CN102311502B - 一种抑制血管新生或生长的融合蛋白及其医疗应用 - Google Patents
一种抑制血管新生或生长的融合蛋白及其医疗应用 Download PDFInfo
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Abstract
本发明涉及一种抑制血管新生或生长的融合蛋白，以及其药物用途或药物制剂。该融合蛋白能够同时抑制PDGF和VEGF，并能有效的治疗由血管新生或生长引起的各种疾病，如肿瘤、眼疾等。
Description
技术领域
本发明涉及基因工程和蛋白质工程技术领域，具体地说，涉及一种能有效抑制血管新生或生长的融合蛋白，以及其药物用途或药物制剂。
背景技术
在正常生理条件下，血管的生成是一个受多个环节严密调节的过程，对修复和维持机体的正常功能具有重要的作用。然而，如肿瘤内部血管快速生长，则是临床常见的现象之一。大量的动物模型和人体临床试验表明，阻断肿瘤内新生血管的形成可以有效地阻止肿瘤的生长和引发肿瘤细胞的死亡，从而达到对肿瘤的治疗效果。因此，抑制血管新生是目前抗肿瘤新药研究的重要进展之一，大分子抗新生血管药物如Avastin经过美国FDA的上市批准，同时还有更多的药物处于不同的临床前和临床研究阶段。另外，对于老年性视网膜血管病变（Age-related macular degeneration，简称为AMD）、糖尿病视网膜病变、关节炎等多种与血管新生相关的疾病，抑制血管新生治疗新药也有广泛和重要的作用。
血管新生是一个有多种活性生物因子进行调控的复杂过程，其中一个关键环节是内皮细胞表面受体由多种生长因子相结合从而被激活，然后经细胞内酪氨酸磷酸化的信号传递***控制内皮细胞活动，从而促使血管新生。多种生长因子中，血管内皮细胞生长因子（Vascular endothelial cell growth factor，简称为VEGF）和血小板衍生生长因子( platelet derived growth factor, 简称为PDGF)是控制血管新生最重要的两类因子，是诱导和促进血管形成作用最强、最专一的血管生长因子，在几乎所有的人体肿瘤中都过量表达，是抗癌研究的一个重要分子靶标。
PDGF主要通过与PDGF受体( PDGFR)结合, 进而激活蛋白激酶信号转导通路而发挥作用。PDGFR由α和β两种亚基构成, 共有3 种二聚体(PDGFR-αα、αβ、ββ), 其中ββ二聚体受体( PDGFR-β)最为重要, 其分子量约为180～190 kd, 属于酪氨酸激酶受体( receptor tyrosine kinase, RTK)家族。PDGFR在肿瘤形成和发展过程中起着重要的作用。PDGFR-β的过度表达或过度活化均能刺激肿瘤内血管生成, 促进肿瘤生长。PDGFR-β是肿瘤血管内皮细胞的分子标志之一, 在肿瘤新生血管内皮细胞中高表达, 并与某些肿瘤的生长、转移及预后密切相关。因此PDGFR-β是一个较为理想的肿瘤靶向治疗的靶标。
VEGF在血管内皮细胞表面上有多种受体，其中包括VEGFR-1（又称为Flt-1）和VEGFR-2（又称为KDR或Flk-1），它们的细胞外部分均由七个可与VEGF相结合的免疫球蛋白样区域 （D1-D7）组成，细胞内部分均包括酪氨酸激酶基团，当受体被VEGF激活后，细胞内的酪氨酸激酶基团发生磷酸化，并导致一系列的信号传递，最终引发血管新生。由于VEGF信号传递对血管新生的重要性，阻断VEGF或VEGF受体从而达到抑制血管新生具有重要的抗癌作用，同时也对其他包括视网膜血管病变等与血管新生相关的疾病有着重要的治疗作用。目前唯一被美国FDA批准的针对血管新生的抗癌药物（Avastin）就是特异性结合VEGF-A的单克隆抗体，其机理就是通过结合VEGF达到阻断VEGF与其受体结合的目的。另一类可能效果更好的VEGF阻断剂是VEGF受体的细胞外片段，其具有天然的针对VEGF的高特异性。
目前大量的研究已经证实血管的生成是受多因子调控的复杂过程，而且PDGF和VEGF是控制血管新生最重要的两类因子。但是目前的药物，尤其是大分子抗体和融合蛋白类药物，都是针对其中一个作为治疗靶点的，考虑到血管新生是一个复杂的过程，为了改进目前仅针对单靶点治疗药物的缺陷，本发明提供了一个针对血管新生最重要的两个靶点同时抑制的融合蛋白，在大大提高生物活性的基础上，保证其在体外和动物内都能更有效地结合或中和VEGF和PDGF，阻断血管新生和生长，从而达到更好的治疗效果。
发明内容
本发明目的之一是提供一种经优化、且包含VEGF和PDGF受体细胞外片段和人免疫球蛋白Fc的融合蛋白，其在体内外都具有优良的稳定性和生物活性，并能同时阻断VEGF和PDGF信号传递，从而抑制血管新生和生长的作用。
为了实现上述目的，本发明提供了以下技术方案：
本发明一方面提供了一种融合蛋白，该融合蛋白是由PDGF受体和VEGF受体的不同片段与人免疫球蛋白Fc构成，其中PDGF受体片段，VEGF受体片段或Fc之间还可以包括一个或多个优化肽连接片段，该肽连接片段选自以下的组：
a. (Gly Gly Gly Gly Ser) n, n可以为1，2，3，4，5；
b. (Gly Gly Gly) n, n可以为1，2，3，4，5；
其中所述PDGF受体片段选自以下的组：
a. PDGF α 受体的细胞外免疫球蛋白样区域1、2、3或4，其氨基酸序列如序列表中SEQ ID NO.1 所述；或
b. PDGF β受体的细胞外免疫球蛋白样区域1、2、3或4，其氨基酸序列如序列表中SEQ ID NO.2 所述；
所述的VEGF受体片段选自以下的组：
a. VEGF-1受体的细胞外第2免疫球蛋白样区域，其氨基酸序列如序列表中SEQ ID NO.3 所述；
b. VEGF-2受体的细胞外第3免疫球蛋白样区域，其氨基酸序列如序列表中SEQ ID NO.4所述；
c. VEGF-1受体的细胞外第4免疫球蛋白样区域，其氨基酸序列如序列表中SEQ ID NO.5 所述。
本发明还进一步提供了具有以下结构的融合蛋白：
a. KH-001：α RD d1 － α RD d2 －α RD d3 － α RD d4 － FLT-1 d2 － KDR d3 －Fc;
b. KH-002：β RD d1 － β RD d2 － β RD d3 － β RD d4 －FLT-1 d2 －KDR d3－Fc;
c. KH-003：α RD d2 －α RD d3－ FLT-1 d2 － KDR d3 －FLT-1 d4－Fc;
d. KH-004： β RD d2 － β RD d3－FLT-1 d2 －KDR d3－FLT-1 d4－Fc;
e. KH-005：α RD d2 －α RD d3－ FLT-1 d2 － KDR d3－Fc;
f. KH-006： β RD d2 － β RD d3－FLT-1 d2 －KDR d3－Fc;
g. KH-007：α RD d2 －α RD d3－GS－ FLT-1 d2 － KDR d3 －GS－Fc;
h. KH-008： β RD d2 － β RD d3－GS－FLT-1 d2 －KDR d3－GS－Fc;
i. KH-009：α RD d2 －α RD d3－ FLT-1 d2 － KDR d3 －GS－Fc;
j. KH-010： β RD d2 － β RD d3－FLT-1 d2 －KDR d3－GS－Fc;
k. KH-011：α RD d2 －α RD d3－GS－ FLT-1 d2 － KDR d3－Fc;
l. KH-012： β RD d2 － β RD d3－GS－FLT-1 d2 －KDR d3－Fc;
其中，
α RD d1、α RD d2、α RD d3、α RD d4的氨基酸序列如序列表中SEQ ID NO.1所述；
β RD d1、β RD d2、β RD d3、β RD d4的氨基酸序列如序列表中SEQ ID NO.2所述；
FLT-1 d2的氨基酸序列如序列表中SEQ ID NO.3 所述；
KDR d3的氨基酸序列如序列表中SEQ ID NO.10 所述；
FLT-1 d4的氨基酸序列如序列表中SEQ ID NO.11 所述；
Fc的氨基酸序列如序列表中SEQ ID NO.12 所述；
GS的氨基酸序列为(Gly Gly Gly Gly Ser) n, n可以为1，2，3，4，5。
其中，更优选为
KH-001的编码DNA如序列表中SEQ ID NO.13 所述；
KH-002的编码DNA如序列表中SEQ ID NO.14所述；
KH-003的编码DNA如序列表中SEQ ID NO.15 所述；
KH-004的编码DNA如序列表中SEQ ID NO.16所述;
KH-005的编码DNA如序列表中SEQ ID NO.17 所述；
KH-006的编码DNA如序列表中SEQ ID NO.18所述；
KH-007的编码DNA如序列表中SEQ ID NO.19 所述；
KH-008的编码DNA如序列表中SEQ ID NO.20所述;
KH-009的编码DNA如序列表中SEQ ID NO.21 所述；
KH-010的编码DNA如序列表中SEQ ID NO.22所述；
KH-011的编码DNA如序列表中SEQ ID NO.23 所述；
KH-01的编码DNA2如序列表中SEQ ID NO.24所述。
本发明还提供了上述融合蛋白的载体，其载体优选为质粒，病毒或DNA片段。本发明还提供了上述融合蛋白载体的细胞，优选为原核或真核细胞。
本发明还提供了上述融合蛋白以及药学上可接受的载体或赋形剂组成的药物组合物；其中制剂形式优选为注射剂、注射用冻干粉针或眼用凝胶。
本发明又提供了上述融合蛋白在制备治疗由血管新生或生长引起的疾病的药物中的应用；其中所述的由血管新生或生长引起的疾病优选为肿瘤或眼部新生血管疾病，所述的眼部新生血管疾病更优选为年龄相关性黄斑病变。
本发明的要点在于现有技术中抑制新生血管药物仅通过抑制VEGF来控制血管新生，但不能使已经生成的血管消退的缺陷，设计并构建了一系列双靶点的融合蛋白，同时也可通过加入肽连接段（peptide linker）插于VEGF受体，PDGF受体的细胞外片段或Fc之间，使得VEGF受体的细胞外片段，PDGF受体的细胞外片段或Fc之间具有足够高的结构弹性和可塑性，从而大大增加了融合蛋白的稳定性，更重要的是，显著提高了与多种VEGF和PDGF结合的亲和力。这种包含肽连接段的优化融合蛋白，和没有包含该肽连接段的融合蛋白相比，具有更高的生物活性和更长的血清半衰期，从而能够更有效地中和VEGF和PDGF，抑制血管新生和肿瘤发展。
PDGF是一个二聚体类的生长因子，其受体分为α和β两种。两种受体细胞外片段中都包含5个免疫球蛋白样的区域，其中D2和D3主要参与对PDGF的结合，D4主要参与受体－受体之间的相互作用(Olli et al. Biochemistry 2000, 39, 2370-2375) 。我们的研究结果表明，不包含D2和D3的多种PDGF受体融合蛋白损失了绝大部分的PDGF亲和力，而包含D2和D3的多种PDGF受体融合蛋白则保存了对PDGF的亲和力，因此，本发明结合PDGF的α和β受体结构域2和3一个或者多个的组合。
本发明涉及的蛋白分子至少具有两个单独的功能结构域，即与VEGF和PDGF结合的受体区域。US610071公开了单独的VEGF片段或Fc的融合蛋白丧失了大部分结合VEGF的活性。在此失败的D2-Fc融合蛋白中，只有一个包含二个氨基酸（FE）的连接段位于D2片段和Fc之间，这可能就是其失败的原因。VEGF, PDGF和Fc是三个个独立的具有不同功能的区域，均必须形成各自稳定有活性的三维空间结构，特别是在二个Fc区域二聚化后，二个生长因子结合区域必须保持其结构稳定性和生物活性，从而保证其对VEGF和PDGF的亲和力。而在此失败的D2-Fc融合蛋白中，D2和Fc间的二个氨基酸很可能不足以提供足够的结构空间来满足D2和Fc稳定结构的需要，使得D2、或Fc、或二者均不能形成稳定的结构，最终大大影响D2-Fc融合蛋白对VEGF的亲和力。本发明则提供了一系列弹性结构的优化肽连接段以连接蛋白中的两个（或三个）相对独立的功能区域，使之都有足够的空间来形成稳定的结构，从而保证了的亲和力和生物活性。
本发明描述的PDGF, VEGF受体细胞外片段和Fc的优化融合蛋白是通过常规或为分子生物学领域研究人员所熟悉的基因重组技术所建造，具体实验步骤如<<分子克隆>>第三版(Joseph Sambrook，科学出版社)。
本发明所涉及的蛋白主要结构如图1所示，其编码DNA可通过常规的基因重组技术获得。所需编码PDGF受体、VEGF受体和Fc片段的DNA序列由NCBI(National Center for Biotechnology Information)的GenBank中获得后，将编码上述优化融合蛋白的DNA序列由PCR合成后分别克隆到载体中，所用载体可以是分子生物学所常用的质粒、病毒或DNA片段。在编码上述优化融合蛋白的DNA序列的末端前加上蛋白分泌信号序列，以保证蛋白质从细胞中分泌出来。载体序列中包括用于驱动基因表达的启动子、蛋白质翻译起始和终止信号、以及多聚腺苷酸(PolyA)序列。载体中有抗菌素抗性基因，以利于载体在宿主细胞，如细菌中繁殖。另外，载体中还包括真核细胞选择性基因，用于稳定转染宿主细胞株的选择。
由于PDGF受体和VEGF受体中各个免疫球蛋白样区域的氨基酸序列之间并没有绝对的分界，因此各免疫球蛋白样区域的氨基酸序列的长度可以有一定的变化。所以，本发明所涉及的优化融合蛋白质的氨基酸序列也可以有一定的变化，它们都属于本发明的范围。
上述优化融合蛋白中的Fc来自人免疫球蛋白IgG1，也可以是亚型IgG2、IgG3、IgG4、或者人免疫球蛋白IgM和IgA，该免疫球蛋白Fc片段可以为Fc全长或部分Fc序列，如选自CH2片断、CH3片断或绞合区域片段，它们都属于本发明的范围。
本发明中涉及的肽连接段目的在于提供更好的弹性和空间的独立性，因此其氨基酸序列和长度都可以允许一定的变化，也可以由一个完全随机的肽链文库中筛选而得，它们都属于本发明的范围。
在完成含编码上述优化融合蛋白的DNA序列的质粒构建以后，即可用该重组载体转染或转化宿主细胞，表达相应的融合蛋白质。能够用于表达这些融合蛋白的表达***有多种，可以是真核细胞也可以是原核细胞，它们包括(但不限于)哺乳动物细胞、细菌、酵母、昆虫细胞等。由于本发明的融合蛋白质的氨基酸序列中包括可糖基化的氨基酸，因此，哺乳动物细胞是表达这些蛋白质的最佳***。可用于蛋白质大规模表达的哺乳动物细胞有多种，例如293细胞、CHO细胞、SP20细胞、NS0细胞、COS细胞、BHK细胞或PerC6细胞等，许多其他细胞也可用于这些蛋白质的表达和生产，因此都包括在本发明所能使用的细胞之列。含有编码上述优化融合蛋白的重组质粒可经转染进入宿主细胞，转染细胞的方法有多种，其中包括但不限于：电转、脂质体转染、钙介导法等。
一种较佳的表达方法是在已稳定转染的宿主细胞中对重组载体进行基因扩增，以提高相应重组融合蛋白的表达量，例如用含有二氢叶酸还原酶（DHFR）的重组载体稳定转染缺乏DHFR的宿主细胞后，可在细胞培养液中增加氨甲喋呤（MTX）的浓度以扩增重组载体在宿主细胞中的拷贝数量；又例如对表达谷氨酰胺合成酶(GS)的稳定转染宿主细胞，利用增加培养液中甲硫氨酸亚砜(MSX)的浓度可扩增重组载体的拷贝数量。哺乳动物细胞以外的其他表达***，例如细菌、酵母或昆虫细胞等也能用于表达这些优化融合蛋白，它们也包括在本发明所能使用的细胞之列。这些表达***的蛋白质产量比哺乳动物细胞的为高，但是，所表达的蛋白质缺乏糖基化或者所形成的糖链与哺乳动物细胞不同。
优化融合蛋白质表达后，可用酶联免疫吸附试验(ELISA)或其他方法测定细胞培养液中融合蛋白质的浓度。对于包含Fc片段的蛋白，可用蛋白A亲和层析法来提纯。对于蛋白，可以根据载体中加入的纯化标签选择响应的纯化方法。
从重组体培养液中获得相应的优化融合蛋白质后，可利用PDGF和VEGF体外结合实验来检测和比较各种蛋白质亲和力。实验结果证明，与现有技术公开的仅仅含有VEGF或PDGF受体结构域融合蛋白相比，本发明所构建的优化融合蛋白质大大同时具备较高的PDGF和VEGF的亲和力（见图2）。相对于单靶点PDGF或VEGF的蛋白，本发明所构建的优化融合蛋白对具有更好的阻断作用，从而可以更有效地抑制血管新生，治疗与血管新生的疾病，它们包括但不限于各种肿瘤、视网膜血管病变AMD、糖尿病视网膜病变、关节炎、贫血或子宫内膜增殖症等。本发明还提供了动物实验以证明本发明提供的优化融合蛋白质在活体内的抗血管新生作用。实验证明，在HCC Hep3B肝癌和Lovo大肠癌的小鼠模型中，本发明提供的融合蛋白都充分抑制了肿瘤的生长，而且效果明显好于现有技术的单独靶点的融合蛋白，因此，本发明所构建的融合蛋白具有高效的抗癌能力。目前，抗VEGF单克隆抗体治疗已经成为治疗湿AMD的最佳治疗方案，但是接受治疗的病人中仅有约三分之一的视力可以提高。该种治疗方案的最主要的缺点仍然是只能抑制新血管的发生，不能使已有血管缩小甚至消失。因此，开发出能使使新生血管消退的治疗方案可以极大地提升药物的治疗效果。.本发明还提供的融合蛋白，就可以通过抑制血管外周细胞，更令人惊奇的是可以使已有新生血管萎缩。在治疗AMD中，血管外周细胞可以通过同时分泌生长因子（如VEGF）和其它前生长因子促使新生血管的形成。因此，仅仅通过抗VEGF治疗，很难使已经形成的血管消退。PDGF作为调节外周细胞聚集和成熟的细胞因子，其表达水平的提高可以促进新生血管的产生。反之，通过抑制PDGF，可以使外周细胞脱落，新生血管消退。另外，在激光诱发脉络膜新生血管（choroidal neovascularization，简称为CNV）小鼠模型中，本发明设计的蛋白质，不但可以抑制新生血管的产生，而且可以使治疗以前产生的新生血管萎缩，使出血面积缩小，因此，对视网膜血管病变拥有良好的医疗前景。
本发明还提供了含有本发明融合蛋白和药用载体的药物组合物。该药物组合物可以按照制剂学常规技术制成各种形式的药物制剂，优选的是注射剂，最优选的是冷冻干燥注射剂。
本发明的药物组合物，其中还包括任何一种或几种其他的具有协同作用的抑制血管新生的药物，所述组合物可以和其他治疗方法一起治疗血管新生相关疾病，所述其他治疗方法选自化学疗法、反射疗法、基因疗法。
采用了C端融合人Fc蛋白的融合蛋白，其目的一方面是为了延长该可溶性融合蛋白的半衰期，使其不易被降解，另一方面是在下游的纯化中能够较方便地通过亲和层析获得目的蛋白。
附图说明
图1 各种蛋白的结构组成。
PDGF α受体的第1免疫球蛋白样区域（表示为α RD d1），第2免疫球蛋白样区域（表示为α RD d2），第3免疫球蛋白样区域（表示为α RD d3），第4免疫球蛋白样区域（表示为α RD d4）；PDGF β受体的第1免疫球蛋白样区域（表示为β RD d1），第2免疫球蛋白样区域（表示为β RD d2），第3免疫球蛋白样区域（表示为β RD d3），第4免疫球蛋白样区域(表示为β RD d4)；VEGFR-1的第2免疫球蛋白样区域(表示为FLT-1d2)，VEGFR-2的第3免疫球蛋白样区域(表示为KDR d3)；人免疫球蛋白Fc区域(表示为Fc)，三种优化肽连接段(表示为G9、G12、和GS)。
图2-A．各种融合蛋白在体外分别结合PDGF的亲和力。
图2-B. 各种融合蛋白在体外分别结合VEGF的亲和力。
图3 融合蛋白有效地抑制HCC Hep3B肝癌在小鼠体内的生长。
图4 为融合蛋白在小鼠眼内抑制由激光诱发脉络膜新生血管CNV小鼠模型中能抑制CNV的生成。
具体实施方式
以下实施例对本发明所涉及的优化融合蛋白构建、试验和应用作了详细说明。但是本发明的内容及用途并不限制于实例的范围。
实施例1、克隆编码优化融合蛋白的DNA序列及构建重组载体
以本公司已构建的FP2和FP3(ZL200510073595.4)分别扩增FLT-1D2、KDRD3、FLT-1D4和Fc，编码PDGFRα和PDGFRβ的免疫球蛋白样的结构域是从人胚胎肝脏细胞，再用不同的引物利用聚合酶链反应（PCR）扩增获得需要的片段，最后将相应的序列融合，从而构建不同融合蛋白质的DNA序列。本优选实施例所构建的12种融合蛋白的结构见附图1。
收集T-175培养瓶中生长状体良好的提取人胚胎肝脏细胞(来自美国标准细胞库)，大约为1×107个细胞，应用RNA提取试剂盒（QIAGEN公司）提取细胞总RNA, 并用Invitrogen公司cDNA试剂盒逆转录成cDNA, -80℃冻存备用。FP2和FP3质粒按照常规培养细菌方法培养，并以质粒提起试剂盒制备质粒，最后以质粒和肝脏cDNA作为模板，采用下列特异引物扩增目的基因片段。
KH-001扩增引物
P1
S: 5’ CAGGATTCGCAAGGACACCATGGGGACTTC 3’
AS: 5’ ACGAAAGGTCTACCTAACTTGAGTTAACAGTTC 3’
P2
S: 5’ CTGTTAACTCAAGTTAGGTAGACCTTTCGTAGAG 3’
AS: 5’ ATTCTGCAGTCATTTACCCGGAGACAGGGAG 3’。
KH-002扩增引物
P3
S: 5’ ATCGGATCCAAGGACACCATGCGGCTTCCGGG 3’
AS: 5’ ACGAAAGGTCTACCTGACATTGATCTGTAGCTGGAAGG 3’
P4
S: 5’ CCAGCTACAGATCAATGTCAGGTAGACCTTTCGTAGAGATG 3’。
KH-003扩增引物
P5
S: 5’ GTTAGATCTAGCTATGGGGACTTCCCATC 3’
AS: 5’ CTACG AAAGGTCTACCTGGCCCGCACCTCTACAACAA 3’
P6
S: 5’ TTTTGTTGTAGAGGTGCGGGCCAGGTACACCTTTCGTAG 3’。
KH-004扩增引物
P7
S: 5’ ATTGGATCCACCATGCGGCTTCCGGGTGCG 3’
AS: 5’ CTACG AAAGGTCTACCTGTAGCCGCTCTCAACCACGGTG 3’
P8
S: 5’ GGTTGAGAGCGGCTACAGGTAGACCTTTCGTAGAGATG 3’。
KH-005扩增引物
PCR引物同KH-003,以FP2为模板。
KH-006扩增引物
PCR引物同KH-004,以FP2为模板。
KH-007扩增引物
P9
S: 5’ CCAGATCCGCCACCTCCGGCCCGCACCTCTACAAC 3’
P10
S: 5’ GTGGCGGATCTGGAGGTGGCGGATCTAGGTAGACCTTTCG3’
AS: 5’ AGATCCGCCACCTCCTTTTCATGGACCCTGACAAATGT 3’
P11
S: 5’ GGAGGTGGCGGATCTGGAGGTGGCGGATCTGACAAAACTCAC 3’。
KH-008扩增引物
P9
S: 5’ CCACCTCCAGATCCGCCACCTCCGTAGCCGCTCTCAACCA 3’。
KH-009
以P5、P6、P10AS及P11联用扩增产物，最后以P5S和P2AS并以上三种扩增产物拼接成KH-009。
KH-0010
以P7、P8、P10AS及P12联用扩增产物，最后以P7S和P2AS并以上三种扩增产物拼接成KH-0010。
KH-0011
以P5、P9、P10S及P12联用扩增产物，最后以P5S和P2AS并以上三种扩增产物拼接成KH-0011。
KH-0012
以P7S、P12及P2AS联用扩增产物，最后以P7S和P2AS并以上三种扩增产物拼接成KH-0012。
在95℃变性3分钟，退火56℃，45秒，72℃延伸2分钟的条件下，进行PCR扩增，30个循环后，获得PDGFR α和PDGFR β，FLT-1，KDR免疫球蛋白样结构域和人免疫球蛋白1的Fc段PCR产物， 采用TA Cloning试剂盒，把PCR产物克隆导入至pCR2.1质粒， 并转化大肠杆菌，挑出平皿上过夜长出的白色耐药菌落，加入到LB液体培养基中过夜扩增。 Qiagen（Mini）质粒抽提试剂盒抽提质粒后酶切及测序鉴定，获得的编码DNA无缺码和移码突变。
采用拼接PCR（sewing PCR）方法，按照图1所设计的蛋白序列，将相应的编码cDNA连接在一起，并在引物中加入EcoRI酶切位点，EcoRI酶切后，用QIAGENE酶切试剂盒纯化DNA片段并***pCDNA3.1质粒，用连接产物转化大肠杆菌，挑出平皿上过夜长出的白色阳性菌落，加入到LB液体培养基中，培养过夜。Qiagen（Mini）质粒抽提试剂盒抽提质粒后酶切及测序鉴定，获得的编码DNA无缺码和移码突变。
KH-007到KH-012类似于KH-001，但是它们是在Fc前分别包含了GS (或G9,G12等)肽连接段的优化融合蛋白。它们的建造是以KH-001质粒为模板，通过拼接PCR（Bridge PCR）的方法构造而得。
实施例2、融合蛋白在CHO细胞中的稳定表达
将编码KH-001到KH-008的重组高纯度质粒利用电转的方式导入CHO细胞（Invitrogen公司）中，在无血清培养液基二天后加入G148，待G148抗体集落形成后，采用有限稀释法进行克隆培养，大约2周后挑取单克隆细胞并转入培养皿中扩大培养。
实施例3融合蛋白的纯化
收集含融合蛋白的细胞培养液，离心取出细胞碎片，采取葡萄球菌A蛋白亲和层析的方法进行纯化。将蛋白A-Sepharose 层析柱以PBS缓冲液洗以平衡后，以2毫升/分钟的速度将超滤器浓缩过的培养液上样，用PBS缓冲液洗直至未结合的蛋白全被洗脱（以A280进行监测），然后用100mM的柠檬酸（pH=3）洗脱结合蛋白，立刻以足够多的2M Tris 进行中和。
实施例4 融合蛋白的体外结合实验
本发明利用高特异性的VEGF和PDGF检测试剂盒（R&D Systems公司）通过检测游离的VEGF和PDGF确定各融合蛋白和的亲和力。实验中，将不同浓度（0到1000 pM）的KH-001到KH-012融合蛋白分别和10 pM的人VEGF165和PDGF（Sigma公司）在室温下相混合，培养过夜后用检测试剂盒来检测没有被融合蛋白结合的游离PDGF和VEGF浓度。Fc和KH-001到KH012的12个融合蛋白分别与PDGF相结的半数抑制浓度（IC50）为6.4pM、6.3pM、6.2pM、5.6pM、5.4pM、4.9pM、4.4pM、3.8pM、2.7pM、1.4pM、1.3pM、1.2pM、0.8 pM，具体结果如图2A显示, 所有融合蛋白都保持着对PDGF的很好亲和力, 其中包含肽连接段的融合蛋白KH-007到KH-012显示了更优的亲和力；Fc和KH-001到KH012的12个融合蛋白分别与VEGF相结的半数抑制浓度（IC50）为6.2pM、5.4pM、4.8pM、4.4pM、3.3pM、2.4pM、1.5pM、1.2pM、1.1pM、0.8pM、0.7pM、0.6pM、0.5 pM，具体结果如图2B显示, 所有融合蛋白都保持着对VEGF的很好亲和力, 其中包含肽连接段的融合蛋白KH-007到KH-012显示了更优的亲和力。该实验进一步说明含有双靶点的融合蛋白具有高效，专一和能结合两个靶分子的特点。
实施例5 制备含融合蛋白的注射剂
优化融合蛋白的注射剂可由多种配方制备而成，通常该融合蛋白的浓度为0.1 mg/ml到100 mg/ml，另外的成分包括但不限于各种载体蛋白、缓冲剂、电解质离子、和缓释剂。其中一种较佳的配方为：20 mg/ml的优化融合蛋白、5 mM Phosphate、5 mM Citrate、100 mM NaCl、0.1% Tween 20、20% Sucrose、pH 6.0，按照注射剂的常规制备方法制成。
实施例6 融合蛋白对Lovo大肠癌细胞在裸鼠内的生长的抑制作用
Lovo大肠癌细胞(来源ATCC)常规培养于37oC，当生长密度达到所需要求后，以5 X 106 Lovo大肠癌细胞/每只数量背部皮下注射BalB/C裸鼠，成瘤后尾静脉注射提各种融合蛋白，用量为2 mg/kg，每周两次一直到第七周，对照组注射相同剂量的人免疫球蛋白Fc。肿瘤的体积每周测量一次，实验结果如图3所示。Fc对照组没有抗癌作用，KH-002, KH-004, KH-006和KH-008都显示了良好的抗癌效果，而KH-008则显示了最优的抗癌效果，完全抑制了肿瘤细胞在小鼠体内的生长。
实施例7 融合蛋白对激光诱导的小鼠脉络膜新生血管（CNV）的抑制作用
采用红色离子激光（能量为120-150mw,光斑直径70-100μm，时间0.1秒）选择视网膜后级部，避开视网膜大血管，在9、12、3和6方位点各激射一发，击破bruch膜，诱导CNV形成。bruch膜破裂产生的气泡是一个诱发脉络膜新生血管（CNV）的重要标志，4个点位均有气泡产生才能入选本实验。小鼠的右眼由玻璃体腔注射500ug融合蛋白，左眼作为对照组不接受任何处理。二个星期后，小鼠被处死并如文献（Mori et al，Investigative Ophthalomology & Visual Science，2002，43 (6)：1994-2000）所述方法检测脉络膜新生血管的程度，结果如图4所显示，融合蛋白能够抑制激光诱发CNV的生成。
SEQUENCE LISTING
<110> 成都康弘生物科技有限公司
<120> 一种抑制血管新生或生长的融合蛋白及其医疗应用
<130> CN1706867A
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Trp Phe Lys Asp Asn Arg Thr Leu Gly Asp Ser Ser Ala Gly Glu Ile
325 330 335
Ala Leu Ser Thr Arg Asn Val Ser Glu Thr Arg Tyr Val Ser Glu Leu
340 345 350
Thr Leu Val Arg Val Lys Val Ala Glu Ala Gly His Tyr Thr Met Arg
355 360 365
Ala Phe His Glu Asp Ala Glu Val Gln Leu Ser Phe Gln Leu Gln Ile
370 375 380
Asn Val
385
<210> 3
<211> 93
<212> PRT
<213> FLT-1 d2
<400> 3
Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr
1 5 10 15
Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile
20 25 30
Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly
35 40 45
Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala
50 55 60
Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly
65 70 75 80
His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr
85 90
<210> 4
<211> 102
<212> PRT
<213> KDR d3
<400> 4
Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys
1 5 10 15
Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp
20 25 30
Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val
35 40 45
Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu
50 55 60
Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr
65 70 75 80
Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe
85 90 95
Val Arg Val His Glu Lys
100
<210> 5
<211> 87
<212> PRT
<213> FLT d4
<400> 5
Thr Val Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys
1 5 10 15
Arg Ser Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu
20 25 30
Val Val Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu Lys Ser Ala Arg
35 40 45
Tyr Leu Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp Val Thr Glu Glu
50 55 60
Asp Ala Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln Ser Asn Val
65 70 75 80
Phe Lys Asn Leu Thr Ala Thr
85
<210> 6
<211> 227
<212> PRT
<213> Fc
<400> 6
Asp Lys Thr His Thr Cys Pro Leu Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
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 Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Ala 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> 7
<211> 818
<212> PRT
<213> KH-001
<400> 7
Gln Leu Ser Leu Pro Ser Ile Leu Pro Asn Glu Asn Glu Lys Val Val
1 5 10 15
Gln Leu Asn Ser Ser Phe Ser Leu Arg Cys Phe Gly Glu Ser Glu Val
20 25 30
Ser Trp Gln Tyr Pro Met Ser Glu Glu Glu Ser Ser Asp Val Glu Ile
35 40 45
Arg Asn Glu Glu Asn Asn Ser Gly Leu Phe Val Thr Val Leu Glu Val
50 55 60
Ser Ser Ala Ser Ala Ala His Thr Gly Leu Tyr Thr Cys Tyr Tyr Asn
65 70 75 80
His Thr Gln Thr Glu Glu Asn Glu Leu Glu Gly Arg His Ile Tyr Ile
85 90 95
Tyr Val Pro Asp Pro Asp Val Ala Phe Val Pro Leu Gly Met Thr Asp
100 105 110
Tyr Leu Val Ile Val Glu Asp Asp Asp Ser Ala Ile Ile Pro Cys Arg
115 120 125
Thr Thr Asp Pro Glu Thr Pro Val Thr Leu His Asn Ser Glu Gly Val
130 135 140
Val Pro Ala Ser Tyr Asp Ser Arg Gln Gly Phe Asn Gly Thr Phe Thr
145 150 155 160
Val Gly Pro Tyr Ile Cys Glu Ala Thr Val Lys Gly Lys Lys Phe Gln
165 170 175
Thr Ile Pro Phe Asn Val Tyr Ala Leu Lys Ala Thr Ser Glu Leu Asp
180 185 190
Leu Glu Met Glu Ala Leu Lys Thr Val Tyr Lys Ser Gly Glu Thr Ile
195 200 205
Val Val Thr Cys Ala Val Phe Asn Asn Glu Val Val Asp Leu Gln Trp
210 215 220
Thr Tyr Pro Gly Glu Val Lys Gly Lys Gly Ile Thr Met Leu Glu Glu
225 230 235 240
Ile Lys Val Pro Ser Ile Lys Leu Val Tyr Thr Leu Thr Val Pro Glu
245 250 255
Ala Thr Val Lys Asp Ser Gly Asp Tyr Glu Cys Ala Ala Arg Gln Ala
260 265 270
Thr Arg Glu Val Lys Glu Met Lys Lys Val Thr Ile Ser Val His Glu
275 280 285
Lys Gly Phe Ile Glu Ile Lys Pro Thr Phe Ser Gln Leu Glu Ala Val
290 295 300
Asn Leu His Glu Val Lys His Phe Val Val Glu Val Arg Ala Tyr Pro
305 310 315 320
Pro Pro Arg Ile Ser Trp Leu Lys Asn Asn Leu Thr Leu Ile Glu Asn
325 330 335
Leu Thr Glu Ile Thr Thr Asp Val Glu Lys Ile Gln Glu Ile Arg Tyr
340 345 350
Arg Ser Lys Leu Lys Leu Ile Arg Ala Lys Glu Glu Asp Ser Gly His
355 360 365
Tyr Thr Ile Val Ala Gln Asn Glu Asp Ala Val Lys Ser Tyr Thr Phe
370 375 380
Glu Leu Leu Thr Gln Val Pro Gly Pro Pro Phe Val Glu Met Tyr Ser
385 390 395 400
Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile
405 410 415
Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe
420 425 430
Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser
435 440 445
Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu
450 455 460
Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr
465 470 475 480
Leu Thr His Arg Gln Thr Val Val Leu Ser Pro Ser His Gly Ile Glu
485 490 495
Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu
500 505 510
Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His
515 520 525
Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser
530 535 540
Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg
545 550 555 560
Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr
565 570 575
Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Pro Gly Pro Asp
580 585 590
Lys Thr His Thr Cys Pro Leu Cys Pro Ala Pro Glu Leu Leu Gly Gly
595 600 605
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
610 615 620
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
625 630 635 640
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
645 650 655
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
660 665 670
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
675 680 685
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
690 695 700
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
705 710 715 720
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
725 730 735
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
740 745 750
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Ala Thr Pro Pro Val
755 760 765
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
770 775 780
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
785 790 795 800
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
805 810 815
Gly Lys
<210> 8
<211> 814
<212> PRT
<213> KH-002
<400> 8
Ser Gln Gly Leu Val Val Thr Pro Pro Gly Pro Glu Leu Val Leu Asn
1 5 10 15
Val Ser Ser Thr Phe Val Leu Thr Cys Ser Gly Ser Ala Pro Val Val
20 25 30
Trp Glu Arg Met Ser Gln Glu Pro Pro Gln Glu Met Ala Lys Ala Gln
35 40 45
Asp Gly Thr Phe Ser Ser Val Leu Thr Leu Thr Asn Leu Thr Gly Leu
50 55 60
Asp Thr Gly Glu Tyr Phe Cys Thr His Asn Asp Ser Arg Gly Leu Glu
65 70 75 80
Thr Asp Glu Arg Lys Arg Leu Tyr Ile Phe Val Pro Asp Pro Thr Val
85 90 95
Gly Phe Leu Pro Asn Asp Ala Glu Glu Leu Phe Ile Phe Leu Thr Glu
100 105 110
Ile Thr Glu Ile Thr Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val
115 120 125
Val Thr Leu His Glu Lys Lys Gly Asp Val Ala Leu Pro Val Pro Tyr
130 135 140
Asp His Gln Arg Gly Phe Ser Gly Ile Phe Glu Asp Arg Ser Tyr Ile
145 150 155 160
Cys Lys Thr Thr Ile Gly Asp Arg Glu Val Asp Ser Asp Ala Tyr Tyr
165 170 175
Val Tyr Arg Leu Gln Val Ser Ser Ile Asn Val Ser Val Asn Ala Val
180 185 190
Gln Thr Val Val Arg Gln Gly Glu Asn Ile Thr Leu Met Cys Ile Val
195 200 205
Ile Gly Asn Glu Val Val Asn Phe Glu Trp Thr Tyr Pro Arg Lys Glu
210 215 220
Ser Gly Arg Leu Val Glu Pro Val Thr Asp Phe Leu Leu Asp Met Pro
225 230 235 240
Tyr His Ile Arg Ser Ile Leu His Ile Pro Ser Ala Glu Leu Glu Asp
245 250 255
Ser Gly Thr Tyr Thr Cys Asn Val Thr Glu Ser Val Asn Asp His Gln
260 265 270
Asp Glu Lys Ala Ile Asn Ile Thr Val Val Glu Ser Gly Tyr Val Arg
275 280 285
Leu Leu Gly Glu Val Gly Thr Leu Gln Phe Ala Glu Leu His Arg Ser
290 295 300
Arg Thr Leu Gln Val Val Phe Glu Ala Tyr Pro Pro Pro Thr Val Leu
305 310 315 320
Trp Phe Lys Asp Asn Arg Thr Leu Gly Asp Ser Ser Ala Gly Glu Ile
325 330 335
Ala Leu Ser Thr Arg Asn Val Ser Glu Thr Arg Tyr Val Ser Glu Leu
340 345 350
Thr Leu Val Arg Val Lys Val Ala Glu Ala Gly His Tyr Thr Met Arg
355 360 365
Ala Phe His Glu Asp Ala Glu Val Gln Leu Ser Phe Gln Leu Gln Ile
370 375 380
Asn Val Pro Gly Pro Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu
385 390 395 400
Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val
405 410 415
Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr
420 425 430
Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe
435 440 445
Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu
450 455 460
Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg
465 470 475 480
Gln Thr Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly
485 490 495
Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly
500 505 510
Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys
515 520 525
Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys
530 535 540
Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly
545 550 555 560
Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser
565 570 575
Thr Phe Val Arg Val His Glu Lys Pro Gly Pro Asp Lys Thr His Thr
580 585 590
Cys Pro Leu Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
595 600 605
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
610 615 620
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
625 630 635 640
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
645 650 655
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
660 665 670
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
675 680 685
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
690 695 700
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
705 710 715 720
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
725 730 735
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
740 745 750
Gln Pro Glu Asn Asn Tyr Lys Ala Thr Pro Pro Val Leu Asp Ser Asp
755 760 765
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
770 775 780
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
785 790 795 800
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
805 810
<210> 9
<211> 755
<212> PRT
<213> KH-003
<400> 9
Tyr Asn His Thr Gln Thr Glu Glu Asn Glu Leu Glu Gly Arg His Ile
1 5 10 15
Tyr Ile Tyr Val Pro Asp Pro Asp Val Ala Phe Val Pro Leu Gly Met
20 25 30
Thr Asp Tyr Leu Val Ile Val Glu Asp Asp Asp Ser Ala Ile Ile Pro
35 40 45
Cys Arg Thr Thr Asp Pro Glu Thr Pro Val Thr Leu His Asn Ser Glu
50 55 60
Gly Val Val Pro Ala Ser Tyr Asp Ser Arg Gln Gly Phe Asn Gly Thr
65 70 75 80
Phe Thr Val Gly Pro Tyr Ile Cys Glu Ala Thr Val Lys Gly Lys Lys
85 90 95
Phe Gln Thr Ile Pro Phe Asn Val Tyr Ala Leu Lys Ala Thr Ser Glu
100 105 110
Leu Asp Leu Glu Met Glu Ala Leu Lys Thr Val Tyr Lys Ser Gly Glu
115 120 125
Thr Ile Val Val Thr Cys Ala Val Phe Asn Asn Glu Val Val Asp Leu
130 135 140
Gln Trp Thr Tyr Pro Gly Glu Val Lys Gly Lys Gly Ile Thr Met Leu
145 150 155 160
Glu Glu Ile Lys Val Pro Ser Ile Lys Leu Val Tyr Thr Leu Thr Val
165 170 175
Pro Glu Ala Thr Val Lys Asp Ser Gly Asp Tyr Glu Cys Ala Ala Arg
180 185 190
Gln Ala Thr Arg Glu Val Lys Glu Met Lys Lys Val Thr Ile Ser Val
195 200 205
His Glu Lys Gly Phe Ile Glu Ile Lys Pro Thr Phe Ser Gln Leu Glu
210 215 220
Ala Val Asn Leu His Glu Val Lys His Phe Val Val Glu Val Arg Ala
225 230 235 240
Pro Gly Pro Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile
245 250 255
His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser
260 265 270
Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile
275 280 285
Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile
290 295 300
Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr
305 310 315 320
Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr
325 330 335
Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys
340 345 350
Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp
355 360 365
Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val
370 375 380
Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu
385 390 395 400
Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr
405 410 415
Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe
420 425 430
Val Arg Val His Glu Lys Thr Val Lys His Arg Lys Gln Gln Val Leu
435 440 445
Glu Thr Val Ala Gly Lys Arg Ser Tyr Arg Leu Ser Met Lys Val Lys
450 455 460
Ala Phe Pro Ser Pro Glu Val Val Trp Leu Lys Asp Gly Leu Pro Ala
465 470 475 480
Thr Glu Lys Ser Ala Arg Tyr Leu Thr Arg Gly Tyr Ser Leu Ile Ile
485 490 495
Lys Asp Val Thr Glu Glu Asp Ala Gly Asn Tyr Thr Ile Leu Leu Ser
500 505 510
Ile Lys Gln Ser Asn Val Phe Lys Asn Leu Thr Ala Thr Pro Gly Pro
515 520 525
Asp Lys Thr His Thr Cys Pro Leu Cys Pro Ala Pro Glu Leu Leu Gly
530 535 540
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
545 550 555 560
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
565 570 575
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
580 585 590
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
595 600 605
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
610 615 620
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
625 630 635 640
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
645 650 655
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
660 665 670
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
675 680 685
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Ala Thr Pro Pro
690 695 700
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
705 710 715 720
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
725 730 735
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
740 745 750
Pro Gly Lys
755
<210> 10
<211> 758
<212> PRT
<213> KH-004
<400> 10
His Asn Asp Ser Arg Gly Leu Glu Thr Asp Glu Arg Lys Arg Leu Tyr
1 5 10 15
Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu Pro Asn Asp Ala Glu
20 25 30
Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr Glu Ile Thr Ile Pro Cys
35 40 45
Arg Val Thr Asp Pro Gln Leu Val Val Thr Leu His Glu Lys Lys Gly
50 55 60
Asp Val Ala Leu Pro Val Pro Tyr Asp His Gln Arg Gly Phe Ser Gly
65 70 75 80
Ile Phe Glu Asp Arg Ser Tyr Ile Cys Lys Thr Thr Ile Gly Asp Arg
85 90 95
Glu Val Asp Ser Asp Ala Tyr Tyr Val Tyr Arg Leu Gln Val Ser Ser
100 105 110
Ile Asn Val Ser Val Asn Ala Val Gln Thr Val Val Arg Gln Gly Glu
115 120 125
Asn Ile Thr Leu Met Cys Ile Val Ile Gly Asn Glu Val Val Asn Phe
130 135 140
Glu Trp Thr Tyr Pro Arg Lys Glu Ser Gly Arg Leu Val Glu Pro Val
145 150 155 160
Thr Asp Phe Leu Leu Asp Met Pro Tyr His Ile Arg Ser Ile Leu His
165 170 175
Ile Pro Ser Ala Glu Leu Glu Asp Ser Gly Thr Tyr Thr Cys Asn Val
180 185 190
Thr Glu Ser Val Asn Asp His Gln Asp Glu Lys Ala Ile Asn Ile Thr
195 200 205
Val Val Glu Ser Gly Tyr Val Arg Leu Leu Gly Glu Val Gly Thr Leu
210 215 220
Gln Phe Ala Glu Leu His Arg Ser Arg Thr Leu Gln Val Val Phe Glu
225 230 235 240
Pro Gly Pro Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile
245 250 255
His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser
260 265 270
Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile
275 280 285
Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile
290 295 300
Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr
305 310 315 320
Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr
325 330 335
Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys
340 345 350
Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp
355 360 365
Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val
370 375 380
Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu
385 390 395 400
Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr
405 410 415
Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe
420 425 430
Val Arg Val His Glu Lys Thr Val Lys His Arg Lys Gln Gln Val Leu
435 440 445
Glu Thr Val Ala Gly Lys Arg Ser Tyr Arg Leu Ser Met Lys Val Lys
450 455 460
Ala Phe Pro Ser Pro Glu Val Val Trp Leu Lys Asp Gly Leu Pro Ala
465 470 475 480
Thr Glu Lys Ser Ala Arg Tyr Leu Thr Arg Gly Tyr Ser Leu Ile Ile
485 490 495
Lys Asp Val Thr Glu Glu Asp Ala Gly Asn Tyr Thr Ile Leu Leu Ser
500 505 510
Ile Lys Gln Ser Asn Val Phe Lys Asn Leu Thr Ala Thr Pro Gly Pro
515 520 525
Pro Gly Pro Asp Lys Thr His Thr Cys Pro Leu Cys Pro Ala Pro Glu
530 535 540
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
545 550 555 560
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
565 570 575
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
580 585 590
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
595 600 605
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
610 615 620
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
625 630 635 640
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
645 650 655
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
660 665 670
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
675 680 685
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Ala
690 695 700
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
705 710 715 720
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
725 730 735
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
740 745 750
Ser Leu Ser Pro Gly Lys
755
<210> 11
<211> 668
<212> PRT
<213> KH-005
<400> 11
Tyr Asn His Thr Gln Thr Glu Glu Asn Glu Leu Glu Gly Arg His Ile
1 5 10 15
Tyr Ile Tyr Val Pro Asp Pro Asp Val Ala Phe Val Pro Leu Gly Met
20 25 30
Thr Asp Tyr Leu Val Ile Val Glu Asp Asp Asp Ser Ala Ile Ile Pro
35 40 45
Cys Arg Thr Thr Asp Pro Glu Thr Pro Val Thr Leu His Asn Ser Glu
50 55 60
Gly Val Val Pro Ala Ser Tyr Asp Ser Arg Gln Gly Phe Asn Gly Thr
65 70 75 80
Phe Thr Val Gly Pro Tyr Ile Cys Glu Ala Thr Val Lys Gly Lys Lys
85 90 95
Phe Gln Thr Ile Pro Phe Asn Val Tyr Ala Leu Lys Ala Thr Ser Glu
100 105 110
Leu Asp Leu Glu Met Glu Ala Leu Lys Thr Val Tyr Lys Ser Gly Glu
115 120 125
Thr Ile Val Val Thr Cys Ala Val Phe Asn Asn Glu Val Val Asp Leu
130 135 140
Gln Trp Thr Tyr Pro Gly Glu Val Lys Gly Lys Gly Ile Thr Met Leu
145 150 155 160
Glu Glu Ile Lys Val Pro Ser Ile Lys Leu Val Tyr Thr Leu Thr Val
165 170 175
Pro Glu Ala Thr Val Lys Asp Ser Gly Asp Tyr Glu Cys Ala Ala Arg
180 185 190
Gln Ala Thr Arg Glu Val Lys Glu Met Lys Lys Val Thr Ile Ser Val
195 200 205
His Glu Lys Gly Phe Ile Glu Ile Lys Pro Thr Phe Ser Gln Leu Glu
210 215 220
Ala Val Asn Leu His Glu Val Lys His Phe Val Val Glu Val Arg Ala
225 230 235 240
Pro Gly Pro Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile
245 250 255
His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser
260 265 270
Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile
275 280 285
Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile
290 295 300
Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr
305 310 315 320
Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr
325 330 335
Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys
340 345 350
Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp
355 360 365
Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val
370 375 380
Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu
385 390 395 400
Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr
405 410 415
Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe
420 425 430
Val Arg Val His Glu Lys Pro Gly Pro Asp Lys Thr His Thr Cys Pro
435 440 445
Leu Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
450 455 460
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
465 470 475 480
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
485 490 495
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
500 505 510
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
515 520 525
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
530 535 540
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
545 550 555 560
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
565 570 575
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
580 585 590
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
595 600 605
Glu Asn Asn Tyr Lys Ala Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
610 615 620
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
625 630 635 640
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
645 650 655
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
660 665
<210> 12
<211> 668
<212> PRT
<213> KH-006
<400> 12
His Asn Asp Ser Arg Gly Leu Glu Thr Asp Glu Arg Lys Arg Leu Tyr
1 5 10 15
Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu Pro Asn Asp Ala Glu
20 25 30
Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr Glu Ile Thr Ile Pro Cys
35 40 45
Arg Val Thr Asp Pro Gln Leu Val Val Thr Leu His Glu Lys Lys Gly
50 55 60
Asp Val Ala Leu Pro Val Pro Tyr Asp His Gln Arg Gly Phe Ser Gly
65 70 75 80
Ile Phe Glu Asp Arg Ser Tyr Ile Cys Lys Thr Thr Ile Gly Asp Arg
85 90 95
Glu Val Asp Ser Asp Ala Tyr Tyr Val Tyr Arg Leu Gln Val Ser Ser
100 105 110
Ile Asn Val Ser Val Asn Ala Val Gln Thr Val Val Arg Gln Gly Glu
115 120 125
Asn Ile Thr Leu Met Cys Ile Val Ile Gly Asn Glu Val Val Asn Phe
130 135 140
Glu Trp Thr Tyr Pro Arg Lys Glu Ser Gly Arg Leu Val Glu Pro Val
145 150 155 160
Thr Asp Phe Leu Leu Asp Met Pro Tyr His Ile Arg Ser Ile Leu His
165 170 175
Ile Pro Ser Ala Glu Leu Glu Asp Ser Gly Thr Tyr Thr Cys Asn Val
180 185 190
Thr Glu Ser Val Asn Asp His Gln Asp Glu Lys Ala Ile Asn Ile Thr
195 200 205
Val Val Glu Ser Gly Tyr Val Arg Leu Leu Gly Glu Val Gly Thr Leu
210 215 220
Gln Phe Ala Glu Leu His Arg Ser Arg Thr Leu Gln Val Val Phe Glu
225 230 235 240
Pro Gly Pro Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile
245 250 255
His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser
260 265 270
Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile
275 280 285
Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile
290 295 300
Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr
305 310 315 320
Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr
325 330 335
Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys
340 345 350
Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp
355 360 365
Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val
370 375 380
Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu
385 390 395 400
Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr
405 410 415
Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe
420 425 430
Val Arg Val His Glu Lys Pro Gly Pro Asp Lys Thr His Thr Cys Pro
435 440 445
Leu Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
450 455 460
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
465 470 475 480
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
485 490 495
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
500 505 510
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
515 520 525
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
530 535 540
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
545 550 555 560
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
565 570 575
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
580 585 590
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
595 600 605
Glu Asn Asn Tyr Lys Ala Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
610 615 620
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
625 630 635 640
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
645 650 655
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
660 665
<210> 13
<211> 672
<212> PRT
<213> KH-007
<400> 13
His Asn Asp Ser Arg Gly Leu Glu Thr Asp Glu Arg Lys Arg Leu Tyr
1 5 10 15
Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu Pro Asn Asp Ala Glu
20 25 30
Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr Glu Ile Thr Ile Pro Cys
35 40 45
Arg Val Thr Asp Pro Gln Leu Val Val Thr Leu His Glu Lys Lys Gly
50 55 60
Asp Val Ala Leu Pro Val Pro Tyr Asp His Gln Arg Gly Phe Ser Gly
65 70 75 80
Ile Phe Glu Asp Arg Ser Tyr Ile Cys Lys Thr Thr Ile Gly Asp Arg
85 90 95
Glu Val Asp Ser Asp Ala Tyr Tyr Val Tyr Arg Leu Gln Val Ser Ser
100 105 110
Ile Asn Val Ser Val Asn Ala Val Gln Thr Val Val Arg Gln Gly Glu
115 120 125
Asn Ile Thr Leu Met Cys Ile Val Ile Gly Asn Glu Val Val Asn Phe
130 135 140
Glu Trp Thr Tyr Pro Arg Lys Glu Ser Gly Arg Leu Val Glu Pro Val
145 150 155 160
Thr Asp Phe Leu Leu Asp Met Pro Tyr His Ile Arg Ser Ile Leu His
165 170 175
Ile Pro Ser Ala Glu Leu Glu Asp Ser Gly Thr Tyr Thr Cys Asn Val
180 185 190
Thr Glu Ser Val Asn Asp His Gln Asp Glu Lys Ala Ile Asn Ile Thr
195 200 205
Val Val Glu Ser Gly Tyr Val Arg Leu Leu Gly Glu Val Gly Thr Leu
210 215 220
Gln Phe Ala Glu Leu His Arg Ser Arg Thr Leu Gln Val Val Phe Glu
225 230 235 240
Gly Gly Gly Gly Ser Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu
245 250 255
Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val
260 265 270
Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr
275 280 285
Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe
290 295 300
Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu
305 310 315 320
Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg
325 330 335
Gln Thr Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly
340 345 350
Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly
355 360 365
Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys
370 375 380
Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys
385 390 395 400
Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly
405 410 415
Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser
420 425 430
Thr Phe Val Arg Val His Glu Lys Gly Gly Gly Gly Ser Asp Lys Thr
435 440 445
His Thr Cys Pro Leu Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
450 455 460
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
465 470 475 480
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
485 490 495
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
500 505 510
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
515 520 525
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
530 535 540
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
545 550 555 560
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
565 570 575
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
580 585 590
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
595 600 605
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Ala Thr Pro Pro Val Leu Asp
610 615 620
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
625 630 635 640
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
645 650 655
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
660 665 670
<210> 14
<211> 672
<212> PRT
<213> KH-008
<400> 14
Tyr Asn His Thr Gln Thr Glu Glu Asn Glu Leu Glu Gly Arg His Ile
1 5 10 15
Tyr Ile Tyr Val Pro Asp Pro Asp Val Ala Phe Val Pro Leu Gly Met
20 25 30
Thr Asp Tyr Leu Val Ile Val Glu Asp Asp Asp Ser Ala Ile Ile Pro
35 40 45
Cys Arg Thr Thr Asp Pro Glu Thr Pro Val Thr Leu His Asn Ser Glu
50 55 60
Gly Val Val Pro Ala Ser Tyr Asp Ser Arg Gln Gly Phe Asn Gly Thr
65 70 75 80
Phe Thr Val Gly Pro Tyr Ile Cys Glu Ala Thr Val Lys Gly Lys Lys
85 90 95
Phe Gln Thr Ile Pro Phe Asn Val Tyr Ala Leu Lys Ala Thr Ser Glu
100 105 110
Leu Asp Leu Glu Met Glu Ala Leu Lys Thr Val Tyr Lys Ser Gly Glu
115 120 125
Thr Ile Val Val Thr Cys Ala Val Phe Asn Asn Glu Val Val Asp Leu
130 135 140
Gln Trp Thr Tyr Pro Gly Glu Val Lys Gly Lys Gly Ile Thr Met Leu
145 150 155 160
Glu Glu Ile Lys Val Pro Ser Ile Lys Leu Val Tyr Thr Leu Thr Val
165 170 175
Pro Glu Ala Thr Val Lys Asp Ser Gly Asp Tyr Glu Cys Ala Ala Arg
180 185 190
Gln Ala Thr Arg Glu Val Lys Glu Met Lys Lys Val Thr Ile Ser Val
195 200 205
His Glu Lys Gly Phe Ile Glu Ile Lys Pro Thr Phe Ser Gln Leu Glu
210 215 220
Ala Val Asn Leu His Glu Val Lys His Phe Val Val Glu Val Arg Ala
225 230 235 240
Gly Gly Gly Gly Ser Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu
245 250 255
Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val
260 265 270
Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr
275 280 285
Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe
290 295 300
Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu
305 310 315 320
Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg
325 330 335
Gln Thr Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly
340 345 350
Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly
355 360 365
Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys
370 375 380
Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys
385 390 395 400
Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly
405 410 415
Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser
420 425 430
Thr Phe Val Arg Val His Glu Lys Gly Gly Gly Gly Ser Asp Lys Thr
435 440 445
His Thr Cys Pro Leu Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
450 455 460
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
465 470 475 480
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
485 490 495
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
500 505 510
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
515 520 525
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
530 535 540
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
545 550 555 560
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
565 570 575
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
580 585 590
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
595 600 605
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Ala Thr Pro Pro Val Leu Asp
610 615 620
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
625 630 635 640
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
645 650 655
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
660 665 670
<210> 15
<211> 670
<212> PRT
<213> KH-009
<400> 15
His Asn Asp Ser Arg Gly Leu Glu Thr Asp Glu Arg Lys Arg Leu Tyr
1 5 10 15
Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu Pro Asn Asp Ala Glu
20 25 30
Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr Glu Ile Thr Ile Pro Cys
35 40 45
Arg Val Thr Asp Pro Gln Leu Val Val Thr Leu His Glu Lys Lys Gly
50 55 60
Asp Val Ala Leu Pro Val Pro Tyr Asp His Gln Arg Gly Phe Ser Gly
65 70 75 80
Ile Phe Glu Asp Arg Ser Tyr Ile Cys Lys Thr Thr Ile Gly Asp Arg
85 90 95
Glu Val Asp Ser Asp Ala Tyr Tyr Val Tyr Arg Leu Gln Val Ser Ser
100 105 110
Ile Asn Val Ser Val Asn Ala Val Gln Thr Val Val Arg Gln Gly Glu
115 120 125
Asn Ile Thr Leu Met Cys Ile Val Ile Gly Asn Glu Val Val Asn Phe
130 135 140
Glu Trp Thr Tyr Pro Arg Lys Glu Ser Gly Arg Leu Val Glu Pro Val
145 150 155 160
Thr Asp Phe Leu Leu Asp Met Pro Tyr His Ile Arg Ser Ile Leu His
165 170 175
Ile Pro Ser Ala Glu Leu Glu Asp Ser Gly Thr Tyr Thr Cys Asn Val
180 185 190
Thr Glu Ser Val Asn Asp His Gln Asp Glu Lys Ala Ile Asn Ile Thr
195 200 205
Val Val Glu Ser Gly Tyr Val Arg Leu Leu Gly Glu Val Gly Thr Leu
210 215 220
Gln Phe Ala Glu Leu His Arg Ser Arg Thr Leu Gln Val Val Phe Glu
225 230 235 240
Pro Gly Pro Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile
245 250 255
His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser
260 265 270
Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile
275 280 285
Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile
290 295 300
Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr
305 310 315 320
Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr
325 330 335
Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys
340 345 350
Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp
355 360 365
Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val
370 375 380
Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu
385 390 395 400
Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr
405 410 415
Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe
420 425 430
Val Arg Val His Glu Lys Gly Gly Gly Gly Ser Asp Lys Thr His Thr
435 440 445
Cys Pro Leu Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
450 455 460
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
465 470 475 480
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
485 490 495
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
500 505 510
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
515 520 525
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
530 535 540
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
545 550 555 560
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
565 570 575
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
580 585 590
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
595 600 605
Gln Pro Glu Asn Asn Tyr Lys Ala Thr Pro Pro Val Leu Asp Ser Asp
610 615 620
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
625 630 635 640
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
645 650 655
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
660 665 670
<210> 16
<211> 670
<212> PRT
<213> KH-010
<400> 16
Tyr Asn His Thr Gln Thr Glu Glu Asn Glu Leu Glu Gly Arg His Ile
1 5 10 15
Tyr Ile Tyr Val Pro Asp Pro Asp Val Ala Phe Val Pro Leu Gly Met
20 25 30
Thr Asp Tyr Leu Val Ile Val Glu Asp Asp Asp Ser Ala Ile Ile Pro
35 40 45
Cys Arg Thr Thr Asp Pro Glu Thr Pro Val Thr Leu His Asn Ser Glu
50 55 60
Gly Val Val Pro Ala Ser Tyr Asp Ser Arg Gln Gly Phe Asn Gly Thr
65 70 75 80
Phe Thr Val Gly Pro Tyr Ile Cys Glu Ala Thr Val Lys Gly Lys Lys
85 90 95
Phe Gln Thr Ile Pro Phe Asn Val Tyr Ala Leu Lys Ala Thr Ser Glu
100 105 110
Leu Asp Leu Glu Met Glu Ala Leu Lys Thr Val Tyr Lys Ser Gly Glu
115 120 125
Thr Ile Val Val Thr Cys Ala Val Phe Asn Asn Glu Val Val Asp Leu
130 135 140
Gln Trp Thr Tyr Pro Gly Glu Val Lys Gly Lys Gly Ile Thr Met Leu
145 150 155 160
Glu Glu Ile Lys Val Pro Ser Ile Lys Leu Val Tyr Thr Leu Thr Val
165 170 175
Pro Glu Ala Thr Val Lys Asp Ser Gly Asp Tyr Glu Cys Ala Ala Arg
180 185 190
Gln Ala Thr Arg Glu Val Lys Glu Met Lys Lys Val Thr Ile Ser Val
195 200 205
His Glu Lys Gly Phe Ile Glu Ile Lys Pro Thr Phe Ser Gln Leu Glu
210 215 220
Ala Val Asn Leu His Glu Val Lys His Phe Val Val Glu Val Arg Ala
225 230 235 240
Pro Gly Pro Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile
245 250 255
His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser
260 265 270
Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile
275 280 285
Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile
290 295 300
Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr
305 310 315 320
Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr
325 330 335
Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys
340 345 350
Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp
355 360 365
Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val
370 375 380
Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu
385 390 395 400
Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr
405 410 415
Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe
420 425 430
Val Arg Val His Glu Lys Gly Gly Gly Gly Ser Asp Lys Thr His Thr
435 440 445
Cys Pro Leu Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
450 455 460
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
465 470 475 480
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
485 490 495
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
500 505 510
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
515 520 525
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
530 535 540
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
545 550 555 560
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
565 570 575
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
580 585 590
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
595 600 605
Gln Pro Glu Asn Asn Tyr Lys Ala Thr Pro Pro Val Leu Asp Ser Asp
610 615 620
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
625 630 635 640
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
645 650 655
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
660 665 670
<210> 17
<211> 670
<212> PRT
<213> KH-011
<400> 17
His Asn Asp Ser Arg Gly Leu Glu Thr Asp Glu Arg Lys Arg Leu Tyr
1 5 10 15
Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu Pro Asn Asp Ala Glu
20 25 30
Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr Glu Ile Thr Ile Pro Cys
35 40 45
Arg Val Thr Asp Pro Gln Leu Val Val Thr Leu His Glu Lys Lys Gly
50 55 60
Asp Val Ala Leu Pro Val Pro Tyr Asp His Gln Arg Gly Phe Ser Gly
65 70 75 80
Ile Phe Glu Asp Arg Ser Tyr Ile Cys Lys Thr Thr Ile Gly Asp Arg
85 90 95
Glu Val Asp Ser Asp Ala Tyr Tyr Val Tyr Arg Leu Gln Val Ser Ser
100 105 110
Ile Asn Val Ser Val Asn Ala Val Gln Thr Val Val Arg Gln Gly Glu
115 120 125
Asn Ile Thr Leu Met Cys Ile Val Ile Gly Asn Glu Val Val Asn Phe
130 135 140
Glu Trp Thr Tyr Pro Arg Lys Glu Ser Gly Arg Leu Val Glu Pro Val
145 150 155 160
Thr Asp Phe Leu Leu Asp Met Pro Tyr His Ile Arg Ser Ile Leu His
165 170 175
Ile Pro Ser Ala Glu Leu Glu Asp Ser Gly Thr Tyr Thr Cys Asn Val
180 185 190
Thr Glu Ser Val Asn Asp His Gln Asp Glu Lys Ala Ile Asn Ile Thr
195 200 205
Val Val Glu Ser Gly Tyr Val Arg Leu Leu Gly Glu Val Gly Thr Leu
210 215 220
Gln Phe Ala Glu Leu His Arg Ser Arg Thr Leu Gln Val Val Phe Glu
225 230 235 240
Gly Gly Gly Gly Ser Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu
245 250 255
Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val
260 265 270
Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr
275 280 285
Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe
290 295 300
Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu
305 310 315 320
Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg
325 330 335
Gln Thr Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly
340 345 350
Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly
355 360 365
Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys
370 375 380
Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys
385 390 395 400
Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly
405 410 415
Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser
420 425 430
Thr Phe Val Arg Val His Glu Lys Pro Gly Pro Asp Lys Thr His Thr
435 440 445
Cys Pro Leu Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
450 455 460
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
465 470 475 480
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
485 490 495
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
500 505 510
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
515 520 525
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
530 535 540
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
545 550 555 560
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
565 570 575
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
580 585 590
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
595 600 605
Gln Pro Glu Asn Asn Tyr Lys Ala Thr Pro Pro Val Leu Asp Ser Asp
610 615 620
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
625 630 635 640
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
645 650 655
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
660 665 670
<210> 18
<211> 670
<212> PRT
<213> KH-012
<400> 18
Tyr Asn His Thr Gln Thr Glu Glu Asn Glu Leu Glu Gly Arg His Ile
1 5 10 15
Tyr Ile Tyr Val Pro Asp Pro Asp Val Ala Phe Val Pro Leu Gly Met
20 25 30
Thr Asp Tyr Leu Val Ile Val Glu Asp Asp Asp Ser Ala Ile Ile Pro
35 40 45
Cys Arg Thr Thr Asp Pro Glu Thr Pro Val Thr Leu His Asn Ser Glu
50 55 60
Gly Val Val Pro Ala Ser Tyr Asp Ser Arg Gln Gly Phe Asn Gly Thr
65 70 75 80
Phe Thr Val Gly Pro Tyr Ile Cys Glu Ala Thr Val Lys Gly Lys Lys
85 90 95
Phe Gln Thr Ile Pro Phe Asn Val Tyr Ala Leu Lys Ala Thr Ser Glu
100 105 110
Leu Asp Leu Glu Met Glu Ala Leu Lys Thr Val Tyr Lys Ser Gly Glu
115 120 125
Thr Ile Val Val Thr Cys Ala Val Phe Asn Asn Glu Val Val Asp Leu
130 135 140
Gln Trp Thr Tyr Pro Gly Glu Val Lys Gly Lys Gly Ile Thr Met Leu
145 150 155 160
Glu Glu Ile Lys Val Pro Ser Ile Lys Leu Val Tyr Thr Leu Thr Val
165 170 175
Pro Glu Ala Thr Val Lys Asp Ser Gly Asp Tyr Glu Cys Ala Ala Arg
180 185 190
Gln Ala Thr Arg Glu Val Lys Glu Met Lys Lys Val Thr Ile Ser Val
195 200 205
His Glu Lys Gly Phe Ile Glu Ile Lys Pro Thr Phe Ser Gln Leu Glu
210 215 220
Ala Val Asn Leu His Glu Val Lys His Phe Val Val Glu Val Arg Ala
225 230 235 240
Gly Gly Gly Gly Ser Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu
245 250 255
Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val
260 265 270
Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr
275 280 285
Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe
290 295 300
Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu
305 310 315 320
Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg
325 330 335
Gln Thr Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly
340 345 350
Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly
355 360 365
Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys
370 375 380
Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys
385 390 395 400
Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly
405 410 415
Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser
420 425 430
Thr Phe Val Arg Val His Glu Lys Pro Gly Pro Asp Lys Thr His Thr
435 440 445
Cys Pro Leu Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
450 455 460
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
465 470 475 480
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
485 490 495
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
500 505 510
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
515 520 525
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
530 535 540
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
545 550 555 560
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
565 570 575
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
580 585 590
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
595 600 605
Gln Pro Glu Asn Asn Tyr Lys Ala Thr Pro Pro Val Leu Asp Ser Asp
610 615 620
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
625 630 635 640
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
645 650 655
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
660 665 670
Claims (6)
1.一种融合蛋白，其特征在于，由PDGF受体和VEGF受体的不同片段与人免疫球蛋白Fc构成，其具体结构如下：
KH-001的氨基酸序列如序列表中SEQ ID NO.7所述；
KH-002的氨基酸序列如序列表中SEQ ID NO.8所述；
KH-003的氨基酸序列如序列表中SEQ ID NO.9所述；
KH-004的氨基酸序列如序列表中SEQ ID NO.10所述；
KH-005的氨基酸序列如序列表中SEQ ID NO.11所述；
KH-006的氨基酸序列如序列表中SEQ ID NO.12所述；
KH-007的氨基酸序列如序列表中SEQ ID NO.13所述；
KH-008的氨基酸序列如序列表中SEQ ID NO.14所述；
KH-009的氨基酸序列如序列表中SEQ ID NO.15所述；
KH-010的氨基酸序列如序列表中SEQ ID NO.16所述；
KH-011的氨基酸序列如序列表中SEQ ID NO.17所述；
KH-012的氨基酸序列如序列表中SEQ ID NO.18所述。
2.一种药物组合物，其特征在于由权利要求1中所述融合蛋白与药学上可接受的载体或赋形剂组成。
3.根据权利要求2所述的药物组合物，其特征在于所述药物组合物的制剂形式为注射剂、注射用冻干粉针或眼用凝胶。
4.根据权利要求1所述的融合蛋白，其特征在于所述融合蛋白在制备治疗由血管新生或生长引起的疾病的药物中的应用。
5.根据权利要求4所述的融合蛋白，其特征在于所述血管新生或生长引起的疾病为肿瘤或眼部新生血管引起的疾病。
6.根据权利要求5所述的融合蛋白，其特征在于所述的眼部新生血管引起的疾病为脉络膜新生血管引起的疾病。
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| EP3161000A4 (en) * | 2014-06-28 | 2018-05-02 | Kodiak Sciences Inc. | Dual pdgf/vegf antagonists |
| EP3710483A4 (en) * | 2017-11-16 | 2021-10-20 | XL-protein GmbH | PASYLATED VEGFR / PDGFR FUSION PROTEINS AND THEIR USE IN THERAPY |
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| CN102633883B (zh) * | 2012-02-24 | 2014-06-25 | 上海白泽生物科技有限公司 | 一种能与egfr、her2、vegf高效结合的融合蛋白、其编码序列及用途 |
| CN103965363B (zh) * | 2013-02-06 | 2021-01-15 | 上海白泽生物科技有限公司 | 与pd-1和vegf高效结合的融合蛋白、其编码序列及用途 |
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| WO2016005381A1 (en) * | 2014-07-10 | 2016-01-14 | Bayer Pharma Aktiengesellschaft | Pdgfrbeta-fc fusion proteins and uses thereof |
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| US6833349B2 (en) * | 1999-06-08 | 2004-12-21 | Regeneron Pharmaceuticals, Inc. | Methods of treating inflammatory skin diseases |
| CN1304427C (zh) * | 2004-06-08 | 2007-03-14 | 成都康弘生物科技有限公司 | 抑制血管新生的融合蛋白质及其用途 |
| CN101721699B (zh) * | 2008-10-13 | 2012-11-07 | 成都康弘生物科技有限公司 | Vegf受体融合蛋白在制备治疗伴随vegf升高的炎症反应的药物中的应用 |
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| EP3161000A4 (en) * | 2014-06-28 | 2018-05-02 | Kodiak Sciences Inc. | Dual pdgf/vegf antagonists |
| EP3710483A4 (en) * | 2017-11-16 | 2021-10-20 | XL-protein GmbH | PASYLATED VEGFR / PDGFR FUSION PROTEINS AND THEIR USE IN THERAPY |
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