED APPLICATIONS
This application claims the benefit of the filing date of PCT Application No. PCT/CN2015/071434 filed 23 Jan. 2015 which claims priority to Chinese Patent Application No. 201410035738.1 filed 25 Jan. 2014, the disclosures of which are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
The present invention relates to the field of genetic engineering. Particularly, the present application relates to a fusion protein inhibiting angiogenesis or vascular growth.
BACKGROUND OF THE INVENTION
Under normal physiological condition, angiogenesis is a process tightly regulated by several factors and it plays a vital part in repairing and maintaining the normal function of the body. However, rapid growth of blood vessels inside tumor is a common clinical phenomenon. It is demonstrated in many animal models and clinical studies that the inhibition of angiogenesis in tumor can inhibit the growth of tumor and induce the death of tumor cell, so that therapeutic effect is achieved. Therefore, the inhibition of angiogenesis has become an important trend for the development of anti-tumor drugs. Macromolecule anti-angiogenesis drugs, such as Avastin, have been approved by the FDA. There are also drugs under pre-clinical and clinical studies. In addition, new anti-angiogenesis drugs are also widely used in the treatment of angiogenesis-related diseases such as age-related macular degeneration (AMD), diabetic retinopathy, and result in significant therapeutic effect.
Angiogenesis is a complex process regulated by many active biological factors. A key process in angiogenesis is the binding and the activation of endothelial cell surface receptor by various growth factors, which control the activity of endothelial cell via intracellular tyrosine phosphorylation signalling and improve angiogenesis. Among these growth factors, vascular endothelial cell growth factor (VEGF) is the most important factor involved in the control of angiogenesis. VEGF is the most potent and the most specific factor in the induction and improvement of angiogenesis. It is overexpressed in almost all human tumors and thus has become an important molecular target in the development of anti-tumor therapy. VEGF has many receptors on the surface of vascular endothelial cell, including VEGFR-1 (also named as Flt-1) and VEGFR-2 (also named as KDR or Flk-1). VEGFR-1 and VEGFR-2 both comprise an extracelluar portion, composed of 7 immunoglobulin-like domains (D1-D7) capable of binding VEGF, and an intracellular portion comprising tyrosine kinase group. Once the receptor is activated by VEGF, the intracellular tyrosine kinase gene is phosphorylated, which results in a signaling cascade and eventually leads to angiogenesis. Due to the importance of VEGF signaling to angiogenesis, blocking VEGF or VEGF receptor to inhibit angiogenesis has significant anticancer effect and is also important for the treatment of other angiogenesis-related diseases such as retinal vasculopathy.
The stability of fusion protein agents has an important influence on their biotechnical applications, and is also vital for the improvement of drug quality and realization of industrial production. The spacial structure of a protein changes under certain physical and chemical conditions, which in turn leads to the change in physical and chemical properties and loss of biological activity. This is called protein denaturation, which is used to describe the process that a protein changes its intramolecular structure and properties when subjected to the influence of physical or chemical factors. Detection of thermal stability of a protein can be used to determine the denaturation effectively and thus is used as a stability test for protein during the development of therapeutic proteins. The detection of thermal stability is mainly carried out by irradiating a protein sample with UV light so that the protein sample emits fluorescence, and determining the denaturation temperature (Tm) of the protein sample by measuring the wavelength and the light intensity of emitted fluorescence, which reflect the change in its structure. Also, protein aggregation is detected by UV excitation using static light scattering. The light intensity of a protein sample changes significantly between aggregation and non-aggregation states. The aggregation onset temperature (Tagg) of a protein sample can be detected accurately according to the change in light intensity. The stability of a protein is reflected by denaturation temperature (Tm) and aggregation onset temperature (Tagg) of the protein.
The constraint conditions for development of macromolecular drugs include the therapeutic effect, the stability, and the feasibility of industrial production of the therapeutic agent. It is necessary to find a drug inhibiting angiogenesis or vascular growth with determined therapeutic effect and high stability.
DESCRIPTION OF THE INVENTION
An object of the present invention is to provide a fusion protein for the inhibition of angiogenesis or vascular growth with determined therapeutic effect and high stability, wherein the fusion protein inhibits angiogenesis or vascular growth by blocking VEGF signaling.
To achieve the above object, the following technical solutions are provided.
In one aspect, the present invention provides a fusion protein for the inhibition of angiogenesis and vascular growth, composed of a human VEGF receptor fragment and an Fc fragment of human immunoglobulin linked thereto, wherein the amino acid sequence of the VEGF receptor fragment is as shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.
In one embodiment, the Fc fragment of human immunoglobulin of the fusion protein is selected from the group consisting of the following sequences: IgG1 Fc (the amino acid sequence of which is SEQ ID NO: 7), IgG2 Fc (the amino acid sequence of which is SEQ ID NO: 8), IgG3 Fc (the amino acid sequence of which is SEQ ID NO: 9), and IgG4 Fc (the amino acid sequence of which is SEQ ID NO: 10).
In a further embodiment, the fusion protein is,
KH02, the amino acid sequence of which is as shown in SEQ ID NO: 14;
KH03, the amino acid sequence of which is as shown in SEQ ID NO: 16; or
KH04, the amino acid sequence of which is as shown in SEQ ID NO: 18.
In another aspect, the present invention provides a nucleotide sequence encoding the fusion protein. Preferably, the nucleotide sequence is,
kh02 encoding fusion protein KH02, the nucleotide sequence of which is as shown in SEQ ID NO: 13;
kh03 encoding fusion protein KH03, the nucleotide sequence of which is as shown in SEQ ID NO: 15; or
kh04 encoding fusion protein KH04, the nucleotide sequence of which is as shown in SEQ ID NO: 17.
In another aspect, the present invention provides an expression vector or a host cell expressing the fusion protein, wherein the expression vector comprises the above nucleotide sequence of the fusion protein. The expression vector can be a recombinant eukaryotic expression vector, preferably mammalian cell expression vector. The expression vector can also be a recombinant viral expression vector, preferably adeno-associated virus or adenovirus vector. The expression vector is capable of replication and expression in the transformed host cell. Preferably, the host cell is CHO cell or its subline, or 293 cell or its subline.
In another aspect, the present invention provides a method for preparing the fusion protein, comprising introducing the above expression vector into a suitable host cell and expressing the fusion protein.
In another aspect, the present invention provides a pharmaceutical composition comprising the fusion protein of the present invention and pharmaceutically acceptable carrier or excipient, which is conventionally used in the art. Preferably, the dosage form of the formulation of the pharmaceutical composition is injection, freeze-dried injection powder or ophthalmic gel. The formulation can be prepared by methods known in the art.
In another aspect, the present invention provides use of the above fusion protein in the preparation of a medicament for treating diseases caused by angiogenesis or vascular growth, wherein the diseases caused by angiogenesis or vascular growth are preferably tumor or diseases caused by angiogenesis in eye, where the diseases caused by angiogenesis in eye are preferably age-related macular degeneration, diabetic retinopathy, chorioretinopathy, etc.
In another aspect, the present invention provides a method for treating diseases caused by angiogenesis or vascular growth comprising administrating the fusion protein or pharmaceutical composition of the present invention to a patient in need thereof, wherein the diseases caused by angiogenesis or vascular growth are preferably tumor or diseases caused by angiogenesis in eye, where the diseases caused by angiogenesis in eye are preferably age-related macular degeneration, diabetic retinopathy, chorioretinopathy, etc.
The fusion protein of the present invention has high thermal stability, and thereby the formation of protein aggregates during fermentation is significantly reduced and the purity and yield of the protein is significantly improved. Also, the present fusion protein has good biological activities.



DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the tendency for thermal denaturation of the VEGF receptor fusion protein. The curve in the graph reflects the increase of internal fluorescence peak of the protein along with the rise of the temperature. The ordinate BCM (nm) shows internal fluorescence peak, while the abscissa shows the temperature.
FIG. 2 is a graph showing the tendency for thermal aggregation of the VEGF receptor fusion protein. The curve in the graph reflects the change of intensity of static light scattering of the protein along with the rise of the temperature. The ordinate SLS at 266 nm shows intensity of static light scattering, while the abscissa shows the temperature.
FIG. 3 is a graph showing the inhibition of VEGF-induced proliferation of HUVEC cell by the VEGF receptor fusion protein. The ordinate shows the concentration of the fusion protein, while the abscissa is absorbance showing cell viability tested by CCK-8 method.
FIG. 4 is a graph showing the inhibition of VEGF-induced migration of HUVEC cell by the VEGF receptor fusion protein. The abscissa shows the molar ratio of the fusion protein to VEGF, while the ordinate is overall migration % (overall migration %=(Ffusion protein−Fbasal)/(Ftotal−Fbasal), Fbasal is the mean fluorescence of culture medium group, Ftotal is the mean fluorescence of VEGF group, Ffusion protein is the mean fluorescence of fusion protein group with different molar ratios).



EXAMPLES
The present invention will be further described through the following Examples. It is to be understood that these Examples are only included to illustrate the present invention and the present invention is not limited to these Examples. Any modification a person skilled in the art could make in light of the present disclosure falls into the scope covered by the claims.
Description of the Sequences
Fragment 1 of human VEGF receptor: the amino acid sequence is SEQ ID NO: 1, the nucleotide sequence is SEQ ID NO: 2;
Fragment 2 of human VEGF receptor: the amino acid sequence is SEQ ID NO: 3, the nucleotide sequence is SEQ ID NO: 4;
Fragment 3 of human VEGF receptor: the amino acid sequence is SEQ ID NO: 5, the nucleotide sequence is SEQ ID NO: 6;
Fusion protein KH01: the amino acid sequence is SEQ ID NO: 12, the nucleotide sequence is SEQ ID NO: 11;
Fusion protein KH02: the amino acid sequence is SEQ ID NO: 14, the nucleotide sequence is SEQ ID NO: 13;
Fusion protein KH03: the amino acid sequence is SEQ ID NO: 16, the nucleotide sequence is SEQ ID NO: 15;
Fusion protein KH04: the amino acid sequence is SEQ ID NO: 18, the nucleotide sequence is SEQ ID NO: 17;
Fusion protein KH05: the amino acid sequence is SEQ ID NO: 20, the nucleotide sequence is SEQ ID NO: 19.
Example 1: Preparation of Fusion Proteins
Material or Reagents
PCR kit (comprising 5×buffer, dNTP and Phusion enzyme): M0530L, NEB Corp.
Agarose gel electrophoresis: IQ300, GE Corp.
Buffer 4 (lot no.: 0101201):NEB Corp.
Avrll: R0174L, NEB Corp.
BstZ17I: R0594L, NEB Corp.
PCR product purification kit: QIAGEN Corp., CAT: 28106
10×T4 buffer: B0202S, NEB Corp.
T4 DNA ligase: M0202L, NEB Corp.
Top10 E. coli: CB104, TIANGEN Corp.
2YT(KAN) plate culture medium: Shanghai Rui Cong Laboratory Equipment Co., Ltd.
FreedomTM CHO-STM Kit: A13696-01, LIFE TECHNOLOGIES CORPORATION.
Clone Pix FL: Genetix Corp.
HiTrap protein A agarose affinity chromatography column: HiTrap protein A HP, 5×1 ml; GE Corp.
PBS buffer (20 mM phosphate, pH 7.4): SD117-500 ml, Shanghai Biotech Co., Ltd.
ForteBio bio-molecular interaction detector: Octet QKe, Pall Corp.
Analyzer for thermal stability of protein: Optim2, Avacta Corp.
VEGF: R&D Systems Inc.
NHS-LCLC-Biotin: Thermo Corp.
1. Construction of Plasmid Comprising Sequences Encoding Fusion Protein.
1.1 Synthesis of Genes and Primers
Synthetic fragments 1 (SEQ ID NO: 21) and 2 (SEQ ID NO: 22) and primers P1-P10 (sequences are shown in SEQ ID NO: 23-32) were synthesized by Beijing GENEWIZ, Inc. Synthetic fragments 1 and 2 were recombined into plasmid vector pUC19 (Beijing GENEWIZ, Inc.). Synthetic fragment 1 comprises nucleotide sequence encoding human VEGF receptor fragment and signal peptide sequence, synthetic fragment 2 comprises coding sequence for human IgG1 Fc. The coding sequences for the present fusion proteins were constructed by PCR using the below primers and synthetic fragments 1 and 2 as the template for constructing respective fusion proteins.
1.2 Obtaining the Coding Sequences for Fusion Proteins.
Coding sequence for each fusion protein was amplified in two parts, wherein the first part is human VEGF receptor fragment; and the second part is human IgG1 Fc fragment. The respective target fragment for each part was obtained by the specific primers, and then the human VEGF receptor fragment and human IgG1 Fc fragment were linked by overlapping PCR, giving the final full gene sequence. The reaction system for the amplification PCR of both the first and the second parts was (total volume 50 μl): 10 μl of 5×buffer, 2 μl of dNTP, 1 μl of specific forward and reverse primers each, 1 μl of template (the above synthetic fragment 1 or 2), and 0.5 μl of Phusion enzyme (PCR fidelity enzyme), adjusted to 50 μl with double distilled water. The reaction condition was as follows: initial denaturation at 98° C. for 30 s, followed by 10 cycles of 98° C. for 10 s and 68° C. for 2 min, followed by 30 cycles of 98° C. for 10 s, 55° C. for 30 s, and 72° C. for 50 s, and finally 72° C. for 5 min. Particularly, for kh02, the primers for the first part were P1 and P6, the template was synthetic fragment 1; the primers for the second part were P5 and P2, the template was synthetic fragment 2. For kh03, the primers for the first part were P1 and P8, the template was synthetic fragment 1; the primers for the second part were P7 and P2, the template was synthetic fragment 2. For kh04, the primers for the first part were P1 and P10, the template was synthetic fragment 1; the primers for the second part were P9 and P2, the template was synthetic fragment 2. For the coding sequence of protein KH01 (kh01), the primers for the first part were P1 and P4, the template was synthetic fragment 1; the primers for the second part were P3 and P2, the template was synthetic fragment 2. The gene products were tested by agarose gel electrophoresis. A total of 8 fragments, i.e. kh01-Q, kh01-H, kh02-Q, kh02-H, kh03-Q, kh03-H, kh04-Q and kh04-H, were obtained. The reaction system for overlapping PCR was (total volume 50 μl): 10 μl of 5×buffer, 2 μl of dNTP, 1 μl of the above amplified first and second fragment as template respectively (for example, for amplification of the full length of kh01, 1 μl of kh01-Q PCR recovery product and 1 μl of kh01-H PCR recovery product were used), 1 μl of forward and reverse primers (P1, P2) respectively, and 0.5 μl of Phusion enzyme (PCR fidelity enzyme), adjusted to 50 μl with double distilled water. The reaction condition was as follows: initial denaturation at 98° C. for 30 s, followed by 30 cycles of 98° C. for 10 s, 55° C. for 30 s, and 72° C. for 50 s, and finally 72° C. for 5 min. The gene products were tested by agarose gel electrophoresis (IQ300, GE). A total of 4 gene fragments, named as kh01-1 (corresponding to SEQ ID NO: 11, but with additional signal peptide encoding sequence), kh02-1 (corresponding to SEQ ID NO: 13, but with additional signal peptide encoding sequence), kh03-1 (corresponding to SEQ ID NO: 15, but with additional signal peptide encoding sequence), and kh04-1 (corresponding to SEQ ID NO: 17, but with additional signal peptide encoding sequence), were obtained. The amplified fragments were found to have the expected size by electrophoresis.
1.3 Enzyme Digestion of Vectors and Gene Fragments
pCHO1.0 plasmid (from Life Technologies, catalog no.: A13696-01), kh01-1, kh02-1, kh03-1, and kh04-1 were subjected to double enzyme digestion respectively. The system for enzyme digestion was as follows. 40 μl of pCHO1.0 plasmid or kh01-1, kh02-1, kh03-1, or kh04-1 amplified fragment, 10 μl of 10×buffer 4 (NEB), 5 μl of Avrll (R0174L, NEB) and BstZ17I (R0594L, NEB) each, and 45 μl sterile water were added to 1.5 ml EP tube, and the mixture was incubated at 37° C. for 5 h after being mixed evenly. The product was recovered by PCR product purification kit (CAT: 28106, QIAGEN).
1.4 Ligation and Transformation of Recombinant Plasmid
The recovered pCHO1.0 fragment (larger fragment obtained by Avrll and BstZ17I digestion, about 13 kb) and the recovered kh01-1, kh02-1, kh03-1, or kh04-1 fragment (Avrll and BstZ17I digested), obtained from digestion by the same enzymes, were ligated together in the presence of T4 DNA ligase. The reaction system for this reaction was as follows. 2 μl of pCHO1.0 fragment (Avrll and BstZ17I digested), 6 μl of kh01-1, kh02-1, kh03-1, or kh04-1 (Avrll and BstZ17I digested) fragment, 1 μl of 10×T4 buffer (B0202S, NEB), and 1 μl of T4 DNA ligase (M0202L, NEB) were added to 1.5 ml EP tube, the mixture was mixed evenly, and then incubated at room temperature (around 20° C.) for 4 h. The ligation product was transformed to competent Top 10 E. coli cell (CB104, TIANGEN) and plated on 2YT(KAN) plate (Shanghai Ruicong Laboratory Equipment Co., Ltd) for overnight incubation at 37° C. The plates were identified as kh01, kh02, kh03, and kh04.
1.5 Colony PCR Screening of Recombinant Plasmids.
Single recombinant colonies were picked from kh01, kh02, kh03, and kh04 plates and were incubated at 37° C. for 3-5 h. After incubation, these colonies were used as PCR templates for PCR screening. The reaction system (total volume 20 μl) for this PCR amplification was as follows. 10 μL of 2×Taq HS (R013A, TAKATA), 2 μL of bacterial liquid as template, and 1 μL of forward primer and reserve primer (P1 and P2, each has a final concentration 0.3 μmol/L), adjusted to 20 μL with double distilled water. The condition for the reaction was: 94° C. for 3 min, followed by 30 cycles of 94° C. for 60 s, 53° C. for 60 s, and 72° C. for 120 s, and finally 72° C. for 5 min. The results showed that a target band of about 1.6 Kbp was amplified from all colonies, suggesting that these colonies are all positive clones.
1.6 Identification of Recombinant Plasmid by Enzymatic Digestion.
The colonies identified as positive by the colony PCR were inoculated, followed by plasmid extraction and identification by enzymatic digestion. Firstly, plasmids were extracted from recombinant bacteria and then analysed by enzymatic digestion. The system for enzymatic digestion was as follows. 2 μl of Plasmid, 1 μl of 10×buffer 4, 1 μl of Avrll and 1 μl of BstZ17I were added to 1.5 ml EP tube, and sterile water was added to adjust the total volume to 10 μl, and then the mixture was mixed evenly and then reacted at 37° C. for 4 h. Agarose gel electrophoresis confirmed that a band around 1.6 kb was obtained after enzymatic digestion for all colonies, suggesting that the picked clones are all positive clones.
1.7 Identification of Recombinant Plasmid by Sequencing
Colonies identified by the colony PCR and enzymatic digestion as positive were sequenced (Suzhou GENEWIZ biotech Co. Ltd.). The results of the sequencing were as expected. These expression plasmids were stored for further usage. Clones with positive sequencing results were numbered as the following, kh01-1 as 610, kh02-1 as 711, kh03-1 as 812, kh04-1 as 915.
2. Transfection of Plasmid and Screening of Cells
Transfection was conducted using host cell CHO-S in FreedomTM CHO-STM Kit (A13696-01, LIFE TECHNOLOGIES) as suggested by the manufacturer. Four plasmids were transfected in this experiment: 610, 915, 812, and 711. Cells transfected with plasmids were incubated by shake-flask culturing. The culture was performed in CD FortiCHO (from Life Technologies) as the culture medium under 37° C., 8% CO2, and 110 rpm/min for 48 h. Viability and count of the cells were detected by cell counter.
48 hours after the transfection, the two-phase selection scheme was conducted: 10P/100M, 20P/200M (P=10 μg/mL puromysin, M=nM methotrexate (MTX)); 30P/500M, 50P/1000M, with CD-FortiCHO being used as the culture medium. Cells obtained after the first screening were 610, 915, 812 and 711 (i.e., comprising the above respective plasmid). The single clone screening was performed at a seeding density of 500 viable cells/ml, and 48 hour after the transfection, each cell pool was seeded to 8 six-well plates and incubated for 1 week in incubator. The growth of cell clone in each plate was observed under fluorescence microscope. Single clone was picked by Clone Pix FL (Genetix). Protein expression and purity were detected to select clone for scale-up culture.
3. Expression, Purification and Identification of Proteins
Clone cells with high yield were picked for scale-up culture from 96-well plate to 24-well plate, then to 6-well plate and then to 50 ml shake-flask.
Supernatant of cell culture incubated for 4-6 days was collected and centrifuged to remove cell debris. The collected supernatant was filtered by 0.45 μm filter. PH was adjusted to 7.4. Fusion proteins were purified by HiTrap protein A affinity chromatography column (HiTrap protein A HP, 5×1 ml; GE). The column was rinsed by 5×deionized water, and balanced by 5×PBS buffer (20 mM phosphate, pH 7.4) (SD117-500 ml, Shanghai Biotech Co., Ltd). The column was loaded with samples and eluate was collected for detection. The column was washed with ten column volume of PBS buffer (0.02 mol/L phosphate, pH 7.4) to remove non-target protein and target protein was eluted from the column by 0.1M glycine buffer (pH3). The purities of the proteins were all detected to be above 90% by SDS-PAGE (polyacrylamide gel electrophoresis).
Example 2: Analysis of Purity and Yield of the Protein
Purities of proteins in fermentation broth were detected by SEC-HPLC. Also, the expression of each protein obtained in Example 1 was detected by ForteBio bio-molecular interaction detector (Octet QKe, Pall). The results are shown in Table 1. It can be seen that the expression levels and purities of fusion proteins KH02-KH04 are better than the same of KH01 protein. Among these proteins, fusion protein KH02 is the best in terms of purity and expression level. The purity of this protein is still maintained above 80% on day 9 of the incubation without addition of nutriment.







TABLE 1







Comparison of purity of cell culture supernatant of the proteins










Purity (%)
Expression level (mg/L)











Culture supernatant
Day 7
Day 9
Day 7
Day 9














KH01
53.5
40.1
173.54
305.27


KH02
84.3
83.5
381.85
578.15


KH03
55.5
42.8
201.3
300.85


KH04
61.5
47.7
136.55
342.10









Example 3: Detection of Thermal Stability of VEGF Receptor Fusion Protein
(1) Detection of Tendency for Thermal Denaturation of the VEGF Receptor Fusion Protein
The spacial conformation of a protein will be unfolded when the protein is subjected to heat treatment. Hydrophobic amino acid residues (such as tryptophan, tyrosine, and phenylalanine) containing aromatic group will be exposed. The degree of protein unfolding can be reflected by fluorescence intensity (IF) inside the aromatic groups. During protein unfolding, fluorescence spectrum of the internal fluorophores will change. Protein with natural structure (normally folded) has lower internal fluorescence intensity and the peak is at around 330 nm. In contrast, denatured protein has significantly increased internal fluorescence intensity and the peak will be shifted to around 350 nm. Half thermal denaturation temperature Tm can be calculated by analysing the change of internal fluorescence intensity and the shift of peak of the protein, and indirectly reflects the tendency for thermal denaturation.
The tendency for thermal denaturation of VEGF receptor fusion protein was detected by analyzer for thermal stability of protein (Optim2, Avacta). About 15 μl of each sample to be detected (1 mg/ml PBS, pH7.2, purity above 90%) was added to Optim2 reaction tube. The range for temperature scanning was set to 25° C.-95° C. Samples were incubated at each temperature point for 60 s. The data were processed by Optim2 analysis software. The results are shown in FIG. 1 and Table 2. It is shown that among the recombinant proteins expressed in Example 1, KH02 has the highest denaturation temperature, suggesting the lowest tendency for thermal denaturation. That is, KH02 has the highest thermal stability.







TABLE 2







Parameters for thermal denaturation of the VEGF receptor fusion protein













Denaturation
Denaturation
Denaturation



Samples
temperature 1
temperature 2
temperature 3






KH01
43.9° C.
61.2° C.
71.2° C.



KH02
48.0° C.
63.7° C.
77.6° C.



KH03
/
63.4° C.
76.7° C.



KH04
/
63.3° C.
77.1° C.









(2) Analysis of Tendency for Thermal Aggregation of the VEGF Receptor Fusion Protein
Light scattering will occur when protein is exposed to UV light. In a certain range, intensity of static light scattering is linearly related to the size of the protein (10-600 KD). Detecting the intensity of light scattering SCS (Static Light Scattering) can show the change in protein size. Tendency for protein aggregation can be indirectly reflected by calculating aggregation onset temperature (Tagg) of the protein.
Tendency for thermal aggregation of the VEGF receptor fusion protein was detected by analyzer for thermal stability of protein (Optim2, Avacta). About 15 μl of each sample to be detected (1 mg/ml PBS, pH7.2, purity above 90%) was added to Optim2 reaction tube. The range for temperature scanning was set to 25° C.-95° C. Samples were incubated at each temperature point for 60 s. The data were processed by Optim2 analysis software. The results are shown in FIG. 2 and Table 3. It is shown that the intensity of static light scattering increases along with the increase of temperature, and among the recombinant proteins, KH02 has the highest aggregation onset temperature (Tagg), suggesting the lowest possibility for thermal aggregation.







TABLE 3







Tendency for thermal aggregation of the VEGF receptor fusion protein










Sample
Tagg






KH01
65.6° C.



KH02
71.8° C.



KH03
69.8° C.



KH04
68.8° C.









Example 4: Detection of Biological Activities of VEGF Receptor Fusion Protein
(1) Proliferation of HUVEC cell
Human umbilical venous endothelial cells (HUVEC, ScienCell) that grew well were seeded to 96-well plate at 3×103 cell/well and 100 μl/well at 37° C., 5% CO2 for 20 hours. ECM medium (Endothelial Cell Medium, catalog no. 1001, Sciencell) containing 2% fetal bovine serum was used to prepare VEGF receptor fusion protein with different molar concentrations (0.0023, 0.007, 0.023, 0.065, 0.19, 0.57, 1.7, 5, 15, 45, 135 nM), which was then mixed evenly with 40 ng/ml VEGF (R&D SYSTEMS), incubated for 2 h. 100 μl of the mixture was then added to HUVEC cells in 96-well plate per cell in triplet. The plate was continuously incubated with 5% CO2 for 96 hours. At the end of incubation, the CCK8 (Dojindo) was added. The inhibitory effect of VEGF receptor fusion protein was shown in EC50. The results are shown in FIG. 3 and Table 4. The present VEGF receptor fusion proteins could all inhibit the VEGF-stimulated proliferation of HUVEC cells effectively, suggesting the present VEGF receptor fusion proteins have good biological activity in terms of the inhibition of VEGF.







TABLE 4







VEGF receptor fusion proteins inhibit the VEGF-stimulated proliferation


of HUVEC cells










Sample
EC50 (nM)













KH01
1.93



KH02
1.26



KH03
2.11



KH04
1.43









(2) Migration of HUVEC Cells
The effect of the present fusion protein on the migration of HUVEC cells was tested by using modified Boyden chamber (FluoroBlokTM Biocoat angiogenesis system: Endothelial cell migration, BD). HUVEC cells that grew well were seeded to upper compartment of the Boyden chamber at 3×105 cell/ml and 75 μl/well. ECM basic medium (catalog no. 1001, Sciencell) containing 2% fetal bovine serum was used to prepare VEGF receptor fusion protein with different molar concentrations (13333 nM, 4444 nM, 1481 nM, 494 nM, 164.5 nM, 54.8 nM, 18.3 nM, 6.1 nM), which was then mixed evenly with 500 μM VEGF (R&D SYSTEMS), incubated for 2 h, and then added to the lower compartment-of the Boyden chamber at 225 μl/well. The whole chamber was incubated at 37° C., 5% CO2 for 20-24 h. The culture medium in the lower compartment was removed. Fluorescent dye Calcein AM (Anaspec) with a final concentration of 5 μg/ml formulated by using HBSS buffer (Hanks Balanced Salt Solution) was added. The chamber was incubated at 37° C. with 5% CO2 for 90 minutes in the dark, and then the fluorescence value was detected at excitation wavelength of 494 nm and detection wavelength of 517 nm on a multi-mode reader. Relative migration of the cells was calculated. As shown in FIG. 4, VEGF receptor fusion proteins of the present invention are substantially the same in the inhibition of VEGF induced migration of HUVEC cells. This confirms again that fusion protein KH02 of the present invention has better biological activity in terms of the inhibition of VEGF.
Example 5: Test of Binding Affinity Between VEGF Receptor Fusion Protein and VEGF
The binding affinity between human VEGF receptor fusion protein and human VEGF was detected on ForteBio bio-molecular interaction detector (Octet QKe, Pall) by Biolayer-Interferometry (BLI). Human VEGF (catalog no. 293-VE-010, R&D SYSTEMS) and NHS-LCLC-biotin (catalog no. 21338, Thermo) were mixed evenly at 1:3 molar ratio, and placed at room temperature for 1 h, then the remaining NHS-LCLC-biotin was removed, giving the final labelled product biotin-hVEGF at 50 μg/ml. 50 μg/ml biotin-hVEGF was loaded on streptavidin sensor. Samples to be tested were formulated to different concentrations (600 nM, 200 nM, 66.7 nm, 22.2 nM, 7.4 nm, 2.46 nM, 0.82 nM, respectively) by sample diluting buffer (PBS, 0.1% BSA, 0.02% Tween-20, 0.003% NaN3). The sample diluting buffer was served as blank control. Parameters for binding kinetics between hVEGF and receptor fusion protein were detected under kinetics analysis mode. The results are shown in Table 5. It is shown that the VEGF receptor fusion proteins expressed in Example 1 all bind significantly with human VEGF. Among these proteins, the KD value of KH02 is about 0.33 nM. Especially, the dynamic dissociation rate of KH02 complex with VEGF is lower than other fusion proteins, suggesting the highest binding affinity.







TABLE 5







Parameters for binding affinity between VEGF receptor fusion protein


and human VEGF











KD (nM)
kon (1/Ms)
kdis (1/s)





KH01
0.51 ± 0.04
6.91 ± 0.09 × 104
3.53 ± 0.06 × 10−5


KH02
0.33 ± 0.02
5.16 ± 0.05 × 104
1.70 ± 0.05 × 10−5


KH03
1.17 ± 0.05
8.67 ± 0.02 × 104
1.01 ± 0.04 × 10−4


KH04
0.35 ± 0.02
2.06 ± 0.05 × 105
7.27 ± 0.02 × 10−5





Notes:


Kon: association constant,


Kids: dissociation constant






Example 6: Affinity Experiment of Fusion Proteins
1.1 Reagents







TABLE 6







list of the reagents used










Name
Manufacturer
Catalog no.
Lot No.





rhVEGF165
R&D Systems
293-VE
II4113062



Inc.




Human IgG-Fc
BETHYL Corp.
A80-104P
A80-104P-78


antibody HRP





TMB substrate
R&D Systems
DY999
308030


solution
Inc.




BSA
BOVOGEN
BSA S1.0
269



Corp.




Tween 20
Riedel-deHaën
63158
41620



Corp.




20xPBS buffer
Biotech Co.
SD8117
13042099Z
















TABLE 7







list of instruments











Name
Brand
Device no.






Microplate reader
Molecular Devices Corp.
JC084



Mini vortex mixer
Shanghai LUXI Corp.
GY227



Electronic balance
Sartorius Corp.
JC082









1.2 Protocol
1) Reagent Preparation
i. 10×PBS buffer: 80.1 g of NaCl, 2.0 g of KCl, 2.0 g of KH2PO4, and 29.0 g of Na2HPO4.12H2O were dissolved in pure water and the final volume was set to 1000 ml;
ii. 1×PBS buffer: 100 ml 10×PBS buffer was dissolved in 850 ml pure water, pH was adjusted to pH 7.2-7.4, and the final volume was set to 1000 ml;
iii. Carbonate buffer: 1.59 g of Na2CO3 and 2.93 g of NaHCO3 were dissolved in 1000 ml ultrapure water, pH was 9.6-9.8, and the solution was stored at room temperature, and filtered with 0.22 μm filter before use.
iv. BSA (bovine serum albumin): stored at 4° C.;
v. Rinsing buffer: 1×PBS containing 0.05% (v/v) polysorbate 20;
vi. Blocking solution and diluent: 1×PBS containing 1% (w/v) BSA;
vii. Stop solution (2N H2SO4): 27.8 ml concentrated sulfuric acid was added slowly to 472.2 ml pure water. Concentrated sulfuric acid is a strong corrosive liquid and should be added with stirring by glass rod and handled with extra care;
viii. rhVEGF165 (R&D SYSTEMS, 293-VE, 50 μg/vial) stock solution: 3 ml 1×PBS was filtered by 0.22 μm filter. 800 μl filtered PBS was added to a previously unopened vial of rhVEGF165. When the visible solid material in the vial was dissolved, 200 μl additional filtered PBS was added. The vial was placed at room temperature for 10 min to fully dissolve rhVEGF165. The concentration of the dissolved rhVEGF165 stock solution was 50 μ/ml. The stock solution was divided into aliquots of 25 μl/vial. The stock solution can be stored at −20° C. for 6 months.
ix. Human IgG-Fc antibody HRP detection antibody (BETHYL A80-104P): 1 mg/ml, stored at 4° C.
2) Coating the Plate
20 μl rhVEGF165 stock solution was added to 7980 μl carbonate buffer, the mixture was mixed evenly and named as coating solution A and its concentration was 125 ng/ml. 2500 μl coating solution A was added to 2500 μl carbonate buffer, the mixture was mixed evenly and named as coating solution B. A microplate was coated in columns 1-6 with coating solution A, and in columns 7-12 with coating solution B, wherein the volume used in the coating was 100 μl/well. The plate was sealed by sealing gel and incubated overnight at room temperature.
3) Rinsing the Plate
The plate was washed with 250 μl rinsing buffer per well and soaked for 120 s, which was repeated for three times. The remaining drops were removed by patting the plate on a paper towel until there was no obvious water mark on the paper towel.
4) Blocking the Plate
Blocking solution was added at 300 μl/well by an 8-channel pipette. Then the plate was sealed by sealing gel and incubated at 37° C. for 2 hours.
5) Preparation of Sample
Samples (fusion proteins KH02 and KH05) were diluted to 1600 ng/ml according to initial protein concentration. The volume of the 1600 ng/ml sample should be at least above 800 μl. A 4× serial dilution was conducted by adding 600 μl diluting solution to 200 μl sample, and such a diluting operation was repeated in series to obtain 8 different concentration gradients (including 1600 ng/ml).
6) Adding the Sample
The plate was rinsed as described above. 100 μl/well samples were added to microplate successively and the plate was sealed by sealing gel. The loading was proceeded from high concentration to low concentration in duplicate. After the loading, the plate was incubated at 37° C. for 1 h.
7) Adding Test Antibody
The plate was rinsed as described above. Human IgG-Fc antibody HRP 0.5 μl was diluted by 10 ml blocking solution and the mixture was mixed evenly. 100 μl/well diluted test antibody was added to wells, and the plate was incubated at 37° C. for 1 h. 8) Color Development
The plate was rinsed as described above. 100 μl/well TMB substrate solution was added. The plate was incubated under dark and at room temperature for 5 min.
9) Stopping and Obtaining Readout
50 μl/well stop solution was added and the reaction was terminated. The microplate was placed under microplate reader to get a readout under 450 nm.
1.3 Results of Affinity Test







TABLE 8







Results of affinity test of samples











Sample
treatment
Kd (unit: pM)














KH05
Day 0
27.253




5 days at high
65.471




temperature





10 days at high
174.983




temperature




KH02
Day 0
22.491




5 days at high
46.420




temperature





10 days at high
70.504




temperature









From the results in Table 8, it can be seen that the affinity of KH02 after a 10-day high temperature treatment is still within acceptable range (22-84 pM). However, affinity of KH05 after a 10-day high temperature treatment exceeds far beyond the acceptable range. Thus, the stability of KH02 in terms of activity is better than KH05.
Example 7: Purity Test at High Temperature
1.1 Reagents and Devices







TABLE 9







list of reagents used










Name
Catalog No.
Specification
Manufacturer













Na2HPO4•12H2O
20130527
500 g/vial
Guangdong





Guanhua Corp.


NaCl
201100905
500 g/vial
Guangdong





Guanhua Corp.


Arg-HCl
20110318
N/A
Shanghai Yuanju





Biotech Corp.


Concentrated HCl
20121101
N/A
Chengdu Kelong





Corp.


Chromatographic
S0534
TSK G3000 SWxl, 
TOSOH Corp.


column

5 μm, 7.8 × 300 mm



TSK guard
R1479
40 * 60
TOSOH Corp.


column
















TABLE 10







list of main devices used










Name
Device No.
Manufacturer
Pattern No.





High performance
JC073
Agilent Technologies
1200


liquid spectrometry

Inc.



Mini vortex mixer
GY265
Shanghai LUXI Corp.
WH-3


Electronic balance
JC080
Sartorius Corp.
BS224S


pH meter
JC077
METTLER Corp.
TOLEDO





S40K









1.2 Protocol
1) Reagent Preparation
i. PBS mobile phase: 7.16 g Na2HPO4.12H2O, 8.77 g NaCl and 42.2 g Arg were dissolved in 800 ml ultrapure water, the pH was adjusted to pH7.2 by HCl, the final volume was set to 1000 ml and the resulting solution was filtered by Φ0.22 μm filter.
ii. Guarding solution for chromatographic column (0.05% NaN3): NaN30.5 g was dissolved in 1000 ml ultrapure water and the resulting solution was filtered by Φ0.22 μm filter.
iii. Ultrapure water: it was filtered by Φ0.22 μm filter.
2) Preparation of Sample
Since the concentration of each sample (KH02, KH05) was 1 mg/ml, these samples can be loaded directly.
3) Conditions for Chromatographic Analysis
Mobile phase: PBS mobile phase;
Chromatographic column: TSK G3000 SWXL (5 μm, 7.8*300 mm);
Temperature of the column: 25° C.; flow rate: 0.5 ml/min; detection wavelength: 280 nm; loading volume: 50 μl.
1.3 Results







TABLE 11







Results of SEC-HPLC purity test of the samples











Sample
Treatment
Detected purity (%)














KH05
Day 0
99.19




5 days at high
54.06




temperature





10 days at high
25.20




temperature




KH02
Day 0
96.75




5 days at high
94.29




temperature





10 days at high
65.15




temperature









As can be seen from Table 11, the purity of KH05 dropped to 54% after a 5-day high temperature treatment. The purity of KH05 was only 25% after a 10-day high temperature treatment. The decrease of purity of KH02 was inapparent after a 5-day high temperature treatment. The purity of KH02 sample was up to 65% after a 10-day high temperature treatment.

## Claims

1. A fusion protein for the inhibition of angiogenesis or vascular growth, the fusion protein comprising a human VEGF receptor fragment and an Fc fragment of human immunoglobulin linked thereto, wherein the amino acid sequence of the VEGF receptor fragment is SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, and wherein the fusion protein is
KH02, the amino acid sequence of which is as shown in SEQ ID NO: 14;
KH03, the amino acid sequence of which is as shown in SEQ ID NO: 16; or
KH04, the amino acid sequence of which is as shown in SEQ ID NO: 18.

2. A nucleotide sequence encoding the fusion protein according to claim 1.

3. The nucleotide sequence of claim 2, wherein the nucleotide sequence is the sequence shown in SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17.

4. An expression vector comprising a nucleotide sequence encoding the fusion protein according to claim 1.

5. The expression vector according to claim 4, wherein, the expression vector is a eukaryotic expression vector or a viral expression vector.

6. The expression vector according to claim 5, wherein, the expression vector is a mammalian cell expression vector or adeno-associated virus vector or adenovirus vector.

7. A pharmaceutical composition comprising the fusion protein according to claim 1 and pharmaceutically acceptable carrier or excipient.

8. A method for treating a disease caused by angiogenesis or vascular growth, the method comprising administering the pharmaceutical composition of claim 7 to a patient in need thereof to inhibit angiogenesis or vascular growth.

9. The method for treating disease caused by angiogenesis or vascular growth according to claim 8, wherein the disease caused by angiogenesis or vascular growth is selected from the group consisting of tumor, age-related macular degeneration, diabetic retinopathy, and chorioretinopathy.

10. The pharmaceutical composition according to claim 7, in the form of a formulation for injection, a freeze-dried injection powder, or an ophthalmic gel.

11. A host cell comprising a nucleotide sequence encoding the fusion protein according to claim 1.

12. The host cell according to claim 11, wherein, the host cell is a CHO cell or a 293 cell.