CN1871258A - 融合多肽及其在抗血管肿瘤治疗中的用途 - Google Patents
融合多肽及其在抗血管肿瘤治疗中的用途 Download PDFInfo
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- CN1871258A CN1871258A CNA2004800313848A CN200480031384A CN1871258A CN 1871258 A CN1871258 A CN 1871258A CN A2004800313848 A CNA2004800313848 A CN A2004800313848A CN 200480031384 A CN200480031384 A CN 200480031384A CN 1871258 A CN1871258 A CN 1871258A
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Abstract
公开了融合的多肽，包括a)容许该融合多肽选择性结合肿瘤血管内皮细胞的3至30个氨基酸的肽，和b)组织因子TF或其片段，该组织因子和片段的特征在于当该融合多肽结合肿瘤血管内皮细胞时能够活化凝血。肽a)和b)直接相互偶联或通过包括至多15个氨基酸的接头相互偶联，同时容许该融合多肽选择性结合肿瘤血管内皮细胞的肽与当该融合多肽结合肿瘤血管内皮细胞时能够活化凝血的肽之C末端偶联。
Description
本发明涉及包括至少两个肽的融合多肽。一个肽包括3至30个氨基酸并且容许该融合多肽选择性地结合肿瘤血管中的内皮细胞。另一个肽由组织因子(TF)或其片段组成，该组织因子和片段的特征在于当融合多肽结合肿瘤血管中的内皮细胞时它们能够活化血液凝固。该肽可直接连接在一起或通过具有至多15个氨基酸的接头(Linker)连接在一起。本发明进一步涉及这些融合多肽在肿瘤疾病的抗血管治疗中的用途及其在制备用于治疗肿瘤疾病的药物中的用途。
发明背景
足够的新血管形成是进行性肿瘤生长的先决条件(1)。新血管生成对于维持扩张的肿瘤生长是特别需要的，因为只有足够的氧合作用将保证为肿瘤供给养分并从肿瘤除去肿瘤降解作用的产物。
在针对肿瘤治疗的现有技术中，已经开发了抗血管治疗性策略，其目标在于破坏肿瘤血管和使相关肿瘤梗死，此外开发了侵袭血管生长和分化的复杂过程的抗血管生成治疗性策略。
这些策略的先决条件是鉴定肿瘤血管内皮中、但不存在于正常组织静息内皮细胞上的靶结构。为了将细胞生长抑制剂和某些毒素以相对于肿瘤细胞本身较少的程度施用于肿瘤的血管内皮细胞而使用这些特异的靶结构。可用于这个目的的靶结构是bFGF(碱性成纤维细胞生长因子)、VEGF(血管内皮生长因子)和VEGFR-2(VEGF受体2)、内皮糖蛋白、内皮唾液酸蛋白、纤粘连蛋白同工型(ED-B结构域)、整合素αvβ3、αvβ5、α1β1和α1β2、氨基肽酶N、NG2蛋白多糖和基质金属蛋白酶2和9(MMP 2和9)(2-13)。例如，Arap等(8)将特异结合α1-整合素的肽与用于现有化疗(阿霉素)领域的活性物质偶联。在动物模型中证明可通过与该肽偶联而增加阿霉素的抗肿瘤效果。
备选的抗血管治疗方法包括肿瘤血管中血液凝固的选择性活化以诱导肿瘤坏死。例如，生产双特异的F(ab’)2抗体片段，其指向截短的组织因子(tTF)和MHC II类抗原。肿瘤内皮细胞中抗原的实验性诱导后，可通过在鼠神经母细胞瘤模型中施用抗体证明抗血管治疗(14)。在相同团队的第二研究中，使用使tTF选择性地偶联于肿瘤血管内皮的天然存在标记物，VCAM-1(血管细胞粘附分子-1)的免疫共轭物(15)。
在非常相似的方法中，将特异于癌胚ED-B结构域的抗体片段(scFv)与tTF融合。在小鼠模型中产生的该融合蛋白，ScFv-tTF导致多种肿瘤中完全的和选择性的梗死(16)。
备选地，将tTF偶联于前列腺-特异的膜抗原的抑制剂(17)。静脉内给药后这种融合蛋白在大鼠前列腺模型中诱导选择性的梗塞坏死。这种融合多肽与细胞毒性物质(阿霉素)组合低剂量给药导致大块肿瘤退化，甚至完全的肿瘤消除(17)。最近已经描述了由针对VEGFR、内皮糖蛋白和VCAM-1的抗体片段组成的其他tTF融合蛋白(18)。
然而现有技术中为了抗血管肿瘤治疗而生产的分子具有缺陷。特别地必须假定这些分子由于其大小而产生免疫原性。因此用这些分子治疗哺乳动物将触发针对这些分子的免疫反应，所以重复施用这些分子变得不可行。
借助于可活化血液凝固的肽部分的偶联配偶体尺寸将用于肿瘤组织，可进一步对血液凝固而言重要的大分子因子VIIa/FX酶-底物复合物的形成导致空间阻碍。当能够活化血液凝固的肽由于相对大的融合配偶体而具有改变的构象时也可妨碍该复合物的形成。
现有技术中(WO 03/035688)，使已知具有选择性结合结构域的融合多肽与组织因子多肽的N-末端偶联，其中选择性的结合结构域为例如纤粘连蛋白的结构域，其结合例如包括RGD肽，或结合PSMA(前列腺特异的膜抗原)的D-β-E二肽的整合素。尽管该构建体在体外证明了酰胺分解和蛋白水解效果，即使与因子VIIa组合，在体内只显示了极弱的抗肿瘤作用。只在与强力霉素组合中动物存活较长。
Hu等(46)描述了多种融合蛋白及其在肿瘤血管中产生血栓的用途，包括来自偶联于截短形式的组织因子的、含有RGD序列的、具有9个氨基酸的寡肽的融合蛋白。再次将RGD肽与tTF的N-末端连接以获得RGD-tTF。功能分析显示含有RGD的融合蛋白对肿瘤生长不产生任何显著的抑制。
因此构建现有技术中已知的构建体以使得选择性的结合结构域与组织因子多肽的N-末端连接。甚至强调必须选择这种结构，因为认为结构模型基础上的N-末端是特别有利于连接的位点，其不会抑制血栓形成的起始。
发明概述
鉴于这种现有技术，因此问题在于提供在体内可有效抑制肿瘤生长的备选血栓形成物质。
现在通过融合多肽解决了这个问题，该融合多肽包括容许该融合多肽选择性结合肿瘤血管内皮细胞的3-30个氨基酸的肽，和组织因子(TF)或其片段，该组织因子和片段的特征在于当融合多肽结合肿瘤血管内皮时它们能够活化血液凝固，这些肽相互直接偶联，或通过具有至多15个氨基酸的接头相互偶联。将能够使融合多肽选择性结合肿瘤血管内皮细胞的肽与当该融合多肽结合肿瘤血管内皮细胞时可活化血液凝固的肽的C-末端偶联。本发明进一步涉及含有相应融合多肽的药物组合物及其用于治疗肿瘤的用途。
附图简述
图1：表示tTF-RGD和tTF-NGR融合蛋白结合αvβ3和CD13的图解。由于RGD序列对αvβ3-整合素和NGR序列对CD13(氨基肽酶N)的特异性而获得肿瘤的选择性。在肿瘤内皮细胞上以高密度选择性地和特异地表达这些受体，但不在正常组织的静息内皮细胞(除了一些例外之外)上表达。融合蛋白的表示是高度示意性的并且不提供关于基本序列的任何信息。
图2：重组tTF1-218和tTF融合蛋白的SDS-PAGE和Western印迹分析。通过在从大肠杆菌(Ecoli)(BL21 DE3)提取和经线性尿素梯度(6M-1M)再折叠后进行SDS-PAGE并用考马斯蓝染色来检验tTF和tTF融合蛋白的纯度。利用单克隆抗-组织-因子抗体(clone V1C7American Diagnostics)通过Western印迹验证蛋白质的鉴定。加样到单独的泳道中：1＝tTF；2＝tTF-RGD；3＝tTF-NGR；4＝tTF-环形NGR1；5＝tTF-环形NGR2；6＝tTF-环形NGR3；7＝tTF-GALNGRSHAG；M＝分子量标记。
图3：FVIIa/tTF1-218或FVIIa/tTF1-218融合蛋白活化FX的Michaelis常数(Km)的测定。利用Ruf(45)描述的方法计算Michaelis-Menten动力学的参数。
图4：tTF、tTF-RGD和tTF-NGR与整合素αvβ3的结合。在ELISA中用针对人TF(American Diagnostica)的多克隆抗体定量0.1μMtTF、tTF-RGD和tTF-NGR与固定的αvβ3的结合。结果表示为中值和四分值的范围。tTF-RGD和tTF之间或tTF-NGR和tTF之间结合的差异是统计学上显著的(p＜0.001，Mann-Whitney测试)。
图5：tTF-RGD结合整合素αvβ3的特异性。通过用合成肽GRGDSP(1-10μM)竞争性抑制来显著抑制tTF-RGD(0.1μM)与固定的αvβ3的结合(对于两个RGD肽浓度的Mann-Whitney测试，p＜0.001)。
图6：tTF和tTF-RGD与人内皮细胞的结合。A：FACS分析用0.1μMtTF(2)或用0.1μMtTF-RGD(3)在4℃培养60min的内皮细胞。B：通过用1μM GRGDSP(4)竞争性抑制tTF-RGD融合蛋白证明75％的结合减少。A和B中的曲线1显示阴性对照。
图7：与用生理盐水溶液(NaCl，n＝8)或tTF(n＝1)灌注的肿瘤生长相比，通过用tTF融合蛋白(tTF-RGD，n＝6；tTF-NGR，n＝6)静脉内治疗对在无胸腺的裸鼠中作为异种移植物生长的人肺癌(CCL185)的抑制。垂直箭头表示用各物质注射的时间点。
图8：与用生理盐水溶液(NaCl，n＝4)或tTF(n＝4)灌注的肿瘤生长相比，通过用tTF融合蛋白(tTF-RGD，n＝3；tTF-NGR，n＝3)静脉内治疗对在无胸腺的裸鼠中作为异种移植物生长的人恶性黑色素瘤(M21)的抑制和部分缓和。垂直箭头表示用注射各种物质的时间点。
图9：注射tTF-NGR融合蛋白(A，图片的左半侧)或NaCl(A，图片的右半侧)后20min的患有肿瘤小鼠的体内肉眼照片。注射tTF-NGR后肿瘤的用蓝-青着色的肉眼图片指示了肿瘤坏死。60min后，使两只小鼠放血，完全切下肿瘤，并进行组织学检查。在B中，我们可看到用tTF-NGR处理的肿瘤的出血性吸胀，这是作为初始肿瘤坏死的结果的次级出血迹象。相比之下，NaCl处理的肿瘤看来是活的(C)。
图10：在患有肿瘤的裸鼠的尾静脉中静脉内注射tTF-RGD(A和B)、tTF-NGR(C和D)和普通盐(E和F)后1小时黑色素瘤的组织学。在用tTF融合蛋白处理的肿瘤中，血管看起来是血栓溶解闭塞的(箭头)。在由血液凝块闭塞的血管的供给区域中可观察到广泛的肿瘤坏死(A-D)。照片是肿瘤的代表性区域(A、C和E：200×放大，B、D和F 400×放大；HE染色(例如，在H.C.Burck，HistologischeTechnik-Leitfaden für die Herstellung mikroskopischer Prparate inUnterricht und Praxis，第5版，Thieme Verlag，Stuttgart 1982，109页及以下中描述的染色)。
图11：注射4mg/kg BW tTF-NGR后1小时，心(A)、肾(B)、肝(C)和肺(D)的代表性组织学。在任何的这些器官中显微镜术没有检测到形成血栓或坏死。(HE染色；200×放大)。
图12：人组织因子(TF)的氨基酸序列。
图13：截短的人组织因子tTF1-218的氨基酸序列(在本申请的范围内也指定简称为tTF)。
图14：融合多肽tTF-GRGDSP的氨基酸序列(也缩写为tTF-RGD)。
图15：融合多肽tTF-GNGRAHA的氨基酸序列(也缩写为tTF-NGR)。
图16：融合多肽tTF-GALNGRSHAG的氨基酸序列。
图17：融合多肽tTF-GCNGRCG的氨基酸序列(也缩写为tTF-环形NGR1)。
图18：融合多肽tTF-GCNGRCVSGCAGRC的氨基酸序列(也缩写为tTF-环形NGR2)。
图19：融合多肽tTF-GCVLNGRMEC的氨基酸序列(也缩写为tTF-环形NGR3)。
图20：截短的人组织因子tTF1-218的核苷酸序列(在本申请的范围内也指定简称为tTF)。
图21：融合多肽tTF-GRGDSP的核苷酸序列(也缩写为tTF-RGD)。
图22：融合多肽tTF-GNGRAHA的核苷酸序列(也缩写为tTF-NGR)。
图23：融合多肽tTF-GALNGRSHAG的核苷酸序列。
图24：融合多肽tTF-GCNGRCG的核苷酸序列(也缩写为tTF-环形NGR1)。
图25：融合多肽tTF-GCNGRCVSGCAGRC的核苷酸序列(也缩写为tTF-环形NGR2)。
图26：融合多肽tTF-GCVLNGRMEC的核苷酸序列(也缩写为tTF-环形NGR3)。
图27：用于产生tTF1-218的寡核苷酸的核苷酸序列。
A：5’-引物；B：3’-引物。
图28：用于产生tTF-GRGDSP的寡核苷酸的核苷酸序列。
A：5’-引物；B：3’-引物。
图29：用于产生tTF-GNGRAHA的寡核苷酸的核苷酸序列。
A：5’-引物；B：3’-引物。
图30：用于产生tTF-GCNGRCG的寡核苷酸的核苷酸序列。
A：5’-引物；B：3’-引物。
图31：用于产生tTF-GCNGRCVSGCAGRC的寡核苷酸的核苷酸序列。A：5’-引物；B：3’-引物。
图32：用于产生tTF-GCVLNGRMEC的寡核苷酸的核苷酸序列。
A：5’-引物；B：3’-引物。
图33：用于产生tTF-GALNGRSHAG的寡核苷酸的核苷酸序列。
A：5’-引物；B：3’-引物。
图34：a：与用生理盐水溶液(NaCl，n＝9)或tTF(n＝11)灌注的肿瘤生长相比，通过用tTF融合蛋白(tTF-RGD，n＝7)静脉内治疗对在无胸腺的裸鼠中作为异种移植物生长的人恶性黑色素瘤(M21)的抑制和部分缓和。垂直箭头表示注射各种物质的时间点。
b：与用生理盐水溶液(NaCl，n＝15)或tTF(n＝14)灌注的肿瘤生长相比，通过用tTF融合蛋白(tTF-RGD，n＝12)静脉内治疗对在无胸腺的裸鼠中作为异种移植物生长的人纤维肉瘤(HT1080)的抑制。垂直箭头表示注射各种物质的时间点。
c：与用生理盐水溶液(NaCl，n＝10)或tTF(n＝5)灌注的肿瘤生长相比，通过用tTF融合蛋白(tTF-RGD，n＝11)静脉内治疗对在无胸腺的裸鼠中作为异种移植物生长的人肺癌(CCL185)的抑制。垂直箭头表示注射各种物质的时间点。在各个情况中对于独立的组用Mann-Whitney测试研究统计学上的显著性，p值在0.05以下认为是显著的，*显示tTF-RGD和缓冲液之间统计学上的差异显著性。
图35：用tTF-RGD融合蛋白(A、C)或NaCl(B、D)处理结束时(第7天)，患有M21肿瘤小鼠的肉眼照片。与外观上活的对照肿瘤相比，显示明显坏死迹象的tTF-RGD-处理肿瘤的大小差异和外观差异是可容易辨别的。
图36：用tTF-RGD和生理盐水溶液处理的小鼠肿瘤和器官的H-E染色
在用tTF-RGD处理的动物中观察到肿瘤细胞的严重血栓形成和坏死(A：200倍，B：400倍)。箭头显示肿瘤血管中形成血栓的实例。在用盐水处理的动物中没有观察到明显的血栓形成或坏死(C：200倍，D：400倍)。箭头显示具有一些红血球的完整肿瘤血管。用tTF-RGD处理的动物的心(E)、肺(F)、肝(G)和肾没有显示任何可见的形成血栓或坏死。
图37：tTF-NGR在纤维肉瘤模型中的作用
没有(tTF-NGR前)和静脉注射给药tTF-NGR后6小时(tTF-NGR后)通过磁共振成像(MRI)研究患有纤维肉瘤(HT1080)的小鼠。显示了高或低的血管体积分数。
发明详述
现在通过包括下列肽的融合多肽克服现有技术中观察到的问题：
a)能够使该融合多肽选择性结合肿瘤血管内皮细胞的3至30个氨基酸的肽；和
b)组织因子(TF)或其片段，该组织因子和片段的特征在于当该融合多肽结合肿瘤血管内皮细胞时，它们能够活化血液凝固，
其中肽a)和b)直接相互偶联或通过具有至多15个氨基酸的接头相互偶联，其特征在于能够使该融合多肽选择性结合肿瘤血管内皮细胞的肽与当该融合多肽结合肿瘤血管内皮细胞时能够活化血液凝固的肽之C末端偶联。本发明进一步涉及含有相应融合蛋白的药物及其用于治疗肿瘤的用途。
除序列a)和b)外，本发明所述的融合多肽可包括附加的序列，只要这些序列对融合多肽的空间构象没有不良影响，并且不会妨碍形成触发血液凝固的酶-底物复合物。本发明所述的融合多肽可例如包含简化肽的重组表达和纯化的His-Tag序列(参考实施例)。然而这些序列的存在不是必须的。根据本发明的优选实施方案，融合多肽因此包括：
a)能够使该融合多肽选择性结合肿瘤血管内皮细胞的3至30个氨基酸的肽；和
b)组织因子(TF)或其片段，该组织因子和片段的特征在于当该融合多肽结合肿瘤血管内皮细胞时，它们能够活化血液凝固，
其中肽a)和b)直接相互偶联或通过具有至多15个氨基酸的接头相互偶联。根据本发明特别优选的实施方案，融合多肽包括：
a)能够使该融合多肽选择性结合肿瘤血管内皮细胞的3至30个氨基酸的肽；和
b)组织因子(TF)或其片段，该组织因子和片段的特征在于当该融合多肽结合肿瘤血管内皮细胞时，它们能够活化血液凝固，
其中肽a)和b)相互偶联。
根据本发明，令人惊讶地显示，来自能够使融合多肽选择性结合肿瘤血管内皮细胞的特别小的肽的融合多肽，和当该融合多肽结合肿瘤血管内皮细胞时能够活化血液凝固的肽对于抗血管肿瘤治疗是特别有利的。容许结合肿瘤血管内皮细胞的小尺寸多肽改善了融合蛋白对内皮细胞磷脂膜的定向。为血液凝固所必需的酶/底物复合物的形成没有受到空间位阻，并且可活化血液凝固的组织因子TF没有经受构象变化。
根据本发明优选的实施方案，当融合多肽结合肿瘤血管内皮细胞时能够活化血液凝固的肽是具有如SEQ ID NO：1(图12)所示氨基酸序列的组织因子TF。本发明进一步包括与SEQ ID NO：1(图12)具有至少70％或至少80％氨基酸同源性的组织因子序列，具有至少95％同源性的序列是特别优选的。通过上下书写两个序列，为了获得待比较序列最大可能的一致，可使100个氨基酸长度上有4个缺口来确定同源性的程度(参考Dayhoff，Atlas of Protein Sequence and Structure，5，124，1972)。然后确定两个氨基酸链中较短链的氨基酸残基的百分数，这与另一链上的相同氨基酸残基相对。
此外当融合多肽结合肿瘤血管内皮细胞时能够活化肿瘤血管中血液凝固的肽可以是组织因子TF的片段或TF同源序列的片段。优选该片段具有如SEQ ID NO：2(图13)所示的序列。如SEQ ID NO：2(图13)所示的序列(tTF1-218或简称tTF)包括TF的N-末端218个氨基酸。此外，根据本发明，也可能使用相对于tTF在N-末端或C-末端缺少几个氨基酸的tTF片段。例如，可能使用在N-末端缺少至多10个氨基酸的片段(tTF11-218)。另外，可使用在C-末端缺少至多8个氨基酸的片段(tTF1-210)，例如(tTF1-214)。
本发明涉及融合多肽，其中能够选择性结合肿瘤血管内皮细胞的肽偶联于能够活化血液凝固的肽的C-末端。根据本发明，使用术语“肿瘤血管内皮细胞”和术语“肿瘤血管中的内皮细胞”来指覆盖肿瘤血管的细胞。根据本发明证实上述排列保证融合蛋白垂直于内皮细胞磷脂膜的方向，这对于触发血液凝固是特别有利的。这个方向相应于诱导血液凝固期间TF的天然方向。如图3所示，对于以这种方式产生的所有构建体，关于由FVIIa/tTF1-218或FVIIa/tTF1-218融合蛋白活化的因子X发现了非常相似的Michaelis-Menten动力学。在现有技术中，相比之下活化血凝块的肽偶联于靶分子的C-末端(参考(16))。因此本发明所述的融合多肽在根本上不同于现有技术中所使用的肽。
能够使融合多肽选择性结合肿瘤血管内皮细胞的肽可以是长度为3-30个氨基酸并且以高特异性结合肿瘤血管内皮细胞的任何肽。可通过现有技术中的常规方法从肽文库分离相应的肽。根据所选择的肽文库，它们可具有线性或环状结构。
根据本发明的一个实施方案，容许融合多肽选择性结合肿瘤血管内皮细胞的肽包括氨基酸序列RGD或NGR。现有技术中已知两个序列特异结合整合素，特别是αvβ3和αvβ5整合素(RGD肽)，以及细胞粘附模体(NGR肽)(参考(8))。根据本发明令人惊讶地显示这些肽特别适于是融合多肽的部分，而该融合多肽的另外部分是当融合多肽结合肿瘤血管内皮细胞时能够活化血液凝固的肽。
使用序列GRGDSP、GNGRAHA和GALNGRSHAG的线性肽以及序列GCNGRCG、GCNGRCVSGCAGRC和GCVLNGRMEC的环状肽获得特别有利的效果。证明包括这些序列和人TF的最初218个氨基酸序列的融合多肽高度适于抗血管肿瘤治疗。特别地，显示这些融合多肽导致肿瘤生长的显著抑制或肿瘤尺寸减小(参见图7和8)。所观察到的肿瘤部分缓和的诱导(参考图8)指向基于小鼠模型高度预测功效的人肿瘤治疗中的阳性结果预期(42、43、44)。
本发明进一步包括具有环状RGD肽的融合蛋白，因为环化提高了对整合素的亲合力(例如如参考文献21中所描述的)。
本发明进一步涉及具有如SEQ ID NO：3-8(图14-19)所示序列之一的融合多肽。
根据另一个实施方案，本发明涉及编码如上所述的融合多肽的核酸。相应的核酸可例如具有如SEQ ID NO：10-15(图21-26)所示的序列之一。
在另一个方面，本发明涉及包括前述核酸之一的载体。相应的载体通常也包括核酸的表达调控序列。在现有技术中全面描述了所述的载体，并且可从大量的公司商业获得这种载体。
在进一步的实施方案中，本发明涉及包括上述核酸或载体之一的细胞。该细胞一般用于表达核酸和重组生产本发明所述的融合多肽。为此目的，可发现大量细胞的应用，包括大肠杆菌(E.coli)、酵母细胞和动物细胞系，例如CHO-或COS-细胞。在现有技术中广泛描述了适当的细胞及其用途。
可进一步通过其他合适的方法，例如通过个别肽的化学偶联生产权利要求1所述的本发明的多肽。因此可通过现有技术中的常规方法，例如通过化学合成或通过异源表达来生产个别的肽，然后通过偶联连接在一起。
最后，本发明也涉及包括上述融合多肽、核酸、载体或细胞的药物组合物。该药物组合物可进一步包括药物学上可接受的相容载体、赋形剂或佐剂。此外，所述药物组合物中的多肽可以修饰的状态，例如peg化(pegyliert)，即与聚乙二醇分子偶联的状态存在。
本发明所述的融合多肽或包含这些融合多肽的药物组合物可用于治疗肿瘤疾病，特别用于抗血管肿瘤治疗。被认为可用本发明所述的融合多肽或包含这些融合多肽的药物组合物辅助治疗的肿瘤疾病包括例如支气管肺癌和胸腔及纵隔腔的其他肿瘤、乳腺癌和其他妇科肿瘤、结肠直肠癌、胰腺癌和胃肠道的其他肿瘤、恶性黑色素瘤和其他皮肤肿瘤、头颈区中的肿瘤、前列腺癌和其他泌尿生殖器肿瘤、肉瘤、内分泌活化的肿瘤、白血病和脊髓发育不良综合症和霍奇金(Hodgkin)淋巴瘤以及非霍奇金淋巴瘤。
此外，也可治疗良性肿瘤，例如血管瘤，和糖尿病性视网膜病中的新血管化作用。
除静脉内给药外，皮下和腹膜内施用融合多肽或药物组合物也是可行的。通过包装在防止融合多肽在胃肠道中断裂的药物载体中，也可口服给药该融合多肽或药物组合物。
进一步的，与其它的治疗途径，例如与细胞毒的化疗或放射组合施用本发明所述的融合多肽可能是有益的。具有其他活性物质的组合物，例如具有因子VIIa或强力霉素的组合物也是可行的，但优选本发明所述的多肽与因子VIIa或强力霉素的组合物不是必须的。
基于下列实施例更详细地描述本发明：
实施例
实施例1：tTF和tTF融合蛋白的表达和纯化
利用SEQ ID NO：16和SEQ ID NO：17(图27)所示的引物通过聚合酶链式反应(PCR)合成编码组织因子TF(下文标明为tTF)N-末端218个氨基酸的cDNA，并克隆到表达载体pET-30a(+)(Novagen)中。将重组质粒转化到大肠杆菌(BL21)中，表达并纯化(Qiagen PlasmidKit)。
沿着截短的组织因子tTF，构建tTF肽融合蛋白，其中靶向肽首先结合可溶组织因子tTF的羧基末端。构建下列线性融合蛋白：
tTF-GRGDSP(SEQ ID NO：3；图14；下文标明为tTF-RGD；使用PCR引物SEQ ID NO：18和SEQ ID NO：19(图28))；
tTF-GNGRAHA(SEQ ID NO：4；图15；下文标明为tTF-NGR；使用PCR引物SEQ ID NO：20和SEQ ID NO：21(图29))；
tTF-GALNGRSHAG(SEQ ID NO：5；图16；使用PCR引物SEQID NO：28和SEQ ID NO：29(图33))；
另外，合成下列环状融合蛋白：
tTF-GCNGRCG(SEQ ID NO：6；图17；下文标明为tTF-环状NGR1；使用PCR引物SEQ ID NO：22和SEQ ID NO：23(图30))；
tTF-GCNGRCVSGCAGRC(SEQ ID NO：7；图18；下文标明为tTF-环状NGR2；使用PCR引物SEQ ID NO：24和SEQ ID NO：25(图31))；
tTF-GCVLNGRMEC(SEQ ID NO：8；图19；下文标明为tTF-环状NGR3；使用PCR引物SEQ ID NO：26和SEQ ID NO：27(图32))
在介导附加表达6个组氨酸残基的N-末端亲合标记和一些载体编码氨基酸的pET30a(+)载体中表达所有的构建体(包括tTF)。在该亲合标记的帮助下，可通过镍-次氮基三乙酸柱(Ni-NTA，Novagen)上的亲合层析纯化构建体。亲合标记显示在SEQ ID NO：30中。作为实施例，SEQ ID NO：31和SEQ ID NO：32显示了具有亲合标记的tTF-GRGDSP和tTF-GNGRAHA的完整氨基酸序列。
选择构建体以使得根据tTF：FVIIa复合物的已知X-射线晶体结构(19)，保证tTF融合蛋白对内皮细胞磷脂膜的垂直方向，这相应于天然TF的方向。进一步考虑所选择的结构不会导致tTF产生对FVIIa和大分子底物FX相互作用的任何空间位阻。由于RGD序列对αvβ3整合素和NGR序列对CD13(氨基肽酶N)的特异性，获得肿瘤选择性，正如在肿瘤内皮细胞上以高密度选择性地和特异地表达这些受体，但除了一些例外之外，不在正常组织的静止内皮细胞上表达(参见图1)。
在大肠杆菌(BL21)中借助pET30a(+)转化和表达tTF和描述为tTF-RGD、tTF-NGR、tTF-GALNGRSHAG和tTF-环状NGR1-3的融合蛋白。离心转化的、IPTG-诱导的大肠杆菌BL21DE3，将每g沉淀颗粒吸入5-7ml的裂解缓冲液(10mM Tris-HCl，pH 7.5；150mMNaCl；1mM MgCl2；10μg/ml抑肽酶；2mg/ml溶菌酶)，并加入20μl Benzonase(Novagen)。室温(RT)下孵育90min后，于4℃以12,000g离心20min，将沉淀颗粒重悬在洗涤缓冲液(10mM Tris/HCl，pH7.5；1mM EDTA；3％Triton X-100)中并通过超声波处理匀质化。在每g沉淀颗粒2-4ml变性缓冲液(6M盐酸胍，0.5M NaCl，20mMNaH2PO4，1mM DTT)中于室温溶解包涵体过夜。用0.22μg的滤器过滤来自离心(5000g，30min，4℃)的上清液。纯化构建体直到借助于另外导入的构建体His-Tag序列在镍-次氮基三乙酸柱(Ni-NTA，Novagen)上均匀化。用His结合缓冲液试剂盒(Novagen)进行蛋白质的纯化和折叠。然后用TBS缓冲液(20mM Tris，150mM NaCl，pH7.4)透析。
通过SDS-PAGE、Western印迹和质谱鉴定该蛋白质(参见图2)。
实施例2：tTF和tTF融合蛋白的功能特性
关于因子X借助因子VIIa活化为因子Xa中的辅助因子活性，通过Michaelis-Menten分析体外证明这些融合蛋白的功能活性。在由Ruf描述的(45)略微改进的方法中确定tTF和tTF融合蛋白在存在磷脂时，借助FVIIa强化FX的特异蛋白水解活化的能力。为此，在微滴定板中吸入20μl下列各种试剂：(a)在TBS-BSA中的50nM重组FVIIa(Novo-Nordisk)；(b)在TBS-BSA中的0.16nM-1.6μM tTF/tTF融合多肽；(c)25mM CaCl2和500μM磷脂泡囊(卵磷脂/磷脂酰丝氨酸，70/30，M/M；Sigma)。在室温孵育10min后，加入5μM浓度的20μl天然底物FX(Enzyme Research Laboratories)。然后以1分钟间隔用移液管吸取样品，并通过加入100mM EDTA溶液终止反应。通过加入生色底物Spectrozyme FXa，在微平板阅读仪中测定405nm处吸光值的变化来测量形成的FXa数量，通过Ruf描述的方法分析Michaelis-Menten动力学的参数。结果显示tTF和tTF融合多肽在这些条件下都是功能性活化的(图3)。发现融合多肽的Michaelis常数(Km)在0.12-1.2nM的范围中(图3)，并且如此在为tTF公开的较低范围中。可因此推测功能性活性不受tTF与肽融合的影响。
实施例3：tTF融合蛋白与αvβ3的体外和体内结合
通过将纯化的αvβ3固定在微滴定平板上，在ELISA(酶联免疫吸附测定)中证明tTF-RGD和tTF-NGR结合αvβ3整合素(参见图4)。通过具有序列GRGDSP的合成肽(来自Gibco公司)在这个测试系统中竞争性抑制tTF-RGD结合αvβ3的事实强调了tTF-RGD结合αvβ3的特异性(参见图5)。
然后评估tTF-RGD特异性结合内皮细胞上的αvβ3。为此，通过FACS(荧光激活细胞分拣法)分析悬浮物中生物素酰化的tTF和tTF-RGD与内皮细胞的差异结合。在实验中利用组织培养中保持的所有内皮细胞被活化，即表达αvβ3分子的事实。这可通过多种免疫组化学方法检测。从而使培养的内皮细胞关于其αvβ3的表达模式方面与肿瘤内皮细胞相对应。因此培养的内皮细胞可用作底物特异结合肿瘤内皮细胞的模型系统，并且也容许作出关于所期望毒性的预测。
使用链霉抗生物素-藻红蛋白作为检测方法。测量的tTF-RGD荧光强度比tTF高出8倍(图6A)。此外，通过施用1μM的合成肽GRGDSP竞争性降低0.1μM tTF-RGD与内皮细胞结合的75％(图6B)。这强调了tTF-RGD结合内皮细胞表面上类似于αvβ3的RGD-结合受体的特异性。
实施例4：动物模型中tTF融合蛋白的抗肿瘤效果
在无胸腺的裸鼠中评估tTF-RGD和tTF-NGR融合蛋白对人肿瘤异种移植物的效果和不良影响。对此使用我们实验室已建立的模型(33，34)。将细胞系CCL185(人肺腺癌)和M-21(人黑色素瘤)皮下注射到雄性BALB/c裸鼠(9-12周龄)的侧腹。当肿瘤体积达到大约50-100mm3(CCL185)或400-600mm3(M-21)时，将小鼠随机分配为4组。第1组只接受生理盐水溶液(NaCl)，第2组tTF，第3组tTF-RGD，以及第4组tTF-NGR(在每个情况中，每kg体重(BW)1.5-2.0mg的蛋白质)。间隔1-3天(取决于特定细胞系的生长速率)对动物进行尾静脉注射。观察到相当可观的融合蛋白治疗活性。用tTF-RGD和tTF-NGR融合蛋白处理的小鼠的肿瘤与tTF和NaCl相比较，其生长受到显著抑制或尺寸减小直到部分缓和(参见图7和8)。
为了验证肿瘤血管中形成血栓诱导的作用机制，进行下列试验：将人黑色素瘤细胞系注射到2只雄性BALB/c裸鼠的侧腹。当肿瘤尺寸达到大约500mm3时，尾静脉注射2.0mg/kg BW的tTF-NGR或NaCl。图9A显示了注射tTF-NGR融合蛋白(图片左半部分)或NaCl(图片右半部分)20min后的患有肿瘤小鼠的体内内眼图。注射tTF-NGR后肿瘤的用蓝-青着色的肉眼图片指示了肿瘤坏死。60min后，使两只小鼠放血，完全切下肿瘤，并进行组织学研究。图9B显示了tTF-NGR处理的肿瘤的出血性吸胀，这是作为初始肿瘤坏死的结果的次级出血迹象。相比之下，NaCl处理的肿瘤看来是活的(图9C)。
黑色素肿瘤的组织学分析显示血管中显微镜下可见的血栓形成(图10A-D)。这个发现验证了tTF-NGR的抗肿瘤效果的暗含机制，即诱导血管中的血栓。通过在正常的组织，例如心、肾、肝和肺中组织学上检测不到凝固和坏死证明tTF-NGR对肿瘤血管的高度选择性(图11A-D)。甚至重复高剂量的tTF-NGR(4mg/kg BW)也不能产生任何可见的凝固形成或器官毒性。
实施例5：HT1080肿瘤动物模型中tTF融合蛋白的抗肿瘤效果
也用纤维肉瘤(HT1080)在BALB/c裸鼠中研究了tTF-RGD融合蛋白的抗肿瘤活性。这些肿瘤快速生长并且可很好地形成血管。两个实验的结果显示在表2和图34中。第二次注射tTF-RGD后，与对照组相比，观察到HT1080肿瘤生长的明显抑制。持续这种作用直到第7天实验结束(P＝0.021，tTF-RGD相对于缓冲液对照(生理盐水溶液)；P＝0.005，tTF-RGD相对于tTF)。在更早期的实验中，在这个模型中观察到肿瘤体积的部分衰退。
表1：
tTF-RGD对小鼠中M21肿瘤生长的影响
| 处理 | 平均肿瘤体积(mm3) | 相对于缓冲液的P | 相对于tTF的P | n | |
| 第0天 | 第7天 | ||||
| 缓冲液 | 590±77 | 994±140 | ns | 9 | |
| tTF | 558±47 | 931±147 | ns | 11 | |
| tTF-RGD | 585±85 | 514±81 | ＜0.01 | ＜0.05 | 7 |
ns：非显著
表2：
tTF-RGD对小鼠中HT1080肿瘤生长的影响
| 处理 | 平均肿瘤体积(mm3) | 相对于缓冲液的P | 相对于tTF的P | n | |
| 第0天 | 第7天 | ||||
| 缓冲液 | 1671±296 | 2431±559 | ns | 15 | |
| tTF | 1751±269 | 2335±398 | ns | 14 | |
| tTF-RGD | 1725±197 | 1241±122 | ＜0.05 | ＜0.01 | 12 |
ns：非显著
表3：
tTF-RGD对小鼠中CCL185肿瘤生长的影响
| 处理 | 平均肿瘤体积(mm3) | 相对于缓冲液的P | 相对于tTF的P | n | |
| 第0天 | 第7天 | ||||
| 缓冲液 | 39±3 | 467±137 | ns | 9 | |
| tTF | 44±8 | 764±148 | ns | 5 | |
| tTF-RGD | 45±5 | 130±19 | ＜0.01 | ＜0.01 | 10 |
ns：非显著
本领域的技术人员可根据本发明公开的内容构建其他的tTF融合蛋白而没有任何问题。潜在的候选物是结合NG2，人黑色素瘤蛋白多糖的鼠同系物的肽TAASGVRSMH和LTLRWVGLMS(12)。NG2的表达限于肿瘤细胞和肿瘤生成血管的血管(35)。另一个候选物是产生基质金属蛋白酶-2(MMP-2)的选择性和潜在抑制的合成肽TTHWGFTL(13)。因为整合素αvβ3也以RGD非依赖的方式明显结合MMP-2，这意味着活化的酶位于生成血管的血管表面(36)。由tTF和该MMP-2抑制肽组成的构建体可相似的介导tTF1-218选择性结合肿瘤血管的内皮细胞膜。
参考文献
1.Folkman J，Watson K，ingber D，Hanahan D：Induction of angiogenesis during the transitionfrom hyperplasia to neoplasia.自然(Nature)339：58-61，1989
2.Dvrorak HJ，Sioussat TM，Brown LF，Berse B，Nagy JA，Sotrel A，Manseau EJ：Distributionof vascular permeability factor(vascular endothelial growth factor)in tumors-Concentrationin tumor blood vessels.J Exp Med 174：1275-1278，1991
3.Dvorak HJ，Brown LF，Detmar M，Dvorak AM：Vascular permeability factor/vascular endo-thelial growth factor，microvascular hyperpermeability，and angiogenesis.Am J Pathol 146：1029-1039，1995
4.Terman BJ，Dougher-Vermazen M：Biological properties of VEGFNPF receptors.CancerMetastasis Rev 15：159-163，1996
5.Burrows FJ，Derbyshire EJ，Tazzari PL，Amlot P，Gazdzar AF，King SW，Letarte M，VitettaES，Thorpe PE：Upregulation of endoglin on vascular endothelial cells in human solid tu-mors：Implications for diagnosis and therapy.Clin Cancer Res 1：1623-1634，1995
6.Rettig WJ，Garlnchesa P，Healey JH，Su SL，Jaffe EA，Old LJ：Identification of endosialin，acell surface glycoprotein of vascular endothelial cells in human cancer.Proc Natl Acad SciUSA 89：10832-10836，1992
7.Carnemolla B，Balza E，Siri A，Zardi L，Nicrotra MR，Bigotti A，Natali PG：A tumor-associatedfibronectin isoform generated by alternative splicing of messenger RNA precursors.J CellBlol 108：1139-1148，1989
8.Arap W，Pasqualini R，Ruoslahti E：Cancer treatment by targeted drug delivery to tumorvasculature in a mouse model.科学(Science)279：377-380，1998
9.Senger DR，Claffey KP，Benes JE，Peruzzi CA，Sergiou AP，Detmar M：Angiogenesis pro-moted by vascular endothelial growth factor：regulation through α1β1 and α2β1 integrins.Proc Natl Acad Sci USA 94：13612-13617，1997
10.Olson TA，Mohanraj D，Roy S，Ramakrishnan S：Targeting the tumor vasculature：inhlbitionof tumor growth by a vascular endothelial growth factor-toxin conjugate.Int J Cancer 73：865-870，1997
11.Bhagwat SV，Lahdenranta J，Giordano R，Arap W，Pasqualini R，Shapiro LH：CD13/APN isactivated by angiogenic signals and is essential for capiliary tube formation.Blood 97：652-659，2001
12.Burg MA，Pasqualini R，Arap W，Ruoslahti E，Stallcup WB：NG2 Proteoglycan-binding pep-tides target tumor neovasculature.癌症研究(Cancer Res)59：2869-2874，1999
13.Koivunen E，Arap W，Valtanen H，Rainisalo A，Medina OP，Heikkila P，Kantor C，GahmbergCG，Salo T，Konttinen YT，Sorsa T，Ruoslahti E，Pasqualini R：Tumor targeting with a selec-tive gelatinase inhibitor.Nat Biotechnol 17：768-774，1999
14.Huang X，Molema G，King S，Watkins L.Edgington TS，Thorpe PE：Tumor infarction in miceby antibody-directed targeting of tissue factor to tumor vasculature.Science 275：547-550，1997
15.Ran S，Gao B，Duffy S，Watkins L，Rote N，Thorpe PE：Infarction of solid Hodgkin’s tumorsin mice by antibody-directed targeting of tissue factor to tumor vasculature.Cancer Res 58：4646-4653，1998
16.Nilsson F，Kosmehl H，Zardi L，Neri D：Targeted delivery of tissue factor to the ED-B domainof fibronectin，a marker of angiogenesis，mediates the infarction of solid tumors in mlce.Cancer Res 61：711-716，2001
17.Liu C，Huang H，Donate F，Dickinson C，Santucci R，El-Sheikh A，Vessella R，Edgington TS.Prostate-specific membrane antigen directed selective thrombotic infarction of tumors.Can-cer Res 62：5470-5475，2002
18.Gottstein C，Wels W，Ober B，Thorpe PE：Generation and characterisation of recombinantvascular targeting agents from hybridoma cell lines.BioTechniques 30：190-200，2001
19.Morrissey JH，Macik BG，Neuenschwander PF，Comp PC：Quantitation of activated factorVll levels in plasma using a tissue factor mutant selectively deficient in promoting factor Vllactivation.血液(Blood)81：734-744，1993
20.Banner DW，D′Arcy A，Chène C，Winkler FK，Guha A，Konigsberg WH，Nemerson Y，Kirchhofer D：The crystal structure of the complex of blood coagulation factor Vlla with solu-ble tissue factor.自然(Nature)380：41-46，1996
21.Koivunen E，Gay DA，Ruoslahti E：Selection of peptides binding to the α5β1 integrin fromphage display library.J Biol Chem 268：20205-20210，1993
22.Healy JM，Murayama O，Maeda T，Yoshino K，Sekiguchi K，Kikuchi M：Peptide ligands forintegrin αvβ3 selected from random phage display libraries.生物化学(Biochemistry)34：3948-3955，1995
23.Pasqualini R，Koivunen E，Kain R，Lahdenranta J，Sakamoto M，Stryhn A，Ashmun RA，Shapiro LH，Arap W，Ruoslahti E：Aminopeptidase N is a receptor for tumor-homing pep-tides and a target for inhibiting angiogenesis.Cancer Res 60：722-727，2000
24.Curnis F，Sacchi A，Borgna L，Magni F，Gasparri A，Corti A：Enhancement of tumor necro-sis factor αantitumor immunotherapeutic properties by targeted delivery to aminopeptidaseN(CD 13).Nature Biotechnology 18：1185-1190，2000
25.Ellerby HM，Arap W，Ellerby LM，Kain R，Andrusiak R，Del Rio G，Krajewski S，LombardoCR，Rao R，Ruoslahti E，Bredesen DE，Pasqualini R：Anti-cancer activity of targeted pro-apoptotic peptides.Nature Med 5：1032-1038，1999
26.Ruoslahti E：Targeting tumor vasculature with homing peptides from phage display.CancerBiol 10：435-442，2000
27.Pasqualini R，Koivunen E，Kain R，Lahdenranta J，Sakamoto M，Stryhn A，Ashmun RA，Shapiro LH，Arap W，Ruohslahti E.Aminopeptidase N is a receptor for tumor-homing pep-tides and a traget for inhibiting angiogenesis.Cancer Res 60：722-727，2000
28.Curnis F，Arrigoni G，Sacchi A，Fischetti L，Arap W，Pasqualini R，Corti A.Differential bind-ing of drugs containing the NGR motif to CD13 isoforms in tumor vessels，epithelia，andmyeloid cells.Cancer Res 62：867-874，2002
29.Senger DR，Claffey KP，Benes JE，Perruzzi CA，Sergiou AP，Detmar M：Angiogenesis pro-moted by vascular endothelial growth factor：regulation through □1□1 and □2□1 integrins.Proc Natl Acad Sci USA 94：13612-13617，1997
30.Yun Z，Menter DG，Nicolson GL：Involvement of integrin αvβ3 in cell adhesion，motility andliver metastasis of murine RAW117 large cell lymphoma.Cancer Res 56：3103-3111，1996
31.Brooks PC，Clark RAF，Cheresh DA：Requirement of vascular integrin αvβ3 for angiogene-sis.Science 264；569-571，1994
32.Brooks PC，Montgomery AM，Rosenfeld M，Reisfeld RA，Hu T，Klier G，Cheresh DA：Inte-grin αvβ3 antagonists promote tumor regression by inducing apoptosis of angiogenic bloodvessels.Cell 92：391-400，1998
33.Topp MS，Koenigsmann M，Mire-Sluis A，Oberberg D，Eitelbach F，von Marschall Z，NotterM，Reufi B，Stein H，Thiel E，Berdel WE：Recombinant human interleukin-4 inhibits growth ofsome human lung tumor cell lines in vitro and in vivo.Blood 82：2837-2844，1993
34.Topp MS，Papadimitriou CA，Eitelbach F，Koenigsmann M，Oelmann E，Koehler B，Ober-berg D，Reufi B，Stein H，Thiel E，Berdel WE：Recombinant human interleukin 4 has antipro-liferative activity on human tumor cell lines derived from epithelial and non-epithelial histolo-gies.Cancer Res 55：2173-2176，1995
35.Schrappe M，Klier FG，Spiro RC，Gladson CL：Correlation of chondroitin sulfate proteogly-can expression on proliferating brain capillary endothelial cells with the malignant phenotypeof astroglial cells.Cancer Res 51：4986-4993，1991
36.Brooks PC：Localization of matrix metalloproteinase MMP-2 to the surface of invasive cellsby interaction with integrin αvβ3.细胞(Cell)85：683-693，1996
37.Brooks PC，Silletti S，von Schalscha TL，Friedlander M，Cheresh DA：Disruption of Angio-genesis by PEX，a noncatalytic metalloproteinase fragment with integrin binding activity.Cell 92：391-400，1g98
38.Schnurch H，Risau W：Expression of tie2，a member of a novel family of receptor tyrosinekinase in the endothelial cell lineage.Development 119：957-968，1993
39.Peters KG，Coogan A，Berry D，Marks J，lglehart JD，Kontos CD，Rao P，Sankar S，TroganE：Expression of tie2/tek in breast tumor vasculature provides a new marker for evaluationof tumor angiogenesis.Br J Cancer 77：51-56，1998
40.Suri C，Jones PF，Patan S，Bartunkova S，Maisonpierre PC，Davis S，Sato TN YancopoulosGD：requisite role of angiopoietin-1，a ligand for the tie2 receptor，during embryonic angio-genesis.Cell 87：1171-1180，1996
41.Maisonpierre PC，Suri C，Jones PF，Bartunkova S，Wiegand SJ，Radziejewski C，ComptonD，McClain J，Aldrich TH，Papadopoulos N，Daly TH，Davis S，Sato TN，Yancopoulos GD：Angiopoietin-2，a natural antagonist for tie2 that disrupts in vivo angiogenesis，Science 277：55-60，1997
42.Scholz CC，Berger DP，Winterhalter BR，HenβH，Fiebig HH：Correlation of drug response inpatients and in the clonogenic assay with solid human tumour xenografts.Eur J Cancer 26：901-905，1990
43.Fiebig HH，Berger DP，Dengler WA，Wallbrecher E，Winterhalter BR：Combined in vitro/invivo test procedure with human tumor xenografts for new drug development.Contrib.On-col.Basel，Karger 42：321-351，1992.
44.Fiebig HH，Burger AM：Human tumor xenografts and explants.Tumor Models in CancerResearch，eds B.A.Teiche r，Humana Press Inc.，Totowa，NJ，2002.
45.Ruf W，Rehemtulla A，Edglngton TS：Phospholipid-independent and-dependent Interac-tions required for tissue factor receptor and cofactor function.生物化学杂志(J Biol Chem)266：2158-2166，1991.
46.Hu P，Yan J，Sharifi J，Bai T，Khawla LA，Epstein AL：Comparison of three different targetedtissue factor fusion proteins for inducing tumor vessel thrombosis.Cancer Research 63：5046-5053.
序列表
<110>Medizinische Klinik und Poliklinik A des Universittsklinikums Münster
<120>用于抗血管肿瘤治疗的融合多肽
<130>P 51875
<160>31
<170>PatentIn version 3.1
<210>1
<211>263
<212>PRT
<213>Homo sapiens
<220>
<221>人TF的氨基酸序列
<400>1
Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser
1 5 10 15
Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln
20 25 30
Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys
35 40 45
Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val
50 55 60
Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala
65 70 75 80
Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn
85 90 95
Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr
100 105 110
Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu
115 120 125
Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg
130 135 140
Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser
145 150 155 160
Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu
165 170 175
Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val
180 185 190
Ile Pro Ser Arg Thr yal Asn Arg Lys Ser Thr Asp Ser Pro Val Glu
195 200 205
Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu Ile Phe Tyr Ile Ile
210 215 220
Gly Ala Val Val Phe Val Val Ile Ile Leu Val Ile Ile Leu Ala Ile
225 230 235 240
Ser Leu His Lys Cys Arg Lys Ala Gly Val Gly Gln Ser Trp Lys Glu
245 250 255
Asn Ser Pro Leu Asn Val Ser
260
<210>2
<211>2
<212>PRT
<213>Homo sapiens
<220>
<221>tTF1-218的氨基酸序列
<400>2
Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser
1 5 10 15
Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln
20 25 30
Va1 Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys
35 40 45
Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val
50 55 60
Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala
65 70 75 80
Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn
85 90 95
Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr
100 105 110
Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu
115 120 125
Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg
130 135 140
Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser
145 150 155 160
Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu
165 170 175
Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val
180 185 190
Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu
195 200 205
Cys Met Gly Gln Glu Lys Gly Glu Phe Arg
210 215
<210>3
<211>224
<212>PRT
<213>人工
<220>
<221>tTF-GRGDSP的氨基酸序列
<400>3
Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser
1 5 10 15
Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln
20 25 30
Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys
35 40 45
Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val
50 55 60
Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala
65 70 75 80
Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn
85 90 95
Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr
100 105 110
Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu
115 120 125
Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg
130 135 140
Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser
145 150 155 160
Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu
165 170 175
Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val
180 185 190
Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu
195 200 205
Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Gly Arg Gly Asp Ser Asp
210 215 220
<210>4
<211>225
<212>PRT
<213>人工
<220>
<221>tTF-GNGRAHA的氨基酸序列
<400>4
Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser
1 5 10 15
Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln
20 25 30
Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys
35 40 45
Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val
50 55 60
Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala
65 70 75 80
Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn
85 90 95
Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr
100 105 110
Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu
115 120 125
Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg
130 135 140
Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser
145 150 155 160
Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu
165 170 175
Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val
180 185 190
Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu
195 200 205
Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Gly Asn Gly Arg Ala His
210 215 220
Ala
225
<210>5
<211>228
<212>PRT
<213>人工
<220>
<221>tTF-GALNGRSHAG的氨基酸序列
<400>5
Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser
1 5 10 15
Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln
20 25 30
Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys
35 40 45
Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val
50 55 60
Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala
65 70 75 80
Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn
85 90 95
Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr
100 105 110
Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu
115 120 125
Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg
130 135 140
Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser
145 150 155 160
Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu
165 170 175
Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val
180 185 190
Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu
195 200 205
Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Gly Ala Leu Asn Gly Arg
210 215 220
Ser His Ala Gly
225
<210>6
<211>225
<212>PRT
<213>人工
<220>
<221>tTF-GCNGRCG的氨基酸序列
<400>6
Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser
1 5 10 15
Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln
20 25 30
Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys
35 40 45
Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val
50 55 60
Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala
65 70 75 80
Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn
85 90 95
Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr
100 105 110
Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu
115 120 125
Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg
130 135 140
Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser
145 150 155 160
Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu
165 170 175
Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val
180 185 190
Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu
195 200 205
Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Gly Cys Asn Gly Arg Cys
210 215 220
Gly
225
<210>7
<211>232
<212>PRT
<213>人工
<220>
<221>tTF-GCNGRCVSGCAGRC的氨基酸序列
<400>7
Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser
1 5 10 15
Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln
20 25 30
Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys
35 40 45
Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val
50 55 60
Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala
65 70 75 80
Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn
85 90 95
Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr
100 105 110
Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu
115 120 125
Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg
130 135 140
Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser
145 150 155 160
Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu
165 170 175
Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val
180 185 190
Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu
195 200 205
Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Gly Cys Asn Gly Arg Cys
210 215 220
Val Ser Gly Cys Ala Gly Arg Cys
225 230
<210>8
<211>228
<212>PRT
<213>人工
<220>
<221>tTF-GCVLNGRMEC的氨基酸序列
<400>8
Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser
1 5 10 15
Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln
20 25 30
Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys
35 40 45
Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val
50 55 60
Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala
65 70 75 80
Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn
85 90 95
Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr
100 105 110
Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu
115 120 125
Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg
130 135 140
Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser
145 150 155 160
Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu
165 170 175
Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val
180 185 190
Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu
195 200 205
Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Gly Cys Val Leu Asn Gly
210 215 220
Arg Met Glu Cys
225
<210>9
<211>654
<212>DNA
<213>人工
<220>
<221>tTF1-218的核苷酸序列
<400>9
tcaggcacta caaatactgt ggcagcatat aatttaactt ggaaatcaac taatttcaag 60
acaattttgg agtgggaacc caaacccgtc aatcaagtct acactgttca aataagcact 120
aagtcaggag attggaaaag caaatgcttt tacacaacag acacagagtg tgacctcacc 180
gacgagattg tgaaggatgt gaagcagacg tacttggcac gggtcttctc ctacccggca 240
gggaatgtgg agagcaccgg ttctgctggg gagcctctgt atgagaactc cccagagttc 300
acaccttacc tggagacaaa cctcggacag ccaacaattc agagttttga acaggtggga 360
acaaaagtga atgtgaccgt agaagatgaa cggactttag tcagaaggaa caacactttc 420
ctaagcctcc gggatgtttt tggcaaggac ttaatttata cactttatta ttggaaatct 480
tcaagttcag gaaagaaaac agccaaaaca aacactaatg agtttttgat tgatgtggat 540
aaaggagaaa actactgttt cagtgttcaa gcagtgattc cctcccgaac agttaaccgg 600
aagagtacag acagcccggt agagtgtatg ggccaggaga aaggggaatt caga 654
<210>10
<211>672
<212>DNA
<213>人工
<220>
<221>tTF-GRGDSP的核苷酸序列
<400>10
tcaggcacta caaatactgt ggcagcatat aatttaactt ggaaatcaac taatttcaag 60
acaattttgg agtgggaacc caaacccgtc aatcaagtct acactgttca aataagcact 120
aagtcaggag attggaaaag caaatgcttt tacacaacag acacagagtg tgacctcacc 180
gacgagattg tgaaggatgt gaagcagacg tacttggcac gggtottctc ctacccggca 240
gggaatgtgg agagcaccgg ttctgctggg gagcctctgt atgagaactc cccagagttc 300
acaccttacc tggagacaaa cctcggacag ccaacaattc agagttttga acaggtggga 360
acaaaagtga atgtgaccgt agaagatgaa cggactttag tcagaaggaa caacactttc 420
ctaagcctcc gggatgtttt tggcaaggac ttaatttata cactttatta ttggaaatct 480
tcaagttcag gaaagaaaac agccaaaaca aacactaatg agtttttgat tgatgtggat 540
aaaggagaaa actactgttt cagtgttcaa gcagtgattc cctcccgaac agttaaccgg 600
aagagtacag acagcccggt agagtgtatg ggccaggaga aaggggaatt cagaggaaga 660
ggtgattctc ca 672
<210>11
<211>675
<212>DNA
<213>人工
<220>
<221>tTF-GNGRAHA的核苷酸序列
<400>11
tcaggcacta caaatactgt ggcagcatat aatttaactt ggaaatcaac taatttcaag 60
acaattttgg agtgggaacc caaacccgtc aatcaagtct acactgttca aataagcact 120
aagtcaggag attggaaaag caaatgcttt tacacaacag acacagagtg tgacctcacc 180
gacgagattg tgaaggatgt gaagcagacg tacttggcac gggtcttctc ctacccggca 240
gggaatgtgg agagcaccgg ttctgctggg gagcctctgt atgagaactc cccagagttc 300
acaccttacc tggagacaaa cctcggacag ccaacaattc agagttttga acaggtggga 360
acaaaagtga atgtgaccgt agaagatgaa cggactttag tcagaaggaa caacactttc 420
ctaagcctcc gggatgtttt tggcaaggac ttaatttata cactttatta ttggaaatct 480
tcaagttcag gaaagaaaac agccaaaaca aacactaatg agtttttgat tgatgtggat 540
aaaggagaaa actactgttt cagtgttcaa gcagtgattc cctcccgaac agttaaccgg 600
aagagtacag acagcccggt agagtgtatg ggccaggaga aaggggaatt cagaggtaac 660
ggaagagcac atgca 675
<210>12
<211>684
<212>DNA
<213>人工
<220>
<221>tTF-GALNGRSHAG的核苷酸序列
<400>12
tcaggcacta caaatactgt ggcagcatat aatttaactt ggaaatcaac taatttcaag 60
acaattttgg agtgggaacc caaacccgtc aatcaagtct acactgttca aataagcact 120
aagtcaggag attggaaaag caaatgcttt tacacaacag acacagagtg tgacctcacc 180
gacgagattg tgaaggatgt gaagcagacg tacttggcac gggtcttctc ctacccggca 240
gggaatgtgg agagcaccgg ttctgctggg gagcctctgt atgagaactc cccagagttc 300
acaccttacc tggagacaaa cctcggacag ccaacaattc agagttttga acaggtggga 360
acaaaagtga atgtgaccgt agaagatgaa cggactttag tcagaaggaa caacactttc 420
ctaagcctcc gggatgtttt tggcaaggac ttaatttata cactttatta ttggaaatct 480
tcaagttcag gaaagaaaac agccaaaaca aacactaatg agtttttgat tgatgtggat 540
aaaggagaaa actactgttt cagtgttcaa gcagtgattc cctcccgaac agttaaccgg 600
aagagtacag acagcccggt agagtgtatg ggccaggaga aaggggaatt cagaggtgct 660
ttaaatggaa gatctcacgc tggt 684
<210>13
<211>675
<212>DNA
<213>人工
<220>
<221>tTF-GCNGRCG的核苷酸序列
<400>13
tcaggcacta caaatactgt ggcagcatat aatttaactt ggaaatcaac taatttcaag 60
acaattttgg agtgggaacc caaacccgtc aatcaagtct acactgttca aataagcact 120
aagtcaggag attggaaaag caaatgcttt tacacaacag acacagagtg tgacctcacc 180
gacgagattg tgaaggatgt gaagcagacg tacttggcac gggtcttctc ctacccggca 240
gggaatgtgg agagcaccgg ttctgctggg gagcctctgt atgagaactc cccagagttc 300
acaccttacc tggagacaaa cctcggacag ccaacaattc agagttttga acaggtggga 360
acaaaagtga atgtgaccgt agaagatgaa cggactttag tcagaaggaa caacactttc 420
ctaagcctcc gggatgtttt tggcaaggac ttaatttata cactttatta ttggaaatct 480
tcaagttcag gaaagaaaac agccaaaaca aacactaatg agtttttgat tgatgtggat 540
aaaggagaaa actactgttt cagtgttcaa gcagtgattc cctcccgaac agttaaccgg 600
aagagtacag acagcccggt agagtgtatg ggccaggaga aaggggaatt cagaggctgc 660
aacggtagat gtggt 675
<210>14
<211>696
<212>DNA
<213>人工
<220>
<221>tTF-GCNGRCVSGCAGRC的核苷酸序列
<400>14
tcaggcacta caaatactgt ggcagcatat aatttaactt ggaaatcaac taatttcaag 60
acaattttgg agtgggaacc caaacccgtc aatcaagtct acactgttca aataagcact 120
aagtcaggag attggaaaag caaatgcttt tacacaacag acacagagtg tgacctcacc 180
gacgagattg tgaaggatgt gaagcagacg tacttggcac gggtcttctc ctacccggca 240
gggaatgtgg agagcaccgg ttctgctggg gagcctctgt atgagaactc cccagagttc 300
acaccttacc tggagacaaa cctcggacag ccaacaattc agagttttga acaggtggga 360
acaaaagtga atgtgaccgt agaagatgaa cggactttag tcagaaggaa caacactttc 420
ctaagcctcc gggatgtttt tggcaaggac ttaatttata cRctttatta ttggaaatct 480
tcaagttcag gaaagaaaac agccaaaaca aacactaatg agtttttgat tgatgtggat 540
aaaggagaaa actactgttt cagtgttcaa gcagtgattc cctcccgaac agttaaccgg 600
aagagtacag acagcccggt agagtgtatg ggccaggaga aaggggaatt cagaggttgt 660
aatggaagat gtgtttctgg atgtgcagga cgatgt 696
<210>15
<211>684
<212>DNA
<213>人工
<220>
<221>tTF-GCVLNGRMEC的核苷酸序列
<400>15
tcaggcacta caaatactgt ggcagcatat aatttaactt ggaaatcaac taatttcaag 60
acaattttgg agtgggaacc caaacccgtc aatcaagtct acactgttca aataagcact 120
aagtcaggag attggaaaag caaatgcttt tacacaacag acacagagtg tgacctcacc 180
gacgagattg tgaaggatgt gaagcagacg tacttggcac gggtcttctc ctacccggca 240
gggaatgtgg agagcaccgg ttctgctggg gagcctctgt atgagaactc cccagagttc 300
acaccttacc tggagacaaa cctcggacag ccaacaattc agagttttga acaggtggga 360
acaaaagtga atgtgaccgt agaagatgaa cggactttag tcagaaggaa caacactttc 420
ctaagcctcc gggatgtttt tggcaaggac ttaatttata cactttatta ttggaaatct 480
tcaagttcag gaaagaaaac agccaaaaca aacactaatg agtttttgat tgatgtggat 540
aaaggagaaa actactgttt cagtgttcaa gcagtgattc cctcccgaac agttaaccgg 600
aagagtacag acagcccggt agagtgtatg ggccaggaga aaggggaatt cagaggatgc 660
gtcttaaatg gtaggatgga atgc 684
<210>16
<211>45
<212>DNA
<213>人工
<220>
<221>用于制备tTF1-218的5′寡核苷酸引物
<400>16
catgccatgg gatcaggcac tacaaatact gtggcagcat ataat 45
<210>17
<211>40
<212>DNA
<213>人工
<220>
<221>用于制备tTF1-218的3′寡核苷酸引物
<400>17
cgggatccta ttatctgaat tcccctttct cctggcccat 40
<210>18
<211>45
<212>DNA
<213>人工
<220>
<221>用于制备tTF-GRGDSP的5′寡核苷酸引物
<400>18
catgccatgg gatcaggcac tacaaatact gtggcagcat ataat 45
<210>19
<211>43
<212>DNA
<213>人工
<220>
<221>用于制备tTF-GRGDSP的3′寡核苷酸引物
<400>19
cgggatccta ttatggagaa tcacotcttc ctctgaattc coc 43
<210>20
<211>45
<212>DNA
<213>人工
<220>
<221>用于制备tTF-GNGRAHA的5′寡核苷酸引物
<400>20
catgccatgg gatcaggcac tacaaatact gtggcagcat ataat 45
<210>21
<211>46
<212>DNA
<213>人工
<220>
<221>用于制备tTF-GNGRAHA的3′寡核苷酸引物
<400>21
cgggatccta ttatgcatgt gctcttccgt tacctctgaa ttcccc 46
<210>22
<211>45
<212>DNA
<213>人工
<220>
<221>用于制备tTF-GCNGRCG的5′寡核苷酸引物
<400>22
catgccatgg gatcaggcac tacaaatact gtggcagcat ataat 45
<210>23
<211>46
<212>DNA
<213>人工
<220>
<221>用于制备tTF-GCNGRCG的3′寡核苷酸引物
<400>23
cgggatccta ttaaccacat ctaccgttgc agcctctgaa ttcccc 46
<210>24
<211>45
<212>DNA
<213>人工
<220>
<221>用于制备tTF-GCNGRCVSGCAGRC的5′寡核苷酸引物
<400>24
catgccatgg gatcaggcac tacaaatact gtggcagcat ataat 45
<210>25
<211>67
<212>DNA
<213>人工
<220>
<221>用于制备tTF-GCNGRCVSGCAGRC的3′寡核苷酸引物
<400>25
cgggatccta ttaacatcgt cctgcacatc cagaaacaca tcttccatta caacctctga 60
attcccc 67
<210>26
<211>45
<212>DNA
<213>人工
<220>
<221>用于制备tTF-GCVLNGRMEC的5′寡核苷酸引物
<400>26
catgccatgg gatcaggcac tacaaatact gtggcagcat ataat 45
<210>27
<211>55
<212>DNA
<213>人工
<220>
<221>用于制备tTF-GCVLNGRMEC的3′寡核苷酸引物
<400>27
cgggatccta ttagcattcc atcctaccat ttaagacgca tcctctgaat tcccc 55
<210>28
<211>45
<212>DNA
<213>人工
<220>
<221>用于制备tTF-GALNGRSHAG的5′寡核苷酸引物
<400>28
catgccatgg gatcaggcac tacaaatact gtggcagcat ataat 45
<210>29
<211>55
<212>DNA
<213>人工
<220>
<221>用于制备tTF-GALNGRSHAG的3′寡核苷酸引物
<400>29
cgggatccta ttaaccagcg tgagatcttc catttaaagc acctctgaat tcccc 55
<210>30
<211>45
<212>PRT
<213>人工
<220>
<221>亲和标记的氨基酸序列
<400>30
His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser Gly
1 5 10 15
Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser
20 25 30
Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Gly
35 40 45
<210>31
<211>269
<212>PRT
<213>人工
<220>
<221>具有N-末端亲和标记的tTF-GRGDSP的氨基酸序列
<400>31
His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser Gly
1 5 10 15
Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser
20 25 30
Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Gly Ser Gly Thr
35 40 45
Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn Phe
50 55 60
Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr Thr
65 70 75 80
Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe Tyr
85 90 95
Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys Asp Val
100 105 110
Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly Asn Val
115 120 125
Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser Pro Glu
130 135 140
Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln Ser
145 150 155 160
Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu Arg
165 170 175
Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Val Phe
180 185 190
Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser Ser
195 200 205
Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu Ile Asp Val
210 215 220
Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val Ile Pro Ser
225 230 235 240
Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys Met Gly
245 250 255
Gln Glu Lys Gly Glu Phe Arg Gly Arg Gly Asp Ser Asp
260 265
<210>32
<211>270
<212>PRT
<213>人工
<220>
<221>具有N-末端亲和标记的tTF-GNGRAHA的氨基酸序列
<400>32
His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser Gly
1 5 10 15
Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser
20 25 30
Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Gly Ser Gly Thr
35 40 45
Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn Phe
50 55 60
Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr Thr
65 70 75 80
Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe Tyr
85 90 95
Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys Asp Val
100 105 110
Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly Asn Val
115 120 125
Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser Pro Glu
130 135 140
Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln Ser
145 150 155 160
Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu Arg
165 170 175
Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Val Phe
180 185 190
Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser Ser
195 200 205
Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu Ile Asp Val
210 215 220
Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val Ile Pro Ser
225 230 235 240
Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys Met Gly
245 250 255
Gln Glu Lys Gly Glu Phe Arg Gly Asn Gly Arg Ala His Ala
260 265 270
Claims (18)
1.融合多肽，包括
a)能够使该融合多肽选择性结合肿瘤血管内皮细胞的3至30个氨基酸的肽，和
b)组织因子(TF)或其片段，该组织因子和片段的特征在于当该融合多肽结合肿瘤血管内皮细胞时，它们能够活化血液凝固，
其中肽a)和b)直接相互偶联或通过具有至多15个氨基酸的接头相互偶联，其特征在于能够使该融合多肽选择性结合肿瘤血管内皮细胞的肽与当该融合多肽结合肿瘤血管内皮细胞时能够活化血液凝固的肽之C末端偶联。
2.权利要求1所述的融合多肽，由肽a)和b)以及具有至多15个氨基酸的接头组成。
3.权利要求1所述的融合多肽，其中肽a)和b)直接相互偶联。
4.权利要求1至3之一项所述的融合多肽，其特征在于当该融合多肽结合肿瘤血管内皮细胞时能够活化血液凝固的肽是组织因子TF，其具有如SEQ ID NO：1所示的序列。
5.权利要求1至3之一项所述的融合多肽，其特征在于当该融合多肽结合肿瘤血管内皮细胞时能够活化血液凝固的肽是组织因子TF的片段，其优选具有如SEQ ID NO：2所示的序列。
6.权利要求1至5之一项所述的融合多肽，其特征在于能够使该融合多肽选择性结合肿瘤血管内皮细胞的3至30个氨基酸的肽具有线性或环状的结构。
7.权利要求1至6之一项所述的融合多肽，其特征在于能够使该融合多肽选择性结合肿瘤血管内皮细胞的3至30个氨基酸的肽包括氨基酸序列RGD或NGR。
8.权利要求7所述的融合多肽，其特征在于能够使该融合多肽选择性结合肿瘤血管内皮细胞的肽选自GRGDSP和GNGRAHA。
9.权利要求7所述的融合多肽，其特征在于能够使该融合多肽选择性结合肿瘤血管内皮细胞的肽选自GCNGRCG、GCNGRCVSGCAGRC、GCVLNGRMEC和GALNGRSHAG。
10.权利要求1至9所述的融合多肽，其特征在于它具有SEQ IDNO：3-8所示序列之一。
11.编码权利要求1至10之一项所述融合多肽的核酸。
12.权利要求11所述的核酸，其特征在于它具有SEQ ID NO：10-15所示序列之一。
13.包括权利要求11或12所述核酸的载体。
14.包括权利要求11或12所述核酸或权利要求14所述载体的细胞。
15.包括权利要求1至10之一项所述融合多肽、权利要求11或12所述核酸、权利要求13所述载体或权利要求14所述细胞的药物组合物。
16.权利要求15所述的药物组合物，其进一步包括药物学上可接受的载体、赋形剂或佐剂。
17.权利要求15或16所述的药物组合物用于治疗肿瘤疾病的用途。
18.权利要求17所述的用途，其特征在于肿瘤疾病选自支气管肺癌和胸腔及纵隔腔的其他肿瘤、乳腺癌和其他妇科肿瘤、结肠直肠癌、胰腺癌和胃肠道的其他肿瘤、恶性黑色素瘤和皮肤的其他肿瘤、头颈区中的肿瘤、前列腺癌和其他泌尿生殖器肿瘤、肉瘤、内分泌活化的肿瘤、白血病和脊髓发育不良综合症和霍奇金淋巴瘤以及非霍奇金淋巴瘤。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10338733.1 | 2003-08-22 | ||
| DE10338733A DE10338733A1 (de) | 2003-08-22 | 2003-08-22 | Fusionspolypeptide und deren Verwendung für die antivaskuläre Tumortherapie |
| PCT/EP2004/009364 WO2005021593A1 (de) | 2003-08-22 | 2004-08-20 | Fusionspolypeptide und deren verwendung für die antivaskuläre tumortherapie |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1871258A true CN1871258A (zh) | 2006-11-29 |
| CN1871258B CN1871258B (zh) | 2012-05-30 |
Family
ID=34201891
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2004800313848A Expired - Lifetime CN1871258B (zh) | 2003-08-22 | 2004-08-20 | 融合多肽及其在抗血管肿瘤治疗中的用途 |
Country Status (14)
| Country | Link |
|---|---|
| US (1) | US7618943B2 (zh) |
| EP (1) | EP1660532B1 (zh) |
| JP (1) | JP5149506B2 (zh) |
| CN (1) | CN1871258B (zh) |
| AU (1) | AU2004268031B2 (zh) |
| CA (1) | CA2535856C (zh) |
| DE (1) | DE10338733A1 (zh) |
| DK (1) | DK1660532T3 (zh) |
| ES (1) | ES2397922T3 (zh) |
| HK (1) | HK1087418A1 (zh) |
| PL (1) | PL1660532T3 (zh) |
| PT (1) | PT1660532E (zh) |
| SI (1) | SI1660532T1 (zh) |
| WO (1) | WO2005021593A1 (zh) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102153653A (zh) * | 2010-12-30 | 2011-08-17 | 厦门大学 | 肿瘤血管靶向多肽与组织因子的融合蛋白及其制备方法 |
| CN104387474A (zh) * | 2014-11-17 | 2015-03-04 | 国家纳米科学中心 | 一种肿瘤血管梗塞剂多肽、基因、表达载体及其应用 |
| CN116600809A (zh) * | 2020-08-26 | 2023-08-15 | 安图瑞克制药有限公司 | 包含用于治疗软组织肉瘤的tTF-NGR的组合物 |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090098529A1 (en) * | 2006-10-16 | 2009-04-16 | Nanhai Chen | Methods for attenuating virus strains for diagnostic and therapeutic uses |
| KR100879239B1 (ko) * | 2007-04-19 | 2009-01-16 | (주)케어젠 | Ｔｇｆｐ－ｃａｐ 펩타이드 및 그의 용도 |
| SG11201501145SA (en) | 2012-08-13 | 2015-04-29 | Otago Innovation Ltd | NT-proCNP AS A BIOMARKER OF VASCULAR DISORDERS AND PREGNANCY COMPLICATION |
| WO2024102818A1 (en) * | 2022-11-09 | 2024-05-16 | Wisconsin Alumni Research Foundation | Combination tumor therapy with thrombosis initiation and platelet recruitment |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6004555A (en) * | 1992-03-05 | 1999-12-21 | Board Of Regents, The University Of Texas System | Methods for the specific coagulation of vasculature |
| US6093399A (en) * | 1992-03-05 | 2000-07-25 | Board Of Regents, The University Of Texas System | Methods and compositions for the specific coagulation of vasculature |
| US5858724A (en) * | 1996-07-16 | 1999-01-12 | Pel-Freez | Recombinant rabbit tissue factor |
| PT988056E (pt) * | 1997-01-22 | 2003-09-30 | Univ Texas | Metodos e composicoes de factor tecidual para o tratamento da coagulacao e de tumores |
| ATE230614T1 (de) * | 1998-07-13 | 2003-01-15 | Univ Texas | Krebsbehandlung mit aminophospholipide bindenden, therapeutischen konjugaten |
| US7622437B2 (en) * | 2000-11-20 | 2009-11-24 | The Board Of Trustees Of The University Of Illinois | Tissue factor compositions and methods |
| US7399743B2 (en) * | 2001-10-26 | 2008-07-15 | The Scripps Research Institute | Targeted thrombosis |
| US7425328B2 (en) * | 2003-04-22 | 2008-09-16 | Purdue Pharma L.P. | Tissue factor antibodies and uses thereof |
-
2003
- 2003-08-22 DE DE10338733A patent/DE10338733A1/de not_active Ceased
-
2004
- 2004-08-20 DK DK04764346.5T patent/DK1660532T3/da active
- 2004-08-20 PT PT47643465T patent/PT1660532E/pt unknown
- 2004-08-20 CA CA2535856A patent/CA2535856C/en not_active Expired - Lifetime
- 2004-08-20 ES ES04764346T patent/ES2397922T3/es not_active Expired - Lifetime
- 2004-08-20 SI SI200431981T patent/SI1660532T1/sl unknown
- 2004-08-20 JP JP2006523616A patent/JP5149506B2/ja not_active Expired - Fee Related
- 2004-08-20 AU AU2004268031A patent/AU2004268031B2/en not_active Expired
- 2004-08-20 CN CN2004800313848A patent/CN1871258B/zh not_active Expired - Lifetime
- 2004-08-20 EP EP04764346A patent/EP1660532B1/de not_active Expired - Lifetime
- 2004-08-20 PL PL04764346T patent/PL1660532T3/pl unknown
- 2004-08-20 WO PCT/EP2004/009364 patent/WO2005021593A1/de active Application Filing
- 2004-08-20 US US10/569,076 patent/US7618943B2/en active Active
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102153653A (zh) * | 2010-12-30 | 2011-08-17 | 厦门大学 | 肿瘤血管靶向多肽与组织因子的融合蛋白及其制备方法 |
| CN102153653B (zh) * | 2010-12-30 | 2012-08-15 | 厦门大学 | 肿瘤血管靶向多肽与组织因子的融合蛋白及其制备方法 |
| CN104387474A (zh) * | 2014-11-17 | 2015-03-04 | 国家纳米科学中心 | 一种肿瘤血管梗塞剂多肽、基因、表达载体及其应用 |
| CN116600809A (zh) * | 2020-08-26 | 2023-08-15 | 安图瑞克制药有限公司 | 包含用于治疗软组织肉瘤的tTF-NGR的组合物 |
Also Published As
| Publication number | Publication date |
|---|---|
| PL1660532T3 (pl) | 2013-03-29 |
| ES2397922T3 (es) | 2013-03-12 |
| PT1660532E (pt) | 2013-01-29 |
| CN1871258B (zh) | 2012-05-30 |
| AU2004268031B2 (en) | 2009-09-03 |
| JP2007528722A (ja) | 2007-10-18 |
| US20070032419A1 (en) | 2007-02-08 |
| DK1660532T3 (da) | 2013-01-21 |
| JP5149506B2 (ja) | 2013-02-20 |
| CA2535856C (en) | 2016-06-07 |
| AU2004268031A1 (en) | 2005-03-10 |
| EP1660532B1 (de) | 2012-10-24 |
| EP1660532A1 (de) | 2006-05-31 |
| DE10338733A1 (de) | 2005-03-24 |
| CA2535856A1 (en) | 2005-03-10 |
| WO2005021593A1 (de) | 2005-03-10 |
| SI1660532T1 (sl) | 2013-02-28 |
| US7618943B2 (en) | 2009-11-17 |
| HK1087418A1 (en) | 2006-10-13 |
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