干扰素α2b变体的制作方法

发明领域本发明涉及包含靶向针对细胞表面抗原的配体和去糖基化干扰素α2b(ifnα2b)的多肽以及这些多肽在治疗癌症中的用途。发明背景已经描述了许多肽和多肽分子通过与细胞表面上的受体相互作用起作用，从而刺激、抑制或以其他方式调节生物应答，并且通常涉及携带所述受体的细胞内的信号转导途径。这些分子的实例包括肽和多肽激素、细胞因子、趋化因子、生长因子、细胞凋亡诱导因子等。这些分子可以是可溶的或可以连接到另一个细胞的表面。由于此类分子的生物活性，一些具有作为治疗剂的潜在用途。若干肽或多肽分子已被监管机构批准为治疗产品，包括例如人生长激素、胰岛素、干扰素ifnα2b、ifnα2a、ifnβ、促红细胞生成素、g-csf和gm-csf。这些和其他肽中的许多已经证明在治疗应用中具有潜力，但是当施用于人类患者时也显示出毒性。毒性的一个原因是这些分子大多数触发各种细胞(包括除介导治疗效果的细胞以外的细胞)上的受体。例如，当ifnα2b用于治疗多发性骨髓瘤时，其效用至少部分地在其与骨髓瘤细胞上的i型干扰素受体的结合中，其进而引起减少的增殖并因此限制疾病进展。然而，遗憾的是，该ifn也与体内许多其它的正常细胞结合，引发各种其他细胞应答，其中一些是有害的(例如流感样症状、中性粒细胞减少、抑郁)。这样的肽的“脱靶(offtarget)”活性的结果是许多肽不适合作为候选药物。在这种情况下，“脱靶活性”是指肽的天然受体的活性，但是作用于除了介导治疗有益作用的细胞以外的细胞表面。尽管一些肽(例如ifnα2b)被批准用于治疗医学病症，但是由于其“脱靶”生物活性，它们耐受性差。脱靶活性和相关的耐受性差也意味着这些基于肽的药物中的一些不能以足够高的剂量施用以对介导治疗效果的靶细胞产生最佳治疗效果。类似地，自20世纪80年代中期以来已知干扰素、特别是ifnα能够增加某些癌细胞的细胞凋亡并降低其增殖。这些生物活性由癌细胞表面的i型干扰素受体介导，当被刺激时，它们引发导致减少的增殖和/或诱导终末分化或细胞凋亡的各种信号转导途径。ifnα已经被美国fda批准用于治疗几种癌症，包括黑色素瘤、肾细胞癌、b细胞淋巴瘤、多发性骨髓瘤、慢性骨髓性白血病(cml)和毛细胞白血病。ifnα对肿瘤细胞的“直接”作用是通过直接与这些细胞上的i型ifn受体结合的ifnα介导的，并且刺激细胞凋亡、终末分化或减少增殖。ifnα对非癌细胞的一个“间接”作用是刺激免疫系统，这可能通过使免疫系统排斥肿瘤而产生额外的抗癌作用。不幸的是，i型干扰素受体也存在于大多数非癌细胞上。ifnα对此类细胞上这种受体的活化导致许多促炎细胞因子和趋化因子的表达，导致毒性。这样的毒性阻止将ifnα以对癌细胞发挥最大的抗增殖和促凋亡活性的水平向受试者给药。ozzello等人(breastcancerresearchandtreatment25：265-76,1993)描述了将人ifnα共价连接到靶向肿瘤的抗体上，从而将ifnα直接抑制活性定位于肿瘤作为降低肿瘤生长速度的方式，并证明此类缀合物在人类癌症的异种移植模型中具有抗肿瘤活性。所观察到的抗癌活性的机制归因于ifnα对癌细胞的直接作用，因为实验中使用的人ifnα与鼠i型ifn受体没有明显的相互作用，这可能导致间接抗癌作用。然而，由于这缺乏人ifnα与鼠细胞的结合，作者无法评估抗体-ifnα缀合物相对于游离infα的毒性。这些作者使用化学方法将ifnα与抗体连接。alkan等人(journalofinterferonresearch,volume4,number3,p.355-63,1984)证明将人ifnα连接到结合eb病毒(epstein-barrvirus，ebv)膜抗原(ma)的抗体将增加其对表达ebv-ma抗原的细胞的抗增殖活性。这种增加的效力取决于靶细胞的抗原表达和抗体的结合特异性。所测试的细胞系是癌细胞系qimr-wil，一种粒细胞白血病。作者提出，ifnα与抗体的连接可用作癌症的治疗，因为它可以减少肿瘤生长。alkan等人没有解决由于它们与正常的抗原阴性细胞相互作用而引起的这些抗体-ifnα缀合物的潜在毒性的问题。还已知抗体和ifnα之间的连接可以通过制备融合蛋白构建体来实现。例如，idec(wo01/97844)公开了人ifnα与靶向肿瘤抗原cd20的igg的重链的c末端的直接融合。其他组已经公开了在igg重链的c末端和ifnα之间使用各种接头。例如，us7,456,257公开了抗体重链恒定区的c末端可以通过序列(ggggs)n的中间富含丝氨酸-甘氨酸(s/g)接头连接到ifnα，其中n可以是1、2或3，并且不管接头长度如何，融合蛋白构建体的ifnα活性没有显著性差异。morrison等人(us2011/0104112a1；和xuanc,stewardkk,timmermanjm,morrisonsl.targeteddeliveryofinterferon-αviafusionto抗cd20resultsinpotentantitumoractivityagainstb-celllymphoma.blood2010；115:2864–71)还公开了连接于靶向癌症的igg抗体重链的c末端的ifnα与中间s/g接头，并且观察到igg和接头与ifnα的融合降低了ifnα对细胞表面上未表达相应抗原的细胞上活性。当与作用于人类细胞上的人非融合蛋白ifnα(游离ifnα)相比时，这些融合蛋白构建体的ifn活性降低是适度的，但对鼠细胞中的鼠ifnα似乎更显著。morrison等人和在us7,456,257中观察到的将其融合到抗体的c末端导致的人ifnα的活性的降低是适度的，并且通常被认为是缺点，因为它降低了ifn的效力。例如，rossi等人(bloodvol.114,no.18,pp3864-71)指出了这个缺点，他们使用将ifnα连接到靶向肿瘤的抗体的替代策略，使得没有观察到ifnα活性损失。一般来说，现有技术教导使用有效的ifn并将该ifn靶向癌细胞。虽然这种方法导致ifn针对癌细胞的活性增加，但是它并没有解决ifn对正常“脱靶”细胞的活性问题。在上述现有技术实例中，当暴露于在其细胞表面上不表达相应抗原的人细胞时，抗体-ifnα融合蛋白的人ifnα部分保持高比例的天然ifnα活性。这种活性可能导致由融合蛋白的ifnα部分活化非癌、正常(“脱靶”)细胞引起的毒性。因此，需要降低基于ifn的药物的“脱靶(off-target)”活性，同时保留这些药物的“在靶(on-target)”治疗效果。维持这些类型治疗剂的靶特异性活性和同时降低其非靶毒性将为治疗有用的肽产生更大的治疗浓度窗口。例如，以使其活性可以针对癌细胞同时最小化其对正常人细胞的作用的形式使用人ifnα是理想的。理想地，癌细胞上的i型干扰素受体将被最大限度地刺激，而非癌细胞上的相同受体将经历最小的刺激。需要将人ifnα靶向癌细胞，使得其对显示抗原的癌细胞比不显示抗原的正常细胞具有显著更多的活性。相同的逻辑适用于其他潜在的治疗分子，例如其他细胞因子、肽和多肽激素、趋化因子、生长因子、细胞凋亡诱导因子等。在wo2013/059885和wo2014/178820中已经证明了这种方法的逻辑，其各自的公开内容通过交叉引用并入本文。发明概述第一方面，本发明提供了包含第一结构域和第二结构域的融合多肽，其中第一结构域包含与细胞表面相关抗原结合的多肽配体，第二结构域包含去糖基化的干扰素α2b(ifnα2b)，去糖基化的ifnα2b具有seqidno：1或seqidno：2的序列，并且其中去糖基化的ifnα2b进一步包含一种或多种氨基酸置换或缺失，其减弱去糖基化的ifnα2b的活性。另一方面，本发明提供了包含选自seqidno：31、61-77、83和87的序列以及选自seqidno：81、82和84的序列的融合多肽。另一方面，本发明提供了包含seqidno：87和seqidno：81的融合多肽。另一方面，本发明提供了包含seqidno：79和seqidno：85的融合多肽。另一方面，本发明提供了包含seqidno：80和seqidno：86的融合多肽。另一方面，本发明提供了包含seqidno：78的融合多肽。另一方面，本发明提供了包含本发明的融合多肽和药学上可接受的载体或稀释剂的组合物。另一方面，本发明提供了治疗受试者中肿瘤的方法，包括向受试者施用本发明的融合多肽或本发明的组合物，其中融合多肽的第一结构域与肿瘤细胞结合。另一方面，本发明提供了本发明的融合多肽在治疗肿瘤中的用途，其中融合多肽的第一结构域与肿瘤结合。另一方面，本发明提供了编码本发明的一种或多种融合多肽的一种或多种分离的多核苷酸。另一方面，本发明提供了包含本发明的一种或多种多核苷酸的载体。另一方面，本发明提供了包含本发明载体的转化细胞。另一方面，本发明提供了在哺乳动物细胞中产生多肽配体-减弱的ifnα2b融合多肽的方法，其中多肽配体-减弱的ifnα2b融合多肽具有降低的异质性和/或增强的fcrn结合和/或改善的靶选择性，该方法包括培养包含编码多肽配体-减弱的ifnα2b融合多肽的多核苷酸的重组哺乳动物细胞，其中ifnα2b序列的t106被另一氨基酸替换或缺失，使得其在哺乳动物细胞中表达时，融合蛋白的ifnα2b组分是去糖基化的。附图简要说明图1：用具有和不具有ifnα2b的o-连接的糖基化的igg1或igg4形式的抗cd38-减弱的ifnα2b的融合蛋白处理(a)arp1和(b)nci-h929细胞的抗增殖活性。图2a、b、c、d和e：用不同氨基酸置换从减弱的ifnα2b中除去o连接的糖基化位点的抗cd38-减弱的ifnα2b的融合蛋白的抗增殖活性。图3：具有(t106t)和不具有(t106a)ifnα2b的o-连接的糖基化的(a)a10.21和(b)a10.43抗cd38-减弱的ifnα2b的融合蛋白的在靶活性。图4：具有(t106t)和不具有(t106a)ifnα2b的o-连接的糖基化的(a)a10.21和(b)a10.43抗cd38-减弱的ifnα2b的融合蛋白的脱靶活性。图5：具有(t106t)和不具有(t106a或δt106)ifnα2b的o-连接的糖基化的抗cd38-减弱的ifnα2b的融合蛋白的脱靶活性。图6：a、b、c、d、e和f：用不同氨基酸置换从减弱的ifnα2b去除o-连接的糖基化位点的抗cd38-减弱的ifnα2b的融合蛋白的脱靶活性。图7：具有(t106t)或不具有(t106a)ifnα2b的o-连接的糖基化的a10.43抗cd38-减弱的ifnα2b的融合蛋白的脱靶活性。图8：具有(t106t)或不具有(t106a)o-连接的糖基化的抗cd38-减弱的ifnα2b的融合蛋白的次优剂量在多发性骨髓瘤的鼠模型中治疗肿瘤的效力。图9：具有(t106t)或不具有(t106a、δt106、t106s、t106v、t106g、t106e)的ifnα2b的o-连接的糖基化的a10.21抗cd38-减弱的ifnα2b的融合蛋白的带电物质的数量，如通过ief凝胶上的条带数量所评估的。图10：具有(t106t)或不具有(t106a)ifnα2b的o连接的糖基化的和具有不同fc同种型的a10.21抗cd38-减弱的ifnα2b的融合蛋白的带电物质的数量，如通过ief凝胶上的条带数量所评估的。图11：在抗体恒定区存在yte置换的情况下，具有(t106t)或不具有(t106a)ifnα2b的o-连接的糖基化的a10.21(具有s228p的igg4)抗cd38-减弱的ifnα2b的融合蛋白的带电物质的数量，如通过ief凝胶上的条带数量所评估的。图12：在各种减弱置换的存在下，具有(t106t)或不具有(t106a)ifnα2b的o-连接的糖基化的a10.21(具有s228p的igg4)抗cd38-减弱的ifnα2b的融合蛋白的带电物质的数量，如通过ief凝胶上的条带数量所评估的。图13：具有不同靶特异性的抗体；与具有(t106t)或不具有(t106a)ifnα2b的o-连接的糖基化的减弱的ifnα2b融合的抗cd138抗体、抗hla抗体和抗cd38抗体(a02.12)(所有具有s228p的igg4)的带电物质的数量，如通过ief凝胶上的条带数量所评估的。图14：具有(t106t)和不具有(t106a、δt106、t106s、t106v、t106g、t106e)ifnα2b的o-连接的糖基化的抗cd38-减弱的ifnα2b的融合蛋白(a10.21igg4(s228p)ifn(a145d))的“在靶”活性。图15：两种不同的结合cd38上的不同表位的抗cd38抗体-减弱的ifnα2b的融合蛋白(a02.12和a10.21，两者具有s228p的igg4)的“在靶”活性，其具有(t106t)或不具有(t106a)ifnα2b的o-连接的糖基化。图16：具有(t106t)和不具有(t106a)ifnα2b的o-连接的糖基化的a10.21抗cd38-减弱的ifnα2b的融合蛋白(a10.21igg4(s228p)ifn)的“在靶”活性，其具有各种ifn减弱置换(r33a、r144i、r145q、a145k或a145g)。图17：具有不同靶特异性的抗体的“在靶”活性；与具有(t106t)或不具有(t106a)ifnα2b的o-连接的糖基化的减弱的ifnα2b融合的抗cd138抗体和抗hla抗体(均为具有s228p的igg4)。图18：在抗体重链中存在yte置换的情况下，具有(t106t)和不具有(t106a)ifnα2b的o-连接糖基化的a10.21抗cd38-减弱的ifnα2b的融合蛋白(a10.21igg4(s228p)ifn(a145d))的“靶向”活性。图19：具有(t106t)和不具有(t106a)o-连接的糖基化ifnα2b的a10.21抗cd38-减弱的ifnα2b(a145d)的融合蛋白的“靶向”活性，其具有各种免疫球蛋白fc同种型。图20：在存在多种氨基酸置换以除去ifn、免疫球蛋白恒定区yte置换以延长半衰期、减弱ifn和fc同种型的情况下，具有和不具有ifnα2b的o-连接的糖基化的a10.21抗cd38-减弱的ifnα2b的融合蛋白的选择性指数。发明详述在整个说明书中，除非上下文另有要求，单词“包含(comprise)”或诸如“comprises”或“comprising”的变体将被理解为暗示包含所述元素或整数或元素组或整数组但不排除任何其他元素或整数或元素组或整数组。本说明书中对任何在先出版物(或从其衍生的信息)或任何已知的事项的引用不是也不应被视为确认或承认或任何形式的表明在先出版物(或从其衍生的信息)或已知事物构成本说明书所涉及的努力领域的公知常识的一部分。本说明书中提及的所有出版物的全部内容通过引用并入本文。必须指出，如本说明书中所使用的那样，单数形式“一(a)”、“一(an)”和“该(the)”包括多个方面，除非上下文另有明确规定。因此，例如，提及“一种试剂”包括单一试剂以及两种或更多种试剂；提及“一个分子”包括单个分子以及两个或更多个分子；等等。本发明的构建体是多肽配体减弱的去糖基的ifnα2b融合构建体，其由于靶向感兴趣的细胞上细胞表面受体的配体和对细胞表面ifn受体的亲和力降低的减弱的ifnα2b二者的作用而显示相对于活化信号传导途径升高的抗原选择性指数。这些构建体基于wo2013/059885中概述的发现，在抗体-ifn融合构建体的上下文中，可以这样的方式突变ifn部分：使得对抗原阴性细胞的ifn活性显著减弱，而对抗原阳性细胞的ifn活性只有适度(ifatall)减弱。与抗原阴性细胞相比，这样的构建体对抗原阳性细胞显示出比游离ifn更强的效力，即1个、2个、3个、4个或5个数量级。在一个实施方案中，抗体减弱的ifn构建体对抗原阳性细胞保留非减弱游离(即不连接至抗体)ifn的效力的至少1％、至少10％、至少20％、至少30％、至少40％或至少50％。此外，在一个实施方案中，抗体减弱的ifn构建体保留非减弱游离(即不连接至抗体)的最大活性的至少30％、至少50％、至少75％或至少90％ifn；在这种情况下，“最大活性”应理解为意指在剂量-反应曲线的高平台部分的信号传导活性(或其下游作用)的量，其中进一步增加试剂不会进一步增加应答。本发明人现在已经发现，与包含o-糖基化的ifnα2b的构建体相比，通过使用包含去糖基化的ifnα2b的构建体获得了意想不到的优点。在一些实施方案中，这些优点包括以下优点中的一个或多个：在靶活性增加、增加的靶选择性和增强的对fcrn的亲和力，同时在哺乳动物细胞表达系统中产生时，提供比o-糖基化的ifnα2b异质性产物更少。增强的fcrn结合是期望的，以改善包含fc区的生物治疗剂的pk。增加的靶选择性是可取的，因为它潜在地降低脱靶毒性，同时实质上保持在靶活性。异质性的降低允许来自哺乳动物细胞培养系统的纯化产物的产量增加。因此，第一方面，本发明提供了包含第一和第二结构域的融合多肽，其中第一结构域包含与细胞表面相关抗原结合的多肽配体，第二结构域包含去糖基化的干扰素α2b(ifnα2b)，去糖基化的ifnα2b具有seqidno：1或seqidno：2的序列，并且其中去糖基化的ifnα2b进一步包含一种或多种氨基酸置换或缺失，其减弱去糖基化的ifnα2b的活性。在本发明的一个实施方案中，去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基是a、c、d、e、f、g、h、i、k、l、m、n、p、q、r、s、v、w或y。这代表用天然存在的氨基酸置换通常存在于人ifnα2b中的t106，在哺乳动物细胞培养中产生时在该位置的氨基酸不允许o-连接的糖基化。在本发明的另一个实施方案中，去糖基化的ifnα2b的序列是seqidno：2。这代表在正常人ifnα2b中发现的残基t106的缺失，其也去除了在该分子中发现的o-连接的糖基化位点。如本文所证实的，每个置换或缺失从人减弱的ifnα2b中去除o-糖基化位点，并且当由cho细胞表达时，降低如在ief凝胶中测量的分子的异质性，并且至少基本上维持减弱的ifnα2b结合细胞表面ifn受体并启动下游信号传导。在另外的实施方案中，去糖基化的ifnα2b的序列是通过一个或多个减弱突变修饰的seqidno：1，减弱突变选自l15a、r22a、r23a、s25a、l26a、f27a、l30a、l30v、k31a、d32a、r33a、r33k、r33q、h34a、q40a、d114r、l117a、r120a、r120e、r125a、r125e、k131a、e132a、k133a、k134a、m148a、r149a、s152a、l153a、n156a、(l30a、h57y、e58n和q61s)、(m148a、h57y、e58n和q61s)、(l153a、h57y、e58n和q61s)、(r144a、h57y、e58n和q61s)、(n65a、l80a、y85a和y89a)、(n65a、l80a、y85a、y89a和d114a)、(n65a、l80a、y85a、y89a和l117a)、(n65a、l80a、y85a、y89a和r120a)、(y85a、y89a和d114a)、(d114a和r120a)、(l117a和r120a)、(l117a、r120a和k121a)、(r120a和k121a)、(r120e和k121e)、用a、d、e、g、h、i、k、l、n、q、s、t、v或y替换位置144处的r、用d、e、g、h、i、k、l、m、n、q、s、t、v或y替换位置145处的a和残基l161-e165的缺失。在另外的实施方案中，去糖基化的ifnα2b的序列是通过一个或多个减弱突变修饰的seqidno：2，减弱突变选自l15a、r22a、r23a、s25a、l26a、f27a、l30a、l30v、k31a、d32a、r33a、r33k、r33q、h34a、q40a、d113r、l116a、r119a、r119e、r124a、r124e、k130a、e131a、k132a、k133a、m147a、r148a、s149a、l152a、n155a、(l30a、h57y、e58n和q61s)、(m147a、h57y、e58n和q61s)、(l152a、h57y、e58n和q61s)、(r143a、h57y、e58n和q61s)、(n65a、l80a、y85a和y89a)、(n65a、l80a、y85a、y89a和d113a)、(n65a、l80a、y85a、y89a和l116a)、(n65a、l80a、y85a、y89a和r119a)、(y85a、y89a和d113a)、(d113a和r119a)、(l116a和r119a)、(l116a、r119a和k120a)、(r119a和k120a)、(r119e和k120e)、用a、d、e、g、h、i、k、l、n、q、s、t、v或y替换位置143处的r、用d、e、g、h、i、k、l、m、n、q、s、t、v或y替换位置144处的a和残基l160-e164的缺失。在另一个实施方案中，通过减弱突变修饰的去糖基化的ifnα2b的序列选自seqidno：3-30和seqidno：32-47。在另一个实施方案中，细胞表面相关抗原选自cd38、cd138、rank配体、hm1.24、cd56、cs1、cd20、cd74、il-6r、blys(baff)、bcma、hla-sr、hla-dr、激肽原、β2微球蛋白、fgfr3、icam-1、matriptase、cd52、egfr、gm2、α4整合素、ifg-1r、kir、cd3、cd4、cd8、cd24、cd44、cd69、cd71、cd79、cd83、cd86、cd96、hla、pd-1、icos、cd33、cd115、cd11c、cd19、cd52、cd14、fsp1、fap、pdgfrα、pdgfrβ、asgr1、asgr2、fsp1、rti140/ti-α、hti56、vegf受体、cd241rche基因的产物、cd117(c-kit)、cd71(转铁蛋白受体)、cd36(血小板应答蛋白受体)、cd34、cd45ro、cd45ra、cd115、cd168、cd235、cd236、cd237、cd238、cd239和cd240。在某些实施方案中，多肽配体是抗体或其抗原结合部分。在另一个实施方案中，多肽配体是结合cd38的抗体。抗体的vh序列选自seqidno：48-56和58，并且抗体的vl序列选自seqidno：81、82和84。在另一个实施方案中，多肽配体是结合cd138的抗体。优选抗体的vh序列为seqidno：59，抗体的vl序列为seqidno：85。在另一个实施方案中，多肽配体结合rank配体。优选多肽配体的序列是seqidno：57。在另一个实施方案中，第一结构域经由肽键与第二结构域连接。第一结构域可以通过肽键(“零长度接头”)直接与第二结构域连接，或者通过长度为1至20个氨基酸的肽接头与第二结构域连接。接头可以是(sggggs)n，其中n为1至3。接头的实例包括sggggs和sggggsggggsggggs。在另一个实施方案中，第一结构域的c末端连接到第二结构域的n末端。在另一个实施方案中，第一结构域的氨基酸序列是糖基化的。另一方面，本发明提供了融合多肽，其包含选自seqidno：31、61-77、83和87的序列以及选自seqidno：81、82和84的序列。另一方面，本发明提供了包含seqidno：87和seqidno：81的融合多肽。另一方面，本发明提供了包含seqidno：79和seqidno：85的融合多肽。另一方面，本发明提供了包含seqidno：80和seqidno：86的融合多肽。另一方面，本发明提供了包含seqidno：78的融合多肽。从上面的讨论可以理解，本发明的融合多肽的具体形式如下：a.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基为a。b.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基为c。c.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基为d。d.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基为e。e.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基为f。f.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基为g。g.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基为h。h.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基为i。i.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基为k。j.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基为l。k.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基为m。l.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基为n。m.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基为p。n.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基为q。o.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基为r。p.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基为v。q.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基为w。r.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1，其中位置106处的残基为y。s.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变l15a修饰的seqidno：1或seqidno：2。t.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变a19w修饰的seqidno：1或seqidno：2。u.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变r22a修饰的seqidno：1或seqidno：2。v.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变r23a修饰的seqidno：1或seqidno：2。w.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变s25a修饰的seqidno：1或seqidno：2。x.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变l26a修饰的seqidno：1或seqidno：2。y.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变f27a修饰的seqidno：1或seqidno：2。z.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变l30a或l30v修饰的seqidno：1或seqidno：2。aa.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变k31a修饰的seqidno：1或seqidno：2。bb.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变d32a修饰的seqidno：1或seqidno：2。cc.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变r33a、r33k或r33q修饰的seqidno：1或seqidno：2。dd.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变h34a修饰的seqidno：1或seqidno：2。ee.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变q40a修饰的seqidno：1或seqidno：2。ff.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变d114r修饰的seqidno：1或通过减弱突变d113r修饰的seqidno：2。gg.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变l117a修饰的seqidno：1或通过减弱突变l116a修饰的seqidno：2。hh.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变r120a或r120e修饰的seqidno：1或通过减弱突变r119a或r119e修饰的seqidno：2。ii.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变r125a或r125e修饰的seqidno：1或通过减弱突变r124a或r124e修饰的seqidno：2。jj.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变k131a修饰的seqidno：1或通过减弱突变k130a修饰的seqidno：2。kk.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变e132a修饰的seqidno：1或通过减弱突变e131a修饰的seqidno：2。ll.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变k133a修饰的seqidno：1或通过减弱突变k132a修饰的seqidno：2。mm.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变k134a修饰的seqidno：1或通过减弱突变k133a修饰的seqidno：2。nn.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变m148a修饰的seqidno：1或通过减弱突变m147a修饰的seqidno：2。oo.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变r149a修饰的seqidno：1或通过减弱突变r148a修饰的seqidno：2。pp.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变s152a修饰的seqidno：1或通过减弱突变s151a修饰的seqidno：2。qq.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变l153a修饰的seqidno：1或通过减弱突变l152a修饰的seqidno：2。rr.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变n156a修饰的seqidno：1或通过减弱突变n155a修饰的seqidno：2。ss.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1或通过减弱突变l30a、h57y、e58n和q61s修饰的seqidno：2。tt.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变m148a、h57y、e58n和q61s修饰的seqidno：1或通过减弱突变m147a、h57y、e58n和q61s修饰的seqidno：2。uu.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变l153a、h57y、e58n和q61s修饰的seqidno：1或通过减弱突变l152a、h57y、e58n和q61s修饰的seqidno：2。vv.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变r144a、h57y、e58n和q61s修饰的seqidno：1或通过减弱突变r143a、h57y、e58n和q61s修饰的seqidno：2。ww.融合多肽，其中去糖基化的ifnα2b的序列是seqidno：1或通过减弱突变n65a、l80a、y85a和y89a修饰的seqidno：2。xx.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变n65a、l80a、y85a、y89a和d114a修饰的seqidno：1或通过减弱突变n65a、l80a、y85a、y89a和d113a修饰的seqidno：2。yy.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变n65a、l80a、y85a、y89a和l117a修饰的seqidno：1或通过减弱突变n65a、l80a、y85a、y89a和l116a修饰的seqidno：2。zz.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变n65a、l80a、y85a、y89a和r120a修饰的seqidno：1或通过减弱突变n65a、l80a、y85a、y89a和r119a修饰的seqidno：2。aaa.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变y85a、y89a和d114a修饰的seqidno：1或通过减弱突变y85a、y89a和d113a修饰的seqidno：2。bbb.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变d114a和r120a修饰的seqidno：1或通过减弱突变d113a和r119a修饰的seqidno：2。ccc.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变l117a和r120a修饰的seqidno：1或通过减弱突变l116a和r119a修饰的seqidno：2。ddd.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变l117a、r120a和k121a修饰的seqidno：1或通过减弱突变l116a、r119a和k120a修饰的seqidno：2。eee.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变r120a和k121a修饰的seqidno：1或通过减弱突变r119a和k120a修饰的seqidno：2。fff.融合多肽，其中去糖基化的ifnα2b的序列是通过减弱突变r120e和k121e修饰的seqidno：1或通过减弱突变r119e和k120e修饰的seqidno：2。ggg.融合多肽，其中去糖基化的ifnα2b的序列seqidno：1是通过用a、d、e、g、h、i、k、l、n、q、s、t、v或y置换位置144处的r进行修饰或者seqidno：2是通过用a、d、e、g、h、i、k、l、n、q、s、t、v或y置换位置143处的r进行修饰。hhh.融合多肽，其中去糖基化的ifnα2b的序列seqidno：1是通过用d、e、g、h、i、k、l、m、n、q、s、t、v或y置换位置145处的a进行修饰或者seqidno：2是通过用d、e、g、h、i、k、l、m、n、q、s、t、v或y置换位置144处的a进行修饰。。iii.融合多肽，其中去糖基化的ifnα2b是通过残基l161-e165的缺失修饰的seqidno：1或者通过残基l161-e165的缺失修饰的seqidno：2。jjj.根据权利要求1的融合多肽，其中去糖基化的ifnα2b的序列选自seqidno：3-30和seqidno：32-47。本文所用的术语“抗体”广义地指由四条多肽链(两条重(h)链和两条轻链(l)链)组成的任何免疫球蛋白(ig)分子或其保留ig分子的必需表位结合特征的任何功能性片段、突变体、变体或衍生物。此类突变体、变体或衍生抗体形式是本领域已知的，其非限制性实施方案在下文中进行讨论。在全长抗体中，每个重链由重链可变区(本文缩写为hcvr或vh)和重链恒定区组成。重链恒定区由三个结构域ch1、ch2和ch3组成。每条轻链由轻链可变区(本文缩写为lcvr或vl)和轻链恒定区组成。轻链恒定区由一个结构域cl组成，在人类中可以是κ或λ类。vh和vl区可以进一步细分为高变区域，称为互补决定区(cdr)，散布于更保守的区域，称为框架区(fr)。每个vh和vl由以下顺序从氨基末端到羧基末端排列的三个cdr和四个fr组成：fr1、cdr1、fr2、cdr2、fr3、cdr3、fr4。免疫球蛋白分子可以是任何类型(例如igg、ige、igm、igd、iga和igy)、类(例如igg1、igg2、igg3、igg4、iga1和iga2)或亚类。如本文所用，抗体的“抗原结合结构域”或“抗原结合部分”是指保留特异性结合抗原(例如cd38)的能力的抗体或蛋白质的一个或多个片段。已经显示抗体的抗原结合功能可以通过全长抗体的片段进行。这样的抗体实施方案还可以是双特异性(bispecific)、双特异性(dualspecific)或多特异性形式，特异性结合两种或多种不同抗原。包含在术语抗体的“抗原结合部分”内的结合片段的实例包括(i)fab片段，由vl、vh、cl和ch1结构域组成的单价片段；(ii)f(ab')2片段，除铰链区的一部分外还包含两个fab片段的二价片段，通过在铰链区域的二硫桥连接；(iii)由vh和ch1结构域组成的fd片段；(iv)由抗体单臂的vl和vh结构域组成的fv片段，(v)结构域抗体(dab)(ward等人1989nature341544-6，winter等人，pct公开wo90/05144a1，其通过引用并入本文)，其包含单个可变结构域；和(vi)分离的互补决定区(cdr)。此外，尽管fv片段的两个结构域vl和vh被分开的基因编码，但是它们可以通过合成接头使用重组方法连接，使得它们能够被制成单个的蛋白质链，其中vl和vh区配对以形成单价分子(称为单链fv(scfv))；参见例如bird等人1988science242423-6；huston等人1988procnatlacadsciusa855879-83)。这样的单链抗体也意在被包括在术语抗体的“抗原结合部分”内。还包括其它形式的单链抗体，例如双抗体。双抗体是二价双特异性抗体，其中vh和vl结构域在单个多肽链上表达，但使用太短的接头而不允许同一链上的两个结构域之间的配对，从而迫使结构域与另一条链的互补结构域配对并产生两个抗原结合位点(参见例如holliger，p.等人，1993，proc.natl.acad.sci.usa90:6444-6448；poljak，r.j.等人，1994，structure2:1121-1123)。这样的抗体结合部分是本领域已知的(kontermann和dubel编辑，antibodyengineering2001springer-verlag.newyork.790pp.,isbn3-540-41354-5)。在一个实施方案中，抗体结合部分是fab片段。本文所述的抗体可以是人源化抗体。术语“人源化抗体”应理解为包含人类可变区的蛋白质，其包括来自移植到来自人抗体的fr上或插入其中的非人物种(例如，小鼠或大鼠或非人灵长类动物)的抗体的cdr(这种抗体也称为“cdr移植抗体”)。人源化抗体还包括其中人蛋白质的一个或多个残基被一个或多个氨基酸置换修饰和/或人蛋白质的一个或多个fr残基替换为相应的非人残基的蛋白质。人源化抗体还可以包含在人抗体或非人抗体中都没有发现的残基。蛋白质的任何附加区域(例如fc区)通常是人的。可以使用本领域已知的方法，例如us5,225,539、us6,054,297、us7,566,771或us5,585,089进行人源化。术语“人源化抗体”还包括超人源化抗体，例如如在us7,732,578中描述的。本文的抗体可以是人的。本文所用的术语“人抗体”是指具有在人体中发现的可变抗体区域和任选地恒定抗体区域的蛋白质，例如，在人种系或体细胞中或从使用这些区域产生的文库中发现的。“人”抗体可以包括未被人序列编码的氨基酸残基，例如，通过体外随机突变或位点定向突变引入的突变(特别是涉及蛋白质少量残基中的保守置换或突变的突变，例如蛋白质残基中的1、2、3、4或5个)。这些“人抗体”不一定需要作为人的免疫应答的结果产生，而是可以使用重组手段(例如，筛选噬菌体展示文库)和/或通过转基因动物(例如，小鼠)，其包含编码人抗体恒定区和/或可变区的核酸和/或使用引导选择(guideselection)(例如，如us5,565,332中所述的)。该术语还包括这种抗体的亲和力成熟形式。为了本公开的目的，人类蛋白质也将被认为包括包含来自人抗体的fr或包含来自人fr共有序列的序列的fr的蛋白质，其中一个或多个cdr是随机的或半随机的，例如，如us6,300,064和/或us6,248,516中所述。本发明多肽的抗体部分可以是任何类的全长抗体，优选igg1、igg2或igg4。这些抗体的恒定结构域优选为人的。此类抗体的可变区可以是非人源性的，或者优选地是人来源的或人源化的。也可以使用抗体片段代替全长抗体。术语“抗体”还包括工程改造的抗体。如将理解的是，有工程改造的抗体的许多变体(例如小鼠单克隆抗体、嵌合型、人源化和人单克隆抗体、单链可变抗体片段(scfv)、微型抗体、适配体以及如上所述的双特异性抗体和双抗体)。例如，(ward等人，1989，nature341：544-546；hamers-casterman等人，1993，nature363：446-448；davies&riechmann,1994,febslett.339:285-290)中公开了单可变区结构域(称为dab)。微型抗体是全抗体的小型形式，其在单链中编码整个抗体的必需元件。合适地，微型载体由与免疫球蛋白分子的铰链区和ch3结构域融合的天然抗体的vh和vl结构域组成，例如美国专利号5,837,821中所公开的。在替代实施方案中，工程改造的抗体可以包含非免疫球蛋白衍生的蛋白质框架。例如，可以参考(ku&schutz,1995,proc.natl.acad.sci.usa92:6552-6556)，其公开了具有随机化以产生cdr的两个环的四螺旋束蛋白细胞色素b562，其已被选择用于抗原结合。存在大量非抗体识别蛋白或蛋白质结构域支架，其可以用作本发明构建体中的抗原结合结构域。这些包括基于以下的支架：细胞毒性t淋巴细胞相关抗原4(ctla-4)(evibody；us7,166,697)；人转铁蛋白(trans-body)；来自蛋白a的z-结构域的三螺旋束(affibody)；单体或三聚体人类c型凝集素结构域(tetranectin)；第十个人纤连蛋白iii型结构域(adnectin)；人类或牛胰蛋白酶抑制剂的kunitz型结构域；昆虫防御素a(iica29)、appi(kuntiz结构域)；脂质运载蛋白、fabp、bilin结合蛋白、apolopropteind(anticalins)；人α-晶体蛋白或泛素分子(affilin)；胰蛋白酶抑制剂ii(微体)；α2p8或锚蛋白重复(重复基序蛋白)、charybdotoxin(蝎毒素)、min-23、纤维素结合结构域(knottins)；新制癌菌素、cbm4-2和tendamistat。此外，除了如上由抗体衍生结构域或非抗体折叠提供的支架之外，还有天然存在的配体结合蛋白或蛋白结构域可用作本发明中的配体结合结构域。例如，具有配体结合特性的蛋白质结构域包括受体的细胞外结构域、信号蛋白的pdz模块，例如ras结合蛋白af-6、粘附分子和酶。使用本领域众所周知的方法通过例如亲和力成熟来增加结合，或通过除去预测的mhcii类结合基序降低免疫原性。通过调节其功能特征，例如抗体依赖性细胞介导的细胞毒性(adcc)、补体依赖性细胞毒性(cdc)、血清半衰期、生物分布和与fc受体的结合或任何这些的组合，可以进一步增强本文的抗体的治疗效用。这种调节可以通过蛋白质工程、糖工程或化学方法来实现。取决于所需的治疗应用，增加或减少这些任意活性可能是有利的。糖工程的实例使用如shinkawat.等人，2003(jbiolchem278：3466-73)中所述的方法。用于抗体亲和力成熟的许多方法是本领域已知的。其中许多基于通过诱变产生变体蛋白的组或文库的一般策略，随后选择和/或筛选以改善亲和力。诱变通常在dna水平上进行，例如通过易错pcr(thie，voedisch等人，2009，methodsmolbiol525：309-322)，通过基因改组进行(kolkman和stemmer2001，natbiotechnol.may；19(5):423-8)，通过使用致突变性化学物质或照射，通过使用具有易错复制机制的“变异体”株(greener1996，invitromutagenesisprotocols.humanapress,nj)或通过利用亲和力成熟机制的体细胞超突变方法(peled，kuang等人，2008，annurevimmunol.26:481-511)。诱变也可以在rna水平上进行，例如通过使用qβ复制酶(kopsidas，roberts等人，2006，immunollett.2006nov15；107(2):163-8)进行。允许筛选改进的变体蛋白质的基于文库的方法可以基于各种展示技术，例如噬菌体、酵母、核糖体、细菌或哺乳动物细胞，并且是本领域众所周知的(benhar2007，expertopinbiolther.may；7(5):763-79)。亲和力成熟可以通过更有针对性/预测性的方法来实现，例如通过定点诱变或基于3d蛋白建模的发现引导的基因合成(参见例如queen，schneider等人1989，pnas，86(24)：10029-33或美国专利6,180,370或美国专利5,225,539)。已经由以下内容描述了增加adcc的方法：ferrara,brunker等人2006,biotechnolbioeng；93:851-61；li,sethuraman等人2006,natbiotechnol；24:210-5；stavenhagen,gorlatov等人2007,cancerres；67:8882-90；shields,namenuk等人2001,jbiolchem；276:6591-604；shinkawa,nakamura等人2003,jbiolchem；278:3466-73和wo2008/006554。已经由以下内容描述了增加cdc的方法：idusogie,wong等人2001,jimmunol；176:346-56；dall'acqua,cook等人2006,jbiolchem；281:23514-24；michaelsen,aase等人1990,scandjimmunol；32:517-28；brekke,bremnes等人1993,molimmunol；30:1419-25；tan,shopes等人1990,pnas；87:162-6；和norderhaug,brekke等人1991,eurjimmunol；21:2379-84。描述增加adcc和cdc的方法的参考文献包括natsume,in等人2008,cancerres；68:3863-72。这些参考文献中的每一篇的公开内容通过交叉引用包括在本文中。在某些实施方案中，减少或消除本发明多肽的抗体组分的adcc和cdc活性可能是有利的，使得ifnα2b活性是调节靶细胞存活的多肽的主要活性。用于调节抗体血清半衰期和生物分布的许多方法基于修饰抗体和新生儿fc受体(fcrn)之间的相互作用，该fc受体具有保护igg免于分解代谢的关键作用，并保持高血清抗体浓度。dall'acqua等人描述了增强对fcrn的结合亲和力的igg1的fc区的置换，从而增加血清半衰期(dall'acqua,woods等人2002,jimmunol；169:5171-80)，并进一步证实了增强具有m252y/s254t/t256e(根据euindex的残基编号)或m265y/s267t/t269(根据kabat编号系统进行残基编号)的三置换的adcc活性的生物利用度和调节(dall'acqua，kiener等。2006，jbiolchem；279:6213-6)。另见美国专利号6,277,375；6821505；和7,083,784。hinton等人已经描述了赋予体内半衰期增加的位置250和428处的恒定结构域氨基酸置换(hinton，johlfs等人，2004，jbiolchem；279:6213-6；hinton，xiong等人2006，jimmunol；176:346-56)。另见美国专利号7,217,797。petkova等人已经描述了赋予体内半衰期增加的位置307、380和434处的恒定结构域氨基酸置换(petkova，akilesh等人，2006，intimmunol；18:1759-69)。还参见shields等人2001，jbiolchem；276:6591-604和wo2000/42072。调节与fc受体的结合和由这些受体介导的随后功能(包括fcrn结合和血清半衰期)的恒定结构域氨基酸置换的其它实例描述于美国专利申请号20090142340；20090068175和20090092599。根据如kabat中的euindex编号的“s228p”在本文中也称为根据kabat等人的“s241p”(1987sequencesofproteinsofimmunologicalinterest.unitedstatesdepartmentofhealthandhumanservices,washingtondc.)。该置换稳定了igg4分子的铰链区，其具有使铰链区的核心序列与igg1或igg2同种型抗体相同的作用。这导致重链的自发解离和重新缔合的减少，这通常导致异二聚体igg4抗体的产生。与抗体分子连接的聚糖已知会影响抗体与fc受体和聚糖受体的相互作用，从而影响抗体活性，包括血清半衰期(kaneko,nimmerjahn等人2006,science；313:670-3；jones,papac等人2007,glcobiology；17:529-40；和kanda,yamada等人2007,glycobiology；17:104-18)。因此，调节所需抗体活性的某些糖型可以赋予治疗优势。用于产生工程改造的糖型的方法是本领域已知的，并且包括但不限于在美国专利号us6,602,684；us7,326,681；us7,388,081和wo2008/006554中所述的那些。通过添加聚乙二醇(peg)延长半衰期已被广泛用于延长蛋白质的血清半衰期，如fishburn2008,jpharmsci；97:4167-83所综述的。如将认识到的，可以在本发明的序列内进行保守的氨基酸置换。“保守置换”是指具有相似性质的氨基酸。如本说明书中所用，以下氨基酸组被视为保守置换：h、r和k；d、e、n和q；v、i和l；c和m；s、t、p、a和g；以及f、y和w。然而，并不意图在减弱和/或糖基化的位点处进行除了具体叙述的那些之外的置换。本文所用的术语“细胞表面相关抗原”广义地指在细胞(包括但不限于恶性细胞或感染性或外来细胞)表面上表达的任何抗原。在本发明的某些方面，本发明的融合多肽构建体或组合物可用于治疗患有癌症的患者。本文考虑的癌症包括：特征在于不受控制的细胞生长(例如肿瘤形成)的一组疾病和病症，而没有将这些细胞区分为专门和不同的细胞。这些疾病和病症包括abl1原癌基因、aids相关癌症、声学神经瘤、急性淋巴细胞性白血病、急性骨髓性白血病、腺样囊性癌、肾上腺皮质癌、致癌性髓样组织转化、脱发、肺泡软部分肉瘤、肛门癌、血管肉瘤、再生障碍性贫血、星形细胞瘤、共济失调毛细血管扩张症、基底细胞癌(皮肤)、膀胱癌、骨癌、肠癌、脑干神经胶质瘤、脑和cns肿瘤、乳腺癌、类癌肿瘤、子宫颈癌、儿童脑肿瘤、儿童期癌症、儿童期白血病、儿童期软组织肉瘤、软骨肉瘤、绒毛膜癌、慢性淋巴细胞性白血病、慢性骨髓性白血病、结肠直肠癌、皮肤t细胞淋巴瘤、隆凸性皮肤纤维肉瘤、结缔组织增生性小圆细胞肿瘤、导管癌、内分泌癌、子宫内膜癌、室管膜瘤、食管癌、尤文氏肉瘤、肝外胆管癌、眼癌、眼睛：黑色素瘤、视网膜母细胞瘤、输卵管癌、范科尼贫血、纤维肉瘤、胆囊癌、胃癌、胃肠癌、胃肠类癌肿瘤、泌尿生殖系统癌、生殖细胞肿瘤、妊娠滋养层疾病、神经胶质瘤、妇科癌症、血液恶性肿瘤、毛细胞白血病、头颈癌、肝细胞癌、遗传性乳腺癌、组织细胞增多症、霍奇金病、人乳头状瘤病毒、葡萄胎、高钙血症、下咽癌、眼内黑色素瘤、胰岛细胞癌、卡波西氏肉瘤、肾癌、兰格汉氏细胞组织细胞增多症、喉癌、平滑肌肉瘤、白血病、利-弗劳梅尼综合征、唇癌、脂肪肉瘤、肝癌、肺癌、淋巴水肿、淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、男性乳腺癌、肾的恶性横纹肌样肿瘤(malignant-rhabdoid-tumor-of-kidney)、髓母细胞瘤、黑色素瘤、梅克尔细胞癌、间皮瘤、转移性癌症、口癌、多发性内分泌肿瘤、蕈样肉芽肿病、骨髓增生异常综合征、多发性骨髓瘤、骨髓增生性疾病、鼻癌、鼻咽癌、肾母细胞瘤、神经母细胞瘤、神经纤维瘤病、nijmegen断裂综合征、非黑色素瘤皮肤癌、非小细胞肺癌(nsclc)、眼癌、食管癌、口腔癌、口咽癌、骨肉瘤、吻合术卵巢癌、胰腺癌、鼻旁癌、甲状旁腺癌、腮腺癌、阴茎癌、外周神经外胚瘤、垂体癌、真性红细胞增多症、前列腺癌、罕见癌症及相关疾病、肾细胞癌、视网膜母细胞瘤、横纹肌肉瘤、罗斯曼-汤姆森综合征、唾液腺癌、肉瘤、神经鞘瘤、塞扎里综合征、皮肤癌、小细胞肺癌(sclc)、小肠癌、软组织肉瘤、脊髓肿瘤、鳞状细胞癌(皮肤)、胃癌、滑膜肉瘤、睾丸癌、胸腺癌、甲状腺癌、移行细胞癌(膀胱)、移行细胞癌(肾-肾盂-/-输尿管)、滋养层癌、尿道癌、泌尿系统癌、尿斑素、子宫肉瘤、子宫癌、阴道癌、外阴癌、瓦氏巨球蛋白血症和肾母细胞瘤。在一个实施方案中，肿瘤选自多发性骨髓瘤或非霍奇金淋巴瘤。如预期的治疗癌症，本发明的融合构建体的抗体部分可以结合肿瘤相关抗原，即由癌细胞选择性表达或在癌细胞中相对大多数正常细胞过度表达的细胞表面抗原。本领域已知有许多肿瘤相关抗原(taa)。taa的非限制性实例包括酶酪氨酸酶；黑色素瘤抗原gm2；甲胎蛋白(afp)；癌胚抗原(cea)；粘蛋白1(muc1)；人表皮生长因子受体(her2/neu)；t细胞白血病/淋巴瘤1(tcl1)癌蛋白。与许多不同癌症相关的示例性taa是端粒酶(htert)；前列腺特异性膜抗原(psma)；尿激酶纤溶酶原激活物及其受体(upa/upar)；血管内皮生长因子及其受体(vegf/vegfr)；细胞外基质金属蛋白酶诱导剂(emmprin/cd147)；表皮生长因子(egfr)；血小板衍生生长因子及其受体(pdgf/pdgfr)和c-kit(cd117)。在us2010/0297076中提供了其他taa的列表，其公开内容通过引用包含在本文中。特别感兴趣的是与多发性骨髓瘤白血病或淋巴瘤细胞相关的细胞表面抗原，包括但不限于cd38、cd138、cd79、cs1和hm1.24。在一个实施方案中，配体减弱的ifn构建体例如抗体减弱的干扰素构建体的抗原是cd38。cd38是46kda的ii型跨膜糖蛋白。它具有20个氨基酸的短n末端胞质尾、单个跨膜螺旋和256个氨基酸的长胞外结构域(bergsagel,p.,blood；85:436,1995和liu,q.,structure,13:1331,2005)。它在许多免疫细胞的表面上表达，包括cd4和cd8阳性t细胞、b细胞、nk细胞、单核细胞、浆细胞和相当大比例的正常骨髓前体细胞(malavasi,f.,hum.immunol.9:9,1984)。然而，在淋巴细胞中，表达似乎取决于细胞的分化和活化状态。静息的t和b细胞是阴性的，而未成熟和活化的淋巴细胞主要是cd38表达阳性的(funaro,a.,j.immunol.145:2390,1990)。另外的研究表明在非造血器官如胰腺、脑、脾和肝中的mrna表达(koguma,t.,biochim.biophys.acta1223:160,1994.)cd38是涉及跨膜信号传导和细胞粘附的多功能胞外酶。它也被称为循环adp核糖水解酶，因为取决于细胞外ph该酶可以将nad+和nadp+转化成cadpr、adpr和naadp。这些产物诱导细胞内的ca2+动员，这可能导致细胞的酪氨酸磷酸化和活化。cd38也是可以与配体cd31相互作用的受体。通过cd31活化受体导致细胞内事件，包括ca2+动员、细胞活化、增殖、分化和迁移(在deaglio,s.,trendsinmol.med.14:210,2008中进行了综述)在多发性骨髓瘤细胞中cd38以高水平表达，在大多数情况下为t-和b-谱系急性淋巴细胞性白血病、一些急性骨髓性白血病、卵泡中心细胞淋巴瘤和t淋巴细胞淋巴瘤。(malavasi,f.,j.clinlabres.22:73,1992)。最近，cd38表达已成为b-谱系慢性淋巴细胞性白血病(b-cll)中可靠的预后标志物(ibrahim,s.,blood.98:181,2001anddurig,j.,leuk.res.25:927,2002)。独立小组已经证明，呈现cd38+克隆的b-cll患者的特征在于患有更晚期的疾病的不利的临床过程，对化学疗法的反应性差和生存时间更短(morabito,f.,haematologica.87:217,2002)。cd38对淋巴样肿瘤的一致和增强的表达使其成为治疗性抗体技术的有吸引力的靶标。用于开发靶向癌症的抗体减弱的去糖基化的ifnα2b融合蛋白构建体的优选抗原是在体内与大多数其他未转化细胞相比对癌细胞显示出选择性或更大表达的抗原。此类抗原的非蛋白质实例包括鞘脂、神经节苷脂gd2(saleh等人，1993，j.immunol.,151,3390-3398)、神经节苷脂gd3(shitara等人，1993，cancerimmunol.immunother.36:373-380)、神经节苷脂gm2(livingston等人，1994，j.clin.oncol.12:1036-1044)、神经节苷脂gm3(hoon等人，1993，cancerres.53:5244-5250)和可以显示在蛋白质或糖脂上的lewisx、lewisy和lewisxy碳水化合物抗原。蛋白质抗原的实例是her-2/neu，人乳头瘤病毒-e6或-e7，muc-1；ks1/4pan-癌抗原(perezandwalker,1990,j.immunol.142:3662-3667；bumal,1988,hybridoma7(4):407-415)；卵巢癌抗原ca125(yu等人，1991，cancerres.51(2):468-475)；前列腺酸性磷酸盐(tailor等人，1990，nucl.acidsres.18(16):4928)；前列腺特异性抗原(henttu和vihko，1989，biochem.biophys.res.comm.160(2):903-910；israeli等，1993，cancerres.53:227-230)；黑色素瘤相关抗原p97(estin等人，1989，j.natl.cancerinstit.81(6):445-446)；黑色素瘤抗原gp75(vijayasardahl等人，1990，j.exp.med.171(4):1375-1380)；前列腺特异性膜抗原；癌胚抗原(carcinoembryonicantigen，cea)(foon等人，1994，proc.am.soc.clin.oncol.13:294)，muc16(抗体包括mj-170，mj-171，mj-172和mj-173[us7,202,346]，3a5[us7,723,485])，nmb(us8,039,593)，恶性人淋巴细胞抗原-apo-1(bernhard等人，1989，science245:301-304)；高分子量黑色素瘤抗原(hmw-maa)(natali等人，1987，cancer59:55-63；mittelman等人，1990，j.clin.invest.86:2136-2144)；伯基特淋巴瘤抗原-38.13；cd19(ghetie等人，1994，blood83:1329-1336)；人类b淋巴瘤抗原-cd20(reff等人，1994，blood83:435-445)；gica19-9(herlyn等人，1982，j.clin.immunol.2:135)，cta-1和lea；cd33(sgouros等人，1993，j.nucl.med.34:422-430)；癌胚抗原(oncofetalantigen)如肝癌的甲胎蛋白或膀胱肿瘤癌胚抗原(hellstrom等人，1985，cancer.res.45:2210-2188)；分化抗原如人肺癌抗原l6或l20(hellstrom等人，1986，cancerres.46:3917-3923)；纤维肉瘤抗原；人白血病t细胞抗原-gp37(bhattacharya-chatterjee等人，1988，j.immunol.141:1398-1403)；肿瘤特异性移植类型的细胞表面抗原(tsta)，如病毒诱导的肿瘤抗原，包括t型抗原、dna肿瘤病毒和rna肿瘤病毒的包膜抗原；拟糖蛋白，乳腺癌抗原如egfr(表皮生长因子受体)，多形性上皮粘蛋白(pem)(hilkens等人，1992，trendsinbio.chem.sci.17:359)；多形性上皮粘蛋白抗原；人乳脂肪球抗原；结直肠肿瘤相关抗原如tag-72(yokata等人，1992，cancerres.52:3402-3408)，co17-1a(ragnhammar等人，1993，int.j.cancer53:751-758)；在胃腺癌中发现的分化抗原(feizi，1985，nature314:53-57)例如i(ma)，在骨髓细胞中发现的ssea-1，在乳腺上皮中发现的vep8、vep9、myl、vim-d5、m18和m39，在结肠直肠癌中发现的癌症d156-22，tra-1-85(血型h)，在结肠腺癌中发现的c14，在肺腺癌中发现的f3，在胃癌中发现的ah6，在胚胎癌细胞中发现的y半抗原，tl5(血型a)，在胰腺癌中发现的e1系列(血型b)抗原，在胚胎癌细胞中发现的fc10.2，胃腺癌抗原，在腺癌中发现的co-514(血型lea)，在腺癌中发现的ns-10，co-43(血型leb)，在a431细胞中发现的g49，在结肠癌中发现的19.9；胃癌粘蛋白；在黑色素瘤中发现的r24，在结肠腺癌中发现的mh2(血型aleb/ley),在胚胎癌细胞中发现的4.2，d1.1，ofa-1，gm2，ofa-2和m1:22:25:8，和ssea-3和ssea-4。hmw-maa(seqidno：433)(也称为黑色素瘤硫酸软骨素蛋白聚糖)是2322个残基的膜结合蛋白，其在超过90％的手术移除的良性痣和黑色素瘤病变过表达(camploi等人，critrevimmunol.；24:267,2004)。因此，它可能是潜在的靶细胞表面相关抗原。用于用本发明的融合蛋白构建体靶向的其它实例的癌抗原包括(示例性癌症显示在括号中)：cd5(t细胞白血病/淋巴瘤)，ca15-3(癌)，ca19-9(癌)，l6(癌)，ca242(结肠直肠癌)，胎盘碱性磷酸酶(癌)，前列腺酸性磷酸酶(前列腺癌)，mage-1(癌)，mage-2(癌)，mage-3(癌)，mage-4(癌)，转铁蛋白受体(癌)，p97(黑色素瘤)，muc1(乳腺癌)，mart1(黑色素瘤)，cd20(非霍奇金淋巴瘤)，cd52(白血病)，cd33(白血病)，人绒毛膜促性腺激素(癌)，cd38(多发性骨髓瘤)，cd21(b细胞淋巴瘤)，cd22(淋巴瘤)，cd25(b细胞淋巴瘤)，cd37(b细胞淋巴瘤)，cd45(急性粒细胞白血病)，hla-dr(b细胞淋巴瘤)，il-2受体(t细胞白血病和淋巴瘤)，cd40(淋巴瘤)，cd79(b细胞白血病或淋巴瘤，霍奇金淋巴瘤)，各种粘蛋白(癌)，p21(癌)，mpg(黑色素瘤)，ep-cam(上皮肿瘤)，叶酸受体α(卵巢癌)，a33(结肠直肠癌)，g250(肾癌)，铁蛋白(霍奇金淋巴瘤)，de2-7egfr(胶质母细胞瘤，乳腺癌和肺癌)，成纤维细胞活化蛋白(上皮)生腱蛋白金属蛋白酶(胶质母细胞瘤)。一些具体有用的抗体包括但不限于br64(trail等人，1997，cancerresearch57:100105)，br96mab(trail等人，1993，science261:212-215)，抗cd40抗原的mab，例如s2c6mab(francisco等人，2000，cancerres.60:3225-3231)或其他抗cd40抗体，例如在美国专利公开号2003-0211100和2002-0142358中公开的那些；抗cd30抗原的mab，例如ac10(bowen等人，1993，j.immunol.151:5896-5906；wahl等人，2002cancerres.62(13):3736-42)或mdx-0060(美国专利公开号2004-0006215)和抗cd70抗原的mab，例如1f6mab和2f2mab(参见例如美国专利公开号2006-0083736)或抗体2h5、10b4、8b5、18e7、69a7(us8,124,738)。其他抗体已在其他地方进行了综述(franke等人，2000，cancerbiother.radiopharm.15:45976；murray，2000，semin.oncol.27:6470；breitling,f.,和dubel,s.,recombinantantibodies,johnwiley,andsons,newyork,1998)。在某些实施方案中，有用的抗体可以结合靶细胞上表达的受体或受体复合物。受体或受体复合物可包含免疫球蛋白基因超家族成员、主要组织相容性蛋白、细胞因子受体、tnf受体超家族成员、趋化因子受体、整联蛋白、凝集素、补体对照蛋白、生长因子受体、激素受体或神经递质受体。适当的免疫球蛋白超家族成员的非限制性实例是cd2、cd3、cd4、cd8、cd19、cd22、cd79、cd90、cd152/ctla-4、pd-1、b7-h4、b7-h3和icos。合适的tnf受体超家族成员的非限制性实例是taci、bcma、cd27、cd40、cd95/fas、cd134/0x40、cd137/4-1bb、tnfr1、tnfr2、rank、骨保护素、apo3、apo2/trailr1、trailr2、trailr3和trailr4。合适的整联蛋白的非限制性实例是cd11a、cd11b、cd11c、cd18、cd29、cd41、cd49a、cd49b、cd49c、cd49d、cd49e、cd49f、cd103和cd104。合适的凝集素的非限制性实例是s型、c型和i型凝集素。cea抗体的实例示于表1中。表1结合在人b细胞上表达的cd22抗原的抗体包括例如hd6、rfb4、uv22-2、to15、4kb128和人源化抗cd22抗体(hll2)(参见例如li等人(1989)cell.immunol.111:85-99；mason等(1987)blood69:836-40；behr等(1999)clin.cancerres.5:3304s-3314s；bonardi等(1993)cancerres.53:3015-3021)。cd33的抗体包括例如hum195(参见例如kossman等人(1999)clin.cancerres.5:2748-2755；us5693761)和cma-676(参见例如sievers等人,(1999)blood93:3678-3684)。示例性抗muc-1抗体包括但不限于mc5(参见例如peterson等人(1997)cancerres.57:1103-1108；ozzello等人(1993)breastcancerres.treat.25:265-276)和hctmo1(参见例如vanhof等人(1996)cancerres.56:5179-5185)。示例性抗tag-72抗体包括但不限于cc49(参见例如pavlinkova等人(1999)clin.cancerres.5:2613-2619)、b72.3(参见例如，divgi等人(1994)nucl.med.biol.21:9-15)和在美国专利号5,976,531在公开的那些。示例性抗hm1.24抗体包括但不限于小鼠单克隆抗hm1.24和人源化抗hm1.24igg1κ抗体(参见例如ono等人(1999)mol.immuno.36:387-395)。在某些实施方案中，靶向部分包含抗her2抗体。更常称为(her-2/neu)的erbb2基因是编码跨膜受体的癌基因。已经开发了几种针对her-2/neu的抗体，其中一些已被临床应用。这些包括曲妥珠单抗(例如herceptintm；fornir等人(1999)oncology(huntingt)13:647-58)、tab-250(rosenblum等(1999)clin.cancerres.5:865-874)、bach-250(同上)、ta1(maier等人(1991)cancerres.51:5361-5369)以及在美国专利号5,772,997；5,770,195(mab4d5；atcccrl10463)；和美国专利号5,677,171在描述的mab。其它完全人的抗her2/neu抗体是本领域技术人员众所周知的。此类抗体包括但不限于c6抗体，例如c6.5,dpl5,g98a,c6mh3-b1,b1d2,c6vlb,c6vld,c6vle,c6vlf,c6mh3-d7,c6mh3-d6,c6mh3-d5,c6mh3-d3,c6mh3-d2,c6mh3-d1,c6mh3-c4,c6mh3-c3,c6mh3-b9,c6mh3-b5,c6mh3-b48,c6mh3-b47,c6mh3-b46,c6mh3-b43,c6mh3-b41,c6mh3-b39,c6mh3-b34,c6mh3-b33,c6mh3-b31,c6mh3-b27,c6mh3-b25,c6mh3-b21,c6mh3-b20,c6mh3-b2,c6mh3-b16,c6mh3-b15,c6mh3-b11,c6mh3-b1,c6mh3-a3,c6mh3-a2,和c6ml3-9。这些和其它抗her2/neu抗体描述于美国专利号6,512,097和5,977,322，在pct公开wo97/00271，在schier等人(1996)jmolbiol255:28-43，schier等人(1996)jmolbiol263:551-567等。更一般地，针对表皮生长因子受体家族的各种成员的抗体在本发明的构建体中非常适合用作靶向抗体或其抗原结合部分。这样的抗体包括但不限于抗-egfr抗体，如美国专利号5,844,093和5,558,864以及欧洲专利号706,799a中所述的。其他说明性的抗egfr家族抗体包括但不限于抗体，例如c6.5,c6ml3-9,c6mh3-b1,c6-b1d2,f5,her3.a5,her3.f4,her3.h1,her3.h3,her3.e12,her3.b12,egfr.e12,egfr.c10,egfr.b11,egfr.e8,her4.b4,her4.g4,her4.f4,her4.a8,her4.b6,her4.d4,her4.d7,her4.d11,her4.d12,her4.e3,her4.e7,her4.f8和her4.c7等(参见例如美国专利公开us2006/0099205a1和us2004/0071696a1，它们是通过引用并入本文)。作为本发明的融合蛋白构建体的抗体靶标，cd38是特别有意义的。cd38的抗体包括例如at13/5(参见例如ellis等人(1995)j.immunol.155:925-937)、hb7等。本发明还提供包含本发明的融合多肽的组合物。这些组合物可进一步包含任何合适的辅助剂，例如但不限于稀释剂、粘合剂、稳定剂、缓冲剂、盐、亲脂性溶剂、防腐剂、佐剂等中的至少一种。药学上可接受的辅助剂是优选的。制备这种无菌溶液的非限制性实例和方法是本领域众所周知的，例如但不限于，gennaro,ed.,remington'spharmaceuticalsciences,18thedition,mackpublishingco.(easton,pa.)1990。可以常规选择适合于本领域已知或如本文的抗体组合物的给药方式、溶解度和/或稳定性的药学上可接受的载体。可用于本发明组合物的药物赋形剂和添加剂包括但不限于蛋白质、肽、氨基酸、脂质和碳水化合物(例如糖，包括单糖、二、三、四和寡糖；衍生化的糖如糖醇、醛糖酸、酯化糖等；以及多糖或糖聚合物)，其可以单独或组合存在，包含单独或组合的1-99.99％重量或体积。示例性的蛋白质赋形剂包括血清白蛋白，例如人血清白蛋白(hsa)、重组人白蛋白(rha)、明胶、酪蛋白等。还可以起缓冲作用的代表性氨基酸包括丙氨酸、甘氨酸、精氨酸、甜菜碱、组氨酸、谷氨酸、天冬氨酸、半胱氨酸、赖氨酸、亮氨酸、异亮氨酸、缬氨酸、甲硫氨酸、苯丙氨酸、阿斯巴甜等。一个优选的氨基酸是组氨酸。第二个优选的氨基酸是精氨酸。适用于本发明的碳水化合物赋形剂包括例如单糖，例如果糖、麦芽糖、半乳糖、葡萄糖、d-甘露糖、山梨糖等；二糖，如乳糖、蔗糖、海藻糖、纤维二糖等；多糖，如棉子糖、松三糖、麦芽糖糊精、葡聚糖、淀粉等；和糖醇，如甘露糖醇、木糖醇、麦芽糖醇、乳糖醇、木糖醇山梨醇(葡萄糖醇)、肌醇等。用于本发明的优选的碳水化合物赋形剂是甘露醇、海藻糖和棉子糖。抗体组合物还可以包括缓冲液或ph调节剂；通常，缓冲液是由有机酸或碱制备的盐。代表性缓冲剂包括有机酸盐，例如柠檬酸、抗坏血酸、葡萄糖酸、碳酸、酒石酸、琥珀酸、乙酸或邻苯二甲酸的盐；tris、盐酸氨丁三醇、磷酸盐缓冲液或氨基酸缓冲液。用于本发明组合物的优选缓冲液是有机酸盐，例如柠檬酸盐或氨基酸。另外，本发明的组合物可以包括聚合物赋形剂/添加剂，例如聚乙烯吡咯烷酮、ficoll(聚合糖)、糊精(例如环糊精，如2-羟丙基-β-环糊精)、聚乙二醇、调味剂、抗微生物剂、甜味剂、抗氧化剂、抗静电剂、表面活性剂(例如聚山梨酸酯如“20”和“80”)、脂质(例如磷脂、脂肪酸)、类固醇(例如胆固醇)和螯合剂(如edta)。适用于根据本发明的抗体组合物的这些和另外已知的药用赋形剂和/或添加剂是本领域已知的，例如列于“remington:thescience&practiceofpharmacy”,19thed.,williams&williams,(1995)和“physician'sdeskreference”,52nded.,medicaleconomics,montvale,n.j.(1998)中的，其公开内容通过引用完全并入本文。优选的载体或赋形剂材料是碳水化合物(例如糖和糖醇)和缓冲液(例如柠檬酸盐)或聚合物试剂。本领域技术人员将理解，除了具体描述的那些之外，本文描述的本发明易于变化和修改。应当理解，本发明包括落在本发明的精神和范围内的所有这些变化和修改。本发明还包括在本说明书中单独或共同地引用或指出的所有步骤、特征、组合物和化合物，以及任何两个或更多个所述步骤或特征的任何和所有组合。除非另有定义，否则本文使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常理解的相同的含义。虽然可以使用与本文类似或等同的任何材料和方法来实践或测试本发明，但是现在描述优选的材料和方法。实施例一般方法在hek-293e细胞中产生抗体-融合构建体本文融合多肽中的多个结构域的dna序列在并入本文的序列表中提供。使用hispeedplasmidmaxikit(qiagen，valencia，ca)制备编码蛋白质构建体(抗体-减弱的ifnα2b融合构建体)的dna质粒，然后使用市售转染试剂和optimem培养基(invitrogen，carlsbad，ca)转染入hek293e细胞(cnrc，montreal，canada)，其在添加有0.45％(w/v)d-(+)-葡萄糖(sigma，castlehill，nsw)、25μg/ml遗传霉素(invitrogen，carlsbad，ca)和1×glutamax(invitrogen，carlsbad，ca)的f17合成培养基中生长。在供应5％co2和120rpm摇动的培养箱中表达6天后，分离培养基，并使用proteinamabselectsuretm琼脂糖珠(gehealthcare，piscataway，nj)进行亲和纯化。使用pdmidi-trapg-25柱(gehealthcare，piscataway，nj)或hiprep26/10脱盐柱(hitrap脱盐hiprep26/10脱盐)将纯化的蛋白质构建体缓冲液交换为0.2m精氨酸hcl、25mm柠檬酸、71.5mm氢氧化钠，ph6.0。然后使用50kdaamiconultra离心过滤装置(millipore，billerica，ma)将纯化的蛋白质构建体浓缩，然后通过读取280nm处的吸光度进行蛋白质浓度测定。在expi293细胞中产生抗体-融合构建体使用hispeedplasmidmaxikit(qiagen，valencia，ca)制备编码蛋白质构建体(抗体-ifnα2b相关构建体)的dna质粒，然后使用在expi293转染试剂盒中提供的转染试剂和optimem培养基(invitrogen，carlsbad，ca)转染入生长在expi表达培养基(lifetechnologies，carlsbad，ca)中的expi293细胞(lifetechnologies，carlsbad，ca)。在供应5％co2和125rpm摇动的培养箱中表达3天后，分离培养基，并使用proteinamabselectsuretm琼脂糖珠(gehealthcare，piscataway，nj)进行亲和纯化。使用pdmidi-trapg-25柱(gehealthcare，piscataway，nj)或hiprep26/10脱盐柱(hitrap脱盐hiprep26/10脱盐)将纯化的蛋白质构建体缓冲液交换为ph6.0的0.2m精氨酸hcl。然后使用50kdaamiconultra离心过滤装置(millipore，billerica，ma)将纯化的蛋白质构建体浓缩，然后通过读取280nm处的吸光度进行蛋白质浓度测定。在cho细胞中产生抗体-融合构建体使用hispeedplasmidmaxikit(qiagen，valencia，ca)制备编码蛋白质构建体(抗体-ifnα2b相关构建体)的dna质粒，然后使用市售转染试剂和optiprosfmtm培养基(invitrogen，carlsbad，ca)转染入生长在freestyletmcho表达培养基(invitrogen，carlsbad，ca)中的cho细胞(lonza)。在供应10％co2和120rpm摇动的培养箱中表达6天后，分离培养基，并使用proteinamabselectsuretm琼脂糖珠(gehealthcare，piscataway，nj)进行亲和纯化。使用pdmidi-trapg-25柱(gehealthcare，piscataway，nj)或hiprep26/10脱盐柱(hitrap脱盐hiprep26/10脱盐)将纯化的蛋白质构建体缓冲液交换为0.2m精氨酸.hcl、25mm柠檬酸、71.5mm氢氧化钠，ph6.0。然后使用50kdaamiconultra离心过滤装置(millipore，billerica，ma)将纯化的蛋白质构建体浓缩，然后通过读取280nm处的吸光度进行蛋白质浓度测定。测量抗体-ifnα2b融合蛋白构建体的抗原靶向活性的方法“在靶(ontarget)(daudi)测定”：该测定用于定量显示ifn受体和ifnα2b融合的抗体靶向的抗原的细胞上的ifnα2b和抗体-iffα2b融合蛋白构建体的抗增殖活性。daudi细胞表达cd20和cd38作为细胞表面相关抗原，以及细胞表面ifn受体。使用来自promega(madison，wisconsin)的试剂celltiter-cat#g7570测量daudi细胞的活力。这是基于发光的测定，其基于atp的定量来确定培养物中细胞的活力。信号强度与微量滴定板孔中的活细胞数成正比。测定的细节如下：将daudi细胞(获自atcc，manassas，va)在t75瓶(tpp，trasadingen，switzerland，cat#90076)中培养至优选的密度为具有10％胎牛血清(fbs；hyclone，logan，utcat#sh30070.03)的rpmi1640(mediatech，inc.，manassas，va，cat#10-040-cv)中0.5×105至0.8×105个活细胞/ml。通过在400g离心5分钟收集细胞，倾析上清液，并将细胞沉淀重悬于rpmi1640+10％fbs中。然后计数细胞，并将密度调节至在rpmi1640+10％fbs中3.0×105个细胞/ml。然后，将50μl细胞悬浮液等分到96孔圆底组织培养板(以下称为“实验板”)(tpp，cat#92067)的每个孔中。在分开的无菌96孔板(以下称为“稀释板”；costar，corning，nycat#3879)上，将测试品一式两份在rpmi1640+10％fbs中连续稀释。然后将50μl/孔从稀释板转移到实验板。然后将实验板在37℃、5％co2孵育4天。将制造商提供的测定缓冲液和测定底物的混合物(以下称为“celltiterglo试剂”，根据制造商的说明书混合)以100μl/孔加入到实验板中。将板摇动两分钟。然后，将100μl/孔从实验板转移到96孔平底白色不透明板(以下称为“测定板”；bdbiosciences，franklinlakes，njcat#353296)。然后使测定板的内容物在室温在黑暗中稳定15分钟。将该板在victor3vmultilabelcounter(perkinelmer，waltham，ma，型号1420-041)上在发光测量通道上读取，并测量发光。结果表示为“相对发光单位(rlu)”。使用prism5(graphpad，sandiego，ca)分析数据，使用非线性回归和三参数曲线拟合来确定曲线的中点(ec50)。对于每个测试品，相对于游离ifnα2b(或具有相对于ifnα2b的已知效力的一些其它形式的ifn)的效力被计算为ec50的比率。本领域普通技术人员将理解，还可以使用许多其它常用的用于测量细胞活力的测定。“在靶(arp)测定”(本文中有时也称为“靶向测定”)：多发性骨髓瘤细胞系arp-1是来自bartbarlogiemd，phd，阿肯色大学医学中心骨髓瘤研究所(littlerock，ak)所长的礼物。这在hardinj.等人(interleukin-6preventsdexamethasone-inducedmyelomacelldeath，blood；84：3063,1994)进行了描述。使用arp-1细胞(cd38+)测试cd38靶向抗体-ifn融合蛋白构建体。培养和测定条件与上文概括的基于daudi的测定除以下不同之处之外相同：将arp-1培养至4.0×105至6.0×105个细胞/ml的密度。在测定前将arp-1浓度调节至1.0×104个细胞/ml。实施例1抗cd38抗体减弱的ifnα2b融合蛋白的等电点收获表达抗cd38抗体-减弱的ifnα2b融合构建体的各种瞬时转染的细胞(表2)，并使用mabselectsureproteina柱进行纯化。使用hiloadsuperdex200柱将样品脱盐至200mm精氨酸、25mm组氨酸ph6.5。表2：构建体的表使用等电聚焦凝胶来确定融合多肽的等电点(pi)，并且检测蛋白质中由于翻译后修饰如磷酸化和糖基化的微小变化。将预制的ief凝胶设置在凝胶槽中，确保凝胶和缓冲液之间的紧密密封。然后将200ml1x阴极缓冲液倒入内室，确保没有缓冲液进入外室。然后将500ml1x阳极缓冲液倒入外室并填充槽的3/4。将样品和ladder加载到凝胶上后，然后在100伏下电泳1小时、在200伏下电泳1小时、在500伏下电泳1/2小时。凝胶电泳完成后，将凝胶取出并在玻璃容器中的tca溶液中固定30分钟。然后立即用去离子水洗涤凝胶3次。将凝胶在simplybluesafestain(invitrogenlifetechnologies)中染色整整一小时，并在水中放置过夜以脱色。使用扫描仪扫描最终的图像。通过将干扰素的苏氨酸106(t106)置换为丙氨酸(显示为t106a)、丝氨酸(t106s)、缬氨酸(t106v)、甘氨酸(t106g)或谷氨酸(t106e)或通过缺失t106(显示为δt106)来去除抗体-减弱的干扰素融合构建体的ifnα2b部分的o-连接的糖基化位点。通过在ief凝胶上分离比较具有和不具有o-连接的糖基化的构建体来研究这些变化对融合构建体的pi和异质性的影响。在每种情况下，缺失t106或用丙氨酸、丝氨酸、缬氨酸、甘氨酸或谷氨酸置换t106减少了ief凝胶上观察到的带电物质的数量，如与未修饰的t106直接比较的条带数减少所证明的那样，因此融合构建体的异质性降低(图9)。ief凝胶上的带电物质减少，因此并入t106s的分子的异质性降低与ifnα2b残基106处o-连接的糖基化的去除是一致的。去除抗体融合构建体的减弱的ifn部分中o-连接的糖基化位点导致相对于o-连接的糖基化蛋白质增加的pi。使用a10.21相同的抗体前端，无论抗体的同种型是igg4、igg1、igg1aa(igg1l235a，g237a，igg1的效应子功能降低形式)、igg2或igg2ss(igg2(a330s，p331s))，该趋势是一致的(图10)。已经显示yte置换(m252y、s254t、t256e)赋予增加的对fcrn的亲和力，推测可增加抗体的半衰期。进一步的实验检查了抗体ifn融合构建体的其他部分中的置换是否会影响由t106缺失或置换导致的减少的异质性。在ief凝胶上评估糖基化的a10.21抗cd38-减弱的ifnα2b融合构建体(yte、t106t)和具有yte置换的非糖基化ifn融合构建体(yte、t106a)的异质性。去除具有yte突变的融合构建体的ifnα2b组分的糖基化降低了异质性(图11)。ifnα2b的减弱是通过置换负责结合ifn受体的关键氨基酸残基来实现。评估了在ifnα2b中具有各种减弱氨基酸置换的a10.21igg4(s228p)ifn构建体以及ifnα2b组分的具有o-连接的糖基化(t106t)或不具有o-连接的糖基化(t106a)的带电物质的数量。ifnα2b的单个氨基酸残基精氨酸-33、精氨酸-144和丙氨酸-145一次一个地用丙氨酸置换残基33(r33a)、异亮氨酸置换残基144(r144i)或赖氨酸、甘氨酸或谷氨酰胺置换残基145(a145k、a145g、a145q)。当直接比较去糖基化的ifn融合构建体始终比其糖基化对应物更不均一(图12)。所显示的异质性的减少与构建体的抗体部分无关。去除抗体(igg4(s228p))-减弱的ifn融合构建体的减弱的ifnα2b部分中的o-连接的糖基化位点也导致ief检测到的异质性下降，融合构建体特异性针对hla、cd138和cd38(针对抗体a10.21-抗体a02.12的cd38上的不同表位)(图13)。实施例2抗体减弱的干扰素α2b融合蛋白的抗增殖活性ifnα2b的抗增殖作用由直接和间接的活性组成。直接活性通过由细胞周期阻滞的癌细胞生长抑制(matsui等人，2003)、由死亡受体依赖性(crowder等人2005)和非依赖性(otsuki等人1998)细胞途径的凋亡或分化(matsui等人2003)而发生。使用发光细胞活力测定针对靶阳性细胞系测量抗体减弱的干扰素融合蛋白的靶特异性直接细胞毒性。抗cd38-ifn前导序列样品稳定克隆或瞬时转染构建体(表3)并收获它们，以及使用mabselectsureproteina柱进行纯化。使用hiloadsuperdex200柱将样品脱盐至200mm精氨酸、25mm组氨酸ph6.5。表3：构建体的表市售ifnα2b将a(来自schering-plough的市售细菌生产的ifnα2b)作为阳性对照使用。抗增殖活性测量使用上述“daudi细胞增殖测定”和“arp-1细胞增殖测定”测量抗增殖活性。使用arp-1细胞增殖测定方法与另外的细胞系nci-h929测量抗体-减弱的ifnα2b融合蛋白的抗增殖活性。在一些实验中，使用专用的96微孔板发光计读取平板，并使用celltiter-2.0试剂代替原来的celltiter-glo，这两者都不影响结果。使用arp-1/nci-h929或daudi细胞增殖测定来定量ifn和抗体-减弱的ifnα2b融合蛋白构建体对展示cd38的细胞的抗增殖活性。daudi、arp1和nci-h929细胞表达cd38作为细胞表面相关抗原。测定的细节如下。在arp-1/nci-h929细胞增殖测定中，使用来自promega(madison，wisconsin)的试剂celltiter-2.0(cat#g9242)测量细胞活力。这是基于发光的测定，其基于atp的定量来确定培养物中细胞的活力。信号强度与微量滴定板孔中的活细胞数成正比。细胞(来自atcc，manassas，va的nci-h929和arp-1，来自bartbarlogiemd，phd，阿肯色大学医学中心的骨髓瘤研究所所长的礼物，littlerock，ak)在t75瓶(tpp，trasadingen，switzerland，cat#90076)中培养至优选密度为具有10％胎牛血清(fbs；hyclone，logan，utcat#sh30070.03)的rpmi1640(mediatech，inc.，manassas，va，cat#10-040-cv)中0.5×105至0.8×105个活细胞/ml。通过在400×g离心5分钟收集细胞，倾析上清液，并将细胞沉淀重悬于rpmi1640+10％fbs中。然后计数细胞，并将密度调节至在rpmi1640+10％fbs中3.0×105个细胞/ml。将50μl细胞悬浮液接种到96孔圆底组织培养板(以下称为“实验板”)(tpp，cat#92067)的每个孔中。在加入测试化合物之前，将细胞在4℃孵育1小时。在分开的无菌96孔板(以下称为“稀释板”；costar，corning，nycat#3879)上，将测试品一式两份在rpmi1640+10％fbs中连续稀释。将50μl/孔从稀释板转移到实验板。然后将实验板在37℃、5％co2孵育4天。将“celltiter-试剂2.0”以100μl/孔加入到实验板中。将板摇动两分钟。将100μl/孔从实验板转移到96孔平底白色不透明板(以下称为“测定板”；bdbiosciences，franklin5lakes，njcat#353296)。然后使测定板的内容物在室温在黑暗中稳定15分钟。在96微孔板发光计上读板。结果表示为“相对发光单位”(rlu)。使用prism5(graphpad，sandiego，ca)分析数据，使用非线性回归和三参数曲线拟合确定ic50。通过将苏氨酸106(t106)置换为丙氨酸(t106a)、丝氨酸(t106s)、缬氨酸(t106v)、甘氨酸(t106g)或谷氨酸(t106e)或缺失t106(显示为δt106)去除抗cd38抗体减弱的干扰素融合构建体的o-连接的糖基化位点。通过比较具有和不具有o-连接的糖基化的与减弱的ifnα2b融合的a10.21抗-cd38抗体来研究对细胞增殖的影响。去除抗体融合构建体的减弱ifn部分中o-连接的糖基化位点导致增加的抗增殖活性，如通过arp1(图1a)和nci-h929细胞(图1b)中相对于相应o-连接的糖基化融合蛋白即a10.21igg4(s228p))ifn(a145d，t106t)和a10.21igg1ifn(a145d，t106t)较低的ic50(nm)所示。无论抗体同种型是igg4和igg1，这种趋势是一致的。将苏氨酸106(t106)置换为丙氨酸(t106a)、丝氨酸(t106s)、缬氨酸(t106v)、甘氨酸(t106g)或谷氨酸(t106e)导致增加的抗增殖活性，如通过相对于o-连接的糖基化融合蛋白较低的ic50(nm)所示。所有非糖基化构建体相对于其在nci-h929细胞中的糖基化对应物显示更高的“在靶”效力(图14)。在一系列抗cd38抗体-减弱的ifnα2b融合蛋白中检查了从减弱的ifnα2b组分中去除o-连接的糖基化位点对抗增殖活性的影响。检查了在t106处具有不同氨基酸置换以去除ifn组分上的o-连接的糖基化位点的抗cd38抗体-减弱的ifnα2b融合蛋白r10a2igg4(s228p)ifn(a145d)的变体(t106a,t106g,t106n,t106f,t106r,t106d,t106e,t106q,t106h,t106i,t106l,t106k,t106m,t106f,t106p,t106s,t106v,t106y和t106w)(表4)，结果如图2所示。虽然效力水平在t106位点处的不同置换之间变化，但预期导致去除o-连接的糖基化的所有置换相对于相应的o-连接的糖基化蛋白质增加效力(图2)。表4：在t106处具有各种氨基酸置换以去除o-连接的糖基化的r10a2抗cd38抗体-减弱的ifn融合构建体变体。以类似的方式评估以下之间的效力的相对变化：结合cd38上不同表位的两种不同的抗cd38抗体干扰素融合蛋白(a02.12和a10.21)，具有或不具有o-连接的糖基化(a02.12igg4(s228p)ifn(a145d，t106t)、a02.12igg4(s228p)ifn(a145d，t106a)、a10.21igg4(s228p)ifn(a145d，t106t)和a10.21igg4(s228p)ifn(a145d，t106a)。不管抗体部分的靶标如何，去除具有抗cd38的不同特异性的抗体(igg4(s228p))融合构建体的减弱的ifn部分中o-连接的糖基化位点导致抗增殖活性的增加(图15)。通过各种减弱置换实现ifnα2b的减弱。还评估了具有(t106t)和不具有o-连接的糖基化(t106a)的ifnα2b中具有各种减弱置换的a10.21igg4(s228p)ifn构建体的抗增殖活性(表5)。在具有多种减弱置换的构建体的ifnα2b组分中去除o-连接的糖基化位点都比其糖基化对应物具有更高的效力(图16)。表5：具有(t106t)和不具有o-连接的糖基化(t106a)的ifnα2b中具有各种减弱置换的a10.21igg4(s228p)ifn构建体。为了研究具有不同结合靶标的其它减弱的ifnα2b融合抗体/蛋白质是否作为去除o-连接的糖基化的结果调节了抗增殖活性，产生了2种构建体(抗cd138抗体和抗hla抗体并与具有或不具有o-连接的糖基化的ifn(a145d)融合(表6)，并测试其抗增殖活性。去除具有抗hla和cd138的特异性的抗体(igg4(s228p))融合构建体的减弱ifn部分中o-连接的糖基化位点导致抗增殖活性增加，而与构建体的抗体部分的靶标无关。这在针对hla和cd138的抗体融合构建体中得到证实(图17)。表6：各种ifn融合蛋白的构建体-与具有或不具有o-连接的糖基化的ifn(a145d)融合的抗cd138抗体和抗hla抗体从包含fc区域中的置换以延长半衰期或降低的效应子功能的融合蛋白中去除o-连接的糖基化对抗增殖活性的影响已经显示yte置换赋予对fcrn增加的亲和力，推测可增加抗体的半衰期。在存在和不存在o-连接的糖基化的情况下，测试含有yte置换的ifn融合抗体的抗增殖活性。以下变体由a10.21抗cd38抗体减弱的ifn融合蛋白制成(表7)。对a10.21igg4(s228p)(a145d，t106a)引入yte置换不影响由减弱的ifn去除o-连接的糖基化引起的效力的增加(图18)。igg1变体的fc部分中的l235a和g237a置换以及igg2变体的fc部分中的a330s和p331s置换导致效应子功能降低。在存在和不存在o-连接的糖基化的情况下，测试减弱的ifn融合含有l235a和g237a置换的igg1抗体和减弱的ifn融合含有a330s和p331s置换的igg2抗体的抗增殖活性。这些变体由a10.21抗cd38抗体减弱的ifn融合蛋白制成(表7)。去除抗体融合构建体的减弱ifn部分中o-连接的糖基化位点导致效力增加，而不管抗体构建体中抗体的同种型是igg1还是igg1aa(igg1(l235a，g237a))、igg2或igg2ss(igg2(a330s，p331s))(图19)。表7：由延长半衰期或降低效应子功能的fc区域中的置换构成的a10.21融合蛋白通过t106的氨基酸置换或缺失从抗cd38抗体干扰素融合蛋白的减弱干扰素部分去除o-连接的糖基化位点导致靶标cd38、cd138或hla阳性细胞的抗增殖活性增加(1.3-12倍)。实施例3抗体-减弱的ifnα2b融合蛋白的在靶/脱靶活性使用萤光素酶生成生物发光进行ilitetm报告基因测定用于定量测定人干扰素α(ifnα2b)生物活性(iu/ml)。该测定中使用的细胞表达cd38并用于测量抗cd38-减弱的ifnα2b的融合蛋白的“在靶(on-target)”活性。当用识别相同表位的抗cd38抗体阻断cd38时，这些细胞也可以用于测量“脱靶(off-target)”活性。这些测定可用于确定选择性指数(si)，其为抗cd38ifn融合蛋白针对cd38+靶细胞具有怎样的选择性活性和针对cd38被阻断的细胞(模拟cd38-细胞)无活性的量度。si越大，试剂对靶标更具有选择性，而接近1的数表示针对靶标或非靶标没有选择性。introna用作阳性对照，因为其针对表达干扰素α受体ifnar1/2的细胞具有活性，而对于其它细胞表面表达的抗原(即cd38)没有选择性，具有约1的si。抗体-减弱的ifn构建体所用构建体的序列列于序列表中，并列于下表8中。表8a用作阳性对照。在靶/脱靶活性测量使用与上述“ilite报告基因测定”相同的方法测量在靶/脱靶活性ilite报告基因测定(pblinterferonsource，piscataway，nj，cat#51100)主要如制造商所述进行，并加入人igg封闭步骤。制造商将ilite细胞系描述为“源自市售的前单核细胞人细胞系的稳定转染细胞系，其特征在于mhcii类抗原，特别是人淋巴细胞抗原(hladr)在细胞表面上的表达”。细胞系表达cd38并含有稳定转染的萤光素酶基因，其表达由干扰素应答元件(ire)驱动，其允许基于发光输出定量干扰素活性。制造商提供的ilite板(以下称为测定板)和稀释剂从-80℃冷冻器中取出并使其平衡至室温。每孔加入50μl稀释液至测定板。将制造商提供的报告细胞的小瓶从-80℃冷冻器中取出并在37℃水浴中解冻。将25μl等分的细胞分配到测定板的每个孔中。接下来，每孔加入12.5μl在rpmi1640+10％fbs(sigmachemicals，st.louis，mo；cat#i4506)中稀释的8mg/ml人igg。将内容物混合并在37℃下孵育15分钟。在分开的“稀释板”上，将测试品一式两份在rpmi1640+10％fbs中连续稀释。然后，将12.5μl测试品从稀释板转移到测定板。然后将测定板在37℃、5％co2孵育17小时。将制造商提供的测定缓冲液和底物从-80℃冷冻器中取出，使其平衡至室温2小时。将制造商提供的测定缓冲液加入到制造商提供的底物小瓶中，并根据制造商的说明书充分混合以产生“发光溶液”。然后，将100μl的发光溶液加入到测定板的每个孔中。将板摇动2分钟。然后将板在暗处在室温孵育5分钟，并在光度测量通道上的victor3vmultilabel计数器上读取，并且测量发光并表示为rlu。为了在ilite测定中测试抗cd38抗体-ifn融合蛋白构建体的脱靶活性，制造商提供的稀释液补充有0.25mg/ml抗cd38抗体(与被测试的抗体-ifn融合蛋白构建体识别cd38上相同表位的抗体，以阻止抗cd38抗体-ifn融合蛋白构建体与ilite细胞上表达的cd38的任何结合)。该阻断阶段之后用抗cd38抗体-ifn融合蛋白或ifnα2b处理。使用prism5(graphpad，sandiego，ca)分析数据，使用非线性回归和三参数曲线拟合确定曲线的中点(ec50)。通过ec50(脱靶活性)/ec50(在靶活性)计算选择性指数(si)。选择性指数(si)是抗cd38ifn构建体针对表达cd38的细胞具有怎样的选择性活性和针对无cd38的细胞无活性的量度。数字越大，它对靶标更具有选择性，而接近1的数字表示针对靶标没有选择性。introna用作阳性对照，其si约为1。通过将苏氨酸106(t106)置换为丙氨酸(显示为t106a)去除抗cd38抗体减弱的干扰素融合构建体的o-连接的糖基化位点。通过比较与具有和不具有o-连接的糖基化的ifnα2b融合的a10.21和a10.43抗-cd38抗体来研究该活性。去除抗体融合构建体的减弱ifn部分中的o连接的糖基化位点(显示为t106a)相对于o-连接的糖基化蛋白质导致在靶活性的轻微增加(图3和表9)以及脱靶活性轻微增加至不增加。o连接的糖基化和非糖基化蛋白质显示出对cd38+细胞的高选择性，而introna不显示选择性。表9：具有和不具有o-连接的糖基化的抗cd38抗体减弱的ifnα2b融合蛋白a10.21和a10.43的ec50和si也可以类似的方式检查从融合蛋白的减弱的ifn去除o-连接的糖基化位点对在靶/脱靶活性的影响。进行ilitetm报告基因在靶-脱靶活性测定以证明抗cd38减弱的ifn融合蛋白对靶cd38+细胞的选择性活性，以及对当用抗cd38抗体(识别相同的表位)阻断cd38模拟靶细胞上预期的活性时的细胞的有限的活性。去除抗体融合构建体的减弱ifn部分中的o-连接的糖基化位点(通过t106a置换)导致相对于o-连接的糖基化蛋白在靶活性略微增加以及脱靶活性轻微增加至不增加。o连接的糖基化和非糖基化抗cd38抗体减弱的干扰素融合蛋白均对cd38+细胞显示出高选择性，而introna没有显示选择性。为了进一步检查作为抗体融合构建体的减弱ifn部分中o-连接的糖基化位点的去除的结果的选择性指数的调节，在一系列构建体中检查在靶-脱靶活性。所检查的变化基于a10.21igg4(s228p)ifn(a145d，t106t)构建体。这些包括苏氨酸106的缺失(δt106)、t106置换为丝氨酸(t106s)、yte置换(ytet106t和ytet106a)，通过从丙氨酸145置换为谷氨酰胺(a145q)的ifn减弱以及改变抗体同种型为igg1(igg1t106t和igg1t106a)。大多数构建体显示选择性指数>1，对靶向cd38+细胞的选择性的大小根据抗cd38ifn融合构建体而变化(图20)。在a10.21igg4(s228p)ifn(a145d，t106a)中观察到最高的选择性。实施例4抗cd38抗体-减弱ifnα2b融合蛋白的脱靶活性通过监测isgf3途径的活化，hek-bluetmifn-α/β细胞允许检测生物活性的人i型ifn。用人ifn-α刺激hek-bluetmifn-α/β细胞活化jak/stat/isgf3途径，随后诱导产生seap(在ifn-α/β诱导型isg54启动子控制下的报告基因)。上清液中seap的水平可以用quanti-bluetm轻松测定。使用该报告基因测定评估从抗体干扰素融合蛋白的减弱干扰素部分去除o-连接的糖基化的作用。抗体/fc融合蛋白构建体的产生收获表达干扰素融合蛋白的各种瞬时转染细胞，并使用mabselectsureproteina柱进行纯化。使用hiloadsuperdex200柱将样品脱盐至200mm精氨酸、25mm组氨酸ph6.5。表10a用作阳性对照。通过hek-blue测定测量脱靶活性除了所使用的接种细胞、稀释抗体和上清液体积之外，使用与“hek-blue脱靶测定”中相同的方法测量抗体-ifn融合物的脱靶活性。测定的细节如下。hek-blue脱靶测定用于使用hek-bluetmifn-α/β细胞系(invivogen，sandiego，ca)来定量抗体-ifn融合构建体结合干扰素-α/β受体(ifnar)的能力。主要按照hek-blueifn-α/β细胞系的制造商所述进行“脱靶(hb-ifn)测定”。hek-bluetmifn-α/β细胞是专门设计用于监测由i型ifn诱导的jak-stat途径的活化。通过将人stat2和irf9基因导入hek293细胞产生细胞，以获得完全活性的i型ifn信号传导途径。hek-bluetmifn-α/β细胞在isg54启动子的控制下稳定表达报告基因：分泌型胚胎碱性磷酸酶(seap)。isg54是众所周知的通过isre依赖机制由i型ifn活化的isg。在ifn-α或ifnβ刺激下，hek-bluetmifn-α/β细胞活化jak-stat途径和随后seap报告基因的表达。seap被分泌到培养基中，可以使用比色试剂quanti-bluetm进行定量。简而言之，将hek-blueifn-α/β细胞(invivogen，sandiegocacat#hkb-ifnab)解冻并在dmem培养基(mediatech，manassasva，cat#10-013-cv)+10％已经热灭活的fbs(hyclone，loganut，cat#sh30070.03)(hifbs)。当细胞达到60-80％汇合时，用cellstripper(mediatech，cat#25-056-cl)提起细胞。细胞在dmem+hifbs中洗涤两次并计数。将细胞调节至在dmem+hifbs中2.77×105个活细胞/ml，并以每孔180μl接种到平底96孔组织培养板(以下称为“实验板”)中。然后，每孔加入20μl稀释入dmem+hifbs的ifnα2b或融合蛋白构建体。将板在37℃、5％co2孵育16-24小时。quanti-blue(invivogen，cat#rep-qb1)是根据制造商的指导准备的。将quanti-blue(180μl)接种到平底板(以下称为“测定板”)的每个孔中。然后，将来自实验板的每孔20μl上清液转移至测定板。然后将测定板在37℃温育1-3小时。在来自moleculardevices的spectramaxplus384型微孔板读数器上读取在620nm处的测定板吸光度。使用graphpadprism分析数据。评估了ifnα2b中o-连接的糖基化的存在或不存在对减弱的ifn融合抗cd38抗体的脱靶活性的影响。通过将苏氨酸106(t106)替换为丙氨酸(显示为t106a)或缺失t106(显示为δt106)去除具有减弱的干扰素融合构建体的抗cd38抗体的o-连接的糖基化位点。通过比较具有和不具有o-连接的糖基化与减弱的ifnα2b融合的x10.21抗-cd38抗体研究对脱靶活性的影响。去除抗体融合构建体的减弱ifn部分中o-连接的糖基化位点导致相对于o-连接的糖基化蛋白即a10.21igg4ifn(145d，t106t)和a10.21igg1ifn(145d，t106t)脱靶活性略微增加(图5)。无论同种型如何，这种趋势是一致的(图5)。然而，ifnα2b(a145d)如此减弱，使得即使在所用的最高剂量下，该测定中seap的诱导水平也受到限制。因此，ec50只是近似值。检查了与减弱的ifn(r10a2igg4(s228p)ifn(a145d))融合的抗体的脱靶活性，其具有不同的氨基酸置换以去除o-连接的糖基化。测试所有可能的氨基酸置换，并将构建体列于表11中。表11已经显示，相对于o-糖基化的ifn融合抗体r10a2igg4(s228p)ifn(a145d，t106t)，这些置换中的每一个都表现出相似或稍微偏少的脱靶活性(增加的ec50)。然而，ifnα2b(a145d)如此减弱，使得即使在所用的最高剂量下，该测定中seap的诱导水平也受到限制。因此，ec50只是近似值。研究了另一种抗cd38抗体减弱的ifnα2b构建体的脱靶活性和通过t106a置换去除o-连接的糖基化的影响。与a10.21相比，a10.43的重链有7个氨基酸变化，而两个构建体具有相同的轻链序列(见序列表)。结果表明，通过t106a置换去除o-连接的糖基化位点略微增加了a10.43的脱靶活性。然而，ifnα2b(a145d)如此减弱，使得即使在所用的最高剂量下，该测定中seap的诱导水平也受到限制。因此，ec50只是近似值。去除抗体干扰素融合构建体的减弱ifn部分中的o-连接的糖基化位点导致相对于o连接的糖基化的蛋白质脱靶活性轻微增加至不增加以及在靶活性略有增加。o连接的糖基化和非糖基化抗cd38抗体减弱的干扰素融合蛋白均对cd38+细胞显示出高选择性，而introna没有显示选择性。实施例5人类新生儿fc受体(fcrn)结合抗cd38-减弱的ifn的评估使用胺偶联操作方案将多聚组氨酸标记的fcrn固定在biacoret200增强仪器中的cm5传感器芯片的指定活性流通池上，同时对参考流动池进行空白固定。多聚组氨酸标记的fcrn的脉冲被注入到活性表面上以确保溶液预先浓缩到流动池上。然后用50mmnaoh洗涤表面。参考表面和活性表面二者用50:50edc/nhs的混合物活化7分钟。之后是仅在活性表面上的10mm醋酸钠ph5.0中的2μg/ml多聚组氨酸标记的fcrn的一系列脉冲。一旦达到150ru的目标，用1m乙醇胺ph8.5使两个表面失活7分钟。该操作方案导致大约150ru多聚组氨酸标记的fcrn固定在活性表面上。每个活性表面用于一次运行。使用zebaspin脱盐柱，7kmwco，0.5ml(pierce产品编号89882)，将测试抗体在pbs-p运行缓冲液(dpbs，ph7.4和0.005％tween-20，在25℃用hcl调节至ph6.0)脱盐。脱盐后，将每个样品的浓度调节至1mg/ml。在测定当天，将1mg/ml溶液在运行缓冲液中进一步稀释以制备最高浓度，然后以1:2稀释以产生浓度系列。测试样品以50μl/分钟的流速通过表面。缔合阶段为100秒，而所有测试浓度的解离阶段为300秒。使用60秒注射的ph8.0的100mmtris、50mmnacl以除去测试抗体再生活性和参考表面。在25℃测定结合常数。使用两个状态反应模型评价抗体多聚组氨酸fcrn结合相互作用，其中将rmax设置为局部，并将ri(折射率)参数设定为局部。所有数据均减去两次参考：第一，减去由抗体与葡聚糖基质结合而产生的参考细胞(空白固定)中的信号，第二，减去活性表面上0nm抗体的信号。对每个抗体的至少两个分开的运行测定结合常数。与抗cd38/ifn样品一起测试prolialot1035726作为每次运行的阳性对照。测定了ka1、kd1、ka2、kd2和kd的平均值。测量了4种抗cd38/ifn(a10.21igg4(s228p)ifn(a145d，t106a)、a10.21igg4(s228p)ifn(a145d，t106t)、a10.43igg4(s228p)ifn(a145d，t106a)、a10.43igg4(s228p)ifn(a145d，t106t))的fcrn结合的平均动力学和亲和力值。在该样品集中，克隆a10.21igg4(s228p)ifn(a145d，t106a)显示出对fcrn的最高亲和力。实施例6在小鼠nci-h929多发性骨髓瘤模型中具有和不具有o链接的糖基化的与减弱的干扰素α2b融合的抗cd38抗体的功效药物与处理表12#-对照组程序:·用溶于50％matrigel的1x107h929肿瘤细胞以sc在侧腹设置cr雌性cb.17scid小鼠。·细胞注射体积是0.2ml/只小鼠。·开始日的鼠龄：8至12周。·当肿瘤达到170-350mm3的平均大小时进行对匹配，并且开始处理。·给药体积＝0.2ml/只小鼠。不针对体重进行调整。·体重：qdx5然后biwk至结束·卡尺测量：biwk至结束·终点tgd。动物要单独监测。实验的终点是肿瘤体积为2000mm3或60天，以先到者为准。应答者可以跟踪更长时间。当达到终点时，动物将按照sop#687进行安乐死。结果示于图8中。在具有和不具有o-连接的糖基化的情况下，观察到大致相当的功效，其中去糖苷基化形式在阻止肿瘤生长中稍微更有效。实施例7在评估人类新生儿fc受体(fcrn)与具有和不具有o-连接的糖基化的减弱ifn融合的抗cd38抗体结合时，没有o-连接的糖基化的蛋白质显示出对fcrn的最高亲和力。这样的作用可以在食蟹猴和人源化fcrn小鼠中进行评估。食蟹猴研究猴研究设计——比较+/-o-glyc·通过留置导管单次静脉输注3mg/kg(1小时)–a10.21igg4(s228p)ifn(145d,t106a)(n＝4)–a10.21igg4(s228p)ifn(145d,t106t)(n＝4)–a10.21igg4(s228p)ifn(145d,t106a)(n＝4)–a10.21igg4(s228p)ifn(145d,t106t)(n＝4)·比较pk和pd(生物效应，即血清新蝶呤水平)·pk：所有猴子，≤1ml，11个时间点(预给药、0分钟(输注结束后即时)、2、6、12、24、48、96、120、168和输注后240小时。将样品(80)通过elisa进行分析。·tk建模：winnonlintableassembly{非分区(non-compartmental)分析)·临床病理学：血液学和血液化学-所有猴子，3个时机(预处理、给药后24小时和第8天)·血清新蝶呤·所有猴子，约0.5ml3个时机在5个时间点(给药前、给药后12、24、96、168小时)人源化fcrn小鼠的药代动力学研究1.在第1天给予32只b6.cg-fcgrtm1dcr'tg(cag-fcgrt)276dcr/dcrj(jaxstock#004919)雌性小鼠通过ip注射1mg/kg的：–a10.21igg4(s228p)ifn(145d,t106a)(n＝8)–a10.21igg4(s228p)ifn(145d,t106t)(n＝8)–a10.43igg4(s228p)ifn(145d,t106a)(n＝8)–a10.43igg4(s228p)ifn(145d,t106t)(n＝8)2.给药前3天、给药日然后每周测量体重。3.每天进行笼侧观察，每周进行临床观察。4.药代动力学血液采集：小鼠在给药前3天和给药后(25μi)1、12、24、48和72小时以及5、7、10和14天取血。小鼠在2个群组(4只小鼠/组/群组)中取血。5.在第14天处死所有小鼠。进行终末心脏穿刺术以收集血液。6.将血液收集在锂肝素管中，并在4℃以10,000rpm离心2分钟。7.将血浆样品在pbs中以1:10稀释并冷冻，然后通过结合elisa分析a10.21igg4ifn(145d)a106、a10.21igg4ifn(145d)t106、a10.43igg4ifn(145d)a106或a10.43igg4ifn(145d)t106。序列表<110>梯瓦制药澳大利亚股份有限公司<120>干扰素α2b变体<130>35241105/wjp<160>112<170>patentinversion3.5<210>1<211>165<212>prt<213>人工序列<220><223>氨基酸位置106处具有突变的人干扰素α2b<220><221>变体<222>(106)..(106)<223>xaa可以是选自a,c,d,e,f,g,h,i,k,l,m,n,p,q,r,v,w,y的任意氨基酸<400>1cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalxaagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>2<211>164<212>prt<213>人工序列<220><223>在位置106具有氨基酸缺失的人干扰素α2b<400>2cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalgluthrproleumetlysglu100105110aspserileleualavalarglystyrpheglnargilethrleutyr115120125leulysglulyslystyrserprocysalatrpgluvalvalargala130135140gluilemetargserpheserleuserthrasnleuglngluserleu145150155160argserlysglu<210>3<211>165<212>prt<213>人工序列<220><223>包含减毒突变l15a的去糖基化（t106a）人干扰素α2b<400>3cysaspleuproglnthrhisserleuglyserargargthralamet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>4<211>165<212>prt<213>人工序列<220><223>包含减毒突变a19w的去糖基化（t106a）人干扰素α2b<400>4cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleutrpglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>5<211>165<212>prt<213>人工序列<220><223>包含减毒突变r22a的去糖基化（t106a）人干扰素α2b<400>5cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetalaargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>6<211>165<212>prt<213>人工序列<220><223>包含减毒突变r23a的去糖基化（t106a）人干扰素α2b<400>6cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargalaileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>7<211>165<212>prt<213>人工序列<220><223>包含减毒突变s25a的去糖基化（t106a）人干扰素α2b<400>7cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargilealaleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>8<211>165<212>prt<213>人工序列<220><223>包含减毒突变l26a的去糖基化（t106a）人干扰素α2b<400>8cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileseralaphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>9<211>165<212>prt<213>人工序列<220><223>包含减毒突变f27a的去糖基化（t106a）人干扰素α2b<400>9cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleualasercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>10<211>165<212>prt<213>人工序列<220><223>包含减毒突变l30x的去糖基化（t106a）人干扰素α2b，其中x可以选自a，v的任意氨基酸<220><221>变体<222>(30)..(30)<223>xaa可以选自a，v的任意氨基酸<400>10cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysxaalysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>11<211>165<212>prt<213>人工序列<220><223>包含减毒突变k31a的去糖基化（t106a）人干扰素α2b<400>11cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleualaasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>12<211>165<212>prt<213>人工序列<220><223>包含减毒突变d32a的去糖基化（t106a）人干扰素α2b<400>12cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysala202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>13<211>165<212>prt<213>人工序列<220><223>包含减毒突变r33x的去糖基化（t106a）人干扰素α2b，其中x可以是选自k,a,q的任意氨基酸<220><221>变体<222>(33)..(33)<223>xaa可以是选自k,a,q的任意氨基酸<400>13cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530xaahisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>14<211>165<212>prt<213>人工序列<220><223>包含减毒突变h34a的去糖基化（t106a）人干扰素α2b<400>14cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530argalaasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>15<211>165<212>prt<213>人工序列<220><223>包含减毒突变q40a的去糖基化（t106a）人干扰素α2b<400>15cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproalagluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>16<211>165<212>prt<213>人工序列<220><223>包含减毒突变d114r的去糖基化（t106a）人干扰素α2b<400>16cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluargserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>17<211>165<212>prt<213>人工序列<220><223>包含减毒突变l117a的去糖基化（t106a）人干扰素α2b<400>17cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserilealaalavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>18<211>165<212>prt<213>人工序列<220><223>包含减毒突变r120x的去糖基化（t106a）人干扰素α2b，其中x可以是选自a,e的任意氨基酸<220><221>变体<222>(120)..(120)<223>xaa可以是选自a,e的任意氨基酸<400>18cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalxaalystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>19<211>165<212>prt<213>人工序列<220><223>包含减毒突变r125x的去糖基化（t106a）人干扰素α2b，其中x可以是选自a,e的任意氨基酸<220><221>变体<222>(125)..(125)<223>xaa可以是选自a,e的任意氨基酸<400>19cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnxaailethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>20<211>165<212>prt<213>人工序列<220><223>包含减毒突变k131a的去糖基化（t106a）人干扰素α2b<400>20cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleualaglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>21<211>165<212>prt<213>人工序列<220><223>包含减毒突变e132a的去糖基化（t106a）人干扰素α2b<400>21cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysalalyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>22<211>165<212>prt<213>人工序列<220><223>包含减毒突变k133a的去糖基化（t106a）人干扰素α2b<400>22cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglualalystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>23<211>165<212>prt<213>人工序列<220><223>包含减毒突变k134a的去糖基化（t106a）人干扰素α2b<400>23cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulysalatyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>24<211>165<212>prt<213>人工序列<220><223>包含减毒突变r144x的去糖基化（t106a）人干扰素α2b，其中x可以是选自a,d,e,g,h,i,k,l,n,q,s,t,v,y的任意氨基酸<220><221>变体<222>(144)..(144)<223>xaa可以是选自a,d,e,g,h,i,k,l,n,q,s,t,v,y的任意氨基酸<400>24cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalxaa130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>25<211>165<212>prt<213>人工序列<220><223>包含减毒突变a145x的去糖基化（t106a）人干扰素α2b，其中x可以是选自d,e,g,h,i,k,l,m,n,q,r,s,t,v,y的任意氨基酸<220><221>变体<222>(145)..(145)<223>xaa可以是选自d,e,g,h,i,k,l,m,n,q,r,s,t,v,y的任意氨基酸<400>25cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140xaagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>26<211>165<212>prt<213>人工序列<220><223>包含减毒突变m148a的去糖基化（t106a）人干扰素α2b<400>26cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilealaargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>27<211>165<212>prt<213>人工序列<220><223>包含减毒突变r149a的去糖基化（t106a）人干扰素α2b<400>27cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetalaserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>28<211>165<212>prt<213>人工序列<220><223>包含减毒突变s152a的去糖基化（t106a）人干扰素α2b<400>28cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserphealaleuserthrasnleuglngluser145150155160leuargserlysglu165<210>29<211>165<212>prt<213>人工序列<220><223>包含减毒突变l153a的去糖基化（t106a）人干扰素α2b<400>29cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheseralaserthrasnleuglngluser145150155160leuargserlysglu165<210>30<211>165<212>prt<213>人工序列<220><223>包含减毒突变n156a的去糖基化（t106a）人干扰素α2b<400>30cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthralaleuglngluser145150155160leuargserlysglu165<210>31<211>612<212>prt<213>人工序列<220><223>a10.21igg4(s228p)ifn(a592d,t553x)，其中x可以是选自a,g,e,s,v的任意氨基酸<220><221>变体<222>(553)..(553)<223>xaa可以是选自a,g,e,s,v的任意氨基酸<400>31gluvalglnleuvalglnserglyalagluvallyslysproglyala151015thrvallysilesercyslysvalserglytyrthrphethraspser202530valmetasntrpvalglnglnalaproglylysglyleuglutrpmet354045glytrpileaspproglutyrglyargthraspvalalaglulysphe505560glnglyargvalthrilethralaaspthrserthraspthralatyr65707580metgluleuserserleuargsergluaspthralavaltyrtyrcys859095alaargthrlystyrasnserglytyrglypheprotyrtrpglygln100105110glythrthrvalthrvalserseralaserthrlysglyproserval115120125pheproleualaprocysserargserthrsergluserthralaala130135140leuglycysleuvallysasptyrpheprogluprovalthrvalser145150155160trpasnserglyalaleuthrserglyvalhisthrpheproalaval165170175leuglnserserglyleutyrserleuserservalvalthrvalpro180185190serserserleuglythrlysthrtyrthrcysasnvalasphislys195200205proserasnthrlysvalasplysargvalgluserlystyrglypro210215220procysproprocysproalaproglupheleuglyglyproserval225230235240pheleupheproprolysprolysaspthrleumetileserargthr245250255progluvalthrcysvalvalvalaspvalserglngluaspproglu260265270valglnpheasntrptyrvalaspglyvalgluvalhisasnalalys275280285thrlysproargglugluglnpheasnserthrtyrargvalvalser290295300valleuthrvalleuhisglnasptrpleuasnglylysglutyrlys305310315320cyslysvalserasnlysglyleuproserserileglulysthrile325330335serlysalalysglyglnproarggluproglnvaltyrthrleupro340345350proserglngluglumetthrlysasnglnvalserleuthrcysleu355360365vallysglyphetyrproseraspilealavalglutrpgluserasn370375380glyglnprogluasnasntyrlysthrthrproprovalleuaspser385390395400aspglyserphepheleutyrserargleuthrvalasplysserarg405410415trpglngluglyasnvalphesercysservalmethisglualaleu420425430hisasnhistyrthrglnlysserleuserleuserleuglylyscys435440445aspleuproglnthrhisserleuglyserargargthrleumetleu450455460leualaglnmetargargileserleuphesercysleulysasparg465470475480hisasppheglypheproglngluglupheglyasnglnpheglnlys485490495alagluthrileprovalleuhisglumetileglnglnilepheasn500505510leupheserthrlysaspserseralaalatrpaspgluthrleuleu515520525asplysphetyrthrgluleutyrglnglnleuasnaspleugluala530535540cysvalileglnglyvalglyvalxaagluthrproleumetlysglu545550555560aspserileleualavalarglystyrpheglnargilethrleutyr565570575leulysglulyslystyrserprocysalatrpgluvalvalargasp580585590gluilemetargserpheserleuserthrasnleuglngluserleu595600605argserlysglu610<210>32<211>165<212>prt<213>人工序列<220><223>包含减毒突变l30a,h57y,e58n和q61s的去糖基化（t106a）人干扰素α2b<400>32cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysalalysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleutyrasnmetileserglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>33<211>165<212>prt<213>人工序列<220><223>包含减毒突变r33a,h57y,e58n和q61s的去糖基化（t106a）人干扰素α2b<400>33cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530alahisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleutyrasnmetileserglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>34<211>165<212>prt<213>人工序列<220><223>包含减毒突变m148a,h57y,e58n和q61s的去糖基化（t106a）人干扰素α2b<400>34cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleutyrasnmetileserglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilealaargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>35<211>165<212>prt<213>人工序列<220><223>包含减毒突变l153a,h57y,e58n和q61s的去糖基化（t106a）人干扰素α2b<400>35cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleutyrasnmetileserglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheseralaserthrasnleuglngluser145150155160leuargserlysglu165<210>36<211>165<212>prt<213>人工序列<220><223>包含减毒突变r144a,h57y,e58n和q61s的去糖基化（t106a）人干扰素α2b<400>36cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleutyrasnmetileserglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalala130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>37<211>165<212>prt<213>人工序列<220><223>包含减毒突变n65a,l80a,y85a,y89a的去糖基化（t106a）人干扰素α2b<400>37cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560alaleupheserthrlysaspserseralaalatrpaspgluthrala65707580leuasplysphealathrgluleualaglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>38<211>165<212>prt<213>人工序列<220><223>包含减毒突变n65a,l80a,y85a,y89a和d114a的去糖基化（t106a）人干扰素α2b<400>38cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560alaleupheserthrlysaspserseralaalatrpaspgluthrala65707580leuasplysphealathrgluleualaglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110glualaserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>39<211>165<212>prt<213>人工序列<220><223>包含减毒突变n65a,l80a,y85a,y89aandl117a的去糖基化（t106a）人干扰素α2b<400>39cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560alaleupheserthrlysaspserseralaalatrpaspgluthrala65707580leuasplysphealathrgluleualaglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserilealaalavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>40<211>165<212>prt<213>人工序列<220><223>包含减毒突变n65a,l80a,y85a,y89aandr120a的去糖基化（t106a）人干扰素α2b<400>40cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560alaleupheserthrlysaspserseralaalatrpaspgluthrala65707580leuasplysphealathrgluleualaglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalalalystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>41<211>165<212>prt<213>人工序列<220><223>包含减毒突变y85a,y89aandr120a的去糖基化（t106a）人干扰素α2b<400>41cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphealathrgluleualaglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalalalystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>42<211>165<212>prt<213>人工序列<220><223>包含减毒突变d114a,r120a的去糖基化（t106a）人干扰素α2b<400>42cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110glualaserileleualavalalalystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>43<211>165<212>prt<213>人工序列<220><223>包含减毒突变l117a,r120a的去糖基化（t106a）人干扰素α2b<400>43cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserilealaalavalalalystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>44<211>165<212>prt<213>人工序列<220><223>包含减毒突变l117a,r120a,k121a的去糖基化（t106a）人干扰素α2b<400>44cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserilealaalavalalaalatyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>45<211>165<212>prt<213>人工序列<220><223>包含减毒突变r120a,k121a的去糖基化（t106a）人干扰素α2b<400>45cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalalaalatyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>46<211>165<212>prt<213>人工序列<220><223>包含减毒突变r120e,k121e的去糖基化（t106a）人干扰素α2b<400>46cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalgluglutyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>47<211>160<212>prt<213>人工序列<220><223>包含残基l161-e165的减毒缺失的去糖基化（t106a）人干扰素α2b<400>47cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalalagluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160<210>48<211>447<212>prt<213>人工序列<220><223>形式为并入铰链稳定化置换s228p的igg4的10.21的vh序列<400>48gluvalglnleuvalglnserglyalagluvallyslysproglyala151015thrvallysilesercyslysvalserglytyrthrphethraspser202530valmetasntrpvalglnglnalaproglylysglyleuglutrpmet354045glytrpileaspproglutyrglyargthraspvalalaglulysphe505560glnglyargvalthrilethralaaspthrserthraspthralatyr65707580metgluleuserserleuargsergluaspthralavaltyrtyrcys859095alaargthrlystyrasnserglytyrglypheprotyrtrpglygln100105110glythrthrvalthrvalserseralaserthrlysglyproserval115120125pheproleualaprocysserargserthrsergluserthralaala130135140leuglycysleuvallysasptyrpheprogluprovalthrvalser145150155160trpasnserglyalaleuthrserglyvalhisthrpheproalaval165170175leuglnserserglyleutyrserleuserservalvalthrvalpro180185190serserserleuglythrlysthrtyrthrcysasnvalasphislys195200205proserasnthrlysvalasplysargvalgluserlystyrglypro210215220procysproprocysproalaproglupheleuglyglyproserval225230235240pheleupheproprolysprolysaspthrleumetileserargthr245250255progluvalthrcysvalvalvalaspvalserglngluaspproglu260265270valglnpheasntrptyrvalaspglyvalgluvalhisasnalalys275280285thrlysproargglugluglnpheasnserthrtyrargvalvalser290295300valleuthrvalleuhisglnasptrpleuasnglylysglutyrlys305310315320cyslysvalserasnlysglyleuproserserileglulysthrile325330335serlysalalysglyglnproarggluproglnvaltyrthrleupro340345350proserglngluglumetthrlysasnglnvalserleuthrcysleu355360365vallysglyphetyrproseraspilealavalglutrpgluserasn370375380glyglnprogluasnasntyrlysthrthrproprovalleuaspser385390395400aspglyserphepheleutyrserargleuthrvalasplysserarg405410415trpglngluglyasnvalphesercysservalmethisglualaleu420425430hisasnhistyrthrglnlysserleuserleuserleuglylys435440445<210>49<211>447<212>prt<213>人工序列<220><223>形式为并入铰链稳定化置换s228p和分别在位置252,254,256置换yte残基的igg4的10.21的vh序列<400>49gluvalglnleuvalglnserglyalagluvallyslysproglyala151015thrvallysilesercyslysvalserglytyrthrphethraspser202530valmetasntrpvalglnglnalaproglylysglyleuglutrpmet354045glytrpileaspproglutyrglyargthraspvalalaglulysphe505560glnglyargvalthrilethralaaspthrserthraspthralatyr65707580metgluleuserserleuargsergluaspthralavaltyrtyrcys859095alaargthrlystyrasnserglytyrglypheprotyrtrpglygln100105110glythrthrvalthrvalserseralaserthrlysglyproserval115120125pheproleualaprocysserargserthrsergluserthralaala130135140leuglycysleuvallysasptyrpheprogluprovalthrvalser145150155160trpasnserglyalaleuthrserglyvalhisthrpheproalaval165170175leuglnserserglyleutyrserleuserservalvalthrvalpro180185190serserserleuglythrlysthrtyrthrcysasnvalasphislys195200205proserasnthrlysvalasplysargvalgluserlystyrglypro210215220procysproprocysproalaproglupheleuglyglyproserval225230235240pheleupheproprolysprolysaspthrleutyrilethrargglu245250255progluvalthrcysvalvalvalaspvalserglngluaspproglu260265270valglnpheasntrptyrvalaspglyvalgluvalhisasnalalys275280285thrlysproargglugluglnpheasnserthrtyrargvalvalser290295300valleuthrvalleuhisglnasptrpleuasnglylysglutyrlys305310315320cyslysvalserasnlysglyleuproserserileglulysthrile325330335serlysalalysglyglnproarggluproglnvaltyrthrleupro340345350proserglngluglumetthrlysasnglnvalserleuthrcysleu355360365vallysglyphetyrproseraspilealavalglutrpgluserasn370375380glyglnprogluasnasntyrlysthrthrproprovalleuaspser385390395400aspglyserphepheleutyrserargleuthrvalasplysserarg405410415trpglngluglyasnvalphesercysservalmethisglualaleu420425430hisasnhistyrthrglnlysserleuserleuserleuglylys435440445<210>50<211>450<212>prt<213>人工序列<220><223>形式为igg1的10.21的vh序列<400>50gluvalglnleuvalglnserglyalagluvallyslysproglyala151015thrvallysilesercyslysvalserglytyrthrphethraspser202530valmetasntrpvalglnglnalaproglylysglyleuglutrpmet354045glytrpileaspproglutyrglyargthraspvalalaglulysphe505560glnglyargvalthrilethralaaspthrserthraspthralatyr65707580metgluleuserserleuargsergluaspthralavaltyrtyrcys859095alaargthrlystyrasnserglytyrglypheprotyrtrpglygln100105110glythrthrvalthrvalserseralaserthrlysglyproserval115120125pheproleualaproserserlysserthrserglyglythralaala130135140leuglycysleuvallysasptyrpheprogluprovalthrvalser145150155160trpasnserglyalaleuthrserglyvalhisthrpheproalaval165170175leuglnserserglyleutyrserleuserservalvalthrvalpro180185190serserserleuglythrglnthrtyrilecysasnvalasnhislys195200205proserasnthrlysvalasplyslysvalgluprolyssercysasp210215220lysthrhisthrcysproprocysproalaprogluleuleuglygly225230235240proservalpheleupheproprolysprolysaspthrleumetile245250255serargthrprogluvalthrcysvalvalvalaspvalserhisglu260265270aspprogluvallyspheasntrptyrvalaspglyvalgluvalhis275280285asnalalysthrlysproargglugluglntyrasnserthrtyrarg290295300valvalservalleuthrvalleuhisglnasptrpleuasnglylys305310315320glutyrlyscyslysvalserasnlysalaleuproalaproileglu325330335lysthrileserlysalalysglyglnproarggluproglnvaltyr340345350thrleuproproserargaspgluleuthrlysasnglnvalserleu355360365thrcysleuvallysglyphetyrproseraspilealavalglutrp370375380gluserasnglyglnprogluasnasntyrlysthrthrproproval385390395400leuaspseraspglyserphepheleutyrserlysleuthrvalasp405410415lysserargtrpglnglnglyasnvalphesercysservalmethis420425430glualaleuhisasnhistyrthrglnlysserleuserleuserpro435440445glylys450<210>51<211>450<212>prt<213>人工序列<220><223>形式为并入置换l238a和g240a以减少效应子功能的igg1的10.21的vh序列<400>51gluvalglnleuvalglnserglyalagluvallyslysproglyala151015thrvallysilesercyslysvalserglytyrthrphethraspser202530valmetasntrpvalglnglnalaproglylysglyleuglutrpmet354045glytrpileaspproglutyrglyargthraspvalalaglulysphe505560glnglyargvalthrilethralaaspthrserthraspthralatyr65707580metgluleuserserleuargsergluaspthralavaltyrtyrcys859095alaargthrlystyrasnserglytyrglypheprotyrtrpglygln100105110glythrthrvalthrvalserseralaserthrlysglyproserval115120125pheproleualaproserserlysserthrserglyglythralaala130135140leuglycysleuvallysasptyrpheprogluprovalthrvalser145150155160trpasnserglyalaleuthrserglyvalhisthrpheproalaval165170175leuglnserserglyleutyrserleuserservalvalthrvalpro180185190serserserleuglythrglnthrtyrilecysasnvalasnhislys195200205proserasnthrlysvalasplyslysvalgluprolyssercysasp210215220lysthrhisthrcysproprocysproalaprogluleualaglyala225230235240proservalpheleupheproprolysprolysaspthrleumetile245250255serargthrprogluvalthrcysvalvalvalaspvalserhisglu260265270aspprogluvallyspheasntrptyrvalaspglyvalgluvalhis275280285asnalalysthrlysproargglugluglntyrasnserthrtyrarg290295300valvalservalleuthrvalleuhisglnasptrpleuasnglylys305310315320glutyrlyscyslysvalserasnlysalaleuproalaproileglu325330335lysthrileserlysalalysglyglnproarggluproglnvaltyr340345350thrleuproproserargaspgluleuthrlysasnglnvalserleu355360365thrcysleuvallysglyphetyrproseraspilealavalglutrp370375380gluserasnglyglnprogluasnasntyrlysthrthrproproval385390395400leuaspseraspglyserphepheleutyrserlysleuthrvalasp405410415lysserargtrpglnglnglyasnvalphesercysservalmethis420425430glualaleuhisasnhistyrthrglnlysserleuserleuserpro435440445glylys450<210>52<211>450<212>prt<213>人工序列<220><223>形式为并入置换l235a和g237a（减少效应子功能）和m255y,s257t,t259e的igg1的10.21的vh序列<400>52gluvalglnleuvalglnserglyalagluvallyslysproglyala151015thrvallysilesercyslysvalserglytyrthrphethraspser202530valmetasntrpvalglnglnalaproglylysglyleuglutrpmet354045glytrpileaspproglutyrglyargthraspvalalaglulysphe505560glnglyargvalthrilethralaaspthrserthraspthralatyr65707580metgluleuserserleuargsergluaspthralavaltyrtyrcys859095alaargthrlystyrasnserglytyrglypheprotyrtrpglygln100105110glythrthrvalthrvalserseralaserthrlysglyproserval115120125pheproleualaproserserlysserthrserglyglythralaala130135140leuglycysleuvallysasptyrpheprogluprovalthrvalser145150155160trpasnserglyalaleuthrserglyvalhisthrpheproalaval165170175leuglnserserglyleutyrserleuserservalvalthrvalpro180185190serserserleuglythrglnthrtyrilecysasnvalasnhislys195200205proserasnthrlysvalasplyslysvalgluprolyssercysasp210215220lysthrhisthrcysproprocysproalaprogluleualaglyala225230235240proservalpheleupheproprolysprolysaspthrleutyrile245250255thrarggluprogluvalthrcysvalvalvalaspvalserhisglu260265270aspprogluvallyspheasntrptyrvalaspglyvalgluvalhis275280285asnalalysthrlysproargglugluglntyrasnserthrtyrarg290295300valvalservalleuthrvalleuhisglnasptrpleuasnglylys305310315320glutyrlyscyslysvalserasnlysalaleuproalaproileglu325330335lysthrileserlysalalysglyglnproarggluproglnvaltyr340345350thrleuproproserargaspgluleuthrlysasnglnvalserleu355360365thrcysleuvallysglyphetyrproseraspilealavalglutrp370375380gluserasnglyglnprogluasnasntyrlysthrthrproproval385390395400leuaspseraspglyserphepheleutyrserlysleuthrvalasp405410415lysserargtrpglnglnglyasnvalphesercysservalmethis420425430glualaleuhisasnhistyrthrglnlysserleuserleuserpro435440445glylys450<210>53<211>446<212>prt<213>人工序列<220><223>形式为igg2主链上的10.21的vh序列<400>53gluvalglnleuvalglnserglyalagluvallyslysproglyala151015thrvallysilesercyslysvalserglytyrthrphethraspser202530valmetasntrpvalglnglnalaproglylysglyleuglutrpmet354045glytrpileaspproglutyrglyargthraspvalalaglulysphe505560glnglyargvalthrilethralaaspthrserthraspthralatyr65707580metgluleuserserleuargsergluaspthralavaltyrtyrcys859095alaargthrlystyrasnserglytyrglypheprotyrtrpglygln100105110glythrthrvalthrvalserseralaserthrlysglyproserval115120125pheproleualaprocysserargserthrsergluserthralaala130135140leuglycysleuvallysasptyrpheprogluprovalthrvalser145150155160trpasnserglyalaleuthrserglyvalhisthrpheproalaval165170175leuglnserserglyleutyrserleuserservalvalthrvalpro180185190serserasnpheglythrglnthrtyrthrcysasnvalasphislys195200205proserasnthrlysvalasplysthrvalgluarglyscyscysval210215220glucysproprocysproalaproprovalalaglyproservalphe225230235240leupheproprolysprolysaspthrleumetileserargthrpro245250255gluvalthrcysvalvalvalaspvalserhisgluaspprogluval260265270glnpheasntrptyrvalaspglyvalgluvalhisasnalalysthr275280285lysproargglugluglnpheasnserthrpheargvalvalserval290295300leuthrvalvalhisglnasptrpleuasnglylysglutyrlyscys305310315320lysvalserasnlysglyleuproalaproileglulysthrileser325330335lysthrlysglyglnproarggluproglnvaltyrthrleupropro340345350serarggluglumetthrlysasnglnvalserleuthrcysleuval355360365lysglyphetyrproseraspilealavalglutrpgluserasngly370375380glnprogluasnasntyrlysthrthrproprometleuaspserasp385390395400glyserphepheleutyrserlysleuthrvalasplysserargtrp405410415glnglnglyasnvalphesercysservalmethisglualaleuhis420425430asnhistyrthrglnlysserleuserleuserproglylys435440445<210>54<211>446<212>prt<213>人工序列<220><223>形式为igg2主链上并入置换m251y,s253t和t255e的10.21的vh序列<400>54gluvalglnleuvalglnserglyalagluvallyslysproglyala151015thrvallysilesercyslysvalserglytyrthrphethraspser202530valmetasntrpvalglnglnalaproglylysglyleuglutrpmet354045glytrpileaspproglutyrglyargthraspvalalaglulysphe505560glnglyargvalthrilethralaaspthrserthraspthralatyr65707580metgluleuserserleuargsergluaspthralavaltyrtyrcys859095alaargthrlystyrasnserglytyrglypheprotyrtrpglygln100105110glythrthrvalthrvalserseralaserthrlysglyproserval115120125pheproleualaprocysserargserthrsergluserthralaala130135140leuglycysleuvallysasptyrpheprogluprovalthrvalser145150155160trpasnserglyalaleuthrserglyvalhisthrpheproalaval165170175leuglnserserglyleutyrserleuserservalvalthrvalpro180185190serserasnpheglythrglnthrtyrthrcysasnvalasphislys195200205proserasnthrlysvalasplysthrvalgluarglyscyscysval210215220glucysproprocysproalaproprovalalaglyproservalphe225230235240leupheproprolysprolysaspthrleutyrilethrargglupro245250255gluvalthrcysvalvalvalaspvalserhisgluaspprogluval260265270glnpheasntrptyrvalaspglyvalgluvalhisasnalalysthr275280285lysproargglugluglnpheasnserthrpheargvalvalserval290295300leuthrvalvalhisglnasptrpleuasnglylysglutyrlyscys305310315320lysvalserasnlysglyleuproalaproileglulysthrileser325330335lysthrlysglyglnproarggluproglnvaltyrthrleupropro340345350serarggluglumetthrlysasnglnvalserleuthrcysleuval355360365lysglyphetyrproseraspilealavalglutrpgluserasngly370375380glnprogluasnasntyrlysthrthrproprometleuaspserasp385390395400glyserphepheleutyrserlysleuthrvalasplysserargtrp405410415glnglnglyasnvalphesercysservalmethisglualaleuhis420425430asnhistyrthrglnlysserleuserleuserproglylys435440445<210>55<211>447<212>prt<213>人工序列<220><223>形式为igg4主链上并入铰链稳定化置换s228p的10.43的vh序列<400>55glnvalglnleuvalglnserglyalagluvallyslysproglyala151015servallysvalsercyslysvalserglytyrthrleuthraspser202530valmetasntrpvalargglnalaproglylysglyleuglutrpmet354045glytrpileaspproglutyrglyargthraspvalalaglnlysphe505560glnglyargvalthrmetthralaaspthrserthraspthralatyr65707580metgluleuserserleuargsergluaspthralavaltyrtyrcys859095alaargthrlystyrasnserglytyrglypheprotyrtrpglygln100105110glythrthrvalthrvalserseralaserthrlysglyproserval115120125pheproleualaprocysserargserthrsergluserthralaala130135140leuglycysleuvallysasptyrpheprogluprovalthrvalser145150155160trpasnserglyalaleuthrserglyvalhisthrpheproalaval165170175leuglnserserglyleutyrserleuserservalvalthrvalpro180185190serserserleuglythrlysthrtyrthrcysasnvalasphislys195200205proserasnthrlysvalasplysargvalgluserlystyrglypro210215220procysproprocysproalaproglupheleuglyglyproserval225230235240pheleupheproprolysprolysaspthrleumetileserargthr245250255progluvalthrcysvalvalvalaspvalserglngluaspproglu260265270valglnpheasntrptyrvalaspglyvalgluvalhisasnalalys275280285thrlysproargglugluglnpheasnserthrtyrargvalvalser290295300valleuthrvalleuhisglnasptrpleuasnglylysglutyrlys305310315320cyslysvalserasnlysglyleuproserserileglulysthrile325330335serlysalalysglyglnproarggluproglnvaltyrthrleupro340345350proserglngluglumetthrlysasnglnvalserleuthrcysleu355360365vallysglyphetyrproseraspilealavalglutrpgluserasn370375380glyglnprogluasnasntyrlysthrthrproprovalleuaspser385390395400aspglyserphepheleutyrserargleuthrvalasplysserarg405410415trpglngluglyasnvalphesercysservalmethisglualaleu420425430hisasnhistyrthrglnlysserleuserleuserleuglylys435440445<210>56<211>447<212>prt<213>人工序列<220><223>形式为并入铰链稳定化置换s228p的igg4主链的10.152的vh序列<400>56gluvalglnleuvalglnserglyalagluvallyslysproglyala151015thrvallysilesercyslysvalserglytyrthrphethraspser202530valmetasntrpvalglnglnhisproglylysglyleuglutrpmet354045glytrpileaspproglutyrglyargthraspvalalaglulysphe505560glnglyargvalthrilethralaaspthrserthraspthralatyr65707580metgluleuserserleuargsergluaspthralavaltyrtyrcys859095alaargthrlystyrasnserglytyrglypheprotyrtrpglygln100105110glythrthrvalthrvalserseralaserthrlysglyproserval115120125pheprol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erargthr245250255progluvalthrcysvalvalvalaspvalserglngluaspproglu260265270valglnpheasntrptyrvalaspglyvalgluvalhisasnalalys275280285thrlysproargglugluglnpheasnserthrtyrargvalvalser290295300valleuthrvalleuhisglnasptrpleuasnglylysglutyrlys305310315320cyslysvalserasnlysglyleuproserserileglulysthrile325330335serlysalalysglyglnproarggluproglnvaltyrthrleupro340345350proserglngluglumetthrlysasnglnvalserleuthrcysleu355360365vallysglyphetyrproseraspilealavalglutrpgluserasn370375380glyglnprogluasnasntyrlysthrthrproprovalleuaspser385390395400aspglyserphepheleutyrserargleuthrvalasplysserarg405410415trpglngluglyasnvalphesercysservalmethisglualaleu420425430hisasnhistyrthrglnlysserleuserleuserleuglylyscys435440445aspleuproglnthrhisserleuglyserargargthrleumetleu450455460leualaglnmetargargileserleuphesercysleulysasparg465470475480hisasppheglypheproglngluglupheglyasnglnpheglnlys485490495alagluthrileprovalleuhisglumetileglnglnilepheasn500505510leupheserthrlysaspserseralaalatrpaspgluthrleuleu515520525asplysphetyrthrgluleutyrglnglnleuasnaspleugluala530535540cysvalileglnglyvalglyvalalagluthrproleumetlysglu545550555560aspserileleualavalarglystyrpheglnargilethrleutyr565570575leulysglulyslystyrserprocysalatrpgluvalvalargasp580585590gluilemetargserpheserleuserthrasnleuglngluserleu595600605argserlysglu610<210>88<211>495<212>dna<213>人工序列<220><223>ifnalpha-2-beta<400>88tgtgatctgcctcaaacccacagcctgggtagcaggaggaccttgatgctcctggcacag60atgaggagaatctctcttttctcctgcttgaaggacagacatgactttggatttccccag120gaggagtttggcaaccagttccaaaaggctgaaaccatccctgtcctccatgagatgatc180cagcagatcttcaatctcttcagcacaaaggactcatctgctgcttgggatgagaccctc240ctagacaaattctacactgaactctaccagcagctgaatgacctggaagcctgtgtgata300cagggggtgggggtgacagagactcccctgatgaaggaggactccattctggctgtgagg360aaatacttccaaagaatcactctctatctgaaagagaagaaatacagcccttgtgcctgg420gaggttgtcagagcagaaatcatgagatctttttctttgtcaacaaacttgcaagaaagt480ttaagaagtaaggaa495<210>89<211>1341<212>dna<213>人工序列<220><223>作为并入s228p的igg2的10.21多核苷酸<400>89gaagtgcagctggtgcagtctggcgccgaagtgaagaaacccggcgctaccgtgaagatc60tcctgcaaggtgtccggctacaccttcaccgactccgtgatgaactgggtgcagcaggcc120cctggcaagggcctggaatggatgggctggatcgaccccgagtacggcagaaccgacgtg180gccgagaagttccagggcagagtgaccatcaccgccgacacctccaccgacaccgcctac240atggaactgtcctccctgcggagcgaggacaccgccgtgtactactgcgcccggaccaag300tacaactccggctacggcttcccctactggggccagggcaccaccgtgaccgtgtcctcc360gcctccaccaagggcccctccgtgttccctctggccccttgctcccggtccacctccgag420tctaccgccgctctgggctgcctggtcaaggactacttccccgagcctgtgacagtgtcc480tggaactctggcgccctgacctccggcgtgcacaccttccctgccgtgctgcagtcctcc540ggcctgtactccctgtcctccgtcgtgaccgtgccttccagctccctgggcaccaagacc600tacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagcgggtggaatct660aagtacggccctccctgccccccctgccctgcccctgaatttctgggcggaccttccgtg720ttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacc780tgcgtggtggtggacgtgtcccaagaggaccccgaggtgcagttcaattggtacgtggac840ggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaactccacctac900cgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagaatacaag960tgcaaagtctccaacaagggcctgccctccagcatcgaaaagaccatctccaaggccaag1020ggacagccccgcgagccccaggtgtacaccctgccccctagccaagaggaaatgaccaag1080aaccaggtgtccctgacctgcctcgtgaagggcttctacccctccgatatcgccgtggaa1140tgggagtccaacggccagcccgagaacaactacaagaccaccccccctgtgctggactcc1200gacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtggcaagagggc1260aacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtcc1320ctgtccctgagcctgggcaag1341<210>90<211>1341<212>dna<213>人工序列<220><223>并入s228p的igg4的10.43的多核苷酸序列<400>90caggtgcagctggtgcagtctggcgccgaagtgaagaaacctggcgcctccgtgaaggtg60tcctgcaaggtgtccggctacaccctgaccgactccgtgatgaactgggtccgacaggcc120cctggcaagggcctggaatggatgggctggatcgaccccgagtacggcagaaccgacgtg180gcccagaaattccagggcagagtgaccatgaccgccgacacctccaccgacaccgcctac240atggaactgtcctccctgcggagcgaggacaccgccgtgtactactgcgcccggaccaag300tacaactccggctacggcttcccctactggggccagggcaccaccgtgaccgtgtcctct360gcttccaccaagggcccctccgtgttccctctggccccttgctcccggtccacctccgag420tctaccgccgctctgggctgcctggtcaaggactacttccccgagcctgtgacagtgtcc480tggaactctggcgccctgacctccggcgtgcacaccttccctgccgtgctgcagtcctcc540ggcctgtactccctgtcctccgtcgtgaccgtgccttccagctccctgggcaccaagacc600tacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagcgggtggaatct660aagtacggccctccctgccccccctgccctgcccctgaatttctgggcggaccttccgtg720ttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacc780tgcgtggtggtggacgtgtcccaagaggaccccgaggtgcagttcaattggtacgtggac840ggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaactccacctac900cgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagaatacaag960tgcaaagtctccaacaagggcctgccctccagcatcgaaaagaccatctccaaggccaag1020ggacagccccgcgagccccaggtgtacaccctgccccctagccaagaggaaatgaccaag1080aaccaggtgtccctgacctgcctcgtgaagggcttctacccctccgatatcgccgtggaa1140tgggagtccaacggccagcccgagaacaactacaagaccaccccccctgtgctggactcc1200gacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtggcaagagggc1260aacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtcc1320ctgtccctgagcctgggcaag1341<210>91<211>1344<212>dna<213>人工序列<220><223>形式为并入s228p的igg4的2.12的多核苷酸<400>91cagctgcagctgcaggaatctggccctggcctcgtgaagcccagcgagacactgagcctg60acctgtaccgtgtccggcggcagcatcagcagcagctcctactactggtcctggatcaga120cagcaccccggcaagggcctggaatggatcggctacatctactacagcggcagcaccaac180tacaaccccagcctgaagtccagagtgaccatcagcgtggacaccagcaagaaccagttc240tccctgaagctgagcagcgtgacagccgccgataccgccgtgtactactgtgccagagtg300ggcggagctggcggctggcctctggatgtgtggggacagggcaccaccgtgacagtgtcc360tcagctagcaccaagggccccagcgtgttccctctggccccttgtagcagaagcaccagc420gagtctacagccgccctgggctgcctcgtgaaggactactttcccgagcccgtcaccgtg480tcctggaactctggcgctctgacaagcggcgtgcacacctttccagccgtgctgcagagc540agcggcctgtactctctgagcagcgtcgtgaccgtgcccagctctagcctgggcaccaag600acctacacctgtaacgtggaccacaagcccagcaacaccaaggtggacaagcgggtggaa660tctaagtacggccctccctgccctccttgcccagcccctgaatttctgggcggaccctcc720gtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaagtg780acctgcgtggtggtggatgtgtcccaggaagatcccgaggtgcagttcaattggtacgtg840gacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaacagcacc900taccgggtggtgtccgtgctgacagtgctgcaccaggactggctgaacggcaaagagtac960aagtgcaaggtgtccaacaagggcctgcctagcagcatcgagaaaaccatcagcaaggcc1020aagggccagccccgcgaacctcaggtgtacacactgccccctagccaggaagagatgacc1080aagaaccaggtgtccctgacctgtctcgtgaaaggcttctacccctccgatatcgccgtg1140gaatgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggac1200agcgacggctcattcttcctgtacagcagactgaccgtggacaagagccggtggcaggaa1260ggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaag1320tccctgtccctgtctctgggcaag1344<210>92<211>1341<212>dna<213>人工序列<220><223>形式为并入s228p的igg4的r10a2的多核苷酸<400>92gaagtccagctgcagcagtctggccccgaagtgggcagacctggctcctccgtgaagatc60tcctgcaaggcctccggctacaccttcaccgactccgtgatgaactgggtcaagcagtct120cccggccagggcctggaatggatcggatggatcgaccccgagtacggcagaaccgacgtg180gccgagaagttcaagaagaaggccaccctgaccgccgactcctccagctccaccgcctac240atctacctgtccggcctgacctccgaggacaccgccacctacttttgcgcccggaccaag300tacaacagcggctacggcttcccctactggggacagggctctctcgtgacagtgtcctca360gcctccaccaagggcccctccgtgttccctctggccccttgctcccggtccacctccgag420tctaccgccgctctgggctgcctggtgaaagactacttccccgagcctgtgaccgtgagc480tggaactctggcgccctgacctccggcgtgcacaccttccctgccgtgctgcagtcctcc540ggcctgtactccctgtcctccgtggtgacagtgccctcctccagcctgggcaccaagacc600tacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagcgggtggaatct660aagtacggccctccctgccccccctgccctgcccctgaatttctgggcggaccttccgtg720tttctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacc780tgcgtggtggtggacgtgtcccaggaagatccagaggtgcagttcaattggtacgtggac840ggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaactccacctac900cgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaag960tgcaaggtgtccaacaagggcctgccctccagcatcgaaaagaccatctccaaggccaag1020ggccagccccgcgagccccaggtgtacaccctgccccctagccaggaagagatgaccaag1080aaccaggtgtccctgacctgtctggtgaaaggcttctacccctccgacattgccgtggaa1140tgggagtccaacggccagcccgagaacaactacaagaccaccccccctgtgctggactcc1200gacggctccttcttcctgtactctcggctgacagtggataagtcccggtggcaggaaggc1260aacgtgttctcctgcagcgtgatgcacgaggccctgcacaaccactatacccagaagtcc1320ctgtccctgagcctgggcaag1341<210>93<211>642<212>dna<213>人工序列<220><223>r10a2vk的多核苷酸序列<400>93gacatcgtgatgacccagagccccacctccatctccatcagcgtgggcgagcgcgtgacc60atgaactgcaaggcctcccagaacgtggacagcgacgtggactggtatcagcagaaaacc120ggccagtcccccaagctgctgatctacaaggccagcaacagatacaccggcgtgcccgac180aggttcaccggctctggctctggaaccgacttcaccttcaccatcagcaacatgcaggcc240gaggatctggccgtgtactactgtatgcagtccaacacccacccccggaccttcggcgga300ggcaccaaactggaactgaagcggaccgtggccgctcccagcgtgttcatcttccccccc360agcgacgagcagctgaagagcggcaccgccagcgtggtctgcctgctgaacaacttctac420ccccgggaggccaaggtgcagtggaaggtggacaacgccctgcagtctggcaacagccag480gaaagcgtcaccgagcaggacagcaaggatagcacctacagcctgagcagcaccctgacc540ctgagcaaggccgactacgagaagcacaaggtgtacgcctgcgaggtcacccaccagggc600ctgtctagccccgtcaccaagagcttcaaccggggcgagtgc642<210>94<211>642<212>dna<213>人工序列<220><223>用于产生抗体10.21和抗体10.43的vk的多核苷酸序列<400>94gacatccagatgacccagtccccctccagcctgtccgcttccgtgggcgacagagtgacc60atcacatgcaaggcctcccagaacgtggactccgacgtggactggtatcagcagaagccc120ggcaaggcccccaagctgctgatctacaaggcctccaacgactacaccggcgtgccctcc180agattctccggctccggctctggcaccgacttcaccttcaccatctccagcctgcagccc240gaggatatcgccacctactactgcatgcagagcaacacccacccccggaccttcggcgga300ggcaccaaggtggaaatcaagcggaccgtggccgctccctccgtgttcatcttcccaccc360tccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctac420ccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaa480gagtccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgacc540ctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggc600ctgtccagccccgtgaccaagtccttcaaccggggcgagtgc642<210>95<211>1341<212>dna<213>人工序列<220><223>形式为并入s228p的igg4的10.152的多核苷酸序列<400>95gaagtgcagctggtgcagtctggcgccgaagtgaagaaacccggcgctaccgtgaagatc60tcctgcaaggtgtccggctacaccttcaccgactccgtgatgaactgggtgcagcagcat120cccggcaagggcctggaatggatgggctggatcgaccccgagtacggcagaaccgacgtg180gccgagaagttccagggcagagtgaccatcaccgccgacacctccaccgacaccgcctac240atggaactgtcctccctgcggagcgaggacaccgccgtgtactactgcgcccggaccaag300tacaactccggctacggcttcccctactggggccagggcaccaccgtgaccgtgtcctct360gcctccaccaagggcccctccgtgttccctctggccccttgctcccggtccacctccgag420tctaccgccgctctgggctgcctggtcaaggactacttccccgagcctgtgacagtgtcc480tggaactctggcgccctgacctccggcgtgcacaccttccctgccgtgctgcagtcctcc540ggcctgtactccctgtcctccgtcgtgaccgtgccttccagctccctgggcaccaagacc600tacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagcgggtggaatct660aagtacggccctccctgccccccctgccctgcccctgaatttctgggcggaccttccgtg720ttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacc780tgcgtggtggtggacgtgtcccaagaggaccccgaggtgcagttcaattggtacgtggac840ggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaactccacctac900cgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagaatacaag960tgcaaagtctccaacaagggcctgccctccagcatcgaaaagaccatctccaaggccaag1020ggacagccccgcgagccccaggtgtacaccctgccccctagccaagaggaaatgaccaag1080aaccaggtgtccctgacctgcctcgtgaagggcttctacccctccgatatcgccgtggaa1140tgggagtccaacggccagcccgagaacaactacaagaccaccccccctgtgctggactcc1200gacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtggcaagagggc1260aacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtcc1320ctgtccctgagcctgggcaag1341<210>96<211>214<212>prt<213>人工序列<220><223>10.152轻链的氨基酸序列<400>96aspileglnmetthrglnserproserserleuseralaservalgly151015aspargvalthrilethrcysglnalaserglnasnvalaspserasp202530valasptrptyrglnglnlysproglylysalaprolysleuleuile354045tyrlysalaserasnasptyrthrglyvalproserargphesergly505560serglyserglythraspphethrphethrileserserleuglnpro65707580gluaspilealathrtyrtyrcysalaglnserasnthrhisproarg859095thrpheglyglyglythrlysvalgluilelysargthrvalalaala100105110proservalpheilepheproproseraspgluglnleulyssergly115120125thralaservalvalcysleuleuasnasnphetyrproarggluala130135140lysvalglntrplysvalaspasnalaleuglnserglyasnsergln145150155160gluservalthrgluglnaspserlysaspserthrtyrserleuser165170175serthrleuthrleuserlysalaasptyrglulyshislysvaltyr180185190alacysgluvalthrhisglnglyleuserserprovalthrlysser195200205pheasnargglyglucys210<210>97<211>642<212>dna<213>人工序列<220><223>10.152轻链的多核苷酸序列<400>97gacatccagatgactcagagcccgtcctcgctttcggcttccgtcggcgaccgcgtgacc60atcacttgtcaggcgtcgcagaacgtcgattccgacgtggactggtaccaacagaagccg120gggaaagcgcccaagctgctgatctacaaggcctccaatgattacactggagtgcctagc180cggttcagcggatcagggtcgggaacggacttcacttttaccatctcaagcctccaacca240gaagatattgccacctattactgcgcacaatcaaacacccacccgagaaccttcggcgga300ggaaccaaggtggagatcaaacgtacggtggcggcgcccagcgtgttcatcttcccaccc360agcgacgagcagctgaagtccggcacagccagcgtggtgtgcctgctgaacaacttctac420ccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagagcggcaacagccag480gaaagcgtgaccgagcaggacagcaaggactccacctacagcctgagcagcaccctgacc540ctgagcaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggc600ctgtccagccccgtgaccaagagcttcaaccggggcgagtgc642<210>98<211>666<212>dna<213>人工序列<220><223>2.12轻链的多核苷酸序列<400>98caggccgtgctgacccagcctgcctccctgtctgcctctcctggcgagtccgccagactg60acctgcaccctgccctccgacatcaacgtgcggtactacaacatctactggtatcagcag120aagcccggcagcccccccagatacctgctgtactactactccgactcccacaagggccag180ggctccggcgtgccctccagattctccggctccaaggacgtgtccaccaactccggcatc240ctgctgatctccggcctgcagtccgaggacattgccacctactactgcatgacttggagc300agcaacggcagcggcgtgttcggcggaggcacccagctgaccgtcctaggtcagcccaag360gccgctcccagcgtgaccctgttccccccaagcagcgaggaactgcaggccaacaaggcc420accctggtgtgcctgatcagcgacttctaccctggggccgtgaccgtggcctggaaggcc480gatagcagccctgtgaaggccggcgtggaaaccaccaccccctccaagcagagcaacaac540aaatacgccgccagcagctacctgtccctgacccccgagcagtggaagtcccaccggtcc600tacagctgccaggtgacacacgagggcagcaccgtggaaaagaccgtggcccccaccgag660tgcagc666<210>99<211>642<212>dna<213>人工序列<220><223>抗cd138κ轻链的多核苷酸序列<400>99gacatccagatgacccagagcacatcctctctgagcgcctccctgggcgatcgcgtgact60atcagttgcagcgcttcccaagggattaacaattacctcaactggtaccagcagaagccc120gacggaaccgtcgagctgctcatctattacacatctacgctgcaaagcggcgtgccttcc180aggttctcagggagcggttccggaactgattactctctgaccattagcaatctcgaacca240gaagacatcggcacatattactgtcagcagtactccaagctgccccggacttttggggga300ggcaccaaactggagatcaagcgtacggtggcggcgcccagcgtgttcatcttcccaccc360agcgacgagcagctgaagtccggcacagccagcgtggtgtgcctgctgaacaacttctac420ccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagagcggcaacagccag480gaaagcgtgaccgagcaggacagcaaggactccacctacagcctgagcagcaccctgacc540ctgagcaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggc600ctgtccagccccgtgaccaagagcttcaaccggggcgagtgc642<210>100<211>1347<212>dna<213>人工序列<220><223>抗cd138igg4(s228p)的多核苷酸序列<400>100caggtgcagctccaacagagcggctccgaactgatgatgcctggggcctctgtcaagatt60tcctgcaaagctaccggctacacattcagcaactattggattgagtgggtgaagcagcgc120ccagggcacggtctggagtggatcggagagatcctgccaggcaccgggaggactatttac180aatgaaaagtttaaaggaaaggccacattcaccgcagacatctctagcaacactgttcaa240atgcagctctcctctctgacctccgaggatagcgccgtgtattactgtgctcggagagac300tactatggcaatttttactatgctatggattactggggacagggcacatctgtgaccgtc360agctccgctagcaccaagggccccagcgtgttccccctggccccttgtagcagaagcacc420agcgagagcacagccgccctgggctgcctggtgaaagactacttccccgagcccgtcacc480gtgtcctggaacagcggagccctgaccagcggcgtgcacacctttccagccgtgctgcag540agcagcggcctgtacagcctgagcagcgtggtgacagtgccctccagcagcctgggcacc600aagacctacacctgtaacgtggaccacaagcccagcaacaccaaggtggacaagcgggtg660gaatctaagtacggcccaccctgccccccctgccctgcccctgaatttctgggcggaccc720tccgtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaa780gtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtccagttcaattggtac840gtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaacagc900acctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagag960tacaagtgcaaagtctccaacaagggcctgcccagctccatcgagaaaaccatcagcaag1020gccaagggccagccccgcgagcctcaggtgtacacactgccccccagccaggaagagatg1080accaagaaccaggtgtccctgacctgtctggtgaaaggcttctaccccagcgatatcgcc1140gtggaatgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctg1200gacagcgacggcagcttcttcctgtactcccggctgaccgtggacaagagccggtggcag1260gaaggcaacgtcttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccag1320aagtccctgagcctgagcctgggcaag1347<210>101<211>1341<212>dna<213>人工序列<220><223>抗hlaigg4(s228p)的多核苷酸序列<400>101caggtccaactcaagcagtccggtccggggctggtccagccatctcaatcactgtctctt60acttgcaccgtgtccggattcagcctgacctcatacggagtgcattgggtgcggcagcct120cccgggaaaggactggagtggctcggagtcatttggtccggtggatcaactgactacaat180gccgctttcatcagcaggctgagcattcggaaggacaactctaagagccaagtgttcttc240aagatgaactcactccaggccgatgacaccgccatctactattgtgccagaaccttcacc300accagcacctctgcatggtttgcatactggggccagggcactcttgtgaccgtgtcagct360gctagcaccaagggccccagcgtgttccccctggccccttgtagcagaagcaccagcgag420agcacagccgccctgggctgcctggtgaaagactacttccccgagcccgtcaccgtgtcc480tggaacagcggagccctgaccagcggcgtgcacacctttccagccgtgctgcagagcagc540ggcctgtacagcctgagcagcgtggtgacagtgccctccagcagcctgggcaccaagacc600tacacctgtaacgtggaccacaagcccagcaacaccaaggtggacaagcgggtggaatct660aagtacggcccaccctgccccccctgccctgcccctgaatttctgggcggaccctccgtg720ttcctgttccccccaaagcccaaggacaccctgatgatcagccggacccccgaagtgacc780tgcgtggtggtggacgtgtcccaggaagatcccgaggtccagttcaattggtacgtggac840ggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaacagcacctac900cgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaag960tgcaaagtctccaacaagggcctgcccagctccatcgagaaaaccatcagcaaggccaag1020ggccagccccgcgagcctcaggtgtacacactgccccccagccaggaagagatgaccaag1080aaccaggtgtccctgacctgtctggtgaaaggcttctaccccagcgatatcgccgtggaa1140tgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagc1200gacggcagcttcttcctgtactcccggctgaccgtggacaagagccggtggcaggaaggc1260aacgtcttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagtcc1320ctgagcctgagcctgggcaag1341<210>102<211>645<212>dna<213>人工序列<220><223>抗hlavk的多核苷酸序列<400>102tctatcgtcatgacccagaccccgaagttcctcctggtgtcagctggtgatcgggtgacc60atcacttgtaaggcctctcagtctgtctcaaacgacgtcgcatggtaccaacaaaagcct120gggcagtcacctaagcttctgatctactatgcttccaatcgctacaccggcgtgcccgac180aggttcaccggatcagggtacggaaccgacttcacctttactatttccaccgtgcaggcc240gaggacctcgccgtgtatttctgccagcaagattacagcagcccaccctggactttcggt300ggaggaactaaactggaaattagacgtacggtggcggcgcccagcgtgttcatcttccca360cccagcgacgagcagctgaagtccggcacagccagcgtggtgtgcctgctgaacaacttc420tacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagagcggcaacagc480caggaaagcgtgaccgagcaggacagcaaggactccacctacagcctgagcagcaccctg540accctgagcaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccag600ggcctgtccagccccgtgaccaagagcttcaaccggggcgagtgc645<210>103<211>1203<212>dna<213>人工序列<220><223>opg-fc的多核苷酸序列<400>103gaaacctttccgccgaaatatctgcattatgatgaagaaaccagccatcagctgctgtgc60gataaatgcccgccgggcacctatctgaaacagcattgcaccgcgaaatggaaaaccgtg120tgcgcgccgtgcccggatcattattataccgatagctggcataccagcgatgaatgcctg180tattgcagcccggtgtgcaaagaactgcagtatgtgaaacaggaatgcaaccgcacccat240aaccgcgtgtgcgaatgcaaagaaggccgctatctggaaggcgaattttgcctgaaacat300cgcagctgcccgccgggctttggcgtggtgcaggcgggcaccccggaacgcaacaccgtg360tgcaaacgctgcccggatggcttttttagcaacgaaaccagcagcaaagcgccgtgccgc420aaacataccaactgcagcgtgtttggcctgctgctgacccagaaaggcaacgcgacccat480gataacatttgcagcggcaacagcgaaagcacccagaaagaacgcaaatgctgcgtggaa540tgcccgccgtgcccggcgccgccggtggcgggcccgagcgtgtttctgtttccgccgaaa600ccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtg660agccatgaagatccggaagtgcagtttaactggtatgtggatggcgtggaagtgcataac720gcgaaaaccaaaccgcgcgaagaacagtttaacagcacctttcgcgtggtgagcgtgctg780accgtggtgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaa840ggcctgccggcgccgattgaaaaaaccattagcaaaaccaaaggccagccgcgcgaaccg900caggtgtataccctgccgccgagccgcgaagaaatgaccaaaaaccaggtgagcctgacc960tgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccag1020ccggaaaacaactataaaaccaccccgccgatgctggatagcgatggcagcttttttctg1080tatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagc1140gtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggc1200aaa1203<210>104<211>1350<212>dna<213>人工序列<220><223>形式为igg1的10.21的多核苷酸序列<400>104gaagtgcagctggtgcagtctggcgccgaagtgaagaaacctggcgccaccgtgaagatc60agctgcaaggtgtccggctacaccttcaccgacagcgtgatgaactgggtgcagcaggcc120cctggcaagggcctggaatggatgggatggatcgaccccgagtacggcagaaccgacgtg180gccgagaagttccagggcagagtgaccatcaccgccgacaccagcaccgacaccgcctac240atggaactgagcagcctgcggagcgaggacaccgccgtgtactactgtgcccggaccaag300tacaacagcggctacggcttcccctactggggccagggcacaaccgtgacagtgtcctca360gctagcaccaagggacccagcgtgttccctctggcccctagcagcaagagcacatctggc420ggaacagccgccctgggctgcctcgtgaaggactactttcccgagcctgtcaccgtgtct480tggaactctggcgccctgacaagcggcgtgcacacctttccagccgtgctgcagagcagc540ggcctgtactctctgagcagcgtcgtgacagtgcccagctctagcctgggcacccagacc600tacatctgcaacgtgaaccacaagcccagcaacaccaaggtggacaagaaggtggaaccc660aagagctgcgacaagacccacacctgtcccccttgtcctgcccccgaactgctgggaggc720ccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatcagccggaccccc780gaagtgacctgcgtggtggtggatgtgtcccacgaggaccctgaagtgaagttcaattgg840tacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagtacaac900agcacctaccgggtggtgtccgtgctgacagtgctgcaccaggactggctgaacggcaaa960gagtacaagtgcaaggtgtccaacaaggccctgcctgcccccatcgagaaaaccatcagc1020aaggccaagggccagccccgcgaaccccaggtgtacacactgcccccaagcagggacgag1080ctgaccaagaaccaggtgtccctgacctgtctcgtgaaaggcttctacccctccgatatc1140gccgtggaatgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtg1200ctggacagcgacggctcattcttcctgtacagcaagctgaccgtggacaagtcccggtgg1260cagcagggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacc1320cagaagtccctgagcctgagccccggcaag1350<210>105<211>1338<212>dna<213>人工序列<220><223>10.21igg2的多核苷酸序列<400>105gaagtgcagctggtgcagtctggcgccgaagtgaagaaacctggcgccaccgtgaagatc60agctgcaaggtgtccggctacaccttcaccgacagcgtgatgaactgggtgcagcaggcc120cctggcaagggcctggaatggatgggatggatcgaccccgagtacggcagaaccgacgtg180gccgagaagttccagggcagagtgaccatcaccgccgacaccagcaccgacaccgcctac240atggaactgagcagcctgcggagcgaggacaccgccgtgtactactgtgcccggaccaag300tacaacagcggctacggcttcccctactggggccagggcacaaccgtgacagtgtcctca360gccagcaccaagggccccagcgtgttccccctggccccctgcagcagaagcaccagcgag420agcacagccgccctgggctgcctggtgaaggactacttccccgagcccgtgacagtgagc480tggaacagcggagccctgacctccggtgtacacaccttccccgccgtgctgcagagcagc540ggcctgtactccctgagcagcgtggtgaccgtgcccagcagcaacttcggcacccagacc600tacacctgcaacgtggaccacaagcccagcaacaccaaggtggacaagaccgtggagagg660aagtgctgcgtggagtgccccccctgcccagcccccccagtggccggaccctccgtgttt720ctgttcccccccaagcccaaggacaccctgatgatcagcaggacccccgaggtgacctgc780gtggtggtggacgtgagccacgaggatccggaggtgcagttcaactggtacgtggacggc840gtggaggtgcacaacgccaagaccaagcccagagaggagcagtttaacagcaccttcagg900gtggtgtccgtgctgaccgtggtgcaccaggactggctgaacggcaaggaatacaagtgc960aaggtctccaacaagggcctgccagcccccatcgagaaaaccatcagcaagaccaagggc1020cagccacgggagccccaagtgtataccctgccccccagccgggaggagatgaccaagaac1080caggtgtccctgacctgtctggtgaagggcttctaccccagcgacatcgccgtggagtgg1140gagagcaacggccagcccgagaacaactacaagaccaccccccccatgctggacagcgac1200ggcagcttcttcctgtactccaagctgacagtggacaagtccaggtggcagcagggcaac1260gtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagagcctg1320agcctgtcccccggcaag1338<210>106<211>1491<212>dna<213>人工序列<220><223>10.21igg3的多核苷酸序列<400>106gaagtgcagctggtgcagtctggcgccgaagtgaagaaacctggcgccaccgtgaagatc60agctgcaaggtgtccggctacaccttcaccgacagcgtgatgaactgggtgcagcaggcc120cctggcaagggcctggaatggatgggatggatcgaccccgagtacggcagaaccgacgtg180gccgagaagttccagggcagagtgaccatcaccgccgacaccagcaccgacaccgcctac240atggaactgagcagcctgcggagcgaggacaccgccgtgtactactgtgcccggaccaag300tacaacagcggctacggcttcccctactggggccagggcacaaccgtgacagtgtcctca360gcgagcaccaaaggcccgagcgtgtttccgctggcgccgtgcagccgcagcaccagcggc420ggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagc480tggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagc540ggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacc600tatacctgcaacgtgaaccataaaccgagcaacaccaaagtggataaacgcgtggaactg660aaaaccccgctgggcgataccacccatacctgcccgcgctgcccggaaccgaaaagctgc720gataccccgccgccgtgcccgcgctgcccggaaccgaaaagctgcgataccccgccgccg780tgcccgcgctgcccggaaccgaaaagctgcgataccccgccgccgtgcccgcgctgcccg840gcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagatacc900ctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagat960ccggaagtgcagtttaaatggtatgtggatggcgtggaagtgcataacgcgaaaaccaaa1020ccgcgcgaagaacagtttaacagcacctttcgcgtggtgagcgtgctgaccgtgctgcat1080caggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcg1140ccgattgaaaaaaccattagcaaaaccaaaggccagccgcgcgaaccgcaggtgtatacc1200ctgccgccgagccgcgaagaaatgaccaaaaaccaggtgagcctgacctgcctggtgaaa1260ggcttttatccgagcgatattgcggtggaatgggaaagcagcggccagccggaaaacaac1320tataacaccaccccgccgatgctggatagcgatggcagcttttttctgtatagcaaactg1380accgtggataaaagccgctggcagcagggcaacatttttagctgcagcgtgatgcatgaa1440gcgctgcataaccgctttacccagaaaagcctgagcctgagcccgggcaaa1491<210>107<211>165<212>prt<213>人工序列<220><223>包含减毒突变a145x的糖基化（t106t）人干扰素α2b，其中x可以是选自d,e,g,h,i,k,l,m,n,q,r,s,t,v,y的任意氨基酸<220><221>变体<222>(145)..(145)<223>xaa可以是选自d,e,g,h,i,k,l,m,n,q,r,s,t,v,y的任意氨基酸<400>107cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalthrgluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140xaagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>108<211>165<212>prt<213>人工序列<220><223>包含减毒突变r144x的糖基化（t106t）人干扰素α2b，其中x可以是选自a,d,e,g,h,i,k,l,n,q,s,t,v,y的任意氨基酸<220><221>变体<222>(144)..(144)<223>xaa可以是选自a,d,e,g,h,i,k,l,n,q,s,t,v,y的任意氨基酸<400>108cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalthrgluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalxaa130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>109<211>165<212>prt<213>人工序列<220><223>包含减毒突变r33x的糖基化（t106t）人干扰素α2b，其中x可以是选自k,a,q的任意氨基酸<220><221>变体<222>(33)..(33)<223>xaa可以是选自k,a,q的任意氨基酸<400>109cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530xaahisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalthrgluthrproleumetlys100105110gluaspserileleualavalarglystyrpheglnargilethrleu115120125tyrleulysglulyslystyrserprocysalatrpgluvalvalarg130135140alagluilemetargserpheserleuserthrasnleuglngluser145150155160leuargserlysglu165<210>110<211>164<212>prt<213>人工序列<220><223>包含位置106的氨基酸缺失且包含减毒突变a144x的人干扰素α2b，其中x可以是选自d,e,g,h,i,k,l,m,n,q,r,s,t,v,y的任意氨基酸<220><221>变体<222>(144)..(144)<223>xaa可以是选自d,e,g,h,i,k,l,m,n,q,r,s,t,v,y的任意氨基酸<400>110cysaspleuproglnthrhisserleuglyserargargthrleumet151015leuleualaglnmetargargileserleuphesercysleulysasp202530arghisasppheglypheproglngluglupheglyasnglnphegln354045lysalagluthrileprovalleuhisglumetileglnglnilephe505560asnleupheserthrlysaspserseralaalatrpaspgluthrleu65707580leuasplysphetyrthrgluleutyrglnglnleuasnaspleuglu859095alacysvalileglnglyvalglyvalgluthrproleumetlysglu100105110aspserileleualavalarglystyrpheglnargilethrleutyr115120125leulysglulyslystyrserprocysalatrpgluvalvalargxaa130135140gluilemetargserpheserleuserthrasnleuglngluserleu145150155160argserlysglu<210>111<211>450<212>prt<213>人工序列<220><223>形式为并入置换m255y,s257t,t259e的igg1的10.21的vh序列<400>111gluvalglnleuvalglnserglyalagluvallyslysproglyala151015thrvallysilesercyslysvalserglytyrthrphethraspser202530valmetasntrpvalglnglnalaproglylysglyleuglutrpmet354045glytrpileaspproglutyrglyargthraspvalalaglulysphe505560glnglyargvalthrilethralaaspthrserthraspthralatyr65707580metgluleuserserleuargsergluaspthralavaltyrtyrcys859095alaargthrlystyrasnserglytyrglypheprotyrtrpglygln100105110glythrthrvalthrvalserseralaserthrlysglyproserval115120125pheproleualaproserserlysserthrserglyglythralaala130135140leuglycysleuvallysasptyrpheprogluprovalthrvalser145150155160trpasnserglyalaleuthrserglyvalhisthrpheproalaval165170175leuglnserserglyleutyrserleuserservalvalthrvalpro180185190serserserleuglythrglnthrtyrilecysasnvalasnhislys195200205proserasnthrlysvalasplyslysvalgluprolyssercysasp210215220lysthrhisthrcysproprocysproalaprogluleuleuglygly225230235240proservalpheleupheproprolysprolysaspthrleutyrile245250255thrarggluprogluvalthrcysvalvalvalaspvalserhisglu260265270aspprogluvallyspheasntrptyrvalaspglyvalgluvalhis275280285asnalalysthrlysproargglugluglntyrasnserthrtyrarg290295300valvalservalleuthrvalleuhisglnasptrpleuasnglylys305310315320glutyrlyscyslysvalserasnlysalaleuproalaproileglu325330335lysthrileserlysalalysglyglnproarggluproglnvaltyr340345350thrleuproproserargaspgluleuthrlysasnglnvalserleu355360365thrcysleuvallysglyphetyrproseraspilealavalglutrp370375380gluserasnglyglnprogluasnasntyrlysthrthrproproval385390395400leuaspseraspglyserphepheleutyrserlysleuthrvalasp405410415lysserargtrpglnglnglyasnvalphesercysservalmethis420425430glualaleuhisasnhistyrthrglnlysserleuserleuserpro435440445glylys450<210>112<211>446<212>prt<213>人工序列<220><223>形式为igg2主链上并入置换a330s和p331s的10.21的vh序列<400>112gluvalglnleuvalglnserglyalagluvallyslysproglyala151015thrvallysilesercyslysvalserglytyrthrphethraspser202530valmetasntrpvalglnglnalaproglylysglyleuglutrpmet354045glytrpileaspproglutyrglyargthraspvalalaglulysphe505560glnglyargvalthrilethralaaspthrserthraspthralatyr65707580metgluleuserserleuargsergluaspthralavaltyrtyrcys859095alaargthrlystyrasnserglytyrglypheprotyrtrpglygln100105110glythrthrvalthrvalserseralaserthrlysglyproserval115120125pheproleualaprocysserargserthrsergluserthralaala130135140leuglycysleuvallysasptyrpheprogluprovalthrvalser145150155160trpasnserglyalaleuthrserglyvalhisthrpheproalaval165170175leuglnserserglyleutyrserleuserservalvalthrvalpro180185190serserasnpheglythrglnthrtyrthrcysasnvalasphislys195200205proserasnthrlysvalasplysthrvalgluarglyscyscysval210215220glucysproprocysproalaproprovalalaglyproservalphe225230235240leupheproprolysprolysaspthrleumetileserargthrpro245250255gluvalthrcysvalvalvalaspvalserhisgluaspprogluval260265270glnpheasntrptyrvalaspglyvalgluvalhisasnalalysthr275280285lysproargglugluglnpheasnserthrpheargvalvalserval290295300leuthrvalvalhisglnasptrpleuasnglylysglutyrlyscys305310315320lysvalserasnlysglyleuproserserileglulysthrileser325330335lysthrlysglyglnproarggluproglnvaltyrthrleupropro340345350serarggluglumetthrlysasnglnvalserleuthrcysleuval355360365lysglyphetyrproseraspilealavalglutrpgluserasngly370375380glnprogluasnasntyrlysthrthrproprometleuaspserasp385390395400glyserphepheleutyrserlysleuthrvalasplysserargtrp405410415glnglnglyasnvalphesercysservalmethisglualaleuhis420425430asnhistyrthrglnlysserleuserleuserproglylys435440445当前第1页12当前第1页12