CN114729020A - 长效gdf15融合蛋白及包含其的药物组合物 - Google Patents
长效gdf15融合蛋白及包含其的药物组合物 Download PDFInfo
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Abstract
本发明涉及包含具有增加的生理活性和体内稳定性的GDF15变体的融合蛋白，以及包含其的药物组合物。根据本发明的GDF15变体或长效GDF15融合蛋白在体内效力、对GDF15受体的结合亲和力和体重减轻作用方面优于常规GDF15变体。因此，包含作为活性成分的本发明的GDF15变体、长效GDF15融合蛋白或融合蛋白二聚体的药物组合物引起食欲抑制，由此可以有效地用作用于代谢疾病或肥胖的治疗剂。此外，包含作为活性成分的GDF15变体、长效GDF15融合蛋白或融合蛋白二聚体的药物组合物可以用于与用于代谢疾病的化学药物和其他治疗剂的组合治疗等，并且可以有效地用于与用于代谢疾病或肥胖的常规治疗剂的组合治疗。
Description
技术领域
本发明涉及包含具有增加的生理活性和体内稳定性的GDF15变体的融合蛋白，以及包含其的药物组合物。
背景技术
生长分化因子-15(growth differentiation factor-15，GDF15)也称为巨噬细胞抑制性细胞因子-1(macrophage inhibitory cytokine-1，MIC-1)、胎盘骨形态发生蛋白(placental bone morphogenetic protein，PBMP)或非甾体抗炎药物活化基因-1(nonsteroidal anti-inflammatory drug-activated gene-1，NAG-1)，是作为转化生长因子-β(transforming growth factor-beta，TGF-β)超家族成员的蛋白质。
最近，研究结果表明，GDF15通过与脑组织中特异性表达的GDNF家族受体α样(GDNFfamily receptor alpha-like，GFRAL)和ret原癌基因(ret proto-oncogene，RET)结合来抑制饮食摄入，从而导致体重减轻(Tsai VW,et al.,PLoS One 2013；8(2):e55174；US 8,192,735)。此外，多项研究表明，在向多种肥胖动物模型施用GDF15的情况下，GDF15具有优异的体重减轻作用，并已确定除了具有这种作用外，GDF15还具有例如降低血糖水平、改善血脂水平和改善胰岛素抵抗的代谢优势。
然而，野生型GDF15的问题在于，在将其用于医学的情况下，由于其体内半衰期短，因此需要高频率的施用。因此，正在努力开发旨在增加GDF15体内半衰期的长效制剂。
同时，在生产长效制剂的多种技术中，免疫球蛋白Fc融合技术使用最广泛，因为它导致体内半衰期增加，并且几乎不担心副作用例如毒性或诱导免疫应答。为了将免疫球蛋白Fc融合的GDF15蛋白开发成长效的治疗性药物，必须满足以下几个条件。
第一，应使由融合引起的体外活性降低最小化。已知GDF15融合蛋白的活性变化很大地取决于融合位点。因此，已将突变引入至GDF15的Fc融合GDF15蛋白的活性变化可取决于是否发生融合或融合位点。第二，融合应导致体内半衰期增加，并且体内半衰期增加应体现能够在人中以每周一次的间隔施用的药物代谢动力学谱。第三，考虑到大多数生物制药可引起患者中的免疫原性，应使由融合接头或突变引起的免疫原性的风险最小化。第四，不应存在由于融合位点或突变引入的稳定性问题。第五，由于取决于融合免疫球蛋白的同种型可发生不期望的免疫应答，因此需要替代实施方案。
当努力改善GDF15的生理活性和稳定性时，本发明人已经确定，在GDF15的特定位置引入突变并且免疫球蛋白Fc区与其结合的情况下，GDF15具有增强的活性和增加的体内半衰期，从而完成了本发明。
发明的公开内容
技术问题
本发明的一个目的是提供具有改善的生理活性和稳定性的GDF15变体以及长效GDF15融合蛋白。
本发明的另一个目的是提供用于预防或治疗糖尿病、肥胖、血脂异常或代谢综合征的药物组合物，其包含作为活性成分的GDF15变体或长效GDF15融合蛋白。
问题的解决方案
为了实现上述目的，在本发明的一个方面中，提供了式(I)所示的GDF15变体。
N端延伸结构域-核心结构域(I)
在本发明的另一个方面中，提供了长效GDF15融合蛋白，其中GDF15变体与人IgGFc或其变体结合。
在本发明的又一个方面中，提供了融合蛋白二聚体，其包含两种长效GDF15融合蛋白。
在本发明的又一个方面中，提供了分离的核酸分子，其编码GDF15变体或GDF15融合蛋白。
在本发明的又一个方面中，提供了表达载体，其包含所述核酸分子。
在本发明的又一个方面中，提供了宿主细胞，其包含所述表达载体。
在本发明的又一个方面中，提供了用于预防或治疗糖尿病、肥胖、血脂异常或代谢综合征的药物组合物，其包含作为活性成分的GDF15变体、长效GDF15融合蛋白或融合蛋白二聚体。
发明的有益作用
根据本发明的GDF15变体或长效GDF15融合蛋白在体外效力、对GDF15受体的结合亲和力和体重减轻作用方面优于常规GDF15变体。
因此，包含作为活性成分的GDF15变体、长效GDF15融合蛋白或融合蛋白二聚体的本发明的药物组合物引起食欲抑制，因此可有效地用作用于代谢疾病或肥胖的治疗剂。
此外，包含作为活性成分的GDF15变体、长效GDF15融合蛋白或融合蛋白二聚体的药物组合物可以用于与用于代谢疾病的化学药物和其他治疗剂的组合治疗等，并且可以有效地用于与用于代谢疾病或肥胖的常规治疗剂的组合治疗。
附图简述
图1示出了通过在活性方面比较长效GDF15融合蛋白(二聚体：FM4、FM4-1、FM4-2和FM4-3)获得的结果，其中成熟GDF15中第56位氨基酸天冬酰胺(N)和/或成熟GDF15中第103位氨基酸天冬氨酸(D)被另外的氨基酸替换。
图2示出了通过在活性方面比较长效GDF15融合蛋白(二聚体：FM9、FM13、FM14、FM15和FM16)获得的结果，其中成熟GDF15中第64位氨基酸丝氨酸(S)被另外的氨基酸替换。
图3示出了通过在活性方面比较长效GDF15融合蛋白(二聚体：FM4、FM5和FM9)获得的结果。
图4示出了通过在对GDF15受体(GFRAL和RET)的结合亲和力方面比较长效GDF15融合蛋白(二聚体：FM4、FM5和FM9)获得的结果。
图5示出了通过在活性方面比较长效GDF15融合蛋白(二聚体：FM1和FM10)获得的结果。
图6示出了通过在活性方面比较长效GDF15融合蛋白(二聚体：FM2和FM11)获得的结果。
图7示出了通过在活性方面比较长效GDF15融合蛋白(二聚体：FM3和FM12)获得的结果。
图8示出了通过在对GDF15受体(GFRAL和RET)的结合亲和力方面比较长效GDF15融合蛋白(二聚体：FM10和FM11)获得的结果。
图9示出了通过在取决于接头类型和长度的活性方面比较长效GDF15融合蛋白(二聚体：FM9-1、FM9-2、FM9-3、FM9-4、FM9-5和FM9-6)获得的结果。
图10示出了通过在取决于接头类型和长度的活性方面比较长效GDF15融合蛋白(二聚体：FM11-1、FM11-2、FM11-3、FM11-4、FM11-5和FM11-6)获得的结果。
图11示出了通过在饮食诱导的肥胖小鼠(diet-induced obese mice，DIO小鼠)的体重变化(％)方面比较通过重复施用长效GDF15融合蛋白(二聚体；FM9-4、FM9-6、FM11-4和FM11-6)获得的结果。
图12示出了通过在饮食诱导的肥胖小鼠(DIO小鼠)的体重变化(％)方面比较通过单次施用长效GDF15融合蛋白(二聚体；FM9-6)获得的结果。
图13示出了通过在ob/ob小鼠的体重变化(％)方面比较通过重复施用长效GDF15融合蛋白(二聚体；FM9-6)获得的结果。
实施本发明的最佳方式
在下文中，将更详细地描述本发明。
GDF15变体
在本发明的一个方面中，提供了式(I)所示的GDF15变体：
N端延伸结构域-核心结构域(I)。
在式(I)中，
N端延伸结构域是由SEQ ID NO:3至5的任一氨基酸序列组成的多肽；以及
核心结构域是SEQ ID NO:20所示的多肽，或者是衍生自SEQ ID NO:20的多肽，其中选自SEQ ID NO:20的氨基酸序列中的第15、50、58、97位的氨基酸及其组合的任一氨基酸被另外的氨基酸替换；
其中第15位氨基酸精氨酸(R)可被丙氨酸(A)、天冬氨酸(D)、天冬酰胺(N)、半胱氨酸(C)、谷氨酸(E)、谷氨酰胺(Q)、甘氨酸(G)、组氨酸(H)、异亮氨酸(I)、亮氨酸(L)、赖氨酸(K)、甲硫氨酸(M)、苯丙氨酸(F)、脯氨酸(P)、丝氨酸(S)、苏氨酸(T)、色氨酸(W)、酪氨酸(Y)或缬氨酸(V)替换，
第50位氨基酸天冬酰胺(N)可被丙氨酸、精氨酸(R)、天冬氨酸、半胱氨酸、谷氨酸、谷氨酰胺、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸或缬氨酸替换，
第58位氨基酸丝氨酸(S)可被丙氨酸、精氨酸、天冬氨酸、天冬酰胺、半胱氨酸、谷氨酸、谷氨酰胺、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、苏氨酸、色氨酸、酪氨酸或缬氨酸替换，以及
第97位氨基酸天冬氨酸(D)可被丙氨酸、精氨酸、天冬氨酸、半胱氨酸、谷氨酸、谷氨酰胺、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸或缬氨酸替换。
如本文使用的术语“核心结构域”是指具有在SEQ ID NO:1的GDF15的氨基酸序列中的第7至112位的氨基酸序列的多肽，其包含SEQ ID NO:20所示的多肽，或衍生自SEQ IDNO:20的多肽，其中选自SEQ ID NO:20的氨基酸序列中第15、50、58、97位的氨基酸及其组合中的任一氨基酸被替换为另外的氨基酸。第一核心结构域可由SEQ ID NO:2的氨基酸序列组成。
具体地，核心结构域可包含选自以下改变(1)至(6)的任一改变：
(1)SEQ ID NO:20的氨基酸序列中第15位氨基酸精氨酸(R)被天冬酰胺(N)替换的改变；
(2)SEQ ID NO:20的氨基酸序列中第50位氨基酸天冬酰胺(N)被亮氨酸(L)替换的改变；
(3)SEQ ID NO:20的氨基酸序列中第58位氨基酸丝氨酸(S)被赖氨酸(K)、精氨酸(R)、天冬酰胺(N)、天冬氨酸(D)、谷氨酸(E)、半胱氨酸(C)或亮氨酸(L)替换的改变；
(4)SEQ ID NO:20的氨基酸序列中第97位氨基酸天冬氨酸(D)被亮氨酸(L)替换的改变；
(5)SEQ ID NO:20的氨基酸序列中第50位氨基酸天冬酰胺(N)和SEQ ID NO:20的氨基酸序列中第97位氨基酸天冬氨酸(D)各自被半胱氨酸(C)或丝氨酸(S)替换的改变；以及
(6)SEQ ID NO:20的氨基酸序列中第15位氨基酸精氨酸(R)被天冬酰胺(N)替换并且SEQ ID NO:20的氨基酸序列中第58位氨基酸丝氨酸(S)被赖氨酸(K)或精氨酸(R)替换的改变。
此处，核心结构域可由选自SEQ ID NO:6至19中的任一氨基酸序列组成。
N端延伸结构域是与上述核心结构域的N端结合的结构域，并且可以是由SEQ IDNO:3至5中的任一氨基酸序列组成的多肽。
本文使用的表述“ΔN2”也可表示为“delta N2”，意指在SEQ ID NO:1所示的人GDF15的氨基酸序列中，第1位和第2位的氨基酸缺失。在将ΔN2表示为N端延伸结构域的情况下，其可表示为“NGDH”。
本文使用的表述“ΔN3、WS插入、G4N、D5S、H6T”也可表示为“delta N3、WS插入、G4N、D5S、H6T”，意指在SEQ ID NO:1所示的人GDF15的氨基酸序列中，第1至3位的氨基酸缺失，并在其中插入色氨酸和丝氨酸；第4位氨基酸甘氨酸被天冬酰胺替换；第5位氨基酸天冬氨酸被丝氨酸替换；以及第6位氨基酸组氨酸被苏氨酸替换。在将ΔN3、WS插入、G4N、D5S、H6T表示为N端延伸结构域的情况下，其可表示为“WSNST”。
本文使用的表述“ΔN3、G4N、D5S、H6T”也可表示为“delta N3、G4N、D5S、H6T”，意指在SEQ ID NO:1所示的人GDF15的氨基酸序列中，第1至3位的氨基酸缺失；第4位氨基酸甘氨酸被天冬酰胺替换；第5位氨基酸天冬氨酸被丝氨酸替换；以及第6位氨基酸组氨酸被苏氨酸替换。在将“ΔN3、G4N、D5S、H6T”表示为N端延伸结构域的情况下，其可表示为“NST”。
GDF15变体可包含由SEQ ID NO:3所示的氨基酸序列组成的N端延伸结构域和由选自SEQ ID NO:6至20的任一氨基酸序列组成的核心结构域。此外，GDF15变体可包含由SEQID NO:4所示的氨基酸序列组成的N端延伸结构域和由选自SEQ ID NO:6至20的任一氨基酸序列组成的核心结构域。此外，GDF15变体可包含由SEQ ID NO:5所示的氨基酸序列组成的N-端延伸结构域，以及由选自SEQ ID NO:6至19的任一氨基酸序列组成的核心结构域。
优选地，GDF15变体可包含由SEQ ID NO:3所示的氨基酸序列组成的N端延伸结构域和由SEQ ID NO:8、9或20所示的氨基酸序列组成的核心结构域。此外，GDF15变体可包含由SEQ ID NO:4所示的氨基酸序列组成的N端延伸结构域和由SEQ ID NO:8、9或20所示的氨基酸序列组成的核心结构域。此外，GDF15变体可包含由SEQ ID NO:5所示的氨基酸序列组成的N端延伸结构域和由选自SEQ ID NO:6、7和10至19的任一氨基酸序列组成的核心结构域。此处，GDF15变体可由选自SEQ ID NO:21至39的任一氨基酸序列组成。
长效GDF15融合蛋白
在本发明的另一个方面中，提供了长效GDF15融合蛋白，其中GDF15变体与人IgGFc或其变体结合。
人IgG Fc或其变体可以是IgG1、IgG2、IgG3或IgG4的Fc，或其变体。具体地，人IgGFc或其变体可以是人IgG1 Fc或其变体，并且人IgG1Fc可由SEQ ID NO:41所示的氨基酸序列组成。
人IgG Fc或其变体可以是与SEQ ID NO:41具有90％、92％、95％、96％、97％、98％、99％或100％同一性的连续氨基酸序列，或包含CH3结构域的Fc片段。在某些实施方案中，人IgG Fc或其变体可以是与SEQ ID NO:41具有90％、92％、95％、96％、97％、98％、99％或100％同一性的连续氨基酸序列，或包含CH2结构域和CH3结构域的Fc片段。在某些实施方案中，人IgG Fc或其变体可以是与SEQ ID NO:41具有90％、92％、95％、96％、97％、98％、99％或100％同一性的连续氨基酸序列，或包含部分铰链区、CH2结构域和CH3结构域的Fc片段。在某些实施方案中，人IgG Fc或其变体可以具有与SEQ ID NO:41具有90％、92％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
IgG Fc或其变体包含：含有IgG1 Fc序列的第一多肽，所述IgG Fc序列包含CH3序列，所述CH3序列包含至少一个工程化突起；和含有IgG1Fc序列的第二多肽，所述IgG Fc序列包含CH3序列，所述CH3序列包含至少一个工程化腔，其中第一多肽通过将第一多肽的突起定位至第二多肽的腔中使第二多肽发生二聚化。
具体地，第一多肽可包含允许与另一个包含工程化腔的IgG Fc多肽(例如，第二多肽)结合的工程化突起。第二多肽可包含允许与另一个包含工程化突起的IgG Fc多肽(例如，第一多肽)结合的工程化腔。此外，第一多肽的突起和第二多肽的腔可各自工程化至IgGFc的CH3结构域中。此处，第一多肽的突起和第二多肽的腔既不连接也不结合至GDF15变体。
工程化突起可包含在具有SEQ ID NO:41所示的氨基酸序列的人IgG1 Fc的氨基酸序列中的至少一个替换。此处，氨基酸位置根据EU编号进行编号。替换可存在于选自氨基酸残基347、366和394的位置。例如，替换可以是选自Q347W/Y、T366W/Y、T394W/Y的任一者及其组合。此外，工程化腔可包含在人IgG1 Fc序列中的相应氨基酸中至少一个替换，并且替换可存在于选自氨基酸残基366、368、394、405和407的位置。例如，替换可以是选自T366S、L368A、T394S、F405T/V/A、Y407T/V/A的任一者及其组合。
优选地，突起可包含替换T366W/Y，并且腔可包含选自T366S、L368A、Y407T/V/A的任一种替换及其组合。例如，突起可包含替换T366W/Y，并且腔可包含替换Y407T/V/A。此外，突起可包含替换T366Y，并且腔可包含替换Y407T。此外，突起可包含替换T366W，并且腔可包含替换Y407A。此外，突起可包含替换T394Y，并且腔可包含替换Y407T。
第一多肽可由选自SEQ ID NO:42、44和46中的任一氨基酸序列组成，第二多肽可由选自SEQ ID NO:43、45和47中的任一氨基酸序列组成。
突起是指“钮(knob)”以及腔是指“扣(hole)”。
第一多肽是包含工程化突起的Fc“钮”，第二多肽是包含工程化突起的Fc“扣”。第一和第二多肽可通过非共价相互作用(例如疏水作用，例如Fc的钮和扣区域之间的疏水相互作用)、共价键(例如二硫键，例如在第一和第二多肽中的Fc的铰链区之间的一个或两个或更多个二硫键)或其两者进行物理缔合。
如本文使用的术语“二聚体”是指包含至少两个多肽的蛋白质复合物。这些多肽中的每一个都包含N端和C端。至少两个多肽可通过共价和非共价(例如，静电、π-效应、范德华力和疏水效应)相互作用中的一种或两种进行缔合。两个多肽可具有相同的氨基酸序列或可彼此不同。在两个多肽彼此相同的情况下，二聚体是指(同)二聚体；在两个多肽彼此不同的情况下，二聚体是指异二聚体。
人IgG Fc或其变体可以是包含第一多肽和第二多肽的异二聚体；并且异二聚体可以是由A-1(SEQ ID NO:42)和A-2(SEQ ID NO:43)形成的异二聚体、由B-1(SEQ ID NO:44)和B-2(SEQ ID NO:45)形成的异二聚体或由C-1(SEQ ID NO:46)和C-2(SEQ ID NO:47)形成的异二聚体。
此外，IgG Fc或其变体可包含另外的突变，以改善长效GDF15融合蛋白的特性。具体地，由第一多肽和第二多肽组成的异二聚体可包含另外的突变。
例如，IgG Fc或其变体可包含废除(例如，降低或消除)IgG效应子功能的突变。具体地，Fc伴侣序列可包含这样的突变，其废除效应子功能，例如补体依赖的细胞毒性(complement-dependent cytotoxicity，CDC)、抗体依赖的细胞毒性(antibody-dependentcellular cytotoxicity，ADCC)和抗体依赖的细胞吞噬作用(antibody-dependentcellular phagocytosis，ADCP)。例如，可将突变E233A和L235A引入由A-1和A-2形成的IgGFc或其变体(其是异二聚体)中，以消除IgG1效应子功能。由B-1和B-2形成的异二聚体，其包含突变N297A，可以用于消除N-连接聚糖。可将突变L234A、L235A和N297A引入由C-1和C-2形成的异二聚体中，以消除IgG1效应子功能和N-连接聚糖。
GDF15变体与IgG Fc或其变体之间的结合可使得IgG Fc或其变体中的第一多肽的C端或第二多肽的C端与N-GDF15变体的N端结合。此外，GDF15变体与IgG Fc或其变体之间的结合可以是这样的：IgG Fc或其变体中的第一多肽的N端或第二多肽的N端与GDF15变体的C端结合。优选地，GDF15变体与IgG Fc或其变体之间的结合可以是这样的：IgG Fc或其变体中的第一多肽的C端与GDF15变体的N端结合。
此外，GDF15变体与IgG Fc或其变体之间的结合可通过接头进行。接头可以是由包含甘氨酸、丝氨酸、丙氨酸和谷氨酸残基的10至50个氨基酸残基组成的肽。接头可包含(G4S)n，其中n可以是1至10的整数或2至7的整数。例如，n可以是2、3、4、5、6或7。在本发明的一个实施方案中，在n是整数5的情况下使用包含(G4S)5的接头。
作为除了包含(G4S)n的接头之外的合适接头的实例，可提及包含GS(G4S)n、GS(EEEA)n、(EEEA)n、GS(EAAAK)n、(EAAAK)n或GSGGSS(PT)n的接头，其中n可以是1至10的整数。然而，合适的接头并不限于此。在本发明的一个实施方案中，在n是整数6的情况下使用包含GS(EEEA)6的接头，或在n是整数5的情况下使用包含GS(EAAAK)5的接头。
具体地，接头可以是GGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO：48)，GSGGGGSGGGGSGGGGS(SEQ ID NO：92)、GSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO：93)、GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO：94)、GSEEEAEEEAEEEAEEEAEEEAEEEA(SEQ ID NO： 95)、GSGGSSPTPTPTPTPTPTPTPTPTPT(SEQ ID NO：96)、或GSEAAAKEAAAKEAAAKEAAAKEAAAK(SEQ ID NO：97)。优选地，接头可以是GGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO：48)、GSEEEAEEEAEEEAEEEAEEEAEEEA(SEQ ID NO：95)或GSEAAAKEAAAKEAAAKEAAAKEAAAK(SEQ ID NO：97)。
长效GDF15融合蛋白包括每个由第一多肽和第二多肽组成的异二聚体一个GDF15变体。GDF15变体可包含至少一个N-连接聚糖。
长效GDF15融合蛋白可包含i)由选自SEQ ID NO：21至39的任一氨基酸序列组成的GDF15变体，ii)由选自SEQ ID NO：42、44和46的任一氨基酸序列组成的第一多肽，以及iii)由选自SEQ ID NO：43、45和47的任一氨基酸序列组成的第二多肽。
优选地，长效GDF15融合蛋白可包含i)由选自SEQ ID NO：21至39的任一氨基酸序列组成的GDF15变体，ii)由SEQ ID NO：48的氨基酸序列组成的接头，iii)由SEQ ID NO：42的氨基酸序列组成的第一多肽，和iv)由SEQ ID NO：43的氨基酸序列组成的第二多肽。
仍优选地，长效GDF15融合蛋白可包含i)由选自SEQ ID NO：21至39的任一氨基酸序列组成的GDF15变体，ii)由选自SEQ ID NO：92至97的任一氨基酸序列组成的接头，iii)由SEQ ID NO：46的氨基酸序列组成的第一多肽，和iv)由SEQ ID NO：47的氨基酸序列组成的第二多肽。
融合蛋白二聚体
本发明的又一个方面中，提供了融合蛋白二聚体，其包含两种长效GDF15融合蛋白。具体地，两种长效GDF15融合蛋白通过GDF15-GDF15相互作用二聚化，这被称为“融合蛋白二聚体”。
核酸分子、表达载体和宿主细胞
本发明的又一个方面中，提供了分离的核酸分子，其编码GDF15变体或长效GDF15融合蛋白。
本文使用的术语“分离的核酸分子”是指本发明的核酸分子，其：在从源细胞中分离出完整的核酸时，已与至少约50％的蛋白质、脂质、碳水化合物或其他与其一起天然存在的物质分离；可操作地连接至其在自然界中不连接的多核苷酸；或在自然界中不作为更大的多核苷酸序列的一部分出现。具体地，本发明的分离的核酸分子基本上不含任何其他污染性核酸分子，或在其自然环境中发现的并且会干扰其在多肽产生中的使用或在其治疗性、诊断性、预防性或研究应用的使用的其他污染物。
此处，编码GDF15变体或长效GDF15融合蛋白的分离的核酸分子由于密码子冗余可具有不同的序列。此外，取决于目的，分离的核酸分子可被适当地修饰或者可具有添加至N端或C端的核苷酸，只要其可以产生GDF15变体或长效GDF15融合蛋白即可。
在本发明的又一个方面中，提供了表达载体，其包含编码GDF15变体或长效GDF15融合蛋白的分离的核酸分子。
如本文使用的术语“表达载体”是指适用于转化宿主细胞并含有指导或控制插入的异源核酸序列表达的核酸序列的载体。载体包含线性核酸、质粒、噬菌粒、黏粒、RNA载体、病毒载体、及其类似物。病毒载体的实例包括但不限于逆转录病毒、腺病毒和腺相关病毒。
如本文使用的术语“异源核酸序列的表达”或靶蛋白的“表达”是指插入的DNA序列的转录、mRNA转录物的翻译以及融合蛋白产物、抗体或抗体片段的产生。
可用的表达载体可以是RcCMV(Invitrogen,Carlsbad)或其变体。可用的表达载体可包含用于促进哺乳动物细胞中靶基因的连续转录的人巨细胞病毒(CMV)启动子，以及用于提高转录后RNA稳定性水平的牛生长激素多腺苷酸化信号序列。
本发明的又一个方面中，提供了包含所述表达载体的宿主细胞。
如本文使用的术语“宿主细胞”是指可以将重组表达载体引入其中的原核或真核细胞。如本文使用的术语“转化的”或“转染的”意指通过本领域已知的多种技术将核酸(例如，载体)引入细胞中。
宿主细胞可用本发明的DNA序列转化或转染，并且可用于靶蛋白的表达和/或分泌。可以用于本发明的宿主细胞的实例可包含永生杂交瘤细胞、NS/0骨髓瘤细胞、293细胞、中国仓鼠卵巢(Chinese hamster ovary，CHO)细胞、HeLa细胞、CAP细胞(人羊水来源细胞)、和COS细胞。
药物组合物
在本发明的又一个方面中，提供了用于预防或治疗糖尿病、肥胖、血脂异常或代谢综合征的药物组合物，其包含作为活性成分的GDF15变体、长效GDF15融合蛋白或融合蛋白二聚体。
本发明的药物组合物可以通过任何途径施用。本发明的组合物可使用任何合适的方式直接(例如，局部地，通过注射、植入或局部施用至组织部位)或全身性地(例如，通过肠胃外或经口施用)提供给动物。在本发明的组合物被肠胃外提供的情况下，例如通过静脉内、皮下、眼部、腹膜内、肌内、直肠内、眶内、脑内、颅内、脊柱内、心室内、鞘内、脑池内、囊内、鼻内或气雾剂施用时，组合物可以是水溶液或包含生理适用的体液混悬液或溶液的一部分。因此，由于载体或载剂是生理上可接受的，其可添加至组合物并递送至患者。因此，通常可包含生理盐水作为制剂的体液样载体。
此外，施用频率可取决于所用制剂中GDF15变体的药代动力学参数而变化。通常，医师会施用药物组合物直到其剂量达到实现期望作用的剂量。因此，药物组合物可作为单剂施用，或以时间间隔的两剂或更多剂(其可以含有或可以不含有等量的靶融合蛋白)施用，或可通过可植入装置或导管作为连续输注施用。适当剂量的进一步细化由本领域技术人员常规地进行并且落入他们常规进行的工作范围内。
此外，融合蛋白在人体内的单位剂量为0.01μg至100mg/kg体重，特别地为1μg至10mg/kg体重。虽然上述量是最佳量，但所述量可取决于待治疗的疾病或有无副作用而变化。最佳剂量可使用常规实验来确定。融合蛋白的施用可通过从外部贮器(例如，静脉袋)或内部贮器(例如，生物可降解的植入物)通过周期性推注注射，或连续静脉内、皮下或腹膜内施用来进行。
此外，本发明的融合蛋白可与其他生物活性分子一起施用于对象接受者。然而，融合蛋白和其他分子的最佳组合，以及其剂型和精确剂量可通过本领域公知的常规实验来确定。
在本发明的又一个方面中，提供了GDF15变体、长效GDF15融合蛋白或融合蛋白二聚体用于预防或治疗糖尿病、肥胖、血脂异常或代谢综合征的用途。
在本发明的又一个方面中，提供了GDF15变体、长效GDF15融合蛋白或融合蛋白二聚体用于制备用于预防或治疗糖尿病、肥胖、血脂异常或代谢综合征的药物的用途。
在本发明的又一个方面中，提供了用于预防或治疗糖尿病、肥胖、血脂异常或代谢综合征的方法，包括向个体施用GDF15变体、长效GDF15融合蛋白或融合蛋白二聚体的步骤。
GDF15变体、长效GDF15融合蛋白或融合蛋白二聚体的剂量、施用频率和施用途径与上述相同。个体可以是患有糖尿病、肥胖、血脂异常或代谢综合征的个体。此外，个体可以是哺乳动物，优选为人。
发明方式
下文中，为了更好地理解本发明，将通过实施例的方式对本发明进行详细描述。然而，根据本发明的实施例可以以多种不同的形式进行修改，并且本发明的范围不应被解释为限于以下实施例。
实施例1：长效GDF15融合蛋白的产生
实施例1.1：基因克隆
一般而言，在物质与长效Fc或白蛋白融合以增加其半衰期的情况下，该融合导致该物质的活性降低。为了改善这一点，设计了多种GDF15变体。
首先，通过对GDF15中的第32、51、56、60、64、90、92、93、97、101和103位的各个氨基酸进行替换来制备第一多肽，通过GDF15的三维结构分析，这些氨基酸预计对蛋白质活性有很大的影响，并导致所得的GDF15与IgG1 Fc_knob结合，这些第一多肽示于下表1中。
[表1]
具体地，为了产生具有Fc_knob-(G4S)5-GDF15变体结构的第一多肽(FM系列)和具有Fc_hole结构的第二多肽，使用pcDNA3.3(Invitrogen)表达载体进行基因克隆，其包含编码由SEQ ID NO:49、60、65和69至90的任一氨基酸序列组成的第一多肽的基因，以及编码由SEQ ID NO:43的氨基酸序列组成第二多肽的基因。此处，编码SEQ ID NO:43、49、60、65和69至90的氨基酸序列的核苷酸序列是通过向Macrogen,Inc.提出请求而合成的。
实施例1.2：长效GDF15融合蛋白(二聚体)的表达和纯化
将实施例1.1中克隆的pcDNA3.3表达载体瞬时转染至ExpiCHO细胞系(Invitrogen)中。然后，在第8天，收获并纯化细胞培养物。为了纯化收获的细胞培养液(Harvest cell culture fluid,HCCF)中的第一多肽和第二多肽，使用蛋白A树脂进行亲和纯化。
具体地，HCCF被加载至用1×PBS(pH 7.4)平衡的MabSelect SuRe蛋白A树脂(GEHealthcare)上以诱导结合。在第一多肽和第二多肽之间的结合完成后，将MabSelect SuRe蛋白A树脂用1×PBS(pH 7.4)洗涤。然后，使用0.1M甘氨酸(pH 3.0)溶液进行洗脱，以获得最终物质。
使用1M Tris-HCl溶液将第一多肽和第二多肽中和至约pH 8.0的水平。第一多肽和第二多肽通过钮进扣(knob-in-hole)相互作用被完全二聚化，并将其命名为“长效GDF15融合蛋白”。长效GDF15融合蛋白的两个分子通过GDF15-GDF15相互作用再次二聚化，并将其命名为“融合蛋白二聚体”。
实施例2：长效GDF15融合蛋白(二聚体)的活性测量
使用包含由SEQ ID NO:49的氨基酸序列组成的成熟GDF15的融合蛋白作为对照，在GDF15活性的方面比较实施例1中产生的长效GDF15融合蛋白。使用Bright-GloTM荧光素酶测定试剂盒(Promega)和过表达GFRAL/RET/SRE-luc的人胚肾293(HEK293)细胞系测量GDF15活性。
具体地，将过表达GFRAL/RET/SRE-luc的1×105个HEK293细胞分配至含有10％FBS的DMEM培养基中的96孔板的每个孔中，然后在37℃和5％CO2下孵育24小时。24小时后，将96孔板中的各培养基更换为50μl无血清培养基，并在37℃和5％CO2下培养4小时。
此外，实施例1中产生的每种长效GDF15融合蛋白是使用无血清培养基从2000nM的浓度开始通过3倍连续稀释制备的。然后，将50μl长效GDF15融合蛋白稀释液添加至含有50μl更换的无血清培养基和GFRAL/RET/SRE-luc细胞系的每孔，使实际浓度通过从1000nM开始3倍连续稀释得到。然后，允许反应在37℃和5％CO2下进行4小时。4小时后，每孔用100μlBright-GloTM溶液处理，该溶液是通过将Bright-GloTM缓冲液添加至Bright-GloTM底物而制备的，并允许反应在室温下进行1分钟。
其后，用能够测量发光(luminescence)的酶标仪(Perkin Elmer,Wallac VictorX5)测量相对光单位(relative light unit，RLU)值。结果示于下表2。此处，基于包含成熟GDF15的融合蛋白(FWT+Fc_hole)(其作为对照)的体外GDF15活性(Emax为100％)，选择了两种改善的长效GDF15融合蛋白。
[表2]
作为结果，选择的两种长效GDF15融合蛋白是具有突变ΔN2、N56C和D103C的长效GDF15融合蛋白(以下是指FM4+Fc_hole)，和具有突变ΔN2和S64K的长效GDF15融合蛋白(以下是指FM5+Fc_hole)；并且其体外GDF15活性(Emax)分别测量为133.3％和147.2％。从这些结果，确定了FM4+Fc_hole和FM5+Fc_hole改善了体外GDF15活性。
实施例3：长效GDF15融合蛋白(二聚体：FM4+Fc_hole)的活性测量
在长效GDF15融合蛋白间的GDF15-GDF15相互作用中，实施例2中确定的引入另外二硫键的FM4+Fc_hole改善了体外GDF15活性。在这些结果的基础上，为了确定二硫键的重要性，另外设计如下表3所示的长效GDF15融合蛋白，其基于FM4+Fc_hole并且其中成熟GDF15中第56位氨基酸天冬酰胺(N)和/或成熟GDF15中的第103位氨基酸天冬氨酸(D)被另外的氨基酸替换，并使该长效GDF15融合蛋白以与实施例1相同的方式产生。然后，以与实施例2相同的方式评价体外GDF15活性。
[表3]
作为结果，如表3所示，鉴定了与FWT+Fc_hole相比，仅FM4+Fc_hole改善了体外GDF15活性(Emax)(图1)。从这些结果，鉴定了半胱氨酸的引入和所得的二硫键的引入在改善FM4+Fc_hole的体外GDF15活性方面发挥了重要作用。
实施例4：长效GDF15融合蛋白(二聚体：FM5+Fc_hole)的活性测量
在实施例2中鉴定的FM5+Fc_hole，其是GDF15的S64K变体，具有改善的体外GDF15活性。基于这些结果，另外设计如下表4所示的长效GDF15融合蛋白，其基于FM5+Fc_hole并且其中成熟GDF15中的第64位氨基酸丝氨酸(S)被另外的氨基酸替换，并使该长效GDF15融合蛋白以与实施例1相同的方式产生。然后，以与实施例2相同的方式评价体外GDF15活性。
[表4]
作为结果，如表4所示，鉴定了与FWT相比，仅FM9+Fc_hole具有改善的体外GDF15活性(Emax)(图2)。
实施例5：长效GDF15融合蛋白(二聚体：FM4+Fc_hole、FM5+Fc_hole和FM9+Fc_hole)的结合亲和力测量
在对作为GDF15受体的GFRAL和RET的结合亲和力方面比较了实施例3和4中具有改善的体外GDF15活性的FM4+Fc_hole、FM5+Fc_hole和FM9+Fc_hole。为了测量对GDF15受体的结合亲和力，使用过表达GFRAL和RET的HEK293细胞系进行基于细胞的酶联免疫吸附测定(enzyme-linked immunosorbent assay，ELISA)。
具体地，将过表达GFRAL/RET/SRE-luc的1×105个HEK293细胞分配至含有10％FBS的DMEM培养基中的96孔板的每个孔中，然后在37℃和5％CO2下孵育24小时。24小时后，从96孔板的每个孔中去除培养基。然后，各培养基用4％多聚甲醛处理并且允许反应在室温下进行20分钟。从中去除多聚甲醛。用0.6％过氧化氢溶液处理，允许反应再次进行20分钟。然后，用具有吐温20(PBST)缓冲液的3％牛血清白蛋白(BSA)-磷酸盐缓冲盐水进行处理，并允许封闭进行2小时。
此外，使用含有1％BSA的PBS缓冲液，从200μg/mL开始，对FM4+Fc_hole、FM5+Fc_hole或FM9+Fc_hole进行2倍连续稀释。将100μlFM4+Fc_hole、FM5+Fc_hole或FM9+Fc_hole分别以不同浓度稀释，并应用至含有GFRAL/RET过表达细胞系的96孔板中，允许反应在室温下进行2小时。然后，用辣根过氧化物酶(HRP)缀合的抗人IgG-Fc抗体(JacksonImmunoResearch#109-035-098)处理每个孔，然后用3,3,5,5-四甲基联苯胺(TMB)缓冲液显色(Bio-Rad#172-1066)。
将每孔用100μl TMB溶液处理，并允许反应在室温下进行10分钟。然后，使用2N硫酸(H2SO4)试剂中止反应。然后，用酶标仪(Perkin Elmer，Wallac Victor X5)在450nm处测量吸光度，以评价长效GDF15融合蛋白(二聚体)中GDF15变体与GDF15受体的结合能力。
作为结果，如图4所示，与作为对照的FWT+Fc_hole相比，FM9+Fc_hole表现出对GDF15受体的显著优异的结合亲和力，并且FM4+Fc_hole和FM5+Fc_hole还表现出对GDF15受体的高结合亲和力。
实施例6：长效GDF15融合蛋白(二聚体)纯化后的纯度改善
为了改善每种长效GDF15融合蛋白在产生时的纯化产率、纯度等，在GDF15的多个位置引入N-连接聚糖。已知GDF15序列中N-连接聚糖的存在会增加在蛋白质分泌过程中的相应蛋白质在内质网和高尔基体中的保留时间，从而使错误折叠的产物减到最少并帮助蛋白质表达。增加的保留时间具有对折叠动力学的有益作用，并且可以导致显著改善的异二聚体(Fc/Fc)钮进扣组装和从哺乳动物组织培养的恢复。
使用尺寸排除色谱(size exclusion chromatography)分析在正确组装的融合蛋白二聚体纯度方面进行对通过将N-连接聚糖引入至长效GDF15融合蛋白中获得的物质与作为对照的融合蛋白FWT+Fc_hole的二聚体相比纯化后纯度改善的评价。
具体地，另外设计了长效GDF15融合蛋白，其中在GDF15的多个位置引入了N-连接聚糖。在这点上，与融合蛋白FWT+Fc_hole的二聚体相比，具有改善的正确组装的融合蛋白二聚体的纯度的变体是在以与实施例1相同的方式产生和进行第一步纯化的情况下获得的，示于表6。
[表6]
作为结果，确定了FWT+Fc_hole经第一步纯化后具有50.9％的正确组装的融合蛋白二聚体纯度，而FM1+Fc_hole、FM2+Fc_hole、FM3+Fc_hole、FM10+Fc_hole、FM11+Fc_hole和FM12+Fc_hole，其各自将N-连接聚糖引入GDF15中，具有改善的正确组装的融合蛋白二聚体纯度，分别为80.2％、73.0％、86.3％、65.2％和70.3％。
此外，美国专利号9920118中的NGM Biopharmaceuticals,Inc.的融合蛋白二聚体(B13a/B13b(其中引入了N连接聚糖)经测量具有75.8％的纯度。
实施例7：长效GDF15融合蛋白(二聚体：FM10+Fc_hole、FM11+Fc_hole和FM12+Fc_hole)的活性评价
使用FM1+Fc_hole、FM2+Fc_hole和FM3+Fc_hole作为对照，其各自在GDF15的多个位置引入N-连接聚糖，将FM10+Fc_hole、FM11+Fc_hole和FM12+Fc_hole以与实施例2相同的方式在GDF15活性方面进行评价。结果示于表7。
[表7]
作为结果，如表7所示，鉴定了与FM1+Fc_hole、FM2+Fc_hole和FM3+Fc_hole相比，FM10+Fc_hole、FM11+Fc_hole和FM12+Fc_hole具有改善的GDF15活性(Emax)(图5至7)。
实施例8：长效GDF15融合蛋白(二聚体：FM10+Fc_hole和FM11+Fc_hole)结合亲和力的测量
将FM10+Fc_hole或FM11+Fc_hole对GDF15受体GFRAL和RET的结合亲和力以与实施例5中相同的方式进行比较和评价。作为结果，如图6所示，与作为对照的FWT+Fc_hole相比，在FM10+Fc_hole和FM11+Fc_hole中测量到对GDF15受体的显著优异的亲和力(图8)。
实施例9：长效GDF15融合蛋白(二聚体：FM6+Fc_hole、FM7+Fc_hole和FM8+Fc_hole)的产生
基于实施例1至8的结果，另外设计了长效GDF15融合蛋白，其中第21和/或64位的氨基酸被替换，并且在GDF15的多个位置引入了N-连接聚糖。在这点上，与融合蛋白FWT+Fc_hole的二聚体相比具有改善的正确组装的融合蛋白二聚体纯度的变体示于表8，其是在以与实施例1相同的方式产生变体并进行第一步纯化的情况下获得的。
[表8]
实施例10：对长效GDF15融合蛋白的融合载体和接头的优化
为了对在纯化后显示出优异的活性和纯度提高的两种变体(FM9+Fc_hole和FM11+Fc_hole)的融合载体和接头进行优化研究，另外设计了长效GDF15融合蛋白，其中融合载体(SEQ ID NO:46和47)和多种接头(SEQ ID NO:92、93、94、95、96和97)被引入各自的GDF15序列中以使效应子功能最小化，并示于表9中。
[表9]
实施例10.1：经优化的长效GDF15融合蛋白(二聚体)的表达和纯化
产生如表9所示的经优化的长效GDF15融合蛋白并将其以与实施例1相同的方式进行第一步纯化。为获得高纯度的长效GDF15融合蛋白，使用阴离子交换(anion exchange，AEX)树脂和阳离子交换(cation exchange，CEX)树脂对通过完成第一步纯化而获得的合并物(pool)进行第二步离子交换(IEX)纯化。
具体地，对于阴离子交换(AEX)，将经过第一步的合并物加载至用1×PBS(pH 7.4)平衡的POROS HQ 50μm强阴离子交换树脂(Thermo Fisher Scientific)上，以诱导结合。在第一多肽与第二多肽之间的结合完成后，将POROS HQ 50μm强阴离子交换树脂用1×PBS(pH7.4)洗涤，并使用含有1M氯化钠的50mM Tris-HCl(pH 8.0)溶液通过浓度梯度进行洗脱，以获得最终物质。使用尺寸排阻色谱分析合并达到纯度为95％或更高的标准的级分。
此外，对于阳离子交换(CEX)，将经过第一步的合并物根据等电点进行pH调节，然后加载至用20mM磷酸钠(pH 6.5)溶液平衡的POROS XS强阳离子交换树脂(Thermo FisherScientific)上，以诱导结合。在第一多肽和第二多肽之间的结合完成后，用20mM磷酸钠(pH6.5)溶液洗涤POROS XS强阳离子交换树脂，然后使用含有1M氯化钠的20mM磷酸钠(pH 6.5)溶液通过浓度梯度进行洗脱，以获得最终物质。使用尺寸排阻色谱分析合并达到纯度为95％或更高的标准的级分。
实施例10.2：经优化的长效GDF15融合蛋白(二聚体)的活性评价
对显示出优异的活性和纯度改善的两种变体(FM9+Fc_hole和FM11+Fc_hole)根据接头类型和长度在活性方面进行了比较和评价。以与实施例2相同的方式评价各长效GDF15融合蛋白的活性，结果示于表10。在此，基于FM9-6+Fc_hole的体外GDF15活性(Emax为100％)，根据GDF15序列以及接头类型和长度在活性方面比较长效GDF15融合蛋白。
[表10]
作为结果，如表10所示，鉴定了各长效GDF15融合蛋白表现出相似活性，除了其中使用接头GS(EEEA)6(SEQ ID NO:95)的那些，并且其中使用接头GS(EEEA)6(SEQ ID NO:95)的长效GDF15融合蛋白(FM9-4+Fc_hole和FM11-4+Fc_hole)表现出相对低的EC50值和相对高的Emax值(图9和10)。
实施例10.3：经优化的长效GDF15融合蛋白(二聚体)的药代动力学评价
在药物处理当天，将购自Orient BIO(韩国)的6周龄雄性C57BL/6小鼠分组(每个采血时间点n＝3)，使每组具有相似的体重平均值，然后分别以1mg/kg的剂量皮下施用FM9-4+Fc_hole、FM9-6+Fc_hole、FM11-4+Fc_hole和FM11-6+Fc_hole一次。分别于施用之后4、24、48、72、96、120、168和240小时采集血液样品。使用免疫测定法测量小鼠血液中每种长效GDF15融合蛋白(二聚体)的浓度。基于测量的浓度值，对各长效GDF15融合蛋白(二聚体)的药代动力学参数结果进行计算，并示于下表11中。
[表11]
实施例10.4：通过重复施用评价经优化的长效GDF15融合蛋白(二聚体)在饮食诱导的肥胖(DIO)小鼠中取决于不同的接头类型的抗肥胖作用
饮食诱导的肥胖(DIO)小鼠是通过在小鼠中饲喂高脂饮食而诱导的，其特征是肥胖、高血糖和胰岛素抵抗。已在C57BL/6N小鼠中饲喂高脂饮食(60千卡％脂肪,ResearchDiets,Cat#D12492,USA)8周的DIO小鼠(Taconic,USA)购自Raon Bio(Animal Inc.,Republic of Korea)。到达后，这些动物被另外饲喂高脂饮食(60％脂肪)5周，然后用于本研究。在给药开始之前一天，基于个体小鼠的平均体重将动物分组(每组n＝6)，然后将FM9-4+Fc_hole、FM9-6+Fc_hole、FM11-4+Fc_hole和FM11-6+Fc_hole分别以10nmol/kg的剂量以2天间隔(Q2D)皮下施用总共四周。以2天间隔(Q2D)皮下施用10nmol/kg的B13a/B13b(美国专利号9920118)和30nmol/kg的索马鲁肽(semaglutide)作为参考物，总共4周。对于载剂处理，以2天间隔(Q2D)皮下施用Dulbecco’s磷酸盐缓冲盐水(DPBS；Gibco,USA)。从药物处理的第1天至第28天，每两天测量一次体重，结果示于下表12。
[表12]
作为结果，证实了与作为参照药的10nmol/kg的B13a/B13b比较，所有具有不同接头类型的供试品(FM9-4+Fc_hole、FM9-6+Fc_hole、FM11-4+Fc_hole和FM11-6+Fc_hole)都表现出显著的体重减轻作用。此外，3个供试品FM9-4+Fc_hole、FM9-6+Fc_hole和FM11-4+Fc_hole显示出与30nmol/kg索马鲁肽处理组相似的体重减轻作用(图11)。
实施例10.5：在饮食诱导的肥胖小鼠中通过单次施用的经优化的长效GDF15融合蛋白(二聚体)的抗肥胖作用的评价
6周龄的雄性C57BL/6N小鼠购自Orient Bio(通过Hallym Lab.Animal Inc.,Republic of Korea)。到达后，C57BL/6N小鼠通过饲喂高脂饮食(60千卡％脂肪,ResearchDiets,Cat#D12492,USA)共13周诱导DIO。在给药开始的前一天，基于个体小鼠的平均体重将动物分组(每组n＝6)，然后将1、3、10和30nmol/kg的FM9-6+Fc_hole经皮下施用一次。作为参考物，30nmol/kg的索马鲁肽经皮下施用一次。对于载剂处理，Dulbecco’s磷酸盐缓冲盐水(DPBS；Gibco,USA)经皮下施用。从药物处理当天至第42天每天测量体重，结果示于下表13中。
[表13]
作为结果，在通过单次施用的体重减轻作用方面中，索马鲁肽30nmol/kg处理组作为参照品，显示出持续2天的药理作用，而单次施用1、3、10和30nmol/kg的FM9-6+Fc_hole证实了各剂量的抗肥胖作用分别持续10天、15天、18天和18天(图12)。
实施例10.6：在ob/ob小鼠中通过重复施用的经优化的长效GDF15融合蛋白(二聚体)的抗肥胖作用的评价
ob/ob小鼠在遗传上缺乏瘦素基因并以高血糖、胰岛素抵抗、食欲过盛和肥胖为特征。5周龄的雄性ob/ob小鼠(Jackson Laboratory,USA)购自Raon Bio(Animal Inc.,Republic of Korea)。用正常食物饮食(Teklad Certified Irradiated Global 18％Protein Rodent Diet,2918C,Harlan Co.,USA)使小鼠适应4周，并在9周龄时开始药物处理。在给药开始之前一天，基于平均体重和通过个体小鼠尾静脉的随机血糖将动物分组(每组n＝6)。然后，以3天间隔(Q3D)皮下施用0.1、1、3nmol/kg的FM9-6+Fc_hole和10nmol/kg的索马鲁肽共10次，并且体重和摄食量分别在实验期间(第1天至第29天)每天或每3天测量一次。对于载剂处理，施用Dulbecco’s磷酸盐缓冲盐水(DPBS；Gibco,USA)。
[表14]
作为结果，证实了FM9-6+Fc_hole以剂量依赖性方式表现出体重减轻作用。0.1nmol/kg FM9-6+Fc_hole处理组显示出与10nmol/kg索马鲁肽处理组相似的体重显著减轻。1nmol/kg或更高的FM9-6+Fc_hole在ob/ob小鼠中表现出最大效力(图13)。
<110> YUHAN CORPORATION
<120> 长效GDF15融合蛋白及包含其的药物组合物
<130> PCC008069YUH
<150> KR 10-2019-0153680
<151> 2019-11-26
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85 90 95
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100 105 110
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Asn Ser Thr Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr
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Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu Gln
85 90 95
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Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His
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Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser Leu His
50 55 60
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85 90 95
Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
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Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
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Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu
20 25 30
Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser
35 40 45
Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Lys Leu His
50 55 60
Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser
65 70 75 80
Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu
85 90 95
Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
100 105 110
<210> 26
<211> 111
<212> PRT
<213> 人工序列
<220>
<223> GDF15变体(ΔN3, WS插入, G4N, D5S, H6T, S64K / FM6)
<400> 26
Trp Ser Asn Ser Thr Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu
1 5 10 15
His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val
20 25 30
Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro
35 40 45
Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Lys Leu
50 55 60
His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala
65 70 75 80
Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser
85 90 95
Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
100 105 110
<210> 27
<211> 109
<212> PRT
<213> 人工序列
<220>
<223> GDF15变体(ΔN3, G4N, D5S, H6T, S64K / FM7)
<400> 27
Asn Ser Thr Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr
1 5 10 15
Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu Ser
20 25 30
Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser Gln
35 40 45
Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Lys Leu His Arg
50 55 60
Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr
65 70 75 80
Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu Gln
85 90 95
Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
100 105
<210> 28
<211> 110
<212> PRT
<213> 人工序列
<220>
<223> GDF15变体(ΔN2, S64R / FM9)
<400> 28
Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His
1 5 10 15
Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu
20 25 30
Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser
35 40 45
Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Arg Leu His
50 55 60
Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser
65 70 75 80
Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu
85 90 95
Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
100 105 110
<210> 29
<211> 111
<212> PRT
<213> 人工序列
<220>
<223> GDF15变体(ΔN3, WS插入, G4N, D5S, H6T, S64R/ FM10)
<400> 29
Trp Ser Asn Ser Thr Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu
1 5 10 15
His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val
20 25 30
Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro
35 40 45
Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Arg Leu
50 55 60
His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala
65 70 75 80
Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser
85 90 95
Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
100 105 110
<210> 30
<211> 109
<212> PRT
<213> 人工序列
<220>
<223> GDF15变体(ΔN3, G4N, D5S, H6T, S64R/ FM11)
<400> 30
Asn Ser Thr Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr
1 5 10 15
Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu Ser
20 25 30
Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser Gln
35 40 45
Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Arg Leu His Arg
50 55 60
Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr
65 70 75 80
Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu Gln
85 90 95
Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
100 105
<210> 31
<211> 110
<212> PRT
<213> 人工序列
<220>
<223> GDF15变体(ΔN2, S64N/ FM13)
<400> 31
Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His
1 5 10 15
Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu
20 25 30
Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser
35 40 45
Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Asn Leu His
50 55 60
Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser
65 70 75 80
Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu
85 90 95
Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
100 105 110
<210> 32
<211> 110
<212> PRT
<213> 人工序列
<220>
<223> GDF15变体(ΔN2,S64D/ FM14)
<400> 32
Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His
1 5 10 15
Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu
20 25 30
Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser
35 40 45
Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Asp Leu His
50 55 60
Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser
65 70 75 80
Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu
85 90 95
Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
100 105 110
<210> 33
<211> 110
<212> PRT
<213> 人工序列
<220>
<223> GDF15变体(ΔN2,S64E/ FM15)
<400> 33
Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His
1 5 10 15
Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu
20 25 30
Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser
35 40 45
Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Glu Leu His
50 55 60
Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser
65 70 75 80
Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu
85 90 95
Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
100 105 110
<210> 34
<211> 110
<212> PRT
<213> 人工序列
<220>
<223> GDF15变体(ΔN2,S64L / FM16)
<400> 34
Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His
1 5 10 15
Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu
20 25 30
Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser
35 40 45
Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Leu Leu His
50 55 60
Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser
65 70 75 80
Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu
85 90 95
Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
100 105 110
<210> 35
<211> 110
<212> PRT
<213> 人工序列
<220>
<223> GDF15变体(ΔN2, D103L / FM4-2)
<400> 35
Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His
1 5 10 15
Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu
20 25 30
Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser
35 40 45
Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser Leu His
50 55 60
Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser
65 70 75 80
Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu
85 90 95
Gln Thr Tyr Asp Leu Leu Leu Ala Lys Asp Cys His Cys Ile
100 105 110
<210> 36
<211> 110
<212> PRT
<213> 人工序列
<220>
<223> GDF15变体(ΔN2, N56C, D103C / FM4)
<400> 36
Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His
1 5 10 15
Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu
20 25 30
Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser
35 40 45
Gln Phe Arg Ala Ala Cys Met His Ala Gln Ile Lys Thr Ser Leu His
50 55 60
Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser
65 70 75 80
Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu
85 90 95
Gln Thr Tyr Asp Cys Leu Leu Ala Lys Asp Cys His Cys Ile
100 105 110
<210> 37
<211> 110
<212> PRT
<213> 人工序列
<220>
<223> GDF15变体(ΔN2, N56S, D103S/ FM4-1)
<400> 37
Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His
1 5 10 15
Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu
20 25 30
Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser
35 40 45
Gln Phe Arg Ala Ala Ser Met His Ala Gln Ile Lys Thr Ser Leu His
50 55 60
Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser
65 70 75 80
Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu
85 90 95
Gln Thr Tyr Asp Ser Leu Leu Ala Lys Asp Cys His Cys Ile
100 105 110
<210> 38
<211> 110
<212> PRT
<213> 人工序列
<220>
<223> GDF15变体(ΔN2, R21N, S64K / FM8)
<400> 38
Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His
1 5 10 15
Thr Val Asn Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu
20 25 30
Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser
35 40 45
Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Lys Leu His
50 55 60
Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser
65 70 75 80
Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu
85 90 95
Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
100 105 110
<210> 39
<211> 110
<212> PRT
<213> 人工序列
<220>
<223> GDF15变体(ΔN2, R21N, S64R / FM12)
<400> 39
Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His
1 5 10 15
Thr Val Asn Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu
20 25 30
Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser
35 40 45
Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Arg Leu His
50 55 60
Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser
65 70 75 80
Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu
85 90 95
Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
100 105 110
<210> 40
<211> 110
<212> PRT
<213> 人工序列
<220>
<223> GDF15 (ΔN2)
<400> 40
Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His
1 5 10 15
Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu
20 25 30
Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser
35 40 45
Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser Leu His
50 55 60
Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser
65 70 75 80
Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu
85 90 95
Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
100 105 110
<210> 41
<211> 227
<212> PRT
<213> 人工序列
<220>
<223> 人IgG1 Fc
<400> 41
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225
<210> 42
<211> 217
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(A-1)
<400> 42
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215
<210> 43
<211> 217
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366S, L368A,
Y407V)-(A-2)
<400> 43
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215
<210> 44
<211> 217
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A, N297A)(T366W)-(B-1)
<400> 44
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215
<210> 45
<211> 217
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A, N297A)(T366S, L368A,
Y407V)-(B-2)
<400> 45
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215
<210> 46
<211> 217
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(L234A, L235A, N297A)(T366W)-(C-1)
<400> 46
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215
<210> 47
<211> 217
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(L234A, L235A, N297A)(T366S, L368A,
Y407V)-(C-2)
<400> 47
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215
<210> 48
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 接头-(G4S)5
<400> 48
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25
<210> 49
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A,
L235A)(T366W)-(G4S)5-GDF15(ΔN2) - FWT
<400> 49
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 50
<211> 353
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN3, WS插入, G4N, D5S, H6T)-FM1
<400> 50
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Trp Ser Asn Ser Thr Cys Pro Leu Gly Pro Gly Arg Cys Cys
245 250 255
Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp
260 265 270
Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala
275 280 285
Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr
290 295 300
Ser Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val
305 310 315 320
Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly
325 330 335
Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys
340 345 350
Ile
<210> 51
<211> 351
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN3, G4N, D5S, H6T)-FM2
<400> 51
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Ser Thr Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu
245 250 255
His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val
260 265 270
Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro
275 280 285
Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser Leu
290 295 300
His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala
305 310 315 320
Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser
325 330 335
Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 52
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, R21N)-FM3
<400> 52
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Asn Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 53
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, N56L)-FM4-3
<400> 53
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Leu Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 54
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, S64K)-FM5
<400> 54
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Lys
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 55
<211> 353
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN3, WS插入, G4N, D5S, H6T, S64K)-FM6
<400> 55
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Trp Ser Asn Ser Thr Cys Pro Leu Gly Pro Gly Arg Cys Cys
245 250 255
Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp
260 265 270
Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala
275 280 285
Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr
290 295 300
Lys Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val
305 310 315 320
Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly
325 330 335
Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys
340 345 350
Ile
<210> 56
<211> 351
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN3, G4N, D5S, H6T, S64K)-FM7
<400> 56
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Ser Thr Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu
245 250 255
His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val
260 265 270
Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro
275 280 285
Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Lys Leu
290 295 300
His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala
305 310 315 320
Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser
325 330 335
Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 57
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, S64R)-FM9
<400> 57
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Arg
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 58
<211> 353
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN3, WS插入, G4N, D5S, H6T, S64R)-FM10
<400> 58
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Trp Ser Asn Ser Thr Cys Pro Leu Gly Pro Gly Arg Cys Cys
245 250 255
Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp
260 265 270
Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala
275 280 285
Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr
290 295 300
Arg Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val
305 310 315 320
Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly
325 330 335
Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys
340 345 350
Ile
<210> 59
<211> 351
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN3, G4N, D5S, H6T, S64R)-FM11
<400> 59
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Ser Thr Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu
245 250 255
His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val
260 265 270
Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro
275 280 285
Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Arg Leu
290 295 300
His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala
305 310 315 320
Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser
325 330 335
Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 60
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, S64N/ FM13)
<400> 60
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Asn
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 61
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2,S64D)-FM14
<400> 61
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Asp
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 62
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2,S64E)-FM15
<400> 62
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Glu
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 63
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2,S64L)-FM16
<400> 63
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Leu
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 64
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, D103L)-FM4-2
<400> 64
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Leu Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 65
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, N56C, D103C)-FM4
<400> 65
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Cys Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Cys Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 66
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, N56S, D103S)-FM4-1
<400> 66
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Ser Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Ser Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 67
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, R21N, S64K)-FM8
<400> 67
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Asn Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Lys
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 68
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, R21N, S64R)-FM12
<400> 68
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Asn Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Arg
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 69
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, W32F)
<400> 69
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Phe
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 70
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, W32H)
<400> 70
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp His
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 71
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, W32Y)
<400> 71
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Tyr
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 72
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, Q51H)
<400> 72
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser His Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 73
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, Q51L)
<400> 73
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Leu Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 74
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, Q51E)
<400> 74
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Glu Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 75
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, Q51N)
<400> 75
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Asn Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 76
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, Q90H)
<400> 76
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile His Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 77
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, Q90E)
<400> 77
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Glu Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 78
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, Q90K)
<400> 78
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Lys Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 79
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, D93E)
<400> 79
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Glu Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 80
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, D93L)
<400> 80
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Leu Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 81
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, D93N)
<400> 81
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asn Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 82
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, D93Q)
<400> 82
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Gln Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 83
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, Q60L)
<400> 83
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Leu Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 84
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2,Q60N)
<400> 84
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Asn Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 85
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, S64Q)
<400> 85
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Gln
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 86
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, T92S)
<400> 86
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Ser Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 87
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, T92E)
<400> 87
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Glu Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 88
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, S97N)
<400> 88
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Asn Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 89
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, S97Q)
<400> 89
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Gln Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 90
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2,Y101F)
<400> 90
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Phe Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 91
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(E233A, L235A)(T366W)-(G4S)5-GDF15
变体(ΔN2, Y101Q)
<400> 91
Ala Pro Ala Leu Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Gln Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 92
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 接头-GS(G4S)3
<400> 92
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10 15
Ser
<210> 93
<211> 27
<212> PRT
<213> 人工序列
<220>
<223> 接头-GS(G4S)5
<400> 93
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10 15
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25
<210> 94
<211> 37
<212> PRT
<213> 人工序列
<220>
<223> 接头-GS(G4S)7
<400> 94
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10 15
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25 30
Gly Gly Gly Gly Ser
35
<210> 95
<211> 26
<212> PRT
<213> 人工序列
<220>
<223> 接头-GS(EEEA)6
<400> 95
Gly Ser Glu Glu Glu Ala Glu Glu Glu Ala Glu Glu Glu Ala Glu Glu
1 5 10 15
Glu Ala Glu Glu Glu Ala Glu Glu Glu Ala
20 25
<210> 96
<211> 26
<212> PRT
<213> 人工序列
<220>
<223> 接头-GSGGSS(PT)10
<400> 96
Gly Ser Gly Gly Ser Ser Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr
1 5 10 15
Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr
20 25
<210> 97
<211> 27
<212> PRT
<213> 人工序列
<220>
<223> 接头-GS(EAAAK)5
<400> 97
Gly Ser Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala
1 5 10 15
Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys
20 25
<210> 98
<211> 344
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(L234A, L235A,
N297A)(T366W)-GS(G4S)3-GDF15变体(ΔN2, S64R / FM9-1)
<400> 98
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Gly Asp His Cys Pro
225 230 235 240
Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr Val Arg Ala Ser Leu
245 250 255
Glu Asp Leu Gly Trp Ala Asp Trp Val Leu Ser Pro Arg Glu Val Gln
260 265 270
Val Thr Met Cys Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn
275 280 285
Met His Ala Gln Ile Lys Thr Arg Leu His Arg Leu Lys Pro Asp Thr
290 295 300
Val Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val Leu
305 310 315 320
Ile Gln Lys Thr Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu
325 330 335
Leu Ala Lys Asp Cys His Cys Ile
340
<210> 99
<211> 354
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(L234A, L235A,
N297A)(T366W)-GS(G4S)5-GDF15变体(ΔN2, S64R / FM9-2)
<400> 99
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
225 230 235 240
Gly Gly Gly Ser Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys
245 250 255
Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala
260 265 270
Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly
275 280 285
Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys
290 295 300
Thr Arg Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys
305 310 315 320
Val Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr
325 330 335
Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His
340 345 350
Cys Ile
<210> 100
<211> 364
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(L234A, L235A,
N297A)(T366W)-GS(G4S)7-GDF15变体(ΔN2, S64R / FM9-3)
<400> 100
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
225 230 235 240
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Gly
245 250 255
Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr Val
260 265 270
Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu Ser Pro
275 280 285
Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser Gln Phe
290 295 300
Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Arg Leu His Arg Leu
305 310 315 320
Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn
325 330 335
Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu Gln Thr
340 345 350
Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
355 360
<210> 101
<211> 353
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(L234A, L235A,
N297A)(T366W)-GS(EEEA)6-GDF15变体(ΔN2, S64R / FM9-4)
<400> 101
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Ser Glu Glu Glu Ala Glu
210 215 220
Glu Glu Ala Glu Glu Glu Ala Glu Glu Glu Ala Glu Glu Glu Ala Glu
225 230 235 240
Glu Glu Ala Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys
245 250 255
Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp
260 265 270
Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala
275 280 285
Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr
290 295 300
Arg Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val
305 310 315 320
Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly
325 330 335
Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys
340 345 350
Ile
<210> 102
<211> 353
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(L234A, L235A,
N297A)(T366W)-GSGGSS(PT)10-GDF15变体(ΔN2, S64R / FM9-5)
<400> 102
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Ser Gly Gly Ser Ser Pro
210 215 220
Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro
225 230 235 240
Thr Pro Thr Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys
245 250 255
Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp
260 265 270
Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala
275 280 285
Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr
290 295 300
Arg Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val
305 310 315 320
Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly
325 330 335
Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys
340 345 350
Ile
<210> 103
<211> 354
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(L234A, L235A,
N297A)(T366W)-GS(EAAAK)5-GDF15变体(ΔN2, S64R / FM9-6)
<400> 103
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Ser Glu Ala Ala Ala Lys
210 215 220
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu
225 230 235 240
Ala Ala Ala Lys Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys
245 250 255
Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala
260 265 270
Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly
275 280 285
Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys
290 295 300
Thr Arg Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys
305 310 315 320
Val Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr
325 330 335
Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His
340 345 350
Cys Ile
<210> 104
<211> 343
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(L234A, L235A,
N297A)(T366W)-GS(G4S)3-GDF15变体(ΔN3, G4N, D5S, H6T,
S64R/ FM11-1)
<400> 104
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Ser Thr Cys Pro Leu
225 230 235 240
Gly Pro Gly Arg Cys Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu
245 250 255
Asp Leu Gly Trp Ala Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val
260 265 270
Thr Met Cys Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met
275 280 285
His Ala Gln Ile Lys Thr Arg Leu His Arg Leu Lys Pro Asp Thr Val
290 295 300
Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val Leu Ile
305 310 315 320
Gln Lys Thr Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu
325 330 335
Ala Lys Asp Cys His Cys Ile
340
<210> 105
<211> 353
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(L234A, L235A,
N297A)(T366W)-GS(G4S)5-GDF15变体(ΔN3, G4N, D5S, H6T,
S64R/ FM11-2)
<400> 105
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
225 230 235 240
Gly Gly Gly Ser Asn Ser Thr Cys Pro Leu Gly Pro Gly Arg Cys Cys
245 250 255
Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp
260 265 270
Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala
275 280 285
Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr
290 295 300
Arg Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val
305 310 315 320
Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly
325 330 335
Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys
340 345 350
Ile
<210> 106
<211> 363
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(L234A, L235A,
N297A)(T366W)-GS(G4S)7-GDF15变体(ΔN3, G4N, D5S, H6T,
S64R/ FM11-3)
<400> 106
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
225 230 235 240
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Ser
245 250 255
Thr Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr Val Arg
260 265 270
Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu Ser Pro Arg
275 280 285
Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser Gln Phe Arg
290 295 300
Ala Ala Asn Met His Ala Gln Ile Lys Thr Arg Leu His Arg Leu Lys
305 310 315 320
Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn Pro
325 330 335
Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu Gln Thr Tyr
340 345 350
Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
355 360
<210> 107
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(L234A, L235A,
N297A)(T366W)-GS(EEEA)6-GDF15变体(ΔN3, G4N, D5S, H6T,
S64R/ FM11-4)
<400> 107
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Ser Glu Glu Glu Ala Glu
210 215 220
Glu Glu Ala Glu Glu Glu Ala Glu Glu Glu Ala Glu Glu Glu Ala Glu
225 230 235 240
Glu Glu Ala Asn Ser Thr Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Arg
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 108
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(L234A, L235A,
N297A)(T366W)-GSGGSS(PT)10-GDF15变体(ΔN3, G4N, D5S,
H6T, S64R/ FM11-5)
<400> 108
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Ser Gly Gly Ser Ser Pro
210 215 220
Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro
225 230 235 240
Thr Pro Thr Asn Ser Thr Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg
245 250 255
Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp
260 265 270
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys
275 280 285
Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Arg
290 295 300
Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
305 310 315 320
Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val
325 330 335
Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile
340 345 350
<210> 109
<211> 353
<212> PRT
<213> 人工序列
<220>
<223> 铰链删除的人IgG1-Fc(L234A, L235A,
N297A)(T366W)-GS(EAAAK)5-GDF15变体(ΔN3, G4N, D5S, H6T,
S64R/ FM11-6)
<400> 109
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
1 5 10 15
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
50 55 60
Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
65 70 75 80
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
115 120 125
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
145 150 155 160
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205
Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Ser Glu Ala Ala Ala Lys
210 215 220
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu
225 230 235 240
Ala Ala Ala Lys Asn Ser Thr Cys Pro Leu Gly Pro Gly Arg Cys Cys
245 250 255
Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp
260 265 270
Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala
275 280 285
Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr
290 295 300
Arg Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val
305 310 315 320
Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly
325 330 335
Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys
340 345 350
Ile
Claims (25)
1.式(I)所示的GDF15变体：
N端延伸结构域-核心结构域(I)
(在式(I)中，
所述N端延伸结构域是由SEQ ID NO:3至5的任一氨基酸序列组成的多肽；以及
所述核心结构域是SEQ ID NO:20所示的多肽，或者是衍生自SEQ ID NO:20的多肽且其中选自在SEQ ID NO:20的氨基酸序列中的第15、50、58、97位的氨基酸及其组合的任一氨基酸被另外的氨基酸替换；
其中第15位氨基酸精氨酸(R)被丙氨酸(A)、天冬氨酸(D)、天冬酰胺(N)、半胱氨酸(C)、谷氨酸(E)、谷氨酰胺(Q)、甘氨酸(G)、组氨酸(H)、异亮氨酸(I)、亮氨酸(L)、赖氨酸(K)、甲硫氨酸(M)、苯丙氨酸(F)、脯氨酸(P)、丝氨酸(S)、苏氨酸(T)、色氨酸(W)、酪氨酸(Y)或缬氨酸(V)替换，
第50位氨基酸天冬酰胺(N)被丙氨酸、精氨酸(R)、天冬氨酸、半胱氨酸、谷氨酸、谷氨酰胺、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸或缬氨酸替换，
第58位氨基酸丝氨酸(S)被丙氨酸、精氨酸、天冬氨酸、天冬酰胺、半胱氨酸、谷氨酸、谷氨酰胺、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、苏氨酸、色氨酸、酪氨酸或缬氨酸替换，以及
第97位氨基酸天冬氨酸(D)被丙氨酸、精氨酸、天冬氨酸、半胱氨酸、谷氨酸、谷氨酰胺、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸或缬氨酸替换)。
2.权利要求1所述的GDF15变体，其中所述核心结构域包含选自以下改变(1)至(6)中的任一改变：
(1)SEQ ID NO:20的氨基酸序列中第15位氨基酸精氨酸(R)被天冬酰胺(N)替换的改变；
(2)SEQ ID NO:20的氨基酸序列中第50位氨基酸天冬酰胺(N)被亮氨酸(L)替换的改变；
(3)SEQ ID NO:20的氨基酸序列中第58位氨基酸丝氨酸(S)被赖氨酸(K)、精氨酸(R)、天冬酰胺(N)、天冬氨酸(D)、谷氨酸(E)、半胱氨酸(C)或亮氨酸(L)替换的改变；
(4)SEQ ID NO:20的氨基酸序列中第97位氨基酸天冬氨酸(D)被亮氨酸(L)替换的改变；
(5)SEQ ID NO:20的氨基酸序列中第50位氨基酸天冬酰胺(N)和SEQ ID NO:20的氨基酸序列中第97位氨基酸天冬氨酸(D)各自被半胱氨酸(C)或丝氨酸(S)替换的改变；以及
(6)SEQ ID NO:20的氨基酸序列中第15位氨基酸精氨酸(R)被天冬酰胺(N)替换并且SEQ ID NO:20的氨基酸序列中第58位氨基酸丝氨酸(S)被赖氨酸(K)或精氨酸(R)替换的改变。
3.权利要求2所述的GDF15变体，其中所述核心结构域由选自SEQ ID NO:6至19的任一氨基酸序列组成。
4.权利要求1所述的GDF15变体，其中所述GDF15变体包含由SEQ ID NO:3所示的氨基酸序列组成的N端延伸结构域和由SEQ ID NO:8、9或20所示的氨基酸序列组成的核心结构域。
5.权利要求1所述的GDF15变体，其中所述GDF15变体包含由SEQ ID NO:4所示的氨基酸序列组成的N端延伸结构域和由SEQ ID NO:8、9或20所示的氨基酸序列组成的核心结构域。
6.权利要求1所述的GDF15变体，其中所述GDF15变体包含由SEQ ID NO:5所示的氨基酸序列组成的N端延伸结构域和由选自SEQ ID NO:6至19的任一氨基酸序列组成的核心结构域。
7.权利要求1所述的GDF15变体，其中所述GDF15变体由选自SEQ ID NO:21至39的任一氨基酸序列组成。
8.长效GDF15融合蛋白，其中权利要求1至7中任一项所述的GDF15变体与人IgG Fc或其变体结合。
9.权利要求8所述的长效GDF15融合蛋白，其中所述IgG Fc或其变体包含：
包含IgG1 Fc序列的第一多肽，所述IgG Fc序列包含CH3序列，所述CH3序列包含至少一个工程化突起；和
包含IgG1 Fc序列的第二多肽，所述IgG Fc序列包含CH3序列，所述CH3序列包含至少一个工程化腔，
其中所述第一多肽通过将所述第一多肽的所述突起定位至所述第二多肽的所述腔中来与所述第二多肽发生二聚化。
10.权利要求8所述的长效GDF15融合蛋白，其中所述结合是这样的：所述IgG Fc或其变体中的所述第一多肽的C端或所述第二多肽的C端与所述GDF15变体的N端结合。
11.权利要求8所述的长效GDF15融合蛋白，其中所述GDF15变体与所述IgG Fc或其变体之间的结合是通过接头进行的。
12.权利要求11所述的长效GDF15融合蛋白，其中所述接头是由包含甘氨酸、丝氨酸、丙氨酸和谷氨酸残基的10至50个氨基酸残基组成的肽。
13.权利要求12所述的长效GDF15融合蛋白，其中所述接头是GGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO：48)、GSGGGGSGGGGSGGGGS(SEQ ID NO：92)、GSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO：93)、GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO：94)、GSEEEAEEEAEEEAEEEAEEEAEEEA(SEQ ID NO：95)、GSGGSSPTPTPTPTPTPTPTPTPTPT(SEQ ID NO：96)或GSEAAAKEAAAKEAAAKEAAAKEAAAK(SEQ IDNO：97)。
14.权利要求9所述的长效GDF15融合蛋白，其中所述第一多肽由选自SEQ ID NO：42、44和46的任一氨基酸序列组成。
15.权利要求9所述的长效GDF15融合蛋白，其中所述第二多肽由选自SEQ ID NO：43、45和47的任一氨基酸序列组成。
16.权利要求8所述的长效GDF15融合蛋白，其中所述GDF15变体包含至少一个N-连接聚糖。
17.权利要求8所述的长效GDF15融合蛋白，其中所述长效GDF15融合蛋白包含i)由选自SEQ ID NO:21至39的任一氨基酸序列组成的GDF15变体，ii)由选自SEQ ID NO:42、44和46的任一氨基酸序列组成的第一多肽，以及iii)由选自SEQ ID NO:43、45和47的任一氨基酸序列组成的第二多肽。
18.融合蛋白二聚体，其包含：
两种权利要求8所述的长效GDF15融合蛋白。
19.分离的核酸分子，其编码权利要求1至7中任一项所述的GDF15变体或权利要求8至17中任一项所述的长效GDF15融合蛋白。
20.表达载体，其包含：
权利要求19所述的核酸分子。
21.宿主细胞，其包含：
权利要求20所述的表达载体。
22.药物组合物，其用于预防或治疗糖尿病、肥胖、血脂异常或代谢综合征，其包含作为活性成分的以下：
权利要求1至7中任一项所述的GDF15变体、权利要求8至17中任一项所述的长效GDF15融合蛋白或权利要求18所述的融合蛋白二聚体。
23.权利要求1至7中任一项所述的GDF15变体、权利要求8至17中任一项所述的长效GDF15融合蛋白或权利要求18所述的融合蛋白二聚体用于预防或治疗糖尿病、肥胖、血脂异常或代谢综合征的用途。
24.权利要求1至7中任一项所述的GDF15变体、权利要求8至17中任一项所述的长效GDF15融合蛋白或权利要求18所述的融合蛋白二聚体用于制备用于预防或治疗糖尿病、肥胖、血脂异常或代谢综合征的药物的用途。
25.用于治疗或预防糖尿病、肥胖、血脂异常或代谢综合征的方法，其包括：
向个体施用权利要求1至7中任一项所述的GDF15变体、权利要求8至17中任一项所述的长效GDF15融合蛋白或权利要求18所述的融合蛋白二聚体的步骤。
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| PCT/KR2020/016842 WO2021107603A2 (en) | 2019-11-26 | 2020-11-25 | Long-acting gdf15 fusion protein and pharmaceutical composition comprising same |
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| KR20240087545A (ko) * | 2022-11-18 | 2024-06-19 | 주식회사유한양행 | 이중 작용 단백질 및 이의 용도 |
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| PT2929891T (pt) | 2004-04-13 | 2020-04-08 | St Vincents Hospital Sydney Ltd | Método para modulação do apetite |
| WO2013113008A1 (en) * | 2012-01-26 | 2013-08-01 | Amgen Inc. | Growth differentiation factor 15 (gdf-15) polypeptides |
| US9161966B2 (en) * | 2013-01-30 | 2015-10-20 | Ngm Biopharmaceuticals, Inc. | GDF15 mutein polypeptides |
| EP3763734A1 (en) * | 2013-07-31 | 2021-01-13 | Amgen Inc. | Growth differentiation factor 15 (gdf-15) constructs |
| KR20170065026A (ko) * | 2014-07-30 | 2017-06-12 | 엔지엠 바이오파마슈티컬스, 아이엔씨. | 대사 장애 치료용으로 이용되는 조성물 및 방법 |
| JP6663428B2 (ja) * | 2014-10-30 | 2020-03-11 | アクセルロン ファーマ， インコーポレイテッド | Ｇｄｆ１５ポリペプチドを使用する、赤血球を増加させるための方法および組成物 |
| MY186702A (en) * | 2014-10-31 | 2021-08-11 | Ngm Biopharmaceuticals Inc | Compositions and methods of use for treating metabolic disorders |
| US10336812B2 (en) * | 2016-05-10 | 2019-07-02 | Janssen Biotech, Inc. | GDF15 fusion proteins and uses thereof |
| KR101727506B1 (ko) * | 2016-07-14 | 2017-05-04 | 충남대학교 산학협력단 | Gdf15 단백질 또는 이를 암호화하는 폴리뉴클레오티드를 유효성분으로 함유하는 지방간의 예방 또는 치료용 약학적 조성물 |
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| US20230002460A1 (en) | 2023-01-05 |
| WO2021107603A3 (en) | 2021-07-15 |
| JP2023503472A (ja) | 2023-01-30 |
| ZA202204624B (en) | 2023-11-29 |
| WO2021107603A2 (en) | 2021-06-03 |
| KR20210065057A (ko) | 2021-06-03 |
| BR112022010227A2 (pt) | 2022-09-13 |
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| EP4065597A4 (en) | 2024-01-24 |
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