CN1406249B - 增加基于抗体的融合蛋白的循环半衰期 - Google Patents
增加基于抗体的融合蛋白的循环半衰期 Download PDFInfo
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Abstract
公开了用于增加基于抗体的融合蛋白循环半衰期的组合物和方法。公开的方法和组合物依赖于改变基于抗体的融合蛋白中抗体部分和融合的蛋白部分之间的接合区的氨基酸序列。接合区带有氨基酸序列改变的基于抗体之融合蛋白当施予哺乳动物时,具有更长的循环半衰期。公开的方法和组合物对于降低哺乳动物肿瘤大小和转移特别有效。
Description
相关申请
本申请权利要求享有2000年2月11日申请的U.S.S.N.60/181,768的优先权,这里引入其公开作为参考。
发明领域
本发明涉及融合蛋白。更特定的,本发明涉及增加基于抗体的融合蛋白的循环半衰期。
发明背景
治疗人类疾病的抗体的使用已经很好地建立,随着遗传工程的引入变得越来越复杂。已开发了几种技术提高抗体的,功用。这些包括:(1)通过细胞融合产生“杂交瘤”,或由产生抗体的细胞,分子克隆抗体重(H)链和轻(L)链而产生的单克隆抗体;(2)其它分子与抗体接合以将它们传递到体内优选部位,如,放射性同位素、毒性药物、蛋白毒素和细胞因子;(3)处理抗体效应物功能以增加或减少生物活性;(4)其它蛋白如毒素和细胞因子与抗体在基因水平连接以产生基于抗体的融合蛋白;和(5)一组或多组抗体联合区在基因水平连接以产生双特异性抗体。
蛋白可通过使用本领域已知的化学或遗传操作方法连接起来。参见,如,Gillies等,Proc.Natl.Acad.Sci.USA 89:14281432(1992);和美国专利号5,650,150。
然而,重组产生的基于抗体的融合蛋白的功用可能受其在体内被从循环中快速清除的限制。例如,已表明抗体-细胞因子融合蛋白在体内的循环半衰期明显低于游离抗体。当检测多种抗体-细胞因子融合蛋白时,Gillies等报道了所有检测的融合蛋白的α期(分配期)半衰期少于1.5小时。的确,大多数基于抗体的融合蛋白两个小时被清除至游离抗体血清浓度的10%。参见,例如,Gillies等,BIOCONJ.CHEM.4:230-235(1993)。近来,已表明与Fc受体结合亲和力降低之基于抗体的融合蛋白的半衰期增加。也表明与Fc受体结合亲和力降低妨碍抗体效应物的某些功能,如抗体依赖性细胞毒性(ADCC),但不妨碍其它功能如补体固定或抗原结合。参见Gillies等,Cancer Res.59(9):2159-66(1999)。
有些情况下,例如癌症或病毒疾病的治疗中,期望维持抗体效应物的功能和长的循环半衰期。因此,本领域需要增加基于抗体的融合蛋白的体内循环半衰期的另外方法。
发明概述
免疫球蛋白G(IgG)分子与多种类型的细胞受体包括特异性针对IgG类抗体的三类Fcγ受体(FcγR),即FcγRI、FcγRII和FcγRIII相互作用。它们也与FcRp类受体以pH依赖性方式相互作用,在中性pH少量结合或不结合,但在pH 6.0时高度结合。
抗体的血清半衰期受抗体所结合Fc受体(FcR)和Fc保护受体(FcRp)能力的影响。免疫球蛋白融合蛋白的血清半衰期也受例如结合到这种受体之能力的影响(Gillies等,Cancer Res.59:2159-66(1999))。
本发明公开了令人惊奇的观察结果,在含有免疫球蛋白(Ig)部分和非免疫球蛋白(非Ig)部分的融合蛋白内,接近两者的连接部分的氨基酸改变戏剧性地增加该融合蛋白的血清半衰期。这个观察结果令人惊奇,因为氨基酸改变影响了不同于Fc区与Fc受体和Fc保护受体相互作用的表面的蛋白表面。此外,甚至在已知Fc受体和Fc保护受体不主要决定融合蛋白的血清半衰期的情况下,本发明的氨基酸改变也具有它们的这种作用。因此,本发明的氨基酸改变可以和影响与Fc受体和/或Fc保护受体相互作用的氨基酸改变相组合以达到协同作用。
本发明提供了含免疫球蛋白的融合蛋白,其中不同于与Fc受体和Fc保护受体(FcRp)相互作用的Fc区表面的位点改变造成血清半衰期的提高。本发明还提供了血清半衰期提高的具有免疫球蛋白部分和第二个非免疫球蛋白蛋白之间融合的蛋白的制备方法。
融合蛋白氨基酸序列的改变主要在Ig部分和非Ig部分连接处。相对于Ig重链和非Ig蛋白天然产生的序列,融合蛋白的连接区含有的改变优选位于连接点的大约10个氨基酸内。更优选,氨基酸改变引起疏水性增加。甚至更优选,氨基酸改变包括抗体部分C末端赖氨酸变为疏水性氨基酸,如丙氨酸或亮氨酸。在优选方案中,本发明的融合蛋白包含Ig重链,优选位于第二个非Ig蛋白的N末端。
在本发明的另一个方案中,通过改变接触FcRp的Fc部分的相互作用表面,优化融合蛋白与FcRp的结合亲和力。已报道IgG与FcRp受体结合的重要序列位于CH2和CH3区。根据本发明,融合蛋白中融合连接的改变和与FcRp相互作用的Fc表面的改变相结合,产生协同作用。有些情况下,增加在pH 6时Fc部分与FcRp的相互作用可能有效,降低在pH 8时Fc部分与FcRp的相互作用可能也有效。这种修饰包括接触Fc受体所需残基的改变,或通过诱导构象改变来改变影响其它重链残基和FcRp受体间接触的其它改变。因此,在优选方案中,通过首先将Ig恒定区的编码序列与第二个非免疫球蛋白的蛋白连接,然后在IgG恒定区中位于选自或接近Ile 253,His 310和His 435的一个或多个氨基酸处引入突变(如点突变、缺失、插入,或基因重排),得到体内循环半衰期增加的基于抗体的融合蛋白。所得基于抗体的融合蛋白具有比未修饰融合蛋白更长的体内循环半衰期。
在某些情况下,使Fc部分的某些效应物功能变异有效。例如,可消除补体固定。此外,在另一组方案中,融合蛋白的Ig成分至少具有与FcγRI,FcγRII或FcγRIII中至少一个结合亲和力降低的IgG恒定区部分。例如,可使用IgG的γ4链代替γ1。这种改变具有的优点是γ4链与融合连接处的一个或多个突变协同作用,产生更长的血清半衰期。因此,也可以用IgG2代替IgG1。在本发明的可供选择的方案中,融合蛋白包括突变IgG1恒定区,例如具有Leu234,Leu235,Gly236,Gly237,Asn297或Pro331中一个或多个突变或缺失的IgG1恒定区。在本发明进一步的方案中,融合蛋白包括突变IgG3恒定区,例如具有Leu281,Leu282,Gly283,Gly284,Asn344,或Pro378中一个或多个突变或缺失的IgG3恒定区。然而,对于有些应用,保留Fc受体结合伴随的效应物功能如ADCC可能有用。
在优选方案中,融合蛋白的第二个非免疫球蛋白部分是细胞因子。这里使用的术语“细胞因子”描述可使具有该种细胞因子受体的细胞发生特异性生物反应的天然产生的或重组蛋白,其类似物,和其片段。优选的,细胞因子是可由细胞产生并分泌的蛋白质。优选细胞因子包括白细胞介素,如白细胞介素2(IL-2),IL-4,IL-5,IL-6,IL-7,IL-10,IL-12,IL-13,IL-14,IL-15,IL-16和IL-18,造血因子如粒细胞-巨噬细胞集落刺激因子(GM-CSF),粒细胞集落刺激因子(G-CSF)和红细胞生成素,肿瘤坏死因子(TNF)如TNFα,淋巴因子如淋巴毒素,代谢过程调节剂如leptin,干扰素α、干扰素例如干扰素β和干扰素γ,和细胞因子。优选,本发明的抗体-细胞因子融合蛋白显示细胞因子生物活性。
在可供选择的优选方案中,融合蛋白的第二个非免疫球蛋白部分是具有生物活性的配体结合蛋白。这种配体结合蛋白可以例如,(1)阻断受体-配体在细胞表面的相互作用;或(2)中和在血液液相中分子(如细胞因子)的生物活性。由此防止它达到其细胞靶。优选,配体结合蛋白包括CD4,CTLA-4,TNF受体,或白细胞介素受体如IL-1和IL-4受体。优选,本发明的抗体-受体融合蛋白显示配体结合蛋白的生物活性。
在另一个可供选择的优选方案中,融合蛋白的第二个非免疫球蛋白部分是蛋白毒素。优选,本发明的抗体-毒素融合蛋白显示蛋白毒素的毒素活性。
在其它优选方案中,融合蛋白的第二个非免疫球蛋白部分是激素、神经营养蛋白、体重调节剂、血清蛋白、凝集因子、蛋白酶、细胞外基质成分、血管生成因子、抗血管生成因子或另一种分泌蛋白或分泌结构域。例如,CD26、IgE受体、聚合IgA受体、其它抗体受体、因子VIII、因子IX、因子X、TrkA、PSA、PSMA、Flt-3配体、endostatin、制管张素和这些蛋白的结构域。
在其它方案中,第二个非免疫球蛋白部分是非人或非哺乳动物蛋白。例如,优选HIV gpl20、HIV Tat、其它病毒(如腺病毒)和RSV的表面蛋白,其它HIV成分,寄生虫表面蛋白(如疟疾抗原)和细菌表面蛋白。这些非人蛋白可以用作例如抗原,或因为它们具有有用活性。例如,第二个非免疫球蛋白部分可以是链激酶、葡激酶、尿激酶、组织纤溶酶原激活剂或具有有用酶活性的其它蛋白。
根据本发明,非免疫球蛋白部分可以是蛋白的一部分。优选,非Ig蛋白部分是基本保留完整蛋白的功能和结构特性的蛋白部分。在优选方案中,非Ig蛋白部分是这里描述的蛋白的功能或结构部分。
在优选方案中,基于抗体的融合蛋白包括靶抗原特异性的可变区和恒定区,二者任何一个通过肽键与第二个非免疫球蛋白的蛋白连接。恒定区可以是正常与可变区相联的恒定区,或不同的一个,如来自不同物种的可变区和恒定区。重链可以包括一个或多个CH1、CH2或CH3结构域的任何组合。优选,重链包括CH1、CH2和CH3结构域,更优选,仅有CH2和CH3结构域。在一个方案中,基于抗体的一种融合蛋白包括与重链和轻链可变区融合的Fv区。也包括在术语“融合蛋白”中的是具有包括来自不同物种构架区和可变区(即互补决定区)的结合域的构建体,如Winter等,英国专利号2,188,638中公开。基于抗体的融合蛋白包括优选地显示抗原结合特异性的可变区。在另一个优选方案中,基于抗体的融合蛋白进一步包括轻链。因此本发明提供了融合蛋白,其中抗体的抗原结合特异性和活性与第二个非免疫球蛋白的蛋白如细胞因子的有力生物活性结合。本发明的融合蛋白可用于将第二个非免疫球蛋白的蛋白传递给体内靶细胞,使第二个非免疫球蛋白的蛋白可以发挥局部的生物作用。
在可供选择的优选方案中,基于抗体的融合蛋白包括通过肽键与第二个非免疫球蛋白的蛋白联结的重链恒定区,但不包括重链可变区。因此本发明进一步提供了保留第二个非免疫球蛋白的蛋白的有力蛋白活性,但缺乏抗体的抗原结合特异性和活性的融合蛋白。
在优选方案中,融合蛋白包括含有二硫键联结的至少部分重链和第二个非Ig蛋白的两个嵌合链。
在优选方案中,本发明的融合蛋白有效治疗癌症、病毒感染、免疫紊乱,和有效增加特殊细胞类型的生长(包括增生)。
本发明的特征也包括编码上述融合蛋白的DNA构建体,和这些构建体转染的细胞系,如骨髓瘤。
这些和其它目的,同这里公开的本发明的优点和特征一起,将由下面的说明书、附图和权利要求变得更加明显。
附图简述
图1显示了KS-IL-2融合蛋白和实施例中描述的含有抗体重链C末端赖氨酸部分置换或其它改变的各种突变融合蛋白的药代动力学行为。采用检测IL-2(图1A)或人Fc(图1B)的ELISA测定抗体或融合蛋白的水平。
图2显示了带有γ1或γ4链的、在抗体重链C末端带有野生型赖氨酸或赖氨酸-丙氨酸突变的KS-IL-2融合蛋白的药代动力学特性。采用检测IL-2部分的ELISA测定抗体或融合蛋白的水平。
图3显示了人抗体与肿瘤坏死因子α(TNFα)融合的药代动力学特性。用检测人Fc区的ELISA测定融合蛋白的水平。所示的是完整抗体-TNFα融合蛋白(黑色菱形)的水平和另一个在抗体C末端部分赖氨酸删除的等同融合蛋白(灰色正方形)的水平。
图4显示了抗体-IL-2融合蛋白与富含FcγR类型受体的固定J774细胞膜的结合。所示的是结合无突变KS-IL-12融合蛋白(黑色菱形)和带有重链C末端赖氨酸变为丙氨酸突变的KS-IL-12融合蛋白(灰色正方形)。
图5显示了抗体-细胞因子融合蛋白对治疗含有CT26结肠癌细胞衍生的改造为表达EpCAM-KS抗原的人皮下肿瘤的Balb/C小鼠的作用。
发明详述
本发明提供了Ig和非Ig部分接合处具有一个或多个突变的抗体融合蛋白,该突变增加该融合蛋白的循环半衰期。本发明的突变融合蛋白具有优越的性能是它们的血清半衰期提高,但不影响抗体部分与体内两个已知的药代动力学决定受体:Fc受体和FcRp的相互作用。
一般来说,本发明基于抗体的融合蛋白包括连接于非免疫球蛋白(非Ig)蛋白之免疫球蛋白(Ig)蛋白的一部分,使得跨越Ig和非Ig蛋白接合区的氨基酸序列,与Ig和非Ig蛋白的野生型氨基酸序列相比,具有至少一个突变。
在一个方案中,至少一个突变在Ig部分的C末端区。在另一个方案中,至少一个突变在非Ig蛋白的N末端区。在进一步的方案中,融合蛋白含有在Ig部分C末端区的至少一个突变和在非Ig蛋白N末端区的至少一个突变。突变可以是点突变、插入、缺失或基因重排。在优选方案中,突变增加接合区的疏水性。例如,用不带电荷或疏水性氨基酸取代带电荷或可电离氨基酸(如用Ala,Leu,Gly,Trp或其它不带电荷或疏水性残基取代Lys,Arg或其它可电离残基)的突变。
在可选择的方案中,间隔肽或接头肽插在Ig和非Ig蛋白之间。优选间隔肽或接头肽是不带电荷,更优选非极性和或疏水性的。优选间隔或接头肽的长度在1个和大约100个氨基酸之间,更优选在1个和大约50个氨基酸之间,或在1个和大约25个氨基酸之间,和甚至更优选在1个和大约15个氨基酸之间。在本发明另一个方案中,融合蛋白的Ig和非Ig部分通过间隔或接头肽连接,且Ig和非Ig部分之一或两个都有至少一个突变。在本发明可供选择的方案中,Ig和非Ig部分被合成的间隔肽分隔,例如PNA间隔肽,优选不带电荷,更优选非极性,和或疏水性的。
根据本发明,免疫球蛋白(Ig)链是免疫球蛋白或包括可变区或恒定区结构域的免疫球蛋白的一部分。优选Ig链是免疫球蛋白重链的一部分,例如,能够结合预选细胞类型的免疫球蛋白可变区。在优选方案中,Ig链包括靶抗原特异性的可变区和恒定区。恒定区可以是正常可变区相联的恒定区,或不同的一个,例如,来自不同物种的可变区和恒定区。在更优选方案中,Ig链包括重链。重链可包括含有一个或多个CH1、CH2或CH3结构域的任何组合。优选,重链包括CH1、CH2和CH3结构域,更优选,仅有CH2和CH3结构域。在一个方案中,免疫球蛋白部分包括重链和轻链可变区融合的Fv区。
根据本发明,非免疫球蛋白的蛋白包括天然产生的蛋白,它不是免疫球蛋白,或合成或重组的不是免疫球蛋白的蛋白,或上述任何的片段。在优选方案中,非免疫球蛋白的蛋白包括功能域如配体结合域,酶域,调节域,或与一个或多个细胞因子相互作用的域。在可供选择的方案中,非免疫球蛋白域包括结构域或表位。
在优选方案中,Ig链通过基因融合与非Ig蛋白连接。优选,基因融合是合成的或重组的,且采用标准的化学合成或分子生物学技术产生。典型地,以部分基因融合构建体形式引入突变。可供选择地,可以使用已知的诱变方法后来引入突变(例如通过使基因融合构建体接触辐射,或化学或生物诱变)。
根据本发明,接合区是融合蛋白中融合蛋白的Ig和非Ig部分之间接合点周围的区域。在优选方案中,接合区包括Ig部分C末端部分和非Ig部分N末端部分。在一个方案中,接合区也包括插在Ig和非Ig部分接合点的间隔或接头肽。
根据本发明的优选方案,Ig部分的突变在Ig部分C末端部分,优选来自Ig部分C末端的大约100个残基内,更优选大约50个残基内,或大约25个残基内,甚至更优选大约10个残基内。
根据本发明的优选方案,非Ig部分的突变在非Ig部分N末端部分,优选来自非Ig部分N末端的大约100个残基内,更优选大约50个残基内,或大约25个残基内,甚至更优选大约10个残基内。
在本发明的优选方案中,突变在Ig部分的C末端部分,但突变不在Ig蛋白与Fc受体(FcR)或FcRp相互作用的部分。
根据本发明的具有突变的抗体融合蛋白,与没有突变的相应融合蛋白的循环半衰期相比,体内循环半衰期增加。抗体融合蛋白的循环半衰期可用检测发挥作用时间的融合蛋白血清水平来测定。实验证据表明本发明的优选突变不依赖于FcR或FcRp的相互作用。首先,增加融合蛋白循环半衰期的优选突变不影响抗体三维结构上结合FcR或FcRp的部分相互作用表面的区域。其次,本发明的优选突变可引起血清半衰期提高,甚至在用IgG-γ4链去除与FcR的相互作用和药代动力学研究FcRp缺陷的β2微球蛋白突变小鼠去除与FcRp的相互作用时亦然。第三,本发明的优选突变不明显影响Ig融合蛋白与J774细胞上FcR的结合。
定点诱变分析表明与Fc受体相互作用的Fc表面接近CH2结构域的铰链区。与FcR相互作用的Fc表面区在三维上与Fc的C末端距离很远。相似的分析表明与氨基酸残基相互作用的FcRp位于CH2和CH3结构域的交界面。
FcRp与其受体在酸性pH(pH 6.0)的结合亲和力比中性或弱碱性pH(pH 7.4)时高得多。这与FcRp在保护含Fc分子如内体细胞内在化的抗体中的作用一致。与溶酶体融合后这些细胞隔室变酸,酸性蛋白酶降解了其蛋白成分。此过程中与结合FcRp的膜结合可防止抗体降解,允许它再循环到细胞外(回到循环中)或穿过细胞层(称作胞转的过程)。后一过程允许在中肠酸性环境下消化牛奶后,IgG通过新生儿肠粘膜。
Fc/FcRp复合体结构表明FcRp与Fc区的侧面结合,接触CH2和CH3结构域,接触区域不是特别接近Fc区的C末端。因此,期望恰好Fc的C末端区的改变不改变与FcRp的相互作用。
不受任何特殊理论的限制,认为根据本发明的增加融合蛋白循环半衰期的融合接合区的突变也减少蛋白酶切割实验中融合蛋白的切割,如实施例15阐述。进一步认为蛋白酶消化可能助于形成完整蛋白(包括融合蛋白)的消失。因此,抵抗蛋白酶可能直接有助于改善蛋白的药代动力学。也进一步认为蛋白酶消化非变性蛋白涉及蛋白酶接近正确构象的暴露序列,和识别氨基酸的特殊序列。因此,影响蛋白的一般构象并因此影响蛋白酶接近其切割位点的融合接合区的突变,可能有助于抵抗蛋白酶并有助于改善药代动力学。此外,改变特定蛋白酶识别序列的突变可能有助于抵抗蛋白酶并有助于改善药代动力学。
本发明突变的一个特征是它们可与抗体部分中的其它突变或置换组合或与Ig部分的其它特性组合,协同调节血清半衰期。例如,本发明增加抗体融合蛋白循环半衰期的一个或多个突变可与影响该抗体融合蛋白和FcR或FcRp间相互作用的突变组合。
此外,本发明的突变可用于多种广泛的抗体部分和多种广泛的非Ig融合伙伴。免疫球蛋白包括IgG,IgM,IgA,IgD和IgE。非Ig融合伙伴包括细胞因子,其它分泌蛋白,酶,或跨膜受体的可溶片段,如配体结合域。
根据本发明,通过Fc部分本身内部的修饰可进一步加强基于抗体的融合蛋白之体内循环半衰期的增加。这些可以是包括或邻近Ile253,His 310或His 435的残基,或当蛋白折叠为其3维结构时能够影响这些残基的离子环境的其它残基。可检测所得蛋白在pH 6和pH7.4-8时的最佳结合,以及选择那些在pH 6时高水平结合的蛋白和在pH 8时低水平结合的蛋白用于体内。
本发明的方法和组合物与血管生成抑制剂如PCT/US99/08335(WO 99/52562)中公开或与前列腺素抑制剂如PCT/US99/08376(WO99/53958)中公开共同给予是有效的。本发明的方法和组合物也可用于多种细胞因子蛋白复合体,如PCT/US00/21715中公开的。本发明的方法和组合物与PCT/US99/03966(WO 99/43713)中公开的增加融合蛋白循环半衰期的其它突变组合也有效。
非限制性合成有效的本发明方案的方法,以及有效检测药代动力学活动的实验,体外和临床前体内动物模型在这里的实施例中描述。优选的编码嵌合链的基因构建体包括:(以5’至3’方向)编码至少部分免疫球蛋白的DNA片段和编码第二个非免疫球蛋白的蛋白的DNA。可供选择的优选基因构建体包括:(以5’至3’方向)编码第二个非免疫球蛋白的蛋白的DNA和编码至少部分免疫球蛋白的DNA。融合基因装配或插入表达载体转染在其中表达的适当受体细胞。
本发明也提供了鉴定增加基于抗体的融合蛋白的循环半衰期的突变的方法。该方法包括将一个或多个突变引入跨越基于抗体的融合蛋白Ig部分和非Ig部分的接合区。测定突变的融合蛋白的循环半衰期,优选通过监测其在体内作用时间的血清水平。
在本发明的一个方案中,增加基于抗体的融合蛋白的循环半衰期的突变是减少蛋白酶切割实验中融合蛋白切割的突变,如实施例15中讨论。优选该突变跨越基于抗体的融合蛋白Ig部分和非Ig部分的接合区(例如,上面讨论的接合区的突变)。可供选择地,该突变可以是融合蛋白中减少蛋白酶切割和增加融合蛋白循环半衰期的任何突变,如实施例16中描述。因此,本发明提供了一般在蛋白中和优选在Ig-细胞因子融合蛋白中筛选突变的方法,以鉴定增加融合蛋白循环半衰期的突变。
通过下列非限制性实施例进一步阐明本发明。这里使用的氨基酸数是指IgG1氨基酸序列。一个本领域普通技术人员将理解融合蛋白包括其它Ig蛋白中相应的突变能有效增加它们的循环半衰期。
因此,这里提出的教导适用于其它Ig分子如IgG2,IgG3,IgG4,IgA,IgM,IgD或IgE。
实施例
实施例1.融合接合处Lys密码子置换的抗体-IL-2基因的构建
抗体-IL-2融合蛋白接合处的氨基酸序列是SerProGlyLys-AlaProThr(SEQ ID NO:1),其中SerProGlyLys(SEQID No.2)是抗体重链的正常羧基末端,AlaProThr是成熟IL-2的N末端序列。为了确定融合接合区的改变对融合蛋白药代动力学的影响,通过诱变使残基置换或缺失,如下所述。
免疫细胞因子的表达载体在Gillies等,(1998)J.Immunol.160:6195-6203中描述。编码重链的人γ1基因中,通过引入一个沉默突变(TCC变为TCA)破坏位于翻译终止密码子上游280bp的XmaI限制性酶切位点。另一个沉默突变(TCT变为TCC)引入重链C末端赖氨酸上游Ser密码三个残基,产生序列TCC CCG GGT AAA(SEQ ID No.3),其中含有一个新的XmaI位点[Lo at al.,(1998)Protein Engineering11:495-500]。在化学合成构建IL-2cDNA,它含有一个新的独特的PvuII限制性酶切位点[Gillies等,(1992)Proc.Natl.Acad.Sci.89:1428-1432]。在表达载体中XmaI和PvuII位点是独特的,它们利于位于CH3和IL-2DNA的接合区的赖氨酸密码子诱变。
用编码期望突变的寡核苷酸双链取代免疫细胞因子表达载体中的XmaI-PvuII片段获得赖氨酸密码子的置换或缺失。在这种情况下,重链和轻链可变区来自人源化KS抗体,识别称作EpCAM(上皮细胞粘附分子)的人抗原。本发明使用的寡核苷酸双链的序列列在下面,其中粗体密码子编码期望突变,斜体序列CCCGG和CAG分别是XmaI位点的粘末端和PvuII位点的平末端。带5’羟基末端的寡核苷酸双链用于连接XmaI-PvuII消化表达载体。使用带5’羟基末端的寡核苷酸消除寡核苷酸双链的自身连接。
1.)Lys置换为Ala
5′T GCA GCA CCT ACT TCA AGT TCT ACA AAGAAA ACA CAG 3′(SEQ ID NO:4)
3′A CGT CGT GGA TGA AGT TCA AGA TGT TTC TTT TGT
5′(SEQ ID NO:5)
2.)Lys置换为Arg
也通过沉默突变引入NarI限制性酶切位点(GGCGCC)以利于重组克隆的筛选。
3.)Lys的缺失
3′A CGT GGA TGA AGT TCA AGA TGT TTC TTT TGT5′(SEQ ID NO:9)
4.)Lys置换为Gly
也通过沉默突变引入ApaI限制性酶切位点(GGGCCC)以利于重组克隆的筛选。
5.)Lys置换为Leu
5′(SEQ ID NO:13)
也通过沉默突变引入NarI限制性酶切位点(GGCGCC)以利于重组克隆的筛选。
6.)Lys置换为AlaAlaAla
(SEQ ID NO:14)
7.)Lys置换为Cys
3′ A ACG CGT GGT TGA AGT TCA AGA TGT TTC TTT TGT
5′(SEQ ID NO:17)
也通过沉默突变引入FspI限制性酶切位点(TGCGCA)以利于重组克隆的筛选。
8.)Lys置换为Asp
3′A CTG CGT GGT TGA AGT TCA AGA TGT TTC TTT TGT5′(SEQ ID NO:19)
DNA测序证实含有lys密码子的各种置换或缺失的重组基因构建体。
实施例2:编码融合接合处额外氨基酸残基的抗体-IL-2基因的构建
分离带含氨基酸残基(如甘氨酸和丝氨酸)的灵活接头的融合蛋白结构域是本领域很普通的。在下列诱变实验中研究了CH3和IL-2之间间隔的重要性。编码不同数量氨基酸残基的平末端寡核苷酸双链连接插入huKS-IL-2表达载体的SmaI限制性内切酶位点(如上提及的XmaI识别位点相同);DNA测序证实插入正确方向。如上讨论,带5’羟基末端的寡核苷酸双链用于消除自身连接。
9.)Lys置换为Cys,带接头连接
下列接头(寡核苷酸双链)连接插入huKS-IL-2表达载体的SmaI限制性内切酶位点。编码SphI限制性酶切位点的序列GCATGC利于含有接头插入的重组体的筛选。
5′G GCATGC GG 3′
3′C CGT ACG CC 5′
接头连接到SmaI位点(CCCGGG)后,融合蛋白接合处的序列变为
C CCG GCA TGC GGG GGT AAA (SEO ID NO:20)(接头序列下划线)
Pro Ala Cys Gly Gly Lys(SEQ ID NO:21)
因此,接头在lys残基的最初位置放入Cys残基,可能形成二硫键。3个氨基酸残基(AlaCysGly)推后最初Lys残基。
10.)编码6个氨基酸残基的接头
下列接头(寡核苷酸双链)连接插入huKS-IL-2表达载体的SmaI限制性内切酶位点。序列GGATCC编码BamHI限制性酶切位点,利于含有接头插入的重组体的筛选。
5′G GGT TCA GGA TCC GGA GG 3′(SEQ ID NO:22)
3′C CCA AGT CCT AGG CCT CC 5′(SEQ ID NO:23)
接头连接到SmaI位点后,融合蛋白接头处的序列变为ProGlySerGlySerGlyGlyGlyLys(SEQ ID NO:24),其中插入的6个氨基酸被下划线。
11.)编码11个氨基酸的接头
下列接头(寡核苷酸双链)连接插入huKS-IL-2表达载体的SmaI限制性内切酶位点。序列GGATCC编码BamHI限制性酶切位点,利于含有接头插入的重组体的筛选。
5′G GGT TCA GGC TCT GGA TCA GGG TCC GGA TCC GG3′(SEQ
ID NO:25)
3′C CCA AGT CCG AGA CCT AGT CCC AGG CCT AGG CC 5′(SEQ
ID NO:26)
接头连接到SmaI位点后,融合蛋白接头处的序列变为ProGlySerGlySerGlySerGlySerGlySerGlyGlyLys(SEQ ID NO:27),其中插入的11个氨基酸被下划线。
实施例3.融合接合处Pro密码子置换的抗体-IL-2的构建
CH3羧基末端的ProGlyLys序列中的脯氨酸突变为Ala,Leu或Gly,和其它氨基酸。这是通过用编码期望改变的寡核苷酸双链取代KS-IL-2表达载体中25个碱基对的SapI-SmaI片段来完成的。下列每条寡核苷酸双链具有SapI粘末端(3-碱基悬挂)和平末端(用于连接限制性片段的SmaI末端)。Pro密码子的置换用粗体表示。这些置换对融合蛋白的药代动力学没有明显的作用,表明Pro残基的结构特性对融合蛋白的药代动力学没有明显作用。
12.)Pro置换为Ala
5′CG CAG AAG AGC CTC TCC CTG TCC GC 3′(SEQ ID NO:28)
3′TC TTC TCG GAG AGG GAC AGG CG 5′(SEQ ID NO:29)
13.)Pro置换为Leu
5′CG CAG AAG AGC CTC TCC CTG TCC CT 3′(SEQ ID NO:30)
3′TC TTC TCG GAG AGG GAC AGG GA 5′(SEQ ID NO:31)
12.)Pro置换为Gly
5′CG CAG AAG AGC CTC TCC CTG TCC GG 3′(SEQ ID NO:32)
3′TC TTC TCG GAG AGG GAC AGG CC 5′(SEQ ID NO:33)
实施例4.hu14.18-(Lvs变为Ala)-IL-2DNA的构建
为了表明Lys置换为Ala对抗体-IL-2融合蛋白药代动力学的影响不限于huKS抗体,我们选择了一个不同的抗体,人源化14.18(hu14.18),它识别GD2,在许多人肿瘤细胞表面过度表达的神经节苷脂。如上所述构建hu14.18-(Lvs变为Ala)-IL-2的表达载体。
实施例5.huKS-(Lys缺失)-TNFαDNA的构建
为了表明Lys残基对抗体-IL-2融合蛋白药代动力学的影响适用于其它细胞因子,我们选择了一个不同的细胞因子TNFα。Nedwin等在Nucleic Acids Res.(1985)13:6361-6373中公开了TNFα的完整cDNA序列,Gillies等在Bioconjugate Chem.(1993)4:230-235中也描述了抗体-TNFα的表达。抗体-TNFα的融合接合处具有序列SerProGlyLys-ValArgSerSerSer(SEQ ID NO:34),其中Val是成熟TNFα的N末端残基。为了与huKS-TNFα比较,用编码Lys残基缺失的诱变引物,采用套式(overlapping)PCR方法[Daugherty等,(1991)Nucleic Acids Res.19:2471-2476]构建编码huKS-(Lys缺失)-TNFα的DNA。因此所得huKS-(Lys缺失)-TNFα的表达载体编码融合蛋白接合处的肽序列SerProGly-ValArgSerSerSer(SEQ ID NO:35)。根据新发明的这个融合蛋白的另外修饰可以包括去除TNF氨基末端序列中的Arg残基以进一步降低接合区的总体电荷。
实施例6.huKS-(EU)-(Lys变为Ala)-IL-2DNA的构建
上面实施例中提及的所有抗体-细胞因子融合蛋白都基于骨髓瘤H链代表的某个异型人IgG1,KOL。为了表明Lys置换为Ala对抗体-IL-2融合蛋白药代动力学的影响不限于KOL,我们选择了一个骨髓瘤H链代表的不同IgG1异型,EU。EU异型在恒定区中3个氨基酸残基与KOL异型不同。EU异型在CH1末含有Lys-229,在CH3起始含有Asp-356和Leu-358。KOL异型在相应位置含有Arg-229,Glu-356和Met-358。采用套式PCR方法,通过诱变KOL DNA得到编码EU异型的DNA。然后,用所得EU DNA取代KOL DNA相应片段产生生产huKS-(EU)-(Lys变为Ala)-IL-2的表达载体。
实施例7.细胞转染和蛋白表达
对于瞬时转染,使用Lipofectamine Plus(Life Technologies,Gaithersburg,MD)根据供应商的方案通过脂质转染将质粒导入婴仓鼠肾(BHK)。
为了得到稳定转染克隆,电穿孔将质粒DNA导入小鼠骨髓瘤NS/0细胞。NS/0细胞在补充10%胎牛血清,2mM谷酰胺和青霉素/链霉素的Dulbecco改良Eagle′s培养基中生长。约5×106个细胞用PBS洗涤一次,并重悬于0.5ml PBS中。然后,10μg线性质粒DNA与细胞在基因脉冲小室(0.4cm电极间距,BioRad)中在冰上孵育10分钟。用基因脉冲标记仪(BioRad,Hercules,CA)电穿孔,设置在0.25V和500μF。允许细胞复苏10分钟。在冰上,它们重悬于生长培养基中后,接着铺在两个96孔板上。在转染后两天加入的100nM氨甲喋呤(MTX)存在下生长筛选稳定转染克隆。每隔3天饲养细胞两到三次以上,在2至3周出现抗MTX克隆。抗Fc-ELISA检测克隆的上清,鉴定高产量者。分离产量高细胞,在含100nM氨甲喋呤的生长培养基中繁殖。
对于凝胶电泳测定常规特性,在蛋白A琼脂糖(Protein ASepharose)(Repligen,Cambridge,MA)上捕获在条件培养基中的抗体-细胞因子融合蛋白,然后在煮沸含或不含2-巯基乙醇的蛋白样本缓冲液中洗脱。在SDS凝胶电泳后,考马斯染色显影蛋白条带。在SDS-PAGE上,抗体重链-IL-2和轻链分别具有明显大约67和28kD的MW。
对于纯化,在硫酸钠缓冲液(100mM NaH2PO4,pH3,和150mMNaCl)中洗脱结合在蛋白A琼脂糖(Protein A Sepharose)上的融合蛋白。然后用0.1N NaOH立即中和这些洗脱液。
实施例8.ELISA步骤
用ELISA测定抗MTX克隆和其它检测样本上清中蛋白产物的浓度。抗huFc ELISA由使用山羊抗人IgG(抗重链和轻链)的捕获步骤和使用辣根过氧化物酶-偶联山羊抗人IgG特异性的F(ab’)2片段的检测步骤组成。因此,抗huFc ELISA测定作为单独抗体或作为细胞因子融合蛋白的人IgG。为了确定完整抗体-IL-2融合蛋白的浓度,使用IL-2检测ELISA。它由使用山羊抗人IgG(抗重链和轻链)的相同捕获步骤组成,但检测步骤使用直接抗IL-2的检测抗体。在有些实验中,用EPCAM代替捕获抗体KS-IL-2检测融合蛋白,因为KS抗体识别EPCAM。在有些实验中,使用市售人IL-2ELISA检测试剂盒(R&D Systems)。包括IL-2检测抗体的所有不同ELISA步骤得到相似的结果。然而,如比较图1A和图1B可见到的,在较后的药代动力学时间点中IL-2免疫反应材料与人Fc免疫反应材料相比,存在进行性损失。这个作用在药代动力学性能最差的融合蛋白最显著。
抗huFc ELISA在下面详细描述。
A.包被平板
用5μg/ml AffiniPure山羊抗人IgG(H+L)(Jackson ImmunoResearch Laboratories,West Grove,PA)的PBS包被ELISA板,96孔板(Nunc-Immuno plate Maxisorp)中每孔100μl。盖上包被的平板并在4℃孵育过夜。然后,用0.05%Tween(Tween 20)的PBS洗涤平板4次,用1%BSA/1%山羊血清的PBS封闭。与封闭缓冲液在37℃孵育2小时后,用0.05%Tween的PBS洗涤4次平板,在纸巾上轻打干燥。
B.检测样本和二抗的孵育
检测样本在样本缓冲液中稀释至适当浓度,样本缓冲液是含有1%BSA/1%山羊血清/0.05%Tween的PBS。用嵌合抗体(带有人Fc)制得标准曲线,抗体浓度已知。为了制得标准曲线,对样本缓冲液做系列稀释得到范围从125ng/mL至3.9ng/mL的标准曲线。稀释的样本和标准加入平板,每孔100μl,平板在37℃孵育2小时。孵育后,用0.05%Tween的PBS洗涤平板8次。然后每孔加入100μl二抗,辣根过氧化物酶-偶联山羊抗人IgG F(ab’)2片段,Fc特异性片段(Jackson Immuno Research),稀释至样本缓冲液中大约1∶120,000。确定每批HRP耦联抗人IgG的二抗的精确稀释度。在37℃孵育2小时后,用0.05%Tween的PBS洗涤平板8次。
C.显影
底物溶液加入平板,每孔100μl。30mg OPD(O-苯二胺二盐酸盐,1片)溶于15mL 0.025M柠檬酸/0.05M Na2HPO4缓冲液中,pH 5,它含有0.03%新鲜加入的H2O2,制备得到底物溶液。允许颜色在室温黑暗中显影30分钟。显影时间改变,依赖于每批和每批包被平板、二抗等的可变性。观察标准曲线中颜色显影以确定何时停止反应。加入4N H2SO4终止反应,每孔100μl。平板阅读器阅读平板,设置490和650nm,编程从490nm OD中减去650nm的背景OD。
实施例9.融合蛋白接合处带有改变的抗体-细胞因子融合蛋白的药代
动力学行为
给Balb/c小鼠静脉内注射后检测融合蛋白的药代动力学行为。后眼眶出血收集小鼠血液,Eppendorf微离心管中保存在4℃。有些情况下,用两种不同的ELISA方法在各种时间点测定血液中存留的人抗体的量和第二个融合的非Ig蛋白的量。可供选择地,Western杂交分析药代动力学时间点推断非Ig部分的存在。
使用在前实施例中描述的技术,给小鼠注射KS(γl)-IL-2融合成熟蛋白,确定对血清半衰期的影响。有些结果在图1和图2中显示。此外,测定抗体具有不同结合特异性的IgG(γl)-IL-2融合的抗体重链C末端赖氨酸缺失的影响。14.18(Lys→Ala)-IL-2的药代动力学性能优于14.18-IL-2,程度类似KS(Lys→Ala)-IL-2与KS-IL-2相比提高的程度。
对于抗体-IL-2融合,影响重链C末端赖氨酸的突变对药代动力学特性的影响等级是(从最好至最差):Lys→Leu~Lys→Ala~Lys→Ala3>Lys→(缺失)>Lys→Asp~Lys→Gly>Lys→(无改变)~Lys→Cys>Lys→Arg。
与KS-TNFα相比,KS(Lys→缺失)-TNFα的药代动力学特性明显提高(图3)。KS-TNFα融合蛋白的药代动力学曲线罕见,因为当用Fc ELISA测定人抗体水平时,在前30分钟内,被检蛋白水平急剧下跌,随后人Fc反应性材料水平缓慢增加。这个作用可重复性高。
当用Western杂交分析药代动力学样本时,发现人Fc交叉反应材料是完整抗体的形式;TNF部分已被切掉和丢失。然而,C末端赖氨酸缺失的KS-TNFα融合蛋白的类似分析表明这个蛋白主要以完整形式幸存,TNF仍存在。
此外,KS-TNFα融合蛋白表达,其中成熟TNFα序列的前八个氨基酸缺失。缺失的KS-TNFα融合蛋白的药代动力学特性优于具有完整成熟TNF序列的相应蛋白。这可能是由于在成熟TNF+2位点的增加接合区疏水性的带电荷Arg残基被去除。
KOL至EU,改变重链恒定区KS(Lys→Ala)-IL-2和KS-IL-2对二者任何一个蛋白的药代动力学特性无影响。
总之,这些结果表明接合处突变引起Ig融合蛋白药代动力学特性的明显提高。在多种抗体,和多种Ig蛋白与Ig部分融合中可见到这个作用。
实施例10.组合融合接合处Ig型从γ1变为γ4的突变导致FcRp单
独作用的血清半衰期协同增加
人γ4Fc区与Fc受体结合差。结果,包含γ4Fc区的融合蛋白与具有γ1链的融合蛋白相比,通常具有优越的药代动力学特性。为了探寻接合处突变是否通过对Fc受体的相互作用,FcRp的相互作用,或二者兼有而影响药代动力学,检测FcRP正常或缺陷的小鼠含有或不含接合处突变的含γ1和γ4融合蛋白的药代动力学特性。这些药代动力学实验结果在图2显示。
图2显示了KS(γl)-IL-2融合蛋白,KS(γ4)-IL-2融合蛋白,KS(γl)(Lys变为Ala)-IL-2融合蛋白和KS(γ4)(Lys变为Ala)-IL-2融合蛋白的药代动力学行为。检测了正常小鼠和β2微球蛋白缺陷的突变小鼠。
这些数据表明,在正常小鼠中,通过在抗体部分C末端导入Lys变为Ala的突变,提高了IgG-γ1抗体-IL-2融合蛋白的药代动力学特性。类似地,抗体部分C末端导入Lys变为Ala的突变,提高了IgG-γ4抗体-IL-2融合蛋白的药代动力学特性。这些数据表明接合处突变能提高由于Fc受体结合下降造成药代动力学改善的融合蛋白的药代动力学特性。
图2也显示了给FcRp的主要亚单位-β2微球蛋白缺乏的突变小鼠注射相同蛋白的药代动力学特性(Junghans和Anderson,Proc.Nat Acad.Sci.(1996)93:5512-5516)。因此,这些突变小鼠FcRp活性缺陷。结果,这种突变小鼠的抗体分解代谢比正常小鼠快大约10倍。
图2的数据表明β2微球蛋白突变小鼠中KS(γl)-IL-2抗体,KS(γ1)-IL-2融合蛋白,KS(γ4)-IL-2融合蛋白,KS(γl)(Lys变为Ala)-IL-2融合蛋白和KS(γ4)(Lys变为Ala)-IL-2融合蛋白的分解代谢均比野生型小鼠更快。然而,两个小鼠株中这些蛋白的血清半衰期的相对顺序也如是:未融合抗体的药代动力学最好,其后是KS(γ4)(Lys变为Ala)-IL-2融合蛋白,KS(γl)(Lys变为Ala)-IL-2融合蛋白,KS(γ4)-IL-2融合蛋白,KS(γ1)-IL-2融合蛋白的药代动力学特性最差。如果接合处突变仅仅通过改变融合蛋白与FcRp的相互作用才具有其作用的话,那么FcRP功能不存在时,接合处突变应该对药代动力学没有影响。
实施例11.完整抗体接合区突变对血清半衰期没有影响
改造C末端赖氨酸变为丙氨酸,使编码完整未融合的KS抗体重链的基因突变。用上述方法表达并纯化野生型和突变型KS,比较药代动力学特性。发现野生型和突变抗体的药代动力学行为不能区别。
实施例12.含或不含融合接合处突变的抗体融合蛋白的Fc受体结合
使用标准步骤,检测KS-IL-2和KS(K-A)-IL-2与Fc受体的结合。没有发现突变的作用。如上述表达和纯化融合蛋白,检测它们结合固定的表达Fc受体的J774细胞的能力。结果在图4中显示。
实施例13.用含有接合处突变的抗体-细胞因子融合蛋白治疗哺乳动
物结肠癌
为了检测接合处突变的抗体-细胞因子融合蛋白对于治疗哺乳动物结肠癌是否有优势,实施下列实验。CT26是来自Balb/C小鼠的结肠癌细胞系。通过标准基因工程技术,改造该细胞系表达能被KS抗体识别的抗原-人上皮细胞粘附分子(EpCAM);这些细胞术语称作CT26/EpCAM细胞(Gillies等,Journal of Immunology(1998)160:6195-6203)。
Balb/C小鼠皮下接种2×106CT26/EpCAM细胞。当肿瘤达到大约50-200立方毫米体积时,将小鼠随机分为7只小鼠的三组进行进一步研究。在第0天,用PBS,大约10微克含IgG1重链(KS-IL2γl)的KS-IL2,或前面实施例中描述的大约10微克含IgG1重链和Lys突变为Ala的KS-IL2(KS-IL2γl[Lys变为Ala])处理带肿瘤小鼠。给小鼠静脉内注射,每天一次,共五天。用测径器测量肿瘤大小。
一个这种实验的结果在图5中显示。在这个实验中,KS-IL2γl使很多但不是所有肿瘤的体积明显减小。在7只KS-IL2γl处理的动物中有6只,在第21天肿瘤仍可测量。然而,KS-IL2γl[Lys变为Ala]处理的动物,肿瘤萎缩,以致到第21天,所有7只动物的肿瘤均不能测量,到第16天,7只小鼠只有2只具有可测量的肿瘤。在图5中,黑色菱形表示在第0,1,2,3和4天,作为对照的用PBS注射小鼠的平均肿瘤体积。实心圆表示用10微克KS-IL2γl处理小鼠的平均肿瘤体积。进行静脉内注射。x轴表示首次注射后过去的天数;y轴以立方毫米表示平均肿瘤体积。
实施例14.用含接合处突变的抗体-细胞因子融合蛋白处理的哺乳动物
的转移抑制
为了检测抗体-细胞因子融合蛋白是否能够抑制肿瘤细胞转移生长,实施下列实验。Lewis肺癌(LLC)是来自C57/B16小鼠的肺肿瘤细胞系。通过标准基因工程技术,改造该细胞系表达能被KS抗体识别的抗原-人上皮细胞粘附分子(EpCAM);这些细胞术语称作LLC/EpCAM细胞。
C57/B16小鼠静脉内注射1×106LLC/EpCAM细胞。五天后,小鼠随机分为6只小鼠的三组,并用PBS,大约20微克KS-IL2,或大约20微克KS-Ala-IL2(Ig部分C末端Lys变为Ala的KS-IL2])处理。第24天对转移定量。如下表表示,PBS处理组大量转移至肺。用KS-γl-IL2处理的动物转移数量明显降低。然而,用KS-γl-Ala-IL2处理的动物比用KS-γl-IL2处理的动物转移更少,有一只动物根本没有检测到转移。
处理组 转移数量 肺重量(g)
PBS >250,>250,>250,>250, 0.92+/-0.14
>250,>250
KS-yl-IL2 62,37,18,17,11,9 0.27+/-0.04
KS-yl-Ala-IL2 4,4,3,3,1,0 0.25+/-0.02
总之,实施例13和14阐明了抗体-细胞因子融合蛋白能抑制发生转移和原发部位肿瘤细胞生长。此外,结果表明抗体-细胞因子融合蛋白能抑制各种不同肿瘤类型,如结肠癌和肺癌造成的疾病。此外,按照本发明的接头区至少一个氨基酸改变的抗体-细胞因子融合蛋白比接头区无氨基酸改变的抗体-细胞因子融合蛋白在抑制转移和肿瘤生长中更有效。
实施例15.接合处突变抵抗蛋白酶的抗体融合蛋白的试验
为了探寻接合处突变的抗体-细胞因子融合蛋白对蛋白酶消化更敏感或更不敏感,用各种蛋白酶在各种时间处理纯化的KS-IL2和KS-Ala-IL2,用SDS-PAGE分析所得产物。
在一个实验中,用0.1mU或0.4mU Cathepsin D(Enzyme Systems,Livermore,California)对4微克KS-IL2和KS-Ala-IL2在37℃处理大约16小时并用SDS-PAGE分析。使用根据制造商指导的缓冲液条件。当用0.4mU Cathepsin D处理KS-IL2时,大约50%KS-IL2重链转换为多种较低分子量的形式。主要消化产物具有稍低于KS-IL2重链但远大约KS重链的分子量。这个结果表明大多数Cathepsin D切割不在重链-IL2接合处发生。
相反,当KS-Ala-IL2与0.4mU Cathepsin D在相同条件下孵育时,Cathepsin D切割的程度大大降低,主要可检测到一条具有主要KS-IL2降解产物分子量的条带。
在第二个实验中,用25mU或50mU Cathepsin L(EnzymeSystems,Livermore,California)对4微克KS-IL2和KS-Ala-IL2在37℃处理大约16小时并用SDS-PAGE分析。使用根据制造商指导的缓冲液条件。当用50mU Cathepsin L处理KS-IL2时,几乎所有KS-IL2重链转换为多种较低分子量的形式。主要消化产物的分子量与KS重链大约相等。这个结果表明大多数Cathepsin L切割在重链-IL2接合处附近或在重链-IL2接合处发生。
相比之下,当KS-Ala-IL2与50mU Cathepsin L在相同条件下孵育时,Cathepsin L切割的程度大大降低,一条具有主要KS-IL2降解产物分子量的条带仍是观察到的主要分子量种。
在第三个实验中,用0.04mU,0.1mU或0.2mU纤溶酶(Sigma,St.Louis,Minnesota)对4微克KS-IL2和KS-Ala-IL2在37℃处理大约16小时并用SDS-PAGE分析。使用根据制造商指导的缓冲液条件。当用0.04mU纤溶酶处理KS-IL2时,大约3/4KS-IL2重链转换为较低分子量的形式,具有比KS重链多大约30个氨基酸的明显分子量。当用0.2mU纤溶酶处理KS-IL2时,基本所有的KS-IL2重链转换为较低分子量的形式,具有比KS重链多大约30个氨基酸的明显分子量。这些结果表明纤溶酶切割KS-IL2在接近重链-IL2接合处发生,但不在重链-IL2接合处。
相比之下,当KS-Ala-IL2与0.04mU纤溶酶在相同条件下孵育时,纤溶酶切割的程度几乎检测不到。当KS-Ala-IL2与0.02mU纤溶酶孵育时,检测到一些未切割的产物。此外,当用纤溶酶切割KS-Ala-IL2时,比KS-IL2重链多大约90个氨基酸分子量大小的种蓄积到明显程度;用纤溶酶消化KS-IL2,这个+90种可能被切割为较低分子量+30种,因此不那蓄积。然而,Lys突变为Ala使完整KS-IL2在纤溶酶存在下明显稳定。每种情况下,在所用条件下,抗体轻链不被切割。
总之,这些结果表明Lys突变为Ala使普遍抵抗蛋白酶切割,甚至对于不在突变部位的切割。不受任何特定理论的限制,Lys突变为Ala可使KS的IL-2部分变得更加抵抗蛋白酶。蛋白酶可能在抗体融合蛋白的药代动力学特性中起着重要的作用。例如,当抗体融合蛋白被含有Fc受体的细胞接受并运输到早期内体,可能抗体部分抵抗使用的蛋白水解条件,但融合伙伴部分更敏感,导致抗体融合蛋白部分或全部消化。
实施例16.使用蛋白酶消化估计抗体融合蛋白的突变
这个实施例提供了提高蛋白药代动力学特性的一般方法。检测蛋白的药代动力学特性及其对蛋白酶的敏感性。产生变异体蛋白并检测对蛋白水解的抵抗。然后检测那些对蛋白水解抵抗增强的变异体的药代动力学特性。发现药代动力学特性提高的抵抗蛋白水解蛋白的比例高于变异体蛋白占总体的比例。一些药代动力学特性提高的变异体蛋白具有一个或多个氨基酸置换,这种置换经检测编码序列可推断不能引起蛋白结构的深刻改变,例如导入N连接糖基化位点。
变异体蛋白的产生可以通过例如表达构建体诱变并分离表达各个变异体蛋白的克隆。使用各种诱变技术的任何一个,包括定点诱变,随机诱变,PCR诱变和从相关序列产生杂种序列的诱变技术。
对于这些试验,使用细胞内蛋白酶有效,如内体蛋白酶。不受任何特定理论的限制,据信某些蛋白,尤其是不被肾过滤清除的蛋白的药代动力学由胞吞发生蛋白水解决定。
使用细胞外蛋白酶,如胰蛋白酶、糜蛋白酶、其它消化蛋白酶、其它血清蛋白酶如凝集因子和组织特异性蛋白酶也有效。例如,使用肿瘤特异性蛋白酶检测突变体蛋白,鉴定药代动力学特性和在肿瘤微环境内稳定性提高的变异体。在另一个实施例中,检测待口服传送的蛋白对胃肠道存在的酶的抵抗性,如胰蛋白酶和糜蛋白酶。发现对胃肠道酶抵抗性增强的变异体蛋白的药代动力学特性提高,如AUC(曲线下的面积)更大。
例如,诱变产生编码含有部分或全部抗体的融合蛋白的表达构建体。产生克隆,表达相应的蛋白,独立或以小量混合检测蛋白对蛋白酶的相对敏感性。然后检测蛋白酶抵抗性增强的变异体抗体融合蛋白的药代动力学特性,大量抵抗蛋白酶的抗体融合蛋白突变体的药代动力学特性提高。测序编码提高的变异体融合蛋白的核酸,发现一些提高的变异体在除融合蛋白接合处之外的位点含有突变,引起抵抗蛋白水解增强和药代动力学提高的表型。
等同物
本发明可以以其它特殊形式体现而不背离其精神或主要特性。因此认为前述方案在所有方面阐述这里描述的本发明,而不是限制它。因此本发明的范围由所附权利要求表示,而不由前述说明书表示,而且意指其中包含权利要求等同的意思和范围内的所有改变。
引入参考文献
这里公开的每篇专利文件和科技出版物以其完整形式通过参考文献引入本申请中。
序列表
<110>Lexigen Pharmaceuticals Corp.
<120>增加基于抗体的融合蛋白的循环半衰期
<130>LEX-011PC
<150>US 60/181,768
<151>2000-02-11
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1 5
Claims (4)
1.一种改变了的基于抗体的融合蛋白,该融合蛋白与无所述改变的相应基于抗体的融合蛋白相比具有更长的体内循环半衰期,所述融合蛋白包含具有CH2和CH3结构域的免疫球蛋白Ig重链,并在其C-末端连接了分泌型非Ig蛋白或其保留了完整非Ig蛋白功能性质的部分的N-末端,其中所述改变是用Ala、Ala-Ala-Ala、Leu、Gly或Trp取代所述Ig链的C-末端残基。
2.权利要求1的基于抗体的融合蛋白,其中所述Ig链包含IgG2或IgG4恒定区。
3.权利要求1或2的基于抗体的融合蛋白,其中所述非Ig蛋白是细胞因子。
4.权利要求3的基于抗体的融合蛋白,其中所述细胞因子是白细胞介素-2。
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US18176800P | 2000-02-11 | 2000-02-11 | |
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PCT/US2001/004455 WO2001058957A2 (en) | 2000-02-11 | 2001-02-09 | Enhancing the circulating half-life of antibody-based fusion proteins |
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Families Citing this family (258)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ATE267215T1 (de) | 1997-12-08 | 2004-06-15 | Lexigen Pharm Corp | Heterodimäre fusionsproteine zur verwendung für gezielte immuntherapie und allgemeine immunerregung |
US20030105294A1 (en) * | 1998-02-25 | 2003-06-05 | Stephen Gillies | Enhancing the circulating half life of antibody-based fusion proteins |
US7387772B1 (en) * | 1999-06-22 | 2008-06-17 | Immunimedics, Inc. | Chimeric, human and humanized anti-CSAP monoclonal antibodies |
CN1763097B (zh) | 1999-01-15 | 2011-04-13 | 杰南技术公司 | 具有改变的效应功能的多肽变体 |
US7183387B1 (en) | 1999-01-15 | 2007-02-27 | Genentech, Inc. | Polypeptide variants with altered effector function |
US6737056B1 (en) | 1999-01-15 | 2004-05-18 | Genentech, Inc. | Polypeptide variants with altered effector function |
EP1187852B1 (en) * | 1999-05-19 | 2007-08-08 | EMD Lexigen Research Center Corp. | EXPRESSION AND EXPORT OF INTERFERON-ALPHA PROTEINS AS Fc FUSION PROTEINS |
US7067110B1 (en) | 1999-07-21 | 2006-06-27 | Emd Lexigen Research Center Corp. | Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens |
SK782002A3 (en) | 1999-07-21 | 2003-08-05 | Lexigen Pharm Corp | FC fusion proteins for enhancing the immunogenicity of protein and peptide antigens |
US20050202538A1 (en) * | 1999-11-12 | 2005-09-15 | Merck Patent Gmbh | Fc-erythropoietin fusion protein with improved pharmacokinetics |
CA2399832C (en) | 2000-02-11 | 2011-09-20 | Stephen D. Gillies | Enhancing the circulating half-life of antibody-based fusion proteins |
ES2288967T3 (es) * | 2000-06-29 | 2008-02-01 | Merck Patent Gmbh | Reforzamiento de las respuestas inmunes mediadas por la proteina de fusion anticuerpo-citoquina por medio del tratamiento combinado por agentes que mejoran la incorporacion de inmunocitoquina. |
US7658921B2 (en) | 2000-12-12 | 2010-02-09 | Medimmune, Llc | Molecules with extended half-lives, compositions and uses thereof |
PT1355919E (pt) | 2000-12-12 | 2011-03-02 | Medimmune Llc | Moléculas com semivida longa, composições que as contêm e suas utilizações |
CA2440221C (en) | 2001-03-07 | 2013-02-05 | Merck Patent Gesellschaft Mit Beschraenkter Haftung | Expression technology for proteins containing a hybrid isotype antibody moiety |
WO2002079415A2 (en) | 2001-03-30 | 2002-10-10 | Lexigen Pharmaceuticals Corp. | Reducing the immunogenicity of fusion proteins |
BR0209177A (pt) | 2001-05-03 | 2004-10-05 | Merck Patent Gmbh | Anticorpo especìfico a tumor recombinante e uso do mesmo |
ES2381025T3 (es) * | 2001-12-04 | 2012-05-22 | Merck Patent Gmbh | Inmunocitocinas con selectividad modulada |
DK1471974T3 (da) * | 2002-02-06 | 2007-12-03 | Ares Trading Sa | Tumornekrose-faktor kombineret med interferon ved demyeliniserende sygdomme |
US7132100B2 (en) | 2002-06-14 | 2006-11-07 | Medimmune, Inc. | Stabilized liquid anti-RSV antibody formulations |
AU2003265866A1 (en) * | 2002-09-03 | 2004-03-29 | Vit Lauermann | Targeted release |
US20060020396A1 (en) * | 2002-09-09 | 2006-01-26 | Rene Gantier | Rational directed protein evolution using two-dimensional rational mutagenesis scanning |
US7365168B2 (en) | 2002-10-15 | 2008-04-29 | Pdl Biopharma, Inc. | Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis |
US7217797B2 (en) | 2002-10-15 | 2007-05-15 | Pdl Biopharma, Inc. | Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis |
US7361740B2 (en) | 2002-10-15 | 2008-04-22 | Pdl Biopharma, Inc. | Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis |
ES2346205T3 (es) | 2002-12-17 | 2010-10-13 | Merck Patent Gmbh | Anticuerpo humanizado (h14.18) del anticurpo 14.18 de raton que se enlaza con gd2 y su fusion con la il-2. |
TWI353991B (en) | 2003-05-06 | 2011-12-11 | Syntonix Pharmaceuticals Inc | Immunoglobulin chimeric monomer-dimer hybrids |
HUE026384T2 (en) | 2003-05-06 | 2016-06-28 | Biogen Hemophilia Inc | VII coagulation factor-FC chimeric proteins for the treatment of hemostasis disorder |
US20050069521A1 (en) * | 2003-08-28 | 2005-03-31 | Emd Lexigen Research Center Corp. | Enhancing the circulating half-life of interleukin-2 proteins |
WO2005037867A1 (en) | 2003-10-15 | 2005-04-28 | Pdl Biopharma, Inc. | ALTERATION OF Fc-FUSION PROTEIN SERUM HALF-LIVES BY MUTAGENESIS OF POSITIONS 250, 314 AND/OR 428 OF THE HEAVY CHAIN CONSTANT REGION OF IG |
WO2005063820A2 (en) | 2003-12-30 | 2005-07-14 | Merck Patent Gmbh | Il-7 fusion proteins |
KR20060124656A (ko) * | 2003-12-31 | 2006-12-05 | 메르크 파텐트 게엠베하 | 개선된 약물동태를 가지는 Fc-에리스로포이에틴 융합단백질 |
WO2005066348A2 (en) | 2004-01-05 | 2005-07-21 | Emd Lexigen Research Center Corp. | Interleukin-12 targeted to oncofoetal fibronectin |
CA2553883C (en) | 2004-01-22 | 2013-04-02 | Merck Patent Gesellschaft Mit Beschraenkter Haftung | Anti-cancer antibodies with reduced complement fixation |
US7670595B2 (en) * | 2004-06-28 | 2010-03-02 | Merck Patent Gmbh | Fc-interferon-beta fusion proteins |
JP5055603B2 (ja) | 2004-08-04 | 2012-10-24 | メントリック・バイオテック・リミテッド・ライアビリティ・カンパニー | 変異Fc領域 |
CA2577370A1 (en) * | 2004-08-16 | 2006-03-02 | Medimmune, Inc. | Integrin antagonists with enhanced antibody dependent cell-mediated cytotoxicity activity |
DE602005020837D1 (de) * | 2004-12-09 | 2010-06-02 | Merck Patent Gmbh | Il-7-varianten mit reduzierter immunogenität |
CN100429233C (zh) * | 2005-02-03 | 2008-10-29 | 上海长海医院 | 一种重组融合蛋白及其制备方法及用途 |
WO2006089133A2 (en) | 2005-02-15 | 2006-08-24 | Duke University | Anti-cd19 antibodies and uses in oncology |
CA2606102C (en) | 2005-04-26 | 2014-09-30 | Medimmune, Inc. | Modulation of antibody effector function by hinge domain engineering |
EP2221316A1 (en) | 2005-05-05 | 2010-08-25 | Duke University | Anti-CD19 antibody therapy for autoimmune disease |
CA2614181A1 (en) * | 2005-07-01 | 2007-01-11 | Medimmune, Inc. | An integrated approach for generating multidomain protein therapeutics |
US20070104689A1 (en) * | 2005-09-27 | 2007-05-10 | Merck Patent Gmbh | Compositions and methods for treating tumors presenting survivin antigens |
CA2625619A1 (en) | 2005-10-14 | 2007-04-26 | Medimmune, Inc. | Cell display of antibody libraries |
US7723477B2 (en) * | 2005-10-31 | 2010-05-25 | Oncomed Pharmaceuticals, Inc. | Compositions and methods for inhibiting Wnt-dependent solid tumor cell growth |
AU2006308870B2 (en) * | 2005-10-31 | 2012-08-16 | Oncomed Pharmaceuticals, Inc. | Compositions and methods for diagnosing and treating cancer |
CA2635618C (en) * | 2005-12-30 | 2015-10-06 | Merck Patent Gesellschaft Mit Beschraekter Haftung | Interleukin-12p40 variants with improved stability |
PT2270050E (pt) | 2005-12-30 | 2013-09-12 | Merck Patent Gmbh | Anticorpos anti-cd19 com imunogenicidade reduzida |
WO2007092772A2 (en) * | 2006-02-03 | 2007-08-16 | Medimmune, Inc. | Protein formulations |
EP1816201A1 (en) | 2006-02-06 | 2007-08-08 | CSL Behring GmbH | Modified coagulation factor VIIa with extended half-life |
EP2650306A1 (en) | 2006-03-06 | 2013-10-16 | Aeres Biomedical Limited | Humanized Anti-CD22 antibodies and their use in treatment of oncology, transplantation and autoimmune disease |
ATE555383T1 (de) | 2006-06-22 | 2012-05-15 | Novo Nordisk As | Lösliche heterodimere rezeptoren und ihre verwendung |
SI1873166T1 (sl) * | 2006-06-30 | 2011-01-31 | Conaris Res Inst Ag | IZBOLJĹ ANI sgp 130Fc DIMERJI |
EP2038417A2 (en) * | 2006-07-06 | 2009-03-25 | Merck Patent GmbH | Compositions and methods for enhancing the efficacy of il-2 mediated immune responses |
EP1878747A1 (en) | 2006-07-11 | 2008-01-16 | greenovation Biotech GmbH | Glyco-engineered antibodies |
TW200817438A (en) * | 2006-08-17 | 2008-04-16 | Hoffmann La Roche | A conjugate of an antibody against CCR5 and an antifusogenic peptide |
PE20120806A1 (es) * | 2006-09-08 | 2012-07-25 | Genentech Inc | Antagonistas del gen wnt y su uso en el diagnostico y tratamiento de trastornos mediados por el wnt |
EP2097096B1 (en) * | 2006-12-22 | 2017-05-31 | CSL Behring GmbH | Modified coagulation factors with prolonged in vivo half-life |
RU2549701C2 (ru) | 2007-05-07 | 2015-04-27 | Медиммун, Ллк | Анти-icos антитела и их применение в лечении онкологических, связанных с трансплантацией и аутоиммунных заболеваний |
CN105131127B (zh) * | 2007-05-30 | 2018-09-07 | 浦项工科大学校产学协力团 | 免疫球蛋白融合蛋白 |
CN105175553B (zh) * | 2007-05-30 | 2019-11-22 | 浦项工科大学校产学协力团 | 免疫球蛋白融合蛋白 |
EP1997830A1 (en) | 2007-06-01 | 2008-12-03 | AIMM Therapeutics B.V. | RSV specific binding molecules and means for producing them |
US20110077383A1 (en) * | 2007-07-03 | 2011-03-31 | Medimmune, Llc | Hinge domain engineering |
AR067537A1 (es) * | 2007-07-17 | 2009-10-14 | Hoffmann La Roche | Purificacion de polipeptidos pegilados |
CL2008002054A1 (es) * | 2007-07-17 | 2009-05-29 | Hoffmann La Roche | Metodo para la regeneracion de una columna de cromatografia de intercambio cationico despues de la elusion de eritropoyetina monopeguilada y metodo para obtener una eritropoyetina monopeguilada, incorporando el metodo de regeneracion de la columna de intercambio cationico. |
CL2008002092A1 (es) | 2007-07-20 | 2009-05-29 | Hoffmann La Roche | Conjugado que contiene dos o mas peptidos antifusogenicos y un anticuerpo anti-cd-4; metodo de produccion; composicion farmaceutica que lo comprende; polipeptidos antifusogenicos y uso del conjugado para tratar infecciones viricas. |
US20090155275A1 (en) | 2007-07-31 | 2009-06-18 | Medimmune, Llc | Multispecific epitope binding proteins and uses thereof |
US8092804B2 (en) | 2007-12-21 | 2012-01-10 | Medimmune Limited | Binding members for interleukin-4 receptor alpha (IL-4Rα)-173 |
WO2009081201A2 (en) | 2007-12-21 | 2009-07-02 | Medimmune Limited | BINDING MEMBERS FOR INTERLEUKIN-4 RECEPTOR ALPHA (IL-4Rα) - 173 |
ES2613963T3 (es) | 2008-01-18 | 2017-05-29 | Medimmune, Llc | Anticuerpos manipulados con cisteína para conjugación específica de sitio |
AU2009211148B2 (en) * | 2008-02-05 | 2014-08-28 | Zymeworks Inc. | Methods for determining correlated residues in a protein or other biopolymer using molecular dynamics |
CN102066575A (zh) * | 2008-04-18 | 2011-05-18 | 雅培制药有限公司 | 结合蛋白体内半衰期的体外估测 |
NZ592338A (en) * | 2008-09-26 | 2012-11-30 | Oncomed Pharm Inc | Frizzled-binding agents and uses thereof |
US20100082438A1 (en) * | 2008-10-01 | 2010-04-01 | Ronnie Jack Garmon | Methods and systems for customer performance scoring |
ES2719496T3 (es) | 2008-11-12 | 2019-07-10 | Medimmune Llc | Formulación de anticuerpo |
WO2010068722A1 (en) | 2008-12-12 | 2010-06-17 | Medimmune, Llc | Crystals and structure of a human igg fc variant with enhanced fcrn binding |
CA2749539C (en) | 2009-01-21 | 2022-07-19 | Amgen Inc. | Compositions and methods comprising interleukin-2 mutants for treating inflammatory and autoimmune diseases |
AU2010238858A1 (en) | 2009-04-22 | 2011-12-08 | Merck Patent Gmbh | Antibody fusion proteins with modified FcRn binding sites |
BRPI1015234A2 (pt) | 2009-06-22 | 2018-02-20 | Medimmune Llc | regiões fc projetadas para conjugação sítio específica. |
US8221753B2 (en) | 2009-09-30 | 2012-07-17 | Tracon Pharmaceuticals, Inc. | Endoglin antibodies |
KR101553740B1 (ko) | 2009-08-17 | 2015-09-17 | 트라콘 파마수티칼즈, 인코포레이티드 | 항엔도글린 항체 및 항vegf 제제를 사용한 암 치료를 위한 병용 요법 |
EP2470559B1 (en) | 2009-08-24 | 2017-03-22 | Amunix Operating Inc. | Coagulation factor ix compositions and methods of making and using same |
US8568726B2 (en) | 2009-10-06 | 2013-10-29 | Medimmune Limited | RSV specific binding molecule |
LT2486141T (lt) | 2009-10-07 | 2018-05-25 | Macrogenics, Inc. | Fc regioną turintys polipeptidai, pasižymintys pagerinta efektorine funkcija dėl fukozilinimo laipsnio pasikeitimų, ir jų naudojimo būdai |
US8722615B2 (en) * | 2009-12-02 | 2014-05-13 | Acceleron Pharma, Inc. | Compositions and methods for increasing serum half-life |
ES2659888T3 (es) | 2009-12-06 | 2018-03-19 | Bioverativ Therapeutics Inc. | Polipéptidos quiméricos e híbridos de Factor VIII-Fc, y métodos de uso de los mismos |
TWI535445B (zh) | 2010-01-12 | 2016-06-01 | 安可美德藥物股份有限公司 | Wnt拮抗劑及治療和篩選方法 |
CN102947341B (zh) | 2010-01-19 | 2018-07-06 | 哈佛大学校长及研究员协会 | 用于病原体检测和治疗的工程化调理素 |
CA2789629A1 (en) | 2010-02-10 | 2011-08-18 | Immunogen, Inc. | Cd20 antibodies and uses thereof |
NZ602700A (en) | 2010-04-01 | 2014-10-31 | Oncomed Pharm Inc | Frizzled-binding agents and uses thereof |
SG186856A1 (en) | 2010-07-09 | 2013-02-28 | Biogen Idec Hemophilia Inc | Factor ix polypeptides and methods of use thereof |
US9611310B2 (en) | 2010-07-09 | 2017-04-04 | Bioverativ Therapeutics Inc. | Systems for factor VIII processing and methods thereof |
US20130177555A1 (en) | 2010-08-13 | 2013-07-11 | Medimmune Limited | Monomeric Polypeptides Comprising Variant FC Regions And Methods Of Use |
WO2012022734A2 (en) | 2010-08-16 | 2012-02-23 | Medimmune Limited | Anti-icam-1 antibodies and methods of use |
RU2566267C2 (ru) | 2010-09-14 | 2015-10-20 | Ф. Хоффманн-Ля Рош Аг | Способ очистки пегилированного эритропоэтина |
HUE041335T2 (hu) | 2011-03-29 | 2019-05-28 | Roche Glycart Ag | Antitest FC-variánsok |
ES2942455T3 (es) | 2011-04-13 | 2023-06-01 | Bristol Myers Squibb Co | Proteínas de fusión Fc que comprenden enlazadores o disposiciones nuevos |
CA2837169C (en) | 2011-05-24 | 2021-11-09 | Zyngenia, Inc. | Multispecific complexes comprising angiopoietin-2-binding peptide and their uses |
EP2718328A4 (en) | 2011-06-08 | 2014-12-24 | Acceleron Pharma Inc | COMPOSITIONS AND METHODS FOR INCREASING THE HALF TIME OF SERUM |
US11492383B2 (en) | 2011-06-24 | 2022-11-08 | Stephen D. Gillies | Light chain immunoglobulin fusion proteins and methods of use thereof |
EP2537933A1 (en) | 2011-06-24 | 2012-12-26 | Institut National de la Santé et de la Recherche Médicale (INSERM) | An IL-15 and IL-15Ralpha sushi domain based immunocytokines |
AU2012280474A1 (en) | 2011-07-01 | 2014-01-16 | Bayer Intellectual Property Gmbh | Relaxin fusion polypeptides and uses thereof |
US10010622B2 (en) | 2011-07-08 | 2018-07-03 | Bioverativ Therapeutics Inc. | Factor VIII chimeric and hybrid polypeptides, and methods of use thereof |
WO2013007563A1 (en) | 2011-07-08 | 2013-01-17 | Bayer Intellectual Property Gmbh | Fusion proteins releasing relaxin and uses thereof |
EP2737311B1 (en) | 2011-07-25 | 2020-12-02 | Bioverativ Therapeutics Inc. | Assays to monitor bleeding disorders |
PE20142361A1 (es) | 2011-09-30 | 2015-01-16 | Dana Farber Cancer Inst Inc | Peptidos terapeuticos |
CN104203982B (zh) | 2011-10-28 | 2018-08-31 | 特瓦制药澳大利亚私人有限公司 | 多肽构建体及其用途 |
CA2854806A1 (en) | 2011-11-07 | 2013-05-16 | Medimmune, Llc | Multispecific and multivalent binding proteins and uses thereof |
ES2721882T3 (es) | 2011-12-23 | 2019-08-06 | Pfizer | Regiones constantes de anticuerpo modificadas por ingeniería genética para conjugación específica de sitio y procedimientos y usos de las mismas |
EP3453402B1 (en) | 2012-01-12 | 2021-07-21 | Bioverativ Therapeutics Inc. | Reducing immunogenicity against factor viii in individuals undergoing factor viii therapy |
PT2814840T (pt) | 2012-02-15 | 2020-01-28 | Bioverativ Therapeutics Inc | Composições de fator viii e métodos de produção e utilização das mesmas |
JP6383666B2 (ja) | 2012-02-15 | 2018-08-29 | バイオベラティブ セラピューティクス インコーポレイテッド | 組換え第viii因子タンパク質 |
IN2014DN08236A (zh) * | 2012-03-03 | 2015-05-15 | Immungene Inc | |
DK3489254T3 (da) | 2012-04-30 | 2022-11-07 | Biocon Ltd | Målrettede/immunmodulatoriske fusionsproteiner og fremgangsmåder til fremstilling deraf |
LT2872534T (lt) | 2012-07-13 | 2018-10-25 | Roche Glycart Ag | Dvigubai specifiniai anti-vegf/anti-ang-2 antikūnai ir jų panaudojimas akių kraujagyslių ligoms gydyti |
NZ630363A (en) | 2012-07-25 | 2018-09-28 | Celldex Therapeutics Inc | Anti-kit antibodies and uses thereof |
BR112015002091A2 (pt) | 2012-08-02 | 2017-12-12 | Hoffmann La Roche | método para a produção de um polipeptídeo, polipeptídeo, uso de um polipeptídeo e composição |
UA115789C2 (uk) | 2012-09-05 | 2017-12-26 | Трейкон Фармасутікалз, Інк. | Композиція антитіла до cd105 та її застосування |
EP2900328A4 (en) | 2012-09-25 | 2016-05-11 | Biogen Ma Inc | PROCESS FOR USE OF FIXED POLYPEPTIDES |
US10391152B2 (en) | 2012-10-18 | 2019-08-27 | Bioverativ Therapeutics Inc. | Methods of using a fixed dose of a clotting factor |
JP2015536933A (ja) | 2012-10-23 | 2015-12-24 | オンコメッド ファーマシューティカルズ インコーポレイテッド | Wnt経路結合剤を用いて神経内分泌腫瘍を処置する方法 |
EP3446700A1 (en) | 2012-10-30 | 2019-02-27 | Bioverativ Therapeutics Inc. | Methods of using fviii polypeptide |
JP2016510411A (ja) | 2013-02-04 | 2016-04-07 | オンコメッド ファーマシューティカルズ インコーポレイテッド | Wnt経路インヒビターによる処置の方法およびモニタリング |
WO2014126871A1 (en) | 2013-02-12 | 2014-08-21 | Bristol-Myers Squibb Company | Tangential flow filtration based protein refolding methods |
EP3299378B1 (en) | 2013-02-12 | 2019-07-31 | Bristol-Myers Squibb Company | High ph protein refolding methods |
US9168300B2 (en) | 2013-03-14 | 2015-10-27 | Oncomed Pharmaceuticals, Inc. | MET-binding agents and uses thereof |
BR112015022123B1 (pt) | 2013-03-15 | 2022-08-09 | Intrinsic Lifesciences, Llc | Anticorpos, fragmentos de ligação ao antígeno dos mesmos que se ligam especificamente à hepcidina ou um peptídeo de hepcidina, uso, meio contentor e kit |
SG10201913874TA (en) | 2013-03-15 | 2020-03-30 | Biogen Ma Inc | Factor ix polypeptide formulations |
US10150800B2 (en) | 2013-03-15 | 2018-12-11 | Zyngenia, Inc. | EGFR-binding modular recognition domains |
EP4122487A1 (en) | 2013-03-15 | 2023-01-25 | Bioverativ Therapeutics Inc. | Factor viii polypeptide formulations |
EA201591559A1 (ru) | 2013-03-15 | 2016-03-31 | Дана-Фарбер Кэнсер Инститьют, Инк. | Терапевтические пептиды |
CN105324124A (zh) | 2013-04-19 | 2016-02-10 | 赛腾制药 | 降低的血管渗漏综合征的细胞因子衍生治疗 |
SG11201508923VA (en) | 2013-04-29 | 2015-11-27 | Teva Pharmaceuticals Australia Pty Ltd | Anti-cd38 antibodies and fusions to attenuated interferon alpha-2b |
US11117975B2 (en) | 2013-04-29 | 2021-09-14 | Teva Pharmaceuticals Australia Pty Ltd | Anti-CD38 antibodies and fusions to attenuated interferon alpha-2B |
CA2913155A1 (en) | 2013-05-21 | 2014-11-27 | President And Fellows Of Harvard College | Engineered heme-binding compositions and uses thereof |
US10548953B2 (en) | 2013-08-14 | 2020-02-04 | Bioverativ Therapeutics Inc. | Factor VIII-XTEN fusions and uses thereof |
LT3052192T (lt) | 2013-10-02 | 2020-12-10 | Medimmune, Llc | Neutralizuojantys antikūnai prieš gripą a ir jų naudojimas |
EP3077504B1 (en) | 2013-12-06 | 2019-08-14 | Dana-Farber Cancer Institute, Inc. | Therapeutic peptides |
EP4332839A2 (en) | 2013-12-06 | 2024-03-06 | Bioverativ Therapeutics Inc. | Population pharmacokinetics tools and uses thereof |
DE202014010499U1 (de) | 2013-12-17 | 2015-10-20 | Kymab Limited | Targeting von humaner PCSK9 zur Cholesterinbehandlung |
US8986691B1 (en) | 2014-07-15 | 2015-03-24 | Kymab Limited | Method of treating atopic dermatitis or asthma using antibody to IL4RA |
US8980273B1 (en) | 2014-07-15 | 2015-03-17 | Kymab Limited | Method of treating atopic dermatitis or asthma using antibody to IL4RA |
EP3083658B1 (en) | 2013-12-18 | 2019-05-08 | President and Fellows of Harvard College | Crp capture/detection of gram positive bacteria |
ES2742682T3 (es) | 2013-12-24 | 2020-02-17 | Argenx Bvba | Antagonistas de FCRN y métodos de uso |
DK3105246T3 (da) | 2014-02-10 | 2021-06-14 | Merck Patent Gmbh | Målrettet TGF-beta-inhibering |
EP2915569A1 (en) | 2014-03-03 | 2015-09-09 | Cytune Pharma | IL-15/IL-15Ralpha based conjugates purification method |
GB201403775D0 (en) | 2014-03-04 | 2014-04-16 | Kymab Ltd | Antibodies, uses & methods |
CA3124243A1 (en) | 2014-03-14 | 2015-09-17 | Dana-Farber Cancer Institute, Inc. | Vaccine compositions and methods for restoring nkg2d pathway function against cancers |
BR112016021777A2 (pt) | 2014-03-24 | 2017-10-03 | Biogen Ma Inc | Formulações de fator ix liofilizado |
UA119352C2 (uk) | 2014-05-01 | 2019-06-10 | Тева Фармасьютикалз Острейліа Пті Лтд | Комбінація леналідоміду або помалідоміду і конструкції анти-cd38 антитіло-атенуйований інтерферон альфа-2b та спосіб лікування суб'єкта, який має cd38-експресуючу пухлину |
RS59643B1 (sr) | 2014-06-06 | 2020-01-31 | Bristol Myers Squibb Co | Antitela na glukokortikoidom indukovani receptor faktora nekroze tumora (gitr) i njihova primena |
WO2016023916A1 (en) | 2014-08-12 | 2016-02-18 | Kymab Limited | Treatment of disease using ligand binding to targets of interest |
NZ730186A (en) | 2014-09-22 | 2020-04-24 | Intrinsic Lifesciences Llc | Humanized anti-hepcidin antibodies and uses thereof |
MA41044A (fr) | 2014-10-08 | 2017-08-15 | Novartis Ag | Compositions et procédés d'utilisation pour une réponse immunitaire accrue et traitement contre le cancer |
KR102639037B1 (ko) | 2014-10-29 | 2024-02-20 | 테바 파마슈티컬즈 오스트레일리아 피티와이 엘티디 | 인터페론 α2b 변이체 |
US20160130324A1 (en) * | 2014-10-31 | 2016-05-12 | Shire Human Genetic Therapies, Inc. | C1 Inhibitor Fusion Proteins and Uses Thereof |
WO2016071701A1 (en) | 2014-11-07 | 2016-05-12 | Kymab Limited | Treatment of disease using ligand binding to targets of interest |
JP2017537084A (ja) | 2014-11-12 | 2017-12-14 | トラコン ファーマシューティカルズ、インコーポレイテッド | 抗エンドグリン抗体及びその用途 |
US9926375B2 (en) | 2014-11-12 | 2018-03-27 | Tracon Pharmaceuticals, Inc. | Anti-endoglin antibodies and uses thereof |
CN113773388A (zh) | 2014-11-21 | 2021-12-10 | 百时美施贵宝公司 | 抗cd73抗体及其用途 |
CN107250157B (zh) | 2014-11-21 | 2021-06-29 | 百时美施贵宝公司 | 包含修饰的重链恒定区的抗体 |
MA41116A (fr) | 2014-12-01 | 2017-10-10 | Ferring Bv | Compositions d'inhibiteur de trans-signalisation par l'il-6 sélectif |
RS61947B1 (sr) | 2014-12-01 | 2021-07-30 | Ferring Bv | Davanje selektivnog inhibitora il-6-trans-signalizacije |
TWI708786B (zh) | 2014-12-23 | 2020-11-01 | 美商必治妥美雅史谷比公司 | 針對tigit之抗體 |
KR20170131463A (ko) | 2015-03-09 | 2017-11-29 | 아르제넥스 비브이비에이 | FcRn 길항제를 사용하여 Fc-함유된 제제의 혈청 수준을 감소시키는 방법 |
MY188430A (en) | 2015-04-10 | 2021-12-08 | Amgen Inc | Interleukin-2 muteins for the expansion of t-regulatory cells |
HUE061253T2 (hu) | 2015-05-29 | 2023-06-28 | Bristol Myers Squibb Co | Antitestek OX40 ellen és azok felhasználásai |
TWI773646B (zh) | 2015-06-08 | 2022-08-11 | 美商宏觀基因股份有限公司 | 結合lag-3的分子和其使用方法 |
EP3328419B1 (en) | 2015-07-30 | 2021-08-25 | MacroGenics, Inc. | Pd-1-binding molecules and methods of use thereof |
MX2018001497A (es) | 2015-08-03 | 2018-05-15 | Bioverativ Therapeutics Inc | Proteinas de fusion de factor ix y metodos para producirlas y usarlas. |
US10435457B2 (en) | 2015-08-06 | 2019-10-08 | President And Fellows Of Harvard College | Microbe-binding molecules and uses thereof |
MX2018004177A (es) | 2015-10-08 | 2018-09-11 | Macrogenics Inc | Terapia de combinacion para el tratamiento de cancer. |
CN108738324B (zh) | 2015-11-19 | 2022-06-21 | 百时美施贵宝公司 | 抗糖皮质激素诱导的肿瘤坏死因子受体(gitr)抗体及其用途 |
CR20180318A (es) | 2015-12-14 | 2018-09-19 | Macrogenics Inc | Moléculas biespecíficas que tienen inmunorreactividad con pd-1 y ctla-4, y métodos de uso de las mismas |
AR108034A1 (es) | 2016-02-17 | 2018-07-11 | Macrogenics Inc | Moléculas de unión a ror1, y métodos de uso de las mismas |
AU2017228470A1 (en) | 2016-03-04 | 2018-08-30 | Bristol-Myers Squibb Company | Combination therapy with anti-CD73 antibodies |
CN108884142A (zh) | 2016-03-16 | 2018-11-23 | 梅里麦克制药股份有限公司 | 用于癌症治疗的工程化trail |
CN109069633A (zh) | 2016-04-15 | 2018-12-21 | 宏观基因有限公司 | 新颖的b7-h3-结合分子、其抗体药物缀合物及其使用方法 |
WO2017196663A1 (en) | 2016-05-09 | 2017-11-16 | Bristol-Myers Squibb Company | Tl1a antibodies and uses thereof |
US9567399B1 (en) | 2016-06-20 | 2017-02-14 | Kymab Limited | Antibodies and immunocytokines |
PE20190418A1 (es) | 2016-07-14 | 2019-03-19 | Bristol Myers Squibb Co | Anticuerpos contra proteina 3 que contiene el dominio de mucina e inmunoglobulina de linfocitos t (tim3) y sus usos |
JP7177035B2 (ja) | 2016-07-15 | 2022-11-22 | エフ.ホフマン-ラ ロシュ アーゲー | Peg化エリスロポエチンを精製するための方法 |
JP2019527706A (ja) | 2016-08-12 | 2019-10-03 | メルク パテント ゲーエムベーハー | 癌の併用療法 |
US10981976B2 (en) | 2016-08-31 | 2021-04-20 | University Of Rochester | Human monoclonal antibodies to human endogenous retrovirus K envelope (HERV-K) and use thereof |
US11779604B2 (en) | 2016-11-03 | 2023-10-10 | Kymab Limited | Antibodies, combinations comprising antibodies, biomarkers, uses and methods |
AU2017363309A1 (en) | 2016-11-23 | 2019-07-11 | Bioverativ Therapeutics Inc. | Mono- and bispecific antibodies binding to coagulation factor IX and coagulation factor X |
US11129906B1 (en) | 2016-12-07 | 2021-09-28 | David Gordon Bermudes | Chimeric protein toxins for expression by therapeutic bacteria |
HUE058664T2 (hu) | 2016-12-23 | 2022-09-28 | Immunogen Inc | ADAM9-et célzó immunkonjugátumok és eljárások azok felhasználására |
JP7128819B2 (ja) | 2016-12-23 | 2022-08-31 | マクロジェニクス,インコーポレーテッド | Adam9結合分子、およびその使用方法 |
WO2018129029A1 (en) | 2017-01-04 | 2018-07-12 | Immunogen, Inc. | Met antibodies and immunoconjugates and uses thereof |
SG11201906947SA (en) | 2017-02-17 | 2019-08-27 | Bristol Myers Squibb Co | Antibodies to alpha-synuclein and uses thereof |
US11459394B2 (en) | 2017-02-24 | 2022-10-04 | Macrogenics, Inc. | Bispecific binding molecules that are capable of binding CD137 and tumor antigens, and uses thereof |
EP3600429B1 (en) * | 2017-03-20 | 2023-10-25 | Bio-Techne Corporation | Il-37 fusion protein and methods of making and using same |
CN111094335B (zh) | 2017-05-15 | 2022-08-23 | 罗切斯特大学 | 广泛中和的抗流感单克隆抗体及其用途 |
CN116333129A (zh) | 2017-05-25 | 2023-06-27 | 百时美施贵宝公司 | 包含经修饰的重链恒定区的抗体 |
WO2019075090A1 (en) | 2017-10-10 | 2019-04-18 | Tilos Therapeutics, Inc. | ANTI-LAP ANTIBODIES AND USES THEREOF |
BR112020007309A2 (pt) | 2017-10-14 | 2020-09-29 | Cytomx Therapeutics, Inc. | anticorpos, anticorpos ativáveis, anticorpos biespecíficos e anticorpos ativáveis biespecíficos e métodos de uso dos mesmos |
AU2018380979B2 (en) | 2017-12-08 | 2023-07-20 | Argenx Bvba | Use of FcRn antagonists for treatment of generalized myasthenia gravis |
US11795226B2 (en) | 2017-12-12 | 2023-10-24 | Macrogenics, Inc. | Bispecific CD16-binding molecules and their use in the treatment of disease |
SG11202005732XA (en) | 2017-12-19 | 2020-07-29 | Xencor Inc | Engineered il-2 fc fusion proteins |
US11802154B2 (en) | 2017-12-20 | 2023-10-31 | Alexion Pharmaceuticals, Inc. | Humanized anti-CD200 antibodies and uses thereof |
US20210087267A1 (en) | 2017-12-20 | 2021-03-25 | Alexion Pharmaceuticals, Inc. | Liquid formulations of anti-cd200 antibodies |
CN111886255A (zh) | 2018-01-12 | 2020-11-03 | 百时美施贵宝公司 | 抗tim3抗体及其用途 |
CN111787949A (zh) | 2018-02-15 | 2020-10-16 | 宏观基因有限公司 | 变体cd3-结合结构域及其在用于治疗疾病的组合疗法中的用途 |
TW201945393A (zh) | 2018-03-21 | 2019-12-01 | 美商戊瑞治療有限公司 | 在酸性pH結合至VISTA之抗體 |
TW202003565A (zh) | 2018-03-23 | 2020-01-16 | 美商必治妥美雅史谷比公司 | 抗mica及/或micb抗體及其用途 |
JOP20200240A1 (ar) | 2018-04-02 | 2020-09-27 | Bristol Myers Squibb Co | أجسام مضادة ضد- trem-1 واستخداماتها |
JP7402541B2 (ja) | 2018-05-03 | 2023-12-21 | ユニバーシティ オブ ロチェスター | 抗インフルエンザノイラミニダーゼモノクローナル抗体およびその使用 |
BR112020025346A2 (pt) | 2018-06-26 | 2021-05-25 | Immunogen, Inc. | imunoconjugados que alvejam adam9 e métodos de uso dos mesmos |
SG11202100096XA (en) | 2018-07-09 | 2021-02-25 | Five Prime Therapeutics Inc | Antibodies binding to ilt4 |
WO2020014306A1 (en) | 2018-07-10 | 2020-01-16 | Immunogen, Inc. | Met antibodies and immunoconjugates and uses thereof |
CA3104536A1 (en) | 2018-07-11 | 2020-01-16 | Bristol-Myers Squibb Company | Antibodies binding to vista at acidic ph |
WO2020072821A2 (en) | 2018-10-03 | 2020-04-09 | Xencor, Inc. | Il-12 heterodimeric fc-fusion proteins |
WO2020076969A2 (en) | 2018-10-10 | 2020-04-16 | Tilos Therapeutics, Inc. | Anti-lap antibody variants and uses thereof |
TW202029980A (zh) | 2018-10-26 | 2020-08-16 | 美商免疫遺傳股份有限公司 | E p C A M 抗體、可活化抗體及免疫偶聯物以及其用途 |
JP2022505923A (ja) | 2018-11-01 | 2022-01-14 | メルク パテント ゲーエムベーハー | 抗tim-3抗体を投与する方法 |
CN113272017A (zh) | 2018-11-01 | 2021-08-17 | 默克专利有限公司 | 抗tim-3抗体 |
WO2020112781A1 (en) | 2018-11-28 | 2020-06-04 | Bristol-Myers Squibb Company | Antibodies comprising modified heavy constant regions |
US20220098310A1 (en) | 2018-12-06 | 2022-03-31 | Alexion Pharmaceuticals, Inc. | Anti-alk2 antibodies and uses thereof |
JP2022515223A (ja) | 2018-12-21 | 2022-02-17 | オーエスイー・イミュノセラピューティクス | ヒトpd-1に対する二機能性分子 |
US11352430B2 (en) | 2018-12-21 | 2022-06-07 | Ose Immunotherapeutics | Humanized anti-human-PD-1 antibody and its use in cancer treatment |
CN113614109A (zh) | 2018-12-21 | 2021-11-05 | Ose免疫疗法公司 | 双功能抗pd-1/il-7分子 |
MX2021007274A (es) | 2018-12-21 | 2021-07-15 | Ose Immunotherapeutics | Molecula bifuncional anti-pd-1/sirpa. |
EP3914622A1 (en) | 2019-01-22 | 2021-12-01 | Bristol-Myers Squibb Company | Antibodies against il-7r alpha subunit and uses thereof |
WO2020165374A1 (en) | 2019-02-14 | 2020-08-20 | Ose Immunotherapeutics | Bifunctional molecule comprising il-15ra |
GB2589049C (en) | 2019-04-11 | 2024-02-21 | argenx BV | Anti-IgE antibodies |
CA3141459A1 (en) | 2019-05-17 | 2020-11-26 | Xencor, Inc. | Il-7-fc-fusion proteins |
CA3138072A1 (en) | 2019-06-07 | 2020-12-10 | Argenx Bvba | Pharmaceutical formulations of fcrn inhibitors suitable for subcutaneous administration |
CN114174536A (zh) | 2019-07-15 | 2022-03-11 | 百时美施贵宝公司 | 抗trem-1抗体及其用途 |
CN114144435A (zh) | 2019-07-15 | 2022-03-04 | 百时美施贵宝公司 | 针对人trem-1的抗体及其用途 |
EP4031575A1 (en) | 2019-09-19 | 2022-07-27 | Bristol-Myers Squibb Company | Antibodies binding to vista at acidic ph |
WO2021067863A2 (en) | 2019-10-03 | 2021-04-08 | Xencor, Inc. | Targeted il-12 heterodimeric fc-fusion proteins |
WO2021122866A1 (en) | 2019-12-17 | 2021-06-24 | Ose Immunotherapeutics | Bifunctional molecules comprising an il-7 variant |
JP2023509195A (ja) | 2020-01-08 | 2023-03-07 | アルジェニクス ビーブイ | 天疱瘡症を治療する方法 |
JP2023514010A (ja) | 2020-01-10 | 2023-04-05 | ブライト ピーク セラピューティクス エージー | 修飾il-2ポリペプチドおよびその使用 |
EP4090674A4 (en) | 2020-01-14 | 2024-01-24 | Synthekine Inc | METHODS AND COMPOSITIONS OF BIASED IL2 MUTEINS |
CA3170570A1 (en) | 2020-04-01 | 2021-10-07 | James J. KOBIE | Monoclonal antibodies against the hemagglutinin (ha) and neuraminidase (na) of influenza h3n2 viruses |
EP4132971A1 (en) | 2020-04-09 | 2023-02-15 | Merck Sharp & Dohme LLC | Affinity matured anti-lap antibodies and uses thereof |
WO2021222935A2 (en) | 2020-04-28 | 2021-11-04 | The Rockefeller University | Neutralizing anti-sars-cov-2 antibodies and methods of use thereof |
US20230192867A1 (en) | 2020-05-15 | 2023-06-22 | Bristol-Myers Squibb Company | Antibodies to garp |
WO2022010798A1 (en) | 2020-07-06 | 2022-01-13 | Kiromic BioPharma, Inc. | Mesothelin isoform binding molecules and chimeric pd1 receptor molecules, cells containing the same and uses thereof |
TWI815194B (zh) | 2020-10-22 | 2023-09-11 | 美商基利科學股份有限公司 | 介白素2-Fc融合蛋白及使用方法 |
JP2023548878A (ja) | 2020-11-04 | 2023-11-21 | ザ ロックフェラー ユニバーシティー | 中和抗sars-cov-2抗体 |
CA3202233A1 (en) | 2020-11-18 | 2022-05-27 | Kiromic BioPharma, Inc. | Gamma-delta t cell manufacturing processes and chimeric pd1 receptor molecules |
IL303745A (en) | 2020-12-17 | 2023-08-01 | Ose Immunotherapeutics | Bifunctional ANTI-PD1/IL-7 molecules |
JP2023554456A (ja) | 2020-12-18 | 2023-12-27 | チューハイ トリノマブ ファーマシューティカル カンパニー リミテッド | 呼吸器合胞体ウイルスに特異的に結合する分子 |
WO2022155324A1 (en) | 2021-01-15 | 2022-07-21 | The Rockefeller University | Neutralizing anti-sars-cov-2 antibodies |
EP4314068A1 (en) | 2021-04-02 | 2024-02-07 | The Regents Of The University Of California | Antibodies against cleaved cdcp1 and uses thereof |
WO2022214652A1 (en) | 2021-04-09 | 2022-10-13 | Ose Immunotherapeutics | Scaffold for bifunctioanl molecules comprising pd-1 or cd28 and sirp binding domains |
CA3213917A1 (en) | 2021-04-09 | 2022-10-13 | Nicolas Poirier | New scaffold for bifunctional molecules with improved properties |
AU2022307460A1 (en) * | 2021-07-09 | 2024-01-04 | Bright Peak Therapeutics Ag | Checkpoint inhibitors conjugated to il-2, and uses thereof |
WO2023281481A1 (en) * | 2021-07-09 | 2023-01-12 | Bright Peak Therapeutics | Antibody conjugates and manufacture thereof |
WO2023147399A1 (en) | 2022-01-27 | 2023-08-03 | The Rockefeller University | Broadly neutralizing anti-sars-cov-2 antibodies targeting the n-terminal domain of the spike protein and methods of use thereof |
WO2023242361A1 (en) | 2022-06-15 | 2023-12-21 | argenx BV | Fcrn binding molecules and methods of use |
WO2024015830A1 (en) | 2022-07-12 | 2024-01-18 | Cytomx Therapeutics, Inc. | Epcam immunoconjugates and uses thereof |
WO2024020579A1 (en) | 2022-07-22 | 2024-01-25 | Bristol-Myers Squibb Company | Antibodies binding to human pad4 and uses thereof |
WO2024028386A1 (en) | 2022-08-02 | 2024-02-08 | Ose Immunotherapeutics | Multifunctional molecule directed against cd28 |
Family Cites Families (173)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4196265A (en) | 1977-06-15 | 1980-04-01 | The Wistar Institute | Method of producing antibodies |
US4522811A (en) | 1982-07-08 | 1985-06-11 | Syntex (U.S.A.) Inc. | Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides |
US4469797A (en) | 1982-09-23 | 1984-09-04 | Miles Laboratories, Inc. | Digoxigenin immunogens, antibodies, labeled conjugates, and related derivatives |
US4737462A (en) | 1982-10-19 | 1988-04-12 | Cetus Corporation | Structural genes, plasmids and transformed cells for producing cysteine depleted muteins of interferon-β |
US4966843A (en) | 1982-11-01 | 1990-10-30 | Cetus Corporation | Expression of interferon genes in Chinese hamster ovary cells |
US4816567A (en) | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
US4703008A (en) * | 1983-12-13 | 1987-10-27 | Kiren-Amgen, Inc. | DNA sequences encoding erythropoietin |
KR850004274A (ko) * | 1983-12-13 | 1985-07-11 | 원본미기재 | 에리트로포이에틴의 제조방법 |
NZ210501A (en) * | 1983-12-13 | 1991-08-27 | Kirin Amgen Inc | Erythropoietin produced by procaryotic or eucaryotic expression of an exogenous dna sequence |
US5082658A (en) | 1984-01-16 | 1992-01-21 | Genentech, Inc. | Gamma interferon-interleukin-2 synergism |
EP0158198A1 (en) | 1984-03-29 | 1985-10-16 | Takeda Chemical Industries, Ltd. | DNA and use thereof |
US5807715A (en) | 1984-08-27 | 1998-09-15 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and transformed mammalian lymphocyte cells for producing functional antigen-binding protein including chimeric immunoglobulin |
US4667016A (en) | 1985-06-20 | 1987-05-19 | Kirin-Amgen, Inc. | Erythropoietin purification |
US4690915A (en) | 1985-08-08 | 1987-09-01 | The United States Of America As Represented By The Department Of Health And Human Services | Adoptive immunotherapy as a treatment modality in humans |
US5679543A (en) | 1985-08-29 | 1997-10-21 | Genencor International, Inc. | DNA sequences, vectors and fusion polypeptides to increase secretion of desired polypeptides from filamentous fungi |
US5643565A (en) | 1985-09-20 | 1997-07-01 | Chiron Corporation | Human IL-2 as a vaccine adjuvant |
US4676980A (en) | 1985-09-23 | 1987-06-30 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Target specific cross-linked heteroantibodies |
US4935233A (en) | 1985-12-02 | 1990-06-19 | G. D. Searle And Company | Covalently linked polypeptide cell modulators |
US5359035A (en) | 1985-12-21 | 1994-10-25 | Hoechst Aktiengesellschaft | Bifunctional proteins including interleukin-2 (IL-2) and granuloctyte macrophage colony stimulating factor (GM-CSF) |
DE3712985A1 (de) | 1987-04-16 | 1988-11-03 | Hoechst Ag | Bifunktionelle proteine |
EP0237019A3 (en) | 1986-03-14 | 1988-03-09 | Toray Industries, Inc. | Interferon conjugate and production thereof using recombinant gene |
GB8607679D0 (en) | 1986-03-27 | 1986-04-30 | Winter G P | Recombinant dna product |
US5225539A (en) | 1986-03-27 | 1993-07-06 | Medical Research Council | Recombinant altered antibodies and methods of making altered antibodies |
DK173067B1 (da) * | 1986-06-27 | 1999-12-13 | Univ Washington | Humant erythropoietin-gen, fremgangsmåde til ekspression deraf i transficerede cellelinier, de transficerede cellelinier sa |
US4894227A (en) | 1986-08-01 | 1990-01-16 | Cetus Corporation | Composition of immunotoxins with interleukin-2 |
US4946778A (en) | 1987-09-21 | 1990-08-07 | Genex Corporation | Single polypeptide chain binding molecules |
US5508031A (en) | 1986-11-21 | 1996-04-16 | Cetus Oncology Corporation | Method for treating biological damage using a free-radial scavenger and interleukin-2 |
US4732683A (en) | 1986-12-02 | 1988-03-22 | Biospectrum, Inc. | Purification method for alpha interferon |
US5019368A (en) | 1989-02-23 | 1991-05-28 | Cancer Biologics, Inc. | Detection of necrotic malignant tissue and associated therapy |
EP0307434B2 (en) | 1987-03-18 | 1998-07-29 | Scotgen Biopharmaceuticals, Inc. | Altered antibodies |
US5258498A (en) | 1987-05-21 | 1993-11-02 | Creative Biomolecules, Inc. | Polypeptide linkers for production of biosynthetic proteins |
DE3856559T2 (de) | 1987-05-21 | 2004-04-29 | Micromet Ag | Multifunktionelle Proteine mit vorbestimmter Zielsetzung |
US5091513A (en) | 1987-05-21 | 1992-02-25 | Creative Biomolecules, Inc. | Biosynthetic antibody binding sites |
ATE122238T1 (de) | 1987-06-10 | 1995-05-15 | Dana Farber Cancer Inst Inc | Bifunktionelle antikörperkonstruktionen und verfahren zur selektiven tötung von zellbeständen. |
US5064646A (en) | 1988-08-02 | 1991-11-12 | The University Of Maryland | Novel infectious bursal disease virus |
ES2054753T3 (es) | 1987-09-02 | 1994-08-16 | Ciba Geigy Ag | Conjugados de citoquinas con inmunoglobulinas. |
EP0308936B1 (en) | 1987-09-23 | 1994-07-06 | Bristol-Myers Squibb Company | Antibody heteroconjugates for the killing of HIV-infected cells |
NZ226414A (en) | 1987-10-02 | 1992-07-28 | Genentech Inc | Cd4 peptide adhesion variants and their preparation and use |
AU2635088A (en) | 1987-12-04 | 1989-06-08 | Du Pont Merck Pharmaceutical Company, The | Immobilized interleukin 2 and interleukin 2 containing a carboxyl-terminal extension |
WO1989006692A1 (en) | 1988-01-12 | 1989-07-27 | Genentech, Inc. | Method of treating tumor cells by inhibiting growth factor receptor function |
CA1341588C (en) | 1988-01-26 | 2009-01-06 | Michel Revel | Human ifn-beta2/i1-6, its purification and use |
US5120525A (en) | 1988-03-29 | 1992-06-09 | Immunomedics, Inc. | Radiolabeled antibody cytotoxic therapy of cancer |
US4975369A (en) | 1988-04-21 | 1990-12-04 | Eli Lilly And Company | Recombinant and chimeric KS1/4 antibodies directed against a human adenocarcinoma antigen |
IT1217724B (it) | 1988-05-26 | 1990-03-30 | Ist Naz Ric Sul Cancro | Anticorpo monoclonale specifico per una sequenza di fibronettina espressa in cellule trasformate ibridoma secernente tale anticorpo e impiego dell'anticorpo monoclonale per la diagnosi di tumori |
IE62463B1 (en) | 1988-07-07 | 1995-02-08 | Res Dev Foundation | Immunoconjugates for cancer diagnosis and therapy |
US5601819A (en) | 1988-08-11 | 1997-02-11 | The General Hospital Corporation | Bispecific antibodies for selective immune regulation and for selective immune cell binding |
US5457038A (en) * | 1988-11-10 | 1995-10-10 | Genetics Institute, Inc. | Natural killer stimulatory factor |
US5242824A (en) | 1988-12-22 | 1993-09-07 | Oncogen | Monoclonal antibody to human carcinomas |
US5530101A (en) | 1988-12-28 | 1996-06-25 | Protein Design Labs, Inc. | Humanized immunoglobulins |
US5116964A (en) | 1989-02-23 | 1992-05-26 | Genentech, Inc. | Hybrid immunoglobulins |
US5225538A (en) | 1989-02-23 | 1993-07-06 | Genentech, Inc. | Lymphocyte homing receptor/immunoglobulin fusion proteins |
US5703055A (en) | 1989-03-21 | 1997-12-30 | Wisconsin Alumni Research Foundation | Generation of antibodies through lipid mediated DNA delivery |
US5166322A (en) | 1989-04-21 | 1992-11-24 | Genetics Institute | Cysteine added variants of interleukin-3 and chemical modifications thereof |
US6750329B1 (en) | 1989-05-05 | 2004-06-15 | Research Development Foundation | Antibody delivery system for biological response modifiers |
ZA902949B (en) | 1989-05-05 | 1992-02-26 | Res Dev Foundation | A novel antibody delivery system for biological response modifiers |
US6291158B1 (en) | 1989-05-16 | 2001-09-18 | Scripps Research Institute | Method for tapping the immunological repertoire |
US5399346A (en) | 1989-06-14 | 1995-03-21 | The United States Of America As Represented By The Department Of Health And Human Services | Gene therapy |
ATE123065T1 (de) | 1989-07-07 | 1995-06-15 | Takeda Chemical Industries Ltd | Proteine und deren herstellung. |
US5073627A (en) | 1989-08-22 | 1991-12-17 | Immunex Corporation | Fusion proteins comprising GM-CSF and IL-3 |
US5856298A (en) | 1989-10-13 | 1999-01-05 | Amgen Inc. | Erythropoietin isoforms |
WO1991005867A1 (en) | 1989-10-13 | 1991-05-02 | Amgen Inc. | Erythropoietin isoforms |
ATE163675T1 (de) | 1989-12-22 | 1998-03-15 | Hoffmann La Roche | Zytotoxischer lymphozyten-reifefaktor |
US5314995A (en) | 1990-01-22 | 1994-05-24 | Oncogen | Therapeutic interleukin-2-antibody based fusion proteins |
US5349053A (en) | 1990-06-01 | 1994-09-20 | Protein Design Labs, Inc. | Chimeric ligand/immunoglobulin molecules and their uses |
US7253264B1 (en) * | 1990-06-28 | 2007-08-07 | Sanofi-Arentideutschland GmbH | Immunoglobulin fusion proteins, their production and use |
US5650150A (en) * | 1990-11-09 | 1997-07-22 | Gillies; Stephen D. | Recombinant antibody cytokine fusion proteins |
US5709859A (en) | 1991-01-24 | 1998-01-20 | Bristol-Myers Squibb Company | Mixed specificity fusion proteins |
US6072039A (en) | 1991-04-19 | 2000-06-06 | Rohm And Haas Company | Hybrid polypeptide comparing a biotinylated avidin binding polypeptide fused to a polypeptide of interest |
DE69233482T2 (de) | 1991-05-17 | 2006-01-12 | Merck & Co., Inc. | Verfahren zur Verminderung der Immunogenität der variablen Antikörperdomänen |
US5199942A (en) | 1991-06-07 | 1993-04-06 | Immunex Corporation | Method for improving autologous transplantation |
ATE173763T1 (de) | 1991-08-30 | 1998-12-15 | Hutchinson Fred Cancer Res | Hybride cytokine |
US20020037558A1 (en) * | 1991-10-23 | 2002-03-28 | Kin-Ming Lo | E.coli produced immunoglobulin constructs |
US6627615B1 (en) | 1991-12-17 | 2003-09-30 | The Regents Of The University Of California | Methods and compositions for in vivo gene therapy |
ATE260971T1 (de) | 1992-04-01 | 2004-03-15 | Univ Rockefeller | Verfahren zur in vitro kultivierung dendritischer vorläuferzellen und deren verwendung zur immunogen herstellung |
DK0615451T3 (da) | 1992-05-26 | 2006-04-24 | Immunex Corp | Hidtil ukendt cytokin der binder til CD30 |
CA2134773A1 (en) | 1992-06-04 | 1993-12-09 | Robert J. Debs | Methods and compositions for in vivo gene therapy |
US5614184A (en) | 1992-07-28 | 1997-03-25 | New England Deaconess Hospital | Recombinant human erythropoietin mutants and therapeutic methods employing them |
CA2142007C (en) | 1992-08-11 | 2007-10-30 | Robert Glen Urban | Immunomodulatory peptides |
DE4228839A1 (de) * | 1992-08-29 | 1994-03-03 | Behringwerke Ag | Verfahren zum Nachweis und zur Bestimmung von Mediatoren |
DE69232604T2 (de) | 1992-11-04 | 2002-11-07 | City Of Hope Duarte | Antikörperkonstrukte |
EP1757694A3 (en) | 1992-11-05 | 2008-02-27 | Sloan Kettering Institute For Cancer Research | Prostate-specific membrane antigen |
US5738849A (en) | 1992-11-24 | 1998-04-14 | G. D. Searle & Co. | Interleukin-3 (IL-3) variant fusion proteins, their recombinant production, and therapeutic compositions comprising them |
US5543297A (en) | 1992-12-22 | 1996-08-06 | Merck Frosst Canada, Inc. | Human cyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2 activity |
US6096331A (en) * | 1993-02-22 | 2000-08-01 | Vivorx Pharmaceuticals, Inc. | Methods and compositions useful for administration of chemotherapeutic agents |
CA2161095A1 (en) | 1993-04-20 | 1994-10-27 | Solis Therapeutics, Inc. | Methods and materials for treatment of individuals infected with intracellular infectious agents |
US5759551A (en) | 1993-04-27 | 1998-06-02 | United Biomedical, Inc. | Immunogenic LHRH peptide constructs and synthetic universal immune stimulators for vaccines |
JPH08509614A (ja) | 1993-04-29 | 1996-10-15 | アボツト・ラボラトリーズ | エリスロポエチン類似体組成物および方法 |
US5554512A (en) | 1993-05-24 | 1996-09-10 | Immunex Corporation | Ligands for flt3 receptors |
CA2125763C (en) | 1993-07-02 | 2007-08-28 | Maurice Kent Gately | P40 homodimer of interleukin-12 |
CN1057534C (zh) | 1993-08-17 | 2000-10-18 | 柯瑞英-艾格公司 | 促红细胞生成素类似物 |
AU692865B2 (en) | 1994-04-26 | 1998-06-18 | Children's Medical Center Corporation | Angiostatin and method of use for inhibition of angiogenesis |
US5639725A (en) | 1994-04-26 | 1997-06-17 | Children's Hospital Medical Center Corp. | Angiostatin protein |
US5837682A (en) | 1996-03-08 | 1998-11-17 | The Children's Medical Center Corporation | Angiostatin fragments and method of use |
CU22615A1 (es) | 1994-06-30 | 2000-02-10 | Centro Inmunologia Molecular | Procedimiento de obtención de anticuerpos monoclonales murinos menos inmunogénicos. anticuerpos monoclonales obtenidos |
US6429199B1 (en) | 1994-07-15 | 2002-08-06 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules for activating dendritic cells |
US6309853B1 (en) | 1994-08-17 | 2001-10-30 | The Rockfeller University | Modulators of body weight, corresponding nucleic acids and proteins, and diagnostic and therapeutic uses thereof |
US5888773A (en) | 1994-08-17 | 1999-03-30 | The United States Of America As Represented By The Department Of Health And Human Services | Method of producing single-chain Fv molecules |
US5541087A (en) | 1994-09-14 | 1996-07-30 | Fuji Immunopharmaceuticals Corporation | Expression and export technology of proteins as immunofusins |
PT706799E (pt) | 1994-09-16 | 2002-05-31 | Merck Patent Gmbh | Imunoconjugados ii |
DE69534265T2 (de) * | 1994-12-12 | 2006-05-04 | Beth Israel Deaconess Medical Center, Inc., Boston | Chimäre zytokine und ihre verwendung |
US6086875A (en) | 1995-01-17 | 2000-07-11 | The Brigham And Women's Hospital, Inc. | Receptor specific transepithelial transport of immunogens |
US6485726B1 (en) | 1995-01-17 | 2002-11-26 | The Brigham And Women's Hospital, Inc. | Receptor specific transepithelial transport of therapeutics |
US5691309A (en) | 1995-01-31 | 1997-11-25 | Eli Lilly And Company | Anti-obesity proteins |
US5552524A (en) | 1995-01-31 | 1996-09-03 | Eli Lilly And Company | Anti-obesity proteins |
US5891680A (en) | 1995-02-08 | 1999-04-06 | Whitehead Institute For Biomedical Research | Bioactive fusion proteins comprising the p35 and p40 subunits of IL-12 |
ES2225874T3 (es) | 1995-03-10 | 2005-03-16 | Genentech, Inc. | Activacion de receptor mediante gas. |
US5719266A (en) | 1995-03-17 | 1998-02-17 | Eli Lilly And Company | Anti-obesity proteins |
US6281010B1 (en) | 1995-06-05 | 2001-08-28 | The Trustees Of The University Of Pennsylvania | Adenovirus gene therapy vehicle and cell line |
GB9511935D0 (en) * | 1995-06-13 | 1995-08-09 | Smithkline Beecham Plc | Novel compound |
WO1997002004A2 (en) | 1995-06-30 | 1997-01-23 | Eli Lilly And Company | Methods for treating diabetes |
US6406689B1 (en) | 1995-10-03 | 2002-06-18 | Frank W. Falkenberg | Compositions and methods for treatment of tumors and metastatic diseases |
US5854205A (en) | 1995-10-23 | 1998-12-29 | The Children's Medical Center Corporation | Therapeutic antiangiogenic compositions and methods |
US6080409A (en) | 1995-12-28 | 2000-06-27 | Dendreon Corporation | Immunostimulatory method |
US5723125A (en) * | 1995-12-28 | 1998-03-03 | Tanox Biosystems, Inc. | Hybrid with interferon-alpha and an immunoglobulin Fc linked through a non-immunogenic peptide |
US6750334B1 (en) * | 1996-02-02 | 2004-06-15 | Repligen Corporation | CTLA4-immunoglobulin fusion proteins having modified effector functions and uses therefor |
CN1136197C (zh) | 1996-05-30 | 2004-01-28 | 霍夫曼-拉罗奇有限公司 | 新的哒嗪酮衍生物 |
US5922685A (en) | 1996-06-05 | 1999-07-13 | Powderject Vaccines, Inc. | IL-12 gene therapy of tumors |
ATE218143T1 (de) | 1996-09-03 | 2002-06-15 | Gsf Forschungszentrum Umwelt | Verwendung bi-und trispezifischer antikörper zur induktion einer tumorimmunität |
US5994104A (en) | 1996-11-08 | 1999-11-30 | Royal Free Hospital School Of Medicine | Interleukin-12 fusion protein |
US6100387A (en) * | 1997-02-28 | 2000-08-08 | Genetics Institute, Inc. | Chimeric polypeptides containing chemokine domains |
US6277375B1 (en) | 1997-03-03 | 2001-08-21 | Board Of Regents, The University Of Texas System | Immunoglobulin-like domains with increased half-lives |
EP0973550B1 (en) | 1997-04-11 | 2002-10-09 | G.D. SEARLE & CO. | Antagonistic anti-avb3 integrin antibodies |
ATE267215T1 (de) | 1997-12-08 | 2004-06-15 | Lexigen Pharm Corp | Heterodimäre fusionsproteine zur verwendung für gezielte immuntherapie und allgemeine immunerregung |
US20030105294A1 (en) * | 1998-02-25 | 2003-06-05 | Stephen Gillies | Enhancing the circulating half life of antibody-based fusion proteins |
BR9908226A (pt) * | 1998-02-25 | 2000-10-24 | Lexigen Pharm Corp | Melhoramento da meia vida de circulação de proteìnas de fusão com base em anticorpo |
PL343462A1 (en) | 1998-04-15 | 2001-08-13 | Lexigen Pharm Corp | Enhancement of antibody-cytokine fusion protein mediated immune responses by co-administration with angiogenesis inhibitor |
EP1071469A2 (en) * | 1998-04-17 | 2001-01-31 | Lexigen Pharmaceuticals Corp. | Enhancement of antibody-cytokine fusion protein mediated immune responses by co-administration with prostaglandin inhibitor |
WO1999054484A1 (en) * | 1998-04-20 | 1999-10-28 | The Regents Of The University Of California | Modified immunoglobulin molecules and methods for use thereof |
WO1999058662A1 (fr) | 1998-05-14 | 1999-11-18 | Merck Patent Gmbh | Proteine fusionnee |
US6620382B1 (en) * | 1998-05-22 | 2003-09-16 | Biopheresis Technologies, Llc. | Method and compositions for treatment of cancers |
US20020142374A1 (en) | 1998-08-17 | 2002-10-03 | Michael Gallo | Generation of modified molecules with increased serum half-lives |
CA2339331C (en) * | 1998-08-25 | 2011-03-01 | Lexigen Pharmaceuticals Corporation | Expression and export of angiogenesis inhibitors as immunofusins |
US6646113B1 (en) * | 1998-09-17 | 2003-11-11 | The Trustees Of The University Of Pennsylvania | Nucleic acid molecule encoding human survival of motor neuron-interacting protein 1 (SIP1) deletion mutants |
US6335176B1 (en) * | 1998-10-16 | 2002-01-01 | Pharmacopeia, Inc. | Incorporation of phosphorylation sites |
KR20020007287A (ko) * | 1999-01-07 | 2002-01-26 | 추후보정 | Fc 융합 단백질로서 항-비만 단백질의 발현 및 이출 |
KR100922809B1 (ko) | 1999-05-06 | 2009-10-21 | 웨이크 포리스트 유니버시티 | 면역 반응을 유발하는 항원을 동정하기 위한 조성물과 방법 |
US6348192B1 (en) | 1999-05-11 | 2002-02-19 | Bayer Corporation | Interleukin-2 mutein expressed from mammalian cells |
EP1187852B1 (en) * | 1999-05-19 | 2007-08-08 | EMD Lexigen Research Center Corp. | EXPRESSION AND EXPORT OF INTERFERON-ALPHA PROTEINS AS Fc FUSION PROTEINS |
JO2291B1 (en) * | 1999-07-02 | 2005-09-12 | اف . هوفمان لاروش ايه جي | Erythropoietin derivatives |
CZ299516B6 (cs) * | 1999-07-02 | 2008-08-20 | F. Hoffmann-La Roche Ag | Konjugát erythropoetinového glykoproteinu, zpusobjeho výroby a použití a farmaceutická kompozice sjeho obsahem |
SK782002A3 (en) | 1999-07-21 | 2003-08-05 | Lexigen Pharm Corp | FC fusion proteins for enhancing the immunogenicity of protein and peptide antigens |
US7067110B1 (en) | 1999-07-21 | 2006-06-27 | Emd Lexigen Research Center Corp. | Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens |
WO2001010912A1 (en) * | 1999-08-09 | 2001-02-15 | Lexigen Pharmaceuticals Corp. | Multiple cytokine-antibody complexes |
US20050202538A1 (en) | 1999-11-12 | 2005-09-15 | Merck Patent Gmbh | Fc-erythropoietin fusion protein with improved pharmacokinetics |
WO2001036489A2 (en) | 1999-11-12 | 2001-05-25 | Merck Patent Gmbh | Erythropoietin forms with improved properties |
CA2399832C (en) | 2000-02-11 | 2011-09-20 | Stephen D. Gillies | Enhancing the circulating half-life of antibody-based fusion proteins |
PT1257297E (pt) * | 2000-02-24 | 2006-12-29 | Philogen Spa | Composições e método para tratamento da angiogénese em lesões patológicas |
US6586398B1 (en) * | 2000-04-07 | 2003-07-01 | Amgen, Inc. | Chemically modified novel erythropoietin stimulating protein compositions and methods |
MXPA02011016A (es) * | 2000-05-12 | 2004-03-16 | Neose Technologies Inc | Glicopeptidos recombinantes de glucolisacion in vitro. |
ES2288967T3 (es) | 2000-06-29 | 2008-02-01 | Merck Patent Gmbh | Reforzamiento de las respuestas inmunes mediadas por la proteina de fusion anticuerpo-citoquina por medio del tratamiento combinado por agentes que mejoran la incorporacion de inmunocitoquina. |
JP2004525621A (ja) * | 2001-01-18 | 2004-08-26 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフトング | グルコセレブロシダーゼ活性を有する二機能性融合タンパク質 |
JP2004532620A (ja) * | 2001-02-19 | 2004-10-28 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフトング | 免疫原性の低減された人工タンパク質 |
CA2440221C (en) | 2001-03-07 | 2013-02-05 | Merck Patent Gesellschaft Mit Beschraenkter Haftung | Expression technology for proteins containing a hybrid isotype antibody moiety |
WO2002079415A2 (en) | 2001-03-30 | 2002-10-10 | Lexigen Pharmaceuticals Corp. | Reducing the immunogenicity of fusion proteins |
BR0209177A (pt) | 2001-05-03 | 2004-10-05 | Merck Patent Gmbh | Anticorpo especìfico a tumor recombinante e uso do mesmo |
US6900292B2 (en) * | 2001-08-17 | 2005-05-31 | Lee-Hwei K. Sun | Fc fusion proteins of human erythropoietin with increased biological activities |
ES2381025T3 (es) | 2001-12-04 | 2012-05-22 | Merck Patent Gmbh | Inmunocitocinas con selectividad modulada |
CN101143221A (zh) | 2002-03-15 | 2008-03-19 | 布赖汉姆妇女医院 | 适合治疗剂全身性递送的中央气道给药 |
ES2346205T3 (es) | 2002-12-17 | 2010-10-13 | Merck Patent Gmbh | Anticuerpo humanizado (h14.18) del anticurpo 14.18 de raton que se enlaza con gd2 y su fusion con la il-2. |
US20050069521A1 (en) | 2003-08-28 | 2005-03-31 | Emd Lexigen Research Center Corp. | Enhancing the circulating half-life of interleukin-2 proteins |
WO2005037867A1 (en) * | 2003-10-15 | 2005-04-28 | Pdl Biopharma, Inc. | ALTERATION OF Fc-FUSION PROTEIN SERUM HALF-LIVES BY MUTAGENESIS OF POSITIONS 250, 314 AND/OR 428 OF THE HEAVY CHAIN CONSTANT REGION OF IG |
WO2005063820A2 (en) | 2003-12-30 | 2005-07-14 | Merck Patent Gmbh | Il-7 fusion proteins |
KR20060124656A (ko) | 2003-12-31 | 2006-12-05 | 메르크 파텐트 게엠베하 | 개선된 약물동태를 가지는 Fc-에리스로포이에틴 융합단백질 |
CA2553883C (en) | 2004-01-22 | 2013-04-02 | Merck Patent Gesellschaft Mit Beschraenkter Haftung | Anti-cancer antibodies with reduced complement fixation |
US7670595B2 (en) | 2004-06-28 | 2010-03-02 | Merck Patent Gmbh | Fc-interferon-beta fusion proteins |
DE602005020837D1 (de) | 2004-12-09 | 2010-06-02 | Merck Patent Gmbh | Il-7-varianten mit reduzierter immunogenität |
US20070104689A1 (en) | 2005-09-27 | 2007-05-10 | Merck Patent Gmbh | Compositions and methods for treating tumors presenting survivin antigens |
CA2635618C (en) | 2005-12-30 | 2015-10-06 | Merck Patent Gesellschaft Mit Beschraekter Haftung | Interleukin-12p40 variants with improved stability |
PT2270050E (pt) | 2005-12-30 | 2013-09-12 | Merck Patent Gmbh | Anticorpos anti-cd19 com imunogenicidade reduzida |
KR20080090484A (ko) | 2005-12-30 | 2008-10-08 | 메르크 파텐트 게엠베하 | GP130 에 대한, IL-6Rα 와 복합체를 형성한 IL-6의 결합을 방해하는 항-IL-6 항체 |
AU2007229698B9 (en) | 2006-03-24 | 2012-11-08 | Merck Patent Gmbh | Engineered heterodimeric protein domains |
EP2038417A2 (en) | 2006-07-06 | 2009-03-25 | Merck Patent GmbH | Compositions and methods for enhancing the efficacy of il-2 mediated immune responses |
US7622522B2 (en) | 2007-09-27 | 2009-11-24 | Sabic Innovative Plastics Ip B.V. | Flame-retardant poly(arylene ether) composition and its use as a covering for coated wire |
-
2001
- 2001-02-09 CA CA2399832A patent/CA2399832C/en not_active Expired - Lifetime
- 2001-02-09 AT AT01916083T patent/ATE336514T1/de active
- 2001-02-09 DK DK01916083T patent/DK1252192T3/da active
- 2001-02-09 AU AU4314801A patent/AU4314801A/xx active Pending
- 2001-02-09 MX MXPA02007733A patent/MXPA02007733A/es active IP Right Grant
- 2001-02-09 JP JP2001558103A patent/JP5179689B2/ja not_active Expired - Fee Related
- 2001-02-09 CN CN018058639A patent/CN1406249B/zh not_active Expired - Lifetime
- 2001-02-09 US US09/780,668 patent/US7091321B2/en not_active Expired - Lifetime
- 2001-02-09 EP EP01916083A patent/EP1252192B1/en not_active Expired - Lifetime
- 2001-02-09 HU HU0204475A patent/HUP0204475A2/hu unknown
- 2001-02-09 DE DE60122286T patent/DE60122286T2/de not_active Expired - Lifetime
- 2001-02-09 WO PCT/US2001/004455 patent/WO2001058957A2/en active IP Right Grant
- 2001-02-09 ES ES01916083T patent/ES2269366T3/es not_active Expired - Lifetime
- 2001-02-09 PT PT01916083T patent/PT1252192E/pt unknown
-
2002
- 2002-08-09 NO NO20023774A patent/NO20023774L/no not_active Application Discontinuation
-
2005
- 2005-11-07 US US11/268,191 patent/US7507406B2/en not_active Expired - Lifetime
-
2006
- 2006-10-24 CY CY20061101528T patent/CY1105725T1/el unknown
-
2009
- 2009-01-27 US US12/360,682 patent/US7790415B2/en not_active Expired - Fee Related
-
2012
- 2012-05-10 JP JP2012108394A patent/JP5572665B2/ja not_active Expired - Lifetime
Non-Patent Citations (1)
Title |
---|
RADZICKA A等.Comparing the Polarities of the Amino Acids: Side-ChainDistribution Coefficients between the Vapor Phase,Cyclohexane, 1-Octanol, and Neutral Aqueous Solution.Biochemistry27.1988,表II. * |
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US7091321B2 (en) | 2006-08-15 |
JP5572665B2 (ja) | 2014-08-13 |
CA2399832A1 (en) | 2001-08-16 |
HUP0204475A2 (en) | 2003-04-28 |
MXPA02007733A (es) | 2004-09-10 |
ES2269366T3 (es) | 2007-04-01 |
WO2001058957A3 (en) | 2002-05-02 |
AU4314801A (en) | 2001-08-20 |
US20060034836A1 (en) | 2006-02-16 |
DE60122286T2 (de) | 2007-08-02 |
US20090264627A1 (en) | 2009-10-22 |
ATE336514T1 (de) | 2006-09-15 |
DK1252192T3 (da) | 2006-11-20 |
JP2012176969A (ja) | 2012-09-13 |
US20020147311A1 (en) | 2002-10-10 |
EP1252192B1 (en) | 2006-08-16 |
JP5179689B2 (ja) | 2013-04-10 |
WO2001058957A2 (en) | 2001-08-16 |
NO20023774D0 (no) | 2002-08-09 |
DE60122286D1 (de) | 2006-09-28 |
CN1406249A (zh) | 2003-03-26 |
CA2399832C (en) | 2011-09-20 |
US7507406B2 (en) | 2009-03-24 |
US7790415B2 (en) | 2010-09-07 |
NO20023774L (no) | 2002-08-09 |
CY1105725T1 (el) | 2010-12-22 |
PT1252192E (pt) | 2007-01-31 |
EP1252192A2 (en) | 2002-10-30 |
JP2003522200A (ja) | 2003-07-22 |
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