CN101405017A - 具有磷脂酰丝氨酸结合结构域的嵌合蛋白 - Google Patents
具有磷脂酰丝氨酸结合结构域的嵌合蛋白 Download PDFInfo
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Abstract
描述了含可溶性组织因子(sTF)和另一亚基(例如膜联蛋白V)的嵌合蛋白。该蛋白通过将sTF靶向至特定受体,例如激活细胞的磷脂酰丝氨酸(PS),而促进血液凝结和/或抑制癌症。这些嵌合蛋白用于治疗因先天因素、药物治疗、外伤或手术所引起的过量出血患者,和/或用作抗癌治疗剂,例如通过使供应癌症的血管凝结,而阻止充足血液流进肿瘤,由此导致肿瘤抑制和死亡,或者可用于治疗使血管内发生凝结,其中所述血管在非癌状态下对受试者产生威胁。
Description
关于联邦赞助的研究或开发的声明。
[0001]不适用。
发明背景
[0002]在多种情况下,止血(引发血液凝结以停止出血的过程)可能会异常而可能危及生命,然而仍需要在特定条件下诱导止血的改良方法。止血由血小板引发,血小板是引发血管创口阻塞的血细胞。在此过程中,血小板被“激活”。其激活的一方面表现在,将磷脂酰丝氨酸(PS)从胞质膜内侧转移至外侧。一旦该过程发生,某些血液凝结蛋白就结合到血小板表面,并在该过程中相互作用,该过程称为“血液凝结”。该过程的最终结果是形成凝血酶(也称为IIa因子)。凝血酶有多种重要作用。首先,它将血蛋白——纤维蛋白原转化为纤维蛋白。每个纤维蛋白结合多个其它纤维蛋白分子,形成纤维蛋白凝块,所述凝块支撑血小板,用于阻止出血。其次,凝血酶也激活血小板,增加参与止血过程的血小板数量。再次,凝血酶以加速血液凝结生化过程的方式影响其它血浆蛋白,例如XI因子。在血小板(或其它PS外露的细胞)表面相互作用的第一个蛋白是组织因子(TF),激活形式的VII因子(称为VIIa因子),和X因子。
[0003]凝血酶的产生,是由血浆丝氨酸蛋白酶VIIa因子与其蛋白辅因子TF相互作用而引发。TF是膜结合蛋白,其在与血流接触的细胞表面并不表达,直至它们被激活。在表达后,TF结合VII因子(促使其激活为VIIa因子)或VIIa因子,提高其将X因子转化为Xa因子的催化效率。TF胞外结构域(全蛋白1-219或3-219氨基酸残基部分)在大肠杆菌中的表达,产生保留结合VIIa因子并变构激活该因子能力的约26kDa多肽。该截短TF(称为可溶性TF或sTF)不结合细胞膜,因此通常在促进VII因子自激活或VIIa因子介导的X因子激活中,效率比天然TF低得多。sTF编码cDNA的工程化使其在哺乳动物细胞表面以糖磷脂酰肌醇锚定蛋白形式表达,这导致与天然TF有相同特异性促凝活性的蛋白,这表明该蛋白膜结合相当重要。
[0004]很久已经意识到,先天VIII因子缺乏,在血液凝结由低浓度TF引发的情况下,以凝血酶产生方面的异常为特征。最近认识到,血小板功能紊乱也与凝血酶产量减少相关。维他命K-依赖性血液凝结蛋白,其是凝血酶产生所需,其通过结合阴离子磷脂、特别是PS,聚集于激活血小板、内皮细胞和/或单核细胞表面。凝血酶,强大的血小板激动剂,其可放大血小板激活,所述激活由接触内皮下的胶原暴露物而引发。因此,延迟的凝血酶产生,可能是出血倾向的基础或可放大出血倾向,其中所述出血倾向伴随着血浆凝结因子紊乱或血小板紊乱。
[0005]其它文献报道,给动物静脉注射促凝血酶原激酶(膜结合的TF)产生凝血的普遍激活,该结果与同时注射sTF和VIIa因子相同,在实验动物出血中可产生有益效果,这表明sTF可作为设计的减少出血治疗的基础。
[0006]期望在有需要的个体中有效增强止血和凝血的治疗蛋白。
附图简述
[0007]图1.重组蛋白的蛋白纯化和western印记。上图,SDS-PAGE和考马斯亮蓝染色。中图,小鼠单抗与人TF的western印记。下图,小鼠单抗与His6的western印记。M,分子量标记;第1-5道,在A,B,C电泳图中相同:第1道,sTF-膜联蛋白V(sTF-annV);第2道,sTFAA-膜联蛋白V(sTFAA-annV);第3道,膜联蛋白V;第4道,sTF;第5道,重组TF。第6道,电泳图A:牛血清白蛋白;电泳图B:重组TF(Dade Behring,Newark,DE)。
[0008]图2.重组蛋白对血浆凝结的影响。A,柠檬酸盐人血浆随不同浓度天然TF(■),sTF()或sTF-annV(●)的凝结时间。加至每样品的PC/PS总量为10mM(PC∶PS摩尔比,4∶1)。显示的是平均值±SEM。B,用稀释偏促凝血酶原激酶时间方案(a dilute partial thromboplastin time protocol)测量的sTF(),膜联蛋白V(▲),sTF+膜联蛋白V(◆),或sTF-annV(●)血浆凝结时间,所述实验方案在实验步骤中描述。C,在所加磷脂缺乏情况下,不同浓度sTF(),膜联蛋白V(▲),sTF-annV(●),或sTFAA-annV(o)的血浆凝结时间。显示的是平均值±SEM。
[0009]图3.sTF-annV和sTFAA-annV的磷脂结合。通过检测每蛋白从磷脂悬浮液取代FITC-膜联蛋白V的能力(该实验方法在实验步骤中描述),将sTF-annV(●)和sTFAA-annV(o)结合PC/PS颗粒能力与膜联蛋白V(▲)的能力进行比较。曲线用非线性回归和单位点配体结合方程式(a one site ligand binding equation)计算机模拟。数据以EDTA--洗脱荧光百分数表示(平均值±SEM)。
[0010]图4.天然TF,sTF-annV或sTF存在情况下的VIIa因子自动激活。在PC/PS(810mM,PC∶PS摩尔比=4∶1)和CaCl2存在情况下,将VII因子(80nM)加入至等摩尔浓度的天然TF(■),sTF(),或sTF-annV(●)中。在指定时间,移出等份试样,通过稀释于含EDTA的溶液中而终止反应。在所加sTF和CaCl2存在情况下,通过测量Chromozym t-PA水解率,确定每样品的VIIa因子含量。显示的是平均值±SEM。
[0011]图5.在重组蛋白存在情况下,VIIa因子介导的Xa因子产生。A,在5pM天然TF(■),sTF(),sTF-annV(●),或sTFAA-annV(o)存在情况下,将不同浓度的X因子,加入至1nM VIIa因子,5mM PC/PS(摩尔比,4∶1),5mM CaCl2和Chromozym X底物。按实验步骤所记载方法,测量底物水解初始速率,该水解速率拟合Michaelis-Menten方程。B,在含28nM X因子,5mM CaCl2,1nMVIIa因子,0.347mM PC/PS和不同浓度的sTF()或sTF-annV(●)的溶液中,测量Xa因子的产生的初始速率。
[0012]图6.sTF-annV或VIIa因子对肝素-处理的血浆aPTT的效应。A,含肝素钠(1单位/mL)血浆的aPTT分析,在不同浓度VIIa因子(●)或sTF-annV(■)存在情况下进行。B,含依诺肝素钠(enoxaparin sodium)(1单位/mL)的血浆aPTT,在不同浓度VIIa因子(●)或sTF-annV(■)存在情况下测量。虚线显示在缺乏肝素或依诺肝素情况下,aPTT分析的平均凝结时间。显示的是平均值±SEM(大多数误差框过小而不可见)。
[0013]图7.sTF-annV对尾出血时间的效应。图A:将小鼠(n=13)给予皮下注射依诺肝素钠(20mg/kg),2小时后将其麻醉,按实验步骤中所记载的方法测量尾出血时间。出血一停止,立即给动物静脉注射位于TBS中的sTF-annV(90mcg/kg)。五分钟后,再次测量出血时间。差异有显著性,p=0.01(成对双尾t-test)。图B:按上述方法,测量尾出血时间。给予SQ依诺肝素(或盐水)2小时后,将动物静脉注射sTF-annV(90mcg/kg)或者等摩尔量的sTF,膜联蛋白V或sTF+膜联蛋白V。10min后测量尾出血时间。sTF-annV缩短了未给予依诺肝素小鼠(p>0.2)和给予依诺肝素小鼠(p<0.0001,Welch′s校正、双尾非成对t-test)的出血时间。在未给予依诺肝素组的每组中,研究两只动物,在依诺肝素处理组的每组中,研究四只动物。
发明详述
[0014]本发明构思递送嵌合蛋白至血管损伤部位,其中所述嵌合蛋白包含可溶性组织因子(sTF)或具有足以增强VIIa因子胺降解活性至少10倍的活性的其有效部分,在所述血管损伤部位其用作止血剂,使诱导分散的血管内凝结(disseminated intravascular coagulation,DIC)的机会降至最低。虽然可以使用激活的血小板、内皮细胞或单核细胞的特异性抗体进行靶向,但是优选能结合所有三种细胞类型的靶向部分,至此,因为它对任意给定个体中可能存在的靶细胞数量改变更不敏感。因为每种感兴趣细胞类型在激活时都在表面表达磷脂酰丝氨酸(PS),所以本发明构思用sTF嵌合蛋白(例如,sTF-annV)将sTF靶向至含PS膜上,其中将sTF与PS结合结构域例如膜联蛋白V(AnnV)偶联,膜联蛋白V为人PS结合蛋白,其显示可结合激活的血小板、内皮细胞和单核细胞。对膜联蛋白V以该方式使用的争论在于它是抗凝蛋白,因为它能与维他命K-依赖性蛋白竞争结合含PS的膜。
[0015]在本发明优选的实施方案中,sTF包含与AnnV氨基末端连接的人TF的217个氨基酸(SEQ ID NO:2)(它也可以连接在羧基末端,也可以通过连接子序列连接在氨基末端或羧基末端)。如前所述,TF常规结合于细胞膜,因为它含有膜内锚定区,然而,sTF缺乏锚定区且10年前已克隆表达,其比天然蛋白(TF)活性低约1000倍。sTF例如由包含SEQ ID NO:1的cDNA编码。
[0016]本文构思的可溶性组织因子(sTF)包括SEQ ID NO:2,或其207位有半胱氨酸且保留了足以增强VIIa因子胺降解活性(amidolytic activity)至少10倍的任意子序列(subsequence)。因此,本文所用的构思的sTF包括,包含氨基酸1-207,5-207,10-207,15-207,20-207,25-207,30-207,35-207,40-207,45-207,50-207,55-207,60-207,65-207,70-207,75-207,80-207,85-207,90-207,95-207,100-207,或105-207的sTF截短部分,以及包含在1-207至105-207之间的所有序列,例如包括2-207和104-207。本发明进一步包括编码SEQ ID NO:2或如上其功能活性截短部分的任意备选cDNA,例如编码SEQ ID NO:2的备选cDNA,可包括与SEQ ID NO:1相似但其具有保守取代密码子的cDNA(即,具有不同核苷酸序列,但编码相同氨基酸的密码)。
[0017]本发明进一步包括如上所述的任意sTF序列或截短序列,其中所述序列在SEQ ID NO:2 N-末端,进一步包括额外的ser-gly序列或仅gly残基,由此分别产生了219,或218个氨基酸的序列,而且其中在SEQ ID NO:2的207位的半胱氨酸残基由此分别移位至209位或208位。
[0018]sTF-AnnV嵌合蛋白的第二结构域,包含全长人蛋白膜联蛋白V(SEQ ID NO:4)或其有效PS结合片段。AnnV是35kDa蛋白,例如由SEQ ID NO:3编码,其可产生于多种细胞,其可结合某些磷脂,特别是如上所述的磷脂酰丝氨酸。PS常见于细胞膜内侧(所有细胞)。存在能转移PS至细胞膜外侧的相关酶机制,其中在钙存在情况下,PS能被AnnV结合。
[0019]连接子区(例如,但不限于SEQ ID NO:6,其由具有SEQ ID NO:5的cDNA编码)任选地在嵌合蛋白sTF域与AnnV域之间出现。在另一实施方式中,本发明嵌合蛋白也可在羧基和/或氨基末端或者内部,含有非功能标记(tag)序列,其主要包括例如6-组氨酸(SEQ ID NO:8,其由具有SEQ ID NO:7的cDNA编码),其用于辅助嵌合蛋白纯化,而且其优选在嵌合蛋白治疗给药应用之前,从嵌合蛋白切除。
[0020]在备选的实施方式中,将突变形式sTF用于嵌合蛋白。在一实施方式中,sTF突变体在163和164位具有丙氨酸残基,而不是天然sTF该位置所见的赖氨酸残基(例如或者在219氨基酸sTF的165位和166位)。该备选实施方案本文称为″sTFAA-AnnV″(SEQ ID NO:10,例如其由SEQ ID NO:9cDNA编码)。在另两个实施方案中,sTF突变体在163和164位有谷氨酸残基或谷氨酰胺残基取代,本文称为″sTFEE″(SEQ ID NO:12,例如其由SEQ ID NO:11cDNA编码)和″sTFQQ″(SEQ ID NO:14,例如其由SEQ ID NO:13cDNA编码)。这些突变体或原始sTF中的每个,可以包括(但不限于)任意或所有下列取代:在13,131,163,164或183位丙氨酸取代,在42或138位天冬酰胺取代,在48位色氨酸取代,在52位丝氨酸取代,在128位天冬氨酸取代,在163或164位谷氨酸取代,或在129,163或164位谷氨酰胺取代。
[0021]事实上,本发明构思在这些位点中的任意位点上,非极性氨基酸可被极性氨基酸或其它非极性氨基酸所取代,极性氨基酸可被非极性氨基酸或其它极性氨基酸所取代,正电荷氨基酸可被负电荷、极性或非极性氨基酸或者其它正电荷氨基酸所取代,以及负电荷氨基酸可被正电荷、极性或非极性氨基酸或者其它负电荷氨基酸所取代。
[0022]因此,本发明的嵌合蛋白sTF-annV优选包括,例如含SEQ ID NO:2的蛋白或其有效部分,其与含SEQ ID NO:4的蛋白或其有效部分连接。sTF-annV蛋白可进一步包括连接子,包括但不限于,位于sTF和annV序列间的具有SEQID NO:6的连接子。含或不含连接子序列的sTF-annV蛋白,可以包含内部或外部标记序列,例如SEQ ID NO:8,用于辅助sTF-annV蛋白纯化。任何其它有效的连接子序列和/或纯化标记序列,都可用于替代SEQ ID NO:6和/或SEQ IDNO:8。
[0023]在本发明某些实施方案中,可能期望嵌合蛋白具有缩短的血清半衰期使血清滞留时间最短,以限制嵌合蛋白引起所不期望的血栓形成潜能,或者使得嵌合蛋白在特定量的凝血酶形成之后被清除。在本发明这些实施方式中,将裂解位点例如血纤蛋白溶酶(plasmin)裂解位点或者凝血酶(thrombin)裂解位点,置于本文所述任一嵌合蛋白的sTF与annV序列之间。例如,具有SEQ IDNO:16(例如其由SEQ ID NO:15cDNA编码)的血纤蛋白溶酶裂解位点,或例如SEQ ID NO:18(例如其由SEQ ID NO:17cDNA编码)凝血酶裂解位点,可被插入至sTF与annV序列间的位置(含或不含连接子序列和/或纯化标记序列)。
[0024]为延长血清半衰期以增加其活性,本发明也构思含多个与膜联蛋白V氨基或羧基末端连接的sTF域的嵌合蛋白(例如具有SEQ ID NO:19的蛋白,其例如由SEQ ID NO:20cDNA编码)。
[0025]本发明也构思包含sTF或多个sTF域(或本文记载的其衍生物)和蛋白的其它非AnnV PS结合域的嵌合蛋白,所述蛋白例如为突触结合蛋白I(synpatotagmin I),或者本领域技术人员已知的其它蛋白,其用于锚定sTF域至含PS的膜。
[0026]用于取代AnnV的其它PS结合蛋白的实例,包括但不限于,膜联蛋白家族成员,乳凝集素(lactadherin),已知结合PS的蛋白中发现的结构域,所述蛋白例如V/Va因子,X/Xa因子,II/IIa因子,VII/VIIa因子,IX/IXa因子,VIII/VIIIa因子,血影蛋白,B类I型清道夫受体,蛋白激酶C,以及含蛋白激酶C的C2结构域的蛋白(其包括突触结合蛋白)),Rab亲和蛋白(Rabphilin)家族成员,PS受体,内皮凝集素样OxLDL受体-1(LOX-1),PS抗体,磷脂酰丝氨酸脱羧酶,MARCKS(肉豆蔻酰化、富丙氨酸蛋白激酶C底物),PS-p68,肌球蛋白,红细胞蛋白4.1,血红蛋白,钙调蛋白家族成员,S100A,S100B,钙周期素结合蛋白家族成员,乳膜糖蛋白,MFG-E8(乳脂球-EGF因子8),以及本领域普通技术人员已知的其它PS结合基序。
[0027]在本发明的一个实施方式中,在给予sTF-annV(或本文构思的任何其它嵌合蛋白)之前,将患肿瘤个体首先给予化疗注射,所述化疗引起肿瘤或肿瘤血管变化,导致sTF-annV结合增加,例如通过在肿瘤或血管细胞表面增强PS表达而导致sTF-annV结合增加。然后给予sTF-annV,所述sTF-annV将会以增加量结合肿瘤和肿瘤血管。化疗定义为致癌细胞死亡的任何治疗(即药物,化学试剂,激素,维他命,等等),其是本领域普通技术人员公知的。另一方法是给予区域放疗(单独或结合药物治疗),以增强sTF-annV效应和/或其结合。
[0028]癌细胞接受来自环境的信号,其允许它们生长、迁移和侵入正常组织。癌细胞不同于正常细胞之处在于它们许多在其表面,有增加数量的某些“受体”分子,可帮助细胞存活。在一个实施方式中,本发明构思将新蛋白设计为阻断肿瘤细胞表面受体,包括组织因子和尿激酶受体,由此改变肿瘤细胞行为。
[0029]在一个实施方式中,本发明构思将嵌合蛋白设计为抑制通过细胞受体的信号传导(signaling),其中所述受体对癌细胞,也可能对正常的炎症细胞比较重要。特别地,本发明构思(1)新蛋白,其与尿激酶受体结合,从而阻止正常经该受体产生信号的两蛋白(尿激酶和血纤蛋白溶酶原激活剂抑制剂-1,也称PAI-1)的结合,和(2)新蛋白,其与细胞表面结合,且包含细胞表面蛋白——组织因子的胞外结构域,所述组织因子用作血浆蛋白——称为VIIa因子的受体。因为包含(天然)组织因子和VIIa因子的复合物,能产生以组织因子胞质尾依赖方式影响癌细胞行为的信号,所以本文构思的含sTF嵌合蛋白,可作为VIIa因子接收池(sink),从癌细胞表面的组织因子转移VIIa因子。本发明也构思同时结合尿激酶受体和VIIa因子的新蛋白,以阻止癌细胞从其自身细胞表面组织因子和尿激酶受体分子受益。本发明也构思组织因子结构域和/或尿激酶受体结构域的突变体,以增强每种这些结构域对其配体的亲和力。
[0030]尿激酶受体和组织因子也见于,称为慢性炎症细胞的巨噬细胞。本文记载的嵌合蛋白也可用于治疗人类或动物疾病,在该疾病中,持续的巨噬细胞介导的炎症在该持久疾病或不适中起作用。
[0031]在另一实施方案中,本发明包括与ATF连接的sTF(sTF-ATF),该嵌合蛋白可同时阻断细胞的TF和尿激酶受体。ATF是尿激酶的氨基末端蛋白片段(尿激酶A链1-135氨基酸,因此称为ATF)。ATF(SEQ ID NO:22,例如其由具有SEQ ID NO:21的cDNA编码)结合尿激酶受体,但与全长尿激酶不同,其不被内化。
[0032]本发明嵌合蛋白可包括替代AnnV的一种或多种肽,所述肽占领肿瘤脉管和其它器官的受体,包括U.S.6,576,239;6,491,894;6,528,481;6,610,651;和6,933,281中记载的引导性肽(homing peptides),其中所述每一文献在此明确通过援引并入。
[0033]可结合肿瘤脉管并可包括本发明蛋白的其它肽,包括但不限于:HWGF(SEQ ID NO:23)-基序肽(基质金属蛋白酶-2和基质金属蛋白酶-9,也称为明胶酶A和明胶酶B,的选择性抑制剂),例如合成肽CTTHWGFTLC(SEQ IDNO:24)靶向血管生成性血管,抑制人内皮细胞和肿瘤细胞迁移,也在动物模型中阻止肿瘤生长和侵入,提高负荷人类肿瘤的小鼠的存活。
[0034]如上所述,本文构思的任一肿瘤治疗方法,可包括以膜联蛋白V(非sTF-膜联蛋白V)进行筛查的初始步骤,以确定对治疗预期有正常或增强应答的个体。优选的个体为显示膜联蛋白V肿瘤吸收而非肿瘤区无过量吸收的个体。这将会是更有效的治疗,因为某些潜在个体在无先行药物治疗和/或放射治疗的情况下,可能有增强的吸收(由此其不一定受益于这些伴随着副作用的辅助治疗),而其它个体可能没有。后者将更可能从首次化疗或放疗中受益,从而允许在sTF-AnnV和“激发”治疗之间产生协同作用。
[0035]在膜联蛋白V筛查中(在sTF-AnnV给药之前进行),膜联蛋白V结合于血液凝块中捕获的激活血小板。因此,膜联蛋白V筛查可确定有sTF-AnnV结合的提高风险的个体(subjects),所述个体在非肿瘤血管床中可能发生病理性血栓(即,中风,心脏病发作,深静脉血栓,等等)。通过进行膜联蛋白V先期筛查,避免对该并发症有更高风险的个体,使用者安全情况可得到提高。膜联蛋白V筛查可用多种成像技术进行(特别是磁共振成像和核医疗学成像),该技术允许开发新的成像化合物。备选地,在给予sTF-AnnV之前(有或没有化疗和/或放疗),给个体静脉注射膜联蛋白V,以预防所不期望发生血栓的血管与sTF-膜联蛋白结合。
[0036]实验步骤
[0037]材料
[0038]如所述制备重组膜-锚定TF,重组sTF(TF2-219),以及VII和TF的单克隆抗体。与重组蛋白的His6标记反应的单克隆抗体,来自Cell SignalingTechnology(Beverly,MA)。Chromozym t-PA(N-甲基磺酰基-D-苯丙酰甘氨酰-L-精氨酸-4-硝基苯胺醋酸盐)和Chromozym X(N-甲氧基羰基-D-正亮氨酰-甘氨酰-L-精氨酸-4-苯基重氮酸醋酸盐)来自Roche Diagnostics Corp(Indianapolis,IN)。纯化的人X,Xa,VII,和VIIa因子来自Enzyme Research Laboratories(South Bend,IN)。除非另有说明,否则,所有其它试剂均来自Sigma Chemical Co.。
[0039]磷脂以氯仿贮存液形式,获自Avanti Polar Lipid(Alabaster,AL),其用于制备单层磷脂泡囊。将等份试样以摩尔比磷脂酰胆碱(PC)/PS 4∶1混合,氩气下蒸发去除氯仿。将磷脂重悬于0.1M NaCl,0.05M Tris-HCl,pH 7.5,超声直至悬液几近透明,产生PC/PS泡囊。磷脂浓度通过磷酸分析确定。
[0040]TF至磷脂泡囊的再脂化,用辛基-β-D-葡糖吡喃糖苷法进行,通常TF/磷脂摩尔比1∶8700。有效TF浓度通过在含Chromozym t-PA的溶液中,用逐步增加浓度的VIIa因子滴定,并将所得胺降解活性,与已知浓度sTF与VIIa因子温育所得胺降解活性(amidolytic activity)进行比较,而测量。TF制备物磷脂浓度用磷酸分析测量,以使TF与sTF-annV直接比较时,PS存在量相当。
[0041]荧光素异硫氰酸酯(FITC)-膜联蛋白V,通过将FITC与膜联蛋白V以2∶1摩尔比,在0.5M碳酸盐缓冲液,pH 9.5室温暗室下温育1小时而制备。未结合FITC通过150mM NaCl,0.01M Tris-HCl,pH 8.2凝胶过滤,接着用相同缓冲液透析另外的48小时而去除。
[0042]表达载体构建
[0043]膜联蛋白V cDNA(于pET-22b(+)载体中)由Antony Rosen博士惠赠。sTF-annV构建体,通过将TF 2-219氨基酸编码cDNA与膜联蛋白V cDNA 5′端连接而产生。sTF cDNA用正向引物
5′-CATGCCATGGCAGGCGCTTCAGGCACTACAAATAC-3′(SEQ ID NO:25),和反向引物
5′-CCCAAGTCTTGGGTTCTCTGAATTCCCCTTTCTC-3′(SEQ ID NO:26)进行PCR克隆。用下列引物
5 -
GGGGAATTCAGAGAAGGTGGCGGTTCAGGCGGTGGAGGTTCAGGAGGTGGCGGATC
AATGGCACAGGTTCTC-3′(SEQ ID NO:27).
使用Multi Site-Directed Mutagenesis Kit(Stratagene,La JoIIa,CA),将编码(GGGGS)3的连接子序列插入至sTF和膜联蛋白V cDNA之间。将sTF-annV基因克隆至pET-22b(+)载体NcoI和HindIII位点。该载体编码在羧基末端有His6标记的蛋白。由突变形式的sTF与膜联蛋白V连接组成的嵌合体(sTFAA-annV),通过定点突变产生。用Multi Site-Directed Mutagenesis Kit(Stratagene,LaJoIIa,CA)和下列引物
5′-CTTTATTATTGGAAATCTTCAAGTTCAGGAGCCGCAACAGCCAAAAC
AAACACTAATGAGTTTTTG-3′(SEQ ID NO:28).
将sTF的164和165位残基(即219氨基酸长度的sTF的165和166残基)进行赖氨酸至丙氨酸的突变。将sTFAA-annV基因克隆至pET-22b(+)载体的NcoI和HindIII位点。DNA测序证实了所有三种构建体的组成。
[0044]sTF-annV,sTFAA-annV和膜联蛋白V的表达和纯化
[0045]将含sTF-annV,sTFAA-annV或annV基因的pET-22b(+)载体,插入至大肠杆菌菌株BL-21/DE3株(Novagen,Madison,W1)。该载体提供了指导重组蛋白进入周质空间的信号肽。当细菌悬液A600达到0.8时,在25℃添加0.3mM异丙基-β-D-硫代半乳吡喃糖苷(IPTG)15小时诱导表达。收获细胞,进行渗透冲击(5mM MgSO4),收集周质组分。用0.3M NaCl,0.05M NaH2PO4,pH 8.0平衡His-Select柱(Sigma,St.Louis,MO)通过固定金属亲和层析,将重组蛋白纯化。在10和20mM咪唑存在情况下用相同溶液将柱洗涤,然后用250mM咪唑洗脱。洗脱液用0.1M NaCl,50mM Tris-HCl pH 7.4透析。重组蛋白的纯度和身份,用SDS-PAGE和Western印记检测。
[0046]磷脂结合分析
[0047]嵌合体对含PS磷脂泡囊的亲和性,通过使用商购aPTT凝结剂(FSL Dade Behring,Newark,DE)作PS源的改进公开试验方法进行测量。按照制造商使用说明重构试剂,并以1∶10稀释于补加2.5mM Ca++的140mM NaCl,0.01M HEPES,pH 7.5(HBS)溶液中(HBS-Ca++)。将稀释磷脂悬液(10mL)在包被微离心管(Slickseal,National Scientific,Claremont,CA)的HBS-Ca++溶液中,与50nM FITC-膜联蛋白V(使试验中PS饱和的浓度,可实验测定)温育。37℃温育15min后,将混合物16,000xg离心20min。将团块在HBS-Ca++溶液中洗涤,然后重悬于含不同量各未标记的竞争性蛋白(膜联蛋白V,sTF-annV或sTFAA-annV)的HBS-Ca++溶液中。再次37℃温育12hr后,将混合物离心,将团块在HBS-Ca++溶液中洗涤。然后将团块重悬于补加10mM EDTA的HBS中,室温温育16hr,以洗脱结合的FITC-膜联蛋白V。将管离心,移出上清液。通过荧光微板读数仪(Victor2,Wallac,PerkinElmer,Boston,MA)测定荧光(激发波长,485nm;发射波长,535nm),确定上清液中FITC-膜联蛋白量。
[0048]VII因子自激活分析
[0049]嵌合蛋白促进VII因子自激活的能力,通过在含5mM CaCl2的0.1MNaCl,50mM Tris-HCl,0.1%牛血清白蛋白,pH 7.4(TBS)(TBS/Ca2+)溶液中,将各嵌合蛋白(终浓度,80nM)添加至VII因子(终浓度,80nM)进行测量。在使用sTF-annV或sTF的实验中,将PC/PS泡囊以与天然TF制备物相同组成和浓度,添加至温育混合物(总磷脂浓度,810mM,PC∶PS摩尔比=4∶1)。在选定时间间隔,从各温育混合物移取10mL等分试样,转移至含40mL TBS+5mM EDTA的聚苯乙烯管中,终止反应。所产生VIIa因子的量,通过添加150mL TBS/Ca2+,过量sTF,和Chromozym t-PA(终浓度分别为5mM,124nM和5mM),并检测A405的变化而测量。
[0050]VIIa因子结合分析
[0051]sTF/TF诱导的VIIa因子胺降解活性增加用于对这些蛋白与VIIa因子的结合进行定量。将逐步增加浓度的sTF-annV,sTFAA-annV或sTF,在96孔分析板中的TBS/Ca2+溶液中与VIIa因子(5nM)温育。室温10min后,将Chromozym t-PA加入(终浓度1mM),用微板读数仪(Molecular Devices,MenloPark,CA)在405nm处,测量底物水解的初速率。从测量值减去不存在sTF,sTF-annV或sTFAA-annV时的VIIa因子背景活性。动力学参数用Prism 4(GraphPad Software,San Diego,CA)计算。
[0052]X因子激活
[0053]sTF,sTF-annV,sTFAA-annV或天然再脂化(relipidated)TF存在情况下VIIa因子介导的X因子激活,通过连续一期分析(one stage assay)进行。将sTF,sTF-annV,sTFAA-annV或天然TF加入TBS/Ca2+溶液中的1nM VIIa因子,X因子,PC/PS(摩尔比,4∶1)和0.5mM Chromozym X中。20min后检测生色底物水解(A405变化),参照纯化人Xa因子制备的标准曲线,将其转化为Xa因子浓度。取所得抛物线导数derivative of the resulting parabolic progress curve(Softmax Pro,MolecularDevices,Menlo Park,CA),确定Xa因子产生速率,用Prism 4(GraphPad Software,San Diego,CA)计算动力学参数。
[0054]血浆凝结分析
[0055]凝结时间
[0056]不同浓度重组蛋白和PC/PS(摩尔比,4∶1)存在情况下及添加CaCl2后的正常人柠檬酸盐化血浆的凝结时间,用机械凝结测量仪(ST-4,DiagnosticaStago,Parsipanny,NJ)测量,其测量上限为999s。
[0057]稀释激活的偏(partial)促凝血酶原激酶时间
[0058]将商业获得aPTT试剂(Dade Behring)以1∶50稀释于TBS,在各种浓度sTF,膜联蛋白V或sTF-annV存在情况下,用于进行柠檬酸盐化人血浆凝结时间测量。将血浆与稀释aPTT试剂和重组蛋白37℃温育3min,然后添加25mM CaCl2。形成凝块所需时间用机械凝结测量仪测量。
[0059]激活的偏促凝血酶原激酶时间(Activated partial thromboplastin time,aPTT)
[0060]根据制造商使用说明,将含1单位/mL肝素钠(AmericanPharmaceutical Partners,Inc.Los Angeles,CA)或依诺肝素钠(AventisPharmaceuticals,Bridgewater,NJ)与不同浓度的sTF-annV或VIIa因子的血浆的aPTT,用商业获得aPTT试剂(FSL;Dade Behring,Newark,DE)进行测量。
[0061]小鼠尾出血时间
[0062]将小鼠按俄克拉荷马大学健康科学中心机构动物保护和使用委员会(the University of Oklahoma Health Sciences Center Institutional Animal Care andUse Committee)认可的方案,进行圈养和研究。将小鼠皮下注射依诺肝素钠,两小时后用戊巴比妥(60mg/kg,i.p.给予)麻醉。将尾在37℃常规盐水中预暖,用解剖刀在直径2mm处横切,然后将尾置于37℃恒温常规盐水管中。记录出血停止所需时间。在初步剂量探寻实验中,逐步增加给予依诺肝素钠的剂量。一旦合适剂量确定(20mg/kg),首次实验5min后进行第二次出血时间测量。额外的出血时间,通过在邻近首次横切的5mm处,横切尾而进行。与第一次相比,第二次出血时间不具有显著性差异(p=0.3,双尾成对t-test,Prism 4,GraphpadSoftware)。未处理小鼠然后用类似方式处理,但是在首次出血时间确定后,立即静脉注射sTF-annV(90mcg/kg)。注射5min后进行第二次出血时间测量。双尾成对t-test,用于比较两次出血时间。然后研究其它组动物。SQ注射盐水或依诺肝素(20mg/kg)两小时后,给小鼠IV注射sTF-annV(90mcg/kg),盐水,sTF,膜联蛋白V,或者sTF+膜联蛋白V的组合。所有蛋白以sTF-annV的等摩尔量注射。10分钟后测量出血时间。
[0063]结果
[0064]膜联蛋白V和膜联蛋白V嵌合体的表达和纯化
[0065]将编码三种蛋白的cDNA构建体(膜联蛋白V,sTF-annV和sTFAA-annV),在大肠杆菌(E.coli)周质空间表达,将蛋白用固定金属亲和层析纯化(参见图1,纯化蛋白用SDS-PAGE和western印记分析)。
[0066]sTF-annV加速血浆凝结
[0067]在初始研究中,比较了在相同浓度添加的磷脂和CaCl2存在的情况下,sTF,天然TF,和sTF-annV加速血浆凝结的能力。如图2A所示,sTF-annV和天然TF相比sTF,在更大程度上缩短了血浆凝结时间。接着,用稀释aPTT实验,确定sTF-annV效果是否能通过添加sTF至膜联蛋白V复制。在稀释aPTT试剂(用作磷脂源)和sTF,膜联蛋白V,sTF+膜联蛋白V组合,或sTF-annV存在情况下,将血浆复钙(recalcified)。如图2B所示,同时加入sTF和膜联蛋白V,不能重现sTF-annV的促凝效果。在该条件下,sTF-annV显示出血浆凝结的两相效应,在更高浓度下延长凝结时间。
[0068]该结果提示,在更低浓度下因sTF结构域而存在促凝效应,在更高浓度下膜联蛋白V抗凝效应则占主导。为了验证该假说,构建TF/sTF结构域在165位和166位(218aa sTF的164和165位)赖氨酸突变为丙氨酸的构建体,该突变已知会损伤X因子激活。所得嵌合体(sTFAA-annV)也展示出血浆凝结双相效应(图2C),但其比sTF-annV有更低的促凝活性,并且在更低浓度下还展示出抗凝活性。在存在TF-annV和sTFAA-annV能改变血浆凝结证据的情况下,对其组分结构域再进行详细功能鉴定。
[0069]磷脂结合活性
[0070]sTF-annV和sTFAA-annV结合PS的能力,用公开的PS-结合实验之改进进行分析,其中膜联蛋白V,sTF-annV或sTFAA-annV与FITC-膜联蛋白V竞争结合磷脂泡囊。如图3所示,两种嵌合体都展示出与膜联蛋白V相当的PS-结合活性。膜联蛋白V的Kd为12nM;sTF-annV,为13nM,以及sTFAA-annV,为11nM。
[0071]VII因子自激活活性
[0072]先前已显示,TF而不是sTF,在PS和Ca2+存在情况下,促进VII因子自激活。sTF不能增加VII因子自激活的原因在于,sTF:VIIa复合物对含PS膜的低亲和力。由于嵌合体的膜联蛋白V结构域显示出对含PS脂质体的高亲和力,我们推测sTF-annV可促进与TF相当的VII因子自激活,而不与sTF相当。如图4所示,sTF-annV以与天然TF相当的速率,促进了VII因子自激活而不是sTF(其无活性)。该结果也提示,sTF-annV能结合VII因子,与天然TF相当。
[0073]VIIa因子结合活性
[0074]为了测量嵌合体的VIIa因子结合,我们对VIIa因子结合TF/sTF时其胺降解活性的增加,进行了定量。由于sTF结合膜的情况不佳,所以我们在缺乏PS情况下进行分析。sTF-annV和sTFAA-annV都可以与sTF相同的程度,增加VIIa因子的胺降解活性,这表明,膜联蛋白V结构域并不干扰sTF结构域结合VIIa因子的能力(数据未示出)。sTF的VIIa因子结合Kd为8.17nM;sTF-annV为7.05nM;以及sTFAA-annV为3.47nM,所有数据都在不存在磷脂泡囊情况下得出。
[0075]X因子激活
[0076]检测了sTF-annV和sTFAA-annV,对VIIa因子介导X因子激活速率的效果。如图5A所示,在sTF-annV或sTFAA-annV存在情况下,X因子激活速率高于sTF存在情况下的激活速率。如sTFAA在先研究所预期的那样,sTF-annV将比sTFAA-annV更有能力支持X因子的激活。如表1所示,在sTF-annV存在情况下VIIa因子催化效率(Kcat/Km)约为天然TF存在情况下的67%,但其约为sTFAA-annV催化效率的5倍。
[0077]由于高浓度sTF-annV与延长血浆凝结时间有关,所以我们研究了逐步增加浓度的sTF-annV对VIIa因子介导X因子激活速率的效应。如图5B所示,sTF-annV展示出X因子激活的双相效应,这与血浆凝结效应相类似。
[0078]表1
辅因子 | VmaxnM.min-1 | kmnM | kcats-1 | kcat/kmμM-1s-1 |
TF | 0.71±0.008 | 46±1 | 2.37 | 51.5 |
sTF-annV | 0.66±0.03 | 64±7 | 2.2 | 34.4 |
sTFAA-annV | 0.71±0.06 | 389±58 | 2.37 | 6.1 |
sTF,sTF-膜联蛋白V和sTFAA-annV对VIIa因子介导的X因子激活速率的效应。
[0079]sTF-膜联蛋白在肝素存在情况下对凝结的效应
[0080]肝素为常用抗凝剂,其通过增强serpin(一种抗凝血酶蛋白)(主要)抑制Xa因子和凝血酶的能力而起作用。在各种sTF-annV浓度下,我们将未分级肝素钠或依诺肝素,低分子量肝素,加入至柠檬酸盐化血浆中,然后测量血浆aPTT。sTF-annV缩短了含1单位/mL未分级肝素(图6A)或1单位/mL依诺肝素(图6B)血浆的aPTT。VIIa因子,其在多种与凝血酶产量减少相关的临床状况中用作治疗剂治疗出血,该因子也缩短了肝素处理血浆的aPTT,尽管不如sTF-annV那么强大(图6A和6B)。
[0081]sTF-annV对小鼠尾出血时间的效应
[0082]虽然人模型出血时间实验被认为是主要反映了血小板功能,但是小鼠凝血酶生产的缺陷却是由延长的出血时间所反映。因此,我们给予正常小鼠依诺肝素,以确定sTF-annV是否会影响体内方式的凝血酶产生。将小鼠皮下注射依诺肝素钠(20mg/kg),一种已知会抑制凝血酶产生的药物,然后2小时后测量出血时间。该出血时间伤口其出血停止后,将动物静脉注射sTF-annV(90mcg/kg),5min后再次测量出血时间。(初步实验显示,依诺肝素处理的动物的第二次出血时间与第一次出血时间不存在显著差异,p=0.3,成对双尾t-test)。如图7A所示,sTF-annV显著缩短了依诺肝素处理小鼠的出血时间(p=0.01,成对双尾t-test)。依诺肝素处理导致的平均出血时间10.7分钟(中值,6.1分钟)。sTF-annV处理后的平均出血时间为1.4分钟(平均值,1.1分钟)。未处理动物的平均出血时间为1.2分钟。
[0083]在其它组小鼠中测量尾出血时间。sTF-annV缩短了未用依诺肝素处理动物的出血时间(1.5±0.7min VS 3.6±0.2min,p>0.02,Welch′s 校正未成对t-test;图7B)。然后将幼稚动物用依诺肝素(20mg/kg,SQ)处理,2小时后,IN注射sTF-annV(90mcg/kg)或等摩尔量的sTF,膜联蛋白V,或者sTF+膜联蛋白V组合。如图7B所示,缩短的出血时间仅在sTF-annV处理动物中观察到(p<0.0001,Welch′s校正双尾未成对t-test),这表明嵌合体本身而不是其各个成分,缩短了出血时间。
[0084]先前研究表明,分离的TF胞外结构域(sTF)保留了足以允许结合VIIa因子并增强其裂解小分子三肽合成底物的构象。然而,sTF在促进VIIa因子介导的X因子激活方面,效率更低。这似乎是因为sTF与sTF-VIIa复合物对膜表面有相对较低亲和力。通过取代TF跨膜结构域的糖磷脂酰肌醇锚定部分,Paborsky et al能产生有完全促凝活性的膜结合sTF变体,这表明如果适当限制于细胞表面,sTF的功能将与天然TF相当。
[0085]其它人也偶联了sTF与肽部分,并显示了其体内和体外凝结系统的活化。各种研究都利用了特定细胞类型或解剖区特异的靶向结构域,所有这些研究都涉及开发抗癌剂,而不是新的止血化合物。
[0086]在本工作中,含sTF结构域的嵌合体蛋白在优选的实施方式中创建,有下列几种新特征:(1)它对在止血中扮演作用的细胞类型具有“多特异性”;(2)它具有具抗凝结特性的膜靶向结构域。因此,虽然膜联蛋白V作为PS结合蛋白的能力,使其作为靶向部分具有吸引力,但是其抗凝活性却阻碍了它应用于设计为促凝止血剂的构建体中。
[0087]因此,sTF-annV初始研究涉及确定其是否确实为促凝剂,如果是,其促凝活性与sTF和天然TF比较如何。因为我们认识到膜联蛋白V与磷脂的比例是重要的,所以我们在分析中利用磷脂悬液,而不是激活细胞,以保持一致性。在我们发现sTF-annV具有促凝活性后(图2A),我们确定促凝活性是嵌合体特异的,其不能通过添加等摩尔量的独立的sTF和膜联蛋白V混合物来重现(图2B)。我们也制备了嵌合体(本文称为sTFAA-annV),其中sTF 165和166位氨基酸具有赖氨酸至丙氨酸的突变,该改变已知会显著降低TF促凝活性,我们发现,该嵌合体损失了促凝活性(图2C)。最后,我们研究了改变反应混合物中嵌合体/PS比例的后果。我们发现,对sTF-annV和sTFAA-annV而言,蛋白/PS比例增加超过某点时,将产生抗凝效应(图2C)。因此,当存在能结合其它凝结因子的功能性过量PS结合位点时,通过膜联蛋白V结构域将sTF靶向至膜表面,将是促凝的。当膜联蛋白V存在量增加时,这些位点将变得更难以接近凝结因子VII/VIIa,X,VIII,IX,V和II,且凝结减慢。
[0088]接着我们对包含sTF-annV的功能结构域进行了分析,以确定将该它们连接在一起是否将导致两者之一的功能增加或损失。嵌合体结合含PS泡囊能力的实验(图3)、促进VII因子自激活实验(图4)、增强VIIa因子胺降解及X因子裂解活性实验(图5),表明sTF和膜联蛋白V结构域都具有功能,尽管天然TF促进VIIa因子至X因子的催化效率是sTF-annV的两倍(参见表1)。
[0089]这些研究表明,sTF-annV可用作促凝剂,但其促凝活性很大程度上依赖于磷脂浓度。因为体内伤口位置的PS局部浓度并不确切了解,所以体外实验只能有限预测PS结合蛋白(例如sTF-annV)的体内行为。为了在可由身体对损伤的应激确定PS浓度的情况下体内检测sTF-annV,我们进行了小鼠尾出血时间实验。在之前给予依诺肝素,低分子量肝素,用于损伤凝血酶产生的情况下,该方法已知对缺陷的凝血酶制造敏感。在预备该实验中,我们在依诺肝素及未分级肝素存在情况下,研究了sTF-annV对血浆凝结的效应,并且将其与VIIa因子效应相比较,所述VIIa因子是目前用于治疗具有多种出血症患者的蛋白。VIIa因子和sTF-annV都缩短了肝素和依诺肝素处理血浆的凝结时间,尽管sTF-annV比VIIa因子强大几个数量级(图6)。
[0090]我们用逐步升高剂量的依诺肝素处理小鼠,确立了再现性增加两次顺序出血时间的剂量。然后我们在第一次和第二次尾出血时间之间,尾静脉注射sTF-annV。在所有测试的动物中,第二次出血时间显著低于第一次出血时间。用其它动物实验,我们发现sTF-annV的效应,不能由独立注射sTF,膜联蛋白V,或两者的混合物重现。因为人VIIa因子在小鼠血浆中不具有完全活性,所以VIIa因子不能用于直接比较。选用该sTF-annV剂量是因为该剂量(基于体重)与常规推荐重组VIIa因子的剂量相当。
[0091]这些研究显示,似乎拮抗性蛋白结构域可在嵌合分子中组合形成蛋白,所形成蛋白其活性并不简单地由主结构域净效应所确定。蛋白sTF-annV展示出可反映其任一相对结构域的效应,这取决于相关情况。sTF-annV展示出促凝和抗凝活性的能力使其在止血分子中独树一帜。
[0092]因为AnnV结合PS,而PS是血液凝结所需,所以单独使用AnnV可阻止凝结机制蛋白结合PS,因此AnnV实际上抑制血液凝结,因此通常不会将其认为是止血剂。尽管sTF在高浓度下能加速血液凝结,但本发明记载的sTF与annV的复合物,将不会是提高血液凝结显而易见的方法,而且因为AnnV结构域存在,很可能被本领域知识人员预期,其要么不具有止血效应,要么具有抗凝效应。
[0093]本发明已经证明在低浓度下比单独sTF更有效。不希望受理论束缚,该结果可能是因为以下事实:AnnV结构域使sTF靠近膜表面,在那里其结合X因子。
[0094]血浆来源的止血剂已经显示出在某些患者中诱导血栓(不期望的血液凝结),血栓也已经在用重组VIIa因子治疗的某些外伤患者中观察到。
[0095]本发明提供了定位sTF至激活血小板区域的方法(本文记载的AnnV结构域或其它结构域),与在脉管系统中循环的效力相当的止血化合物相比,其可能更安全。
[0096]我们的工作显示,在更高浓度下,sTF-annV蛋白丧失了促凝效应(止血),变成了抗凝剂,这一点可通过血液凝结时间的延长来证明。这可能是因为以下事实:在高浓度下,由于占领了X因子潜在结合位点,AnnV结构域效应变得比其定位sTF结构域至膜表面的效应更重要。没有报道其它止血剂在高浓度下具有抗凝活性。该特征可能使sTF-AnnV比其它当前可获得的止血剂更安全。
[0097]实用性
[0098]本发明嵌合蛋白可用于各种与止血和血栓形成相关的治疗应用。
[0099]用作止血剂
[0100]本发明构思给予嵌合蛋白sTF-annV(或本文记载与其它PS结合元件连接的sTF嵌合体)于出血患者,以在出血区域增加凝血酶产生而使出血停止。通常,按压或缝合能使出血停止。然而,有些时候这些方法不能容易地变成,在此情况下,化合物例如sTF-annV将是有用的。这样的情况包括(1)在抗凝药物存在情况下的出血,(2)缺乏(先天或非先天)一种或多种凝血因子的出血,例如这可以在外伤和大量输血、维他命K缺乏、血友病、血液凝结因子抗体发展中观察到,(3)肝脏疾病,(4)在压迫出血区无效或无法应用情况下的出血,例如损伤弥散区出血,食道脉管曲张、胃炎、膀胱炎、子宫内膜炎、血管损伤或头创伤出血,(5)在血小板不以适当量存在情况下的出血,(6)在血小板不能正确发挥功能情况下的出血,或者(7)在出血症患者治疗中,用作重组VIIa因子(FDA-批准药物)的辅助药。
[0101]用于诱导血栓形成
[0102]也可预期本文记载的sTF-annV或类似嵌合蛋白,用于在不与癌状态相关的血管中诱导血栓形成,但这对宿主可能是有害的。这样的血管收集物包括但不限于,毛细管扩张、动脉畸形、静脉畸形、毛细血管畸形、淋巴管畸形、动静脉畸形、血管瘤、或动脉瘤。此外,本发明预期用该记载蛋白治疗以结构正常的血管生长为特征的区域,其方式或位点可能会危及宿主健康,有时统称其为“新血管生成”区。可以预期,将sTF-annV或其变体给予宿主(通过本文记载的多种途径中的任一种),从而以治疗方式诱导局部血栓形成。在之前或同时利用另一治疗药征以提高sTF-annV效力可能是必要的。这种辅助治疗可能包括药物,化学物,电刺激,或者放射治疗。
[0103]用于癌症治疗
[0104]膜联蛋白V可在肿瘤内结合肿瘤和/或血管细胞。膜联蛋白V(或本文记载的其它PS结合结构)介导的sTF肿瘤定位,将导致引发肿瘤内血液凝结,从而切断运输至肿瘤的营养流,导致肿瘤死亡。本发明利用AnnV结构域或PS结合结构将嵌合蛋白定位至肿瘤。
[0105]用于靶向治疗部分
[0106]本发明也构思将sTF-annV用于递送治疗物质,例如放射性物质或化疗化合物,至肿瘤或异常血管床。在一个实施方式中,例如将治疗性增强的(例如放射性的)sTF-annV注射至患癌宿主。将治疗活性的sTF-annV定位于肿瘤血管中,可能再接着给予化疗、放疗或其它操作以增强sTF-annV对目标区域的靶向,这将增强分子的治疗效果。使用这种修饰的、与放射性或化疗部分连接的sTF-annV分子可用于治疗各种癌症、血管瘤、或血管畸形中的任一种。化疗或放射性物质与蛋白如何连接的实例,在U.S.6,074,643;6,696,478;6,403,771;5,620,675;5,346,686;5,219,556;6,852,318,5,863,538;7,001,991;5,108,987;5,000,935;4,895,714;和4,886,780中有记载,将其在此明确通过援引并入。
[0107]本发明化合物的“治疗有效量”或“生物有效量”,是指可有效控制、抑制、减少或促进止血或血栓形成,或者控制、抑制或减少肿瘤生长的量。术语“控制”意指减慢、打断、阻止或停止疾病或状况进展,且不一定显示出完全消除疾病所有症状的一切过程。
[0108]术语“治疗有效量”或“生物有效量”进一步意在定义产生疾病状况特征性的任何参数或临床症状改进的量。实际剂量针对不同特定分子会有不同,其根据患者整体状况、症状严重程度及相反指示而变化。
[0109]本文使用的术语“个体(subject)”或“患者”是指患有特定疾病、需要本文所记载治疗的温血动物,例如哺乳动物。应该理解,豚鼠、狗、猫、大鼠、小鼠、马、牛、绵羊、动物园动物(200animals)、家畜、以及人类,都是本术语意义范围内的动物实例。
[0110]用于本文记载的治疗的化合物的治疗有效量或生物有效量,可通过使用常规技术和观察类似条件下获得的结果,由参与诊断医师(本领域的技术人员)容易地确定。在确定治疗有效剂量时,参与诊断医师要考虑多种因素,包括但不限于:哺乳动物的种类;动物尺寸、年龄和总体健康状况;所涉及特定疾病或状况;疾病或状况的相关程度或严重程度;个体的反应;给药的特定化合物;给药方式;给药制剂特征性的的生物利用度;所选的剂量方案;伴随药物的使用;以及其它相关情况。
[0111]本发明化合物的治疗有效量或生物有效量,也指治疗本文记载疾病状况或另一状况有效的化合物量。
[0112]本发明组合物的治疗有效量或生物有效量,通常包含足量活性成分(即嵌合蛋白)以递送从约0.1ng/kg至约100mg/kg(活性成分重量/患者体重)。优选地,组合物递送至少10ng/kg至50mg/kg,最优选至少100ng/kg至10mg/kg。
[0113]本发明方法的执行,包括以任意合适系统或局部制剂、以有效递送上文所列剂量的量,给予个体治疗有效量或生物有效量的活性成分。药剂给药可以一次性给药,或(例如)每天1-5次、或每周1次或2次,或者通过静脉滴注连续给药,这取决于所期望的治疗效果。
[0114]显然,优选的给药剂量和给药方式,可由本领域技术人员确定。制剂制备领域的技术人员,可以容易地选择适当的给药形式和给药方式,这取决于所选化合物特定特性,所治疗疾病的状态或状况,疾病或状况所处的阶段,以及使用本领域已知制剂技术的其它相关情况,这些情况例如记载在Remington′sPharmaceutical Sciences,latest edition,Mack Publishing Co.中。
[0115]药物组合物可用本领域的已知技术进行制备。典型地,将治疗有效量化合物与药学可接受的载体进行混合。
[0116]本发明的化合物或组合物可以多种途径给药,例如口服或胃肠外给药(即皮下给药、静脉内给药、肌肉内给药、腹膜内给药、或气管内给药),眼内给药,或颅内给药。
[0117]对于口服给药而言,化合物可制备成固体或液体制剂,例如胶囊、丸剂、片剂、锭剂、熔融物、粉末、悬浮剂、或乳剂。固体单位剂型可为常规明胶形式的胶囊,包含例如表面活性剂、润滑剂以及惰性填充剂,诸如乳糖、蔗糖和玉米淀粉,或者它们可为缓释制剂。药剂可以肠衣包被。
[0118]在另一实施方式中,本发明化合物可用传统片剂基质例如乳糖、蔗糖和玉米淀粉,结合粘合剂例如阿拉伯树胶、玉米淀粉或明胶,崩解剂例如马铃薯淀粉或藻酸,以及润滑剂例如硬脂酸或硬脂酸镁,制成片剂。液体制剂可将活性成分溶解于水性或非水性药学上可接受的溶剂而制备,所述药学上可接受溶剂还可包含悬浮剂、甜味剂、调味剂、以及防腐剂,这些都是本领域已知的。
[0119]对胃肠外给药而言,可将化合物溶解于生理可接受的药物载体,以溶液或悬浮液给予。说明性的合适药物载体是,水、盐水、葡糖溶液、果糖溶液、乙醇、或者动植物或合成来源的油。药物载体也可包含本领域已知的防腐剂和缓冲剂。
[0120]本发明化合物在某些条件下,也可局部给予。这可通过简单配制待给药化合物的溶液进行,在存在或不存在其它赋形剂情况下,优选使用已知促进透皮吸收的溶剂,例如乙醇或二甲基亚砜(DMSO)进行。优选的局部给药,使用贮库和多孔膜型药贴或固体基质类药贴进行。
[0121]如上所述,组合物也可包括合适载体。对于局部应用而言,任何常规赋形剂都可添加,将活性成分制成乳液、软膏、粉末、乳膏、喷雾剂、或气溶胶。对于外科植入而言,活性成分可与任一公知生物可降解和生物可蚀解载体组合,例如聚乳酸和胶原制剂。这些材料可以是固体植入物、缝合物、海绵物、伤口敷料,等等形式。在任何情况下,对于这些材料的局部应用而言,活性成分通常以重量比约1∶1000至1∶20,000在载体或赋形剂中出现,但不限于该范围内的比例。局部用组合物的制备,在Remington′s Pharmaceutical Sciences,latest edition,(Mack Publishing)中有详细记载。
[0122]其它的药物方法可用于调节药效持续期。半衰期增加的制剂和缓释制剂,可通过使用聚合物结合、复合或吸收本文记载的蛋白而获得。控制递送和/或增加半衰期,可通过选择合适大分子(例如多糖、聚酯、聚氨基酸、聚乙烯吡咯烷酮均聚物、乙烯醋酸乙烯酯、甲基纤维素、或羧甲基纤维素,以及丙烯酰胺,例如N-(2-羟丙基)甲基丙烯酰胺、大分子的合适浓度、以及整合的方法而获得,以控制释放。
[0123]另一控制缓释制剂药效持续期或半衰期的可能方法是,将嵌合蛋白或其功能衍生物整合至多聚物材料颗粒中,例如聚酯、聚酰胺、聚氨基酸、水凝胶、聚(乳酸)、乙烯醋酸乙烯共聚物、诸如PEG的共聚物胶束、以及聚(1-天冬酰胺)。
[0124]本文记载蛋白的半衰期,可用本领域已知方法将其与其它分子例如聚合物结合形成药物-聚合物缀合物而增加。例如,蛋白可结合至本领域已知的惰性聚合物分子,例如在称为“PEG化”的方法中的聚乙二醇(PEG)分子。PEG化由此可增加体内半衰期,从而增加蛋白分子治疗效果。PEG化也可降低蛋白分子潜在抗原性。PEG化也能增强蛋白溶解度,从而提高其治疗效果。所用PEGs可为线性或支链。
[0125]PEG分子可被官能团修饰,例如Harris et al.,(9)所示,其全部内容在此明确通过援引引入,而且蛋白的氨基末端或半胱氨酸残基(如果存在)或其中的其它连接氨基酸都可用于连接PEG,其中PEG分子可载有一种或多种类型蛋白中的一个或多个,或者蛋白可载有一个以上PEG分子。
[0126]“PEG化蛋白”是指本发明的蛋白,其具有的聚乙二醇(PEG)部分共价连接在蛋白的氨基酸残基或连接基团上。
[0127]“聚乙二醇”或“PEG”是指聚亚烷基二醇化合物或其衍生物,有或没有偶联剂或偶联/活化部分衍生化(例如硫醇、triflate、tresylate、氮丙啶、环氧乙烷,或者优选马来酰亚胺部分)。化合物,诸如马来酰亚胺单甲氧基PEG,是本发明示例性或活化PEG化合物。其它的聚亚烷基二醇化合物,例如聚丙二醇,可用于本发明中。其它的合适聚合物缀合物包括但不限于例如,非多肽聚合物,下列类型的带电或中性聚合物:葡聚糖、多聚唾液酸或其它糖类聚合物,生物素衍生物,以及树状聚合物。术语PEG也包括其它聚亚烷基氧化物类的聚合物。
[0128]例如,PEG可连接于蛋白的任意N端氨基酸,和/或连接于N端氨基酸的下游氨基酸残基,例如赖氨酸、组氨酸、色氨酸、天冬氨酸、谷氨酸和半胱氨酸,或者本领域技术人员已知的其它此类氨基酸。例如,半胱氨酸-PEG化蛋白可通过将聚乙二醇与蛋白的半胱氨酸残基的含硫基团进行连接而制成。
[0129]化学修饰嵌合蛋白包含至少一个PEG部分,优选至少两个PEG部分直至不消除活性、结合于蛋白的最多数量PEG部分,例如,PEG部分可结合于氨基酸残基,优选在蛋白的N端部分或其附近。
[0130]结合于蛋白的PEG部分,分子量分布可从约200至20,000MW。优选地,PEG部分从约1,000至8,000MW,更优选从约3,250至5,000MW,最优选约5,000MW。
[0131]PEG分子共价结合于每个本发明化学修饰蛋白上的实际数量,可根据所期望的蛋白稳定性(即血清半衰期)而大幅变化。
[0132]本文构思的蛋白可用,例如(但不限于)美国专利Nos.,4,179,337;5,382,657;5,972,885;6,177,087;6,165,509;5,766,897;和6,217,869公开的技术连接于PEG分子;该各文献说明书和附图在此明确通过援引引入。此外,嵌合蛋白的血清半衰期可通过用额外氨基酸或多肽延长嵌合蛋白而增加,例如通过添加数个连续sTF结构域,或者通过附着与治疗个体相容的合适蛋白,例如人类个体的人血清白蛋白。
[0133]此外,也可将嵌合蛋白包埋于制备的微胶囊中,例如,通过团聚技术或界面聚合技术制备(例如分别为羟甲基纤维素或明胶微胶囊和聚-(甲基丙烯酸甲酯)微胶囊),或包埋于胶状药物递送系统(例如脂质体、白蛋白微球、微乳液、纳米颗粒、以及纳米胶囊)或巨乳液(macroemulsions)中。这些技术在最新版本的Remington′s Pharmaceutical Sciences中有公开。
[0134]美国专利4,789,734记载了脂质体中包埋生化物质的方法,其在此明确通过援引引入。总体而言,将物质溶于水溶液,加入适当的磷脂和脂质,如果需要,加入表面活性剂,然后将物质进行必要的透析或超声。G.Gregoriadis(10)对已知方法进行了综述。聚合物或蛋白形成的微球,是本领域技术人员公知的,其能定制以通过胃肠道直接进入血流。或者,可整合试剂,而且微球或微球复合物植入,以缓释一定时期,范围从数天至数月。参见例如美国专利4,906,474;4,925,673;和3,625,214,它们在此引入作为参考。
[0135]将组合物用作可注射物质时,其能制成常规注射载体形式。合适的载体包括生理相容和药学上可接受的磷酸盐缓冲盐水,优选等渗。
[0136]对于本发明冻干产品的重构而言,可使用无菌稀释液,所述稀释液可含有接近生理条件的通常接受的物质和/或官方规定所要求的物质。在这方面,无菌稀释液可包含缓冲剂以获得生理可接受的pH,例如氯化钠、盐水、磷酸盐冲盐水、和/或其它生理学可接受并且/或者可安全使用的物质。一般而言,人体静脉注射物质应与食品药品监督管理局所确立的规则一致,其可由本领域人员获得。
[0137]药物组合物也可以是含上述多种相同物质的水溶液形式,用于重构冻干产品。
[0138]化合物也可以药学可接受的酸或碱加成盐给予,所述盐通过与无机酸反应,例如盐酸、氢溴酸、高氯酸、硝酸、硫氰酸、硫酸、和磷酸,以及与有机酸反应,例如甲酸、乙酸、丙酸、羟基乙酸、乳酸、丙酮酸、草酸、丙二酸、琥珀酸、马来酸、和反丁烯二酸,或者通过与无机碱反应,例如氢氧化钠、氢氧化铵、氢氧化钾,以及通过与有机碱反应,例如单-,二-,三烷基和芳基胺、以及取代的乙醇胺,而形成。
[0139]如上所述,本发明化合物可并入用于治疗目的的药物制剂。然而,术语“药物制剂”在本文中广义意指包括含有本发明嵌合蛋白组合物的制剂,其不仅用于治疗目的,而且用于本领域已知的试剂或诊断目的,或者用于组织培养目的。用于治疗应用的药物制剂,应包含“药学上可接受的”或“治疗有效量的”嵌合蛋白,即该用量是预防性或治疗性健康措施所必需的。如果药物制剂用作试剂或诊断目的,那么它应包含试剂量或诊断量的嵌合蛋白。
[0140]本发明的范围并不受限于本文所记载的具体实施方式,因为这些实施方式意在说明本发明的某一方面,而任何功能等价实施方式都在本发明范围内。的确,本发明化合物和方法的各种变形以及本文记载和显示的那些,对本领域技术人员来说,根据前文记载是显而易见的。
[0141]本文引用的各参考文献、专利或公开文献,将其整体内容在此明确通过援引而引入。
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[0191]Ran S,Gao B,Duffy S,Watkins L,Rote N,Thorpe PE.Infarction of solid Hodgkin′stumors in mice by antibody-directed targeting of tissue factor to tumor vasculature.Cancer Res.1998;58:4646-4653.
[0192]Nilsson F,Kosmehl H,Zardi L,Neri D.Targeted delivery of tissue factor to the ED-Bdomain of fibronectin,a marker of angiogenesis,mediates the Infarction of solid tumorsin mice.Cancer Res.2001;61:711-716.
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[0196]Schatz S,Turecek PL,Fiedler C,et al.Evaluation of the haemostatic potential of factorVIII-heparin cofactor II hybrid proteins in a mouse model.Br J Haematol.2003;123:692-695.
[0197]Nelsestuen GL,Stone M,Martinez MB,Harvey SB,Foster D,Kisiel W.Elevatedfunction of blood clotting factor VIIa mutants that have enhanced affinity formembranes.Behavior in a diffusion-limited reaction.J Biol Chem.2001;276:39825-39831.
[0198]Rippmann JF,Pfizenmaler K,Mattes R,Rettig WJ,Moosmayer D.Fusion of the tissuefactor extracellular domain to a tumour stroma specific single-chain fragment variableantibody resuits in an antigen-specific coagulation-promoting molecule.Biochem J.2000;349 Pt 3:805-812.
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序列表
<110>Lind,Stuart E.
Ding,Wei-Qun
Harrison,Roger G.
<120>具有磷脂酰丝氨酸结合结构域的嵌合蛋白
<130>5835.073wo
<150>60/659,938
<151>2005-03-09
<160>28
<170>Patentin version 3.3
<210>1
<211>651
<212>DNA
<213>人类
<400>1
actacaaata ctgtggcagc atataattta acttggaaat caactaattt caagacaatt 60
ttggagtggg aacccaaacc cgtcaatcaa gtctacactg ttcaaataag cactaagtca 120
ggagattgga aaagcaaatg cttttacaca acagacacag agtgtgacct caccgacgag 180
attgtgaagg atgtgaagca gacgtacttg gcacgggtct tctcctaccc ggcagggaat 240
gtggagagca ccggttctgc tggggagcct ctgtatgaga actccccaga gttcacacct 300
tacctggaga caaacctcgg acagccaaca attcagagtt ttgaacaggt gggaacaaaa 360
gtgaatgtga ccgtagaaga tgaacggact ttagtcagaa ggaacaacac tttcctaagc 420
ctccgggatg tttttggcaa ggacttaatt tatacacttt attattggaa atcttcaagt 480
tcaggagccg caacagccaa aacaaacact aatgagtttt tgattgatgt ggataaagga 540
gaaaactact gtttcagtgt tcaagcagtg attccctccc gaacagttaa ccggaagagt 600
acagacagcc cggtagagtg tatgggccag gagaaagggg aattcagaga a 651
<210>2
<211>217
<2112>PRT
<213>人类
<400>2
Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn
1 5 10 15
Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr
20 25 30
Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe
35 40 45
Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys Asp
50 55 60
Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly Asn
65 70 75 80
Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser Pro
85 90 95
Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln
100 105 110
Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu
115 120 125
Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Val
130 135 140
Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser
145 150 155 160
Ser Gly Ala Ala Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu Ile Asp
165 170 175
Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val Ile Pro
180 185 190
Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys Met
195 200 205
Gly Gln Glu Lys Gly Glu Phe Arg Glu
210 215
<210>3
<211>957
<212>DNA
<213>人类
<400>3
gcacaggttc tcagaggcac tgtgactgac ttccctggat ttgatgagcg ggctgatgca 60
gaaactcttc ggaaggctat gaaaggcttg ggcacagatg aggagagcat cctgactctg 120
ttgacatccc gaagtaatgc tcagcgccag gaaatctctg cagcttttaa gactctgttt 180
ggcagggatc ttctggatga cctgaaatca gaactaactg gaaaatttga aaaattaatt 240
gtggctctga tgaaaccctc tcggctttat gatgcttatg aactgaaaca tgccttgaag 300
ggagctggaa caaatgaaaa agtactgaca gaaattattg cttcaaggac acctgaagaa 360
ctgagagcca tcaaacaagt ttatgaagaa gaatatggct caagcctgga agatgacgtg 420
gtgggggaca cttcagggta ctaccagcgg atgttggtgg ttctccttca ggctaacaga 480
gaccctgatg ctggaattga tgaagctcaa gttgaacaag atgctcaggc tttatttcag 540
gctggagaac ttaaatgggg gacagatgaa gaaaagttta tcaccatctt tggaacacga 600
agtgtgtctc atttgagaaa ggtgtttgac aagtacatga ctatatcagg atttcaaatt 660
gaggaaacca ttgaccgcga gacttctggc aatttagagc aactactcct tgctgttgtg 720
aaatctattc gaagtatacc tgcctacctt gcagagaccc tctattatgc tatgaaggga 780
gctgggacag atgatcatac cctcatcaga gtcatggttt ccaggagtga gattgatctg 840
tttaacatca ggaaggagtt taggaagaat tttgccacct ctctttattc catgattaag 900
ggagatacat ctggggacta taagaaagct cttctgctgc tctgtggaga agatgac 957
<210>4
<211>319
<212>PRT
<213>人类
<400>4
Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe Asp Glu
1 5 10 15
Arg Ala Asp Ala Glu Thr Leu Arg Lys Ala Met Lys Gly Leu Gly Thr
20 25 30
Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg Sar Asn Ala Gln
35 40 45
Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu Phe Gly Arg Asp Leu
50 55 60
Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly Lys Phe Glu Lys Leu Ile
65 70 75 80
Val Ala Leu Met Lys Pro Ser Arg Leu Tyr Asp Ala Tyr Glu Leu Lys
85 90 95
His Ala Leu Lys Gly Ala Gly Thr Asn Glu Lys Val Leu Thr Glu Ile
100 105 110
Ile Ala Ser Arg Thr Pro Glu Glu Leu Arg Ala Ile Lys Gln Vel Tyr
115 120 125
Glu Glu Glu Tyr Gly Ser Ser Leu Glu Asp Asp Val Val Gly Asp Thr
130 135 140
Ser Gly Tyr Tyr Gln Arg Met Leu Val Val Leu Leu Gln Ala Asn Arg
145 150 155 160
Asp Pro Asp Ala Gly Ile Asp Glu Ala Gln Val Glu Gln Asp Ala Gln
165 170 175
Ala Leu Phe Gln Ala Gly Glu Leu Lys Trp Gly Thr Asp Glu Glu Lys
180 185 190
Phe Ile Thr Ile Phe Gly Thr Arg Ser Val Ser His Leu Arg Lys Val
195 200 205
Phe Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr Ile
210 215 220
Asp Arg Glu Thr Ser Gly Asn Leu Glu Gln Leu Leu Leu Ala Val Val
225 230 235 240
Lys Ser Ile Arg Ser Ile Pro Ala Tyr Leu Ala Glu Thr Leu Tyr Tyr
245 250 255
Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr Leu Ile Arg Val Met
260 265 270
Val Ser Arg Ser Glu Ile Asp Leu Phe Asn Ile Arg Lys Glu Phe Arg
275 280 285
Lys Asn Phe Ala Thr Ser Leu Tyr Ser Met Ile Lys Gly Asp Thr Ser
290 295 300
Gly Asp Tyr Lys Lys Ala Leu Leu Leu Leu Cys Gly Glu Asp Asp
305 310 315
<210>5
<211>45
<212>DNA
<213>人类
<400>5
ggtggaggcg gttcaggcgg tggaggttca ggaggtggcg gatca 45
<210>G
<211>15
<212>PRT
<213>人类
<400>6
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210>7
<211>29
<212>DNA
<213>人类
<400>7
ccgctcgaga ccaccaccac caccactga 29
<210>8
<211>9
<212>PRT
<213>人类
<400>8
Pro Leu Glu His His His His His His
1 5
<210>9
<211>652
<212>DNA
<213>人类
<400>9
actacaaata ctgtggcagc atataattta acttggaaat caactaattt caagacaatt 60
ttggagtggg aacccaaacc cgtcaatcaa gtctacactg ttcaaataag cactaagtca 120
ggagattgga aaagcaaatg cttttacaca acagacacag agtgtgacct caccgacgag 180
attgtgaagg atgtgaagca gacgtacttg gcacgggtct tctcctaccc ggcagggaat 240
gtggagagca ccggttctgc tggggagcct ctgtatgaga actccccaga gttcacacct 300
tacctggaga caaacctcgg acagccaaca attcagagtt ttgaacaggt gggaacaaaa 360
gtgaatgtga ccgtagaaga tgaacggact ttagtcagaa ggaacaacac tttcctaagc 420
ctccgggatg tttttggcaa ggacttaatt tatacacttt attattggaa atcttcaagt 480
tcaggaagcc gcaacagcca aaacaaacac taatgagttt ttgattgatg tggataaagg 540
agaaaactac tgtttcagtg ttcaagcagt gattccctcc cgaacagtta accggaagag 600
tacagacagc ccggtagagt gtatgggcca ggagaaaggg gaattcagag aa 652
<210>10
<211>217
<212>PRT
<213>人类
<400>10
Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn
1 5 10 15
Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr
20 25 30
Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe
35 40 45
Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys Asp
50 55 60
Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly Asn
65 70 75 80
Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser Pro
85 90 95
Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln
100 105 110
Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu
115 120 125
Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Val
130 135 140
Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser
145 150 155 160
Ser Gly Ala Ala Thr Ala Lys Thr Asn Thr Asn Glu Ala Ala Ile Asp
165 170 175
Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val Ile Pro
180 185 190
Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys Met
195 200 205
Gly Gln Glu Lys Gly Glu Phe Arg Glu
210 215
<210>11
<211>652
<212>DNA
<213>人类
Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Vel
130 135 140
Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser
145 150 155 160
Ser Gly Glu Glu Thr Ala Lys Thr Asn Thr Asn Glu Ala Ala Ile Asp
165 170 175
Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val Ile Pro
180 185 190
Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys Met
195 200 205
Gly Gln Glu Lys Gly Glu Phe Arg Glu
210 215
<210>13
<211>652
<212>DNA
<213>人类
<400>13
actacaaata ctgtggcagc atataattta acttggaaat caactaattt caagacaatt 60
ttggagtggg aacccaaacc cgtcaatcaa gtctacactg ttcaaataag cactaagtca 120
ggagattgga aaagcaaatg cttttacaca acagacacag agtgtgacct caccgacgag 180
attgtgaagg atgtgaagca gacgtacttg gcacgggtct tctcctaccc ggcagggaat 240
gtggagagca ccggttctgc tggggagcct ctgtatgaga actccccaga gttcacacct 300
tacctggaga caaacctcgg acagccaaca attcagagtt ttgaacaggt gggaacaaaa 360
gtgaatgtga ccgtagaaga tgaacggact ttagtcagaa ggaacaacac tttcctaagc 420
ctccgggatg tttttggcaa ggacttaatt tatacacttt attattggaa atcttcaagt 480
tcaggaacaa cagacagcca aaacaaacac taatgagttt ttgattgatg tggataaagg 540
agaaaactac tgtttcagtg ttcaagcagt gattccctcc cgaacagtta accggaagag 600
tacagacagc ccggtagagt gtatgggcca ggagaaaggg gaattcagag aa 652
<210>14
<211>217
<212>PRT
<213>人类
<400>14
Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn
<400>11
actacaaata ctgtggcagc atataattta acttggaaat caactaattt caagacaatt 60
ttggagtggg aacccaaacc cgtcaatcaa gtctacactg ttcaaataag cactaagtca 120
ggagattgga aaagcaaatg cttttacaca acagacacag agtgtgacct caccgacgag 180
attgtgaagg atgtgaagca gacgtacttg gcacgggtct tctcctaccc ggcagggaat 240
gtggagagca ccggttctgc tggggagcct ctgtatgaga actccccaga gttcacacct 300
tacctggaga caaacctcgg acagccaaca attcagagtt ttgaacaggt gggaacaaaa 360
gtgaatgtga ccgtagaaga tgaacggact ttagtcagaa ggaacaacac tttcctaagc 420
ctccgggatg tttttggcaa ggacttaatt tatacacttt attattggaa atcttcaagt 480
tcaggaagaa gagacagcca aaacaaacac taatgagttt ttgattgatg tggataaagg 540
agaaaactac tgtttcagtg ttcaagcagt gattccctcc cgaacagtta accggaagag 600
tacagacagc ccggtagagt gtatgggcca ggagaaaggg gaattcagag aa 652
<210>12
<211>217
<212>PRT
<213>人类
<400>12
Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn
1 5 10 15
Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr
20 25 30
Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe
35 40 45
Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys Asp
50 55 60
Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly Asn
65 70 75 80
Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser Pro
85 90 95
Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln
100 105 110
Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu
115 120 125
1 5 10 15
Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr
20 25 30
Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe
35 40 45
Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys Asp
50 55 60
Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly Asn
65 70 75 80
Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser Pro
85 90 95
Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln
100 105 110
Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu
115 120 125
Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Val
130 135 140
Phe Gly Lys Asp Lou Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser
145 150 155 160
Ser Gly Gln Gln Thr Ala Lys Thr Asn Thr Asn Glu Ala Ala Ile Asp
165 170 175
Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val Ile Pro
180 185 190
Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys Met
195 200 205
Gly Gln Glu Lys Gly Glu Phe Arg Glu
210 215
<210>15
<211>39
<212>DNA
<213>人类
<400>15
ctcgggaggta gtggcatcta ccgtagccga tcactagag 39
<210>16
<11>12
<212>PRT
<213>人类
<400>16
Leu Gly Gly Ser Gly Ile Tyr Arg Ser Arg Ser Leu
1 5 10
<210>17
<211>18
<212>DNA
<213>人类
<400>17
ctcgttcctc gtggaagt 18
<210>18
<211>6
<212>PRT
<213>人类
<400>18
Leu Val Pro Arg Gly Ser
1 5
<210>19
<211>2400
<212>DNA
<213>人类
<400>19
ggcactacaa atactgtggc agcatataat ttaacttgga aatcaactaa tttcaagaca 60
attttggagt gggaacccaa acccgtcaat caagtctaca ctgttcaaat aagcactaag 120
tcaggagatt ggaaaagcaa atgcttttac acaacagaca cagagtgtga cctcaccgac 180
gagattgtga aggatgtgaa gcagacgtac ttggcacggg tcttctccta cccggcaggg 240
aatgtggaga gcaccggttc tgctggggag cctctgtatg agaactcccc agagttcaca 300
ccttacctgg agacaaacct cggacagcca acaattcaga gttttgaaca ggtgggaaca 360
aaagtgaatg tgaccgtaga agatgaacgg actttagtca gaaggaacaa cactttccta 420
agcctccggg atgtttttgg caaggactta atttatacac tttattattg gaaatcttca 480
agttcaggaa agaaaacagc caaaacaaac actaatgagt ttttgattga tgtggataaa 540
ggagaaaact actgtttcag tgttcaagca gtgattccct cccgaacagt taaccggaag 600
agtacagaca gcccggtaga gtgtatgggc caggagaaag gggaattcag agaaggtgga 660
ggcggttcag gcggtggagg ttcaggaggt ggcggatcaa agcttggcgc ttcaggcact 720
acaaatactg tggcagcata taatttaact tggaaatcaa ctaatttcaa gacaattttg 780
gagtgggaac ccaaacccgt caatcaagtc tacactgttc aaataagcac taagtcagga 840
gattggaaaa gcaaatgctt ttacacaaca gacacagagt gtgacctcac cgacgagatt 900
gtgaaggatg tgaagcagac gtacttggca cgggtcttct cctacccggc agggaatgtg 960
gagagcaccg gttctgctgg ggagcctctg tatgagaact ccccagagtt cacaccttac 1020
ctggagacaa acctcggaca gccaacaatt cagagttttg aacaggtggg aacaaaagtg 1080
aatgtgaccg tagaagatga acggacttta gtcagaagga acaacacttt cctaagcctc 1140
cgggatgttt ttggcaagga cttaatttat acactttatt attggaaatc ttcaagttca 1200
ggaaagaaaa cagccaaaac aaacactaat gagtttttga ttgatgtgga taaaggagaa 1260
aactactgtt tcagtgttca agcagtgatt ccctcccgaa cagttaaccg gaagagtaca 1320
gacagcccgg tagagtgtat gggccaggag aaaggggaat tcagagaagg tggaggcggt 1380
tcaggcggtg gaggttcagg aggtggcgga tcagcacagg ttctcagagg cactgtgact 1440
gacttccctg gatttgatga gcgggctgat gcagaaactc ttcggaaggc tatgaaaggc 1500
ttgggcacag atgaggagag catcctgact ctgttgacat cccgaagtaa tgctcagcgc 1560
caggaaatct ctgcagcttt taagactctg tttggcaggg atcttctgga tgacctgaaa 1620
tcagaactaa ctggaaaatt tgaaaaatta attgtggctc tgatgaaacc ctctcggctt 1680
tatgatgctt atgaactgaa acatgccttg aagggagctg gaacaaatga aaaagtactg 1740
acagaaatta ttgcttcaag gacacctgaa gaactgagag ccatcaaaca agtttatgaa 1800
gaagaatatg gctcaagcct ggaagatgac gtggtggggg ccacttcagg gtactaccag 1860
cggatgttgg tggttctcct tcaggctaac agagaccctg atgctggaat tgatgaagct 1920
caagttgaac aagatgctca ggctttattt caggctggag aacttaaatg ggggacagat 1980
gaagaaaagt ttatcaccat ctttggaaca cgaagtgtgt ctcatttgag aaaggtgttt 2040
gacaagtaca tgactatatc aggatttcaa attgaggaaa ccattgaccg cgagacttct 2100
ggcaatttag agcaactact ccttgctgtt gtgaaatcta ttcgaagtat acctgcctac 2160
cttgcagaga ccctctatta tgctatgaag ggagctggga cagatgatca taccctcatc 2220
agagtcatgg tttccaggag tgagattgat ctgtttaaca tcaggaagga gtttaggaag 2280
aattttgcca cctctcttta ttccatgatt aagggagata catctgggga ctataagaaa 2340
gctcttctgc tgctctgtgg agaagatgac ccgctcgagc accaccacca ccaccactga 2400
<210>20
<211>804
<212>PRT
<213>人类
<400>20
Met Ala Gly Ala Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu
1 5 10 15
Thr Trp Lys Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys
20 25 30
Pro Val Asn Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp
35 40 45
Trp Lys Ser Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr
50 55 60
Asp Glu Ile Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe
65 70 75 80
Ser Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro
85 90 95
Leu Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu
100 105 110
Gly Gln Pro Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn
115 120 125
Val Thr Val Glu Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe
130 135 140
Leu Ser Leu Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr
145 150 155 160
Tyr Trp Lys Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr
165 170 175
Asn Glu Phe Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser
180 185 190
Val Gln Ala Val Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp
195 200 205
Ser Pro Val Glu Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Leu
225 230 235 240
Gly Ala Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp
245 250 255
Lys Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val
260 265 270
Asn Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys
275 280 285
Ser Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu
290 295 300
Ile Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr
305 310 315 320
Pro Ala Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr
325 330 335
Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln
340 345 350
Pro Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr
355 360 365
Val Glu Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser
370 375 380
Leu Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp
385 390 395 400
Lys Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu
405 410 415
Phe Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln
420 425 430
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Ser Ala Ala Phe Lys Thr Leu Phe Gly Arg Asp Leu Leu Asp Asp Leu
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Lys Ser Glu Leu Thr Gly Lys Phe Glu Lys Leu Ile Val Ala Leu Met
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Gln Arg Met Leu Val Val Leu Leu Gln Ala Asn Arg Asp Pro Asp Ala
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Gly Ile Asp Glu Ala Gln Val Glu Gln Asp Ala Gln Ala Leu Phe Gln
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Ala Gly Glu Leu Lys Trp Gly Thr Asp Glu Glu Lys Phe Ile Thr Ile
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Phe Gly Thr Arg Ser Val Ser His Leu Arg Lys Val Phe Asp Lys Tyr
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Ser Gly Asn Leu Glu Gln Leu Leu Leu Ala Val Val Lys Ser Ile Arg
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Thr Ser Leu Tyr Ser Met Ile Lys Gly Asp Thr Ser Gly Asp Tyr Lys
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His His His His
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cagcactgtg aaatagataa gtcaaaaacc 150
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cccaagtctt gggttctctg aattcccctt tctc 34
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Claims (24)
1、在动物中诱导止血或血栓形成的方法,包括:
提供含磷脂酰丝氨酸结合结构域和可溶性组织因子结构域的嵌合蛋白,其中所述可溶性组织因子结构域包含SEQ ID NO:2或其有人组织因子活性的突变体或部分;以及
将该嵌合蛋白以有效量给予所述动物,所述的量有效促进所述动物血管化区域的凝结。
2、权利要求1的方法,其中动物的血管化区域为非癌性的。
3、权利要求1的方法,其中磷脂酰丝氨酸结合结构域包括膜联蛋白。
4、权利要求1的方法,其中磷脂酰丝氨酸结合结构域包括人膜联蛋白V。
5、权利要求1的方法,其中SEQ ID NO:2的嵌合蛋白突变体包括至少一个选自下组的取代:在13,131,163,164或183位丙氨酸取代,在42或138位天冬酰胺取代,在48位色氨酸取代,在52位丝氨酸取代,在128位天冬氨酸取代,在129,163或164位谷氨酰胺取代,以及在163或164位谷氨酸取代。
6、权利要求1的方法,其中嵌合蛋白进一步在SEQ ID NO:2氨基末端包括ser-gly或gly。
7、权利要求1的方法,其中嵌合蛋白进一步包括连接可溶性组织因子结构域和磷脂酰丝氨酸结合结构域的连接子。
8、权利要求1的方法,其中动物血管化区域出血是因为:存在抗凝药物;因外伤、输注、抗体或先天条件而缺乏凝血因子;肝脏疾病;血管或头部损伤;胃肠道状况,包括胃炎、溃疡、和食道脉管曲张;膀胱炎,子宫内膜炎;或者因血小板不足或血小板功能异常而出血。
9、权利要求1的方法,进一步包括将嵌合蛋白与重组VIIa因子一起给药。
10、权利要求1的方法,其中血管化区域包括毛细管扩张、动脉畸形、静脉畸形、毛细血管畸形、淋巴管畸形、动静脉畸形、血管瘤、或动脉瘤。
11、权利要求1的方法,其中嵌合蛋白被局部给予,以诱导局部血栓形成。
12、权利要求1的方法,进一步包括紧接嵌合蛋白给予步骤,给予药物、化学物、电刺激、或辐射。
13、权利要求1的方法,其中嵌合蛋白进一步包括治疗化合物或物质,其中嵌合蛋白用于递送该治疗化合物或物质至需要治疗的位点。
14、权利要求13的方法,其中治疗化合物或物质被递送至的血管化区域为肿瘤或异常血管床。
15、权利要求13的方法,其中治疗物质或化合物是化疗的或放射性的。
16、在动物中诱导止血或血栓形成的药物或组合物,其包括:
含可溶性组织因子结构域和磷脂酰丝氨酸结合结构域的嵌合蛋白,其中所述可溶性组织因子结构域包含SEQ ID NO:2或其有人组织因子活性的突变体或部分,并且,其中嵌合蛋白置于药学上可接受的载体中。
17、权利要求16的药物或组合物,其中磷脂酰丝氨酸结合结构域包括膜联蛋白。
18、权利要求17的药物或组合物,其中膜联蛋白是膜联蛋白V。
19、权利要求16的药物或组合物,其中SEQ ID NO:2的嵌合蛋白突变体包括至少一个选自下组的取代:在13,131,163,164或183位丙氨酸取代,在42或138位天冬酰胺取代,在48位色氨酸取代,在52位丝氨酸取代,在128位天冬氨酸取代,在129,163或164位谷氨酰胺取代,以及在163或164位谷氨酸取代。
20、权利要求16的药物或组合物,其中嵌合蛋白进一步在SEQ ID NO:2氨基末端包括ser-gly或gly。
21、权利要求16的药物或组合物,其中嵌合蛋白进一步包括连接可溶性组织因子结构域和磷脂酰丝氨酸结合结构域的连接子。
22、权利要求16的药物或组合物,用于治疗动物血管化区域,其中动物血管化区域出血是因为:存在抗凝药物;因外伤、输注、抗体或先天条件而缺乏凝血因子;肝脏疾病;血管或头部损伤;胃肠道状况,包括胃炎、溃疡、和食道脉管曲张;膀胱炎,子宫内膜炎;或者因血小板不足或血小板功能异常而出血,或者包括毛细管扩张、动脉畸形、静脉畸形、毛细血管畸形、淋巴管畸形、动静脉畸形、血管瘤、或动脉瘤。
23、权利要求16的药物或组合物,其中嵌合蛋白进一步包括治疗化合物或物质,其中嵌合蛋白用于递送该治疗化合物或物质至需要治疗的位点。
24、权利要求23的药物或组合物,其中所述治疗物质或化合物是化疗的或放射性的。
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EP (1) | EP1877437A4 (zh) |
CN (1) | CN101405017A (zh) |
AU (1) | AU2006220513A1 (zh) |
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Cited By (5)
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CN102294018A (zh) * | 2011-08-01 | 2011-12-28 | 南京大学 | 一种阿霉素与膜联蛋白v偶联物及其制备方法和应用 |
CN110023339A (zh) * | 2016-09-23 | 2019-07-16 | Csl有限公司 | 凝血因子结合蛋白及其应用 |
CN110945020A (zh) * | 2017-03-21 | 2020-03-31 | 布坦坦基金会 | 重组蛋白及其片段、生产所述重组蛋白的方法、合成基因以及重组蛋白的用途 |
WO2021037230A1 (zh) * | 2019-08-30 | 2021-03-04 | 苏州亚宝药物研发有限公司 | 膜联蛋白a5的用途 |
WO2023165624A1 (zh) * | 2022-03-04 | 2023-09-07 | 苏州亚宝药物研发有限公司 | 治疗颅脑损伤的方法 |
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- 2006-03-09 WO PCT/US2006/008541 patent/WO2006096828A2/en active Application Filing
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WO2006096828A3 (en) | 2008-11-20 |
EP1877437A2 (en) | 2008-01-16 |
US20080280831A1 (en) | 2008-11-13 |
CA2608070A1 (en) | 2006-09-14 |
US7393833B2 (en) | 2008-07-01 |
US7807644B2 (en) | 2010-10-05 |
AU2006220513A1 (en) | 2006-09-14 |
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