VEGF-trap polypeptides and uses thereof

Abstract

The present application provides a VEGF-trap polypeptide and its use. The VEGF-trap polypeptide described in the present application comprises a substitution of amino acid at position 148 compared with the sequence shown in SEQ ID NO. 8. The VEGF-trap polypeptide described in the present application comprises a mutant of the second Ig domain of VEGFR1 and the third Ig domain of VEGFR2. The VEGF-trap described in the present application can be used to construct a fusion protein or an immunoconjugate. The present application also provides the use of the VEGF-trap or the fusion protein or the immunoconjugate in treating a disease or a disorder.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a VEGF-trap polypeptide and its application. BACKGROUND

[0002] The uncontrolled invasive growth and metastasis of tumors depend on angiogenesis. Vascular endothelial growth factor (VEGF) is one of the strongest positive regulators of vascularization, which stimulates the formation of new blood vessels by binding to its specific receptor, vascular endothelial growth factor receptor (VEGFR). Therefore, blocking the conduction of VEGFs signaling pathway is one of the effective methods for treating tumors.

[0003] Bevacizumab (i.e. VEGF-trap, trade name, Avastin) developed by Genentech Company is a recombinant human-mouse chimeric antibody, which can block the binding of VEGF-A to vascular endothelial cell growth factor receptor, so that VEGFR cannot be activated to play an anti-angiogenic role. SUMMARY

[0004] The present application provides a VEGF-trap polypeptide and its application. The VEGF-trap described in the present application contains a substitution of amino acid at position 148 compared with the sequence shown in SEQ ID NO. 8. The VEGF-trap described in the present application can be used to construct a fusion protein or an immunoconjugate. The present application also provides the application of the VEGF-trap or the fusion protein or the immunoconjugate in treating diseases or disorders. The stability of the VEGF-trap polypeptide described in the present application is significantly improved, and / or the ability of non-specific binding is significantly reduced.

[0005] In one aspect, the present application provides a VEGF-trap polypeptide, which contains a substitution of amino acid at position 148 compared with the sequence shown in SEQ ID NO. 8.

[0006] In certain embodiments, the VEGF-trap polypeptide contains a substitution of amino acid at position 153 compared with the sequence shown in SEQ ID NO. 8.

[0007] In certain embodiments, the VEGF-trap polypeptide contains a substitution of amino acid at position 156 compared with the sequence shown in SEQ ID NO. 8.

[0008] In certain embodiments, the VEGF-trap polypeptide comprises an amino acid substitution at an amino acid residue selected from the group consisting of: K148, R153, and K156.

[0009] In certain embodiments, the VEGF-trap polypeptide comprises an amino acid substitution set forth as K148A or K148T.

[0010] In certain embodiments, the VEGF-trap polypeptide comprises an amino acid substitution set forth as R153A or R153T.

[0011] In certain embodiments, the VEGF-trap polypeptide comprises an amino acid substitution set forth as K156A or K156T.

[0012] In certain embodiments, the VEGF-trap polypeptide comprises an amino acid substitution selected from the group consisting of:

[0013] a) K148A, R153A, and K156A; and,

[0014] b) K148T, R153T, and K156T.

[0015] In certain embodiments, the VEGF-trap polypeptide comprises a mutant of the second Ig domain of VEGFR1 and the third Ig domain of VEGFR2. In certain embodiments, the mutant comprises an amino acid sequence as set forth in any one of SEQ ID NOs. 2-3.

[0016] In certain embodiments, the second Ig domain of VEGFR1 comprises an amino acid sequence as set forth in SEQ ID NO. 4.

[0017] In certain embodiments, the VEGF-trap polypeptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs. 6-7.

[0018] In certain embodiments, the VEGF-trap polypeptide is capable of binding to VEGF.

[0019] In another aspect, the present application provides a fusion protein or immunoconjugate comprising a VEGF-trap polypeptide described herein.

[0020] In another aspect, the present application provides an isolated nucleic acid molecule encoding a VEGF-trap polypeptide described herein and / or a fusion protein or immunoconjugate described herein.

[0021] In another aspect, the present application provides a vector comprising an isolated nucleic acid molecule described herein.

[0022] In another aspect, the present application provides a cell comprising or expressing a VEGF-trap polypeptide described herein, a fusion protein or immunoconjugate described herein, an isolated nucleic acid molecule described herein, or a vector described herein.

[0023] In another aspect, the present application provides a method of producing a VEGF-trap polypeptide described herein, comprising culturing a cell described herein under conditions wherein the VEGF-trap polypeptide described herein is expressed.

[0024] In another aspect, the present application provides a pharmaceutical composition comprising a VEGF-trap polypeptide described herein, a fusion protein or immunoconjugate described herein, an isolated nucleic acid molecule described herein, or a vector described herein, and / or a cell described herein, and optionally a pharmaceutically acceptable adjuvant.

[0025] In another aspect, the present application provides use of a VEGF-trap polypeptide described herein, a fusion protein or immunoconjugate described herein, and / or a pharmaceutical composition described herein, in the manufacture of a medicament for treating a tumor.

[0026] In another aspect, the present application provides a method of inhibiting growth of blood vessels in a human, comprising administering an effective amount of a VEGF-trap polypeptide described herein, and / or a fusion protein or immunoconjugate described herein.

[0027] In another aspect, the present application provides a method of inhibiting VEGF receptor ligand activity, comprising administering an effective amount of a VEGF-trap polypeptide described herein, and / or a fusion protein or immunoconjugate described herein.

[0028] In another aspect, the present application provides a method of reducing or preventing plasma leakage, comprising administering to a subject in need thereof an effective amount of a VEGF-trap polypeptide described herein, and / or a fusion protein or immunoconjugate described herein.

[0029] Other aspects and advantages of the present application will become apparent to those skilled in the art from the following detailed description. In some instances, any of the examples described herein can be presented in terms of methods comprising the disclosed steps, regardless of and without limitation to the specific implementation of those steps disclosed herein. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make modifications to the specific implementations disclosed, without departing from the spirit and scope of the inventions involved. Accordingly, the description in the specification of the present application is merely exemplary, and is not to be taken in a limiting sense. DETAILED DESCRIPTION

[0030] The following detailed description of the application is presented to enable any person skilled in the art to make and use the application. Various modifications to the embodiments described herein will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the scope of the application.

[0031] Definitions

[0032] In the present application, the term "polypeptide" generally refers to a molecule composed of monomers (amino acids) connected linearly by amide bonds (also known as peptide bonds). The term "polypeptide" can be any chain having two or more amino acids, and does not refer to a product of a particular length. The polypeptides described herein include peptides, dipeptides, tripeptides, oligopeptides, proteins, amino acid chains, or any other term used to refer to a chain having two or more amino acids, and the term "polypeptide" can be used interchangeably with or in place of any of these terms. The term "polypeptide" can also refer to post-expression modified products of the polypeptide including but not limited to glycosylated, acetylated, phosphorylated, acylated, derivatized by known protecting groups, proteolytic cleavage, or by modifications introduced by natural processes such as amino acid oxidation, deamination, dimerization, and other post- translational processes. The polypeptides can be derived from natural biological sources or generated by recombinant techniques.

[0033] In the present application, the term "VEGF" generally refers to vascular endothelial growth factor. VEGF can be involved in regulating normal and abnormal vasculogenesis and neovascularization associated with tumors and ocular disorders (see Ferrara, N. and Davis-Smyth, T., Endocr. Rev. 18, 1997, 4-25, et al.). VEGF can have an important regulatory function in new blood vessel formation during embryonic vasculogenesis and in angiogenesis during adulthood. VEGF can promote tumor growth. VEGF is a highly conserved homodimeric glycoprotein. VEGF has six isoforms: VEGF-A, VEGF-B (including VEGF-B167 and VEGF-B186), VEGF-C, VEGF-D, and VEGF-E. In the present application, the VEGF can be human VEGF.

[0034] In the present application, the term "VEGFR1" generally refers to vascular endothelial growth factor receptor 1. VEGFR1 is one of the VEGFRs. VEGFR belongs to the receptor tyrosine kinase superfamily and is a membrane mosaic protein. The extracellular portion of VEGFR is about 750 amino acid residues and consists of 7 Ig-like domains similar to immunoglobulin structure. The second Ig domain in the extracellular region of VEGFR1 is the region that binds to the ligand. Different isoforms of VEGFR1 can competitively bind VEGF (e.g., can bind VEGF-A, VEGF-B), thereby preventing VEGF from binding to VEGFR2.

[0035] In the present application, the term "VEGFR2" generally refers to vascular endothelial growth factor receptor 2. The third Ig domain of VEGFR2 can play a role in the specificity of binding to ligands. VEGFR2 can bind to VEGF-A, VEGF-E.

[0036] In the present application, the term "VEGF-trap polypeptide" generally refers to a recombinant fusion protein comprising the second Ig domain of VEGFR1 and the third Ig domain of VEGFR2 fused together with the Fc fragment of human IgG1, and modified recombinant fusion proteins modified on this basis. The VEGF-trap polypeptide can be Aflibercept developed by Genentech. The VEGF-trap polypeptide can bind to all subtypes of human VEGF-A. The VEGF-trap polypeptide can also bind to PIGF-2. The VEGF-trap polypeptide can block the process of VEGF-A inducing VEGFR1 activation, and can also block the process of VEGF-A inducing VEGFR2 activation. The VEGF-trap polypeptide can play a role in anti-tumor angiogenesis, and can effectively reduce the volume and metastasis of solid tumors. The VEGF-trap polypeptide can be used alone, or can be used in combination with chemotherapy drugs to treat tumors, for example, can treat recurrent or refractory solid tumors.

[0037] In the present application, the term "amino acid substitution" generally refers to the replacement of at least one existing amino acid residue with another different amino acid residue. The replaced amino acid residue can be a "naturally occurring amino acid residue", for example, can be alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (He), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). The replaced amino acid residue can also be a non-naturally occurring form of an amino acid residue, for example, can be norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogs. In the present application, the amino acid substitution can be a non-conservative substitution. The non-conservative substitution can include changing an amino acid residue in a target protein or polypeptide in a non-conservative manner, for example, changing an amino acid residue having a certain side chain size or a certain property (e.g., hydrophilicity) to an amino acid residue having a different side chain size or a different property (e.g., hydrophobicity). In the present application, the amino acid substitution can also be a conservative substitution. The conservative substitution can include changing an amino acid residue in a target protein or polypeptide in a conservative manner, for example, changing an amino acid residue having a certain side chain size or a certain property (e.g., hydrophilicity) to an amino acid residue having the same or similar side chain size or the same or similar property (e.g., still hydrophilic). Such a conservative substitution generally does not have a great impact on the structure or function of the resulting protein. In the present application, the amino acid sequence variants of the fusion protein or fragments thereof can include conservative amino acid substitutions that do not significantly alter the structure or function of the protein (e.g., a mutant having at least partial activity of human GLP-1).

[0038] In the present application, the term "nucleic acid molecule" generally refers to any nucleic acid that can encode a VEGF-trap polypeptide described herein. The nucleic acid molecules described herein can include naturally occurring nucleic acids, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), as well as artificially designed nucleic acids produced by chemical synthesis or using recombinant genetic techniques, including, for example, nucleic acid analogs, such as peptide nucleic acid (PNA) or locked nucleic acid (LNA), etc. (see Sambrook, J. and Russel, D. W. (2001), Molecular cloning: A laboratory manual (3rd ed.) Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press). Naturally occurring nucleic acids can include DNA sequences (such as genomic DNA or cDNA molecules), as well as RNA sequences (such as hnRNA, mRNA or rRNA molecules) or their reverse complement nucleic acid sequences. The nucleic acid molecules described herein can be single-stranded or double-stranded molecules. For example, they can be 30 to 5000 nucleotides in length, such as 30 to 3000 nucleotides, 45 to 2000 nucleotides, 60 to 1000 nucleotides, or 75 to 500 nucleotides.

[0039] In the present application, the term "vector" generally refers to a vehicle that can transfer genetic material (e.g., a nucleic acid molecule described herein) to a target location (e.g., a cell). The vector can include a plasmid, a virus, a cosmid, and / or an artificial chromosome. For example, the vector can include an engineered vector. The engineered vector can comprise an origin of replication, a multiple cloning site, and a selectable marker. The vector can be a nucleotide sequence, such as a DNA sequence. The vector can comprise a nucleic acid of interest (e.g., a nucleic acid molecule described herein) to be transferred and a nucleic acid fragment that functions as the vehicle. The vector can comprise functional structures that can regulate and / or control the expression of the genetic material. The vector can further comprise one or more functional structures selected from the group consisting of a promoter, a genetic marker, an antibiotic resistance, a reporter gene, and a protein purification tag. The vector can function to express a nucleic acid molecule described herein in a target cell.

[0040] In the present application, the term "cell" generally refers to a cell comprising a nucleic acid molecule described herein, and the progeny of the same. The cell can comprise a primary transformed cell, and can also comprise progeny derived therefrom, no matter how many times the cell has been propagated. The progeny can not be completely identical to the parent cell in nucleic acid content, and can contain mutations.

[0041] In the present application, the term "immunoconjugate" generally refers to a polypeptide (e.g., a VEGF-trap polypeptide described herein) linked to a therapeutic moiety, such as a cytotoxin, a drug, or a radioisotope. The cytotoxin or cytotoxic agent can include any agent that is detrimental (e.g., lethal) to cells. For example, it can include paclitaxel and / or cytochalasin B. The radioisotope can include radioactive iodine.

[0042] In the present application, the term "pharmaceutical composition" generally refers to a composition that can be administered to a subject in need thereof. In certain embodiments, the pharmaceutical composition can include a composition for parenteral, transdermal, intraluminal, intraarterial, intramembranous, and / or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition can be administered to a patient by infusion or injection. For example, it can be administered intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally. The pharmaceutical composition can include a pharmaceutically acceptable carrier. For example, the pharmaceutically acceptable carrier can include a phosphate buffered saline solution, water, emulsions (such as an oil / water emulsion), various types of wetting agents, sterile solutions, liposomes. The compositions can be formulated by conventional methods known in the art. The dosage regimen for the pharmaceutical composition is determined by the attending physician and clinical factors. Dosage amounts for a given patient will depend on many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health status, and other drugs being administered concurrently.

[0043] In the present application, the term "comprising" generally means including, but not limited to.

[0044] In the present application, the term "about" generally means within 0.5%-10% of a specified value, such as within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of a specified value. DETAILED DESCRIPTION

[0046] In one aspect, the present application provides a VEGF-trap polypeptide having the amino acid sequence of K KLVNR DL K TQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO. 8) compared to the sequence set forth in SEQ ID NO. 8 at position 148.

[0047] In the present application, the position of the substitution of the amino acid can be counted from the N-terminus of the amino acid sequence set forth in SEQ ID NO. 8.

[0048] For example, the VEGF-trap polypeptide can comprise 1-3 (e.g., can comprise 1, 2, or 3) amino acid substitutions compared to the sequence set forth in SEQ ID NO. 8.

[0049] For example, the VEGF-trap polypeptide can comprise a substitution of an amino acid at position 153 compared to the sequence set forth in SEQ ID NO. 8.

[0050] For example, the VEGF-trap polypeptide can comprise a substitution of an amino acid at position 156 compared to the sequence set forth in SEQ ID NO. 8.

[0051] In the present application, the VEGF-trap polypeptide can comprise an amino acid substitution at an amino acid residue selected from the group consisting of K148, R153, and K156. For example, the VEGF-trap polypeptide can comprise an amino acid substitution at K148. For example, the VEGF-trap polypeptide can comprise an amino acid substitution at R153. For example, the VEGF-trap polypeptide can comprise an amino acid substitution at K156. For example, the VEGF-trap polypeptide can comprise amino acid substitutions at K148 and R153. For example, the VEGF-trap polypeptide can comprise amino acid substitutions at R153 and K156. For example, the VEGF-trap polypeptide can comprise amino acid substitutions at K148 and K156. For example, the VEGF-trap polypeptide can comprise amino acid substitutions at K148, R153, and K156.

[0052] In the present application, the "amino acid substitution Xn" means that an amino acid substitution occurs at the residue X corresponding to the n-th residue in the amino acid sequence shown in SEQ ID NO: 8, wherein n is a positive integer and X is an abbreviation of any amino acid residue. For example, "amino acid substitution K148" means that an amino acid substitution occurs at the residue K corresponding to the 148-th residue in the amino acid sequence shown in SEQ ID NO: 8.

[0053] In the present application, the amino acid substitution includes the selection of the kind of the substituted amino acid. The expression level of the VEGF-trap polypeptide of the present application comprising the amino acid substitution can not be significantly affected. Meanwhile, the stability of the VEGF-trap polypeptide of the present application (e.g., in vivo stability in a subject) can be significantly improved. And / or, the ability of non-specific binding thereof can be significantly reduced.

[0054] In the present application, the kind of the substituted amino acid of each amino acid substitution of the VEGF-trap polypeptide can be independently selected. In the present application, the amino acid substitution "XnY" means that the residue X corresponding to the n-th residue in the amino acid sequence shown in SEQ ID NO: 8 is substituted with the amino acid residue Y, wherein n is a positive integer, X and Y are each independently an abbreviation of any amino acid residue, and X is different from Y. For example, the amino acid substitution "K148A" means that the amino acid residue K corresponding to the 148-th residue in the amino acid sequence shown in SEQ ID NO: 8 is substituted with the amino acid residue A.

[0055] For example, the VEGF-trap polypeptide can comprise the amino acid substitution of K148A or K148T.

[0056] For example, the VEGF-trap polypeptide can comprise an amino acid substitution of R153A or R153T.

[0057] For example, the VEGF-trap polypeptide can comprise an amino acid substitution of K156A or K156T.

[0058] For example, the VEGF-trap polypeptide can comprise an amino acid substitution selected from the group consisting of:

[0059] a) K148A, R153A and K156A; and,

[0060] b) K148T, R153T and K156T.

[0061] For example, the VEGF-trap polypeptide can comprise an amino acid sequence as set forth in any one of SEQ ID NO. 6-7.

[0062] In the present application, the VEGF-trap polypeptide can comprise a mutant of the second Ig domain of VEGFR1 and the third Ig domain of VEGFR2. For example, the mutant can comprise an amino acid sequence as set forth in any one of SEQ ID NO. 2-3. For example, the second Ig domain of VEGFR1 comprises an amino acid sequence as set forth in SEQ ID NO. 4.

[0063] In the present application, the VEGF-trap polypeptide can comprise an immunoglobulin Fc region.

[0064] The immunoglobulin Fc region described in the present application can include a heavy chain constant region 1 (CH1) and a heavy chain constant region 2 (CH2) of an immunoglobulin. In addition, the immunoglobulin Fc region can comprise all or part of the Fc region as long as it has a similar physiological activity to the native protein. There are five types of human immunoglobulins having different effector functions and pharmacokinetic properties, IgA, IgD, IgE, IgG, and IgM. For example, the Fc region of the present application can be an Fc region derived from IgG. IgG has four subtypes (Gl, G2, G3, and G4), each of which has different biological functions called effector functions. These effector functions are generally mediated by interaction with Fc receptors (FcγR) or by binding to Clq and fixing complement. The immunoglobulin Fc region described in the present application can be an Fc region derived from IgGl or IgG4.

[0065] In the present application, the VEGF-trap polypeptide described in the present application can still retain the biological properties possessed by the original VEGF-trap (e.g., Aflibercept). For example, the VEGF-trap polypeptide described in the present application can bind to VEGF.

[0066] In another aspect, the present application provides a fusion protein or immunoconjugate comprising a VEGF-trap polypeptide described herein. The fusion protein or immunoconjugate described herein has a biological activity. The biological activity includes the biological activity possessed by the VEGF-trap polypeptide (e.g., the ability to bind VEGF).

[0067] In another aspect, the present application provides an isolated nucleic acid molecule encoding a VEGF-trap polypeptide described herein and / or a fusion protein or immunoconjugate described herein.

[0068] The nucleic acid molecule described herein can be isolated. For example, it can be produced or synthesized by (i) amplification in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) recombinant production by cloning, (iii) purification, such as by enzymatic cleavage and gel electrophoresis fractionation, or (iv) synthesis, such as by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology. Recombinant DNA and molecular cloning techniques include those described by Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by T.J. Silhavy, M.L. Bennan and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F.M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987). Briefly, the nucleic acids can be prepared from genomic DNA segments, cDNAs, and RNAs, all of which can be extracted directly from cells or recombinantly produced by various amplification methods, including but not limited to PCR and RT-PCR.

[0069] Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York N.Y., 1993.

[0070] In another aspect, the present application provides a vector comprising the isolated nucleic acid molecule described herein.

[0071] For example, the vector can comprise one or more of the nucleic acid molecules described herein. In addition, the vector can comprise other genes, such as marker genes that allow for selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector can comprise expression control elements that allow for proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art, and can include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate transcription or translation of mRNA. In certain embodiments, the expression control sequences are regulatable elements. The specific structure of the expression control sequences can vary depending on the species or the function of the cell type, but generally comprise 5' non-transcribed sequences and 5' and 3' non-translated sequences involved in the initiation of transcription and translation, respectively, such as TATA boxes, capping sequences, CAAT sequences, and the like. For example, the 5' non-transcribed expression control sequences can comprise a promoter region, which can comprise a promoter sequence operably linked to nucleic acids for transcriptional control functions. The expression control sequences can also include enhancer sequences or upstream activator sequences. In the present application, suitable promoters can include, for example, promoters for SP6, T3, and T7 polymerases, human U6 RNA promoters, CMV promoters, and artificial hybrid promoters thereof (e.g., CMV), wherein a portion of the promoter can be fused to a portion of the promoter of a gene for another cellular protein (e.g., human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), which can or can not include additional introns. One or more of the nucleic acid molecules described herein can be operably linked to the expression control elements. The vector can comprise a plasmid, a cosmid, a virus, a phage, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector.

[0072] In another aspect, the present application provides a cell comprising or expressing the VEGF-trap polypeptide described herein, the fusion protein or immunoconjugate described herein, the isolated nucleic acid molecule described herein, or the vector described herein.

[0073] The cell can be a prokaryotic cell (e.g., a bacterial cell), a CHO cell, an NS / 0 cell, a HEK293T cell, or a HEK293A cell, or can be another eukaryotic cell, such as a cell from a plant, a fungus, or a yeast cell, etc. The vectors described herein can be introduced into the cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc. For example, the host cell can be COS, CHO, NSO, sf9, sf21, DH5a, BL21(DE3), or TG1.

[0074] In another aspect, the present application provides a method of producing a VEGF-trap polypeptide described herein, comprising culturing a cell described herein under conditions such that the VEGF-trap polypeptide described herein is expressed.

[0075] For example, such methods are known to those of ordinary skill in the art, for example, by using appropriate media, appropriate temperatures, and incubation times, etc. In some cases, the methods can further comprise the step of harvesting (e.g., isolating and / or purifying) the VEGF-trap polypeptide described herein. For example, the VEGF-trap polypeptide described herein can be purified and isolated by affinity chromatography using protein G-sepharose or protein A-sepharose, and by gel electrophoresis and / or high performance liquid chromatography, etc.

[0076] In another aspect, the present application provides a pharmaceutical composition comprising a VEGF-trap polypeptide described herein, a fusion protein or immunoconjugate described herein, an isolated nucleic acid molecule described herein, or a vector described herein, and / or a cell described herein, and optionally a pharmaceutically acceptable adjuvant.

[0077] The pharmaceutically acceptable adjuvant can include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and / or nonionic surfactants, etc.

[0078] In the present application, the pharmaceutical composition can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at a tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration through a subcutaneous depot. For example, for oral administration, the pharmaceutical composition can be prepared into a small tablet, a tablet, a capsule, an elixir, a suspension, a syrup, or a wafer. For injection preparations, the pharmaceutical composition can be prepared into, for example, an ampoule of a unit dosage form or a unit dosage form of, for example, a multi-dose container. The pharmaceutical composition can also be prepared into a solution, a suspension, a tablet, a pill, a capsule, and a long-acting preparation.

[0079] The frequency and dosage of administration of the pharmaceutical composition can be determined by various factors including the type of disease to be treated, the route of administration, the age, sex, body weight and the severity of the disease of the patient, and the type of drug as an active ingredient. Since the pharmaceutical composition has excellent in vivo efficacy and duration of concentration, it can significantly reduce the frequency and dosage of administration of the drug.

[0080] In another aspect, the present application provides use of a VEGF-trap polypeptide described herein, a fusion protein or immunoconjugate described herein, and / or a pharmaceutical composition described herein in the manufacture of a medicament for treating a tumor.

[0081] A VEGF-trap polypeptide described herein, a fusion protein or immunoconjugate described herein, and / or a pharmaceutical composition described herein for use in treating a tumor.

[0082] The present application provides a method of treating a tumor, comprising administering to a subject in need thereof an effective amount of a VEGF-trap polypeptide described herein, a fusion protein or immunoconjugate described herein, and / or a pharmaceutical composition described herein.

[0083] In the present application, the tumor can be a VEGF-positive tumor. The tumor can be a solid tumor or a non-solid tumor.

[0084] In another aspect, the present application provides a method of inhibiting the growth of a blood vessel (e.g., human), comprising administering an effective amount of a VEGF-trap polypeptide described herein, a fusion protein or immunoconjugate described herein, and / or a pharmaceutical composition described herein.

[0085] In another aspect, the present application provides a method of inhibiting VEGF receptor ligand activity, comprising administering an effective amount of a VEGF-trap polypeptide described herein, a fusion protein or immunoconjugate described herein, and / or a pharmaceutical composition described herein.

[0086] For example, the VEGF receptor ligand activity can include biological activity and / or function of VEGF and / or VEGFR itself. For example, it can include binding of VEGF to VEGFR.

[0087] In another aspect, the present application provides a method of reducing or preventing plasma leakage, comprising administering to a subject in need thereof an effective amount of a VEGF-trap polypeptide described herein, a fusion protein or immunoconjugate described herein, and / or a pharmaceutical composition described herein.

[0088] For example, the VEGF-trap polypeptide described herein, the fusion protein or immunoconjugate described herein, and / or the pharmaceutical composition described herein can alleviate and / or treat capillary leak syndrome (CLS). For example, the disease and / or symptoms such as progressive edema, hypovolemic and distributive shock, and / or non-proteinuric hypoproteinemia can be alleviated and / or treated.

[0089] The present application includes the following embodiments:

[0090] 1. A VEGF-trap polypeptide comprising a substitution of an amino acid at position 148 as compared to the sequence set forth in SEQ ID NO. 8.

[0091] 2. The VEGF-trap polypeptide according to embodiment 1, comprising a substitution of an amino acid at position 153 as compared to the sequence set forth in SEQ ID NO. 8.

[0092] 3. The VEGF-trap polypeptide according to any one of embodiments 1-2, comprising a substitution of an amino acid at position 156 as compared to the sequence set forth in SEQ ID NO. 8.

[0093] 4. The VEGF-trap polypeptide according to any one of embodiments 1-3, comprising a substitution of an amino acid at an amino acid residue selected from the group consisting of K148, R153, and K156.

[0094] 5. The VEGF-trap polypeptide according to any one of embodiments 1-4, comprising a substitution of an amino acid set forth in K148A or K148T.

[0095] 6. The VEGF-trap polypeptide according to any one of embodiments 1-5, comprising a substitution of an amino acid set forth in R153A or R153T.

[0096] 7. The VEGF-trap polypeptide according to any one of embodiments 1-6, comprising a substitution of an amino acid set forth in K156A or K156T.

[0097] 8. The VEGF-trap polypeptide according to any one of embodiments 1-7, comprising a substitution of an amino acid selected from the group consisting of:

[0098] a) K148A, R153A, and K156A; and,

[0099] b) K148T, R153T, and K156T.

[0100] 9. The VEGF-trap polypeptide according to any one of embodiments 1-8, comprising a mutant of the second Ig domain of VEGFR1 and the third Ig domain of VEGFR2, wherein the mutant comprises an amino acid sequence as set forth in any one of SEQ ID NO. 2-3.

[0101] 10. The VEGF-trap polypeptide according to embodiment 9, wherein the second Ig domain of VEGFR1 comprises an amino acid sequence as set forth in SEQ ID NO. 4.

[0102] 11. The VEGF-trap polypeptide according to any one of embodiments 1-10, comprising an amino acid sequence as set forth in any one of SEQ ID NO. 6-7.

[0103] 12. The VEGF-trap polypeptide according to any one of embodiments 1-11, capable of binding VEGF.

[0104] 13. A fusion protein or immunoconjugate comprising the VEGF-trap polypeptide according to any one of embodiments 1-12.

[0105] 14. An isolated nucleic acid molecule encoding the VEGF-trap polypeptide according to any one of embodiments 1-12 and / or the fusion protein or immunoconjugate according to embodiment 13.

[0106] 15. A vector comprising the isolated nucleic acid molecule according to embodiment 14.

[0107] 16. A cell comprising or expressing the VEGF-trap polypeptide according to any one of embodiments 1-12, the fusion protein or immunoconjugate according to embodiment 13, the isolated nucleic acid molecule according to embodiment 14, or the vector according to embodiment 15.

[0108] 17. A method of producing the VEGF-trap polypeptide according to any one of embodiments 1-12, comprising culturing the cell according to embodiment 16 under conditions wherein the VEGF-trap polypeptide according to any one of embodiments 1-12 is expressed.

[0109] 18. A pharmaceutical composition comprising the VEGF-trap polypeptide according to any one of embodiments 1-12, the fusion protein or immunoconjugate according to embodiment 13, the isolated nucleic acid molecule according to embodiment 14, or the vector according to embodiment 15 and / or the cell according to embodiment 16, and optionally a pharmaceutically acceptable adjuvant.

[0110] 19. Use of the VEGF-trap polypeptide according to any one of embodiments 1 to 12, the fusion protein or immunoconjugate according to embodiment 13, and / or the pharmaceutical composition according to embodiment 18 in the preparation of a therapeutic medicament for the treatment of a tumor.

[0111] 20. A method of inhibiting growth of blood vessels in a human comprising administering an effective amount of the VEGF-trap polypeptide according to any one of embodiments 1 to 12 and / or the fusion protein or immunoconjugate according to embodiment 13.

[0112] 21. A method of inhibiting VEGF receptor ligand activity comprising administering an effective amount of the VEGF-trap polypeptide according to any one of embodiments 1 to 12 and / or the fusion protein or immunoconjugate according to embodiment 13.

[0113] 22. A method of reducing or preventing plasma leakage comprising administering to a subject in need thereof an effective amount of the VEGF-trap polypeptide according to any one of embodiments 1 to 12 and / or the fusion protein or immunoconjugate according to embodiment 13.

[0114] Without wishing to be bound by any theory, the following examples are merely intended to illustrate the fusion proteins, methods of preparation, and uses of the present application, and are not intended to limit the scope of the present application.

[0115] Examples

[0116] Example 1: Determination of expression level of the VEGF-trap polypeptide according to the present application

[0117] Based on the amino acid sequence of the VEGF-trap polypeptide according to the present application (SEQ ID NO. 6 or SEQ ID NO. 7), the nucleotide sequence encoding the same was artificially synthesized. The nucleotide sequence was then placed in a pCDNA4.0 expression vector, and the expression vector was transfected into human 293 cells.

[0118] After culturing the human 293 cells capable of expressing the VEGF-trap polypeptide described above for 6 to 7 days, the culture supernatant was collected, and the target protein was obtained after one-step purification with protein A. The expression level of the VEGF-trap polypeptide was determined using the same. Aflibercept (which comprises the amino acid sequence shown in SEQ ID NO. 8) was used as a control group, and was also transfected and expressed in human 293 cells under the same conditions.

[0119] The results show that the expression level of the VEGF-trap polypeptide according to the present application is about 300 mg / L, which is comparable to or slightly higher than the expression level of the control group (200 to 300 mg / L).

[0120] Example 2: Detection of the thermodynamic stability of the VEGF-trap polypeptide of the present application

[0121] Tm value method:

[0122] A small volume of the target protein (i.e. the VEGF-trap polypeptide of the present application) was determined by using the protein thermal shift assay instrument of UNCHAINED LABS.

[0123] The temperature program was as follows: the starting temperature was 15°C, the temperature increasing rate was 0.3°C / min, and the end temperature was 95°C. The fluorescence absorbance value of the sample at each temperature and each wavelength was recorded. The denaturation temperature Tm value was fitted by the software as the highest point of the first derivative at the confidence wavelength BCM; the starting aggregation temperature Tagg value was the tenth value of the first derivative at the static light scattering SLS, 473 nm.

[0124] The results are shown in Tables 1-2, respectively:

[0125] Table 1: Tm value of the VEGF-trap polypeptide of the present application

[0126]

[0127] Table 2: Tagg value of the VEGF-trap polypeptide of the present application

[0128]

[0129] The results show that, compared with the control group, the Tm value of the VEGF-trap polypeptide of the present application is increased from 56-57°C to 61-63°C; and the Tagg value is increased from 65°C to 67°C. Since the more stable the protein is, the higher the Tm value is, therefore the stability of the VEGF-trap polypeptide of the present application is higher than that of the control group.

[0130] Example 3: Detection of the binding ability of the VEGF-trap polypeptide of the present application

[0131] BLI (K2) method:

[0132] The binding ability of VEGF-trap polypeptide to recombinant human VEGFa was detected by biofilm interference technology BLI. The Octet K2 instrument was used for measurement. First, the recombinant camel anti-human Fc antibody was immobilized on the AHC biosensor. Then, human VEGFa was gradiently diluted, injected for 150 s, the dissociation time was 900 s, and 10 mM glycine-HCl (pH 1.7) was regenerated for 5 s. The simple one-to-one Languir binding model (Octet K2 data analysis software 9.0) was used to calculate the binding rate (kon) and dissociation rate (Kdis). The equilibrium dissociation constant (kD) was calculated as the ratio of kdis / kon. Aflibercept (which comprises the amino acid sequence shown in SEQ ID NO. 8) was used as a control group.

[0133] The results show that the K D values of the control group and the VEGF-trap polypeptides described in the present application (the amino acid sequences of which are shown in SEQ ID NO. 6 or SEQ ID NO. 7, respectively) specifically binding to VEGFa are all less than 10 -12 M.

[0134] Example 4: Detection of the in vivo half-life of the VEGF-trap polypeptides described in the present application

[0135] In vivo half-life detection method:

[0136] SPF level SD rats were used for the experiment, and the animals were about 6-8 weeks old and weighed about 200-220 g. The experiment was carried out in a SPF animal room, and the animal room environment temperature was 23±2℃, the relative humidity was 40-70%, and the light and dark alternated for 12 hours. The experimental animals were adapted for 7 days before the experiment. During this period, the experimental animals were kept healthy and adapted to the environment, and free to eat and drink water.

[0137] The SD rats were randomly divided into groups, and the VEGF-trap polypeptides described in the present application (the amino acid sequences of which are shown in SEQ ID NO. 6 or SEQ ID NO. 7, respectively) were injected into the SD rats at a single dose of 10 mg / kg. SD rats administered with Aflibercept (which comprises the amino acid sequence shown in SEQ ID NO. 8) in the same manner were used as a control group. The blood sampling points were before administration and several predetermined time points after administration, and about 100 μL of blood was collected from the jugular vein. The collected blood was added to a centrifuge tube, and after standing at room temperature for 30-60 min, it was centrifuged at low temperature, and the serum was quickly separated and stored at -80℃. The concentration of VEGF-trap polypeptide in the serum of the test rats was determined by ELISA.

[0138] The results show that the AUC value of the VEGF-trap polypeptide of the present application is about 2.5 times that of the control group, and the T1 / 2 value is about 2 times that of the control group.

[0139] The foregoing detailed description has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the claims to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. It is intended that the scope of the claims and the patent be limited not with the detailed description. SEQUENCE LISTING <110> Suzhou Conexus Gerui Biotechnology Co., Ltd. <120> VEGF-trap polypeptide and application thereof <130> 0041-PA-024 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 105 <212> PRT <213> Homo sapiens <400> 1 Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val 1 5 10 15 Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val 20 25 30 Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys 35 40 45 Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys 50 55 60 Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln 65 70 75 80 Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn 85 90 95 Ser Thr Phe Val Arg Val His Glu Lys 100 105 <210> 2 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> K148A, R153A and K156A <400> 2 Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val 1 5 10 15 Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val 20 25 30 Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Ala 35 40 45 Lys Leu Val Asn Ala Asp Leu Ala Thr Gln Ser Gly Ser Glu Met Lys 50 55 60 Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln 65 70 75 80 Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn 85 90 95 Ser Thr Phe Val Arg Val His Glu Lys 100 105 <210> 3 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> K148T, R153T and K156T <400> 3 Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val 1 5 10 15 Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val 20 25 30 Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Thr 35 40 45 Lys Leu Val Asn Thr Asp Leu Thr Thr Gln Ser Gly Ser Glu Met Lys 50 55 60 Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln 65 70 75 80 Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn 85 90 95 Ser Thr Phe Val Arg Val His Glu Lys 100 105 <210> 4 <211> 100 <212> PRT <213> Homo sapiens <400> 4 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 1 5 10 15 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 20 25 30 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 35 40 45 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 50 55 60 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 65 70 75 80 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 85 90 95 Gln Thr Asn Thr 100 <210> 5<​​​​​​​​​​​​​​​​Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 6 <211> 431 <212> PRT <213> Artificial Sequence <220> <223> corresponds to K148A, R153A and K156A <400> 6 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 1 5 10 15 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 20 25 30 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 35 40 45 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 50 55 60 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 65 70 75 80 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 85 90 95 Gln Thr Asn Thr lie lie Asp Val Val Leu Ser Pro Ser His Gly lie 100 105 110 Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr 115 120 125 Glu Leu Asn Val Gly lie Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys 130 135 140 His Gin His Ala Lys Leu Val Asn Ala Asp Leu Ala Thr Gin Ser Gly 145 150 155 160 Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr lie Asp Gly Val Thr 165 170 175 Arg Ser Asp Gin Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met 180 185 190 Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr 195 200 205 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 210 215 220 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg 225 230 235 240 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 245 250 255 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 260 265 270 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 275 280 285 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 290 295 300 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 305 310 315 320 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 325 330 335 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 340 345 350 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 355 360 365 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 370 375 380 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 385 390 395 400 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 405 410 415 Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 420 425 430 <210> 7 <211> 431 <212> PRT <213> Artificial Sequence <220> <223> corresponds to K148T, R153T and K156T <400> 7 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 1 5 10 15 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 20 25 30 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 35 40 45 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 50 55 60 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 65 70 75 80 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 85 90 95 Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile 100 105 110 Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr 115 120 125 Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys 130 135 140 His Gln His Thr Lys Leu Val Asn Thr Asp Leu Thr Thr Gln Ser Gly 145 150 155 160 Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr 165 170 175 Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met 180 185 190 Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr 195 200 205 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 210 215 220 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 225 230 235 240 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 245 250 255 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 260 265 270 Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val 275 280 285 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 290 295 300 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr 305 310 315 320 lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 325 330 335 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys 340 345 350 Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser 355 360 365 Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 370 375 380 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 385 390 395 400 Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 405 410 415 Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 420 425 430 <210> 8 <211> 431 <212> PRT <213> Artificial Sequence <220> <223> Original VEGF-trap <400> 8 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 1 5 10 15 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 20 25 30 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 35 40 45 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 50 55 60 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 65 70 75 80 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 85 90 95 Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile 100 105 110 Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr 115 120 125 Glu Leu Asn Val Gly lie Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys 130 135 140 His Gin His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gin Ser Gly 145 150 155 160 Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr lie Asp Gly Val Thr 165 170 175 Arg Ser Asp Gin Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met 180 185 190 Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr 195 200 205 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 210 215 220 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg 225 230 235 240 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 245 250 255 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 260 265 270 Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val 275 280 285 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 290 295 300 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 305 310 315 320 Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 325 330 335 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys 340 345 350 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 355 360 365 Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 370 375 380 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 385 390 395 400 Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 405 410 415 Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 420 425 430

Claims

1. A VEGF-trap polypeptide comprising a mutant of the second Ig domain of VEGFR1 and the third Ig domain of VEGFR2, wherein the polypeptide, compared with the sequence shown in SEQ ID NO. 8, simultaneously has amino acid substitutions at positions 148, 153 and 156, wherein the mutant of the third Ig domain of VEGFR2 of the substituted VEGF-trap polypeptide consists of an amino acid sequence as shown in any one of SEQ ID NO. 2-3.

2. The VEGF-trap polypeptide according to claim 1, wherein the second Ig domain of VEGFR1 is composed of the amino acid sequence shown in SEQ ID NO.

4.

3. The VEGF-trap polypeptide according to claim 1, comprising the amino acid sequence shown in any one of SEQ ID NO. 6-7.

4. The VEGF-trap polypeptide according to claim 1, which is capable of binding VEGF.

5. An isolated nucleic acid molecule encoding the VEGF-trap polypeptide according to any one of claims 1-4.

6. A vector comprising the isolated nucleic acid molecule of claim 5.

7. A cell comprising or expressing the VEGF-trap polypeptide of any one of claims 1-4, the isolated nucleic acid molecule of claim 5, or the vector of claim 6.

8. A method for preparing the VEGF-trap polypeptide according to any one of claims 1-4, comprising culturing the cells of claim 7 under conditions expressing the VEGF-trap polypeptide according to any one of claims 1-4.

9. A pharmaceutical composition comprising the VEGF-trap polypeptide of any one of claims 1-4, the isolated nucleic acid molecule of claim 5 or the carrier of claim 6 and / or the cell of claim 7, and a pharmaceutically acceptable adjuvant.

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