US20100215655A1

US20100215655A1 - Angiogenesis-inhibiting chimeric proteins and the use

Info

Publication number: US20100215655A1
Application number: US12/773,315
Authority: US
Inventors: Jianmin Fang; Zheng Liu; DeChao Yu
Original assignee: Chengdu Kanghong Biotechnologies Co Ltd
Current assignee: Chengdu Kanghong Biotechnologies Co Ltd
Priority date: 2004-06-08
Filing date: 2010-05-04
Publication date: 2010-08-26
Also published as: PT1767546E; CA2569108A1; EP1767546B1; JP2008503243A; RU2006146995A; EP1767546A4; RU2355414C2; CA2569108C; KR100897379B1; US20080206238A1; ES2381014T3; KR20070029204A; BRPI0512286B1; BRPI0512286A; EP1767546A1; US7750138B2; BRPI0512286B8; DK1767546T3; PL1767546T3; WO2005121176A1

Abstract

The present invention is directed to DNA sequence encoding angionenesis-inhibiting recombinant chimeric protein, the chimeric protein per se, the pharmaceutical use of the chimeric protein, and to the pharmaceutical composition containing the recombinant protein and the formulation thereof.

Description

This application is a divisional application of U.S. patent application Ser. No. 11/628,735 filed Dec. 7, 2006, which was filed under 35 U.S.C. 371 as a national stage of PCT/CN2005/000802, with an international filing date of Jun. 8, 2005, and claims priority to patent application CN 200410044965.7 filed Jun. 8, 2004, the content of each of which is hereby incorporated by reference in its entirety.

FIELD OF INVENTION

The present invention relates to gene engineering technology, more specifically to DNA sequences encoding angiogenesis-inhibiting recombinant chimeric proteins, the encoded chimeric proteins herein, therapeutic applications thereof, medical composition and formulation containing the chimeric proteins.

BACKGROUND OF INVENTION

Angiogenesis is a process of growing new blood vessels from existing blood vessels. Most adult vascular system is quiescence, angiogenesis only occurs in some physiological and pathological mechanisms, such as tumor, diabetic retinopathies, arthritis, anemia organs, endometrial hyperplasia, etc. Angiogenesis plays key roles in rapid growth of tumor cells during tumor development (Hanahan and Folkman: Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis, Cell, 1996, 86:353-364). Studies of animal cancer models and human clinical trials have already proved that inhibition of tumor angiogenesis could effectively inhibit tumor growth and development, therefore prolong patient's life. Angiogenesis is mediated and regulated by many biological factors. Main cells mediating angiogenesis are vascular endothelial cells that form the inside wall of blood vessels. Various growth factors can bind to relevant receptors on the surface of vascular endothelial cells, regulate cellular processes via intracellular signal transduction, and therefore mediate angiogenesis.
Among various growth factors, VEGF (vascular endothelial cell growth factor) is the most important angiogenesis factor (Ferrara: VEGF and the quest for tumor angiogenesis factor, Nat. Rev. Cancer, 2002, 10: 795-803; Ferrara: Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications, Semin. Oncol., 2002, 29 (6 sumppl): 10-14). VEGF could be secreted by many types of cells, but often over-expressed in tumor cells. VEGF functions by binding to appropriate receptors. There are mainly two kinds of VEGF receptors: FLT-1 (fms-like tyrosine kinase) and KDR. In terms of molecular structures, these two receptors both consist of three different functional regions: the first region is the extracellular region, consisting of seven immunoglobulin-like (Ig-like) domains (d1-d7), which has specific affinity to VEGF, and is the key region for binding VEGF; the second region is the trans-membrane region containing hydrophobic amino acid residues; the third region is the intracellular domain that contains tyrosine kinase functioning group, which gets phosphorylated after the receptor is activated by VEGF, triggering the intracellular signal transduction, leading to functional effects of endothelial cells and angiogenesis.
FLT-1 and KDR are mainly distributed in vascular endothelial cells. Thus, VEGF's mediating activity to vascular endothelial cells is highly specific. VEGF promotes endothelial cell differentiations, guides endothelial cell migrations, inhibits apoptosis, induces vascular morphological changes, and is a highly effective pro-angiogenesis factor.
The expression level of VEGF in tumor tissues is higher than that in the normal tissues. In addition, rapid growth of tumor cells often leads to hypoxia inside the tumor, and hypoxia further induces expression of VEGF. Thus, VEGF is the key factor promoting tumor angiogenesis. Many animal studies have shown that inhibiting binding of VEGF to its receptors could effectively inhibit tumor angiogenesis, and therefore inhibit tumor growth. In other angiogenesis-related diseases, such as diabetic retinopathies and arthritis, etc, VEGF is also closely involved in the development of these diseases (Ferrara: Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications. Semin. Oncol. 2002, 29 (6 sumppl): 10-14).
Because of the critical roles of VEGF in cancers and other diseases, proteins or chemicals that specifically inhibit VEGF have therapeutic potentials. For example, studies have shown that neutralizing antibody against VEGF could effectively inhibit tumor growth (Jain: Tumor angiogenesis and accessibility: role of vascular endothelial growth factor, Semin. Oncol., 2002, 29 (6 suppl): 3-9). Therefore, developing novel effective VEGF inhibitors is important in clinical research. Since FLT-1 and KDR are natural binders of VEGF, there were studies that investigated the anti-angiogenesis roles of the soluble FLT-1 (the extracellular domain of FLT-1) and the soluble KDR (the extracellular domain of KDR) (Yoko Hasumi: Soluble FLT-1 Expression Suppresses Carcinomatous Ascites in Nude Mice Bearing Ovarian Cancer. Cancer Research 62, 2002: 2019-2023). The soluble FLT-1 could effectively inhibit growth of vascular endothelial cells in vitro, but it has a short serum half-life and can not reach effective serum concentration. Similarly, the soluble KDR was also able to inhibit growth of vascular endothelial cells in vitro, but its anti-tumor activity in animal models was not satisfactory (Yoko Hasumi: Soluble FLT-1 Expression Suppresses Carcinomatous Ascites in Nude Mice Bearing Ovarian Cancer. Cancer Research 62, 2002: 2019-2023).
To overcome the shortcomings of the prior art, the present invention provides novel chimeric proteins containing different fragments of FLT-1 and KDR to effectively block the biological activity of VEGF and inhibit angiogenesis.

SUMMARY OF INVENTION

The first aspect of the invention is to provide novel recombinant chimeric proteins that block the biological activity of VEGF and inhibit angiogenesis.
The second aspect of the invention is to provide DNA sequences encoding the above-mentioned chimeric proteins.
The third aspect of the invention is to provide vectors containing the coding DNA sequences of the chimeric proteins and recombinant hosts thereof.
The fourth aspect of the invention is to provide the use of the chimeric proteins in preparing medicaments that block the VEGF activity and inhibit angiogenesis, and medical composition containing the chimeric proteins and appropriate medical carriers and dosage form thereof, as well as therapeutic applications of the medical composition.
Key points of the invention are to design and construct a series of chimeric proteins with different FLT-1 or KDR fragments, which preferably contain human immunoglobulin Fc (construction method is shown in FIG. 1), and then to select the chimeric protein with high affinity to VEGF using assays including the VEGF binding assay, and finally to obtain the proper VEGF inhibitor. Construction of the chimeric protein is based on the conventional molecular cloning technologies. Detailed experimental methodology could be found in laboratory manuals such as Molecular Cloning, the 2^ndor the 3^rdedition (Joseph Sambrook).
According to the present invention, the chimeric proteins made via recombinant DNA technology contain different fragments of the VEGF receptors FLT-1 and KDR, wherein the chimeric proteins are selected from the following groups:

- a. Consisting of the 1^stIg-like domain of KDR, the 2^ndIg-like domain of FLT-1, and the 3^rdIg-like domain of KDR, designated as KDRd1-FLTd2-KDRd3;
- b. Consisting of the 2^ndIg-like domain of FLT-1, and the 3^rdand the 4^thIg-like domains of KDR, designated as FLTd2-KDRd3,4;
- c. Consisting of the 2^ndIg-like domain of FLT-1, the 3^rdIg-like domain of KDR, and the 4^thIg-like domain of FLT-1, designated as FLTd2-KDRd3-FLTd4;
- d. Consisting of the 2^ndIg-like domain of FLT-1, and the 3^rd, the 4^thand the 5^thIg-like domains of KDR, designated as FLTd2-KDRd3,4,5;
- e. Consisting of the 2^ndIg-like domain of FLT-1, the 3^rdIg-like domain of KDR, and the 4^thand the 5^thIg-like domains of FLT-1, designated as FLTd2-KDRd3-FLTd4,5.

The amino acid sequence of FLTd2 is shown as SEQ ID NO.1. The amino acid sequence of FLTd4 is shown as SEQ ID NO.2. The amino acid sequence of KDRd1 is shown as SEQ ID NO.3. The amino acid sequence of KDRd3 is shown as SEQ ID NO.4. The amino acid sequence of KDRd4 is shown as SEQ ID NO.5.
As used herein, FLT refers to the FLT-1 sequence, KDR refers to the KDR sequence; di refers to the i^thIg-like domain in FLT-1 or KDR.
Preferably, the present invention provides a class of chimeric proteins that contain human immunoglobulin Fc, and are preferably selected from the following groups:
FP2′ designated as KDRd1-FLTd2-KDRd3-Fc;
FP3′ designated as FLTd2-KDRd3,4-Fc;
FP4′ designated as FLTd2-KDRd3-FLTd4-Fc;
FP5′ designated as FLTd2-KDRd3,4,5-Fc;
FP6′ designated as FLTd2-KDRd3-FLTd4,5-Fc.
As used herein, Fc refers to the human immunoglobulin Fc fragment derived from human immunoglobulin FC such as IgG, IgM, and IgA, or subclasses IgG1, IgG2, IgG3, and IgG4. The Fc region can be the full length Fc sequence or a fragment of the Fc sequence from CH2, CH3, or the hinge region.
As shown in FIG. 1, the chimeric protein known in the prior art (designated as FP1′) consists of the 2^ndIg-like domain of FLT-1 (FLTd2), the 3^rdIg-like domain of KDR (KDRd3), and the human immunoglobulin Fc. The chimeric protein FP2′ provided in the invention has added amino acid sequence of the 1^stIg-like domain of KDR (KDRd1) into FP1′, which increases binding sites for VEGF and enhances affinity to VEGF. The chimeric proteins FP3′ and FP4′ have added sequences of the 4^thIg-like domain of KDR (KDRd4) or the 4^thIg-like domain of FLT-1 (FLTd4) based on FP1′, respectively. FP5′ and FP6′ have added the 4^thand the 5^thIg-like domains of KDR (KDRd4, 5) and the 4^thand the 5^thIg-like domains of FLT-1 (FLT-1d4, 5) based on FP1′, respectively. These added sequences could help dimerization of the chimeric proteins, folding 3-dimentional structures favorable for VEGF binding, and enhancing affinity to VEGF.
More preferably, the present invention provides a chimeric protein FP3′ with amino acid sequence shown as SEQ ID NO.7.
The chimeric proteins described in the invention can be obtained through conventional recombinant DNA technologies. At first, recombinant DNA coding sequences of the above mentioned chimeric proteins could be obtained, wherein the coding DNA sequences of FLT-1 and KDR are available in GenBank, NCBI (National Center for Biotechnology Information). Secondly, the DNA coding sequences of the above-mentioned chimeric proteins are cloned into vectors after PCR synthesis. The vectors herein could be commonly used plasmids, viruses, or DNA fragments in molecular biology. Secretory signal sequence is inserted into the terminal of the DNA sequence of the aforesaid chimeric peptides to ensure secretion out of cells. The vector sequence includes a promoter region that enables gene transcription, starting and stopping signals for protein translation, and a polyA sequence. The vector contains an antibiotics resistant gene for propagation in bacteria. In addition, the vector contains a eukaryotic cell selection gene for selection of stable transfected cell lines.
Because there is no absolute boundary of the amino acid sequences of all Ig-like domains in FLT-1 and KDR, the sequence length of these domains could have variations. Thus the sequences of the chimeric proteins described in the invention could have similar variations. It should be appreciated that all of these sequence variants are not to be considered as beyond the scope of the invention.
After plasmids construction of the above-mentioned chimeric proteins, the plasmids could be used to transfect host cells to express the chimeric proteins. There are many expression systems for these chimeric proteins, including (but not limited to) mammalian cells, bacteria, yeast, and insect cells. Among them, mammalian and insect cells are eukaryotic cells, whereas bacteria and yeast cells are prokaryotic cells. Proteins expressed from mammalian cells are glycosylated. Since the chimeric proteins of the invention contain glycosylation sites, mammalian cells are the best cells to express them. There are many mammalian cell types suitable for large scale protein productions, such as 293 cells, CHO cells, SP20 cells, NS=b 0 =l cells, COS cells, BHK cells, PerC6 cells, and etc. Many other types of cells could also be used to express and produce these proteins, and they are all within the scope of the invention. Plasmids encoding the above-mentioned chimeric proteins could be transfected into the cells. Methods of transfection include, but not limited to, electroporation, liposome-mediated transfection, Calcium precipitation, and etc.
Expression systems other than mammalian cells could also be used to express these chimeric proteins, such as bacteria, yeast, insect cells, and etc. They should all be considered as within the scope of the invention. These expression systems have a higher protein production yield comparing with that of mammalian cells, however, they produce proteins with no glycosylation or with carbohydrate chains glycosylated different from that of mammalian cells.
After expression of the chimeric proteins, the chimeric proteins concentrations in the cell culture media could be measured by ELISA or other assays. Since the chimeric proteins contain the immunoglobulin Fc region, they could be purified using Protein A affinity chromatography.
After various chimeric proteins were obtained from culture media of the recombinant host cells, they were assayed in VEGF binding experiments to compare their affinities to VEGF. Their VEGF inhibition activities were further assayed in a VEGF-induced human vascular endothelial cell proliferation experiment. The experimental results have shown that all chimeric proteins constructed according to the present invention can bind to VEGF with high affinities (FIG. 2), comparing with the FP1′ of the prior art. In addition, they could effectively block the VEGF activation of the vascular endothelial cells and inhibit growth of the endothelial cells. Further experiments have shown that FP3′ has the best blocking activity on VEGF and is the most effective VEGF-blocking chimeric proteins of the invention.
Thus, the chimeric proteins constructed in the invention have supreme blocking activities on VEGF, and all have biological activities of anti-angiogenesis, therefore can be used to treat angiogenesis or VEGF related diseases, including but not limited to various tumor, diabetic retinopathies, arthritis, anemia, endometrial hyperplasia, etc.
In order to further substantiate the anti-angiogenesis effect of the chimeric proteins in vivo, some animal model experiments have also been made. The results of these experiments have shown that in B16F10 melanoma BALB/C nude mouse model and human PC-3 prostate tumor xenograft mouse model, the chimeric proteins of the invention have been much better than FP1′ of the prior art, and effectively inhibited tumor growth and prolonged animal life. Thus, the chimeric proteins of the invention are of high anti-cancer activities.
The present invention also provides medical compositions comprising the above-described chimeric proteins and appropriate medical carriers. Said compositions can be formulated into any dosage forms according to conventional formulation methodologies, preferably into a dosage form for injection, and more preferably into a lyophilized format.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structures of the five chimeric proteins according to a preferred embodiment of the invention and FP1′ of the prior art. They are constructed with different fragments from FLT-1, KDR and the immunoglobulin Fc region using genic engineering technologies.

FIG. 2 exhibits the results of VEGF binding of the five chimeric proteins in a preferred embodiment of the invention, in comparison with that of FP1′ of the prior art, wherein OD readings refer to the binding signals of chimeric proteins to VEGF. The results have shown that all the five chimeric proteins bound to VEGF with much higher affinities than FP1′, among them, FP3′ has the highest affinity.

FIG. 3 shows that the chimeric proteins of the invention could effectively inhibit human vascular endothelial cell growth in vitro, in comparison with FP1′.

FIG. 4 shows that the chimeric protein FP3 could effectively inhibit B16F10 melanoma tumor growth in mice.

FIG. 5 shows that the chimeric protein FP3′ could effectively inhibit human PC-3 prostate tumor growth in mice.

FIG. 6 compares anti-tumor growth activities of the chimeric protein FP3′ of the invention with that of FP1′ of the prior art in mice.

SPECIFIC EMBODIMENTS

The following examples provide detailed description of construction, experimental methodology and application of the chimeric proteins described in the invention. But these examples should not be construed to limit the protection scope of the invention.

Embodiment 1

Cloning of the DNA Sequences Encoding the Chimeric Proteins and Construction of the Recombinant Vectors

Other than the immunoglobulin Fc coding DNA sequence, coding DNA sequences of the various chimeric proteins of the invention come from cDNAs of FLT-1 and KDR. Since FLT-1 and KDR are mainly expressed in vascular endothelial cells, the total RNA were extracted from human umbilical vein endothelial cells (HUVEC) using a RNA purification kit (QIAGEN); then cDNAs were synthesized from the RNA using AMV Reverse Transcriptese (Promega); then various FLT-1 and KDR fragments were PCR amplified with different primers; finally the sequences from FLT-1, KDR, and human immunoglobulin Fc (IgG1 Fc) were fused together by PCR to construct recombinant DNA sequences encoding various chimeric proteins. Structures of all six chimeric proteins (including FP1′ of the prior art) are shown in FIG. 1.

Example 1

Construction of FP3′ Coding Sequence and Recombinant Vector

HUVEC cells (Clonetics) were cultured with EGM-2 media (Clonetics) in T-175 flasks. 1×10⁷cells were collected and subjected to the total RNA extraction using the RNA purification kit from Qiagen, and then cDNA was synthesized using the Invitrogen cDNA kit. The cDNA product was stored at −80° C. until usage.
PCR utilizing specific primers were used to amplify various FLT-1 and KDR domains from the HUVEC cDNA. Likewise, PCR utilizing specific primers from Lymph Nodes cDNA (BD Clontech) were used to amplify human IgG1 Fc was PCR.
Primers:

for FLT-1 d2 forward:
5′-cctttcgtagagatgtacagtga-3′;	(SEQ ID NO: 8)

for FLT-1 d2 reverse:
5′-tatgattgtattggtttgtccat-3′;	(SEQ ID NO: 9)

for KDR d3-4 forward:
5′-gatgtggttctgagtccgtctca-3′;	(SEQ ID NO: 10)

for KDR d3-4 reverse:
5′-cggtgggacatacacaaccaga-3′;	(SEQ ID NO: 11)

for human IgG1 Fc forward:
5′-gacaaaactcacacatgcccact-3′;	(SEQ ID NO: 12)
and

for human IgG1 Fc reverse:
5′-tcatttacccggagacagggagag-3′.	(SEQ ID NO: 13)

The Ig-like domains and the human IgG1 Fc fragment were PCR amplified at conditions of denaturing at 95° C. for 30 seconds, annealing at 56° C. for 45 seconds, extension at 72° C. for 2 minutes, and 30 cycles. The PCR products were then cloned into plasmid pCR2.1 (Invitrogen) using the TA cloning kit. After transformation into E.coli (JM109), white colonies were picked and cultured overnight in LB media. DNA plasmids were prepared using the Qiagen kit and subjected to enzyme digestion and DNA sequencing.
The cDNAs of FLT-1, KDR and IgG Fc were fused together by sewing PCR using primers containing the EcoRI site. After digestion with EcoRI, the DNA fragment was purified with the Qiagen purification kit and cloned into plasmid pcDNA3.1. After transformed into E. coli (JM109), positive colonies were picked and cultured overnight in LB media. DNA plasmids were extracted with the Qiagen plasmid purification kit and then subjected to enzyme digestion and DNA sequencing. The obtained FP3′ DNA coding sequence is shown as SEQ ID NO.6. The confirmed plasmids were used to transfect 293 cells or CHO cells to obtain stable cell lines expressing FP3′. The amino acid sequence of FP3′ is shown as SEQ ID NO.7.

Example 2

Construction of FP1′ Gene and Recombinant Vector

FP1′ was constructed similarly as in Example 1. The only difference was that the targeted recombinant DNA was constructed by fusing together the 2^ndIg-like domain of FLT-1, the 3^rdIg-like domain of KDR, and the same human IgG1 Fc as in Example 1.

Example 3

Construction of FP2′ Gene and Recombinant Vector

FP2′ was constructed similarly as in Example 1. The only difference was that the targeted recombinant DNA was constructed by fusing together the 1^stIg-like domain of KDR, the 2^ndIg-like domain of FLT-1, the 3^rdIg-like domain of KDR, and the same human IgG1 Fc as in Example 1.

Example 4

Construction of FP4′ Gene and Recombinant Vector

FP4′ was constructed similarly as in Example 1. The only difference was that the targeted recombinant DNA was constructed by fusing together the 2^ndIg-like domain of FLT-1, the 3^rdIg-like domain of KDR, the 4^thIg-like domain of FLT-1, and the same human IgG1 Fc as in Example 1.

Example 5

Construction of FP5′ Gene and Recombinant Vector

FP5′ was constructed similarly as in Example 1. The only difference was that the targeted recombinant DNA was constructed by fusing together the 2^ndIg-like domain of FLT-1, the 3^rd-5^thIg-like domain of KDR, and the same human IgG1 Fc as in Example 1.

Example 6

Construction of FP6′ Gene and Recombinant Vector

FP6′ was constructed similarly as in Example 1. The only difference was that the targeted recombinant DNA was constructed by fusing together the 2^ndIg-like domain of FLT-1, the 3^rdIg-like domain of KDR, the 4^th-5^thIg-like domain of FLT-1, and the same human IgG1 Fc as in Example 1.

Embodiment 2

Expression of the Chimeric Proteins in Cells

Example 7

Expression of the Chimeric Proteins FP3′

After construction of above-mentioned recombinant plasmids, high quality plasmid DNAs were obtained using Qiagen's plasmid kit, and then were transfected into 293 cells (ATCC) using FUGEN6 transfection kit (Roche). Two different methods were used to express the chimeric proteins depending on amount of proteins needed.
The first method was a method of transient transfection. A small amount of the chimeric proteins were produced using this method. Firstly, 293 cells were cultured in DMEM media with 10% FBS in tissue culture dishes. At 60-80% cell confluence, the mixture of plasmid DNA and FUGEN6 reagent was added into the culture. The culture media was exchanged to serum-free DMEM in the next day and the cells were continued to culture for 3 more days before media was collected. These media contained the expressed chimeric proteins, and the concentration of the chimeric proteins was assayed by ELISA.
The second method was a method of stable transfection. A stable cell line was established to produce a large amount of the chimeric proteins. The host cells were again 293 cells (ATCC). The step of transfecting recombinant plasmid was the same as that of transient transfection described above. However, at the 2^ndday, the cells were cultured in DMEM with neomycin and cloned by limited dilution. After about 21 days, neomycin resistant clones were picked and cultured in a larger scale. Finally, chimeric proteins were expressed in shaker flasks. The concentration of chimeric proteins was assayed by ELISA.
FP3′ was purified from the cultured media using assays including affinity chromatography and gel filtration, etc. The molecular weight of FP3′ was 140 KD.

Example 8

Expression of other Chimeric Proteins

Chimeric proteins FP1′, FP2′, FP4′, FP5′, and FP6′ were obtained in accordance with the methods of example 7.

Embodiment 3

Binding Experiment of the Chimeric Proteins to VEGF

Affinities of the chimeric proteins to VEGF were determined by the VEGF binding assay in the present invention. Firstly, recombinant VEGF proteins (Chemicom) were coated in a 96-well ELISA plate, and non-specific protein binding sites of the plate were then blocked using 5% milk solution. Secondly, different concentrations of various chimeric proteins were added into each well, and incubated for 2 hours at 37° C. After washing, rabbit anti-human Ig-HRP (Sigma) was added into each well, and finally colorimetric enzyme substrates were added to the plate. The absorption OD readings were recorded with the use of an ELISA plate reader. A higher OD value indicated a stronger binding affinity of the chimeric proteins to VEGF.
As shown in FIG. 2, all five chimeric proteins constructed and expressed in the embodiments of the present invention were able to bind VEGF, and had better affinities than FP1′ of the prior art. The binding was detectable at low concentrations of 1 μg/ml. Preferably, FP3′ had the best affinity and was the best VEGF inhibitor. Its half maximal binding concentration was about 5 times lower than that of FP1′. FP5′ had a little bit lower affinity to VEGF than that of FP3′. This result suggests that the 4^thIg-like domain of KDR could substantially increase the blocking activity of the chimeric protein to VEGF. However, adding more of the KDR domains such as the 5^thIg-like domain could not further enhance the inhibitory effect on VEGF. The other three chimeric proteins exhibited lower affinities comparing to FP3′ and FP5′, but higher affinities than FP1′.

Embodiment 4

The Chimeric Proteins Effectively Inhibited Proliferation of Human Vascular Endothelial Cells In Vitro

This preferred embodiment of the invention is to prove that the chimeric proteins could effectively block VEGF-induced growth of vascular endothelial cells. In the experiment, HUVEC cells (Clonetics) were seeded in a 96-well tissue culture plate in EBM media with 2% FBS and 15 ng/ml of VEGF. Different amounts of the 293 cells supernatant containing the chimeric proteins were added into the plate. Un-transfected 293 cells media containing no chimeric proteins was used as negative control. All HUVEC cells were cultured in 37° C. for 3 days before cell densities were determined by cell counting.
The HUVEC proliferation experiment has shown that all five chimeric proteins constructed according to the invention could inhibit proliferation of vascular endothelial cells more effectively than FP1′ of the prior art (FIG. 3). Since the HUVEC cell proliferation in the experiment was induced by VEGF activation, it is therefore suggested that all five chimeric proteins were able to inhibit the receptor activation of VEGF, and all of them possessed anti-angiogenesis activities. Among which, FP3′ had the best inhibiting effect on the HUVEC cell growth with IC50 of about 3 ng/ml. The IC50 of FP1′ of the prior art was about 12 ng/ml, and the IC50s of FP2′, FP4′, FP5′ and PF6′ were all about 5-8 ng/ml.

Embodiment 5

The Chimeric Polypeptides Inhibited Tumor Growth in Mice

Example 9

Preparation of Injection Formulation Containing the Chimeric Proteins

The injection formulation was prepared according to any conventional methodologies, for injection formulations using 24 mg/ml of FP3′, 5 mM of PB, 100 mM of NaCl, and 20% sucrose.

Example 10

The Chimeric Proteins Effectively Inhibited the Growth of B16F10 Melanoma Cells in Mice

As VEGF inhibitors, one application of the chimeric proteins of the invention is to be used in anti-cancer therapy. Because of its highly effective blocking effect on VEGF, FP3′ was chosen to perform anti-tumor experiments in animal models.
The animal model was murine with B16F10 melanoma cells which is a kind of rapidly growing tumor cells. In the experiment, 1×10⁵B16F10 cells in 0.05 ml were first injected subcutaneously in the back of BALB/C nude mice. Then the purified chimeric protein was injected intraperitomneally with 400 μg each mouse (mice average weight 22 g), twice a week. Same amount of the purified human immunoglobulin Fc was injected into negative control mice. Tumor growth curves were shown in FIG. 4, indicating that the chimeric protein FP3′ effectively inhibited the growth of the melanoma cells (P<0.01).

Example 11

The Chimeric Proteins Effectively Inhibited the Growth of Xenographed Prostate Cancer PC-3 Cells in Mice

Xenograft model of human tumor cells growing in nude mice is one of the animal models, which is most similar with human tumors. Nude mice lack of immune rejection, thus many human tumor cells could grow in nude mice and form tumor. The chimeric protein FP3′ was tested for inhibiting growth of human prostate tumor PC-3 cells (ATCC) in BALB/C nude mice. In this model, 1×10⁵PC-3 cells in 0.05 ml were first injected subcutaneously in the back of mice. Then the purified chimeric protein was injected intraperitomneally with 400 μg each mouse, twice a week. Same amount of the purified human immunoglobulin Fc was injected into negative control mice. The experimental results were shown in FIG. 5. In the control mice, tumor grew more than 1000 mm ³45 days after implantation. However, in the mice administered with the chimeric proteins, FP3′ has almost completely inhibited the tumor growth (P<0.01), demonstrating a significant therapeutic anti-tumor effect.

Embodiment 6

Comparing Study of the Chimeric Protein FP3′ and FP1′ of the Prior Art in Inhibiting Tumor Growth in Mice

In order to further demonstrate the supreme anti-cancer activity of FP3′, the effects of FP1′ and FP3′ were compared in a tumor growth experiment. 10 healthy BALB/C nude mice were chosen and each was injected subcutaneously in the back with 1×10⁵rat glioblastoma C6 cells in 0.05 ml. Then 2.5 mg/kg of purified PF1′ or PF3′ were injected intraperitomneally twice a week, respectively, up to 31 days. The same amount of the purified human immunoglobulin Fc was injected into negative control mice. The experimental results are shown in FIG. 6. FP1′ and FP3′ both had a significant therapeutic effect on the tumor. At day 35, tumor volumes of mice administered with FP1′ and FP3′ were 1167.3 and 557.6, respectively, whereas tumor volume of the control mice treated with Fc has already reached 1312.3 at day 24. Therefore, FP3′ had more significant effect (P<0.05) than FP1′ of the prior art.
All taken together, the chimeric proteins constructed according to the invention had a high affinity to VEGF, were able to inhibit vascular endothelial cell proliferation in vitro, and effectively inhibited tumor growth in vivo. Since angiogenesis is critical in all tumor growth, the chimeric proteins of the invention can be used in therapeutic applications against many tumors.

Claims

1. A type of chimeric proteins consisting of different fragments of the VEGF receptors FLT-1 and KDR, characterized in that the chimeric proteins are selected from the following groups :

a. consisting of the 2^ndIg-like domain of FLT-1, and the 3^rdand the 4^thIg-like domains of KDR, designated as FLTd2-KDRd3,4;

b. consisting of the 2^ndIg-like domain of FLT-1, the 3^rdIg-like domain of KDR, and the 4^thIg-like domain of FLT-1, designated as FLTd2-KDRd3-FLTd4;

c. consisting of the 2^ndIg-like domain of FLT-1, and the 3 ^rd, the 4^thand the 5^thIg-like domains of KDR, designated as FLTd2-KDRd3,4,5;

d. consisting of the 2^ndIg-like domain of FLT-1, the 3^rdIg-like domain of KDR, and the 4^thand the 5^thIg-like domains of FLT-1, designated as FLTd2-KDRd3-FLTd4,5.

2. A type of chimeric proteins consisting of the chimeric proteins of claim 1 and human immunoglobulin Fe, which are selected from the following groups:

FP4′ designated as FLTd2-KDRd3-FLTd4-Fc;

FP5′ designated as FLTd2-KDRd3,4,5-Fc;

FP6′ designated as FLTd2-KDRd3-FLTd4,5-Fc.

3. The chimeric proteins of claim 2, wherein the immunoglobulin Fc is derived from classes or subclasses of human immunoglobulin.

4. The chimeric proteins of claim 3, wherein the immunoglobulin Fc fragment is a full length Fc or a fragment of Fc sequence.

5. An isolated recombinant DNA coding sequence encoding a chimeric protein, comprising fragments of the VEGF receptors FLT-1 and KDR, and optionally Immunoglobulin Fc, wherein the chimeric protein is selected from the group consisting of:

a. a chimeric protein consisting of the 2^ndIg-like domain of FLT-1, and the 3^rdand 4^thIg-like domains of KDR, designated as FLTd2-KDRd3,4;

b. a chimeric protein consisting of the 2^ndIg-like domain of FLT-1, the 3^rdIg-like domain of KDR, and the 4^thIg-like domain of FLT-1, designated as FLTd2-KDRd3-FLTd4;

c. a chimeric protein consisting of the 2^ndIg-like domain of FLT-1, and the 3^rd, 4^thand 5^thIg-like domains of KDR, designated as FLTd2-KDRd3,4,5;

d. a chimeric protein consisting of the 2^ndIg-like domain of FLT-1, the 3^rdIg-like domain of KDR, and the 4^thand the 5^thIg-like domains of FLT-1, designated as FLTd2-KDRd3 -FLTd4, 5;

e. FP4′ designated as FLTd2-KDRd3-FLTd4-Fc;

f. FP5′ designated as FLTd2-KDRd3-FLTd4-Fc; and

g. FP6′ designated as FLTd2-KDRd3-FLTd4,5-Fc.

6. An isolated recombinant vector comprising the recombinant DNA sequences of claim 5, wherein the vector is selected from plasmid, virus, or DNA fragment.

7. An isolated recombinant host cell containing the recombinant vector of claim 6, wherein the host cell is a eukaryotic cell or prokaryotic cell.

8. A pharmaceutical composition which comprises one or more chemeric proteins encoded by the recombinant DNA according to claim 5 and a pharmaceutical acceptable carrier.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is an injection solution.

10. A method of treating an angiogenesis-related disease in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 8, wherein the disease is selected from the group consisting of a tumor, a diabetic retinopathies, arthritis, anemia and endometrial hyperplasia.