Cancer treatment compositions containing vegr deep blockades and methods of making same

Abstract

The present invention relates to a cancer therapeutic composition for inhibiting angiogenesis, and a method for preparing the same. The angiogenesis inhibitor according to the present invention is a cancer therapeutic composition containing a fusion protein including a vascular endothelial growth factor binding domain of vascular endothelial growth factor receptor 1 (VEGFR1) and a b1 domain of neuropilin 1 (NRP1). The novel fusion protein is an angiogenesis inhibitor for blocking the binding of VEGF to its receptor on a cell membrane, and has an effect of inhibiting the proliferation of cancer cells as well as the growth and metastasis of cancer. In addition, the fusion protein can be used as an anticancer agent, and exhibits an effective anticancer effect at a lower dose than conventional angiogenesis inhibitors.

Description

Technical Field

[0001] This invention relates to a novel angiogenesis-inhibiting fusion protein that inhibits cancer growth and metastasis, and its preparation method. Background Technology

[0002] Angiogenesis is an essential process for normal organ growth and repair, and it is also a highly finely regulated process. Imbalances in these processes can lead to inflammation, cardiovascular disease, immune disorders, or malignant diseases. Vascular endothelial growth factor A (VEGFA) is a major inducer of angiogenesis and is known to be involved in cancer growth and progression. The VEGFA gene contains eight exons and produces at least six major VEGFA isoforms: VEGFA-121, VEGFA-145, VEGFA-165, VEGFA-183, VEGFA-189, and VEGFA-206. Among these, three major homologous proteins, VEGFA-121, VEGFA-165, and VEGFA-189, are secreted in cells, but their characteristics, bioavailability, and distribution differ from each other. However, their angiogenic function is generally considered to be regulated by VEGF. 165 adjust.

[0003] VEGFA binds to two tyrosine kinase receptors (VEGF receptor (VEGFR) 1 and VEGFR 2), participating not only in the development of new blood vessels but also preventing apoptosis to maintain blood flow. Angiogenesis signaling is mediated by the binding of VEGFA to VEGFR 2 (KDR) and its co-receptor (neurociliin-1 (NRP1)). Although VEGFR 2 is the main receptor involved in angiogenesis and vasculogenesis, VEGFR 1 has a much higher affinity for VEGFA than VEGFR 2.

[0004] VEGFA expression levels increase under physiologically essential conditions such as wound healing and hypoxia, and are also increased in pathological conditions such as proliferative retinopathy, arthritis, psoriasis, and cancer. Furthermore, VEGFA is an important mediator of tumor angiogenesis because it induces new blood vessel growth from peripheral blood vessels, allowing cancer cells to access oxygen and nutrients, and promoting cancer metastasis.

[0005] Many anti-VEGF drugs have been developed (such as antibody agents, aptamers, and tyrosine phosphorylation inhibitors). Recently, aflibercept, a fusion protein with high affinity for VEGFR1 and VEGFR2, has been highlighted as a next-generation drug. This drug consists of binding sites for both VEGFR1 and VEGFR2.

[0006] As a related prior art, Korean Patent Publication No. 1397088, entitled "Fusion protein for inhibiting angiogenesis and tumor cell proliferation and composition comprising the same", discloses a fusion protein comprising an angiogenesis inhibitor and a cancer-specific antibody, which has therapeutic effects on gastric cancer or breast cancer.

[0007] In relevant literature, Regeneron and Bayer Healthcare have reported on VEGF Trap-Eye (abflibercept; trade name, ...). The results of a Phase III clinical trial for macular degeneration showed that it can achieve therapeutic effects with a lower dose compared to ranibizumab.

[0008] Finely et al. developed a mouse model transplanted with human cancer cells to study cancer characteristics, including cancer growth rate and VEGF secretion, using VEGF-trap to predict VEGF secretion rate, suggesting its potential use as a live cancer model in the preclinical stage.

[0009] The inventors have prepared an angiogenesis inhibitor (VEGF deep blocker) VE GF De ep The invention comprises a VEEP fusion protein, which is a decoy receptor containing the Ig2 domain of VEGFR1 and the b1 domain of NRP1, to bind VEGFA with a higher affinity than VEGF-trap and block VEGFA signal transduction. Summary of the Invention

[0010] Technical issues

[0011] One object of the present invention is to provide a novel angiogenesis-inhibiting fusion protein for inhibiting cancer growth and metastasis, and a method for preparing the same.

[0012] One object of the present invention is to provide a recombinant DNA that encodes a novel angiogenesis-inhibiting fusion protein that inhibits cancer growth and metastasis.

[0013] Technical solution

[0014] Therefore, one aspect of the present invention provides a fusion protein characterized by comprising an Ig2 domain of vascular endothelial growth factor receptor 1 (VEGFR1) and a b1 domain of neuropilin 1 (NRP1). Compositions containing said protein are capable of inhibiting cancer growth and metastasis and can be used to treat cancer.

[0015] Invention Effects

[0016] The novel fusion protein according to the present invention is an angiogenesis inhibitor that blocks the binding of vascular endothelial growth factor to its receptors on the cell membrane and inhibits the proliferation, growth, and metastasis of cancer cells. Compositions containing the fusion protein can be effectively used as anticancer agents. Furthermore, compared to conventional angiogenesis inhibitors, this composition exhibits higher anticancer efficacy at lower doses. Attached Figure Description

[0017] Figure 1 This is a schematic diagram showing the VEGFR1-NRP1-Fc fusion protein.

[0018] Figure 2 The expression of the VEEP fusion protein was shown.

[0019] Figure 3 The results are shown by comparing the anticancer effects of VEEP and the control VEGF-trap in an LLC mouse model.

[0020] Figure 4 The results of the endpoint analysis in the LLC mouse model on day 30 after VEEP treatment are shown. Detailed Implementation

[0021] The mode of the present invention

[0022] The invention will be described in more detail below with reference to embodiments. However, these embodiments are for illustrative purposes only, and the invention is not intended to limit these embodiments.

[0023] A first embodiment of the present invention provides a fusion protein characterized by comprising an Ig2 domain of vascular endothelial growth factor receptor 1 (VEGFR1) and a b1 domain of neuropilin 1 (NRP1). More specifically, VEGFR1 may be, but is not limited to, represented by SEQ ID NO:1. The Ig2 domain and the b1 domain of neuropilin 1 (NRP1) may be in various forms, and preferably, the Ig2 domain and the b1 domain of neuropilin 1 (NRP1) may be represented by SEQ ID NO:2. The fusion protein may further comprise an Fc domain of an immunoglobulin, and various Fc domains may be used. Preferably, the Fc domain may be represented by SEQ ID NO:3. The fusion protein may further comprise a leader sequence for expression, and various leader sequences may be used. Preferably, the leader sequence may be represented by SEQ ID NO:4. More preferably, the fusion protein may have the amino acid sequence of SEQ ID NO:5.

[0024] A second embodiment of the present invention provides a composition for cancer treatment comprising the aforementioned fusion protein. Those skilled in the art can add various forms of drug delivery materials, excipients, stabilizers, etc., to the composition; these formulations are also within the scope of the present invention.

[0025] A third embodiment of the invention provides a DNA fragment encoding the protein. Those skilled in the art can generate various DNA sequences encoding the fusion protein of the present invention based on the degeneracy of the genetic code. Finally, any type of DNA sequence encoding the protein of the present invention is within the scope of this invention.

[0026] A fourth embodiment of the present invention provides a transformant obtained by transformation / transfection using the above-described recombinant vector. Various cells can be used for the transformant; human cells are preferred, with HEK293E cells being the most preferred.

[0027] A fifth embodiment of the present invention provides a method for preparing a protein, comprising culturing transformed cells according to the present invention and separating the fusion protein from the cell culture medium.

[0028] method

[0029] 1. Preparation of VEGFR1-NRP1-Fc fusion protein

[0030] DNA fragments encoding the Ig2 domain of VEGFR1 were obtained by PCR of synthetic DNA (Ezbio; EZbio), and DNA fragments encoding the b1 domain of NRP1 were obtained from the Human Gene Bank of the Korea Institute of Bioscience and Biotechnology. The DNA fragments containing both fragments were ligated to human Fc DNA to generate a fusion protein. The cloned DNA was transfected into HEK293E cells, and the culture medium was purified using Protein A resin. The concentration of the purified protein was calculated by measuring the A280 absorbance.

[0031] 2. In vitro affinity assay (ELISA)

[0032] VEEP and VEGF-trap were added to VEGF-coated 96-well plates. After several washes, HRP-coupled anti-human Fc was added, along with stable TMB, and absorbance was measured at 450 nm.

[0033] 3. VEEP animal experiments

[0034] Animal models of target diseases and solid tumors

[0035] As an animal model to verify the anticancer effect of the drug, 6-week-old male C57BL / 6 mice were purchased from Koatech (Pyeongtaek, South Korea). The animals underwent a one-week acclimatization period in accordance with animal experiment ethics guidelines. LLC (Lewis lung cancer) cell lines were purchased from ATCC and used to induce solid tumors in the mice.

[0036] In vivo determination of the anticancer effect and endpoint of VEEP

[0037] 1x10 6 LLC cells were subcutaneously injected into the right flank region of 6-week-old C57BL / 6 mice. Starting from day 9 post-LLC injection, 25 mg / kg VEEP and 25 mg / kg VEGF-trap were subcutaneously injected every 3 days for a total of 8 administrations. Tumor proliferation was measured every 3–4 days for 30 days, and tumor survival was observed. Tumor size was measured using electronic digital calipers to measure orthogonal length (major axis) and width (minor axis). The result was expressed as Π / 6 × (length). 2 The volume of the solid tumor was calculated using the × width. On day 30, mice were sacrificed, the solid tumors were extracted, and weighed using a microbalance.

[0038] <Example 1> Fusion Protein

[0039] The purified VEGFR1-NRP1-Fc fusion protein was analyzed by SDS-PAGE under both reducing and non-reducing conditions. Monomeric and dimer bands were observed at positions corresponding to their predicted sizes. Figure 2 ).

[0040] Example 2: Affinity Analysis

[0041] To determine the affinity of the fusion protein VEEP for VEGFA, the binding affinity of VEGF to its receptor or blocker was analyzed using ELISA. For the affinity assay, VEGF was selected from VEGFA homologous proteins. 165 For controls, naturally expressed VEGFA receptors in cells (such as VEGFR1, VEGFR2, NRP1, and NRP2) were compared and analyzed with commercially available blockers (such as VEGF-trap, bevacizumab, and ranibizumab). The results showed that VEGF-trap inhibited the activity of VEGF receptors. 165 The binding affinity for VEGF was highest in the control group. Conversely, compared to all controls, the fusion protein VEEP of the present invention showed a high affinity for VEGF. 165 It exhibits the highest binding affinity and shows a binding affinity 10 times higher than VEGF-trap (Table 1).

[0042] [Table 1] Binding Affinity

[0043]

[0044]

[0045] <Example 3> Anticancer Efficacy Test

[0046] To measure the anticancer effect of the VEEP fusion protein prepared and purified according to the present invention, solid tumor growth and survival were observed using an LLC mouse model. As a result of solid tumor growth measurement in LLC mice, the average tumor size in the control group without inhibitor treatment was 13105 mm. 3 In the VEGF-trap treatment group, the average tumor size decreased to 9479 mm. 3 However, the reduction effect varied considerably among different mouse models. Conversely, the average size of solid tumors in VEEP-treated mice was measured to be 5872 mm. 3 Compared with the control group without the blocking agent, it showed a significant increase in anti-cancer effect. Figure 3 Furthermore, VEEP exhibits superior anticancer effects compared to VEGF-trap. The survival rate of mice treated with the VEEP fusion protein was 80%, superior to the VEGF-trap treatment control (80%) or the untreated control (0%). Therefore, it is believed that the VEEP fusion protein prepared in this invention can be effectively applied to patients who cannot be treated with VEGF-trap.

[0047] <Example 4> Endpoint Analysis

[0048] Tumor size and weight were measured on day 30 after VEEP treatment in LLC mice. The solid tumor weight in LLC mice treated with VEEP fusion protein was significantly lower (5.15 g) compared to that in mice treated with VEGF-trap (7.49 g). Figure 3 Mice treated with VEGF-trap exhibited significant differences in tumor size. Mice treated with VEEP had a smaller average tumor size than those treated with VEGF-trap, and the degree of tumor size reduction was similar among the VEEP-treated mice. Figure 4 Therefore, based on the results of the above analysis of anticancer effects, it is considered that the VEEP blocker of the present invention is effective in patients who do not respond to treatment for VEGF-trap.

[0049] References

[0050] 1、Arcondeguy,T.,et al.(2013).VEGF-A mRNA processing,stability andtranslation:a paradigm for intricate regulation of gene expression at thepost-transcriptional level.Nucleic Acids Res 41,7997-8010.

[0051] 2、Carmeliet,P.(2005)Angiogenesis in life,disease and medicine.Nature438,932-936.

[0052] 3、Ferrara,N.(2005)The role of VEGF in the regulation of physiologicaland pathological angiogenesis.EXS 209-231.

[0053] 4、Bhagat,N.,et al.(2009)Diabetic macular edema:pathogenesis andtreatment.Surv.Ophthalmol.54(1),1-32.

[0054] 5、Houck,K.A.,et al.(1991)The vascular endothelial growth factorfamily:identification of a fourth molecular species and characterization ofalternative splicing of RNA.Mol.Endocrinol.5,1806.1814.

[0055] 6、Vempati,P.,Popel,A.S.and Mac Gabhann,F.(2011)Formation of VEGFisoform-specific spatial distributions governing angiogenesis:computationalanalysis.BMC Syst.Biol.5,59.

[0056] 7、Houck,K.A.,et al.(1992)Dual regulation of vascular endothelialgrowth factor bioavailability by genetic and proteolyticmechanisms.J.Biol.Chem.267,26031-26037.

[0057] 8、Carmeliet,P.(2005)VEGF as a key mediator of angiogenesis incancer.Oncology 69(Suppl.3),4-10.

[0058] 9、Ferrara,N.,Gerber,H.P.and LeCouter,J.(2003)The biology of VEGF andits receptors.Nat.Med.9,669-676.

[0059] 10、Ferrara,N.(1999)Role of vascular endothelial growth factor in theregulation of angiogenesis.Kidney Int.56,794-814.

[0060] 11、Ferrara,N.(2002)VEGF and the quest for tumour angiogenesisfactors.Nat.Rev.Cancer 2,795-803.

[0061] 12、Folkman,J.(1995)Angiogenesis in cancer,vascular,rheumatoid andother disease.Nat.Med.1,27-31.

[0062] 13、Stewart,M.W.(2012).Aflibercept(VEGF Trap-eye):the newest anti-VEGFdrug.Br J Ophthalmol 96,1157-1158.

[0063] 14. Finley, SD, Dhar, M., and Popel, AS (2013). Compartment model predicts VEGF secretion and investigates the effects of VEGF trap in tumor-bearing mice. Front Oncol 3,196.

[0064] Sequence Listing 1: Sequences of the VEGFR1 portion

[0065] VSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNT(VEGFR1)

[0066] Sequence Listing 2: Sequences of the Nrp1 portion

[0067] SaiakegfSanysvLQSsvsedfkcmealgmesgeihsdqitassqystnwsaersrlnypengwtpgedsyrewiqvdlgllrfvtavgtqgaisketkkkyyvktykidvssngedwitikegnkpvlfqgntnptdvvvavfpkplitrfvrikpatwetgismrfevygckit

[0068] Sequence Listing 3: Immunoglobulin Fc Domain

[0069] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0070] Sequence Listing 4: Leader Sequence

[0071] MYLGLNYVFIVFLLNGVQS

[0072] Sequence Listing 5: Full-Length Sequence

[0073] MYLGLNYVFIVFLLNGVQSVSDTGPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTSaiakegfSan ysvLQSsvsedfkcmealgmesgeihsdqitassqystnwsaersrlnypengwtpgedsyrewiqvdlgllrfvtavgtqgaisketkkkyyvktykidvssngedwitikegnkpvlfqgntnptdvvv avfpkplitrfvrikpatwetgismrfevygckitDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK <110> Ibanchos LLC <120> Cancer therapeutic composition containing a deep VEGF blocker that inhibits tumor angiogenesis and its preparation method <130> PP160001 <160> 5 <170> KopatentIn 2.0 <210> 1 <211> 101 <212> PRT <213> VEGFR1 in Homo sapiens <400> 1 Val Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro 1 5 10 15 Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg 20 25 30 Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp 35 40 45 Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly 50 55 60 Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys 65 70 75 80 Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His 85 90 95 Arg Gln Thr Asn Thr 100 <210> 2 <211> 177 <212> PRT <213> Homo sapiens partial Nrp1 sequence <400> 2 Ser Ala Ile Ala Lys Glu Gly Phe Ser Ala Asn Tyr Ser Val Leu Gln 1 5 10 15 Ser Ser Val Ser Glu Asp Phe Lys Cys Met Glu Ala Leu Gly Met Glu 20 25 30 Ser Gly Glu Ile His Ser Asp Gln Ile Thr Ala Ser Ser Gln Tyr Ser 35 40 45 Thr Asn Trp Ser Ala Glu Arg Ser Arg Leu Asn Tyr Pro Glu Asn Gly 50 55 60 Trp Thr Pro Gly Glu Asp Ser Tyr Arg Glu Trp Ile Gln Val Asp Leu 65 70 75 80 Gly Leu Leu Arg Phe Val Thr Ala Val Gly Thr Gln Gly Ala Ile Ser 85 90 95 Lys Glu Thr Lys Lys Lys Tyr Tyr Val Lys Thr Tyr Lys Ile Asp Val 100 105 110 Ser Ser Asn Gly Glu Asp Trp Ile Thr Ile Lys Glu Gly Asn Lys Pro 115 120 125 Val Leu Phe Gln Gly Asn Thr Asn Pro Thr Asp Val Val Val Ala Val 130 135 14​​​​​​​​​​​​​​​​​​​​​​​​​​ 20 25 30 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 Glu Glu Met 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 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 4 <211> 19 <212> PRT <213> Homo sapiens pre-sequence <400> 4 Met Tyr Leu Gly Leu Asn Tyr Val Phe Ile Val Phe Leu Leu Asn Gly 1 5 10 15 Val Gln Ser <210> 5 <211> 524 <212> PRT <213> Artificial sequence <220> <223> VEEP6 fusion protein <400> 5 Met Tyr Leu Gly Leu Asn Tyr Val Phe Ile Val Phe Leu Leu Asn Gly 1 5 10 15 Val Gln Ser Val Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser 20 25 30 Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile 35 40 45 Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe 50 55 60 Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser 65 70 75 80 Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu 85 90 95 Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr 100 105 110 Leu Thr His Arg Gln Thr Asn Thr Ser Ala Ile Ala Lys Glu Gly Phe 115 120 125 Ser Ala Asn Tyr Ser Val Leu Gln Ser Ser Val Ser Glu Asp Phe Lys 130 135 140 Cys Met Glu Ala Leu Gly Met Glu Ser Gly Glu Ile His Ser Asp Gln 145 150 155 160 Ile Thr Ala Ser Ser Gln Tyr Ser Thr Asn Trp Ser Ala Glu Arg Ser 165 170 175 Arg Leu Asn Tyr Pro Glu Asn Gly Trp Thr Pro Gly Glu Asp Ser Tyr 180 185 190 Arg Glu Trp Ile Gln Val Asp Leu Gly Leu Leu Arg Phe Val Thr Ala 195 200 205 Val Gly Thr Gln Gly Ala Ile Ser Lys Glu Thr Lys Lys Lys Tyr Tyr 210 215 220 Val Lys Thr Tyr Lys Ile Asp Val Ser Ser Asn Gly Glu Asp Trp Ile 225 230 235 240 Thr Ile Lys Glu Gly Asn Lys Pro Val Leu Phe Gln Gly Asn Thr Asn 245 250 255 Pro Thr Asp Val Val Val Ala Val Phe Pro Lys Pro Leu Ile Thr Arg 260 265 270 Phe Val Arg Ile Lys Pro Ala Thr Trp Glu Thr Gly Ile Ser Met Arg 275 280 285 Phe Glu Val Tyr Gly Cys Lys Ile Thr Asp Lys Thr His Thr Cys Pro 290 295 300 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 305 310 315 320 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 325 330 335 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 340 345 350 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 355 360 365 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 370 375 380 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 385 390 395 400 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 405 410 415 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 420 425 430 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 435 440 445 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 450 455 460 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 465 470 475 480 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 485 490 495 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 500 505 510 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 515 520

Claims

1. A fusion protein comprising an Ig2 domain of vascular endothelial growth factor receptor 1 (VEGFR1), a b1 domain of neuropilin 1 (NRP1), and an Fc domain of an immunoglobulin, wherein the Ig2 domain of VEGFR1, the b1 domain of NRP1, and the Fc domain are fused in order from the N-terminus to the C-terminus, wherein the Ig2 domain of VEGFR1 is composed of the amino acid sequence shown in SEQ ID NO:1, the b1 domain of NRP1 is composed of the amino acid sequence shown in SEQ ID NO:2, and the Fc domain of the immunoglobulin is composed of the amino acid sequence shown in SEQ ID NO:

3.

2. The fusion protein of claim 1, wherein the fusion protein further comprises the amino acid sequence shown in SEQ ID NO:

4.

3. The fusion protein of claim 2, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:

5.

4. A composition for cancer treatment, said composition comprising the fusion protein according to any one of claims 1 to 3.

5. A DNA fragment encoding the fusion protein according to any one of claims 1 to 3.

6. A recombinant vector comprising the DNA fragment of claim 5.

7. Transformed cells, said transformed cells obtained by transforming host cells with the recombinant vector of claim 6.

8. The transformed cells of claim 7, wherein, The host cell was HEK293E.

9. A method for preparing proteins for cancer treatment, the method comprising: (a) Culturing the transformed cells of claim 7 to produce the fusion protein; as well as (b) Isolate the fusion protein from the culture solution of the cells.

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