CN107488233A - 治疗感染性与恶性疾病的标靶化学治疗药物的形成方法 - Google Patents
治疗感染性与恶性疾病的标靶化学治疗药物的形成方法 Download PDFInfo
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Abstract
本发明提供一种融合蛋白，其与前驱药物结合而用于癌症治疗，其中该融合蛋白包含(i)配体，其与生长因子受体特异性结合，(ii)前驱药物酵素，其可将该前驱药物转换为活性药物成分，以及(iii)链结子，其位于该配体与该前驱药物酵素之间。本发明亦提供一种DNA建构体，其包含(i)编码与生长因子受体特异性结合的配体的核苷酸序列，以及(ii)编码前驱药物酵素的核苷酸序列。
Description
本申请是申请人为台北荣民总医院，发明名称为“治疗感染性与恶性疾病的标靶化学治疗药物的形成方法”，国家申请号为201210181043.5的发明专利申请的分案申请。
技术领域
本发明涉及关于一种融合蛋白，其用于结合前驱药物以治疗癌症。
背景技术
人类表皮癌的主要特征在于表皮生长因子受体(EGFR)家族的生长因子与受体的功能活化。由生长因子-生长因子受体(EGF-EGFR axis)所主导的信息传递途径在癌细胞的增生、存活、转移、与血管新生中扮演了重要的角色(Ciardiello,F.,and Tortora,G.EGFRantagonists in cancer treatment,N Engl J Med 358,1160-1174,2008.)。
相对较为安全的前驱药物5-氟胞嘧啶(5-fluorocytosine,5-FC)可通过胞嘧啶脱氨酶(cytosine deaminase)转化为常用的化疗药物5-氟尿嘧啶(5-fluorouracil,5-FU)。相较于5-FC，5-FU的毒性为1000倍(Miller,C.R.,Williams,C.R.,Buchsbaum,D.J.,andGillespie,G.Y.Intratumoral 5-fluorouracil produced by cytosine deaminase/5-fluorocytosine gene therapy is effective for experimental humanglioblastomas,Cancer Res 62,773-780,2002；Hamstra,D.A.,Rice,D.J.,Fahmy,S.,Ross,B.D.,and Rehemtulla,A.Enzyme/prodrug therapy for head and neck cancerusing a catalytically superior cytosine deaminase,Hum Gene Ther 10,1993-2003,1999.)。许多表皮生长因子受体过度表达的癌症，例如头颈癌、胰脏癌、大肠直肠癌等，经常以5-FU治疗。然而5-FU的高全身毒性为重要问题。
因此，需要标靶型前驱药物/药物系统以将化疗药物集中于肿瘤所在位置。
发明内容
本发明提供一种融合蛋白，其与前驱药物结合以治疗癌症，其中所述的融合蛋白包含：
(i)配体，其与选自由促血管生成素(Angiopoietin)、大脑衍生神经营养因子(Brain Derived Neurotrophic Factor)、睫状神经营养因子(Ciliary NeurotrophicFactor)、表皮生长因子(Epidermal Growth Factor)、纤维母细胞生长因子(FibroblastGrowth Factor)、神经胶质衍生神经营养因子(Glial Derived Neurotrophic Factor)、肝细胞生长因子(Hepatocyte Growth Factor)、调蛋白(Heregulin)、类胰岛素生长因子(Insulin-like Growth Factor)、介白素(Interleukin)、角质细胞生长因子(Keratinocyte Growth Factor)、巨噬细胞发炎蛋白(Macrophage InflammatoryProtein)、巨噬细胞趋化蛋白(Macrophage Chemoattractant Protein)、神经生长因子(Nerve growth factor)、神经营养因子(Neurotrophin)、血小板衍生生长因子(PlateletDerived Growth Factor)、色素上皮衍生因子(Pigment Epithelium Derived Factor)、血小板因子(Platelet Factor)、基质细胞衍生因子(Stromal Cell Derived Factor)、干细胞因子(Stem Cell Factor)、基质金属蛋白酶组织抑制因子(Tissue inhibitor ofmetalloproteinase)、转型生长因子(Transforming Growth Factor)、肿瘤坏死因子(Tumor Necrosis Factor)、以及血管内皮生长因子(Vascular Endothelial GrowthFactor)所组成群组的生长因子受体特异性结合；
(ii)前驱药物酵素，其可将所述的前驱药物转化为活性药物成分；以及
(iii)连接子，其位于该配体与该前驱药物酵素之间。
在一实施例中，本发明提供此处所述的融合蛋白与前驱药物结合以制备用于治疗癌症的药物组合物的用途。
在另一实施例中，本发明提供一种DNA建构体，其包含：
(i)一编码与生长因子受体特异性结合的配体的核苷酸序列，其中该配体选自由促血管生成素、大脑衍生神经营养因子、睫状神经营养因子、表皮生长因子、纤维母细胞生长因子、神经胶质衍生神经营养因子、肝细胞生长因子、调蛋白、类胰岛素生长因子、介白素、角质细胞生长因子、巨噬细胞发炎蛋白、巨噬细胞趋化蛋白、神经生长因子、神经营养因子、血小板衍生生长因子、色素上皮衍生因子、血小板因子、基质细胞衍生因子、干细胞因子、基质金属蛋白酶组织抑制因子、转型生长因子、肿瘤坏死因子、以及血管内皮生长因子所组成的群组；以及
(ii)一编码前驱药物酵素的核苷酸序列。
附图说明
实施例显示于附图中用以说明本发明。然而应理解的是，本发明不限于此处所显示的较佳实施例。在附图中：
图1为本发明的酵母菌表达建构体(yeast expression constructs)的示意图。
图2a为pPICZ-α-Fcy-hEGF-myc-his6的DNA建构体与序列(4198bp)。
图2b为pPICZ-α-Fcy-Fur-hEGF-myc-his6的DNA建构体与序列(4951bp)。
图2c为针对酵母菌表达宿主而优化的pPICZ-α-Fcy-(VPGVG)2-hEGF-cMyc-KKKRKV-6His(4213bp)的DNA建构体与序列。
图2d为pPICZ-α-Fcy-hVEGFa-myc-his6的DNA建构体与序列(4384bp)。
图2e为pPICZ-α-Fcy-Fur-hVEGFa-myc-his6的DNA建构体与序列(5137bp)。
图3为本发明的E.coli表达建构体(E.coli expression constructs)的示意图。
图4a为pET56-Fcy-hEGF-His6的DNA建构体与序列(5785bp)。
图4b为pET56-Fcy-Fur-hEGF-His6的DNA建构体与序列(6538bp)。
图4c为pET56-Fcy-hVEGFa-His6的DNA建构体与序列(5971bp)。
图4d为pET56-Fcy-Fur-hVEGFa-His6的DNA建构体与序列(6742bp)。
图4e为pET56-Fcy-Fur-hVEGFc-His6的DNA建构体与序列(6685bp)。
图4f为pET56-Fcy-mVEGFa-His6的DNA建构体与序列(5977bp)。
图4g为pET56-Fcy-Fur-mVEGFa-His6的DNA建构体与序列(6730bp)。
图4h为pET56-Fcy-Fur-mVEGFc-His6的DNA建构体与氨基酸序列(6685bp)。
图5显示经纯化的his6标记的Fcy、hEGF、以及Fcy-hEGF的考马斯蓝(Coomassieblue)染色凝胶与西方墨点法的结果。
图6显示(a)经纯化的蛋白与经固定的表皮生长因子受体(EGFR)于体外结合的示意图，以及(b)his6标记的Fcy、hEGF、以及Fcy-hEGF与EGFR于体外结合的饱和曲线(saturation curves)与亲和力(affinity)。
图7显示流式细胞分析的结果，用以评估抗EGFR抗体、经纯化的his6标记的Fcy、hEGF、以及Fcy-hEGF与A431(a)、MCF-7(b)、MDA-468(c)、以及MDA-231(d)细胞的结合程度。
图8显示通过经纯化的Fcy与Fcy-hEGF于体外将5-FC转化为5-FU的酵素活性结果。
图9a至图9d显示MTT分析的结果，其用以评估受到Fcy-hEGF与5-FC影响的A431、MDA-468、MDA-231、MCF-7、以及HUVEC的细胞存活率。
图10显示通过Fcy-EGF增进5-FC细胞毒性的结果。
图11显示MTT分析的结果，其用以评估在递增浓度的5-FC存在下受到Fcy-hEGF影响的HCT116与LS174T的细胞存活率。
图12显示5-FC结合Fcy-hEGF对于HCT116大肠癌于体内生长的抑制效果。
图13为癌症标靶前驱药物-生长因子融合建构体的克隆策略，其中“生长因子”部分的DNA序列可编码选自下列蛋白所组成群组的任一者：促血管生成素(Angiopoietin)、大脑衍生神经营养因子(Brain Derived Neurotrophic Factor)、睫状神经营养因子(Ciliary Neurotrophic Factor)、表皮生长因子(Epidermal Growth Factor)、纤维母细胞生长因子(Fibroblast Growth Factor)、神经胶质衍生神经营养因子(Glial DerivedNeurotrophic Factor)、肝细胞生长因子(Hepatocyte Growth Factor)、调蛋白(Heregulin)、类胰岛素生长因子(Insulin-like Growth Factor)、介白素(Interleukin)、角质细胞生长因子(Keratinocyte Growth Factor)、巨噬细胞发炎蛋白(MacrophageInflammatory Protein)、巨噬细胞趋化蛋白(Macrophage Chemoattractant Protein)、神经生长因子(Nerve growth factor)、神经营养因子(Neurotrophin)、血小板衍生生长因子(Platelet Derived Growth Factor)、色素上皮衍生因子(Pigment Epithelium DerivedFactor)、血小板因子(Platelet Factor)、基质细胞衍生因子(Stromal Cell DerivedFactor)、干细胞因子(Stem Cell Factor)、基质金属蛋白酶组织抑制因子(Tissueinhibitor of metalloproteinase)、转型生长因子(Transforming Growth Factor)、肿瘤坏死因子(Tumor Necrosis Factor)、以及血管内皮生长因子(Vascular EndothelialGrowth Factor)；以及“前驱药物酵素”部分的DNA序列可编码选自由下列蛋白所组成群组的任一者：酒精脱氢酶(Alcohol dehydrogenase)、碱性磷酸酶(Alkaline phosphatase)、乙型内酰胺酶(β-lactamase)、乙型葡糖苷酸酶(β-glucoronidase)、羧酸酯酶(Carboxyesterases)、羧肽酶A(Carboxypeptidase A)、羧肽酶G2(Carboxypeptidase G2)、胞嘧啶脱氨酶(Cytosine deaminase)、胞嘧啶脱氨酶尿嘧啶磷酸核糖转移酶(Cytosinedeaminase-uracil phosphoribosyltransferase)、糖苷酶(Glycosidases)、硝基还原酶(Nitroreductase)、青霉素酰胺酶(Penicillin amidase)、胸腺嘧啶激酶(thymidinekinase)。
具体实施方式
除非另有定义，此处所使用的技术性与科学性词汇具有本发明所属技术领域的技术人员所共同理解的意义。除非另有定义，当用于此处时下列词汇具有所认定的意义。
此处所使用的冠词“一”或“该”代表其语法上的意义为一或大于一(亦即至少为一)。举例而言，一元素代表一元素或大于一元素。
用于此处时，一“个体”为具有癌症或可能具有癌症的任何动物，例如哺乳类且尤其包含人类。
此处所使用的词汇“聚胜肽”指通过胜肽键连结的氨基酸残基所组成的分子或聚合物。聚胜肽可由使用例如自动聚胜肽合成仪而合成。此处所使用的词汇“蛋白”典型地代表大型聚胜肽。“胜肽”典型地代表较短的聚胜肽。
当用于此处时，“融合蛋白”为透过结合编码二个或多个原始为不同蛋白的基因所创造的蛋白。
当用于此处时，“前驱药物”指在给药时，必须历经前驱药物酵素的化学转化才成为活性药剂的化合物。
当用于此处时，“前驱药物酵素”为能够将前驱药物转化为活性药剂的酵素。
当用于此处时，“连接子”为短片段的聚胜肽。
当用于此处时，“聚核苷酸”或“核酸”指核苷酸单元组成的聚合物。聚核苷酸包含自然产生的核酸，例如脱氧核糖核酸(DNA)与核糖核酸(RNA)以及核酸类似物，包含非自然合成的核酸，例如重组聚核苷酸。聚核苷酸可利用例如自动DNA合成仪来合成。词汇“核酸”典型地代表大型聚核苷酸。将被理解的是，当以DNA序列代表核苷酸序列时(亦即A、T、C、G)，其同时包含RNA序列(亦即A、U、C、G)，其中U取代T。词汇“cDNA”代表与mRNA互补或完全一致的DNA，无论是单股或双股形式。
此处所使用的词汇“编码”代表聚核苷酸中特定核苷酸序列的固有性质(例如基因、cDNA、或mRNA)，以在生物过程中作为其它聚合物与巨分子的模板，该聚合物与巨分子具有核苷酸(亦即rRNA、tRNA、与mRNA)的定义序列、或是氨基酸的定义序列，以及由此产生的生物性质。因此，若由一基因转录所产生的mRNA于细胞或其它生物系统中进行转译而产生蛋白，则该基因可为此蛋白的编码。所属技术领域的技术人员可理解的是，由于基因密码简并的结果，因此许多不同的聚核苷酸与核酸可编码出相同的聚胜肽。亦可理解的是，所属技术领域的技术人员可使用常规使用的技术取代核苷酸而不影响所述聚核苷酸所编译出的聚胜肽序列，此反映了欲表达此聚胜肽的任何特定宿主有机体的密码子使用偏好。因此，除非另有特别指出，否则“可编译氨基酸序列的聚核苷酸”包含可编译出相同氨基酸序列的任何互为简并的聚核苷酸序列。编码蛋白与RNA的聚核苷酸可包含内含子(intron)。
此处所使用的词汇“重组聚核苷酸”意指具有非自然性相互结合序列的聚核苷酸。重组聚核苷酸可以载体(vector)的形式存在。“载体”可包含目标核苷酸序列以及调控序列。载体可用以表达给定的核苷酸序列或维持给定的核苷酸序列以将此序列复制、操控、或是于不同位置间转移(例如于不同有机体之间)。载体可为了上述的目的而导入适合的宿主细胞。
载体的范例包含但不限于质体(plasmid)、黏质体(cosmid)、YAC、或PAC。典型地，在载体中给定的核苷酸序列与调控序列操作性地连结，故当载体导入宿主细胞中时，给定的核苷酸序列可在调控序列的控制下于宿主细胞中表达。举例而言，调控序列可包含促进子(promoter)、起始密码子(start codon)、复制区(replication region)序列、增强子(enhancer)、操纵子(operator)序列、分泌信号(secretion signal)序列(例如IL2信号胜肽)、以及其它调控序列。较佳地，载体可进一步包含标记序列(例如抗生素抗性标记序列)以便于进行筛选。
氨基酸可由三字母或单字母表达。表1列出了标准氨基酸缩写。
表1：标准氨基酸缩写
| 氨基酸 | 3字母 | 1字母 |
| Alanine(丙氨酸) | Ala | A |
| Arginine(精氨酸) | Arg | R |
| Asparagine(天冬酰氨酸) | Asn | N |
| Aspartic acid(天冬氨酸) | Asp | D |
| Cysteine(半胱氨酸) | Cys | C |
| Glutamic acid(谷氨酸) | Glu | E |
| Glutamine(谷氨酰氨) | Gln | Q |
| Glycine(甘氨酸) | Gly | G |
| Histidine(组氨酸) | His | H |
| Isoleucine(异亮氨酸) | Ile | I |
| Leucine(亮氨酸) | Leu | L |
| Lysine(赖氨酸) | Lys | K |
| Methionine(甲硫氨酸) | Met | M |
| Phenylalanine(苯丙氨酸) | Phe | F |
| Proline(脯氨酸) | Pro | P |
| Serine(丝氨酸) | Ser | S |
| Threonine(苏氨酸) | Thr | T |
| Tryptophan(色氨酸) | Trp | W |
| Tyrosine(酪氨酸) | Tyr | Y |
| Valine(缬氨酸) | Val | V |
本发明的特征在于一与前驱药物结合以治疗癌症的融合蛋白，其中该融合蛋白包含(i)与生长因子受体(growth factor receptor)特异性结合的配体(ligand)，其选自由促血管生成素、大脑衍生神经营养因子、睫状神经营养因子、表皮生长因子、纤维母细胞生长因子、神经胶质衍生神经营养因子、肝细胞生长因子、调蛋白、类胰岛素生长因子、介白素、角质细胞生长因子、巨噬细胞发炎蛋白、巨噬细胞趋化蛋白、神经生长因子、神经营养因子、血小板衍生生长因子、色素上皮衍生因子、血小板因子、基质细胞衍生因子、干细胞因子、基质金属蛋白酶组织抑制因子、转型生长因子、肿瘤坏死因子、以及血管内皮生长因子所组成的群组；(ii)前驱药物酵素，其可将该前驱药物转化为活性药物成分；以及(iii)连接子，其位于该配体与该前驱药物酵素之间。
根据本发明的一实施例，该融合蛋白包含的前驱药物酵素选自由酒精脱氢酶、碱性磷酸酶、乙型内酰胺酶、乙型葡糖苷酸酶、羧酸酯酶、羧肽酶A、羧肽酶G2、胞嘧啶脱氨酶、胞嘧啶脱氨酶尿嘧啶磷酸核糖转移酶、糖苷酶、硝基还原酶、青霉素酰胺酶、胸腺嘧啶激酶所组成的群组。
在本发明的一实施例中，该前驱药物酵素位于该融合蛋白的氨基端且该配体位于该融合蛋白的羧基端。
在本发明的特定实施例中，该配体为表皮生长因子(EGF)或血管内皮细胞生长因子(VEGF)。具体地说，此处所使用的”配体”具有选自由SEQ ID NO:1 1(EGF)与SEQ ID NO:2-5(VEGF)所组成群组的氨基酸序列。
根据一实施例，用以治疗癌症的前驱药物为5-FC且前驱药物酵素为胞嘧啶脱氨酶或与尿嘧啶磷酸核糖转移酶融合的胞嘧啶脱氨酶。具体地说，此处所使用的“前驱药物酵素”具有SEQ ID NO:6(“胞嘧啶脱氨酶”或“Fcy”)或SEQ ID NO:7(“与尿嘧啶磷酸核糖转移酶融合的胞嘧啶脱氨酶”或“Fcy-Fur”)的氨基酸序列。
在本发明的一实施例中，该融合蛋白具有选自由SEQ ID NO:8(Fcy-hEGF)、SEQ IDNO:9(Fcy-Fur-hEGF)、SEQ ID NO:10(Fcy-(VPGVG)2-hEGF-cMyc-KKKRKV)、SEQ ID NO:11(Fcy-hVEGFa)、SEQ ID NO:12(Fcy-Fur-hVEGFa)、SEQ ID NO:13(Fcy-hEGF)、SEQ ID NO:14(Fcy-Fur-hEGF)、SEQ ID NO:15(Fcy-hVEGFa)、SEQ ID NO:16(Fcy-Fur-hVEGFa)、SEQ IDNO:17(Fcy-Fur-hVEGFc)、SEQ ID NO:18(Fcy-mVEGFa)、SEQ ID NO:19(Fcy-Fur-mVEGFa)、以及SEQ ID NO:20(Fcy-Fur-mVEGFc)所组成群组的氨基酸序列。
在另一实施例中，本发明提供一种此处所描述的融合蛋白与前驱药物结合以制备用于癌症治疗的医药组合物的用途。
在又一实施例中，本发明提供一种DNA建构体包含(i)一编码与生长因子受体特异性结合的配体的核苷酸序列，其中该配体为选自由促血管生成素、大脑衍生神经营养因子、睫状神经营养因子、表皮生长因子、纤维母细胞生长因子、神经胶质衍生神经营养因子、肝细胞生长因子、调蛋白、类胰岛素生长因子、介白素、角质细胞生长因子、巨噬细胞发炎蛋白、巨噬细胞趋化蛋白、神经生长因子、神经营养因子、血小板衍生生长因子、色素上皮衍生因子、血小板因子、基质细胞衍生因子、干细胞因子、基质金属蛋白酶组织抑制因子、转型生长因子、肿瘤坏死因子、以及血管内皮生长因子所组成的群组；以及(ii)一编码前驱药物酵素的核苷酸序列。
根据本发明另一实施例，该DNA建构体包含编码前驱药物酵素的核苷酸序列，其选自由酒精脱氢酶、碱性磷酸酶、乙型内酰胺酶、乙型葡糖苷酸酶、羧酸酯酶、羧肽酶A、羧肽酶G2、胞嘧啶脱氨酶、胞嘧啶脱氨酶尿嘧啶磷酸核糖转移酶、糖苷酶、硝基还原酶、青霉素酰胺酶、胸腺嘧啶激酶所组成的群组。
在本发明的特定实例中，与生长因子受体特异性结合的配体的核苷酸序列编码为选自由SEQ ID NO:SEQ ID NO:21(EGF)与SEQ ID NO:22-25(VEGF)所组成的群组。
在本发明的特定实例中，编码前驱药物酵素的核苷酸序列为SEQ ID NO:26(Fcy)或SEQ ID NO:27(Fcy-Fur)。
在本发明的实施例中，本发明的DNA建构体具有选自由SEQ ID NO:28(Fcy-hEGF)、SEQ ID NO:29(Fcy-Fur-hEGF)、SEQ ID NO:30(Fcy-(VPGVG)2-hEGF-cMyc-KKKRKV)、SEQ IDNO:31(Fcy-hVEGFa)、SEQ ID NO:32(Fcy-Fur-hVEGFa)、SEQ ID NO:33(Fcy-hEGF)、SEQ IDNO:34(Fcy-Fur-hEGF)、SEQ ID NO:35(Fcy-hVEGFa)、SEQ ID NO:36(Fcy-Fur-hVEGFa)、SEQID NO:37(Fcy-Fur-hVEGFc)、SEQ ID NO:38(Fcy-mVEGFa)、SEQ ID NO:39(Fcy-Fur-mVEGFa)、以及SEQ ID NO:40(Fcy-Fur-mVEGFc)所组成群组的核苷酸序列。
本发明将以接下来的实施例进一步说明，其提供以为示范的目的而非用以限制。
实施例1：于酵母菌表达载体中Fcy-hEGF-myc-his6、Fcy-Fur-hEGF-myc-his6、hEGF-myc-his6、以及Fcy-myc-his6的DNA克隆
表达Fcy与Fur的基因序列使用来自酵母菌的cDNA基因库作为模板并以PCR加以扩增，其中人类EGF与VEGF使用源自人类癌症细胞株SKOV3-ip1的cDNA基因库作为模板并以PCR加以扩增。PCR的产物以限制酶BamHI与EcoRI处理，其目标序列已由PCR引子导入，且连接至以相同限制酶BamHI与EcoRI切割的蛋白表达载体pPICZ-α。Fcy与hEGF是个别克隆到此载体中，位于氨基端的α-分泌信号胜肽(α-secreting signal peptide)之后，其中羧基端为已内建于pPICZ-α载体中的c-myc与六组氨酸(myc-his6)标签以便于蛋白辨识与纯化。另一个Fcy的PCR产物于5’与3’两端均具有BamHI辨识序列，将此产物以BamHI切割后与同样以BamHI切割的pPIC-α-hEGF-myc-his6建构体接合，pPIC-α-hEGF-myc-his6在以BamHI切割后先以小牛肠碱性磷酸酶(CIAP)处理避免载体再度接合。于酵母菌表达载体pPICZ-α中Fcy-hEGF-myc-his6、Fcy-Fur-hEGF-myc-his6、hEGF-myc-his6、以及Fcy-myc-his6的示意图如图1所示。图2a至图2e显示：图2a pPICZ-α-Fcy-hEGF-myc-his6(4198bp)、图2b pPICZ-α-Fcy-Fur-hEGF-myc-his6(4951bp)、图2c针对酵母菌表达优化的建构体pPICZ-α-Fcy-(VPGVG)2-hEGF-cMyc-KKKRKV-6His(4213bp)、图2d pPICZ-α-Fcy-hVEGF-myc-his6(4384bp)、以及图2e pPICZ-α-Fcy-Fur-hVEGF-myc-his6(5137bp)所获得核苷酸与氨基酸序列的图谱。
实施例2：于细菌表达载体中Fcy-hEGF-myc-his6、Fcy-Fur-hEGF-myc-his6、hEGF-myc-his6、以及Fcy-myc-his6的DNA克隆
为了探讨除了酵母菌之外宿主中的蛋白表达，发明人建构了与Fcy或Fcy-Fur融合的生长因子，例如EGF或VEGF。图3显示E.coli表达载体pET56中Fcy-hEGF(或VEGF)-his6、Fcy-Fur-hEGF(或VEGF)-his6、hEGF(或VEGF)-his6、以及Fcy-his6融合基因的示意图。与酵母菌表达建构体的克隆策略相似，所需的融合基因以PCR扩增且以不同组的限制酶NcoI与XhoI切割。经切割的片段克隆到以相同限制酶切割的载体pET56中。所获得的建构体显示于图4a至图4h中。
实施例3：Fcy-hEGF-myc-his6、hEGF-myc-his6、以及Fcy-myc-his6的表达与纯化
于pPICZ-α建构体中的Fcy-hEGF-myc-his6、hEGF-myc-his6、或Fcy-myc-his6转型(transformed)到野生型X-33毕赤巴斯德酵母(Pichia pastoris)中，于含有zeocin抗生素(200μg/mL)的洋菜培养盘上培养3到4天直到出现菌落。以抗c-myc的抗体侦测筛选高蛋白表达的各个菌落。为了大量表达，将筛选出的菌落接种于含有0.5升BMD培养基的摇瓶中、培养至OD600为8-10、且每日添加甲醇以诱导蛋白表达。诱导3天之后，收集含有蛋白的培养基且于注入镍树脂管柱(nickel-resin column)(Qiagen)之前先行过滤。将管柱以10倍管柱体积且含有5mM咪唑(imidazole)的PBS清洗，且使用plus纯化系统(plus purification system)增加咪唑的浓度以洗涤出蛋白。此蛋白以考马斯蓝(Coomassie blue)染色的SDS-PAGE凝胶(图5右侧)以及利用c-myc特异性抗体进行西方墨点分析加以鉴定(图5左侧)。
实施例4：Fcy-hEGF-myc-his6、hEGF-myc-his6、以及Fcy-myc-his6与经纯化的EGFR的体外结合
图6显示测量Fcy-hEGF-myc-his6与固定在ELISA盘上经过纯化的EGFR于体外结合的示意图。Fcy-hEGF-myc-his6、hEGF-myc-his6、以及Fcy-myc-his6与固定在ELISA盘上经过纯化的EGFR的体外结合使用标记HRP(辣根过氧化酶)的抗His6抗体测量。Fcy-hEGF-myc-his6以及hEGF-myc-his6与EGFR的结合亲和力分别为5nM与9nM，而Fcy-myc-his6与EGFR之间则无法辨识有结合(图6右侧)。
实施例5：Fcy-hEGF-myc-his6、hEGF-myc-his6、以及Fcy-myc-his6结合具有不同EGFR表达量的细胞
A431、MDA-468、MDA-231、以及MCF-7细胞中EGFR的表达量以荧光活化细胞分选仪(FACS)进行分析。将细胞表面以抗EGFR抗体cetuximab(尔必得舒(erbitux))染色，接着以标记FITC的抗人类IgG抗体结合cetuximab。为了测试经过纯化的Fcy-hEGF-myc-his6、hEGF-myc-his6、以及Fcy-myc-his6与EGFR的结合能力，将A431、MCF-7、MDA-468、以及MDA-231分别与标记有his6的蛋白共同培养1小时。稍后将细胞与标记FITC的his6特异性抗体共同培养，接着进行FACS分析。荧光强度代表由cetuximab侦测到的EGFR量、或是与癌症细胞连结具his6标记的蛋白量(见图7)。
实施例6：Fcy‐hEGF‐myc‐his6与Fcy-myc‐his6的体外酵素活性
Fcy-hEGF与Fcy的酵素活性通过测量5-FU的产量而测得。将50nanomole的Fcy-hEGF-myc-his6(1.25mg)或Fcy-myc-his6(0.75mg)加入37℃含有递增5-FC浓度(0、0.03、0.1、0.3、1、与3mM)的0.3ml PBS中以启动5-FC转换为5-FU。每3分钟抽取2μl的反应物且使用NanoDrop 2000分光亮度计(Thermo Scientific)测量5-FC与5-FU的荧光强度。5-FU的浓度通过Senter等人推导的公式：[5-FU]mM＝0.185x A255-0.049x A290测量255nm与290nm的吸光值加以计算(Senter,P.D.,Su,P.C.,Katsuragi,T.,Sakai,T.,Cosand,W.L.,Hellstrom,I.,and Hellstrom,K.E.(1991)Generation of 5-fluorouracil from 5-fluorocytosine by monoclonal antibody-cytosine deaminase conjugates,BioconjugChem 2,447-451)。不同浓度的5-FC下5-FU的生成率(V)是使用Graphpad prism 5软件(圣地雅哥，美國)适配至Michaelis-Menten equation，如图8所示。纯化的Fcy-hEGF与Fcy蛋白对5-FC的km值分别为0.25及0.49mM。5-FU生成的Vmax对于Fcy-hEGF与Fcy蛋白分别为177及173min-1。
实施例7：MTT分析以测量经过Fcy-hEGF与5-FC处理的A431、MDA-468、HUVEC、MDA-231、以及MCF-7的细胞存活率
为了验证5-FC/Fcy-hEGF-myc-his6对于细胞存活率的效果，A431、MDA-468、HUVEC、MDA-231、与MCF-7于0.1mg/ml的5-FC存在下与递增浓度的Fcy-hEGF-myc-his6共同培养(图9a与图9d)，同时另一个实验使用固定量的Fcy-hEGF-myc-his6(0.2μg/ml)与递增浓度的5-FC(0-1mg/ml)进行(图9b与图9c)。在添加蛋白与5-FC达3天之后，培养于96孔培养盘中的细胞以MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)分析存活率。将25μl的MTT溶液(5mg/ml于PBS中)添加至96孔培养盘。作用2小时之后，移除MTT溶液、以PBS清洗细胞、且接着添加0.1ml萃取缓冲液(20％十二烷基硫酸钠于50％二甲基甲酰氨中)。在37℃经过4小时作用之后，使用盘式读取仪(Bio-Rad)于570nm测量吸光值，并以萃取缓冲液作为对照。相较于EGFR表达较少的HUVEC、MDA-231、与MCF-7，5-FC与Fcy-hEGF-myc-his6对于EGFR过度表达的MDA-468与A431的IC50较低(见图10)。
实施例8：MTT分析以测量表达EGFR的大肠癌细胞LS174T及HCT116受Fcy-hEGF与5-FC抑制的细胞存活率
与实施例7相似，以MTT分析测量受到Fcy-hEGF(0.2μg/ml)与递增浓度5-FC影响的表达EGFR大肠癌细胞LS174T与HCT116的细胞存活率。当5-FC的IC50分别为2.5μg/ml(10μM)与6.0μg/ml(24μM)时，可观察到对于LS174T与HCT116细胞有显着的效果。具体如图11所示。实施例9：Fcy-hEGF/5-FC于体内抑制HCT116的生长
使用过度表达EGFR的大肠癌细胞HCT116进行动物实验。将细胞以皮下接种于balb/c裸鼠的后背双侧。当肿瘤于2周后到达3-5mm的长度时，每3天以20μg的Fcy或Fcy-hEGF处理小鼠。小鼠每天注射10mg的5-FC(500mg/kg)。2种处理组别的肿瘤尺寸如图12所示。相较于Fcy，Fcy-hEGF呈现较佳的抗肿瘤效果(p＝0.03)。
实施例10：癌症标靶前驱药物-生长因子融合蛋白的示意图
除了显示于图1到图4a-4h中的建构体，亦可建构如图13所示同时包含生长因子与前驱药物酵素的DNA建构体及其所生蛋白。“生长因子”部分的DNA序列可编码选自由促血管生成素、大脑衍生神经营养因子、睫状神经营养因子、表皮生长因子、纤维母细胞生长因子、神经胶质衍生神经营养因子、肝细胞生长因子、调蛋白、类胰岛素生长因子、介白素、角质细胞生长因子、巨噬细胞发炎蛋白、巨噬细胞趋化蛋白、神经生长因子、神经营养因子、血小板衍生生长因子、色素上皮衍生因子、血小板因子、基质细胞衍生因子、干细胞因子、基质金属蛋白酶组织抑制因子、转型生长因子、肿瘤坏死因子、以及血管内皮生长因子所组成群组的其中任一者。“前驱药物酵素”部分的DNA序列可编码选自由酒精脱氢酶、碱性磷酸酶、乙型内酰胺酶、乙型葡糖苷酸酶、羧酸酯酶、羧肽酶A、羧肽酶G2、胞嘧啶脱氨酶、胞嘧啶脱氨酶尿嘧啶磷酸核糖转移酶(在酵母菌中为Fcy-Fur)、糖苷酶、硝基还原酶、青霉素酰胺酶、及胸腺嘧啶激酶所组成群组的其中任一者。
<110> 台北荣民总医院
<120> 治疗感染性与恶性疾病的标靶化学治疗药物的形成方法
<130> 17P05002-TW
<150> 61/492,649
<151> 2011-06-02
<160> 40
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35 40 45
Tyr Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn
50 55 60
Thr Ser Thr Gly Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro
65 70 75 80
Leu Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr
85 90 95
Ser Cys Arg Cys Met Ser Lys Leu Asp
100 105
<210> 6
<211> 158
<212> PRT
<213> 皊艭幻ダ颠Saccharomyces cerevisiae
<400> 6
Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp
1 5 10 15
Ile Ala Tyr Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro
20 25 30
Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg
35 40 45
Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu
50 55 60
Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys
65 70 75 80
Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly
85 90 95
Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Val Gly Glu Asn Val
100 105 110
Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu
115 120 125
Val Val Val Val Asp Asp Glu Arg Cys Lys Lys Ile Met Lys Gln Phe
130 135 140
Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu
145 150 155
<210> 7
<211> 409
<212> PRT
<213> 皊艭幻ダ颠Saccharomyces cerevisiae
<400> 7
Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp
1 5 10 15
Ile Ala Tyr Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro
20 25 30
Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg
35 40 45
Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu
50 55 60
Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys
65 70 75 80
Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly
85 90 95
Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Val Gly Glu Asn Val
100 105 110
Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu
115 120 125
Val Val Val Val Asp Asp Glu Arg Cys Lys Lys Ile Met Lys Gln Phe
130 135 140
Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu Met Asn
145 150 155 160
Pro Leu Phe Phe Leu Ala Ser Pro Phe Leu Tyr Leu Thr Tyr Leu Ile
165 170 175
Tyr Tyr Pro Asn Lys Gly Ser Phe Val Ser Lys Pro Arg Asn Leu Gln
180 185 190
Lys Met Ser Ser Glu Pro Phe Lys Asn Val Tyr Leu Leu Pro Gln Thr
195 200 205
Asn Gln Leu Leu Gly Leu Tyr Thr Ile Ile Arg Asn Lys Asn Thr Thr
210 215 220
Arg Pro Asp Phe Ile Phe Tyr Ser Asp Arg Ile Ile Arg Leu Leu Val
225 230 235 240
Glu Glu Gly Leu Asn His Leu Pro Val Gln Lys Gln Ile Val Glu Thr
245 250 255
Asp Thr Asn Glu Asn Phe Glu Gly Val Ser Phe Met Gly Lys Ile Cys
260 265 270
Gly Val Ser Ile Val Arg Ala Gly Glu Ser Met Glu Gln Gly Leu Arg
275 280 285
Asp Cys Cys Arg Ser Val Arg Ile Gly Lys Ile Leu Ile Gln Arg Asp
290 295 300
Glu Glu Thr Ala Leu Pro Lys Leu Phe Tyr Glu Lys Leu Pro Glu Asp
305 310 315 320
Ile Ser Glu Arg Tyr Val Phe Leu Leu Asp Pro Met Leu Ala Thr Gly
325 330 335
Gly Ser Ala Ile Met Ala Thr Glu Val Leu Ile Lys Arg Gly Val Lys
340 345 350
Pro Glu Arg Ile Tyr Phe Leu Asn Leu Ile Cys Ser Lys Glu Gly Ile
355 360 365
Glu Lys Tyr His Ala Ala Phe Pro Glu Val Arg Ile Val Thr Gly Ala
370 375 380
Leu Asp Arg Gly Leu Asp Glu Asn Lys Tyr Leu Val Pro Gly Leu Gly
385 390 395 400
Asp Phe Gly Asp Arg Tyr Tyr Cys Val
405
<210> 8
<211> 219
<212> PRT
<213>
<220>
<223> Fcy-hEGF
<400> 8
Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp
1 5 10 15
Ile Ala Tyr Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro
20 25 30
Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg
35 40 45
Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu
50 55 60
Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys
65 70 75 80
Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly
85 90 95
Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Val Gly Glu Asn Val
100 105 110
Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu
115 120 125
Val Val Val Val Asp Asp Glu Arg Cys Lys Lys Ile Met Lys Gln Phe
130 135 140
Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu Val Pro
145 150 155 160
Gly Val Gly Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr
165 170 175
Cys Leu His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr
180 185 190
Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg
195 200 205
Asp Leu Lys Trp Trp Glu Leu Arg His Ala Gly
210 215
<210> 9
<211> 470
<212> PRT
<213>
<220>
<223> Fcy-Fur-hEGF
<400> 9
Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp
1 5 10 15
Ile Ala Tyr Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro
20 25 30
Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg
35 40 45
Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu
50 55 60
Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys
65 70 75 80
Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly
85 90 95
Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Val Gly Glu Asn Val
100 105 110
Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu
115 120 125
Val Val Val Val Asp Asp Glu Arg Cys Lys Lys Ile Met Lys Gln Phe
130 135 140
Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu Met Asn
145 150 155 160
Pro Leu Phe Phe Leu Ala Ser Pro Phe Leu Tyr Leu Thr Tyr Leu Ile
165 170 175
Tyr Tyr Pro Asn Lys Gly Ser Phe Val Ser Lys Pro Arg Asn Leu Gln
180 185 190
Lys Met Ser Ser Glu Pro Phe Lys Asn Val Tyr Leu Leu Pro Gln Thr
195 200 205
Asn Gln Leu Leu Gly Leu Tyr Thr Ile Ile Arg Asn Lys Asn Thr Thr
210 215 220
Arg Pro Asp Phe Ile Phe Tyr Ser Asp Arg Ile Ile Arg Leu Leu Val
225 230 235 240
Glu Glu Gly Leu Asn His Leu Pro Val Gln Lys Gln Ile Val Glu Thr
245 250 255
Asp Thr Asn Glu Asn Phe Glu Gly Val Ser Phe Met Gly Lys Ile Cys
260 265 270
Gly Val Ser Ile Val Arg Ala Gly Glu Ser Met Glu Gln Gly Leu Arg
275 280 285
Asp Cys Cys Arg Ser Val Arg Ile Gly Lys Ile Leu Ile Gln Arg Asp
290 295 300
Glu Glu Thr Ala Leu Pro Lys Leu Phe Tyr Glu Lys Leu Pro Glu Asp
305 310 315 320
Ile Ser Glu Arg Tyr Val Phe Leu Leu Asp Pro Met Leu Ala Thr Gly
325 330 335
Gly Ser Ala Ile Met Ala Thr Glu Val Leu Ile Lys Arg Gly Val Lys
340 345 350
Pro Glu Arg Ile Tyr Phe Leu Asn Leu Ile Cys Ser Lys Glu Gly Ile
355 360 365
Glu Lys Tyr His Ala Ala Phe Pro Glu Val Arg Ile Val Thr Gly Ala
370 375 380
Leu Asp Arg Gly Leu Asp Glu Asn Lys Tyr Leu Val Pro Gly Leu Gly
385 390 395 400
Asp Phe Gly Asp Arg Tyr Tyr Cys Val Val Pro Gly Val Gly Asn Ser
405 410 415
Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly
420 425 430
Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val
435 440 445
Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp
450 455 460
Glu Leu Arg His Ala Gly
465 470
<210> 10
<211> 250
<212> PRT
<213>
<220>
<223> Fcy-(VPGVG)2-hEGF-cMyc-KKKRKV
<400> 10
Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp
1 5 10 15
Ile Ala Tyr Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro
20 25 30
Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg
35 40 45
Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu
50 55 60
Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys
65 70 75 80
Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly
85 90 95
Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Val Gly Glu Asn Val
100 105 110
Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu
115 120 125
Val Val Val Val Asp Asp Glu Arg Cys Lys Lys Ile Met Lys Gln Phe
130 135 140
Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu Val Pro
145 150 155 160
Gly Val Gly Val Pro Gly Val Gly Asn Ser Asp Ser Glu Cys Pro Leu
165 170 175
Ser His Asp Gly Tyr Cys Leu His Asp Gly Val Cys Met Tyr Ile Glu
180 185 190
Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu
195 200 205
Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg His Ala Gly
210 215 220
Gly Gly Gly Leu Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Lys Lys
225 230 235 240
Lys Arg Lys Val His His His His His His
245 250
<210> 11
<211> 284
<212> PRT
<213>
<220>
<223> Fcy-hVEGFa
<400> 11
Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp
1 5 10 15
Ile Ala Tyr Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro
20 25 30
Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg
35 40 45
Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu
50 55 60
Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys
65 70 75 80
Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly
85 90 95
Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Val Gly Glu Asn Val
100 105 110
Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu
115 120 125
Val Val Val Val Asp Asp Glu Arg Cys Lys Lys Ile Met Lys Gln Phe
130 135 140
Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu Val Pro
145 150 155 160
Gly Val Gly Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu
165 170 175
Val Val Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile
180 185 190
Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr
195 200 205
Ile Phe Lys Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys
210 215 220
Asn Asp Glu Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr
225 230 235 240
Met Gln Ile Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu
245 250 255
Met Ser Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp
260 265 270
Arg Ala Arg Gln Glu Lys Cys Asp Lys Pro Arg Arg
275 280
<210> 12
<211> 535
<212> PRT
<213>
<220>
<223> Fcy-Fur-hVEGFa
<400> 12
Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp
1 5 10 15
Ile Ala Tyr Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro
20 25 30
Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg
35 40 45
Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu
50 55 60
Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys
65 70 75 80
Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly
85 90 95
Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Val Gly Glu Asn Val
100 105 110
Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu
115 120 125
Val Val Val Val Asp Asp Glu Arg Cys Lys Lys Ile Met Lys Gln Phe
130 135 140
Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu Met Asn
145 150 155 160
Pro Leu Phe Phe Leu Ala Ser Pro Phe Leu Tyr Leu Thr Tyr Leu Ile
165 170 175
Tyr Tyr Pro Asn Lys Gly Ser Phe Val Ser Lys Pro Arg Asn Leu Gln
180 185 190
Lys Met Ser Ser Glu Pro Phe Lys Asn Val Tyr Leu Leu Pro Gln Thr
195 200 205
Asn Gln Leu Leu Gly Leu Tyr Thr Ile Ile Arg Asn Lys Asn Thr Thr
210 215 220
Arg Pro Asp Phe Ile Phe Tyr Ser Asp Arg Ile Ile Arg Leu Leu Val
225 230 235 240
Glu Glu Gly Leu Asn His Leu Pro Val Gln Lys Gln Ile Val Glu Thr
245 250 255
Asp Thr Asn Glu Asn Phe Glu Gly Val Ser Phe Met Gly Lys Ile Cys
260 265 270
Gly Val Ser Ile Val Arg Ala Gly Glu Ser Met Glu Gln Gly Leu Arg
275 280 285
Asp Cys Cys Arg Ser Val Arg Ile Gly Lys Ile Leu Ile Gln Arg Asp
290 295 300
Glu Glu Thr Ala Leu Pro Lys Leu Phe Tyr Glu Lys Leu Pro Glu Asp
305 310 315 320
Ile Ser Glu Arg Tyr Val Phe Leu Leu Asp Pro Met Leu Ala Thr Gly
325 330 335
Gly Ser Ala Ile Met Ala Thr Glu Val Leu Ile Lys Arg Gly Val Lys
340 345 350
Pro Glu Arg Ile Tyr Phe Leu Asn Leu Ile Cys Ser Lys Glu Gly Ile
355 360 365
Glu Lys Tyr His Ala Ala Phe Pro Glu Val Arg Ile Val Thr Gly Ala
370 375 380
Leu Asp Arg Gly Leu Asp Glu Asn Lys Tyr Leu Val Pro Gly Leu Gly
385 390 395 400
Asp Phe Gly Asp Arg Tyr Tyr Cys Val Val Pro Gly Val Gly Ala Pro
405 410 415
Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met
420 425 430
Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp
435 440 445
Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser
450 455 460
Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu
465 470 475 480
Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg
485 490 495
Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln
500 505 510
His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu
515 520 525
Lys Cys Asp Lys Pro Arg Arg
530 535
<210> 13
<211> 219
<212> PRT
<213>
<220>
<223> Fcy-hEGF
<400> 13
Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp
1 5 10 15
Ile Ala Tyr Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro
20 25 30
Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg
35 40 45
Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu
50 55 60
Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys
65 70 75 80
Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly
85 90 95
Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Val Gly Glu Asn Val
100 105 110
Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu
115 120 125
Val Val Val Val Asp Asp Glu Arg Cys Lys Lys Ile Met Lys Gln Phe
130 135 140
Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu Val Pro
145 150 155 160
Gly Val Gly Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr
165 170 175
Cys Leu His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr
180 185 190
Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg
195 200 205
Asp Leu Lys Trp Trp Glu Leu Arg His Ala Gly
210 215
<210> 14
<211> 470
<212> PRT
<213>
<220>
<223> Fcy-Fur-hEGF
<400> 14
Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp
1 5 10 15
Ile Ala Tyr Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro
20 25 30
Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg
35 40 45
Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu
50 55 60
Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys
65 70 75 80
Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly
85 90 95
Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Val Gly Glu Asn Val
100 105 110
Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu
115 120 125
Val Val Val Val Asp Asp Glu Arg Cys Lys Lys Ile Met Lys Gln Phe
130 135 140
Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu Met Asn
145 150 155 160
Pro Leu Phe Phe Leu Ala Ser Pro Phe Leu Tyr Leu Thr Tyr Leu Ile
165 170 175
Tyr Tyr Pro Asn Lys Gly Ser Phe Val Ser Lys Pro Arg Asn Leu Gln
180 185 190
Lys Met Ser Ser Glu Pro Phe Lys Asn Val Tyr Leu Leu Pro Gln Thr
195 200 205
Asn Gln Leu Leu Gly Leu Tyr Thr Ile Ile Arg Asn Lys Asn Thr Thr
210 215 220
Arg Pro Asp Phe Ile Phe Tyr Ser Asp Arg Ile Ile Arg Leu Leu Val
225 230 235 240
Glu Glu Gly Leu Asn His Leu Pro Val Gln Lys Gln Ile Val Glu Thr
245 250 255
Asp Thr Asn Glu Asn Phe Glu Gly Val Ser Phe Met Gly Lys Ile Cys
260 265 270
Gly Val Ser Ile Val Arg Ala Gly Glu Ser Met Glu Gln Gly Leu Arg
275 280 285
Asp Cys Cys Arg Ser Val Arg Ile Gly Lys Ile Leu Ile Gln Arg Asp
290 295 300
Glu Glu Thr Ala Leu Pro Lys Leu Phe Tyr Glu Lys Leu Pro Glu Asp
305 310 315 320
Ile Ser Glu Arg Tyr Val Phe Leu Leu Asp Pro Met Leu Ala Thr Gly
325 330 335
Gly Ser Ala Ile Met Ala Thr Glu Val Leu Ile Lys Arg Gly Val Lys
340 345 350
Pro Glu Arg Ile Tyr Phe Leu Asn Leu Ile Cys Ser Lys Glu Gly Ile
355 360 365
Glu Lys Tyr His Ala Ala Phe Pro Glu Val Arg Ile Val Thr Gly Ala
370 375 380
Leu Asp Arg Gly Leu Asp Glu Asn Lys Tyr Leu Val Pro Gly Leu Gly
385 390 395 400
Asp Phe Gly Asp Arg Tyr Tyr Cys Val Val Pro Gly Val Gly Asn Ser
405 410 415
Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly
420 425 430
Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val
435 440 445
Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp
450 455 460
Glu Leu Arg His Ala Gly
465 470
<210> 15
<211> 284
<212> PRT
<213>
<220>
<223> Fcy-hVEGFa
<400> 15
Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp
1 5 10 15
Ile Ala Tyr Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro
20 25 30
Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg
35 40 45
Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu
50 55 60
Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys
65 70 75 80
Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly
85 90 95
Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Val Gly Glu Asn Val
100 105 110
Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu
115 120 125
Val Val Val Val Asp Asp Glu Arg Cys Lys Lys Ile Met Lys Gln Phe
130 135 140
Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu Val Pro
145 150 155 160
Gly Val Gly Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu
165 170 175
Val Val Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile
180 185 190
Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr
195 200 205
Ile Phe Lys Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys
210 215 220
Asn Asp Glu Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr
225 230 235 240
Met Gln Ile Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu
245 250 255
Met Ser Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp
260 265 270
Arg Ala Arg Gln Glu Lys Cys Asp Lys Pro Arg Arg
275 280
<210> 16
<211> 535
<212> PRT
<213>
<220>
<223> Fcy-Fur-hVEGFa
<400> 16
Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp
1 5 10 15
Ile Ala Tyr Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro
20 25 30
Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg
35 40 45
Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu
50 55 60
Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys
65 70 75 80
Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly
85 90 95
Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Val Gly Glu Asn Val
100 105 110
Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu
115 120 125
Val Val Val Val Asp Asp Glu Arg Cys Lys Lys Ile Met Lys Gln Phe
130 135 140
Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu Met Asn
145 150 155 160
Pro Leu Phe Phe Leu Ala Ser Pro Phe Leu Tyr Leu Thr Tyr Leu Ile
165 170 175
Tyr Tyr Pro Asn Lys Gly Ser Phe Val Ser Lys Pro Arg Asn Leu Gln
180 185 190
Lys Met Ser Ser Glu Pro Phe Lys Asn Val Tyr Leu Leu Pro Gln Thr
195 200 205
Asn Gln Leu Leu Gly Leu Tyr Thr Ile Ile Arg Asn Lys Asn Thr Thr
210 215 220
Arg Pro Asp Phe Ile Phe Tyr Ser Asp Arg Ile Ile Arg Leu Leu Val
225 230 235 240
Glu Glu Gly Leu Asn His Leu Pro Val Gln Lys Gln Ile Val Glu Thr
245 250 255
Asp Thr Asn Glu Asn Phe Glu Gly Val Ser Phe Met Gly Lys Ile Cys
260 265 270
Gly Val Ser Ile Val Arg Ala Gly Glu Ser Met Glu Gln Gly Leu Arg
275 280 285
Asp Cys Cys Arg Ser Val Arg Ile Gly Lys Ile Leu Ile Gln Arg Asp
290 295 300
Glu Glu Thr Ala Leu Pro Lys Leu Phe Tyr Glu Lys Leu Pro Glu Asp
305 310 315 320
Ile Ser Glu Arg Tyr Val Phe Leu Leu Asp Pro Met Leu Ala Thr Gly
325 330 335
Gly Ser Ala Ile Met Ala Thr Glu Val Leu Ile Lys Arg Gly Val Lys
340 345 350
Pro Glu Arg Ile Tyr Phe Leu Asn Leu Ile Cys Ser Lys Glu Gly Ile
355 360 365
Glu Lys Tyr His Ala Ala Phe Pro Glu Val Arg Ile Val Thr Gly Ala
370 375 380
Leu Asp Arg Gly Leu Asp Glu Asn Lys Tyr Leu Val Pro Gly Leu Gly
385 390 395 400
Asp Phe Gly Asp Arg Tyr Tyr Cys Val Val Pro Gly Val Gly Ala Pro
405 410 415
Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met
420 425 430
Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp
435 440 445
Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser
450 455 460
Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu
465 470 475 480
Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg
485 490 495
Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln
500 505 510
His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu
515 520 525
Lys Cys Asp Lys Pro Arg Arg
530 535
<210> 17
<211> 519
<212> PRT
<213>
<220>
<223> Fcy-Fur-hVEGFc
<400> 17
Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp
1 5 10 15
Ile Ala Tyr Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro
20 25 30
Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg
35 40 45
Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu
50 55 60
Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys
65 70 75 80
Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly
85 90 95
Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Val Gly Glu Asn Val
100 105 110
Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu
115 120 125
Val Val Val Val Asp Asp Glu Arg Cys Lys Lys Ile Met Lys Gln Phe
130 135 140
Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu Met Asn
145 150 155 160
Pro Leu Phe Phe Leu Ala Ser Pro Phe Leu Tyr Leu Thr Tyr Leu Ile
165 170 175
Tyr Tyr Pro Asn Lys Gly Ser Phe Val Ser Lys Pro Arg Asn Leu Gln
180 185 190
Lys Met Ser Ser Glu Pro Phe Lys Asn Val Tyr Leu Leu Pro Gln Thr
195 200 205
Asn Gln Leu Leu Gly Leu Tyr Thr Ile Ile Arg Asn Lys Asn Thr Thr
210 215 220
Arg Pro Asp Phe Ile Phe Tyr Ser Asp Arg Ile Ile Arg Leu Leu Val
225 230 235 240
Glu Glu Gly Leu Asn His Leu Pro Val Gln Lys Gln Ile Val Glu Thr
245 250 255
Asp Thr Asn Glu Asn Phe Glu Gly Val Ser Phe Met Gly Lys Ile Cys
260 265 270
Gly Val Ser Ile Val Arg Ala Gly Glu Ser Met Glu Gln Gly Leu Arg
275 280 285
Asp Cys Cys Arg Ser Val Arg Ile Gly Lys Ile Leu Ile Gln Arg Asp
290 295 300
Glu Glu Thr Ala Leu Pro Lys Leu Phe Tyr Glu Lys Leu Pro Glu Asp
305 310 315 320
Ile Ser Glu Arg Tyr Val Phe Leu Leu Asp Pro Met Leu Ala Thr Gly
325 330 335
Gly Ser Ala Ile Met Ala Thr Glu Val Leu Ile Lys Arg Gly Val Lys
340 345 350
Pro Glu Arg Ile Tyr Phe Leu Asn Leu Ile Cys Ser Lys Glu Gly Ile
355 360 365
Glu Lys Tyr His Ala Ala Phe Pro Glu Val Arg Ile Val Thr Gly Ala
370 375 380
Leu Asp Arg Gly Leu Asp Glu Asn Lys Tyr Leu Val Pro Gly Leu Gly
385 390 395 400
Asp Phe Gly Asp Arg Tyr Tyr Cys Val Val Pro Gly Val Gly Ala His
405 410 415
Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys Thr
420 425 430
Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe Gly
435 440 445
Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr Arg
450 455 460
Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr Ser
465 470 475 480
Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu Ser
485 490 495
Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser Cys
500 505 510
Arg Cys Met Ser Lys Leu Asp
515
<210> 18
<211> 283
<212> PRT
<213>
<220>
<223> Fcy-mVEGFa
<400> 18
Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp
1 5 10 15
Ile Ala Tyr Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro
20 25 30
Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg
35 40 45
Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu
50 55 60
Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys
65 70 75 80
Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly
85 90 95
Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Val Gly Glu Asn Val
100 105 110
Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu
115 120 125
Val Val Val Val Asp Asp Glu Arg Cys Lys Lys Ile Met Lys Gln Phe
130 135 140
Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu Val Pro
145 150 155 160
Gly Val Gly Ala Pro Thr Thr Glu Gly Glu Gln Lys Ser His Glu Val
165 170 175
Ile Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys Arg Pro Ile Glu
180 185 190
Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile
195 200 205
Phe Lys Pro Ser Cys Val Pro Leu Met Arg Cys Ala Gly Cys Cys Asn
210 215 220
Asp Glu Ala Leu Glu Cys Val Pro Thr Ser Glu Ser Asn Ile Thr Met
225 230 235 240
Gln Ile Met Arg Ile Lys Pro His Gln Ser Gln His Ile Gly Glu Met
245 250 255
Ser Phe Leu Gln His Ser Arg Cys Glu Cys Arg Pro Lys Lys Asp Arg
260 265 270
Thr Lys Pro Glu Lys Cys Asp Lys Pro Arg Arg
275 280
<210> 19
<211> 534
<212> PRT
<213>
<220>
<223> Fcy-Fur-mVEGFa
<400> 19
Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp
1 5 10 15
Ile Ala Tyr Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro
20 25 30
Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg
35 40 45
Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu
50 55 60
Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys
65 70 75 80
Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly
85 90 95
Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Val Gly Glu Asn Val
100 105 110
Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu
115 120 125
Val Val Val Val Asp Asp Glu Arg Cys Lys Lys Ile Met Lys Gln Phe
130 135 140
Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu Met Asn
145 150 155 160
Pro Leu Phe Phe Leu Ala Ser Pro Phe Leu Tyr Leu Thr Tyr Leu Ile
165 170 175
Tyr Tyr Pro Asn Lys Gly Ser Phe Val Ser Lys Pro Arg Asn Leu Gln
180 185 190
Lys Met Ser Ser Glu Pro Phe Lys Asn Val Tyr Leu Leu Pro Gln Thr
195 200 205
Asn Gln Leu Leu Gly Leu Tyr Thr Ile Ile Arg Asn Lys Asn Thr Thr
210 215 220
Arg Pro Asp Phe Ile Phe Tyr Ser Asp Arg Ile Ile Arg Leu Leu Val
225 230 235 240
Glu Glu Gly Leu Asn His Leu Pro Val Gln Lys Gln Ile Val Glu Thr
245 250 255
Asp Thr Asn Glu Asn Phe Glu Gly Val Ser Phe Met Gly Lys Ile Cys
260 265 270
Gly Val Ser Ile Val Arg Ala Gly Glu Ser Met Glu Gln Gly Leu Arg
275 280 285
Asp Cys Cys Arg Ser Val Arg Ile Gly Lys Ile Leu Ile Gln Arg Asp
290 295 300
Glu Glu Thr Ala Leu Pro Lys Leu Phe Tyr Glu Lys Leu Pro Glu Asp
305 310 315 320
Ile Ser Glu Arg Tyr Val Phe Leu Leu Asp Pro Met Leu Ala Thr Gly
325 330 335
Gly Ser Ala Ile Met Ala Thr Glu Val Leu Ile Lys Arg Gly Val Lys
340 345 350
Pro Glu Arg Ile Tyr Phe Leu Asn Leu Ile Cys Ser Lys Glu Gly Ile
355 360 365
Glu Lys Tyr His Ala Ala Phe Pro Glu Val Arg Ile Val Thr Gly Ala
370 375 380
Leu Asp Arg Gly Leu Asp Glu Asn Lys Tyr Leu Val Pro Gly Leu Gly
385 390 395 400
Asp Phe Gly Asp Arg Tyr Tyr Cys Val Val Pro Gly Val Gly Ala Pro
405 410 415
Thr Thr Glu Gly Glu Gln Lys Ser His Glu Val Ile Lys Phe Met Asp
420 425 430
Val Tyr Gln Arg Ser Tyr Cys Arg Pro Ile Glu Thr Leu Val Asp Ile
435 440 445
Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys
450 455 460
Val Pro Leu Met Arg Cys Ala Gly Cys Cys Asn Asp Glu Ala Leu Glu
465 470 475 480
Cys Val Pro Thr Ser Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile
485 490 495
Lys Pro His Gln Ser Gln His Ile Gly Glu Met Ser Phe Leu Gln His
500 505 510
Ser Arg Cys Glu Cys Arg Pro Lys Lys Asp Arg Thr Lys Pro Glu Lys
515 520 525
Cys Asp Lys Pro Arg Arg
530
<210> 20
<211> 519
<212> PRT
<213>
<220>
<223> Fcy-Fur-mVEGFc
<400> 20
Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp
1 5 10 15
Ile Ala Tyr Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro
20 25 30
Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg
35 40 45
Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu
50 55 60
Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys
65 70 75 80
Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly
85 90 95
Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Val Gly Glu Asn Val
100 105 110
Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu
115 120 125
Val Val Val Val Asp Asp Glu Arg Cys Lys Lys Ile Met Lys Gln Phe
130 135 140
Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu Met Asn
145 150 155 160
Pro Leu Phe Phe Leu Ala Ser Pro Phe Leu Tyr Leu Thr Tyr Leu Ile
165 170 175
Tyr Tyr Pro Asn Lys Gly Ser Phe Val Ser Lys Pro Arg Asn Leu Gln
180 185 190
Lys Met Ser Ser Glu Pro Phe Lys Asn Val Tyr Leu Leu Pro Gln Thr
195 200 205
Asn Gln Leu Leu Gly Leu Tyr Thr Ile Ile Arg Asn Lys Asn Thr Thr
210 215 220
Arg Pro Asp Phe Ile Phe Tyr Ser Asp Arg Ile Ile Arg Leu Leu Val
225 230 235 240
Glu Glu Gly Leu Asn His Leu Pro Val Gln Lys Gln Ile Val Glu Thr
245 250 255
Asp Thr Asn Glu Asn Phe Glu Gly Val Ser Phe Met Gly Lys Ile Cys
260 265 270
Gly Val Ser Ile Val Arg Ala Gly Glu Ser Met Glu Gln Gly Leu Arg
275 280 285
Asp Cys Cys Arg Ser Val Arg Ile Gly Lys Ile Leu Ile Gln Arg Asp
290 295 300
Glu Glu Thr Ala Leu Pro Lys Leu Phe Tyr Glu Lys Leu Pro Glu Asp
305 310 315 320
Ile Ser Glu Arg Tyr Val Phe Leu Leu Asp Pro Met Leu Ala Thr Gly
325 330 335
Gly Ser Ala Ile Met Ala Thr Glu Val Leu Ile Lys Arg Gly Val Lys
340 345 350
Pro Glu Arg Ile Tyr Phe Leu Asn Leu Ile Cys Ser Lys Glu Gly Ile
355 360 365
Glu Lys Tyr His Ala Ala Phe Pro Glu Val Arg Ile Val Thr Gly Ala
370 375 380
Leu Asp Arg Gly Leu Asp Glu Asn Lys Tyr Leu Val Pro Gly Leu Gly
385 390 395 400
Asp Phe Gly Asp Arg Tyr Tyr Cys Val Val Pro Gly Val Gly Ala His
405 410 415
Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys Thr
420 425 430
Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe Gly
435 440 445
Ala Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr Arg
450 455 460
Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr Ser
465 470 475 480
Thr Gly Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu Ser
485 490 495
Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser Cys
500 505 510
Arg Cys Met Ser Lys Leu Asp
515
<210> 21
<211> 168
<212> DNA
<213> 醇Homo sapiens
<400> 21
aatagtgact ctgaatgtcc cctgtcccac gatgggtact gcctccatga tggtgtgtgc 60
atgtatattg aagcattgga caagtatgca tgcaactgtg ttgttggcta catcggggag 120
cgatgtcagt accgagacct gaagtggtgg gaactgcgcc atgctggt 168
<210> 22
<211> 363
<212> DNA
<213> 醇Homo sapiens
<400> 22
gcacccatgg cagaaggagg agggcagaat catcacgaag tggtgaagtt catggatgtc 60
tatcagcgca gctactgcca tccaatcgag accctggtgg acatcttcca ggagtaccct 120
gatgagatcg agtacatctt caagccatcc tgtgtgcccc tgatgcgatg cgggggctgc 180
tgcaatgacg agggcctgga gtgtgtgccc actgaggagt ccaacatcac catgcagatt 240
atgcggatca aacctcacca aggccagcac ataggagaga tgagcttcct acagcacaac 300
aaatgtgaat gcagaccaaa gaaagataga gcaagacaag aaaaatgtga caagccgagg 360
cgt 363
<210> 23
<211> 315
<212> DNA
<213> 醇Homo sapiens
<400> 23
gcacattata atacagagat cttgaaaagt attgataatg agtggagaaa gactcaatgc 60
atgccacggg aggtgtgtat agatgtgggg aaggagtttg gagtcgcgac aaacaccttc 120
tttaaacctc catgtgtgtc cgtctacaga tgtgggggtt gctgcaatag tgaggggctg 180
cagtgcatga acaccagcac gagctacctc agcaagacgt tatttgaaat tacagtgcct 240
ctctctcaag gccccaaacc agtaacaatc agttttgcca atcacacttc ctgccgatgc 300
atgtctaaac tggat 315
<210> 24
<211> 360
<212> DNA
<213> 產鴞公Mus musculus
<400> 24
gcaccaacaa cagagggaga acaaaagagt cacgaggtta tcaaattcat ggacgtctac 60
caaaggtcat attgtagacc aattgaaaca ttggttgaca tattccagga atatcctgat 120
gagatagagt acatcttcaa accatcctgc gtcccactta tgagatgtgc tggttgctgt 180
aatgatgagg cattggagtg tgttcctaca tctgaaagta acattactat gcagattatg 240
agaatcaagc ctcatcaatc acaacatatc ggtgaaatgt cttttctaca acactctaga 300
tgtgaatgta gacctaagaa agatagaact aagcctgaaa agtgtgataa acctaggaga 360
<210> 25
<211> 315
<212> DNA
<213> 產鴞公Mus musculus
<400> 25
gcccattata acacagagat cctgaaaagt attgataatg agtggagaaa gactcaatgc 60
atgccacgtg aggtgtgtat agatgtgggg aaggagtttg gagcagccac aaacaccttc 120
tttaaacctc catgtgtgtc cgtctacaga tgtgggggtt gctgcaacag cgaggggctg 180
cagtgcatga acaccagcac aggttacctc agcaagacgt tgtttgaaat tacagtgcct 240
ctctcacaag gccccaaacc agtcacaatc agttttgcca atcacacttc ctgccggtgc 300
atgtctaaac tggat 315
<210> 26
<211> 474
<212> DNA
<213> 皊艭幻ダ颠Saccharomyces cerevisiae
<400> 26
atggtcacag gaggcatggc ttcaaagtgg gaccagaagg gcatggacat tgcctatgag 60
gaggctgctc tgggctacaa ggagggaggg gtcccaattg gtggctgcct catcaacaac 120
aaggatggca gtgtcctggg caggggccac aacatgaggt tccagaaggg cagtgccacc 180
ctgcatgggg agatcagcac cctggagaac tgtggcaggc tggagggcaa ggtctacaag 240
gacaccactc tgtacaccac cctcagccct tgtgacatgt gcacaggggc catcatcatg 300
tatggcattc ccaggtgtgt ggtgggagag aatgtcaact tcaagtcaaa aggagagaag 360
tacctccaga ccaggggcca tgaggtggtt gtggtggatg atgagaggtg caagaagatt 420
atgaagcagt tcattgatga gagaccccag gactggtttg aggacattgg ggag 474
<210> 27
<211> 1227
<212> DNA
<213> 皊艭幻ダ颠Saccharomyces cerevisiae
<400> 27
atggtcacag gaggcatggc ttcaaagtgg gaccagaagg gcatggacat tgcctatgag 60
gaggctgctc tgggctacaa ggagggaggg gtcccaattg gtggctgcct catcaacaac 120
aaggatggca gtgtcctggg caggggccac aacatgaggt tccagaaggg cagtgccacc 180
ctgcatgggg agatcagcac cctggagaac tgtggcaggc tggagggcaa ggtctacaag 240
gacaccactc tgtacaccac cctcagccct tgtgacatgt gcacaggggc catcatcatg 300
tatggcattc ccaggtgtgt ggtgggagag aatgtcaact tcaagtcaaa aggagagaag 360
tacctccaga ccaggggcca tgaggtggtt gtggtggatg atgagaggtg caagaagatt 420
atgaagcagt tcattgatga gagaccccag gactggtttg aggacattgg ggagatgaac 480
cctctcttct tcctggcctc tcccttcctc tatctcacct acctcatcta ctaccccaac 540
aagggctcct ttgtgtccaa gcccaggaac ctccagaaga tgtccagtga gcccttcaag 600
aatgtgtacc tcctccccca gaccaaccaa ctcctgggac tctacaccat catcaggaac 660
aagaacacca ccaggccaga cttcatcttc tacagtgaca ggatcatcag gctcctggtg 720
gaggagggcc tcaaccacct ccccgtgcag aagcagattg tggagactga caccaatgag 780
aactttgagg gagtgtcttt catgggcaag atttgtgggg tgtccattgt gagggctggg 840
gagagcatgg agcaaggcct gagggactgt tgcaggagtg tgaggattgg caagatcctg 900
atccagaggg atgaggagac tgccctgccc aagctgttct atgagaagct ccctgaagac 960
atctctgaga ggtatgtctt cctcctggac cccatgctgg caactggagg ctctgcaatc 1020
atggccactg aggtgctcat caagagggga gtcaagcctg agaggatcta cttcctcaac 1080
ctcatctgct caaaggaggg cattgagaag taccatgctg ccttccctga agtgaggatt 1140
gtcactgggg ctctggacag gggcctggat gagaacaagt acctggtccc tggcctggga 1200
gactttgggg acagatacta ctgtgtg 1227
<210> 28
<211> 657
<212> DNA
<213>
<220>
<223> Fcy-hEGF
<400> 28
atggtcacag gaggcatggc ttcaaagtgg gaccagaagg gcatggacat tgcctatgag 60
gaggctgctc tgggctacaa ggagggaggg gtcccaattg gtggctgcct catcaacaac 120
aaggatggca gtgtcctggg caggggccac aacatgaggt tccagaaggg cagtgccacc 180
ctgcatgggg agatcagcac cctggagaac tgtggcaggc tggagggcaa ggtctacaag 240
gacaccactc tgtacaccac cctcagccct tgtgacatgt gcacaggggc catcatcatg 300
tatggcattc ccaggtgtgt ggtgggagag aatgtcaact tcaagtcaaa aggagagaag 360
tacctccaga ccaggggcca tgaggtggtt gtggtggatg atgagaggtg caagaagatt 420
atgaagcagt tcattgatga gagaccccag gactggtttg aggacattgg ggaggtacca 480
ggtgttggta atagtgactc tgaatgtccc ctgtcccacg atgggtactg cctccatgat 540
ggtgtgtgca tgtatattga agcattggac aagtatgcat gcaactgtgt tgttggctac 600
atcggggagc gatgtcagta ccgagacctg aagtggtggg aactgcgcca tgctggt 657
<210> 29
<211> 1410
<212> DNA
<213>
<220>
<223> Fcy-Fur-hEGF
<400> 29
atggtcacag gaggcatggc ttcaaagtgg gaccagaagg gcatggacat tgcctatgag 60
gaggctgctc tgggctacaa ggagggaggg gtcccaattg gtggctgcct catcaacaac 120
aaggatggca gtgtcctggg caggggccac aacatgaggt tccagaaggg cagtgccacc 180
ctgcatgggg agatcagcac cctggagaac tgtggcaggc tggagggcaa ggtctacaag 240
gacaccactc tgtacaccac cctcagccct tgtgacatgt gcacaggggc catcatcatg 300
tatggcattc ccaggtgtgt ggtgggagag aatgtcaact tcaagtcaaa aggagagaag 360
tacctccaga ccaggggcca tgaggtggtt gtggtggatg atgagaggtg caagaagatt 420
atgaagcagt tcattgatga gagaccccag gactggtttg aggacattgg ggagatgaac 480
cctctcttct tcctggcctc tcccttcctc tatctcacct acctcatcta ctaccccaac 540
aagggctcct ttgtgtccaa gcccaggaac ctccagaaga tgtccagtga gcccttcaag 600
aatgtgtacc tcctccccca gaccaaccaa ctcctgggac tctacaccat catcaggaac 660
aagaacacca ccaggccaga cttcatcttc tacagtgaca ggatcatcag gctcctggtg 720
gaggagggcc tcaaccacct ccccgtgcag aagcagattg tggagactga caccaatgag 780
aactttgagg gagtgtcttt catgggcaag atttgtgggg tgtccattgt gagggctggg 840
gagagcatgg agcaaggcct gagggactgt tgcaggagtg tgaggattgg caagatcctg 900
atccagaggg atgaggagac tgccctgccc aagctgttct atgagaagct ccctgaagac 960
atctctgaga ggtatgtctt cctcctggac cccatgctgg caactggagg ctctgcaatc 1020
atggccactg aggtgctcat caagagggga gtcaagcctg agaggatcta cttcctcaac 1080
ctcatctgct caaaggaggg cattgagaag taccatgctg ccttccctga agtgaggatt 1140
gtcactgggg ctctggacag gggcctggat gagaacaagt acctggtccc tggcctggga 1200
gactttgggg acagatacta ctgtgtggta ccaggtgttg gtaatagtga ctctgaatgt 1260
cccctgtccc acgatgggta ctgcctccat gatggtgtgt gcatgtatat tgaagcattg 1320
gacaagtatg catgcaactg tgttgttggc tacatcgggg agcgatgtca gtaccgagac 1380
ctgaagtggt gggaactgcg ccatgctggt 1410
<210> 30
<211> 750
<212> DNA
<213>
<220>
<223> Fcy-(VPGVG)2-hEGF-cMyc-KKKRKV
<400> 30
atggttacgg gaggaatggc ttccaagtgg gatcagaagg gtatggatat agcttacgaa 60
gaagcagctt tgggttacaa agaaggaggt gttccaattg ggggttgctt gattaataac 120
aaagacggct ccgttttagg aaggggccat aacatgaggt ttcaaaaggg atcagccacc 180
ttgcacggag agatctcaac tcttgaaaac tgtggcaggt tagagggcaa ggtgtacaaa 240
gatactactt tatacactac cttgtctcct tgtgacatgt gcacgggtgc cataatcatg 300
tatgggattc caagatgcgt cgttggagag aacgttaatt ttaagtctaa gggtgaaaag 360
tatttacaaa caagagggca cgaagtggtg gtggttgatg acgagcgttg taagaagatc 420
atgaaacaat tcattgatga aagaccacaa gactggtttg aggacattgg agaagtacct 480
ggggtaggag taccaggtgt tggaaatagt gattctgaat gtcccctttc ccatgatggc 540
tattgtcttc atgatggtgt ttgcatgtat atcgaagcat tggacaaata cgcatgtaat 600
tgtgtcgtcg gatatatagg tgaaagatgc cagtacagag acttgaaatg gtgggaacta 660
agacatgctg gtggcggtgg attggaacag aaactaattt ctgaagagga cctgaaaaag 720
aaacgtaagg tccaccatca tcaccaccat 750
<210> 31
<211> 852
<212> DNA
<213>
<220>
<223> Fcy-hVEGFa
<400> 31
atggtcacag gaggcatggc ttcaaagtgg gaccagaagg gcatggacat tgcctatgag 60
gaggctgctc tgggctacaa ggagggaggg gtcccaattg gtggctgcct catcaacaac 120
aaggatggca gtgtcctggg caggggccac aacatgaggt tccagaaggg cagtgccacc 180
ctgcatgggg agatcagcac cctggagaac tgtggcaggc tggagggcaa ggtctacaag 240
gacaccactc tgtacaccac cctcagccct tgtgacatgt gcacaggggc catcatcatg 300
tatggcattc ccaggtgtgt ggtgggagag aatgtcaact tcaagtcaaa aggagagaag 360
tacctccaga ccaggggcca tgaggtggtt gtggtggatg atgagaggtg caagaagatt 420
atgaagcagt tcattgatga gagaccccag gactggtttg aggacattgg ggaggtacca 480
ggtgttggtg cacccatggc agaaggagga gggcagaatc atcacgaagt ggtgaagttc 540
atggatgtct atcagcgcag ctactgccat ccaatcgaga ccctggtgga catcttccag 600
gagtaccctg atgagatcga gtacatcttc aagccatcct gtgtgcccct gatgcgatgc 660
gggggctgct gcaatgacga gggcctggag tgtgtgccca ctgaggagtc caacatcacc 720
atgcagatta tgcggatcaa acctcaccaa ggccagcaca taggagagat gagcttccta 780
cagcacaaca aatgtgaatg cagaccaaag aaagatagag caagacaaga aaaatgtgac 840
aagccgaggc gt 852
<210> 32
<211> 1605
<212> DNA
<213>
<220>
<223> Fcy-Fur-hVEGFa
<400> 32
atggtcacag gaggcatggc ttcaaagtgg gaccagaagg gcatggacat tgcctatgag 60
gaggctgctc tgggctacaa ggagggaggg gtcccaattg gtggctgcct catcaacaac 120
aaggatggca gtgtcctggg caggggccac aacatgaggt tccagaaggg cagtgccacc 180
ctgcatgggg agatcagcac cctggagaac tgtggcaggc tggagggcaa ggtctacaag 240
gacaccactc tgtacaccac cctcagccct tgtgacatgt gcacaggggc catcatcatg 300
tatggcattc ccaggtgtgt ggtgggagag aatgtcaact tcaagtcaaa aggagagaag 360
tacctccaga ccaggggcca tgaggtggtt gtggtggatg atgagaggtg caagaagatt 420
atgaagcagt tcattgatga gagaccccag gactggtttg aggacattgg ggagatgaac 480
cctctcttct tcctggcctc tcccttcctc tatctcacct acctcatcta ctaccccaac 540
aagggctcct ttgtgtccaa gcccaggaac ctccagaaga tgtccagtga gcccttcaag 600
aatgtgtacc tcctccccca gaccaaccaa ctcctgggac tctacaccat catcaggaac 660
aagaacacca ccaggccaga cttcatcttc tacagtgaca ggatcatcag gctcctggtg 720
gaggagggcc tcaaccacct ccccgtgcag aagcagattg tggagactga caccaatgag 780
aactttgagg gagtgtcttt catgggcaag atttgtgggg tgtccattgt gagggctggg 840
gagagcatgg agcaaggcct gagggactgt tgcaggagtg tgaggattgg caagatcctg 900
atccagaggg atgaggagac tgccctgccc aagctgttct atgagaagct ccctgaagac 960
atctctgaga ggtatgtctt cctcctggac cccatgctgg caactggagg ctctgcaatc 1020
atggccactg aggtgctcat caagagggga gtcaagcctg agaggatcta cttcctcaac 1080
ctcatctgct caaaggaggg cattgagaag taccatgctg ccttccctga agtgaggatt 1140
gtcactgggg ctctggacag gggcctggat gagaacaagt acctggtccc tggcctggga 1200
gactttgggg acagatacta ctgtgtggta ccaggtgttg gtgcacccat ggcagaagga 1260
ggagggcaga atcatcacga agtggtgaag ttcatggatg tctatcagcg cagctactgc 1320
catccaatcg agaccctggt ggacatcttc caggagtacc ctgatgagat cgagtacatc 1380
ttcaagccat cctgtgtgcc cctgatgcga tgcgggggct gctgcaatga cgagggcctg 1440
gagtgtgtgc ccactgagga gtccaacatc accatgcaga ttatgcggat caaacctcac 1500
caaggccagc acataggaga gatgagcttc ctacagcaca acaaatgtga atgcagacca 1560
aagaaagata gagcaagaca agaaaaatgt gacaagccga ggcgt 1605
<210> 33
<211> 657
<212> DNA
<213>
<220>
<223> Fcy-hEGF
<400> 33
atggtcacag gaggcatggc ttcaaagtgg gaccagaagg gcatggacat tgcctatgag 60
gaggctgctc tgggctacaa ggagggaggg gtcccaattg gtggctgcct catcaacaac 120
aaggatggca gtgtcctggg caggggccac aacatgaggt tccagaaggg cagtgccacc 180
ctgcatgggg agatcagcac cctggagaac tgtggcaggc tggagggcaa ggtctacaag 240
gacaccactc tgtacaccac cctcagccct tgtgacatgt gcacaggggc catcatcatg 300
tatggcattc ccaggtgtgt ggtgggagag aatgtcaact tcaagtcaaa aggagagaag 360
tacctccaga ccaggggcca tgaggtggtt gtggtggatg atgagaggtg caagaagatt 420
atgaagcagt tcattgatga gagaccccag gactggtttg aggacattgg ggaggtacca 480
ggtgttggta atagtgactc tgaatgtccc ctgtcccacg atgggtactg cctccatgat 540
ggtgtgtgca tgtatattga agcattggac aagtatgcat gcaactgtgt tgttggctac 600
atcggggagc gatgtcagta ccgagacctg aagtggtggg aactgcgcca tgctggt 657
<210> 34
<211> 1410
<212> DNA
<213>
<220>
<223> Fcy-Fur-hEGF
<400> 34
atggtcacag gaggcatggc ttcaaagtgg gaccagaagg gcatggacat tgcctatgag 60
gaggctgctc tgggctacaa ggagggaggg gtcccaattg gtggctgcct catcaacaac 120
aaggatggca gtgtcctggg caggggccac aacatgaggt tccagaaggg cagtgccacc 180
ctgcatgggg agatcagcac cctggagaac tgtggcaggc tggagggcaa ggtctacaag 240
gacaccactc tgtacaccac cctcagccct tgtgacatgt gcacaggggc catcatcatg 300
tatggcattc ccaggtgtgt ggtgggagag aatgtcaact tcaagtcaaa aggagagaag 360
tacctccaga ccaggggcca tgaggtggtt gtggtggatg atgagaggtg caagaagatt 420
atgaagcagt tcattgatga gagaccccag gactggtttg aggacattgg ggagatgaac 480
cctctcttct tcctggcctc tcccttcctc tatctcacct acctcatcta ctaccccaac 540
aagggctcct ttgtgtccaa gcccaggaac ctccagaaga tgtccagtga gcccttcaag 600
aatgtgtacc tcctccccca gaccaaccaa ctcctgggac tctacaccat catcaggaac 660
aagaacacca ccaggccaga cttcatcttc tacagtgaca ggatcatcag gctcctggtg 720
gaggagggcc tcaaccacct ccccgtgcag aagcagattg tggagactga caccaatgag 780
aactttgagg gagtgtcttt catgggcaag atttgtgggg tgtccattgt gagggctggg 840
gagagcatgg agcaaggcct gagggactgt tgcaggagtg tgaggattgg caagatcctg 900
atccagaggg atgaggagac tgccctgccc aagctgttct atgagaagct ccctgaagac 960
atctctgaga ggtatgtctt cctcctggac cccatgctgg caactggagg ctctgcaatc 1020
atggccactg aggtgctcat caagagggga gtcaagcctg agaggatcta cttcctcaac 1080
ctcatctgct caaaggaggg cattgagaag taccatgctg ccttccctga agtgaggatt 1140
gtcactgggg ctctggacag gggcctggat gagaacaagt acctggtccc tggcctggga 1200
gactttgggg acagatacta ctgtgtggta ccaggtgttg gtaatagtga ctctgaatgt 1260
cccctgtccc acgatgggta ctgcctccat gatggtgtgt gcatgtatat tgaagcattg 1320
gacaagtatg catgcaactg tgttgttggc tacatcgggg agcgatgtca gtaccgagac 1380
ctgaagtggt gggaactgcg ccatgctggt 1410
<210> 35
<211> 852
<212> DNA
<213>
<220>
<223> Fcy-hVEGFa
<400> 35
atggtcacag gaggcatggc ttcaaagtgg gaccagaagg gcatggacat tgcctatgag 60
gaggctgctc tgggctacaa ggagggaggg gtcccaattg gtggctgcct catcaacaac 120
aaggatggca gtgtcctggg caggggccac aacatgaggt tccagaaggg cagtgccacc 180
ctgcatgggg agatcagcac cctggagaac tgtggcaggc tggagggcaa ggtctacaag 240
gacaccactc tgtacaccac cctcagccct tgtgacatgt gcacaggggc catcatcatg 300
tatggcattc ccaggtgtgt ggtgggagag aatgtcaact tcaagtcaaa aggagagaag 360
tacctccaga ccaggggcca tgaggtggtt gtggtggatg atgagaggtg caagaagatt 420
atgaagcagt tcattgatga gagaccccag gactggtttg aggacattgg ggaggtacca 480
ggtgttggtg cacccatggc agaaggagga gggcagaatc atcacgaagt ggtgaagttc 540
atggatgtct atcagcgcag ctactgccat ccaatcgaga ccctggtgga catcttccag 600
gagtaccctg atgagatcga gtacatcttc aagccatcct gtgtgcccct gatgcgatgc 660
gggggctgct gcaatgacga gggcctggag tgtgtgccca ctgaggagtc caacatcacc 720
atgcagatta tgcggatcaa acctcaccaa ggccagcaca taggagagat gagcttccta 780
cagcacaaca aatgtgaatg cagaccaaag aaagatagag caagacaaga aaaatgtgac 840
aagccgaggc gt 852
<210> 36
<211> 1605
<212> DNA
<213>
<220>
<223> Fcy-Fur-hVEGFa
<400> 36
atggtcacag gaggcatggc ttcaaagtgg gaccagaagg gcatggacat tgcctatgag 60
gaggctgctc tgggctacaa ggagggaggg gtcccaattg gtggctgcct catcaacaac 120
aaggatggca gtgtcctggg caggggccac aacatgaggt tccagaaggg cagtgccacc 180
ctgcatgggg agatcagcac cctggagaac tgtggcaggc tggagggcaa ggtctacaag 240
gacaccactc tgtacaccac cctcagccct tgtgacatgt gcacaggggc catcatcatg 300
tatggcattc ccaggtgtgt ggtgggagag aatgtcaact tcaagtcaaa aggagagaag 360
tacctccaga ccaggggcca tgaggtggtt gtggtggatg atgagaggtg caagaagatt 420
atgaagcagt tcattgatga gagaccccag gactggtttg aggacattgg ggagatgaac 480
cctctcttct tcctggcctc tcccttcctc tatctcacct acctcatcta ctaccccaac 540
aagggctcct ttgtgtccaa gcccaggaac ctccagaaga tgtccagtga gcccttcaag 600
aatgtgtacc tcctccccca gaccaaccaa ctcctgggac tctacaccat catcaggaac 660
aagaacacca ccaggccaga cttcatcttc tacagtgaca ggatcatcag gctcctggtg 720
gaggagggcc tcaaccacct ccccgtgcag aagcagattg tggagactga caccaatgag 780
aactttgagg gagtgtcttt catgggcaag atttgtgggg tgtccattgt gagggctggg 840
gagagcatgg agcaaggcct gagggactgt tgcaggagtg tgaggattgg caagatcctg 900
atccagaggg atgaggagac tgccctgccc aagctgttct atgagaagct ccctgaagac 960
atctctgaga ggtatgtctt cctcctggac cccatgctgg caactggagg ctctgcaatc 1020
atggccactg aggtgctcat caagagggga gtcaagcctg agaggatcta cttcctcaac 1080
ctcatctgct caaaggaggg cattgagaag taccatgctg ccttccctga agtgaggatt 1140
gtcactgggg ctctggacag gggcctggat gagaacaagt acctggtccc tggcctggga 1200
gactttgggg acagatacta ctgtgtggta ccaggtgttg gtgcacccat ggcagaagga 1260
ggagggcaga atcatcacga agtggtgaag ttcatggatg tctatcagcg cagctactgc 1320
catccaatcg agaccctggt ggacatcttc caggagtacc ctgatgagat cgagtacatc 1380
ttcaagccat cctgtgtgcc cctgatgcga tgcgggggct gctgcaatga cgagggcctg 1440
gagtgtgtgc ccactgagga gtccaacatc accatgcaga ttatgcggat caaacctcac 1500
caaggccagc acataggaga gatgagcttc ctacagcaca acaaatgtga atgcagacca 1560
aagaaagata gagcaagaca agaaaaatgt gacaagccga ggcgt 1605
<210> 37
<211> 1557
<212> DNA
<213>
<220>
<223> Fcy-Fur-hVEGFc
<400> 37
atggtcacag gaggcatggc ttcaaagtgg gaccagaagg gcatggacat tgcctatgag 60
gaggctgctc tgggctacaa ggagggaggg gtcccaattg gtggctgcct catcaacaac 120
aaggatggca gtgtcctggg caggggccac aacatgaggt tccagaaggg cagtgccacc 180
ctgcatgggg agatcagcac cctggagaac tgtggcaggc tggagggcaa ggtctacaag 240
gacaccactc tgtacaccac cctcagccct tgtgacatgt gcacaggggc catcatcatg 300
tatggcattc ccaggtgtgt ggtgggagag aatgtcaact tcaagtcaaa aggagagaag 360
tacctccaga ccaggggcca tgaggtggtt gtggtggatg atgagaggtg caagaagatt 420
atgaagcagt tcattgatga gagaccccag gactggtttg aggacattgg ggagatgaac 480
cctctcttct tcctggcctc tcccttcctc tatctcacct acctcatcta ctaccccaac 540
aagggctcct ttgtgtccaa gcccaggaac ctccagaaga tgtccagtga gcccttcaag 600
aatgtgtacc tcctccccca gaccaaccaa ctcctgggac tctacaccat catcaggaac 660
aagaacacca ccaggccaga cttcatcttc tacagtgaca ggatcatcag gctcctggtg 720
gaggagggcc tcaaccacct ccccgtgcag aagcagattg tggagactga caccaatgag 780
aactttgagg gagtgtcttt catgggcaag atttgtgggg tgtccattgt gagggctggg 840
gagagcatgg agcaaggcct gagggactgt tgcaggagtg tgaggattgg caagatcctg 900
atccagaggg atgaggagac tgccctgccc aagctgttct atgagaagct ccctgaagac 960
atctctgaga ggtatgtctt cctcctggac cccatgctgg caactggagg ctctgcaatc 1020
atggccactg aggtgctcat caagagggga gtcaagcctg agaggatcta cttcctcaac 1080
ctcatctgct caaaggaggg cattgagaag taccatgctg ccttccctga agtgaggatt 1140
gtcactgggg ctctggacag gggcctggat gagaacaagt acctggtccc tggcctggga 1200
gactttgggg acagatacta ctgtgtggta ccaggtgttg gtgcacatta taatacagag 1260
atcttgaaaa gtattgataa tgagtggaga aagactcaat gcatgccacg ggaggtgtgt 1320
atagatgtgg ggaaggagtt tggagtcgcg acaaacacct tctttaaacc tccatgtgtg 1380
tccgtctaca gatgtggggg ttgctgcaat agtgaggggc tgcagtgcat gaacaccagc 1440
acgagctacc tcagcaagac gttatttgaa attacagtgc ctctctctca aggccccaaa 1500
ccagtaacaa tcagttttgc caatcacact tcctgccgat gcatgtctaa actggat 1557
<210> 38
<211> 849
<212> DNA
<213>
<220>
<223> Fcy-mVEGFa
<400> 38
atggtcacag gaggcatggc ttcaaagtgg gaccagaagg gcatggacat tgcctatgag 60
gaggctgctc tgggctacaa ggagggaggg gtcccaattg gtggctgcct catcaacaac 120
aaggatggca gtgtcctggg caggggccac aacatgaggt tccagaaggg cagtgccacc 180
ctgcatgggg agatcagcac cctggagaac tgtggcaggc tggagggcaa ggtctacaag 240
gacaccactc tgtacaccac cctcagccct tgtgacatgt gcacaggggc catcatcatg 300
tatggcattc ccaggtgtgt ggtgggagag aatgtcaact tcaagtcaaa aggagagaag 360
tacctccaga ccaggggcca tgaggtggtt gtggtggatg atgagaggtg caagaagatt 420
atgaagcagt tcattgatga gagaccccag gactggtttg aggacattgg ggaggtacca 480
ggtgttggtg caccaacaac agagggagaa caaaagagtc acgaggttat caaattcatg 540
gacgtctacc aaaggtcata ttgtagacca attgaaacat tggttgacat attccaggaa 600
tatcctgatg agatagagta catcttcaaa ccatcctgcg tcccacttat gagatgtgct 660
ggttgctgta atgatgaggc attggagtgt gttcctacat ctgaaagtaa cattactatg 720
cagattatga gaatcaagcc tcatcaatca caacatatcg gtgaaatgtc ttttctacaa 780
cactctagat gtgaatgtag acctaagaaa gatagaacta agcctgaaaa gtgtgataaa 840
cctaggaga 849
<210> 39
<211> 1602
<212> DNA
<213>
<220>
<223> Fcy-Fur-mVEGFa
<400> 39
atggtcacag gaggcatggc ttcaaagtgg gaccagaagg gcatggacat tgcctatgag 60
gaggctgctc tgggctacaa ggagggaggg gtcccaattg gtggctgcct catcaacaac 120
aaggatggca gtgtcctggg caggggccac aacatgaggt tccagaaggg cagtgccacc 180
ctgcatgggg agatcagcac cctggagaac tgtggcaggc tggagggcaa ggtctacaag 240
gacaccactc tgtacaccac cctcagccct tgtgacatgt gcacaggggc catcatcatg 300
tatggcattc ccaggtgtgt ggtgggagag aatgtcaact tcaagtcaaa aggagagaag 360
tacctccaga ccaggggcca tgaggtggtt gtggtggatg atgagaggtg caagaagatt 420
atgaagcagt tcattgatga gagaccccag gactggtttg aggacattgg ggagatgaac 480
cctctcttct tcctggcctc tcccttcctc tatctcacct acctcatcta ctaccccaac 540
aagggctcct ttgtgtccaa gcccaggaac ctccagaaga tgtccagtga gcccttcaag 600
aatgtgtacc tcctccccca gaccaaccaa ctcctgggac tctacaccat catcaggaac 660
aagaacacca ccaggccaga cttcatcttc tacagtgaca ggatcatcag gctcctggtg 720
gaggagggcc tcaaccacct ccccgtgcag aagcagattg tggagactga caccaatgag 780
aactttgagg gagtgtcttt catgggcaag atttgtgggg tgtccattgt gagggctggg 840
gagagcatgg agcaaggcct gagggactgt tgcaggagtg tgaggattgg caagatcctg 900
atccagaggg atgaggagac tgccctgccc aagctgttct atgagaagct ccctgaagac 960
atctctgaga ggtatgtctt cctcctggac cccatgctgg caactggagg ctctgcaatc 1020
atggccactg aggtgctcat caagagggga gtcaagcctg agaggatcta cttcctcaac 1080
ctcatctgct caaaggaggg cattgagaag taccatgctg ccttccctga agtgaggatt 1140
gtcactgggg ctctggacag gggcctggat gagaacaagt acctggtccc tggcctggga 1200
gactttgggg acagatacta ctgtgtggta ccaggtgttg gtgcaccaac aacagaggga 1260
gaacaaaaga gtcacgaggt tatcaaattc atggacgtct accaaaggtc atattgtaga 1320
ccaattgaaa cattggttga catattccag gaatatcctg atgagataga gtacatcttc 1380
aaaccatcct gcgtcccact tatgagatgt gctggttgct gtaatgatga ggcattggag 1440
tgtgttccta catctgaaag taacattact atgcagatta tgagaatcaa gcctcatcaa 1500
tcacaacata tcggtgaaat gtcttttcta caacactcta gatgtgaatg tagacctaag 1560
aaagatagaa ctaagcctga aaagtgtgat aaacctagga ga 1602
<210> 40
<211> 1557
<212> DNA
<213>
<220>
<223> Fcy-Fur-mVEGFc
<400> 40
atggtcacag gaggcatggc ttcaaagtgg gaccagaagg gcatggacat tgcctatgag 60
gaggctgctc tgggctacaa ggagggaggg gtcccaattg gtggctgcct catcaacaac 120
aaggatggca gtgtcctggg caggggccac aacatgaggt tccagaaggg cagtgccacc 180
ctgcatgggg agatcagcac cctggagaac tgtggcaggc tggagggcaa ggtctacaag 240
gacaccactc tgtacaccac cctcagccct tgtgacatgt gcacaggggc catcatcatg 300
tatggcattc ccaggtgtgt ggtgggagag aatgtcaact tcaagtcaaa aggagagaag 360
tacctccaga ccaggggcca tgaggtggtt gtggtggatg atgagaggtg caagaagatt 420
atgaagcagt tcattgatga gagaccccag gactggtttg aggacattgg ggagatgaac 480
cctctcttct tcctggcctc tcccttcctc tatctcacct acctcatcta ctaccccaac 540
aagggctcct ttgtgtccaa gcccaggaac ctccagaaga tgtccagtga gcccttcaag 600
aatgtgtacc tcctccccca gaccaaccaa ctcctgggac tctacaccat catcaggaac 660
aagaacacca ccaggccaga cttcatcttc tacagtgaca ggatcatcag gctcctggtg 720
gaggagggcc tcaaccacct ccccgtgcag aagcagattg tggagactga caccaatgag 780
aactttgagg gagtgtcttt catgggcaag atttgtgggg tgtccattgt gagggctggg 840
gagagcatgg agcaaggcct gagggactgt tgcaggagtg tgaggattgg caagatcctg 900
atccagaggg atgaggagac tgccctgccc aagctgttct atgagaagct ccctgaagac 960
atctctgaga ggtatgtctt cctcctggac cccatgctgg caactggagg ctctgcaatc 1020
atggccactg aggtgctcat caagagggga gtcaagcctg agaggatcta cttcctcaac 1080
ctcatctgct caaaggaggg cattgagaag taccatgctg ccttccctga agtgaggatt 1140
gtcactgggg ctctggacag gggcctggat gagaacaagt acctggtccc tggcctggga 1200
gactttgggg acagatacta ctgtgtggta ccaggtgttg gtgcccatta taacacagag 1260
atcctgaaaa gtattgataa tgagtggaga aagactcaat gcatgccacg tgaggtgtgt 1320
atagatgtgg ggaaggagtt tggagcagcc acaaacacct tctttaaacc tccatgtgtg 1380
tccgtctaca gatgtggggg ttgctgcaac agcgaggggc tgcagtgcat gaacaccagc 1440
acaggttacc tcagcaagac gttgtttgaa attacagtgc ctctctcaca aggccccaaa 1500
ccagtcacaa tcagttttgc caatcacact tcctgccggt gcatgtctaa actggat 1557
Claims (2)
1.一种用于治疗癌症的融合蛋白，其特征在于，用于与癌症治疗的前驱药物5-氟胞嘧啶(5-fluorocytosine)结合，其中该融合蛋白包含：
(i)血管内皮生长因子(Vascular Endothelial Growth Factor)为配体；
(ii)胞嘧啶脱氨酶(cytosine deaminase)为前驱药物酵素；以及
(iii)连接子(linker)，其位于该配体与该前驱药物酵素之间。
2.一种权利要求1所述的融合蛋白结合前驱药物用以制备癌症治疗的医药组合物的用途。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201161492649P | 2011-06-02 | 2011-06-02 | |
| US61/492,649 | 2011-06-02 | ||
| CN201210181043.5A CN102807620B (zh) | 2011-06-02 | 2012-06-04 | 治疗感染性与恶性疾病的标靶化学治疗药物的形成方法 |
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| CN201210181043.5A Division CN102807620B (zh) | 2011-06-02 | 2012-06-04 | 治疗感染性与恶性疾病的标靶化学治疗药物的形成方法 |
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| CN201710840189.9A Pending CN107488233A (zh) | 2011-06-02 | 2012-06-04 | 治疗感染性与恶性疾病的标靶化学治疗药物的形成方法 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003083052A2 (en) * | 2002-03-25 | 2003-10-09 | The Trustees Of Boston University | Method of using anti-apoptotic factors in gene expression |
| US20060239965A1 (en) * | 2005-02-28 | 2006-10-26 | Szoka Francis C Jr | Extracellular matrix binding chimeric proteins and methods of use thereof |
| CN101098882A (zh) * | 2004-11-14 | 2008-01-02 | 脉管生物生长有限公司 | 表现出内皮细胞特异性的启动子和使用其调节血管生成的方法 |
| WO2010036986A2 (en) * | 2008-09-26 | 2010-04-01 | Tocagen Inc. | Recombinant vectors |
-
2012
- 2012-06-04 TW TW101120005A patent/TWI486446B/zh not_active IP Right Cessation
- 2012-06-04 CN CN201210181043.5A patent/CN102807620B/zh not_active Expired - Fee Related
- 2012-06-04 CN CN201710840189.9A patent/CN107488233A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003083052A2 (en) * | 2002-03-25 | 2003-10-09 | The Trustees Of Boston University | Method of using anti-apoptotic factors in gene expression |
| CN101098882A (zh) * | 2004-11-14 | 2008-01-02 | 脉管生物生长有限公司 | 表现出内皮细胞特异性的启动子和使用其调节血管生成的方法 |
| US20060239965A1 (en) * | 2005-02-28 | 2006-10-26 | Szoka Francis C Jr | Extracellular matrix binding chimeric proteins and methods of use thereof |
| WO2010036986A2 (en) * | 2008-09-26 | 2010-04-01 | Tocagen Inc. | Recombinant vectors |
Non-Patent Citations (3)
| Title |
|---|
| ZHANG Z等: "VEGF-dependent tumor angiogenesis requires inverse and reciprocal regulation of VEGFR1 and VEGFR2", 《CELL DEATH AND DIFFERENTIATION》 * |
| 方瑾等: "抗人大肠癌单链抗体一胞嘧啶脱氨酶融合基因的构建及表达", 《世界华人消化杂志》 * |
| 陈建雄: "VEGF启动子驱动的双自杀基因治疗大肠癌的实验研究", 《中国优秀硕士学位论文全文数据库（电子期刊）医药卫生科技辑》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| TWI486446B (zh) | 2015-06-01 |
| CN102807620B (zh) | 2017-10-27 |
| CN102807620A (zh) | 2012-12-05 |
| TW201249990A (en) | 2012-12-16 |
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